Antisense Drug Technology: Principles, Strategies, and Applications, Second Edition

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Second Edition

Antisense Drug Technology

Principles, Strategies, and Applications

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Second Edition

Antisense Drug Technology

Principles, Strategies, and Applications

Edited by

Stanley T. Crooke

Boca Raton London New York

CRC Press is an imprint of the Taylor & Francis Group, an informa business

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CRC Press Taylor & Francis Group 6000 Broken Sound Parkway NW, Suite 300 Boca Raton, FL 33487-2742 © 2008 by Taylor & Francis Group, LLC CRC Press is an imprint of Taylor & Francis Group, an Informa business No claim to original U.S. Government works Printed in the United States of America on acid-free paper 10 9 8 7 6 5 4 3 2 1 International Standard Book Number-10: 0-8493-8796-5 (Hardcover) International Standard Book Number-13: 978-0-8493-8796-8 (Hardcover) This book contains information obtained from authentic and highly regarded sources. Reprinted material is quoted with permission, and sources are indicated. A wide variety of references are listed. Reasonable efforts have been made to publish reliable data and information, but the author and the publisher cannot assume responsibility for the validity of all materials or for the consequences of their use. No part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or retrieval system, without written permission from the publishers. For permission to photocopy or use material electronically from this work, please access www.copyright.com (http:// www.copyright.com/) or contact the Copyright Clearance Center, Inc. (CCC) 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. CCC is a not-for-profit organization that provides licenses and registration for a variety of users. For organizations that have been granted a photocopy license by the CCC, a separate system of payment has been arranged. Trademark Notice: Product or corporate names may be trademarks or registered trademarks, and are used only for identification and explanation without intent to infringe. Library of Congress Cataloging-in-Publication Data Antisense drug technology : principles, strategies, and applications / editor Stanley T. Crooke. -- 2nd ed. p. ; cm. Includes bibliographical references and index. ISBN-13: 978-0-8493-8796-8 (alk. paper) ISBN-10: 0-8493-8796-5 (alk. paper) 1. Antisense nucleic acids--Therapeutic use. I. Crooke, Stanley T. [DNLM: 1. Oligonucleotides, Antisense--therapeutic use. QU 57 A6324 2006] I. Title. RM666.A564A567 2006 615’.31--dc22 Visit the Taylor & Francis Web site at http://www.taylorandfrancis.com and the CRC Press Web site at http://www.crcpress.com

2006101712

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Contents Preface ..............................................................................................................................................ix Acknowledgments............................................................................................................................xi The Editor......................................................................................................................................xiii Contributors....................................................................................................................................xv Part I Introduction ........................................................................................................................................1 Chapter 1 Mechanisms of Antisense Drug Action, an Introduction...................................................................3 Stanley T. Crooke, Timothy Vickers, Walt Lima, and Hongjiang Wu Chapter 2 The RNase H Mechanism ................................................................................................................47 Walt Lima, Hongjiang Wu, and Stanley T. Crooke Chapter 3 Small RNA Silencing Pathways.......................................................................................................75 Alla Sigova and Phillip D. Zamore Chapter 4 Splice Switching Oligonucleotides as Potential Therapeutics.........................................................89 Peter Sazani, Maria A. Graziewicz, and Ryszard Kole Part II The Basics of Oligonucleotide-Based Therapeutics ......................................................................115 Chapter 5 Basic Principles of Antisense Drug Discovery ..............................................................................117 Susan M. Freier and Andrew T. Watt Chapter 6 The Medicinal Chemistry of Oligonucleotides..............................................................................143 Eric E. Swayze and Balkrishen Bhat Chapter 7 Basic Principles of the Pharmacokinetics of Antisense Oligonucleotide Drugs ...........................183 Arthur A. Levin, Rosie Z. Yu, and Richard S. Geary Chapter 8 Routes and Formulations for Delivery of Antisense Oligonucleotides .........................................217 Gregory E. Hardee, Lloyd G. Tillman, and Richard S. Geary Chapter 9 Liposomal Formulations for Nucleic Acid Delivery......................................................................237 Ian MacLachlan v

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Part III A Hybridization-Based Drugs: Basic Properties 2⬘-O-Methoxyethyl Oligonucleotides ..................271 Chapter 10 Pharmacological Properties of 2⬘-O-Methoxyethyl-Modified Oligonucleotides ..........................273 C. Frank Bennett Chapter 11 Pharmacokinetic/Pharmacodynamic Properties of Phosphorothioate 2⬘-O-(2-Methoxyethyl)Modified Antisense Oligonucleotides in Animals and Man .........................................................305 Richard S. Geary, Rosie Z. Yu, Andrew Siwkowski, and Arthur A. Levin Chapter 12 Toxicologic Properties of 2⬘-O-Methoxyethyl Chimeric Antisense Inhibitors in Animals and Man .......................................................................................................................327 Scott P. Henry, Tae-Won Kim, Kimberly Kramer-Stickland, Thomas A. Zanardi, Robert A. Fey, and Arthur A. Levin Chapter 13 An Overview of the Clinical Safety Experience of First- and Second-Generation Antisense Oligonucleotides............................................................................................................365 T. Jesse Kwoh Chapter 14 Manufacturing and Analytical Processes for 2⬘-O-(2-Methoxyethyl)-Modified Oligonucleotides.............................................................................................................................401 Daniel C. Capaldi and Anthony N. Scozzari Part III B Hybridization-Based Drugs: Basic Properties Duplex RNA Drugs ..............................................435 Chapter 15 Utilizing Chemistry to Harness RNA Interference Pathways for Therapeutics: Chemically Modified siRNAs and Antagomirs .............................................................................437 Muthiah Manoharan and Kallanthottathil G. Rajeev Chapter 16 Discovery and Development of RNAi Therapeutics......................................................................465 Antonin R. de Fougerolles and John M. Maraganore Part IV Other Chemical Classes of Drugs ..................................................................................................485 Chapter 17 Optimization of Second-Generation Antisense Drugs: Going Beyond Generation 2.0 ................487 Brett P. Monia, Rosie Z. Yu, Walt Lima, and Andrew Siwkowski Chapter 18 Modulating Gene Function with Peptide Nucleic Acids (PNA)....................................................507 Peter E. Nielsen

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Chapter 19 Locked Nucleic Acid......................................................................................................................519 Troels Koch and Henrik Ørum Chapter 20 Morpholinos ...................................................................................................................................565 Patrick L. Iversen Part V Therapeutic Applications ...............................................................................................................583 Chapter 21 Potential Therapeutic Applications of Antisense Oligonucleotides in Ophthalmology ................585 Lisa R. Grillone and Scott P. Henry Chapter 22 Cardiovascular Therapeutic Applications ......................................................................................601 Rosanne Crooke, Brenda Baker, and Mark Wedel Chapter 23 Developing Antisense Drugs for Metabolic Diseases: A Novel Therapeutic Approach ...............641 Sanjay Bhanot Chapter 24 Inflammatory Diseases...................................................................................................................665 Susan A. Gregory and James G. Karras Chapter 25 Antisense Oligonucleotides for the Treatment of Cancer..............................................................699 Boris A. Hadaschik and Martin E. Gleave Chapter 26 Targeting Neurological Disorders with Antisense Oligonucleotides.............................................721 Richard A. Smith and Timothy M. Miller Chapter 27 Mechanisms and Therapeutic Applications of Immune Modulatory Oligodeoxynucleotide and Oligoribonucleotide Ligands for Toll-Like Receptors ............................................................747 Jörg Vollmer and Arthur M. Krieg Chapter 28 Aptamer Opportunities and Challenges .........................................................................................773 Charles Wilson Index...............................................................................................................................................801

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Preface At the conclusion of the preface for the first edition of this publication, I wrote: The chapters in this volume, recent publications, and recent symposia, such as the meeting sponsored by Nature Biotechnology, provide compelling answers to the questions about the technology. In the aggregate, the data provide ample justification for cautious optimism. It is clearly a remarkably valuable tool to dissect pharmacological processes and confirm the roles of various genes. Perhaps more importantly, even the first-generation compounds— the phosphorothioates—may have sufficient properties to be of use as drugs for selected indications, and new generations of antisense drugs may broaden the therapeutic utility of drugs based on antisense technology. Nevertheless, it is important to remember that we are less than a decade into the aggressive creation and evaluation of antisense technology. And we are attempting to create an entirely new branch of pharmacology: new chemical class, oligonucleotides; a new receptor, RNA; a new drug–receptor binding motif, hybridization; and new postreceptor binding mechanisms. Thus there are still many more questions than answers. Arguably, then, we are at the end of the beginning of this technology. There is a great deal more to do before we understand the true value and limits of antisense, but we are buoyed by the progress to date and look forward to the challenges ahead.

So where do we stand today? This volume speaks to logarithmic progress in oligonucleotide-based therapeutics and, in particular, antisense therapeutics. Advances in every area from medicinal chemistry to clinical evaluations are remarkable, especially when one considers the modest investment that has been made relative to the investments in other new platform opportunities such as monoclorial antibodies or gene therapy. Nevertheless, despite logarithmic progress in advancing and understanding the technology, the process of converting the technology to therapeutically important, commercially successful, and systemically administered new medicines has encountered several substantial disappointments. Although macugen, an aptamer, was approved for the local treatment of age-related macular edema (see Chapter 22), two first-generation antisense drugs administered systemically failed to achieve positive phase 3 studies. The new drug application (NDA) for a third drug, Genasense, was rejected by the Food and Drug Administration (FDA) and a new NDA for use of the drug in chronic lymphocytic leukemia was also rejected. How should we interpret these disappointments with regard to the value of the technology as a whole and in the context of the extraordinary progress that has been reported? This volume provides detailed answers to that enormously complex question. Affinitak, a first-generation antisense inhibitor of protein kinase C
(PkC
) was added to either carboplatinum and taxol or gemcitibine and cisplatinum and its effects on survival in patients with stage III/IV nonsqamous cell carcinoma of the lung (NSCCL) were evaluated. It resulted in no statistically significant survival benefit. Although we were unable to measure drug levels in tumors in the phase 3 studies, based on our experience with other first- and second-generation antisense drugs, we believe it is likely that there was sufficient drug in the tumors to produce a pharmalogic effect. So perhaps, PkC
is not a significant contributor to the maintenance of the malignant phenotype in these patients. Perhaps we underdosed; we certainly didn’t achieve a maximal tolerated dose. Perhaps the disease was so advanced that patients could not benefit. That a new drug added to a two-drug regimen for NSCCL failed to bring benefit is not too surprising as most drugs have failed in this setting. Nevertheless, Affinitak failed and it was a setback for acceptance of the technology. The failure of Affinitak is discussed in some detail in Chapter 26. Because initial phase 2 results obtained in randomized double-blind placebo-controlled clinical trials were quite positive, the failure of Alicaforsen in the treatment of patients with Crohn’s disease is perhaps more disappointing and puzzling. It was also surprising because after a dose of 2 mg/kg three times weekly for one month in a phase 2 trial, we reported reductions of ICAM-1, the Alicaforsen target, in the bowels of patients with Crohn’s disease. In fact, in two phase 3 studies we used doses substantially greater than 2 mg/kg, so it is likely that the drug was dosed adequately. Our ix

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best guess is that ICAM-1 is not critical in maintenance of Crohn’s disease in the patients we studied. Interestingly, as discussed in Chapter 25, the enema formulation of the drugs has demonstrated good efficacy in patients with ulcerative colitis. However, it is gratifying that in studies on Affinitak and Alicaforsen and now many other firstgeneration antisense drugs, we did not encounter severe dose-limiting toxicities. Even in the presence of very toxic chemotherapeutic regimes, in very sick patients, Affinitak’s contribution to toxicity was modest (see Chapters 14 and 26 for review). The third first-generation antisense drug to undergo phase 3 evaluations, an NDA, Genasense, was submitted for approval by the FDA for the treatment of malignant melanoma and was rejected. More recently, an NDA for the treatment of chronic lymphocytic leukemia was submitted. Despite the fact that the trial met its primary endpoint, the oncology advisory panel recommended the NDA be denied. Are there general lessons about antisense technology to be derived from these disappointments? Yes. First and most important, the experiences with these drugs and the many other first-generation antisense drugs demonstrate that even first-generation antisense drugs are reasonably well tolerated. Even Genasense, the most immunotoxic of the first-generation antisense drugs, was adequately tolerated when administered at relatively high doses in the presence of very cytotoxic chemotherapeutic regimens. Second, it is clear that antisense drugs with improved potency, better pharmacokinetic properties, and improved therapeutic index are needed. Of course, that was obvious in 1989 when we began the work on the technology, the evaluation of first-generation drugs, and the creation of second-generation drugs. Importantly, as Part 3 of the volume demonstrates, 2⬘ methoxyethyl chimeras represent a dramatic advance and are performing well. Other new chemistries and mechanisms of action offer the promise of even greater advances (Part 4 of this volume). Third, obviously drugs fail and often we really do not understand why. After more than 100 years of experience with small molecule drug discovery, 9 out of the 10 small molecules that begin development fail. So, it should not be surprising that representatives of a new class of drugs also fail. After all, they are subject to the same challenges and issues that all drugs face in phase 3 trials and in the regulatory process. What is important is amply demonstrated by this volume. Our understanding of the technology has advanced at a remarkable pace. New mechanisms and new opportunities for antisense drugs and other oligonucleotide-based drugs are being identified at an exciting pace. Second-generation antisense drugs are dramatically better, work in vitro, in vivo, and in clinical trials. And the technology is poised to continue logarithmic growth. Therefore, the tasks that remain are to finish the first leg of the journey. Developing a new platform for drug development is much more than a marathon. We must continue to aggressively, but prudently pursue the great opportunity that is presented by the technology and which is now firmly in our grasp. Stanley T. Crooke Isis Pharmaceuticals, Inc.

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Acknowledgments Editing a multiauthor volume is a bit like managing the United Nations. The editor has no authority and must depend on the goodwill and commitment of the authors of the chapters and his ability to twist the occasional arm. In the preparation of this volume, I was fortunate to have outstanding authors who met their commitments. I want to thank all the authors for their scholarly contributions and their commitment to meet the deadlines I imposed. I hope the reader will agree that all the chapters offer excellent value and are more than simply reviews. Rather, individually and in the aggregate, I believe that they constitute a compilation of the information available about this complex topic today and are integrated in a fashion that supports the development of a perspective on the future and an informed agenda with which to continue to advance the science. I want to thank Donna Parrett, who not only was responsible for preparing the two chapters in which I was involved, but was also coordinating the assembly of the book while completing all her other responsibilities. Thanks, Donna. I also want to thank those who will read the book. I appreciate your interest and look forward to the many contributions to the field that will be supported by the knowledge summarized in this volume.

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The Editor Stanley T. Crooke is founder, chairman, and chief executive officer of Isis Pharmaceuticals. Isis is a development-stage biopharmaceutical company that is focused on a new paradigm in drug discovery, antisense oligonucleotides. Since Dr. Crooke and his colleagues founded Isis in 1989, the company has grown rapidly, completing its initial public offering in May 1991, and has reported broad progress in antisense technology and its rapid conversion to therapeutic product opportunities. Isis was the first company to commercialize an antisense drug and has achieved a number of important corporate collaborative relationships. In 2006, Dr. Crooke was named in Nature Biotechnology as one of biotech’s influential individuals. Dr. Crooke is currently a member Northern Arizona University Arts and Sciences Advisory Council, Flagstaff, Arizona and San Diego State University BioScience Center Scientific Advisory Board. He is a member of the Current Drugs Advisory Board; the Editorial Advisory Board of Journal of Drug Targeting and Antisense Research and Development; and the Editorial Board of Gene Therapy and Molecular Biology. He is also editor-in-chief of Current Opinion in Anticancer Drugs and section editor for Biologicals and Immunologicals for Expert Opinion on Investigational Drugs. Prior to founding Isis, Dr. Crooke was president of Research and Development for SmithKline Beckman Corporation (SKB). He also coordinated the research and development activities of SKB including its instruments, diagnostics, animal health, and clinical laboratory businesses. Before joining SKB, Dr. Crooke helped establish the anticancer drug discovery and development program at Bristol Myers, which succeeded in bringing to market a significant number of drugs. During his career, Dr. Crooke has supervised the development of 19 drugs currently on the market and others in development. In addition to his involvement in the pharmaceutical industry, Dr. Crooke also maintains active academic positions. He is an adjunct professor at San Diego State University, and has won a number of teaching awards. He has authored over 440 publications and has edited 20 books. Dr. Crooke is active in molecular and cellular biology and pharmacology of antisense oligonucleotides. Dr. Crooke received his BS in Pharmacy from Butler University, Indianapolis, Indiana, and his MD and PhD from Baylor College of Medicine, Houston, Texas.

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Contributors Brenda Baker Isis Pharmaceuticals, Inc. Carlsbad, California

Maria A. Graziewicz Ercole Biotech Inc. Chapel Hill, North Carolina

C. Frank Bennett Isis Pharmaceuticals, Inc. Carlsbad, California

Susan A. Gregory Isis Pharmaceuticals, Inc. Carlsbad, California

Sanjay Bhanot Isis Pharmaceuticals, Inc. Carlsbad, California

Lisa R. Grillone Aerie Pharmaceuticals, Inc. Research Triangle Park, North Carolina

Balkrishen Bhat Isis Pharmaceuticals, Inc. Carlsbad, California

Boris A. Hadaschik The Prostate Centre Vancouver General Hospital Vancouver, British Columbia, Canada

Daniel C. Capaldi Isis Pharmaceuticals, Inc. Carlsbad, California Rosanne Crooke Isis Pharmaceuticals, Inc. Carlsbad, California Stanley T. Crooke Isis Pharmaceuticals, Inc. Carlsbad, California Antonin R. de Fougerolles Alnylam Pharmaceuticals Cambridge, Massachusetts Robert A. Fey Isis Pharmaceuticals, Inc. Carlsbad, California Susan M. Freier Isis Pharmaceuticals, Inc. Carlsbad, California Richard S. Geary Isis Pharmaceuticals, Inc. Carlsbad, California Martin E. Gleave Department of Urologic Sciences Vancouver General Hospital Vancouver, British Columbia, Canada

Gregory E. Hardee Isis Pharmaceuticals, Inc. Carlsbad, California Scott P. Henry Isis Pharmaceuticals, Inc. Carlsbad, California Patrick L. Iversen AVI BioPharma, Inc. Corvallis, Oregon James G. Karras Isis Pharmaceuticals, Inc. Carlsbad, California Tae-Won Kim Isis Pharmaceuticals, Inc. Carlsbad, California Troels Koch Santaris Pharma Hørsholm, Denmark Ryszard Kole University of North Carolina Lineberger Comprehensive Cancer Center Chapel Hill, North Carolina Kimberly Kramer-Stickland Biogen IDEC San Diego, California xv

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CONTRIBUTORS

Arthur M. Krieg Coley Pharmaceutical Group, Inc. Wellesley, Massachusetts

Peter Sazani Ercole Biotech Inc. Chapel Hill, North Carolina

T. Jesse Kwoh Isis Pharmaceuticals, Inc. Carlsbad, California

Anthony N. Scozzari Isis Pharmaceuticals, Inc. Carlsbad, California

Arthur A. Levin Isis Pharmaceuticals, Inc. Carlsbad, California

Alla Sigova Department of Biochemistry and Molecular Pharmacology University of Massachusetts Medical School Worcester, Massachusetts

Walt Lima Isis Pharmaceuticals, Inc. Carlsbad, California Ian MacLachlan Protiva Biotherapeutics Inc. Burnaby, British Columbia, Canada Muthiah Manoharan Alnylam Pharmaceuticals Cambridge, Massachusetts John M. Maraganore Alnylam Pharmaceuticals Cambridge, Massachusetts Timothy M. Miller Department of Neurosciences University of California San Diego, California Brett P. Monia Isis Pharmaceuticals, Inc. Carlsbad, California

Andrew Siwkowski Isis Pharmaceuticals, Inc. Carlsbad, California Richard A. Smith Center for Neurologic Study La Jolla, California Eric E. Swayze Isis Pharmaceuticals, Inc. Carlsbad, California Lloyd G. Tillman Isis Pharmaceuticals, Inc. Carlsbad, California Timothy Vickers Isis Pharmaceuticals, Inc. Carlsbad, California Jörg Vollmer Coley Pharmaceutical, GmbH Langenfeld, Germany

Peter E. Nielsen The Panum Institute University of Copenhagen Copenhagen, Denmark

Andrew T. Watt Isis Pharmaceuticals, Inc. Carlsbad, California

Henrik Ørum Santaris Pharma Hørsholm, Denmark

Mark Wedel Isis Pharmaceuticals, Inc. Carlsbad, California

Kallanthottathil G. Rajeev Alnylam Pharmaceuticals, Inc. Cambridge, Massachusetts

Charles Wilson Archemix Corp Cambridge, Massachusetts

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CONTRIBUTORS

Hongjiang Wu Isis Pharmaceuticals, Inc. Carlsbad, California Rosie Z. Yu Isis Pharmaceuticals, Inc. Carlsbad, California

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Phillip D. Zamore Department of Biochemistry and Molecular Pharmacology University of Massachusetts Medical School Worcester, Massachusetts Thomas A. Zanardi Isis Pharmaceuticals, Inc. Carlsbad, California

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PART

I

Introduction

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CHAPTER

1

Mechanisms of Antisense Drug Action, an Introduction Stanley T. Crooke, Timothy Vickers, Walt Lima, and Hongjiang Wu

CONTENTS 1.1

1.2

1.3

1.4

Introduction...............................................................................................................................5 1.1.1 The Opportunity ...........................................................................................................5 1.1.2 The Challenge...............................................................................................................5 1.1.3 Phases of Antisense Drug Action .................................................................................7 RNA Intermediary Metabolism ................................................................................................7 1.2.1 Coding RNAs................................................................................................................7 1.2.2 Noncoding RNAs..........................................................................................................9 1.2.2.1 Antisense Transcripts ....................................................................................9 1.2.2.2 Small Noncoding RNAs..............................................................................10 1.2.2.3 Other Noncoding RNAs ..............................................................................12 Factors that Influence the Selectivity of Antisense Drugs .....................................................12 1.3.1 Affinity........................................................................................................................12 1.3.2 Specificity for Nucleic Acid Sequences .....................................................................13 1.3.3 Protein Binding to Target RNA ..................................................................................14 1.3.4 Facilitated Hybridization ............................................................................................14 1.3.5 Levels of Target RNA.................................................................................................15 1.3.6 Terminating Mechanism .............................................................................................15 1.3.7 Posttranscriptional Modifications of RNA .................................................................15 1.3.8 Screening Processes Used to Identify Antisense Inhibitors .......................................16 1.3.9 Therapeutic Specificity (Therapeutic Index)..............................................................19 Occupancy-Only-Mediated Mechanisms ...............................................................................19 1.4.1 Modulation of Splicing...............................................................................................19 1.4.1.1 Can Antisense Drugs Be Used to Alter Splicing in Vitro and in Vivo? ....................................................................................20 1.4.1.2 How Is the Activity of Antisense Drugs Affected by the Strength of the Splicing Signal? .................................................................21 1.4.1.3 Does the Position of the Antisense Drug at a Splice Site Affect the Activity of the Antisense Drug?..........................................24 1.4.1.4 Do the Characteristics of Introns or Exons Affect the Activities of Antisense Agents? .................................................24 3

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1.4.1.5

Can Antisense Agents Designed to Bind Exonic Enhancer or Silencer Sequences Affect Splicing? ......................................25 1.4.1.6 Does Chemical Class Influence Activity?...................................................25 1.4.2 Translation Arrest .......................................................................................................25 1.4.2.1 Is It Feasible to Arrest Translation with Antisense Drugs? ........................25 1.4.2.2 What Are the Optimal Sites in Target RNAs to Induce Translation Arrest? ..........................................................................25 1.4.2.3 What Is the Influence of Chemical Class on Activity?...............................26 1.4.2.4 How Robust Is Translation Arrest? .............................................................26 1.4.3 Disruption of Necessary RNA Structure ....................................................................26 1.5 Occupancy-Activated Destabilization ....................................................................................26 1.5.1 5
Capping ..................................................................................................................27 1.5.2 Inhibition of 3
-Polyadenylation ................................................................................27 1.5.3 Other Mechanisms......................................................................................................27 1.5.4 RNase H......................................................................................................................27 1.5.4.1 Do Antisense Drugs That Use the RNase H Mechanism Work?...................................................................................28 1.5.4.2 What Sites in Target RNAs Are Accessible to RNase H–Based Antisense Drugs? .........................................................................28 1.5.4.3 Can Information about the Enzymology of RNase H1 Be Used to Improve the Performance of RNase H–Based Drugs?..............................................................................30 1.5.4.4 How Robust a Mechanism Is RNase H?.....................................................30 1.5.5 Double-Strand RNase (siRNA) ..................................................................................31 1.5.5.1 siRNA and RNase H Mechanisms Are Similar ..........................................32 1.5.5.2 RISC-Mediated Pathways ...........................................................................33 1.5.5.3 RISC-Mediated Pathways Are Promiscuous with Regard to Hybridization-Based Off-Target Effects.....................................34 1.5.5.4 siRNAs May Induce Transcriptional Repression........................................35 1.5.5.5 siRNAs Have Displayed Activities in Vivo That Are Similar to Those Displayed by RNase H Antisense Drugs—But There Are Also Substantial Differences................................................................................36 1.5.5.6 Structural Features and Medicinal Chemistry of siRNAs ..................................................................................36 1.5.5.7 Unique Challenges of Duplex RNA Drugs.................................................37 1.5.6 Covalent Modifications of Target Nucleic Acids .......................................................38 1.5.7 Oligonucleotide-Induced Cleavage of Target RNA....................................................38 1.5.8 RNase L–Mediated Cleavage .....................................................................................38 1.6 Micro-RNAs ...........................................................................................................................39 1.7 Conclusions and Future Perspectives .....................................................................................39 Acknowledgments ............................................................................................................................40 References ........................................................................................................................................40

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MECHANISMS OF ANTISENSE DRUG ACTION, AN INTRODUCTION

5

1.1 INTRODUCTION 1.1.1

The Opportunity

The antisense concept derives from an understanding of nucleic acid structure and function and depends on Watson–Crick hybridization [1]. Thus, arguably, the demonstration that nucleic acid hybridization is feasible [2] and the advances in situ hybridization and diagnostic probe technology [3] lay the most basic elements of the foundation supporting the antisense concept. The first clear enunciation of the concept of antisense oligonucleotides as therapeutic agents was in the work of Zamecnik and Stephenson [4] in 1978. In this publication, these authors reported the synthesis of a 13 nucleotide long oligodeoxyribonucleotide, which was complementary to a sequence in the respiratory syncytial virus genome. They suggested that this oligonucleotide could be stabilized by 3
- and 5
-terminal modifications and showed evidence of antiviral activity. More important, they discussed possible sites for binding in ribonucleic acid (RNA) and potential mechanisms of action of oligonucleotides. The opportunity suggested by the antisense concept was seductive for a number of reasons. First, it suggested that it might be possible to create gene-selective reagents and drugs. The perceived value of such selectivity has increased as the molecular biology of the cell has become better understood and it has become apparent that most genes are arrayed within multigene families. The concept also suggested the possibility of the rational design of reagents and drugs based on well-understood principles: Watson–Crick hybridization rules. Again, the perceived value of such an approach has increased since the ability to dissect molecular biological processes at ever greater detail has evolved and the productivity of drug discovery exercises using traditional approaches has declined. The demonstration of broad distribution to multiple organs, multiple routes of administration, and excellent therapeutic index of second-generation antisense drugs as well as their potential applicability to a very broad array of diseases have further enhanced the perceived value of the technology. Finally, advances in understanding noncoding RNAs, RNA structure, function, and metabolism have broadened the potential targets and pathways to be exploited, adding further to the perceived value of the platform. In short, the more that has been understood about the RNA world and the performance of the hybridization-based drugs, the broader and more exciting the overall opportunity seems. 1.1.2

The Challenge

The development of antisense technology is, in effect, the creation of a new pharmacology. The receptor for antisense drugs is a specific sequence of nucleotides in a target RNA. Thus, a key step in the development of the platform was the understanding of the structure, functions, and intermediary metabolism of RNA from a pharmacological perspective. Advances in understanding all these aspects of RNA and advances in identifying novel RNA species have contributed to the evolution of antisense technology. Prior to the late 1980s and early 1990s, essentially no medicinal chemistry had been performed on oligonucleotides. In fact, the phosphorothioate modification, which has proven to be a versatile and useful backbone modification, was first synthesized to stabilize polyribonucleotides used to induce interferon [5] and the methylphosphonates were discovered as a part of an effort to evaluate the effects of chemical modifications on hybridization [6]. However, since the early 1990s, extraordinary progress in creating and evaluating modifications of oligonucleotides has been reported (for review, see [7,8]). The pharmacophore, a dinucleotide, and a few of the many hundreds of modifications made and tested are all shown in Figure 1.1. In our laboratories at Isis, we have had the opportunity to evaluate and compare hundreds of modified oligonucleotides in various animal models. Thus, there is today a substantial chemical toolbox and an extraordinarily rich database that extends from synthesis to hybridization and nuclease stability to all the characteristics of interest with regard to drug properties for these modifications.

Figure 1.1

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O O PS O

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As progress in creating the chemical toolbox was effected, these chemicals were used to induce drug effects expected to begin with binding to the target RNA. Once an antisense drug binds to its target sequence, it may induce a variety of events that lead to destruction or inactivation of the RNA. We have referred to these as terminating mechanisms. Again, the progress in understanding terminating mechanisms has been gratifying and will be the subject of this chapter. Of course, contemporaneously, the properties of various types of antisense drugs had to be defined. These properties include ease of synthesis, analytical characteristics, stability, pharmacokinetics, toxicological properties, formulations, and routes of delivery. Remarkable progress has been achieved for many different types of oligonucleotide analogs providing the basis for rational selection of oligonucleotides, formulations, doses, schedules, and the design of preclinical and clinical trials (for review, see [9] and this volume). 1.1.3

Phases of Antisense Drug Action

At the most conceptual level, the effects of hybridization-based drugs can be divided into three phases: pre- or nonhybridization, hybridization, and posthybridization. Because the typical cellular level of target m-RNAs is less than 100 copies per cell, the interaction with target RNA results in de minimis reduction of the total antisense drug concentration. Consequentially, nonhybridization interactions, principally with cellular and extracellular proteins, account for the pharmacokinetics and non-pharmacologically-based toxicological properties of antisense drugs. As our understanding has become more sophisticated, ever larger numbers of subtle variations in these properties due to variations in sequence have been identified. Nevertheless, most pharmacokinetic and toxicologic observations are qualitatively and quantitatively consistent across all members of a chemical class, i.e., class-generic behaviors are observed. Thanks to the extraordinary advances reported, we now understand these properties in detail for several classes of antisense drugs and are beginning to dissect them down to the level of specific interactions between oligonucleotides and proteins that result in specific properties (for review, see [10–14] and this volume). Equally impressive progress has been reported with regard to understanding the posthybridization or terminating mechanisms. In fact, today, for some mechanisms we have sufficient understanding to use the mechanistic information to design improved antisense drugs. The progress in understanding the mechanisms of action of antisense drugs has previously been reviewed [15] and is the subject of this chapter. Rather remarkably, however, very little progress has been reported in understanding how hybridization to a specific target RNA occurs in cells. Although the evidence that antisense agents can specifically hybridize to many specific RNA species and cause reduction of the target RNA is overwhelming in cells and animals, little is understood either about the process by which hybridization takes place or the kinetics of intracellular hybridization in cells. Conceptually, it would seem challenging for antisense drugs to identify and bind to the few copies of their cognate sequences in the midst of substantially greater concentrations of potentially competitive nucleic acid sequences in the cells. This remains an area in which there are inadequate explanations for the observed phenomena and where future research should focus.

1.2 RNA INTERMEDIARY METABOLISM 1.2.1

Coding RNAs

Most oligonucleotides are designed to modulate the information transfer from the gene to protein—in essence, to alter the intermediary metabolism of RNA. Figure 1.2 summarizes these processes. RNA intermediary metabolism is initiated with transcription. The transcription initiation complex contains proteins that recognize specific deoxyribonucleic acid (DNA) sequences and

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Transcriptional arrest

Transcription

CAP Capping/ polyadenylation

AAAA

CAP

Splicing

AAAA Nucleus

CAP

AAAA

Transport CAP

Effects on catabolism of RNA

CAP AAAA

Degradation CAP Translation

Effects on anabolism of mRNA

AAAA

Translational arrest

Protein

Figure 1.2

RNA intermediary metabolism. Steps in the transcription and processing for a pre-mRNA are shown. The thick lines represent exons. The thin lines represent noncoding regions including the 5
and 3
UTRs and introns. Potential sites for intervention are defined by arrows.

locally denature double-stranded DNA, thus allowing a member of the RNA polymerase family to transcribe one strand of the DNA (the antisense strand) into a sense pre-messenger RNA (pre-mRNA) molecule. Usually during transcription, the 5
end of the pre-mRNA is capped by adding a methyl-guanosine, and most often by methylation of one or two adjacent sugar residues. This enhances the stability of the pre-mRNA and may play a role in a number of key RNA processing events [16]. Between the 5
cap and the site at which translation is initiated is usually a stretch of nucleotides referred to as the 5
untranslated region (5
-UTR). This area may play a key role in regulating messenger RNA (mRNA) half-life and transitional efficiency [17]. Similarly, the 3
end of the pre-mRNA usually has a stretch of several hundred nucleotides beyond the translation termination signal. This area often plays an important role in determining mRNA half-life. Moreover, posttranscriptionally, most pre-mRNA species are polyadenylated. Polyadenylation stabilizes the RNA, is important in transport of mature mRNA out of the nucleus, and may play important roles in the cytoplasm as well [18,19]. Because eukaryotic genes usually contain intervening sequences (introns), most pre-mRNA species must have these sequences excised and the mature RNA spliced together. Splicing reactions are complex, highly regulated, and involve specific sequences, small-molecular-weight RNA species, and numerous proteins. Alternative splicing processes are often used to produce different mature mRNAs and, thus, different proteins. Even though introns have been considered waste, important sequences are conserved, and intronic sequences can play important roles including coding for proteins, antisense transcripts, and noncoding RNAs [20]. Mature mRNA is exported to the cytoplasm and engages in translation. mRNA half-lives vary from a few minutes to many hours, and appear to be highly regulated. Each step shown in the pathway is a composite of numerous steps and is theoretically amenable to intervention with oligonucleotides. The pathway is fully defined for virtually no mRNA, and available information is insufficient to determine the rate-limiting steps in the intermediary metabolism of any mRNA species. Alternative splicing is an important biological process, has been implicated in a large number of diseases and has been the subject of intervention with antisense drugs. In a typical multiexon

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RNA, the splicing pattern may be altered in many ways. Although the splicing of most exons is constitutive, the splicing of some exons is inducible and is highly cell- and tissue-context-dependent (for review, see [16]). Although a key determinant of the rate and extent of splicing is the strength of the splicing site consensus sequences, intronic, and exonic sequences that may enhance or inhibit splicing of a particular exon play important roles. As will be discussed, interventions with antisense drugs designed to encourage a particular splicing pattern have been successful. Sites at which success has been observed include splice sites and exonic sequences. 1.2.2

Noncoding RNAs

In the past few years, the prevalence and importance of noncoding RNAs have become much more apparent. Although there are numerous types of noncoding RNAs, two classes, antisense transcripts and micro-RNAs, may be particularly important with regard to antisense therapeutics.

1.2.2.1 Antisense Transcripts Antisense transcripts were identified in eukaryotic cells some time ago, but the extent of antisense transcripts and the breadth of roles they may play have been appreciated only recently (for review, see [21]). Antisense transcripts may code for a variety of proteins or may be noncoding [22]. Noncoding antisense transcripts appear to regulate gene expression at a variety of points in the information transfer process from transcription to translation [21,22]. Of equal importance, however, is the actual production of double-stranded RNA (dsRNA) through binding of an antisense transcript to its sense transcript. Eukaryotic cells contain a variety of enzymatic pathways to process dsRNAs [23,24] and they can induce a number of important processes including posttranscriptional gene silencing [25]. Antisense transcripts can arise from transcription of both strands of a gene. Alternatively in many cases, transcription of two closely positioned genes coding for different proteins and reading in the opposite orientation can lead to an “antisense” transcript for the genes [26]. Importantly, recent studies suggest that 5–10% of the human genome may have antisense transcripts [27,28]. Thus, antisense transcripts may be the result of at least two processes and play important roles in regulating gene function and phenotype via a variety of mechanisms [29]. Given the prevalence and roles of natural antisense manuscripts, it seems likely that they have an effect on the activities of antisense drugs. Clearly, if an antisense transcript were to hybridize to a portion of a target RNA, that portion would be less accessible to an antisense drug, irrespective of chemistry or mechanism of action. Also, any influence of an antisense transcript on the regulation of intermediary metabolism of a targeted RNA could influence the effects induced by an antisense drug designed to bind to that RNA. To date, there has been no report of studies designed to directly assess the effect of antisense transcripts on the activities of antisense drugs. However, at Isis we have created antisense drugs to more than 4000 genes and have never failed to find active drugs to any RNA target based on screening processes that evaluate the effects of scores of antisense drugs designed to bind to multiple sites within the RNA. Nor has it proven unusually difficult to identify antisense drugs designed to bind to the 3
untranslated region (3
-UTR) of targeted RNAs, a site to which many antisense transcripts bind (for review, see [9,30]). As most antisense transcripts are reported to be poly A minus and localized to the nucleus, it may be that some of the potential effects of antisense transcripts on antisense drug activity are avoided when antisense drugs are designed to target mature RNAs and can work in both the nucleus and cytosol. However, many antisense drugs are designed to bind to sites that are excised before the RNA exits the nucleus and have been shown to be active; so drug effects in the cytoplasm cannot fully explain the failure to identify RNA sequences that may be inaccessible because of binding to antisense RNA. Clearly, much more work needs to be done in this area with focused experiments that can directly address the impacts of antisense transcripts on the activities of antisense drugs.

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1.2.2.2 Small Noncoding RNAs The recent discovery of small noncoding RNAs has stimulated one of the most exciting new areas of research in cell biology. Small noncoding RNAs are RNA species that do not code for proteins, but are involved in a host of vital cellular processes. These tiny RNAs add a new layer of regulation of gene function, are involved in processing multiple classes of RNA, respond to viral infections, represent novel RNA targets for antisense drugs, present novel cellular pathways to exploit as new mechanisms for antisense drugs, and in due course may be found to be accountable for some of the limitations and off-target effects of antisense drugs. This group of RNAs is comprised of C or D box containing small RNAs (C/D RNAs), micro-RNAs and small interfering RNA (siRNAs) (for review, see [31]). C/D RNAs are metabolically stable 60–300 nucleotide RNAs that reside in the nucleus and localize to the nucleolus or Cajal bodies and are involved in site-specific methylation of RNAs. They are crucial to processing of preribosomal RNAs as well as pre-mRNAs. Most are transcribed from introns of genes and are processed from the host-gene intron. They contain specific C-box or D-box sequences located near their 5
or 3
termini, respectively. Micro-RNAs are 19–23 nucleotide RNA species. It is estimated that as many as 3% of genes in the mammalian genome have complementary micro-RNAs. Micro-RNAs may form perfect duplexes with their targets, in which case they lead to cleavage in the center of the duplex; or imperfect duplexes, in which case they may lead to translation arrest or cleavage of the targeted RNA. Although micro-RNAs and siRNAs were originally thought to be quite different and result in different cellular responses, in fact the characteristics and behaviors of these RNAs appear to be very similar. Figure 1.3 summarizes the characteristics of the small noncoding RNAs discovered to date.

5′ D/D′

3′

3′

5′

CH3

5 bp Small nucleolar RNAs (snoRNAs)

18S, 5.8S, 28S rRNAs

3′

5′

U6

Other RNAs ? (mRNA ?)

3′ 5′ Perfect DNA duplex

RNA degradation (RNA interference) Figure 1.3

Small Cajal-body-specific RNAs (scaRNAs)

U1, U2, U4, U5

5′

3′ 5′

3′ Irregular RNA duplex

Chromatin modifications

Nonproductive translation

C/D RNA modifications guide and miRNAs. Sequences complementary to the cognate RNA target are depicted in thick lines. (Top) For C/D RNAs, the nucleotide triggered for 2
-O-methylation is denoted by a box. It is always paired to the fifth nucleotide upstream from the D (or D
) box. The different RNA targets of C/D snoRNAs and scaRNAs are shown. (Bottom) For miRNAs, depending on the degree of complementary with their target, they can trigger RNA cleavage in the middle of the duplex (RNA interference) or nonproductive translation of the target mRNA by an unknown mechanism. miRNA can also promote RNA-directed DNA methylation.

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Micro-RNAs can be transcribed from the sense or antisense strands of DNA. They can be located in introns, exons, or intragenic regions. Thus, they may use the transcriptional machinery of annotated genes or they may derive from independent transcriptional units (for review, see [32]). Irrespective of gene location or orientation, the initial transcript, the pre-mir, is typically kilobases long and is processed in the nucleus to a pre-mir, an approximately 70-nucleotide species by an RNase III called drosha. The pre-mir is exported to the cytoplasm and cleaved to the approximately 20-nucleotide mir by another RNase III dicer (for review, see [33]). The mir can then be loaded into the RNA-induced silencing complex (RISC) complex in which it may hybridize perfectly or imperfectly with target RNAs. Perfect hybridization leads to degradation by the RISC complex. Imperfect hybridization may lead to translation arrest or degradation of the RNA after localization in processing bodies [34]. Each of these steps is significantly more complex than described and a more detailed description is beyond the scope of this review, but the pathways are shown in simplified form in Figure 1.4. In addition to these complex pathways, it is now apparent that microRNAs or RASi RNAs may, if their sequences are complementary to repetitive DNA sequences, activate the RNA-induced transcriptional silencing (RITS) complex and lead to gene inactivation and heterochromatin formation (for reviews, see [35–37]). Furthermore, these processes can lead to

miRNA - siRNA Biogenesis dsRNA Cell membrane

RLC TRBP

Dicer

siRNA Ago2

RLC

Exportin 5

TRBP

Dicer

Pre-mir Drosha

mRNA

Mature mir RISC

Ago 1/2/3/4

DGCR8

?

Ago

Dcp

P body

40s 60s

Pre-mir DNA Nucleus

mRNA

Cytoplasm

Figure 1.4 The micro-RNA and siRNA pathways. Large precursor RNAs (pre-mirs) are transcribed. They are processed by human RNase III (Drosha) in the nucleus to pre-mirs. Pre-mirs are exported from the nucleus by exporten. In the cytoplasm pre-mi processing bodies are processed by a complex, the RISC loading complex (RLC), of proteins including the RNase III, dicer, and thyroid hormone receptor-binding protein (TRBP). Mature mirs load RISC and eIF2c2 cleaves the target RNA. This may enter processing bodies (p-bodies). Alternatively, the RISC complex can arrest translation. Exogenous RNAs may also enter the pathways.

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“spreading” and the silencing of additional genes located near the repetitive DNA. Finally, it has been shown that RNA sufficient to induce gene silencing can be processed in the nucleus by transcription of inverted DNA repeats or by cleavage of longer RNA precursors. The potential impacts of small RNAs on antisense therapeutics are manifold and several will be discussed in more detail in later sections of this review and in subsequent chapters. Most directly and obviously micro- and siRNAs reinforce the opportunity suggested in 1998 that RNA-like antisense drugs that activate double-strand RNases could be therapeutically valuable [38]. The direct application of duplex oligoribonuleotides to exploit the RNA interference (RNAi) pathway has been the subject of a great deal of work and is but one means of exploiting double-strand RNases [39]. Perhaps even more important is the identification of multiple new pathways by which regulatory RNAs are produced and used. Each arm of each of these pathways presents unique opportunities and risks. Each protein and each RNA involved presents a potential site for the interaction of antisense drugs that could lead to off-target effects. Micro-RNAs are themselves attractive targets for antisense drugs as demonstrated by several recent publications (for review, see [40]). Again, this area is one that deserves a great deal of attention. It is gratifying to see substantial progress already.

1.2.2.3 Other Noncoding RNAs There are, of course, many other noncoding RNAs, including ribosomal RNA, small-molecular weight nuclear, nuclear, and nucleolar RNAs (for review, see [41]) and RASi RNAs [37]. Although a review of these RNA species is beyond the scope of this chapter, each class of RNA could influence the effects of antisense drug if they were to bind to partially complementary sequences accrued and may represent interesting target opportunities as well.

1.3 FACTORS THAT INFLUENCE THE SELECTIVITY OF ANTISENSE DRUGS 1.3.1

Affinity

The affinity of oligonucleotides for their receptor sequences results from hybridization interactions. The two major contributors to the free energy of binding are hydrogen bonding (usually Watson–Crick base pairing) and base stacking in the double helix that is formed. Affinity is affected by ionic strength, where in general the higher the ionic strength, the higher the affinity of charged oligonucleotides for polynucleotides. As affinity results from hydrogen bond formation between bases and stacking occurs between coplanar bases, affinity increases as the length of the oligonucleotide receptor complex increases. Thus, the affinity per nucleotide unit and the number of hybridizing nucleotide pairs are crucial determinants of overall affinity. Affinity also varies as a function of the sequence in the duplex. Nearest-neighbor rules support the prediction of the free energy of binding for DNA–DNA and RNA–RNA hybrids with relatively high precision [42,43]. A common misconception is that DNA–RNA duplexes are more stable than DNA–DNA duplexes. In fact, the relative stability of these duplexes varies as a function of the sequence. RNA–RNA duplexes are typically the most stable [9]. As with other drug–receptor interactions, activity requires a minimum level of affinity. For many targets and types of oligonucleotides, the minimum length of an oligonucleotide may be 12–14 nucleotides. Although theoretical affinities for oligonucleotide single-strand nucleic acid interactions are very large, in practice, affinity constants are substantially lower. Several factors contribute to the differences between theoretical and realized affinities. Undoubtedly, the most important factor is that RNA can adopt a variety of secondary structures (for review, see [44]). In addition to secondary structure, RNA can adopt tertiary structures. Tertiary structures result from the interactions of secondary structures in an RNA molecule with other secondary structural elements or single-stranded regions [45].

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A third factor that can potentially reduce the affinity of an oligonucleotide for its RNA receptor is that oligonucleotides can form secondary and tertiary structures themselves. To avoid duplex formation, oligonucleotides that contain self-complementary regions are usually not employed. However, other structures that were not well understood or expected have been described. Tetrameric complexes formed by oligonucleotides with multiple guanosines [46–49] and other base sequences [50] can be highly stable, clearly would prevent an antisense interaction, and have a number of biological effects that have confounded interpretation of experiments. Since RNA and oligonucleotide structures are affected by ionic milieu and nonproductive interactions with proteins and polycations, the in vivo situation is, of course, considerably more complicated. Relatively little is understood about the interplay among all these factors and their effects on the true affinities of oligonucleotides for potential RNA targets. Advances in the medicinal chemistry of antisense drugs have resulted in numerous classes of these agents that display substantially enhanced affinity (for review, see [51]). Conceptually, increased affinity should result in increased potency. Indeed, this has been observed in both in vitro and in vivo test systems (e.g., see [52]). However, in our experience, the increase in potency has typically not been as substantial as predicted based on affinity considerations. This has prompted us to begin to search for potential cellular factors that may influence hybridization. A second potential effect of increasing affinity should be that greater numbers of sites within an RNA molecule might be accessible to antisense drugs. Indeed, that has been our experience. For example, we have compared the activities of lower- and higher-affinity antisense drugs that work via an RNase H (RNase H enzymes are double-strand RNA-binding proteins that cleave RNA in an RNA–DNA duplex) mechanism against a number of cellular targets. In addition to the expected increase in potency, many more sites within the RNA were accessible to the higher-affinity class of antisense drugs for all the transcripts studied. 1.3.2

Specificity for Nucleic Acid Sequences

Specificity derives from the selectivity of Watson–Crick or other types of base pairing. The decrease in affinity associated with a mismatched base pair varies as a function of the specific mismatch, the position of the mismatch in a region of complementarity, and the sequence surrounding the mismatch. In a typical interaction between complementary 18-mers, the G37 or change in the Gibbs free energy of binding induced by a single mismatch varies from 0.2 to 4.0 kcal/ mol/modification at 100 mM NaCl. Thus, a single base mismatch could result in a change in affinity of approximately 500-fold [53]. Modifications of oligonucleotides may alter specificity. At the genomic level, any sequence of 17 residues is expected to occur only once [54]. Assuming a random distribution of sequences in RNA, any sequence of 13 residues is expected to occur once in the cellular RNA population and, if the nonrandom nature of mammalian RNA sequence is taken into account, an 11-mer or perhaps smaller oligonucleotide could identify and bind to a unique sequence [55]. To exploit fully the theoretical potential for specificity of an oligonucleotide in a therapeutic context, it is necessary to manipulate the length of the oligonucleotide and its concentration at target. The results of such an exercise have been reported [56]. In this study, phosphorothioate oligodeoxynucleotides were designed to target the normal or codon 12-point mutation of Ha-ras mRNA. Predictions from hybridization experiments suggested that approximately a fivefold specificity for mutant compared to normal Ha-ras RNA was possible. By optimizing oligonucleotide length and the extracellular concentration of the oligonucleotide, nearly theoretical specificity was achieved in cells in tissue culture. Other factors can also be used to enhance specificity. RNA secondary and tertiary structure assures that not all sequences are equally accessible. Design of oligonucleotides to interact with sequences involved in the maintenance of RNA structure can theoretically enhance specificity and, if the structure is essential to the stability or function of the RNA, potency. Furthermore, many RNA

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and DNA sequences interact with proteins, again assuring far more diversity in response to an oligonucleotide and, therefore, greater specificity than might be predicted solely on the basis of differences in nucleic acid sequence. 1.3.3

Protein Binding to Target RNA

Although understanding RNA structure provides crucial information that enhances the identification of optimal binding sites for antisense drugs, it is not sufficient for a number of reasons, including the fact that RNAs bind multiple proteins at multiple sites (for review, see [57]). Obviously, any protein interaction with a target RNA may adversely affect the ability of the antisense drug to interact with the desired sites in the RNA. Despite the progress in understanding factors that influence antisense drug activity, virtually nothing is known about the competition between antisense drugs and proteins that bind to RNA. This is undoubtedly one of the reasons that algorithms designed to select optimal binding sites in RNA based on predicting internal RNA structures have failed to provide significant benefit, as discussed in a later section. 1.3.4

Facilitated Hybridization

Precisely how an antisense drug binds to its cognate site in a target RNA in cells is unknown. However, given the enormous excess of partially complementary sites relative the very few copies of individual mRNAs with the fully complementary binding site, it seems unlikely that the process is as simple as hybridization reactions in which appropriate concentrations of two reactants are mixed in a test tube. This has prompted us to consider the possibility that intracellular hybridization to cognate sites is somehow facilitated. Several proteins have been shown to promote RNA annealing or hybridization [57]. A protein found in messenger ribonucleoprotein particles (mRNP), a member of the Y-box family of proteins P50 (YB-1), has been reported to be involved in a number of DNA repair and replication processes and to promote hybridization of RNA (and DNA) [58]. This protein was reported to enhance RNA hybridization by up to a thousandfold. Its activity was dependent on the ratio of protein to RNA, the length of the RNA, and ionic strength. It did not require adenosine triphosophate (ATP) hydrolysis. It also enhanced hybridization of DNA. The promiscuity of this protein suggests that it can participate in facilitating the hybridization of antisense drugs of various chemistries to target RNAs. Its localization to the polysomes, m-RNPs, and in the nucleus makes it an excellent candidate to facilitate the hybridization of antisense drugs in all cellular compartments. Although the detailed mechanism is not yet understood, it has been proposed that YB-1 works stoichiometrically by melting interfering structures and increasing the local effective concentrations of the two hybridizing strands. As all the proteins in this family have clusters of positively charged amino acids, it is thought that they may serve to neutralize the charge–charge repulsion of the two nucleic acids strands as well. Other proteins display similar properties. For example, the major proteins of the heterogeneous nuclear ribonucleoprotein complexes (hnRNP A1, C1, and U) exhibit annealing activities [59]. More recently, the RISC has been shown to facilitate hybridization of RNA strands in the RISC complex by as much as 400 fold [60]. Thus, there is ample and growing evidence that facilitated hybridization occurs in cells. How might facilitated hybridization affect the activity of antisense drugs? First, it may be the case that the facilitation of hybridization by RISC is simply the first demonstration of the phenomenon and that all interactions of antisense drugs with their cognate sequences are facilitated. It is possible that the substantial differences in potency of antisense drugs designed to bind to different sites in target RNAs could be a function of RNA structure, proteins that are bound, and the ability of the site to participate in facilitated hybridization. Finally, as chemistries diverge substantially from natural nucleic acid structures, e.g., peptide nucleic acids, they may be less able to participate in facilitated hybridization and this might explain the limited activities displayed by some chemical

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classes. It may also impose true structural limitations on the divergence from natural nucleic acid structures that can be tolerated in antisense drugs. At present, the proposition that facilitated hybridization is important in determining the effectiveness of antisense drugs is largely conjectural. However, this area has benefited from virtually no research and now that the tools are available, progress in this important area should be forthcoming. 1.3.5

Levels of Target RNA

Although it is theoretically possible that the concentration, transcriptional rate, or stability of a target RNA may influence the effectiveness of antisense drugs, at least for antisense drugs that work via the RNase H mechanism, it has been experimentally determined that RNA concentration and transcription rate do not affect their performance [61]. In this study the level of RNA transcribed from either an exogenous or induced endogenous gene was varied from 1 to 400 copies per cell and shown to have no effect on the potency or efficacy of antisense drugs transfected into either A549 or HeLa cells. Nor did transcription rate have any effect. Less direct evidence also derives from our broad experience in screening for antisense drug activity. As a general rule, within a specific chemical and mechanistic class of antisense drugs, the maximum potency and efficacy achieved against each RNA target is roughly comparable. Inasmuch as we have identified antisense drugs to approximately 4000 genes, if RNA concentration, transcriptional rate, or RNA stability significantly influenced antisense drug performance it seems likely that greater variability in these parameters would have been observed (for review, see Chapter 5 in this volume). Why do these factors not affect the activities of antisense drugs? Consider the basic equation that defines drug action: D  F & DR → Effect where D is the drug concentration, R the receptor concentration, and DR the drug–receptor complex. Given the low concentrations of pre- and m-RNAs in cells and the high intracellular concentrations of antisense drugs achieved, the receptor concentration can be eliminated and drug effect should be dependant only on drug concentration. Transcriptional rate should have no effect because receptor concentrations are irrelevant. Similarly, m-RNA decay rate should have no effect. 1.3.6

Terminating Mechanism

The first step in the induction of pharmacodymic effects by an antisense drug is hybridization to its cognate sequence or receptor. What happens after binding is also of great importance. Terminating mechanisms can be divided into occupancy only and occupancy-induced degradation classes. Inhibiting RNA function by occupying selected sites in the target RNA has been demonstrated and includes processes such as translation arrest and inhibition of splicing or induction of alternative splicing. Occupancy-induced destabilization of RNA involves the recruitment of nucleases that degrade the target RNA and can include RNases H and double-strand RNases such as those involved in siRNA activities. The “robustness” of the terminating mechanism can be qualitatively characterized using several parameters. These include the ease of identification of active antisense drugs, the ratio of active to inactive sites in target RNAs, and the potency and efficacy of drugs that use the mechanism both in vitro and in vivo. We now have enough experience to compare the robustness of various mechanisms in vitro. For comparisons of robustness in vivo, we have less data, but some trends are emerging. These topics will be considered in some detail in later sections of this review. 1.3.7

Posttranscriptional Modifications of RNA

Although RNAs are subject to far fewer postsynthesis modifications than proteins, posttranscriptional modifications that could influence the activities of antisense drugs do occur.

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Posttranscriptional modifications of RNA include 2
-0 methyl modifications of the ribose, conversion of uridine to pseudouridine and RNA editing (for review, see [62–64]). However, only RNA editing has been shown to modify the sequences of pre- and mature m-RNAs. RNA editing is effected by the deamination of either adenosine to inosine or cytosine to uridine. In both cases, the sequence of the RNA is modified from that originally transcribed from the gene. RNA editing has been shown to play significant roles by introducing new codons that may initiate translation, prematurely stop translation, alter splicing, or influence other steps in the metabolism and utilization of RNAs (for review, see [64,65]). Given how frequently RNA editing occurs and its substantial importance to normal and pathophysiological process, it seems likely that the processes should influence the activities of antisense drugs. Clearly, the introduction of a mismatch in the center of a 2
gap in a second-generation antisense drug should dramatically reduce activity that would be dependant on RNase H1. It is also possible that editing of siRNA duplexes by adenosine deaminase and editing of single-strand oligoribonucleotides by cytosine deaminase could take place even though they would not be ideal substrates for the enzymes. If that were to happen, of course, off-target effects could ensue. To date, however, no systematic studies of the potential impacts of RNA editing on the activities of antisense drugs have been reported. Although it is unlikely that RNA editing could profoundly influence the activities of many antisense drugs, it is possible that a thorough evaluation of this possibility could help explain some anomalous behaviors observed occasionally with specific antisense drugs. 1.3.8

Screening Processes Used to Identify Antisense Inhibitors

In drug discovery, it is axiomatic that the more thorough the search to identify an optimal drug, the better the drug. Antisense drug discovery technology provides additional incentives for thorough screening. First, the design, synthesis, and testing of potential antisense inhibitors is straightforward, rapid, and automatable. Second, a wide array of potential chemical modifications is now available. Third, substantial experience with multiple terminating mechanisms can inform screening processes. Fourth, because all the drugs from a particular chemical class behave similarly, there is great value in databases that can be created from information about the performance of hundreds to thousands of representatives of each chemical class. Fifth, it is now apparent that it is possible to identify problematic sequence motifs such as immunostimulatory consensus sequences, and thus avoid them. At Isis, the screening process begins with the sequence of the target RNA. That sequence is input and algorithms design antisense inhibitors to 40–80 different sites within the RNA excluding known problematic motifs and sequences that may lead to internal structures in the antisense agents. These are then screened at active concentrations in cells. Multiple active antisense inhibitors are then studied in detailed dose–response curves in vitro. For targets of substantial interest, at this stage we often screen antisense inhibitors to as many as several hundred additional sites. Then typically, 5–6 potential antisense drugs are taken into rodents. In fact, we typically evaluate several antisense inhibitors in monkeys (Figure 1.5). Thus, compared to earlier times, the antisense drugs designed today at Isis begin better because of the medicinal chemistry and basic research done earlier and the number of problematic motifs excluded. The final selection of the lead drug for detailed study then benefits from the number of potential drugs evaluated in vitro and in vivo providing better data-driven choices. As can be observed in Figure 1.6, just screening multiple sites in each target RNA greatly enhances the likelihood of enhanced performance by antisense drugs. Consider, for example, the likelihood of success with antisense oligonucleotide 1 (ASO1) or ASO2 compared to some of the less active antisense inhibitors. Moreover, as this screen was performed with higher-affinity more potent second-generation antisense drugs, the ratio of sensitive versus insensitive sites is very high and much higher than observed with most other chemistries or mechanisms of action. Furthermore, each RNA is different. Thus, in our experience algorithms designed [66,67] to identify optimal sites in target RNAs have simply not performed adequately to reduce the need for detailed in vitro screening (see Chapter 5 for more detailed discussion).

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150

FAK mRNA (relative units)

125

100

75

50

25

0

Figure 1.5

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 Oligonucleotide

Routine screening process to identify optimal antisense drugs (2006). The sequence of the target RNA is used to identify potential antisense inhibitors. On the basis of earlier research, many potentially problematic sequences are excluded. The process is enhanced by evaluating multiple candidates in several species. Avoid mouse immunostimulatory motifs

Avoid mouse toxic motifs Oligo design

Avoid human immunostimulatory motifs

Avoid C strings

Avoid toxicity Mouse lean screens/ pharmacology screens

Avoid proinflammatory activity Avoid toxicity

Monkey screens (human candidate) Avoid kidney toxicity

Figure 1.6

Avoid proinflammatory activity

Results of an initial screen of 2
-MOE chimeric antisense inhibitors designed to inhibit superoxide dismutase 1. Each inhibitor was transfected into A549 cells at 100 nM and the effects on SOD1 RNA levels evaluated 24 h after treatment of the cells.

Do these generalizations apply to drugs other than 2
methoxy-ethyl (2
-MOE) chimeras that work via an RNase H mechanism? Yes. For example, consider Figure 1.7, which compares the activities of phosphorothioate oligodeoxynucleotides and 2
-MOE chimeras. Or consider the experience gained comparing siRNAs to second-generation antisense inhibitors. The activities of siRNAs are influenced

Figure 1.7

5′-UTR 3300 nt

ASO 1

3′-UTR

Intron Targeting ASOs

Comparison of antisense inhibition of p125 FAK by first- and second-generation antisense drugs. A549 cells were treated with 400 nM of each of the antisense drugs. Twenty-four hours after transfection, the level of p125 FAK messenger RNA was assessed by real-time polymerase chain reaction (RT-PCR). The black bars represent the level of p125 FAK RNA with 20 mer phosphorothioate oligonucleotides. The grey bars represent the levels of p125 FAK RNA in cells treated with a 20 mer 2
-MOE chimeric second-generation antisense drugs. Both classes of drugs activate human RNase H1.

0

20

40

ASO 2

Negative control oligos

18

60

Untreated control

21:54

80

100

120

140

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% Control expression

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by target structure just as RNase H–based antisense drugs [68]. In fact a thorough and direct comparison of the performance of second-generation antisense inhibitors to siRNA showed very comparable ratios of sensitive to insensitive sites and considerable overlap in active sites [69]. Single-strand antisense RNAs behave similarly as well [70]. For less robust mechanisms such as translation arrest or inhibition of splicing, the need to identify an optimal inhibitor is even greater. 1.3.9

Therapeutic Specificity (Therapeutic Index)

Clearly, in a therapeutic context, the ability of an oligonucleotide to bind selectively to specific sequences in nucleic acid targets is an important factor in determining its therapeutic index. However, oligonucleotide analogs can interact with other cellular components, and these interactions can have significant effects on the therapeutic index of oligonucleotides. The factors that determine the significance of nonnucleic interactions of oligonucleotides on the therapeutic index include the affinities for nonnucleic acid sites versus nucleic acids, the numbers of different nonnucleic acid binding sites, the concentrations of each of the binding sites, the biological importance of various binding sites, and kinetic factors. These are, of course, conceptually equivalent to the factors that affect the therapeutic index of drugs of all classes, but very little is understood about these potential interactions [71–73]. Chemical classes of oligonucleotides differ in their tendency to interact with various nonnucleic acid targets. For example, phosphorothioates tend to bind to a wide range of proteins with relatively low affinity [15]. Nevertheless, detailed in vitro and in vivo toxicological studies have shown that these interactions probably reduce the therapeutic index of phosphorothioates less than perhaps was expected [7,72]. We believe that this is because the phosphorothioates bind with very low affinity to a large number of proteins and their potential toxic effects are consequently “buffered.” As previously discussed, improvements in the performance of antisense drugs effected by advances in chemistry, design, mechanisms of action, and screening processes have all been shown to profoundly influence the therapeutic specificity of antisense drugs. 1.4 OCCUPANCY-ONLY-MEDIATED MECHANISMS Classic competitive antagonists are thought to alter biological processes because they bind to receptors, preventing natural agonists from binding and inducing normal biological processes. Binding of oligonucleotides to specific sequences may inhibit the interaction of the RNA with proteins, other nucleic acids, or other factors required for essential steps in the intermediary metabolism of the RNA or its utilization by the cell. 1.4.1

Modulation of Splicing

A key step in the intermediary metabolism of most mRNA molecules is the excision of introns. These “splicing” reactions are sequence-specific and require the concerted action of spliceosomes. Consequently, oligonucleotides that bind to sequences required for splicing may prevent binding of necessary factors or physically prevent the required cleavage reactions. This then would result in inhibition of the production of the mature mRNA. In the past several years, substantial progress has been reported in the discovery of antisense drugs that inhibit splicing and result in alternative splicing. Not surprisingly, the first compelling demonstration that antisense drugs could affect splicing was the restoration of correct splicing in thalesemic pre-mRNA in a cell-free system [74]. Since that observation, a variety of chemically modified antisense drugs have been shown to alter splicing in vitro and in vivo. Obviously, to induce alternative splicing, antisense drugs that do not induce degradation of the target RNA must be used, so fully modified 2
-methoxy, 2
-MOE, peptide nucleic acid (PNA), morpholino, and fully modified locked nucleic acid (LNA) analogs have been studied (for review, see [75]).

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Although the inhibition of splicing is interesting, alternative splicing is perhaps more exciting because in this setting it is possible to use an antisense drug to induce the production of an alternative protein; in effect to produce “agonist-like” activities. Both the inhibition of splicing and the induction of alternative splicing have been reported for a number of genes in vitro and a few in vivo (for review, see Chapter 4 in this volume). However, alteration of splicing has proven to be very difficult for many genes and no obvious explanation for difference in susceptibility has emerged. Because the use of antisense drugs to alter splicing is reviewed in detail in a subsequent chapter, in this chapter I will address a number of questions concerning the mechanism: 1. Can antisense drugs be used to alter splicing in vitro and in vivo? 2. How is the activity of antisense drugs affected by the a. Strength of splicing signal b. Splicing element to which the drugs binds c. Position of the drug vis-à-vis the splicing element d. Characteristics of the intron or exon e. Presence of exonic enhancers or silencers f. Chemical class or the antisense drug 3. Is it possible to design antisense drugs that affect exonic enhancer or silencer function? 4. Is efficacy influenced by cell or tissue context? 5. How robust a mechanism is alteration of splicing?

1.4.1.1 Can Antisense Drugs Be Used to Alter Splicing in Vitro and in Vivo? Today, there is no question that appropriately designed antisense drugs can alter splicing. The evidence for this is, of course, broader and more compelling in vitro, but there are now also multiple well-documented examples of antisense drugs altering splicing in vivo.

Alteration of Dystrophin Splicing One of the better-characterized examples of alteration in splicing induced by antisense drugs is the exon skipping induced in dystrophin RNA. Dystrophin is an essential protein for the normal function of skeletal and cardiac muscle. The gene for dystrophin is subject to mutations that result in early termination of the protein or frame shift mutations that can result in a dysfunctional protein. When shortened dystrophin is produced, the protein is partially effective and milder muscular dystrophy is observed. As the pre-mRNA for dystrophin has a large number of introns, the goal has been to induce exon skipping, thereby avoiding the frame shift mutations that result in a shortened form of the protein. A number of laboratories have reported that 2
methoxy and morpholino oligomers induced altered splicing in vitro and in vivo [76–80]. Indeed, in dystrophic mice, induction of alternative splicing with a morpholino antisense drug resulted in improved muscle performance.

B-Cell Lymphoma/Leukemia Cell x (Bc1-x) The Bc1-x gene can result in the production of either a long or short form of the protein and this is effected by use of an alternative splice site. Bc1-x long inhibits apoptosis while Bc1-x short induces apoptosis. 2
-MOE antisense agents were shown to induce the production of Bc1-x short in malignant cells and this resulted in apoptosis [81,82].

Alteration of Splicing in -Globin RNA The Kole Laboratory has developed a transgenic mouse that expresses green fluorescent protein and has a -globin intron with an aberrant splice site. When the aberrant splice is blocked, normal splicing ensues and green fluorescent protein is produced and easily detectable in mouse tissues.

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These investigators used this model to demonstrate that 2
-MOE and 4-Lysine PNA antisense agents could potently induce alternative splicing of the transgene while morpholinos were less potent [83].

MyD88 MyD88 is an adapter protein through which proinflammatory cytokines signal. In a very thorough study, the authors demonstrated that 2
-MOE antisense agents could alter splicing of MyD88 in vitro and in several tissues in vivo [84]. This was associated with reduced interleukin 1 beta (IL-1) signaling in vitro and in vivo.

1.4.1.2 How Is the Activity of Antisense Drugs Affected by the Strength of the Splicing Signal? Both 5
and 3
splicing signals are degenerate in mammalian cells and numerous factors influence the relative efficiency of splicing of different introns. Some of these factors include the strength of the 5
and 3
splice sites, the characteristics of the polypyrimidine tract, the size of the intron, the guanosine cytosine (GC) content of the intron, the presence of exonic enhancers and silencers, and the affinity of the two exonic sequences adjacent to the intron for each other (for review see [85]). To some extent, it may be simpler to define the strength of the 5
splice site than other sites. Based only on the sequence of the 5
splice sites itself, 5
splice sites have been divided into strong, intermediate, and weak. The strength of the splice site correlated with the complementarity to the terminus of uridine-rich RNA 1a (U1a RNA) [86]. Only for the weak 5
splice sites did factors other than the sequence of the splice site play a role. In contrast, the efficiency of 3
splice sites appears to be related to the strength of the splice signal, the GC content of the intron, the position and strength of the polypyrimidine tract, and the presence of exonic regulatory elements. Even considering all these factors, the correlations with predicted rates of splicing are only modest [87]. Thus, it is clear that attempting to evaluate the effects of the efficiency of a particular splicing event on the activity of antisense drugs is likely to be challenging. Indeed, that has proven to be the case. To begin to address the effects of splice-site strength on the activities of antisense inhibitors, some years ago we [88] constructed a luciferase receptor gene with -globin or adenovirus introns. Mutating the weaker splice signals in the -globin construct progressively toward the stronger adenovirus signals progressively increased the extent of splicing. Inhibition of splicing was greater when the weaker splice sites were targeted. On the basis of these studies, we suggested that introns with weaker splicing signals might be more sensitive to antisense inhibition. In a thorough examination of multiple introns in dystrophin pre-mRNA, the effects of one hundred and fourteen (114) 2
methoxy antisense inhibitors designed to bind to different sites and induce skipping of many different exons were evaluated [76]. Although there was substantial variability in the ease with which different splicing of events were inhibited, very little correlation between the strength of splice sites and activity was observed. Antisense agents designed to bind to 3
splice sites and branch points were reported to be ineffective. A detailed analysis of antisense agents designed to cause skipping of exon 23 in the dystrophin pre-mRNA also showed that the 5
splice site, but not the 3
splice, was sensitive to the effects of these agents [77]. In contrast, in other pre-mRNA targets, antisense agents designed to bind to either 5
or 3
splice sites were effective [83,84]. On the basis of all the results available then, it appears that alteration in splicing can be achieved by binding to either 5
or 3
splice sites, but there is great variability in the results. In fact, in unpublished studies focused on eIF-4E and survivin, in which we have thoroughly evaluated 5
and 3
splice sites, again, activities were observed at both splice sites. Figure 1.8 exemplifies our experience. In this study, 20-nucleotide full 2
-MOE antisense agents were designed to bind to various regions of the 5
and 3
splice junctions in various pre-mRNA, cells were treated, and the induction of splice-variant RNA species evaluated. For MCL-1, inhibitors of both the 5
and 3
splice sites were effective, with the 5
splice site being slightly more susceptible. In contrast, for

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Acceptor

Donor

Intron

Exon

0

Intron

0

5′ Splice site ASOs

3′ Splice site ASOs

utc

MCL-1

mMyd88

hMyd88

ERalpha exon4

IL4

Syk

Figure 1.8

Effect of 2
MOE ASOs on RNA splicing. A schematic representation of targeting strategy. A series of 5 ASOs were designed to the acceptor or donor site of the exon targeted for skipping. Number indicates ASO start site relative to the intro/exon junction. All ASOs are 20 nucleotides in length and have 2
MOE ASOs targeted to exon splice sites at a single concentration of 200 nM for 4 h using Lipofectin Reagent. ASO was removed and fresh media added to cells. Following overnight incubation, cells were harvested and total RNA purified using RNeasy mini columns (Qiagen). Standard RT-PCR analysis of mRNA was preformed using PCR primers complementary to exonic sequence bracketing the targeted exon. Of total RNA, 5 g were reverse-transcribed in the presence of oligo(dT) using SuperScript II reverse transcriptase according to the manufacturer’s protocol (Invitrogen Life technologies). Following a 1-h incubation at 42°C, the cDNA was diluted by the addition of 80 l of water. Three microliters of the diluted cDNA were combined with 15 l of HotStarTaq mix (Qiagen) and 2.5 l each of 10 uM forward and reverse PCR primer in a final volume of 30 l. The PCR was cycled 30 s at 94°C, 30 s at 72°C, and 2 min at 60°C with 35 repetitions. Products were visualized by electrophoresis on 2% agarose gels stained with ethidium bromide.

splicing in mouse and human, MyD88, only the 3
splice site was susceptible. For the other targets, both 5
and 3
splice sites were sensitive with slight differences in sensitivity between the two sites. So both the 5
and 3
splice sites are amenable to the effects of antisense drugs. Is there then a pattern of sensitivity with regard to the strength of the splicing signal? If there is, it is certainly neither obvious nor universal. One of the problems, of course, is deciding how strong a splice signal is. Using the classification of 5
splice sites proposed by Roca et al. [86], no pattern in responsiveness as a function of splice signal is evident in published data, nor in unpublished information at

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Isis involving many targets. Although it is even more difficult to assign a “strength” value for 3
splice sites, certainly no pattern emerges [89]. What about more complex approaches to assignment of “strength” to splice sites? A good example derives from studies on estrogen receptor  (ER). ER has 8 exons. Skipping of each of the exons has been observed naturally, as has skipping of several exons simultaneously [90]. Because all the splice sites are relatively homologous to consensus sites, a statistical evaluation of sequences 60 nucleotides upstream and downstream of the splice sites was used to assess relative “strength.” Relative strengths of splice signals were then compared to the frequency with which an intron was skipped naturally. Although the correlation was very limited, the study suggested that something abut the intron–exon junctions around exon 4 encouraged more alternative splicing. We evaluated the ability to alter splicing of three introns in ER (Figure 1.9). In this experiment, we evaluated five 2
-MOE inhibitors positioned around the 5
and 3
splice sites of these exons. Figure 1.9 shows that for the frequently alternatively spliced exon 4 that had an intermediate “strength” of splice signal, potent inhibitors were found. In contrast, exon 7 is also frequently alternatively spliced as is shown in Figure 1.9, but no inhibitors were found.

Acceptor

(a)

Donor

Intron

Intron

Exon

0

0

5′ Splice site ASOs

(b) utc

3′ Splice site ASOs 0

0

ER-alpha exon7

ER-alpha exon5

ER-alpha exon4

Figure 1.9

Effects of 2
MOE ASOs on estrogen receptor alpha RNA splicing. (a) Schematic representation of targeting strategy. A series of 5 ASOs was targeted to the acceptor or donor site of the exon targeted for skipping. Numbering indicates ASO start site relative to the intron/exon junction. All ASOs are 20 nucleotides in length and have 2
MOE sugar modifications with phosphorothioate linkages at all positions. (b) MCF-7 cells were treated with 2
MOE ASOs targeted to exon splice sites at a single concentration of 200 nM for 4 h using Lipofectin Reagent. ASO was removed and fresh media added to cells. Following overnight incubation, cells were harvested and total RNA purified; then standard RT-PCR analysis of mRNA was preformed using PCR primers complementary to exonic sequence bracketing the targeted exon as detailed above.

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION

Exon 5 is also frequently alternatively spliced, has a high “strength score” and no inhibitors were identified. On the basis of all the data, we can therefore conclude the following: ●

● ●

The ability to alter splicing of different introns by antisense drugs is variable and no obvious rules have emerged. Both 5
and 3
splice sites are amenable to effects of antisense drugs. There is no correlation between strength of splice sites or propensity to undergo alternative splicing, and the effectiveness of antisense modulation of splicing is apparent.

1.4.1.3 Does the Position of the Antisense Drug at a Splice Site Affect the Activity of the Antisense Drug? The answer to this question is unequivocally yes. This conclusion is supported by an evaluation of published data and exemplified by the data shown in Figure 1.8. Although effectiveness appears to vary as a function of the position of the 20-nucleotide antisense agents vis-à-vis the splice junctions for all targets, the only generalization supported by the observations is that agents that bind to or are adjacent to the junction and extend some distance into the intron or exon tend to be more effective than those that do not include the splice junction.

1.4.1.4 Do the Characteristics of Introns or Exons Affect the Activities of Antisense Agents? Given the wide variation in responsiveness of introns to alteration of splicing by antisense drugs and the lack of obvious explanations for this provided by in the characteristics of the splice site, it is obvious that other intronic or exonic characteristics must contribute [89,90]. However, no generalizations are feasible based on available data. For example, we evaluated the three ER exons shown in Table 1.1 for the presence of several putative exonic splicing enhancers (ESEs). At the 5
and 3
ends of the exons to which the splice antisense agents would have bound, all three exons were endowed with a number of putative ESEs, yet there was no obvious difference that could explain the ease of induction of skipping of exon 4 versus the other exons. Of course, it may be that the putative ESEs are, in fact, not functional or that antisense binding to the SF2 ESE is somehow unique. Nevertheless, based on evidence available today, the identification of ESEs within the binding site of antisense agents designed to bind to splice junctions results in no obvious guidance with regard to the design of the drugs.

Table 1.1

Predicted Exonic Enhancer Sequences in Estrogen Receptor 
5

ESE sites

3

ESE sites

Target

SF2/ASF Thr  1.956

SC35 Thr  2.383

SRp40 Thr  2.67

SRp55 Thr  2.676

SF2/ASF Thr  1.956

SC35 Thr  2.383

SRp40 Thr  2.67

SRp55 Thr  2.676

ER-alpha E4 ER-alpha E5 ER-alpha E7 mMyD88 hMyD88 Syk MCL-1 IL-4

3.233 N N 2.786 2.786 4.018 N 2.922

2.893 N 3.321 3.867 3.299 3.850 N N

3.009 2.995 N N 3.830 2.863 2.762 N

Na 3.405 N 2.858 3.366 N 3.831 N

2.506 N 4.065 3.122 4.115 3.407 N 3.411

N 3.721 2.476 3.186 3.186 4.956 2.572 2.711

3.455 N 3.526 3.176 3.280 3.585 N 2.692

N N 4.087 N N 2.908 N 3.831

Note: ESEfinder was used to locate putative exonic splicing enhancers within or overlapping the first or last 20 bases of each exon. Scores are given for ESEs with values above the threshold for each sequence element. The thresholds are values above which a score for a given sequence is considered to be significant (high-score motif). Where more than one ESE was found for given sequence element, only the highest score has been given. a ESE absent [89].

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1.4.1.5 Can Antisense Agents Designed to Bind Exonic Enhancer or Silencer Sequences Affect Splicing? The answer to the question is yes. Perhaps the most thorough study reported to date examined the effects of a number of 2
-MOE antisense agents that targeted exonic regulatory sequences in the pre-mRNA of the spinal motor neuron 2 (SMN2) gene [91]. Several agents were shown to either induce or inhibit exon inclusion.

1.4.1.6 Does Chemical Class Influence Activity? Both in vitro and in vivo, the answer to this question is yes. Analyses of in vitro data are more robust both because there are more data and because differences in pharmacokinetic and toxicologic properties are less likely to influence the results. Activities have been observed for fully modified 2
methoxy, 2
-MOEs, 4-Lysine and 8-Lysine PNAs, and morpholinos. As a general rule, in vitro potencies correlated with relative affinities for RNA: PNA2
-MOEmorpholinos2
methoxy [76–84]. Only modest experience has been reported in vivo. Nevertheless, 2
methoxy, 2
-MOEs, morpholinos, and Lysine PNAs have been shown to be active [76–84]. Direct comparisons have again suggested that affinity for target RNA is the critical determinant although differences in pharmacokinetics complicate the analyses. 1.4.2

Translation Arrest

Translation arrest is defined as the inhibition of translation secondary to binding (and not inducing cleavage) of an antisense drug to a target RNA in a fashion that inhibits the translation of the message into protein. Because polysomes are capable of “melting” structures in RNA, inhibitors of translation have typically interacted with the 5
-UTR, or the translation initiation codon, or an internal ribosome entry (IRE) sequence (for review, see [7,15]). However, antisense inhibitors designed to bind to sites in coding sequences have also been shown to be active. Here again, meaningful progress has been reported and significant questions remain to be answered.

1.4.2.1 Is It Feasible to Arrest Translation with Antisense Drugs? Yes. Translation arrest can be induced by a variety of chemical classes of antisense drugs in vitro and in vivo. Well-documented examples include inhibition of intercellular adhesion molecule 1 (ICAM-1) [92], Hepatitis C virus (HCV) [93–95], and c-Myc [96]. In vivo activities have been reported for targets such as HCV and c-Myc [94–96]. Morpholino antisense drugs designed to inhibit translation have also been studied in the clinic and these results are reviewed in Chapter 20.

1.4.2.2 What Are the Optimal Sites in Target RNAs to Induce Translation Arrest? The 5
-UTRs of m-RNAs vary in length from a few nucleotides to hundreds, beginning with a 5
cap [92]. Many m-RNAs also contain internal IREs, thus multiple potential sites outside the coding region may be affected. Several studies have attempted to identify optimal sites in target RNAs for translation arrest. For example, in a study on Hepatitis C viral core protein production in hepatocytes, a 2
-MOE antisense agent designed to the translation initiation codon did not inhibit protein production while another antisense drug designed to bind to a loop in the 5
-UTR was effective [97]. In a more detailed study, multiple 2
methoxy antisense agents were designed to bind a variety of sites in the 5
-UTR and the coding region of the core protein. The effects were evaluated in a cell free protein synthesis assay and in hepatocytes [93]. Binding to several sites in the UTR, the translation initiation codon and in the open reading frame resulted in inhibition of core protein synthesis, but the most effective inhibitors were all located near the translation initiation site. In fact, effectiveness declined dramatically as the binding sites were shifted a few nucleotides into the coding sequence from the translation initiation codon.

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In contrast, for mammalian m-RNAs, most studies have suggested that binding in the 5
-UTR tends to be more effective. For example, the inhibition of the translation of ICAM-1 was effected by binding antisense agents to the 5
cap [91]. In this interesting study, the level of ICAM-1 RNA actually increased in the cytoplasm and was associated with subpolysome functions rather than polysomes, suggesting that entry into the polysome cycle was inhibited by binding the antisense drug to the 5
cap. Also of interest was the observation that binding to the area of the m-RNA adjacent to the cap had no effect on the splicing of the first intron.

1.4.2.3 What Is the Influence of Chemical Class on Activity? Very little work has been reported with regard to this question, but two studies [92,93,98] have evaluated the effects of various fully 2
-modified oligonucleotides. Although the correlation is not perfect, if the results from studies with the relatively unstable 2
fluoro (2
F) analog are excluded, there is a trend toward increasing potency with increasing affinity for target RNA.

1.4.2.4 How Robust Is Translation Arrest? Given the amount of work reported on translation arrest, a definitive conclusion about the robustness of the mechanism versus cleavage-moving mechanisms is not possible. However, it is clear that significantly less sequence space is available to antisense agents designed to inhibit translation splicing and the ratio of actives to inactives is lower than for cleavage-based mechanisms. However, in the one study that directly compared translation arrest to RNase H–based target RNA cleavage, potencies were roughly equivalent in vitro [92]. 1.4.3

Disruption of Necessary RNA Structure

RNA adopts a variety of three-dimensional structures induced by intramolecular hybridization, the most common of which is the stem loop. These structures play crucial roles in a variety of functions. They are used to provide additional stability for RNA and as recognition motifs for a number of proteins, nucleic acids, and ribonucleoproteins that participate in the intermediary metabolism and activities of RNA species. Thus, given the potential general utility of the mechanism, it is surprising that occupancy-based disruption of RNA structure has not been more extensively exploited. As an example, we designed a series of oligonucleotides that bind to the important stem-loop present in all RNA species in human immunodeficiency virus (HIV), the transactivating region (TAR) element. We synthesized a number of oligonucleotides designed to disrupt TAR, and showed that several indeed did bind to TAR, disrupt the structure, and inhibit TAR-mediated production of a reporter gene [99,100]. Furthermore, general rules useful in disrupting stem-loop structures were developed [101]. Although designed to induce relatively nonspecific cytotoxic effects, two other examples are noteworthy. Oligonucleotides designed to bind to a 17-nucleotide loop in Xenopus 28 S RNA required for ribosome stability and protein synthesis inhibited protein synthesis when injected into Xenopus oocytes [102]. Similarly, oligonucleotides designed to bind to highly conserved sequences in 5.8 S RNA inhibited protein synthesis in rabbit reticulocyte and wheat germ systems [103].

1.5 OCCUPANCY-ACTIVATED DESTABILIZATION RNA molecules regulate their own metabolism. A number of structural features of RNA are known to influence stability, various processing events, subcellular distribution, and transport. It is likely that as RNA intermediary metabolism is better understood, many other regulatory features and mechanisms will be identified.

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1.5.1

27

5

Capping

A key early step in RNA processing is 5
capping (Figure 1.2). This stabilizes pre-mRNA and is important for the stability of mature mRNA. It also is important in binding to the nuclear matrix and transport of mRNA out of the nucleus. Since the structure of the cap is unique and understood, it presents an interesting target. Several oligonucleotides that bind near the cap site have been shown to be active, presumably by inhibiting the binding of proteins required to cap the RNA. For example, the synthesis of Simian virus 40 (SV40) T-antigen was reported to be most sensitive to an oligonucleotide linked to poly-L-lysine that targeted the 5
-cap site of RNA [102,104]. However, again, in no published study has this putative mechanism been rigorously demonstrated. In fact, in no published study have the oligonucleotides been shown to bind to the sequences for which they were designed. In studies in our laboratory, we have designed oligonucleotides to bind to 5
cap structures and identified reagents to cleave the unique 5
cap structure [105]. These studies demonstrated that 5
-cap-targeted oligonucleotides were capable of inhibiting the binding of the translation initiation factor eIF-4. 1.5.2

Inhibition of 3

-Polyadenylation

In the 3
-UTR of pre-mRNA molecules, there are sequences that result in the posttranscriptional addition of long (hundreds of nucleotides) tracts of polyadenylate. Polyadenylation stabilizes mRNA and may play other roles in the intermediary metabolism of RNA species. Theoretically, interactions in the 3
-terminal region of pre-mRNA could inhibit polyadenylation and destabilize the RNA species. Although there are a number of oligonucleotides that interact in the 3
-UTR and display antisense activities [106], to date, only one study has reported evidence for alterations in polyadenylation. In this study [106], fully modified 2
-MOE antisense regents caused polyadenylation to be redirected, increasing RNA stability and enhanced protein synthesis. This study certainly merits attention and follow up because the mechanism offers the potential to create antisense agents that selectively increase expression of a protein. 1.5.3

Other Mechanisms

In addition to 5
capping and 3
adenylation, there are clearly other sequences in the 5
- and 3
-UTRs of mRNA that affect the stability, localization, and translatability of the molecules. Again, there are a number of antisense drugs that may work by interfering with these processes, but no studies that confirm these possibilities have been reported. 1.5.4

RNase H

Without question, the RNase H mechanism has proven to be the most robust mechanism identified and characterized to date. The experience with first- and second-generation RNase H–based antisense drugs in vitro and in vivo exceeds by many fold the total experience with drugs of all other mechanisms. Several thousand humans have been treated with first- and second-generation RNase H–based antisense drugs. Again, this experience dwarfs the combined experience with antisense drugs designed to work through other mechanisms (for review, see 9; Chapters 2, 3, and 4; and Part 4 of this volume), and this mechanism is reviewed in considerable detail in Chapter 2 of this volume. In this chapter, I will address a number of questions about the RNase H mechanism and consider future experiments. The RNases H cleave RNA only in RNA–DNA duplexes. In human cells, two RNases H have been identified, cloned, and expressed (for review, see [107,108] and Chapter 2 in this volume). We have characterized the enzymological properties of human RNase H1 and to a lesser extent those of human RNase H2 (for review see chapter 2 of this volume). We have demonstrated that the antisense effects

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of DNA-like antisense drugs in cells and animals are due strictly to the activities of RNase H1, even though RNase H2 is substantially more abundant. We have demonstrated that RNase H2 is not involved in the effects of DNA-like antisense drugs because it is inaccessible to the oligonucleotide RNA duplexes formed, probably because it is bound to chromatin (Chapter 2, this volume and [107–109]).

1.5.4.1 Do Antisense Drugs That Use the RNase H Mechanism Work? Yes. In vitro, in vivo, and in humans RNase H–based antisense drugs have produced impressive pharmacological effects in a wide range of cells and tissues when given by a wide range of routes of administration (for review, see [9] and this volume). In fact, in studies in our laboratories on more than 4000 genes, we have never failed to identify multiple potent and selective RNase H–based actives for any target.

1.5.4.2 What Sites in Target RNAs Are Accessible to RNase H–Based Antisense Drugs? Figure 1.7 shows results form an in vitro screen for RNase H–based active sites. Obviously, sites throughout the pre-mRNA are accessible to RNase H1–based antisense drugs. Despite the vast experience in designing and testing RNase H–based antisense drugs, no rules or guides have emerged that enable selection of optimal sites in target RNAs to which to bind to. Thus, we continue to screen against a large number of sites throughout the target RNA as shown in Figure 1.7. That no rules for site selection have emerged despite the enormous experience confirms that the interaction of oligonucleotides with target RNA and the recruitment and activation of RNase H1 are complex processes governed by many factors that are inadequately understood. The demonstration that sites in pre-mRNA and mature mRNA appear to be equally amenable to the effects of RNase H–based antisense drugs is consistent with the observations that human RNase H1 is present in the nucleus, cytosol, and mitochondria (for review, see [15,108,109] and Chapters 2 and 10 in this volume). As we have demonstrated that recruitment of RNase H1 to the antisense drug–RNA duplex is limiting [109], it is likely that in addition to the access of the antisense drug to a site in a target RNA, the access and relative activity of human RNase H1 at the site of drug binding to the duplex is critical. Human RNase H1 consists of an RNA-binding domain, a spacer region, and a catalytic domain (Figure 1.10) (for review, see [110,111] and Chapter 2 in this volume). Two Lysines and a tryptophan in the RNA-binding domain position the enzyme at the first DNA/RNA nucleotide and the catalytic domain cleaves approximately one helical turn from the binding site. The enzyme displays little sequence preference, but within a particular site the enzyme displays preferred cleavage sites. Thus, for any site to be affected by human RNase H1, a binding site and a site with appropriate characteristics to support cleavage must be separated by one helical turn from the RNA binding site. Therefore, a number of sites that might be accessible to antisense drugs might not be optimal sites for RNase H1 resulting in variations in potency. The use of second-generation antisense drugs exaggerates the potential effects of site selection as shown in Figure 1.11. Consequently, the substantial variations in potency from site to site are easily explained. Until much more is known about the influence of sequence on protein binding and subtle changes in RNA structure, it is very unlikely that more sophisticated rules for site selection will be developed, i.e., screening to identify optimal sites will remain essential. Perhaps a more interesting question is why so many sites are amenable to RNase H1–mediated cleavage. The answer to this question awaits much more research. We know that RNase H1 is active as a monomer in contrast to RNase H2, which is only active in a complex of several proteins. We also know that RNase H1 is present in multiprotein complexes, but do not know the identity of any of the RNase H1–associated proteins. Perhaps the associated proteins facilitate access and the induction of the appropriate RNA conformation to support activity.

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Figure 1.10 Schematic showing a simplified view of RNase H and siRNA mechanisms. The left panel shows a single-stranded DNA-like antisense oligonucleotide entering a cell, interacting with its target RNA in the cytoplasm or nucleus of a cell, and then recruiting RNase H1. The right panel shows an siRNA duplex entering a cell, the duplex entering the siRNA pathway, the sense strand being removed, and the RISC complex cleaving the target RNA.

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RNA-BD

Cat

5′ 3′

mRNA ASO

Figure 1.11 Schematic of potential interactions of human RNase H1 with a 2
-MOE- chimeric antisense drug mRNA duplex. Because RNase H1 is unable to cleave RNA opposed to a 2
-MOE modified nucleotide and because subtle conformational changes due to sequence differences result in preferred cleavage sites, the extent of cleavage can vary substantially from site to site in a target RNA.

1.5.4.3 Can Information about the Enzymology of RNase H1 Be Used to Improve the Performance of RNase H–Based Drugs? Yes. This topic is dealt with in considerably more detail in Chapter 2 of this volume. Suffice it to say, we have shown that by extending the RNase H gap and introducing modifications at the junctions of the MOE wings and the gap, we can improve potency [110,112]. For example, we have shown that a gap-widened version of a 2
-MOE chimeric antisense inhibitor of phosphatase and tensin homolog deleted on chromosome ten (PTEN) was significantly more potent in reducing liver PTEN RNA than the parent second-generation antisense drug when administered to mice [111]. Although the extent to which the potency may be increased varies, several of these “generation 2.2” analogs are progressing toward clinical trials.

1.5.4.4 How Robust a Mechanism Is RNase H? The RNase H mechanism is remarkably robust. At Isis alone, RNase H–based inhibitors to as many as 4000 genes have been identified. With second-generation antisense inhibitors, the active to inactive ratio in vitro is typically 1 or greater. Thorough studies comparing second-generation RNase H–based inhibitors to siRNA in vitro have demonstrated that the two approaches result in approximately the same hit rates and similar potencies at most sites [69]. However, for scientists without ready access to 2
-MOE chimeric antisense agents, siRNA is probably a more efficient and easier-to-use approach for in vitro gene functionalization (e.g., see [113]). The fact that there is considerable overlap in active sites in target RNAs for 2
-MOE second-generation antisense inhibitors and siRNA (Figure 1.12) suggests that access to human RNase H1 and to the RISC complex in the cytoplasm to target RNAs must be similar. More important, there is substantial experience with RNase H–based first- and second-generation antisense drugs in animals and in man. Second-generation 2
-MOE antisense inhibitors have proven to be potent and versatile gene-selective inhibitors in vivo. As their pharmacokinetic and toxicological properties are well defined, the design of appropriate controls and interpretation of the results of in vivo experiments are relatively straightforward (for review, see [10–12,114] and Part 3 of this volume). Moreover, they can be administered by multiple routes [115]. Finally, they display important activities in clinical trials (for review, see Part 3 of this volume). Another important consideration with regard to the “robustness” of an antisense mechanism is how well in vitro results obtained with agents that work via the mechanism in question correlate with in vivo observations. We have had the opportunity to address this question in a number of ways and in several species, but the most complete data set compares the potencies of RNase H–based antisense drugs in reducing target RNAs in vitro to their potencies with regard to reducing target RNA levels in the liver of mice treated systemically with the drugs. One interesting means to evaluate this is to systematically adjust the potency of antisense drugs either by introducing mismatches or by introducing chemical modifications, then comparing the rank order potency in vitro versus in vivo. For example, the potency of first-generation RNase H–based

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Figure 1.12 Schematic showing the RNA binding, spacer, and catalytic domains of human RNase H1 and their interactions with an mRNA antisense drug duplex.

inhibitors to c-Raf kinase was varied by introducing mismatches; then the rank order potency in vitro was compared to that observed for reducing c-Raf kinase in RNA levels in mouse livers. There was a one-to-one correlation [116]. These observations have been repeated many times with other targets and other sets of antisense agents. Further, we compared the activities of 129 RNase H–based antisense drugs targeted to 63 different genes in vitro to their relative potencies in reducing the same target RNA in mouse livers after systemic administration. The correlation was highly statistically significant ( p 0.001). Thus, the RNase H mechanism has proven to be extraordinarily robust in vitro and in vivo, and as growing data suggest, in the clinic. In fact, the robustness of the mechanism is even more remarkable on reflection. We know that only RNase H1 is involved in this mechanism. We have shown that although it is expressed ubiquitously, the concentration of RNase H1 in cells is very low [117]. Moreover, phosphorothioate antisense drugs are highly effective competitive antagonists to human RNase H1 [118]. Despite this, for many targets, RNase H–based antisense drugs can reduce the levels of target RNAs by as much as 90%. Why is not the top part of the dose–response curve lost if phosphorothioate-modified oligonucleotides are such effective inhibitors of human RNase H1? We do not know, but we hypothesize that human RNase H1 is located in a multiprotein complex. Perhaps it is protected from inhibition until it binds to a duplex substrate. We also surmise that for most target RNAs, turnover of the enzyme is not terribly important because of the limited number of copies of most RNAs in cells. These questions should be the focus of future research in addition to continuing to use the understanding about human RNase H to guide the development of antisense drugs that form RNA antisense duplexes that are more attractive substrates for human RNase H1. 1.5.5

Double-Strand RNase (siRNA)

In 1998, using stabilized single-strand and duplex oligoribonucleotides, we reported that dsRNases in cells and tissues could degrade RNA–RNA-like duplexes [38]. We also showed that nuclear and cytoplasmic homogenates could degrade RNA–RNA-like duplexes and partially purified the enzyme involved. Subsequently, such activities in mammalian cells were shown to be mediated, at least in part, by the siRNA pathway [70]. The identification of the siRNA pathway in mammalian cells has led to the emergence of the use of siRNAs for gene functionalization, target validation, and therapeutic purposes, and to the elaboration of exciting new areas of cell biology. Chapters 3, 15, and 16 of this volume provide detailed reviews of the

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pathways and progress in the development of siRNA therapeutics. In this chapter, our goals are to the following: Mechanistically compare and contrast the siRNA mechanism to the RNase H–based mechanism. Consider some of the unique opportunities and challenges presented by the siRNA pathway with regard to the therapeutic potential of siRNAs. Consider some of the challenges of using duplex RNAs as therapeutics and potential solutions to these issues.

● ●

●

1.5.5.1 siRNA and RNase H Mechanisms Are Similar The goal of both RNase H–based and siRNA antisense inhibitors is to bind to target RNAs via Watson and Crick hybridization and recruit a cellular nuclease that will degrade the targeted RNA (Figure 1.13). Antisense drugs designed to work via the RNase H mechanism are single-stranded and must have a DNA-like portion that serves as the antisense strand of the duplex that becomes a substrate for RNase H1. As RNase H1 is present in both the nucleus and cytosol, it is possible for these antisense agents to work in both cellular compartments and on sites in the RNA that are excised in the nucleus or are present in the mature m-RNA. siRNA activities are typically effected with duplex RNAs. The simplest way to think of the sense strand is that it meets the basic definition of a drug delivery device: it is used to enhance the stability of the drug, the antisense strand, and the delivery of the antisense strand to the site of

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Target site start position Figure 1.13 Phosphatase and tensin homolog deleted on chromosome ten (PTEN) oligonucleotide screen. A series of 36 chimeric RNase H–dependent oligonucleotides and a series of corresponding siRNA duplexes were administered to T24 cells in the presence of Lipofectin Reagent. Sixteen hours later, total RNA was harvested and PTEN mRNA levels assessed by qRT/PCR as detailed in Materials and Methods. Results are the percent PTEN mRNA relative to untreated control. Solid bars: chimeric RNase H–dependent oligonucleotides; striped bars: siRNAs.

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action, RISC. Unfortunately, it is not yet an optimal drug delivery device because it is not pharmacologically inert and is metabolized to potentially pharmacologically active metabolites. Once the sense strand is removed, the antisense strand is used by the RISC complex to degrade the target RNA. It has been suggested that RISC is localized in the cytoplasm, so siRNA drugs are assumed to work in the cytoplasm. Thus, in both cases, hybridization-based drugs are designed to exploit normal cellular processes to effect cleavage of target RNA. Thanks to the extraordinary progress in understanding the RISC mediated pathways, we know more about the cellular functions and pathways with which siRNAs may interact than RNase H–based antisense drugs, but obviously RNase H has cellular functions and participates in pathways including those involved in de novo and repair DNA synthesis and probably others (for review, see Chapter 2). Similarities between RNase H–based and RISC-based mechanisms extend beyond both simply being RNA cleavage mechanisms. As was discussed previously, both require access to RNA and display similar patterns of sites of activities in target RNAs [119–121]. Both are influenced similarly by RNA structure [119–121]. The variations between the potencies for the most sensitive versus least sensitive sites in RNA are roughly comparable [119]. In fact, eIF2c2, the RISC effector, is an RNase H–type enzyme and generates cleavage products with 5
phosphates and 3
hydroxyls [122,123]. Both mechanisms are quite robust in vitro despite the fact that human RNase H1 and eIF2c2 are present in low amounts in cells. Finally, oligonucleotides that employ both mechanisms can be chemically modified with similar modifications, albeit at different positions and with somewhat different results (for review, see [124–129] and Chapter 15 of this volume). There are also important differences between the RNase H and RISC-based mechanisms.

1.5.5.2 RISC-Mediated Pathways One of the most important differences between the RNase H–based mechanism and the RISCbased mechanism is that pathways can be induced by activation of RISC or other pathways that may be induced by double-strand RNA molecules are remarkably more diverse that those associated with RNase H. In fact, the siRNA-associated pathways are extraordinarily complex and our understanding of the pathways evolves almost monthly as new data are reported. These pathways will be reviewed in much more detail in Chapter 3 and other chapters of this volume. The purpose of this discussion is to highlight therapeutic issues and opportunities related to the diverse siRNA pathways and the pleitropic effects that they may mediate. In contrast to RNase H, RISC-mediated pathways are designed to differentially respond to siRNAs that are fully or partially complementary to target RNA and to induce different outcomes (Figure 1.14). Fully complementary siRNAs are thought to induce an initial cleavage by eIF2c2, then subsequent degradation of the target RNA in cytoplasmic bodies. Partially complementary siRNAs may induce target RNA degradation or translation arrest. Thus, cellular responses to siRNAs are much more promiscuous with regard to hybridization and more versatile than those to RNase H–based antisense drugs, offering interesting opportunities and substantial challenges. From a gene functionalization perspective, the potential to affect multiple RNA targets is highly problematic, but the potential to affect the level of synthesis of unintended proteins is even more problematic because of analytical limitations in measuring proteins. From a therapeutic perspective, the potential to induce multiple hybridization-based effects through at least two different mechanisms is a cause of substantial concern and will require very careful experimentation and controls to (1) define the mechanism by which pharmacological effects are induced and (2) ensure the safety of each siRNA drug candidate. Moreover, since such effects may be very species-specific (if not cell- and tissue-context dependent), they influence considerations about the design of preclinical toxicological evaluation.

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Programmed RISC

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Ribosome Cytoplasmic bodies Figure 1.14 RISC Pathway Programmed RISC contains guide (antisense) strand bound to the slicer enzyme eIF2c2. Guide strand exhibiting full complementarity (siRNA) or partial complementarity (miRNA) hybridize to target mRNA and the glycine/tryptophan-rich GW182 RNA-binding protein localizes programmed RISC to cytoplasmic bodies [168]. eIF2c2 of programmed RISC containing siRNA guide strand cleaves target mRNA. eIF2c2 nicked mRNA is further degraded by the 5
→ 3
exonuclease XRN1 and 3
→ 5
exonuclease of the exosome in cytoplasmic bodies [169]. Exosome cofactors SKI2, 3, and 8 of yeast have also been shown to be important for the degradation of the nicked mRNA [169]. Target mRNA bound to programmed RISC containing miRNA guide strand is degraded in the cytoplasmic bodies by deadenolases and the decapping enzymes DCP1 and 2 [170,171]. Alternatively, target mRNA bound to programmed RISC containing miRNA guide strand promotes ribosomal drop-off, resulting in translational arrest [172].

1.5.5.3 RISC-Mediated Pathways Are Promiscuous with Regard to Hybridization-Based Off-Target Effects Although effects on target RNAs that have sequences that are partially complementary to the desired target are observed on occasion with RNase H–based antisense drugs, this has proven to be a very modest issue. In fact, the dominant cause of off-target effects of RNase H–based antisense drugs is interactions with proteins. In contrast, siRNA-mediated systems have evolved to be promiscuous at the level of RNA sequence. This is a unique and potentially very difficult problem with which to contend. In fact, as suggested by Figure 1.14, the RISC complex tolerates a large number of mismatches outside the seed region and still results in translation arrest and/or target RNA cleavage. This is dramatically different from RNase H, which may tolerate a mismatch or two at the 5
or 3
termini, but is intolerant of mismatches of any sort in the cleavage area (for review, see Chapter 2 in this volume). Nevertheless, specific effects for many siRNAs have been reported; there are probably qualitycontrol processes that limit the number of off-target effects. But there are now several reports that

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demonstrate that hybridization-based off-target effects are common with siRNAs. Two papers published by the same group [130,131] exemplify the challenges related to the promiscuity of siRNAs and the problems of developing an exciting new scientific idea. Despite the common wisdom that all pharmacological agents result in unintended effects and scores of publications dealing with offtarget effects of other hybridization-based drugs, this group initially drew the following conclusion: “These results indicate that siRNA is a highly specific tool for targeted gene knockdown, establishing siRNA mediated gene silencing as a reliable approach to large scale gene functionalization and target validation” [130,131]. One year later, using similar methods but more thorough investigations, the same authors reported very substantial off-target effects that occurred at concentrations near those at which the target RNA was reduced [132]. Such hybridization-based off-target effects have also been reported by other laboratories [133–135]. In one study, RNAs with 3
-UTR sequences complementary to the seven-nucleotide seed region of the antisense strand of siRNA (a large number) were affected [133,135]. This problem is exacerbated by the potential for true nonspecific effects that may not be related to promiscuous hybridization but rather other mechanisms such as induction of interferon [134]. Furthermore, a screen of randomly selected siRNAs showed that a number of siRNAs could result in reduced viability of HeLa cells transfected with 10 nM of the siRNAs. The toxicity appeared to correlate with the presence of a four-base-pair motif [136]. The mechanisms that might underlie the cellular toxicity were not identified. That the problems of specificity associated with promiscuous hybridization may not be insurmountable is suggested by one publication in which modifications at position 2 of a siRNA reduced off-target effects [137]. It is also important to remember that the sense strand is capable of hybridization-based effects as well as the antisense strand. Contending with the potential of promiscuous hybridizationbased effects of two stabilized strands will be particularly challenging. However, as siRNAs are modified to enhance nuclease stability and in vivo pharmacokinetics, those modifications are likely to increase protein binding, exacerbating the non-hybridization-based off-target effects. In short, a great deal of very careful pharmacological work is required to minimize these problems. Specifically, we must develop appropriate chemistries and designs to reduce the promiscuity of hybridization-based effects while minimizing non-hybridization-based effects. We must be able to measure both strands in vivo and create sense strands that are relatively rapidly degraded so as to avoid the potential pharmacological effect of the sense strand. Alternatively, the use of stabilized single-strand antisense oligonucleotides may be of value here.

1.5.5.4 siRNAs May Induce Transcriptional Repression siRNAs can induce heterochromatin and gene silencing, i.e., a heritable repression of gene transcription, in all species including humans (for review, see [35,138]). siRNAs may be generated in the nucleus by the action of the RNA-dependent RNA polymerase on single-stranded RNA or by transcription of inverted DNA repeats [35]. These siRNAs can interact with the RITS complex. Activation of RITS can directly methylate DNA or induce methylation of histones, which can methylate DNA [138]. This then leads to the formation of heterochromatin, silencing of the targeted gene, and in some cases spreading of silencing to contiguous genes [139–144]. Exogenous siRNAs complementary to promoter regions of both an integrated reporter gene and an endogenous gene in human cells have also been reported to be capable of transcriptional gene silencing [144]. The transcriptional silencing activity required transport of the siRNA to the nucleus, suggesting that it interacts with the RITS complex in the nucleus. However, RISC and siRNA have now been reported to be present in the nucleus [145], so the effects might have been mediated via nuclear loading RISC and transfer to RITS. What are the implications of transcriptional gene silencing, heterochromatin formation, and spreading for the therapeutic uses of siRNAs and RISC activation? Although it is theoretically possible to avoid promoter sequences, it is also possible that the RITS complex is imprecise in its

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interactions with nucleic acid sequences. If RITS were to be activated by therapeutic siRNAs, in theory alteration in the genome could result. If the RITS complex displays limited promoter sequence fidelity or if spreading occurs, substantial, entirely unexpected genomic effects could ensue. Thus, until more is understood it seems sensible to attempt to avoid activation of the RITS pathway if possible.

1.5.5.5 siRNAs Have Displayed Activities in Vivo That Are Similar to Those Displayed by RNase H Antisense Drugs—But There Are Also Substantial Differences Gratifying progress has been reported in demonstrating that siRNAs can be active in vivo. However, much remains to be learned before it is clear that siRNAs have properties that will support systemic applications for therapeutic purposes. Unmodified siRNAs designed to reduce TNF superfamily 6 (FAS) message and protein levels were administered to mice via hydrodynamic vein injections. Doses of 2–2.3 mg/kg were administered at 0 h, 8 h, and 24 h; then liver effects were evaluated. Fas RNA and protein were reduced for multiple days after the injections and the mice were protected from ligand-induced hepatitis [146]. These remarkable results may be partially explained by the hydrodynamic injection method. However, it has clearly been shown that unmodified siRNAs are rapidly cleared from blood via glomular filtration and are relatively rapidly degraded in plasma and tissues [147]. Thus, the effects and their prolonged duration are difficult to reconcile with the known pharmacokinetic properties of these chemicals, unless once RISC is loaded, the complex is stable and provides effects for prolonged periods of time. This same group also reported that siRNA bound to a fusion protein that included the fab fragment of an HIV-1 envelope antibody could accumulate in B16 melanoma tumors and reduce tumor growth [148]. Other groups have reported reduction of target RNAs in the livers of mice after delivering unmodified and modified siRNA in liposomes and in the livers of monkeys [149,150]. While these are encouraging results, substantial experience with liposome-formulated oligonucleotides has demonstrated significant limitations including the need to give the drugs by intravenous infusion and significant toxicities. A more important and relevant study was conducted in mice treated with siRNA containing 2
methoxy and partial phosphorothioate modifications and 3
sense conjugated cholesterol. This siRNA was targeted to apoB100 and produced significant reductions in apoB100 and reductions in cholesterol [151]. This work is important because it demonstrates that a chemically modified siRNA duplex can be delivered systemically without liposome formulation and produce effects. However, we have shown multiple times that cholesterol-conjugated oligonucleotides are highly hepatotoxic [152,153]. In conclusion, there is ample evidence of progress toward being able to use siRNAs systemically for therapeutic purposes. However, a great deal of work needs to be accomplished before such approaches can be considered ready for clinical development, in contrast to second-generation RNase H–based antisense drugs.

1.5.5.6 Structural Features and Medicinal Chemistry of siRNAs That it is possible to exploit the RISC pathway using agents that differ substantially from the 21-base-pair duplexes originally shown to be active [154] is now clear. One of our principal areas of focus is to develop stable single-strand RNA-like antisense drugs that can exploit both RISC and non-RISC mechanisms. Our demonstration in 1998 that single-strand stabilized RNA-like antisense agents were active in cells [38] and the demonstration that single-strand antisense RNA activates RISC [70,155,156] are quite exciting. In our laboratories, we have confirmed that chemically modified single-strand antisense oligoribonuleotides can reduce target RNAs and, at least in part, their effects are mediated by the RISC pathway (Balkrishen Bhat unpublished results). These results are perhaps more remarkable than might appear at first blush since we know that even extensively modified single-strand antisense oligoribonuleotides are still relatively unstable. Thus, as more is

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learned about the medicinal chemistry of single-strand oligoribonuleotides, significant improvements in potency should be achievable. This could be quite important if some of the potential limitations of duplex RNA drugs about which we are concerned prove to be difficult to overcome. It is also possible to vary the length and structures of siRNA duplexes and influence the activities of siRNAs. In addition to variations in the ends (blunt versus overhangs), other factors such as GC content, bias toward low internal stability at the 3
terminus of the sense strand, and lack of inverted repeats increase siRNA activities in vitro [157]. siRNAs that were 25–31 nucleotides long and were substrates for dicer-mediated cleavage were also found to be more potent than 21 mers [158]. Additionally, substantial progress in defining potential chemical modifications that might be useful and identifying sites in siRNAs that can be modified without dramatic loss of activity have been reported [124–129,151]. Thus, it seems feasible to introduce a variety of modifications that may improve the drug properties of both single-strand antisense oligoribonuleotides and siRNA. The structure–activity relationships are being worked out for both classes of molecules and they appear to be different. More work is required to identify the modifications and positions to be modified that provide the best properties for systemic applications for therapeutic purposes for both classes of molecules.

1.5.5.7 Unique Challenges of Duplex RNA Drugs In the discovery and development of new drugs, many factors must be considered in addition to the observations of desired pharmacological effects at achievable doses. siRNA duplexes pose drug development challenges that are unique and different from those encountered with single-strand oligonucleotides.

Physical Chemical Properties siRNA duplexes are substantially different from single-strand oligonucleotides. Obviously, they are at least twice the molecular weight of single-strand antisense drugs. This is nontrivial since single-strand antisense drugs have molecular weights of 6000–7000 Daltons, the increase to perhaps greater than 14,000 Daltons should dramatically affect pharmacokinetic and toxicologic properties. It would also substantially increase the cost of manufacturing such drugs. Perhaps more important is the change in the interactions with water. Single-strand oligonucleotides are amphipathic, with the phosphates being very water-soluble and the bases hydrophobic. In a duplex, the phosphate backbones are presented to water and water is somewhat excluded from the more hydrophobic internal portion of the duplex. All these changes mean that extrapolation from the behavior of single-strand oligonucleotides to the behavior of duplexes is not possible.

Manufacturing Large-scale manufacturing of single-strand antisense drugs is well in hand and the advances made over the past 15 years can be employed in the manufacture of the sense and antisense strands of the duplex. The challenge will be to hybridize on large scale and to prove that there is no residual single-strand contamination.

The Sense Strand The sense strand is a drug delivery device. However, it is pharmacologically active. It may bind to unintended transcripts. It may be an immunostimulant. It may bind to a variety of proteins. As the sense strand is modified to enhance the pharmacokinetic properties of the duplex, these properties will likely become more prominent. Certainly, assays will need to be developed to follow the fate of the sense strand and to evaluate the potential effects of this pharmacologically and metabolically active drug delivery device.

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Immune Stimulation Oligonucleotides are immunostimulatory. The immunostimulatory properties of oligonucleotides are affected by dose length, sequence, chemical modifications, internal structure, and other factors (for review, see [9] and Chapters 1, 3, 13, and 27 of this volume). For single-strand antisense drugs, it is now possible to reduce the potential for immunostimulation. It is also possible to optimize to enhance immunostimulatory (see Chapter 27 of this volume). As a general rule, any propensity toward internal structure enhances the immunostimulatory potential of oligonucleotides. Thus, conceptually, duplex RNA might be expected to be at least as immunostimulatory and perhaps much more immunostimulatory than single-strand oligonucleotides once they are sufficiently stable and distribute to a variety of organs. In fact, siRNAs have been shown to be immunostimulatory. They have been shown to induce interferon in mammalian cells [159]. Unmodified siRNAs 23 nucleotide and longer induced interferon, up-regulated the dsRNA receptor and toll-like receptor 2 in a celltype-specific mamer [157]. In addition, dsRNAs may induce innate immunity [87]. Finally, both single strands when liberated from the duplex have the potential to be immunostimulants. Does the potential to induce a wide variety of immunological responses secondary to duplex properties and motifs, single-strand properties and motifs, and a variety of mechanisms pose an insurmountable problem? Probably not. However, it requires careful attention and lessons learned from studies on single-strand antisense drugs should be considered carefully. For example, ensuring that pharmacological observations are truly the result of siRNAs effects on the target RNA and not confounded by immunological effects is crucial and has been the subject of an enormous effort with regard to RNase H antisense-based drugs. For single-strand antisense drugs, we know that the rodent is particularly sensitive to these effects. We know that there are problematic sequence motifs that are species-specific. We know that chemical modifications can alter immunological properties of these drugs. We know that liposome encapsulation may worsen these effects (for review, see [7]). These lessons should be considered as pharmacological effects are reported and decisions to develop siRNA drugs are made. 1.5.6

Covalent Modifications of Target Nucleic Acids

One area of research that was a significant focus for a number of years was the synthesis of oligonucleotides conjugated to alkylating moleties species designed to alkylate the target RNA and result in disablement and degradation, or either of these. Despite all the early excitement and work, this approach has to a large extent been abandoned. This is primarily because there was no evidence that such approaches resulted in drugs as effective as those that were RNase H–based and because of concerns about toxicity (for review, see [15]). 1.5.7

Oligonucleotide-Induced Cleavage of Target RNA

Another approach that has been the subject of very substantial investment was the creation of either oligonucleotides that were conjugated to RNA cleavage reagents or synthetic ribozymes. Although work continues in these areas (for review, see [15]), little progress has been reported in recent years. It now appears that it is pharmacologically more attractive and feasible to create antisense drugs that recruit either RNase H1 or dsRNases such as the RISC complex than to create oligonucleotides that can effect RNA cleavage themselves. 1.5.8

RNase L–Mediated Cleavage

RNase L or 2
-5 adenylate–dependent nuclease is an enzyme that cleaves RNA that contains at least a trimer of 2
-5
linked adenylic acid at the 5
terminus of a target RNA (for review, see [160]). The enzyme is ubiquitous but exists in inactive form until activated by interferon. The enzyme has multiple anykrin motifs that suppress the activity of the enzyme until 2
-5
is bound [124,161]. Failure in the regulation of RNase L or truncation of the enzyme is associated with disease. It is

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therefore quite important to consider RNase L as a potential confounding mechanism for any dsRNA that could activate the interferon pathway. It is also a potential mechanism that could be exploited to induce antisense effects [160]. The challenge to exploitng RNase L as a mechanism has been the synthesis of stable oligonucleotide analogs that contain a 2
-5
moiety. To some extent, this has been accomplished. One set of modifications included full 2
methoxy modifications of the oligonucleotide coupled to 5
and 3
phosphorothioate modifications and a 5
thiophosphate [162]. This molecule displayed improved activity against respiratory syncytial virus infection in monkeys after intranasal administration. Other modifications have also been reported. In short, the 2
-5
adenyllic RNase or RNase L mechanism remains a potentially attractive mechanism. Very little work on this mechanism has been reported and it probably deserves more substantial efforts in the future. 1.6 MICRO-RNAs As previously discussed, micro-RNAs represent a new level of transcriptional and translational control and a very exciting area of research (for review, see [163]). They also present interesting targets for antisense-based therapeutics. Fully modified 2
methoxy antisense drugs designed to bind to several micro-RNAs were administered to mice and shown to produce substantial changes in m-RNA levels in a large number of RNA species [164]. Mir-122 inhibition with 2
-MOE-based antisense drugs substantially reduced serum cholesterol, hepatic fatty acid oxidation, and hepatic synthesis of cholesterol [164,165] demonstrating that alteration of mir function could result in potentially interesting therapeutic effects. More recently, an initial structure activity relationship (SAR) study identified potential approaches to enhance the activities of antisense agents that target mirs [166]. The opportunity to target mirs with antisense drugs opens a number of exciting avenues because the mirs are thought to be involved in relatively large-scale phenotypic shifts including differentiation and dedifferentiation phenomena. Given the number of potential roles of these RNAs in normal and pathophysiologic processes and the relative ease with which they can be targeted with antisense drugs, the therapeutic potential seems quite high. However, careful selection of diseases to be targeted and exploration of the effects on phenotype will be essential given the breadth of potential effects. Furthermore, as components of the micro-RNA pathway are important to the maintenance of a normal phenotype, effects on micro-RNA pathways by single-strand antisense agents or siRNAs could have toxicities. In fact, overexpression of siRNAs has been shown to be toxic to animals possibly secondary to effects on exportin [167]. Therefore, this is an exciting new opportunity that requires prudent but aggressive research. 1.7 CONCLUSIONS AND FUTURE PERSPECTIVES In the 5 years since the publication of the first volume of this series, remarkable progress has been reported. RNase H–based antisense drugs have progressed across a broad front and the information gained about the mechanism is being used to improve their performance. New opportunities such as the use of dsRNases including siRNA and micro-RNAs have emerged and been the subjects of exciting progress. Perhaps equally important, concepts such as transcriptional control though triplex formation, and ribozymes have been thoroughly evaluated and demonstrated to offer too little value using current approaches to warrant further investment. Several areas deserve continued aggressive research. We need to continue to evaluate secondgeneration RNase H–based antisense drugs in the clinic and in animals to better define their strengths and limitations, particularly with regard to chronic administration. We need to continue to use the information on the RNase H mechanism to improve the performance of RNase H–based drugs and determine if generation 2.2 antisense drugs do indeed do perform better than second-generation drugs in the clinic.

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We must continue to invest in understanding the RISC mediated pathways and determine which limbs of the pathway we can safely exploit. We must then create agents that can be specific enough to activate the desired limbs of the RISC pathways only. We must determine if surmounting the challenges posed by duplex RNA drugs is feasible and evaluate the potential of single-strand RNA-based drugs. Splicing is now an even more interesting process to alter as we know it is feasible and have the tools to exploit if. However, we must develop a better understanding of splicing processes so that we can improve the robustness of the mechanism. RNase L represents a mechanism that deserves more investment. At present, it is difficult to know if it offers any advantage over other cleavage mechanisms. We should generate the data to support an informed decision about the value of this mechanism. Finally, micro-RNAs represent an exciting opportunity, but the roles of micro-RNAs, the pathways involved, and risks associated with large phenotypic shifts should be adequately understood before proceeding with the development of anti-mer therapeutics. ACKNOWLEDGMENTS We thank Dr. Frank Bennett, Dr. David Ecker, and Dr. Brenda Baker for critical review and helpful comments; as also Tracy Reigle for preparing the figures and Donna Parrett for excellent typographical assistance. REFERENCES 1. Watson, J., Molecular structure of nucleic acids: a structure for deoxyribose nucleic acid. Nature, 1953. 171: 737–738. 2. Gillespie, D. and S. Spiegelman, A quantitative assay for DNA-RNA hybrids with DNA immobilized on a membrane. J. Mol. Biol., 1965. 12(3): 829–842. 3. Thompson, J.D. and D. Gillespie, Current concepts in quantitative molecular hybridization. Clin. Biochem., 1990. 23(4): 261–266. 4. Zamecnik, P.C. and M.L. Stephenson, Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proceedings of the National Academy of Science USA, 1978. 75(1): 280–284. 5. De Clercq, E., F. Eckstein, and T.C. Merigan, Interferon induction increased through chemical modification of synthetic polyribonucleotide. Science, 1969. 165: 1137–1140. 6. Barrett, J.C., P.S. Miller, and P.O. Ts’o, Inhibitory effect of complex formation with oligodeoxyribonucleotide ethyl phosphotriesters on transfer ribonucleic acid aminoacylation. Biochemistry, 1974. 13(24): 4897–4906. 7. Crooke, S.T., Basic Principles of antisense technologies, in Antisense Drug Technology: Basic Principles, Strategies, and Applications, S.T. Crooke, ed., 2001, Marcel Dekker, Inc.: New York, pp. 1–28. 8. Cook, P.D., Medicinal chemistry of antisense oligonucleotides, in Antisense Drug Technology: Basic Principles, Strategies, and Applications, S.T. Crooke, ed., 2001, Marcel Dekker, Inc.: New York, pp. 29–56. 9. Crooke, S.T., ed., Antisense Drug Technology: Basic Principles, Strategies, and Applications, 2001, Marcel Dekker, Inc.: New York. 10. Geary, R.S. et al., Pharmacokinetic properties in animals, in Antisense Drug Technology: Principles, Strategies, and Applications, S.T. Crooke, ed., 2001, Marcel Dekker, Inc.: New York, pp. 119–154. 11. Crooke, R.M. and M.J. Graham, Suborgan pharmacokinetics, in Antisense Drug Technology: Basic Principles, Strategies, and Applications, S.T. Crooke, ed., 2001, Marcel Dekker, Inc.: New York, pp. 155–182. 12. Yu, R.Z. et al., Pharmacokinetic properties in humans, in Antisense Drug Technology: Principles, Strategies, and Applications, S.T. Crooke, ed., 2001, Marcel Dekker, Inc.: New York, pp. 183–200. 13. Levin, A.A. et al., Toxicity of antisense oligonucleotides, in Antisense Drug Technology: Principles, Strategies, and Applications, S.T. Crooke, ed., 2001, Marcel Dekker, Inc.: New York, pp. 201–267.

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UTR seed matches, but not overall identity, are associated with RNAi offtargets. Nat. Meth., 2006. 3(3): 199–204. 134. Jackson, A.L. et al., Expression profiling reveals off-target gene regulation by RNAi. Nat. Biotechnol., 2003. 21(6): 635–637. 135. Scacheri, P.C. et al., Short interfering RNAs can induce unexpected and divergent changes in the levels of untargeted proteins in mammalian cells. Proceedings of the National Academy of Science USA, 2004. 101(7): 1892–1897. 136. Fedorov, Y. et al., Off-target effects by siRNA can induce toxic phenotype. RNA, 2006. 12: 1–9. 137. Jackson, A.L. et al., Position-specific chemical modification of siRNAs reduces “off-target” transcript silencing. RNA, 2006. 12: 1197–1205. 138. Lippman, Z. and R. Martienssen, The role of RNA interference in heterochromatic silencing. Nature, 2004. 431(7006): 364–370. 139. Noma, K. et al., RITS acts in cis to promote RNA interference-mediated transcriptional and post-transcriptional silencing. Nat. Genet., 2004. 36(11): 1174–1180. 140. Cam, H.P. et al., Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome. Nat. Genet., 2005. 37(8): 809–819. 141. Schramke, V. et al., RNA-interference-directed chromatin modification coupled to RNA polymerase II transcription. Nature., 2005. 435(7046): 1275–1279. 142. Sugiyama, T. et al., RNA-dependent RNA polymerase is an essential component of a self-enforcing loop coupling heterochromatin assembly to siRNA production. Proceedings of the National Academy of Science USA, 2005. 102(1): 152–157. 143. Volpe, T.A. et al., Regulation of heterochromatic silencing and histone H3 lysine-9 methylation by RNAi. Science, 2002. 297(5588): 1833–1837.

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144. Morris, K.V. et al., Small interfering RNA-induced transcriptional gene silencing in human cells. Science, 2004. 305(5688): 1289–1292. 145. Robb, G.B. et al., Specific and potent RNAi in the nucleus of human cells. Nat. Struct. Mol. Biol., 2005. 12(2): 1–4. 146. Song, E. et al., RNA interference targeting Fas protects mice from fulminant hepatitis. Nat. Med., 2003. 347–351. 147. Allerson, C.R., N. Sioufi, R. Jarres, T.P. Prakash, N. Naik, A. Berdeja, L. Wanders, R. Griffey, E.E. Swayze, and B. Bhat, Fully 2
-modified oligonucleotide duplexes with improved in vitro potency and stability compared to unmodified small interfering RNA. J. Med. Chem., 2005 (in press). 148. Song, E. et al., Antibody mediated in vivo delivery of small interf RNAs via cell-surface receptors. Nat. Biotechnol., 2005. 23(6): 709–717. 149. Morrissey, D.V. et al., Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat. Biotechnol., 2005. 23(8): 1002–1007. 150. Zimmermann, T.S. et al., RNAi-mediated gene silencing in non-human primates. Nature, 2006. 441(7089): 111–114. 151. Soutschek, J. et al., Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs. Nature, 2004. 432(7014): 173–178. 152. Crooke, S.T. et al., Pharmacokinetic properties of several novel oligonucleotide analogs in mice. J. Pharmacol. Exp. Ther., 1996. 277(2): 923–937. 153. Henry, S.P. et al., Toxicological properties of several novel oligonucleotide analogs in mice. AntiCancer Drug Des., 1997. 12(1): 1–14. 154. Elbashir, S.M. et al., Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells. Nature, 2001. 411: 494–498. 155. Martinez, J. et al., Single-stranded antisense siRNAs guide target RNA cleavage in RNAi. Cell, 2002. 110: 563–574. 156. Xu, Y. et al., Functional comparison of single- and double-stranded siRNAs in mammaliam cells. Biochem. Biophys. Res. Comm., 2004. 316: 680–687. 157. Reynolds, A. et al., Rational siRNA design for RNA interference. Nat. Biotechnol., 2004. 22(3): 326–30. 158. Kim, D.-H. et al., Synthetic dsRNA Dicer substrates enhance RNAi potency and efficacy. Nat. Biotechnol., 2005. 23(2): 222–226. 159. Bridge, A.J. et al., Induction of an interferon response by RNAi vectors in mammalian cells. Nat. Genet., 2003. 34(3): 263–264. 160. Torrence, P.F. et al., Develpoment of 2
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CHAPTER

2

The RNase H Mechanism Walt Lima, Hongjiang Wu, and Stanley T. Crooke

CONTENTS 2.1

Introduction.............................................................................................................................47 2.1.1 ASO Terminating Mechanisms ..................................................................................47 2.1.2 RNase H Enzymes......................................................................................................48 2.1.3 Human RNases H .......................................................................................................48 2.2 Human RNase H1...................................................................................................................49 2.2.1 Biochemical Properties...............................................................................................49 2.2.2 Structure and Enzymology .........................................................................................49 2.2.2.1 RNA-Binding Domain ................................................................................50 2.2.2.2 Catalytic Domain.........................................................................................52 2.2.3 Biological Roles .........................................................................................................55 2.2.4 Genomics and Regulation...........................................................................................56 2.3 Human RNase H2...................................................................................................................56 2.3.1 Structure and Enzymology .........................................................................................56 2.3.2 Biological Roles .........................................................................................................61 2.3.3 Genomics and Regulation...........................................................................................63 2.4 The Roles of the Human RNases H in the Effects of DNA-Like ASOs................................63 2.5 The Effects of Chimeric ASOs on Human RNase H1 Activity .............................................65 2.6 Implications for Antisense Therapeutics ................................................................................70 References ........................................................................................................................................71

2.1 INTRODUCTION 2.1.1

ASO Terminating Mechanisms

Antisense oligonucleotides (ASOs) are designed to modulate the information transfer from gene to protein—in essence to alter mRNA intermediary metabolism. mRNA intermediary metabolism is extremely complex beginning with transcription and concluding with degradation usually after translation. Each step is complex and in dynamic equilibrium with competing pathways. Although great progress has been made in understanding these processes, much remains unknown, and we

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have only begun to understand the potential impacts of ASOs on these processes and the factors that influence the outcomes. Once ASOs bind to a target RNA, they may induce pharmacological effects by one of two mechanisms: occupancy or occupancy-mediated destabilization [1–3]. Occupancy only mediated mechanisms include alteration of splicing, inhibiting of translation, and the disruption of required RNA structure and miRNA-induced gene silencing. Occupancy-mediated destabilization includes degradation of the target RNA by single- and double-stranded RNases. RNases H are enzymes that cleave the target RNA when bound in a DNA–RNA duplex. In addition, there are double-strand RNases that recognize RNA–RNA duplexes and cleave the target RNA. For example, the RISC endonuclease Argonaute2 combined with the antisense strand of siRNA, cleaves target mRNA at the sequence complementary to the siRNA [4,5]. This enzyme may also be involved in the activity of single-strand RNA-like ASOs. Thanks to the substantial progress reported in understanding the human RNases H, their roles in the effects of DNA-like ASOs, and the factors that influence the activities of DNA-like ASOs; today, we have the intellectual framework for the design of optimized RNase H-active ASOs. In this report, we shall review the progress in this area and discuss the implications of the observations on the design of more potent ASO therapeutics. 2.1.2

RNase H Enzymes

The RNases H hydrolyze RNA in RNA–DNA hybrids [6]. RNase H belongs to a nucleotidyl transferase super family, which includes transposase, retroviral integrase, Holliday junction resolvase, and the RISC nuclease Argonaute2. Proteins with RNase H activity have been isolated from numerous organisms ranging from viruses to mammalian cells and tissues [7–12]. Although RNase H isotypes vary substantially in molecular weight and associated functions, the nuclease properties of the enzymes are similar. All RNase H enzymes, for example, function as endonucleases exhibiting limited sequence specificity, require divalent cations (e.g., Mn2⫹ and Mg2⫹), and generate products with 5⬘-phosphate and 5⬘-hydroxyl termini [8]. In prokaryotes, three classes of RNase H enzymes, RNase H1, H2, and H3, have been identified. RNase H2 and H3 share significant sequence homology, whereas RNase H3 and RNase H1 share similar divalent cation preference and cleavage properties. Of the three classes, RNase H2 appears to be the most ubiquitous [13]. To date no organism has been shown to express active forms of all three classes of RNase H. The best characterized of the prokaryotic enzymes is Escherichia coli RNase H1 [14–18]. This enzyme is believed to be involved in DNA replication [19]. The key amino acids involved in metal binding, substrate binding, and catalysis have been identified and are highly conserved in the RNase H1 family [14,20–22]. Furthermore, the enzyme–substrate interaction has been determined based on the X-ray cocrystal structure for Bacillus halodurans RNase H1 and the heteroduplex substrate [23]. RNase H has also been shown to be involved in viral replication. RNase H domains have been identified in viral reverse transcriptases, and these typically share homology with E. coli RNase H1 [20]. The RNase H portion of the enzyme has been shown to cleave the viral RNA strand producing RNA primers for second-strand DNA synthesis, thereby converting the viral RNA into double-strand DNA [24]. Two classes of RNase H enzymes have been identified in mammalian cells [7,9–12]. They were reported to differ with respect to cofactor requirements and activity. For example, RNase H type 1 has been shown to be activated by both Mg2⫹ and Mn2⫹, and was active in the presence of sulfhydryl reagents, whereas RNase H type 2 was shown to be activated by only Mg2⫹ and inhibited by Mg2⫹ and sulfhydryl reagents [12]. 2.1.3

Human RNases H

Both human RNase H genes have been cloned and expressed [21–22,25]. RNase H1 is a 286 amino acid (aa) protein with a calculated mass of 32 kDa [22]. The enzyme is encoded by a single

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gene that is at least 10 kb in length and is expressed ubiquitously in human cells and tissues. The amino acid sequence of human RNase H1 displays strong homology with RNase H1 from yeast, chicken, E.coli, and mouse [22]. The human RNase H2 enzyme is a 299 aa protein with a calculated mass of 33.4 kDa and has also been shown to be ubiquitously expressed in human cells and tissues ([25]; H. Wu, unpublished data). Human RNase H2 shares strong amino acid sequence homology with RNase H2 from Caenorhabditis elegans, yeast, and E. coli [25].

2.2 HUMAN RNase H1 2.2.1

Biochemical Properties

Human RNase H1 has been cloned, expressed, and purified to electrophoretic homogeneity. The enzyme is active as a single polypeptide and retains activity after it is denatured and refolded [22,26]. The activity of RNase H1 is Mg2⫹-dependent and inhibited by Mn2⫹. Human RNase H1 was also inhibited by increasing ionic strength with optimal activity for both KCl and NaCl observed at 10–20 mM [22,26]. The enzyme exhibited a bell-shaped response to divalent cations and pH, with the optimum conditions for catalysis observed to be 1 mM Mg2⫹ and pH 7–8 [22,26]. The protein was shown to be reversibly denatured under the influence of temperature and destabilizing agents such as urea. Renaturation of human RNase H1 was observed to be highly cooperative and did not require divalent cations. Furthermore, RNase H1 displayed no tendency to form intermolecular disulfides or to form homomultimers. Human RNase H1 was shown to bind selectively to “A-form” duplexes with 10–20-fold greater affinity than that observed for E. coli RNase H1 [22,26]. Finally, human RNase H1 displays a strong positional preference for cleavage, that is, the enzyme cleaves between 8 and 12 nucleotides from the 5⬘-RNA–3⬘-DNA terminus of the duplex [26]. One biochemical property that has been used to classify RNase H enzymes is the sensitivity to sulfhydryl alkylating reagents such as N-ethylmaleimide (NEM) [12,14,22,27]. In general, RNase H1 enzymes are inhibited by NEM and both the E.coli and human enzymes share this property. In the case of E. coli RNase H1, NEM alkylation of C13 and C133 was responsible for the observed loss in enzymatic activity [14]. Alkylation of the cysteines was predicted to sterically interfere with substrate binding, as the E.coli enzyme was shown to be active under both reduced and oxidized conditions and the cysteine residues were not required for endonuclease activity [14]. A similar NEM alkylation pattern was observed for human RNase H1 with alkylation of the conserved C148 (C13 in the E. coli enzyme) resulting in the observed loss in activity (Figure 2.1) [28]. In addition, NEM alkylation of human RNase H1 had no effect on the binding affinity of the enzyme for the substrate. Given that C148 is positioned close to the catalytic site of the enzyme and the phosphate backbone of the substrate, NEM alkylation likely interferes with proper positioning of the enzyme on the substrate. Human RNase H1 is active only under reduced conditions [28]. Site-directed mutagenesis of human RNase H1 indicated that the conserved C148 and adjacent C147 residues were responsible for the observed redox-dependent activity of the enzyme (Figure 2.1) [28]. Tryptic digestion of the enzyme and analysis of the fragments by
HPLC-ESI-FITCR mass spectrometry revealed a unique disulfide bond between the vicinal C147 and C148 residues under oxidized conditions [28]. Oxidation of the enzyme had no effect on the binding affinity of the enzyme for the substrate suggesting that the oxidized enzyme exhibited a conformation that could no longer catalyze the hydrolysis of the RNA. 2.2.2

Structure and Enzymology

The structure of human RNase H1 consists of three domains: a 73-aa region homologous with the RNA-binding domain (RNA-BD) of yeast RNase H1 at the amino-terminus of the protein, the conserved catalytic domain at the carboxy-terminus of the protein, and a 62-aa spacer region that

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION E186 W43 K59-K60

RNA-binding

1

Figure 2.1

C18

C46

D145

Spacer

73

K226-K227 K236 D210 K231

Catalytic

135 C147-C148 C191

286

Schematic showing the structure of human RNase H1. Enzyme consists of three domains. The 73-aa RNA-BD at the amino-terminus of the protein contains the tryptophan and lysine residues involved substrate binding at, respectively, positions 43, 59, and 60 as well as two cysteines at positions 18 and 46. The 62-aa spacer region is positioned between the RNA-BD and the catalytic domain. The 151-aa catalytic domain at the carboxy-terminus of the enzyme contains the glutamic and aspartic acid residues of the catalytic triad at positions 145, 186, and 210; the basic substrate-binding residues at positions 226, 227, 231, and 236; and the cysteines of the redox switch at positions 147 and 148.

separates the RNA-BD from the catalytic domain (Figure 2.1) [29–31]. The roles of each of the regions and a number of the specific amino acids were determined by site-directed mutagenesis of both the enzyme and substrate [26,29,32–35]. Although the specific role of the spacer region remains unclear, this region was shown to be required for the activity [29,36].

2.2.2.1 RNA-Binding Domain The RNA-BD of human RNase H1 is conserved in other eukaryotic RNases H1 [22,31]. The nuclear magnetic resonance (NMR) structure of the RNA-BD of Saccharomyces cerevisiae RNase H1 consists of a three-stranded antiparallel
-sheet sandwiched between two -helices and shares strong structural similarities with the N-terminal domain of the ribosomal RNA-binding protein L9 [31,37]. Two highly conserved lysine residues are located within the third
-strand. In addition, a highly conserved tryptophan at position 22 was shown to project outwards. A solvent exposed aromatic amino acid at this position was also observed in the L9 protein and has been shown to be important for binding to 23S ribosomal RNA [37]. In a 46 aa peptide corresponding to the RNA-BD of S. cerevisiae RNase H1, the conserved lysine residues have been shown to be important for binding to the heteroduplex substrate [30]. Site-directed mutagenesis of human RNase H1 showed that the conserved lysine residues at positions 59 and 60 were involved in binding to the heteroduplex substrate (Figure 2.1) [36]. Alanine substitution of the Lys59 (K59A) and Lys60 (K60A) resulted in a twofold reduction in the binding affinity for the substrate compared with the wild-type enzyme. Alanine substitution of the conserved tryptophan residue (W43A), in contrast, had no effect on the binding affinity for the substrate but exhibited a significantly lower Km and kcat values compared with the wild-type enzyme (Table 2.1) [36]. The lower Km and kcat values for the mutant enzyme are indicative of nonproductive binding interactions between the enzyme and the substrate suggesting that the tryptophan is important for properly positioning the enzyme on the heteroduplex for catalysis. Importantly, human RNase H1 has been shown to exhibit a strong positional preference for cleavage cleaving the heteroduplex substrate 7–12 nucleotides from the 3⬘-DNA/5⬘-RNA terminus (Figure 2.2) [26]. Under single turnover conditions, the positional preference for cleavage was more pronounced suggesting that given a single interaction between human RNase H1 and the heteroduplex, the majority of the RNase H1 proteins bound to the heteroduplex in such a manner so as to cleave the substrate 7–10 nucleotides from the 5⬘-RNA [36]. Mutants in which W43 and K59–K60 of the RNA-BD were substituted with alanine, showed a loss of the positional preference for cleavage (Figure 2.2) [36]. Together these data suggest that the W43, K59, and K60

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Table 2.1 Initial Cleavage Rates for Wild-Type and Mutant Human RNase H1 Proteins Human Enzyme RNase RNase RNase RNase

H1 H1[K59,60A] H1[W43A] H1[W43/K59.60A]

kcat (min⫺1)

Km (nM) 601 1264 129 1084

⫾ ⫾ ⫾ ⫾

42 88 4 96

1.46 1.00 0.04 1.19

⫾ ⫾ ⫾ ⫾

0.05 0.08 0.001 0.11

kcat/Km (M⫺1/min) (2.4 ⫾ 0.04) (7.9 ⫾ 0.6) (3.0 ⫾ 0.2) (8.8 ⫾ 0.1)

⫻ ⫻ ⫻ ⫻

6

10 105 105 105

Kd (nM) 665 1121 412 1556

⫾ ⫾ ⫾ ⫾

13 136 67 119

Note: The kcat, Km, and Kd values were determined as previously described [36]. The kcat, Km, and Kd values are an average of n ⱖ 2 slopes of Lineweaver–Burk and/or Augustisson analysis with estimated errors of CV ⬍ 10%. (A)

r(GGGCGCCGUCGGUGUGG) d(CCCGCGGCAGCCACACC) (B)

r(GGGCGCCGUCGGUGUGG) d(CCCGCGGCAGCCACACC) Figure 2.2

Comparison of cleavage patterns for human and E. coli RNases H1. Digestion of the heteroduplex was performed as previously described [29]. The RNA sequence (5⬘ → 3⬘) is shown above the DNA sequence. The arrows indicate the sites of enzymatic digestion, and the size of the arrows reflects the relative cleavage intensities. (A) Cleavage pattern for human RNase H1. (B) Cleavage pattern for E. coli RNase.

residues constitute an extended nucleic-binding surface for the RNA-BD of the human RNase H1 with the lysine residues forming electrostatic interactions with the phosphate backbone and the solvent-exposed tryptophan forming either stacking interactions or hydrogen bonds with the heterocycle bases of the substrates [36]. In addition, these data suggest that the interaction between the RNA-BD and the substrate takes place at the 3⬘-DNA/5⬘-RNA pole of the heteroduplex (Figure 2.3). Structure–activity relationships at the 3⬘-DNA/5⬘-RNA terminus of the heteroduplex substrate have also been performed. These studies include modifications at the 3⬘, 2⬘, and heterocycle base of the terminal 3⬘-nucleotide in the DNA strand as well as modifications at the 5⬘ and 2⬘ of the terminal 5⬘-nucleotide in the RNA strand [36]. Heteroduplex substrates containing a 3⬘-phosphate at the 3⬘-terminus of the DNA strand or 5⬘-phosphate at the 5⬘-terminus of the RNA strand had no effect on either the cleavage pattern or rate of cleavage compared with the substrate containing hydroxyl groups at the 3⬘-DNA/5⬘-RNA terminus [36]. In contrast, a single ribonucleotide or 2⬘-methoxyethyl (MOE) substitution at the 3⬘-terminus of the DNA strand, with or without a 3⬘-phosphate, resulted in the ablation of the 5⬘-most cleavage site (Figure 2.3). A similar ablation of the 5⬘-most cleavage site was observed for a heteroduplex substrate in which a mismatched base pair was positioned at the 3⬘-DNA/5⬘-RNA terminus [36]. Similarly, heteroduplex substrates in which the DNA strand was successively truncated at the 3⬘-terminus resulted in a concomitant ablation of the 5⬘-most cleavage with a constant 7 base pair separation between the 3⬘-terminus of the DNA and 5⬘-most cleavage site [36]. Conversely, no shift in the cleavage pattern was observed for heteroduplex substrates in which the DNA strand was successively truncated at the 5⬘-terminus. The structure–activity relationships for the 3⬘-DNA/5⬘-RNA terminus of the heteroduplex are consistent with the site-directed mutagenesis of the RNA-BD in which stable base pairing at the 3⬘-DNA/5⬘-RNA terminus would be important for intercalation of the

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION (A)

Spacer RNABD

Cat

5′ 3′

7 base pairs

(B)

Spacer RNABD

Cat

5′ 3′

7 base pairs Figure 2.3

Schematic illustrating the relationship between the position of the RNA-binding domain and the catalytic domain of human RNase H1 on the heteroduplex substrate. (A) Each observed cleavage site on the RNA is coupled to a specific binding interaction between the RNA-binding domain and the 3⬘-DNA/5⬘-RNA pole of the heteroduplex substrate. The distance between the heteroduplex/RNAbinding domain interaction and the catalytic site is ⬃7 base pairs. (B) Crossed-out box represents either an RNA/RNA, 2⬘-MOE/RNA or mismatched base pair at the 3⬘-DNA/5⬘-RNA terminus of the heteroduplex. The alteration in helical geometry or steric interference by the 2⬘-substitutents disrupts the binding interaction between the RNA-binding domain and the heteroduplex resulting in the observed ablation of catalytic activity at the 5⬘-most cleavage site on the RNA.

W43 residue, and the interstrand phosphate distance across the minor groove within this region would be critical for the interaction with K59 and K60 [36]. Taken together these data suggest that the RNA-BD of human RNase H1 binds to the first stable 3⬘-DNA/5⬘-RNA base pair in the heteroduplex substrate and positions the catalytic domain approximately one helical turn 5⬘ on the RNA (Figure 2.3).

2.2.2.2 Catalytic Domain The catalytic domain of human RNase H1 is highly conserved relative to other RNase H1 proteins [15–17,29,38]. The glutamic acid and two aspartic acid residues of the catalytic site, as well as the histidine and aspartic acid residues of the proposed second divalent cation-binding site of the E. coli enzyme are conserved in human RNase H1 (Figure 2.1) [15–17,38]. In addition, the lysine residues within the highly basic -helical substrate-binding region of E. coli RNase H1 are also conserved in the human enzyme (Figure 2.1). The substitution of the conserved catalytic amino acids Asp-145, Glu-186, and Asp-210 of human RNase H1 with respectively, Asn, Gln, and Asn resulted in the complete ablation of the catalytic activity [29]. Furthermore, the ablation of cleavage activity observed for the catalytic site mutations did not appear to be due to a loss in the binding affinity for the heteroduplex substrate, as these substitutions had no effect on the binding affinity of the mutant proteins for the heteroduplex substrate. Alanine substitution of as few as two lysine residues in the basic substrate-binding region of the catalytic domain (e.g., lysines at positions 226, 227, 231, and 236)

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ablated the activity of human RNase H1 [29]. Again, the basic substrate-binding mutants exhibited binding affinities for the substrate comparable to the wild-type enzyme, suggesting that other regions of the human enzyme may be contributing to the overall binding interaction and that the lysine residues may play a more critical role in properly positioning the enzyme on the substrate for cleavage. In fact, the binding affinity for the deletion mutant of human RNase H1 in which the RNA-BD was deleted was fivefold lower compared with the wild-type enzyme [29]. In addition, the catalytic rate for the mutant enzyme without the RNA-BD was twofold faster than the kcat observed for the wild-type enzyme [29]. The deletion mutant demonstrates that the RNA-binding domain increases the affinity for substrate and reduces catalytic efficiency. Structure–activity relationships at the catalytic site of the heteroduplex substrate have also been determined [35]. In this study, modified nucleotides were introduced into the oligodeoxyribonucleotides at the human RNase H1-preferred cleavage sites on the heteroduplex. The modifications consisted of nucleotides exhibiting RNA-like northern, DNA-like southern, and eastern-biased sugars with and without 2⬘-substitutents. In addition, varying degrees of conformational flexibility were introduced into the heteroduplex substrate by incorporating base modifications which -stack with adjacent nucleotides but do not form hydrogen bonds, abasic deoxynucleotides, internucleotide hydrocarbon linkers ranging from three to five residues, and ganciclovir substitution of the deoxyribose. Heteroduplexes containing modifications exhibiting strong northern (e.g., 2⬘-fluorothymidine and 2-thiouridine) or southern (e.g., 2⬘-methylthiothymidine) conformational biases with and without bulky 2⬘-subtituents showed significantly slower site-specific cleavage rates for the ribonucleotide opposing the modification as well as the adjacent ribonucleotides. The nucleotide modifications predicted to mimic the sugar pucker of the deoxyribonucleotide of an RNA/DNA heteroduplex (e.g., heteroduplexes containing the 2⬘-ara-fluoropyrimidines and pseudouridine modifications) exhibited cleavage rates comparable to the rates observed for the unmodified substrate [35]. The 2⬘-ara-fluoro modification has been shown by NMR to form the eastern O4⬘-endo sugar conformation similar to DNA when hybridized to RNA [39]. In addition, the size and position of the 2⬘-ara-substituent, that is, the fluorine is directed upward and away from the minor groove, is predicted not to sterically interfere with the enzyme. These modified heteroduplexes, which suggest variations in minor groove width as a function of sugar conformation, appear to obviate the proper positioning of the enzyme on the heteroduplex substrate. Modifications imparting the greatest degree of conformational flexibility were the poorest substrates, resulting in dramatically slower cleavage rates for the ribonucleotide opposing the modification and the surrounding ribonucleotides. Specifically, heteroduplex substrates containing highly flexible hydrocarbon linkers were among the poorest substrates for RNase H1 activity [35]. The site-specific rates for the ribonucleotide opposing the hydrocarbon linkers as well as the surrounding 3⬘- and 5⬘-ribonucleotides were either significantly reduced or ablated resulting in initial cleavage rates (V0), approximately twofold slower than the unmodified substrate. Heteroduplex substrates containing ganciclovir-, abasic-, and tetrahydrofuran-modified deoxyribonucleotides were also poor substrates for human RNase H1, although the site-specific cleavage rates for these heteroduplexes were slightly faster than the rates observed for the heteroduplexes containing the hydrocarbon linkers [35]. In contrast, the base modifications that -stack with adjacent nucleotides, but do not form hydrogen bonds with the bases of the RNA strand (e.g., 2-fluoro-6-methylbenzoimidazole, 4-methylbenzoimidazole, and 2,4-difluorotoluyl deoxyribonucleotides), better supported human RNase H1 activity. Comparable initial cleavage rates and site-specific cleavage rates were observed for these heteroduplexes compared with the unmodified substrate [35]. Although conformational flexibility of the deoxyribose was preferred, flexibility in the phosphate backbone of the DNA strand inhibited human RNase H1 activity. These data suggest that proper positioning of the phosphate groups

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of the deoxyribonucleotide, presumably for electrostatic contact with the enzyme, is essential for human RNase H1 catalysis. The cleavage rates observed for the -stacking deoxyribonucleotides suggest that stable base stacking independent of hydrogen bond formation between the bases at the catalytic site appeared to offer sufficient rigidity to the phosphate backbone. Taken together these data suggest that sugar conformation, minor groove width, and the relative positions of the intra- and internucleotide phosphates are critical determinants in the selective recognition of the heteroduplex substrate by human RNase H1. In addition, the structure–activity relationships at the catalytic site of the heteroduplex substrate suggest that the preferred properties for the modified oligodeoxyribonucleotide include (1) a conformationally flexible sugar producing an O4⬘-endo pucker when hybridized to RNA; (2) no sterically bulky 2⬘-substituents; and (3) a conformationally rigid phosphate backbone. Clearly, the 2⬘-ara-fluoro-, pseudouridine-, and -stacking-modified deoxyribonucleotides exhibit many of these qualities. The recent X-ray crystal structure of RNase H from B. halodurans bound to the RNA/DNA heteroduplex substrate offers further insights into the observed effects of modified nucleotides on human RNase H1 catalysis [23]. Specifically, the catalytic domain of the enzyme was shown to interact with both the RNA and DNA strands of the heteroduplex substrate including the phosphates of the RNA strand on either sides of the scissile phosphate; the three 2⬘-hydroxyls upstream; and three 2⬘-hydroxyls downstream of the scissile phosphate; the heterocycle bases of both strands upstream of the scissile phosphate; and the DNA backbone upstream of the cleavage site via a phosphate-binding pocket (Figure 2.4). In all, the binding interactions between the enzyme and substrate comprise six base pairs of the heteroduplex substrate. The catalytic domain of human RNase H1 shares strong sequence homology with the catalytic domain of the B. halodurans RNase H, suggesting that human RNase H1 likely interacts in a similar manner with the heteroduplex substrate. Considering the scope of the interactions, modified nucleotides within the DNA strand of the heteroduplex can affect catalysis in a number of different ways. For example, the modified nucleotides could have a direct effect on catalysis if the modifications were positioned at sites on the substrate that interact directly with the enzyme (e.g., modified internucleotide linkages positioned at the phosphate-binding pocket of the enzyme) (Figure 2.4). In addition, modified nucleotides that influence the local substrate structure would have a local effect on catalysis when positioned adjacent to the nucleotides that directly interact with the enzyme (e.g., hydrophobic base modifications) (Figure 2.4). Finally, modified nucleotides that exert a long-range influence on substrate structure would exhibit a distributive or transmission effect on catalysis (e.g., northern- or southern-biased 2⬘-modifications positioned outside the footprint of the enzyme) (Figure 2.4).

3′

5′

5′

3′ PO-binding pocket Figure 2.4

Model for the interaction of RNase H1 with the heteroduplex substrate. The putative enzyme/nucleotide interactions for cleavage at ribonucleotide 7. The arrow indicates the position of the scissile phosphate. Heteroduplex is shown with the RNA strand (upper) oriented from 5⬘ to 3⬘ and the ASO (lower) from 3⬘ to 5⬘. The dark gray and light gray structures indicate the interactions between the enzyme and, respectively, the sugars of the nucleotides and heterocycle bases. The black filled circles represent the enzyme/phosphate interactions.

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2.2.3

55

Biological Roles

Human RNase H1 is ubiquitously expressed in the cell, residing in both the nucleus and cytoplasm [40]. Important insights into the biological roles of mammalian RNase H1 have recently been provided by an RNase H1 knockout mouse [41]. The knockout was embryonically lethal and failed to produce mitochondrial DNA resulting in defective mitochondria and massive apoptosis. Both the mouse and human enzyme have a putative mitochondrial localization signal (MLS). Thus, the authors concluded that the enzyme is likely involved in Okazaki fragment processing in the mitochondria. The biochemical and enzymological data are consistent with the proposed biological role of the enzyme. The enzyme is believed to participate in the generation and/or removal of RNA primers during lagging strand DNA synthesis. These RNA primers form chimeric structures consisting of a 7–14 ribonucleotide region at the 5⬘-terminus and contiguous stretches of DNA extending in the 3⬘-direction (Figure 2.5) [42]. On the basis of the positional preference for cleavage exhibited by human RNase H1, cleavage of the chimeric structure would occur at or near the RNA–DNA junction, effectively removing the RNA primer (Figure 2.5). Alternatively, human RNase H1 cleavage of the RNA/DNA heteroduplex formed during DNA replication would produce the observed 7–14 ribonucleotide primers for lagging strand DNA synthesis. The human RNase H1 activity observed for Okazaki-like substrates was consistent with the proposed biological role for the enzyme [43]. The Okazaki-like substrates consisted of an oligodeoxyribonucleotide annealed to a complementary RNA/DNA chimeric oligonucleotide containing 10 and 15 ribonucleotides at the 5⬘-pole and, respectively, 10 and 5 contiguous deoxyribonucleotides extending 3⬘. The Okazaki-like substrates ablated the 3⬘-most cleavage sites and exhibited enhanced cleavage rates for the remaining cleavage sites. In other words, the successive ablation of the 3⬘-most cleavage sites produced new preferred cleavage sites [43]. Furthermore, given that the short RNA primers are interspersed within long stretches of dsDNA, the model for the interaction between the enzyme and the heteroduplex substrate correlates well with biological role for the enzyme. First, the strong positional preference for cleavage is consistent with the length of the RNA primers. Second, human RNase H1 binds the RNA/DNA heteroduplex ⬃50-fold tighter than dsDNA suggesting that the enzyme would not be trapped in nonproductive interactions within the large field of dsDNA [26]. Third, the limited sequence discrimination exhibited by the enzyme would be beneficial given that the RNA primers comprise mixed sequences. Finally, given that high concentrations of proteins and their cofactors such as manganese superoxide dismutase (SOD2) are required to regulate the highly oxidative environment of the mitochondria, human RNase H1 may have evolved a sensitivity to manganese as well as a redox switch to regulate the activity of the enzyme within this environment.

RNA primer

Lagging strand

5′ Okazaki fragment

RNA primer 5′

Okazaki fragment

Okazaki fragment

Leading DNA strand Figure 2.5

Schematic illustrating the position of human RNase H1 cleavage during lagging strand DNA synthesis. Upper and lower lines represent, respectively, the lagging and leading DNA strands. Lagging strand contains the 7–12 nucleotide RNA primers interdispersed between 200 and 300 nucleotide long Okazaki fragments. Arrows indicate the predicted human RNase H1 cleavage sites based on the positional preference for cleavage exhibited by the enzyme, that is, 7–12 nucleotides from the 5⬘-terminus of the RNA. Human RNase H1 cleavage is predicted to occur at or near the junction between the RNA primer and the Okazaki fragment.

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION

Genomics and Regulation

Human RNase H1 is encoded by a single gene on chromosome 2p25 [22]. The gene spans 13 kb and contains seven introns with the shortest being 333 base pairs and the longest being 4480 base pairs. There are also multiple pseudogenes in chromosomes 1 and 17. Although expressed at low levels, the enzyme is broadly expressed in cells and tissues [22]. In all cells and tissues, we observed a 1.2 kb band that corresponds to the mature message and a 5 kb band that may be an alternatively or incompletely processed message or a transcript from one of the pseudogenes. The promoter region has numerous potential regulatory elements, but to date, we have seen no evidence of transcriptional regulation of the gene (Wu, unpublished data). Obviously, much more work is required before firm conclusions can be drawn about transcriptional regulation or alternative pre-mRNA processing.

2.3 HUMAN RNase H2 Although human RNase H2 has been cloned and expressed by a number of laboratories [25], much less is known about the properties of this enzyme. This is due primarily to the fact that human RNase H2 is inactive as a monomer. Human RNase H2 is also inactive in the gel renaturation assay so there is no assay with which to study the enzyme in situ. Consistent with these observations, recombinant RNase H2 from yeast was also shown to be inactive as a single polypeptide [44]. In fact, affinity purification identified two yeast proteins Ydr279p and Ylr54p associated with RNase H2, which restored RNase H2 activity in a reconstitution assay [44]. 2.3.1

Structure and Enzymology

Human RNase H2 is a 33 Kd protein that is homologous to E. coli RNase H2 (23% amino acid identity). On the basis of the crystal structure and mutational analyses of archaeal RNase H2, human RNase H2 has highly conserved RNA binding and catalytic domains [45,46]. Of the type 2 enzymes, prokaryote RNases HII are the best characterized. The three-dimensional structure has been determined and the key catalytic amino acids identified [47]. Although type 1 and 2 enzymes exhibit very low-sequence homology, the tertiary structures of these enzymes are similar [45]. In fact, the catalytic- and substrate-binding amino acids of type 1 and 2 enzymes are conserved [47]. The prokaryote type 1 and 2 enzymes were also shown to generate similar patterns based on short heteroduplexes [48]. Finally, prokaryotic RNases HI and HII were shown to differ with respect to cofactor requirements. Specifically, RNase H1 can be activated with both Mg2⫹ and Mn2⫹, whereas RNase H2 is active only in the presence of Mn2⫹ [48]. Given that cloned, expressed, and purified human RNase H2 is inactive in the gel renaturation or solution-based assays, we have developed an assay that, for the first time, supports preliminary characterization of the enzymology of human RNase H2 (Wu, unpublished data). To achieve this, we reasoned that the enzyme may require one or more protein cofactors for activity or that refolding of the enzyme could be ineffective. Thus, we prepared highly purified polyclonal antibodies to both human RNase H1 and RNase H2 and used them to immunoprecipitate both native enzymes in protein complexes from cells. We immunoprecipitated the overexpressed cloned enzymes and analyzed the activity of both the native and overexpressed proteins either by the trichloroacetic acid (TCA) precipitation or gel electrophoresis assays. For the overexpression of human RNase H2, a strain of adenovirus containing the full-length H2 cDNA insert was developed (Figure 2.6A). Figure 2.6B shows the Western blot for overexpressed human RNase H2 in Hela and A549 cells. Furthermore, the virally encoded human RNase H2 protein comigrated with the RNase H2 protein from uninfected cells confirming that the overexpressed protein was full length (data not shown). Peak expressions were observed 36–48 h after

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(A)

Full-length human RNase H2 cDNA Met1

Inserted cDNAs

FL_H2

Met27

FL_H1

MLS Full-length human RNase H1 cDNA Met27

NT 26 animo acid minus H1 cDNA

Virus shuttle vector: pACCMVpLpA(-)LoxP-ssp

Adv arm  CMV promoter − MCS  Adv arm (B) Size standard

Native Hela lysate

H2 virus infection

A549 cells Time (h)

0

12

24

36

Hela cells 48

0

12

24

48

Size standard 50 kDa

50 kDa RNase H2

36 kDa

36 kDa 30 kDa

30 kDa H2 Ab immobilized on Agarose Gel

Figure 2.6

36

Western blot with anti-RNase H2 Ab

Development of adenoviruses overexpressing human RNases H. (A) Human RNase H constructs in adenovirus shuttle vectors. Full-length (FL) and N-terminal 26 amino acid (putative mitochondria localization signal, MLS) minus RNase H1, and full-length RNase H2 cDNAs were amplified by PCR and cloned into EcoRI and XhoI sites in the multiple cloning site (MCS) downstream from the CMV promoter in the adenovirus shuttle vector, pACCCMVpLpA(-)Loxp-ssp (core facility of University of Michigan). (B) Western blot analysis of protein lysates from Hela or A549 cells infected with fulllength H2 virus (200 pfu/cell). The cells were harvested at different time points (0, 12, 24, 36, and 48 h) after virus infection. The protein concentrations of the cell lysates were measured. The lysates were subjected to 4–20% gradient SDS-PAGE (20
g/lane) and Western blot analysis with antiRNase H2 antibody (right panel). Immunoprecipitation was performed using uninfected Hela cell lysate with purified H2 Ab which was covalently immobilized to agorose beads. The eluted samples were subjected to western blot analysis with the H2 Ab (left panel).

infection. Human RNase H2 was also overexpressed in T24, MCF7, and HepG2 cells (data not shown). The overexpression of human RNase H1 was performed as previously reported [1]. Because RNase H1 contains a 26 aa putative MLS at the N-terminus of the protein, the 26-aa deletion mutant adenovirus was also developed to evaluate the role of this signal peptide in the subcellular localization of RNase H1. To compliment the overexpression experiments, potent selective DNA-like ASOs and small interference RNAs (siRNA) were identified to reduce cellular RNase H2 [49,50]. A screen of ASOs targeting the mRNA of human RNase H2 revealed ISIS 194186, which was located at nucleotides 1008 to 989 in the 3⬘ UTR of the RNA (accession number AY 363912) as the most potent ASO (Table 2.2). The most potent siRNA for RNase H2 was si-21956 and located at nucleotides 667–686 in the coding region. Both the ASOs and siRNAs targeting human RNase H2 mRNA reduced RNase H2 mRNA and protein levels in a dose-dependent manner and the effects were specific to RNase H2 (Figure 2.7). The duration of effect was greater than 48 h (data not shown). The two RNase H enzymes display different cleavage patterns in the substrate (Figure 2.8). Human RNase H1 preferentially cleaves the heteroduplex 6–7 nucleotides from the 5⬘-RNA/3⬘-DNA terminus of the duplex while RNase H2 preferentially cleaves 11–12 bases from this terminus. Further, the cleavage pattern observed for immunoprecipitated human RNase H1 was identical to that observed with cloned and purified RNase H1 [40]. In fact, there was excellent correlation between the human RNase H1 activities observed for the recombinant protein, the gel renaturation assay, and the immunoprecipitation (IP) assay. Specifically, RNase H1 immunoprecipitated from human cells exhibited similar cleavage site specificity and basic properties such as sensitivity to divalent cations as those observed for both the recombinant protein and gel renaturation

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Table 2.2 ASOs and siRNAs against Human RNase H2 ISIS # 21955 21956 21957 21958 21959 21960 21961 21962 194186

Antisense oligonucleotides O

O B

O O P O -S O

2′-MOE O O

B DNA

O P O -S O

Si21955 si21956 si21957 si21958 si21959 si21960 si21961 si21962

Small interference RNA (siRNA)

Sequences

% Inhibition

CAGTTTCTCCACGAATTGCC TTTTGTCTTGGGATCATTGG AGCTGAACCGGACAAACTGG CCTCTTTCTCCAGGATGGTC ACTCCAGGCCGCGTTCCAGG CCTACGTGTGGTTCTCCTTA GCACACTCCCACCTTGCTTC CAAAAGGAAGTAGCTGGACC CCTACGTGTGGTTCTCCTTA

33.3 63.1 68.1 38.0 81.2 91.7 6.1 36.0

GGCAAUUCGUGGAGAAACUGC CCAAUGAUCCCAAGACAAAAG CCAGUUUGUCCGGUUCAGCUG GACCAUCCUGGAGAAAGAGGC CCUGGAACGCGGCCUGGAGUC UAAGGAGAACCACACGUAGGG GAAGCAAGGUGGGAGUGUGCU GGUCCAGCUACUUCCUUUUGG

19.2 87.6 42.4 45.8 53.8 9.9 15.2 0

Note: Bold text in the ASO sequences represent 2⬘-MOE nucleotides and plain text represent deoxynucleotides. All ASOs contained phosphorothioate linkages throughout the molecules. To the left of the table is the structure of 2⬘-MOE and 2⬘-deoxynucleotides with a phosphorothioate linkage between them. % inhibition: reduction of RNase H2 mRNA level in A549 with ASO (200 nM) or siRNA (100 nM) treatment for 24 h compared to control oligonucleotide or siRNA treatment, detected by Northern blot with 32Plabeled RNase H2 cDNA probe. ISIS194186 shares the same sequence as ISIS21960 (the most potent ASO for RNase H2 in the study), but with only five (instead of six) 2⬘-MOE nucleotides in both wings of the oligonucleotide. For siRNAs, all nucleotides are ribonucleotides and the internucleotide linkages are phosphates. Only the sense strands are shown.

Hela cells

(A) Treatment

Control

Concentration (nM)

0 120 10

ASO194186 25

60 120

Hela cells

A549 cells Control

ASO194186

0 120 10 25

60 120

Control

si21956

0 120 10 25

60 120

A549 cells Control 0

si21956

120 10 25 60 120

RNase H2 (1.4 kb) G3PDH

(B) RNase H2 (~37 kDa) 100% ------------------- <25%

Figure 2.7

100% ------------------- <10%

100% ------------------- <20%

100% ---------------- <10%

ASO ISIS194186 or si21956 reduces RNase H2 mRNA and protein levels in Hela and A549 cell lines. Cells were treated with different amounts of ASO or siRNA for 24 h and total RNA and cell lysates were prepared. (A) RNA was subjected to 1.2% agarase/formaldehyde gel (5
g total RNA/lane) and Northern blot analysis with 32P-labeled human RNase H2 or a G3PDH cDNA probe. (B) 20
g proteins of cell lysate were used for Western blot with anti human RNase H2 Ab.

3 8 24 60

∗

∗ ∗

0 1 3

8 24 60

G−5′

G

G

C

G

C

C

G

U

C

G

G

U

G

U

G

G−3′

RNA sequence

0

10

20

30

40

G G

C

G

C C* G

U C* G*

G

U

G

U

Substrate RAS RNA sequence (5′−3′)

G

G

RNase H2

RNase H1

G

9:30 PM

Relative percentage digestion

Different digestion patterns of human RNase H1 and H2. The 17-mer RAS RNA/DNA substrate was prepared and subjected to digestion by the H1 or H2 Ab immunoprecipitated (IP) samples from Hela cells for different time periods at 37oC. The digested duplexes were subjected to denaturing polyacrylamide gel analysis. (Left) Cleavage of 17-mer RAS duplex. The asterisks indicate the major differences in cleavage sites between RNase H1 and H2. (Right) The relative extent of digestions at each position of the substrate was calculated with the phosphor-imager and the relative percentages of digestion are compared between RNase H1 and H2. The sequence of the target RNA is shown to the right of the gel image and as the abscissa of the graph that quantitates the site-specific cleavage ratio.

1

H2

6/20/2007

Figure 2.8

Time (min) 0

H1

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION

assay [26,28,29,40]. These data also confirm the specificity of the IP assay because different cleavage patterns were observed for the immunoprecititation of human RNase H2 and RNase H1 (Figure 2.8 and Figure 2.13). Finally, these results differ from the cleavage specificities observed for prokaryotic RNases H2 in which similar cleavage patterns were observed for both the type 1 and 2 enzymes [48]. The metal requirements between RNases H2 from human and prokaryotes also differed. Specifically, human RNase H2 is active in the presence of Mg2⫹ and Mn2⫹ whereas the prokaryote RNase H2 was active only in the presence of Mn2⫹ (Wu, unpublished). Figure 2.9 demonstrates that overexpression of the enzymes resulted in greater activity in the Hela cell extracts. The extent of the increase in RNase H1 activity was comparable to the change in the enzyme protein level as shown in the Western blot after H1 Ab IP (Figure 2.9A), while approximately the twofold increase of H2 activity was much less than the change in the level of the protein (six- to eightfold) (Figure 2.9B). In contrast, when the cells were pretreated with H2 siRNA, the H2 activity extracted from cells was significantly reduced (⬃67%) and the extent of the reduction correlated well with the reduction of the H2 protein (Figure 2.9C). Our data suggest that RNase H2 may be part of a multiprotein complex that plays a key role in regulating its activity. For example, the failure to increase activity as greatly as the amount of RNase H2 overexpressed in the cell suggests that other proteins in the putative complex may be rate limiting (Figure 2.9B). In fact, Jeong et al. [44] have shown that S. cerevisiae RNase H2 is in a complex containing at least two other proteins and that these proteins are required for activity. We believe this is likely the case in mammalian cells as well.

(A)

(B) Control FL_H1 Virus virus

Loxp

40 30 20 10 0 0

5

10

Time (min)

Control H1 virus I.R. (nM/min) 1.6± 0.29 12.52± 0.43

Percentage digestion (%)

H1 virus

50

Percentage digestion (%)

60

60

Percentage digestion (%)

RNase H2

RNase H2

RNase H1

Figure 2.9

Control H2 siRNA siRNA treated treated cells cells

(C) Control FL_H2 Virus virus

H2 virus 50

Loxp

40 30 20 10 0

0

5

10

Time (min)

Control H2 virus I.R. (nM/min) 4.59±0.65 7.59 ± 2.66

30 Control siRNA H Ab IP

25

H2 siRNA treatment H2 Ab

20 15 10 5 0 0

5

10

Time (min)

Control siRNA H2 siRNA I.R. (nM/min) 3.02±0.30 0.98 ± 0.24

RNase H Ab immunoprecipitation (IP)-coupled TCA assay. Hela cells were either infected with human RNase H1, H2, control virus (200 pfu/cell) or treated with 100 nM H2, control siRNA in 10 cm plate in quadruplicate for 24 h before harvest. Cell lysates were prepared and protein concentrations measured. A 0.7 mg protein lysate was used for H1 Ab IP (15
g H1 Ab/mg protein lysate) or 0.35 mg per tube for H2 Ab IP (30
g H2 Ab/mg protein). One set of the IP samples was eluted in 2x SDS loading buffer (Invitrogen Inc., San Diego) and subjected to SDS-PAGE and Western blot with H1 or H2 Abs. The other three sets of IP samples were used in the enzyme activity assay with 50 nM of a 17-mer RNA/DNA duplex as the substrate. The RNA/DNA duplex was hybridized and then digested with the IP samples for different lengths of time at 37oC. The digested duplexes were subjected to TCA precipitation and the radioactivity in supernatants was determined for the digested RNA fragments by scintillation counting. The experiments were performed in triplicate. The bars show the standard errors of the mean. The initial rates were calculated and presented under the graph. Comparison of the effects of pretreatment of cells with control or full-length RNase H1 viruses (A), cells infected with control or H2 viruses (B), and cells transfected with control or RNase H2 siRNAs (C).

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61

Thus it is now possible to compare both human enzymes in comparable assays with IP validation that indeed each activity represents the enzyme assumed. Results from the assay demonstrate that the enzymes display different cleavage patterns. Obviously much more work is required with multiple substrates before drawing definitive conclusions about substrate requirements and cleavage patterns for human RNase H2. 2.3.2

Biological Roles

Little is known regarding the biological role of human RNase H2. Our IP data suggest that (1) the majority of the human RNase H2 activity was found in the nuclease; and (2) human RNase H2 may be part of a multiprotein complex that plays a key role in regulating its activity. To test this hypothesis, we evaluated the subcellular localization of human RNase H2 by fluorescence microscopy (Figure 2.10). As a control we also evaluated the subcellular localization of human RNase H1. In uninfected Hela cells, human RNase H2 is localized primarily in the nucleus. For human RNase H1, endogenous enzyme is present in very low levels and mainly in nucleus (Figure 2.10A). RNase H1 was difficult to identify in the cytosol by immunofluorescence, but faint cytoplasmic fluorescence was observed. In cells infected with adenoviral vectors overexpressing RNase H2, again the enzyme was localized primarily in the nucleus. Overexpression of fulllength RNase H1 resulted in extensive nuclear staining and staining of the cytoplasm. Because the N-terminal 26 aa of human RNase H1 contains a putative MLS, we constructed the 26-aa deletion (A)

(B) NR IgG

Anti-H1 Ab

(C) Anti-H1 Ab

Infected with virus 1 H1 rol _H 6(−) ont C 2 FL

us vir

Size standard

50 kDa

Uninfected Hela cell

Anti-H2 Ab

Uninfected Hela cell

Anti-H1 Ab

FL_H1 virus infected Hela cell

RNase H1

36 kDa

infected Hela cell

30 kDa

Mitotracker red Purified H1 Ab

Uninfected Hela cell

26(−)H1 virus infected Hela cell

Anti-H2 Ab

Anti-H1 Ab

FL_H2 virus infected Hela cell

FL_H1 virus infected Hela cell

Figure 2.10

FL_H1 virus infected Hela cell

26(−)H1 virus infected Hela cell

Anti-H1 Ab mitotracker red merge

FL_H1 virus infected Hela cell

26(−)H1 virus infected Hela cell

(See color insert following page 270.) Immunofluoresence staining of human RNases H with purified anti-H1 or H2 antibody. Normal rabbit IgG (NR IgG) was used as control. (A) Human RNase H1 and H2 immunostaining of normal (uninfected) or virus infected Hela cells. (B) RNase H1 staining of Hela cells infected with H1 virus and costaining with mitochondrial-specific stain. (C) Expression of human full length (FL) and N-terminal 26 amino acid (⫺26) minus RNase H1 in Hela cells. Hela cells were infected with FL or ⫺26 minus RNase H1 virus or control virus (LoxP) for 24 h. The cell lysates were prepared and subjected to immunoprecipitation with RNase H1 Ab. The immunoprecipitated samples were then used to the Western blot with same H1 Ab.

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mutant and evaluated its subcellular localization (Figure 2.10C). Clearly, the deletion mutant is localized strictly to the nucleus confirming that the mitochondrial signaling sequence plays a role in the subcellular localization of the full-length enzyme. To evaluate this in more detail, we compared the immunoflourescense of full-length and 26-aa minus RNase H1 enzymes with a mitochondrial-specific staining with mitotracker red (Figure 2.10B). This clearly shows that the full-length enzyme is localized to the nucleus and colocalized with the mitochondrial stain while the 26-aa minus protein is localized strictly to the nucleus, which is consistent with the mouse [41]. In addition, these results suggest that the first 26 aa of the enzyme is indeed an MLS. Inasmuch as human RNase H2 has been thought to be involved in nuclear DNA synthesis, one possibility is that it is bound to chromatin. In these experiments, we cross-linked various proteins to chromatin in Hela cells as described in Figure 2.11. We compared the rates of in vivo cross-linking for human RNase H2 to chromatin to rates of cross-linking of proteins known to be integral members of chromatin and to chromatin-associated proteins. Figure 2.11 shows that RNase H2 readily cross-linked to chromatin at about the same rate as histone H4 or topoisomerase. The two well-known DNA-interacting transcription factors, CREB and NFB are also shown by this cross-linking method. In contrast, RNase H1 did not cross-link at any reproducible level at any time. These observations add support for the concept that RNase H2 is involved in nuclear DNA synthesis, while RNase H1 is involved in mitochondrial DNA synthesis. They also suggest that because RNase H2 is chromatin associated, it cannot participate in the antisense effects of DNA-like ASOs while RNase H1 can. Of course, our data do not exclude the possibility that other cellular interactions may limit the accessibility of RNase H2 to ASO–RNA duplexes, but since the putative RNase

(A)

Size Hela Std. (KDa) lysate MW

(B)

0

Cross-link time (min) 2 4 8 16 32

Abs for Western blot

RNase H2 in chromatin

Histone H3

10

Histone H4

100

DNA Topoisomerase I

36

RNase H2

36

RNase H1

% RNase H2

RNase H2 in Chromatin

17

100 75 50 25 0 0

2 4 8 16 Cross-linking time (min)

32

Histone H4 in chromatin

CREB

65

NFκB

% Histon H4

45

Histone H4 in nulear 100 75 50 25 0

0

2 4 8 16 Cross-linking time (min)

32

Figure 2.11 RNase H2 is associated with chromatin. (A) Formaldehyde cross-linking with the time course of 2, 4, 8, 18, and 32 min was performed in Hela cells preincubated with 3H-thymidine and the DNA/protein complexes. The complexes were boiled for 20 min in SDS loading buffer and the samples with the equivelent DNA concentrations (⬃80
g and 11,000 cpm of 3H/lane or 20
g/lane for histone H3 and H4 analysis) were subjected to SDS-PAGE. The Hela cell lysate (50
g) was loaded as the control. The Western blots were then performed with a variety of Abs. (B) Correlation of the crosslinking time with extent of protein (RNase H2, Fen 1, and histone H4) binding to DNA. The Western blots for RNase H2, Flap endonuclease-1 (Fen 1), and histone H4 were analyzed by image scanning from multiple (ⱖ3 times) cross-linking experiments. The extent of the three proteins binding to DNA was correlated to the cross-linking times. The bars represent standard error of the mean of multiple experiments.

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H2 protein complex is fully capable of degrading substrates in lysates, they do demonstrate that the protein complex does not preclude activity. Moreover, virtually all the nuclear RNase H2 cross-linked to chromatin, so certainly the bulk of the enzyme is likely complexed to chromatin. 2.3.3

Genomics and Regulation

The RNase H2 gene spans ⬃7 kb and has seven introns, the shortest of which is 89 base pairs and the longest 2678 base pairs (Wu, unpublished data). It is located at p13.2 on chromosome 19. In contrast to RNase H1, a single RNA at ⬃1.4 kb is detected with appropriate probes on Northern blots. In contrast to RNase H1, human RNase H2 appears to be transcriptionally regulated. Its expression appears to vary as a function of the cell cycle, being present in greater quantities during S phase (Sharkey and Crooke, unpublished).

2.4 THE ROLES OF THE HUMAN RNases H IN THE EFFECTS OF DNA-LIKE ASOs Although it has been assumed that DNA-like ASOs cause target RNA reduction by binding to the target RNA and creating a DNA/RNA duplex that serves as a substrate for RNase H, definitive proof that this mechanism is responsible for the observed effects in mammalian cells and animals has been lacking [1,3]. In cell-free systems, the addition of E. coli RNase H or human RNase H1 to DNA–RNA duplexes results in degradation of the target RNA [26,39,51]. The ability of DNA-like ASOs to cause a reduction in target RNAs in cells has been demonstrated many times as well [3]. Moreover, changes in the structure of DNA-like ASOs that resulted in loss of the ability of the duplex to serve as a substrate for RNase H in cell-free systems have been reported to result in a loss of target RNA reduction in cells treated with the modified ASOs [3,52,53]. Additionally Giles et al. [54] used reverse ligation polymerase chain reaction (PCR) to identify cleavage products from bcr-abl mRNA in cells treated with a DNA-like ASO. Nevertheless, none of these studies directly demonstrate that DNA-like ASOs reduce target RNA by activating RNase H, nor has the specific RNase H that is responsible been identified. To address this issue, we have overexpressed both human RNase H1 and RNase H2 in several cell lines and mouse liver. Specifically, Hela and A549 cells were infected with either the control (LoxP) or H2 insert containing adenovirus (Figure 2.6) [40]. We have also reduced the levels of each enzyme by using DNA-like ASOs and siRNA designed to reduce each of the enzymes (Figure 2.7) [40]. We have then explored the effects of these manipulations on the potencies of a number of well-characterized DNA-like ASOs to several different target RNAs [55–58]. In every case, increasing the level and activity of human RNase H1 increased the potency of the ASOs, while increasing the level and activity of RNase H2 had no effect on the IC50 values for several DNA-like ASOs to different targets [40]. Moreover, overexpression of human RNase H1 in mouse liver but not RNase H2 increased the potency of a DNA-like ASO targeting Fas after intravenous administration [40]. Further, reducing the level and activity of RNase H1 reduced the potencies of the ASOs, while reducing RNase H2 had no effect [40]. To further confirm that reduction of RNase H2 did not cause a loss of ASO potency, we performed the experiment shown in Figure 2.12. In this experiment, the total siRNA concentration was held constant at 25 nM and the ratio of RNase H1 siRNA to RNase H2 siRNA was varied. The 25 nM siRNA to human RNase H2 had no effect on the potency of the c-Raf ASO. As the ratio of siRNA to RNase H1 versus RNase H2 was increased, there was a progressive loss of potency for the c-Raf ASO. These data represent the first direct demonstration of the involvement of an RNase H in the activity of DNA-like ASOs and that RNase H1, not RNase H2, is responsible for most of the activity.

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% Reduction of cRaf mRNA

64

Page 64

100 90 80 70 60 50 40 30 20 10 0

Control H1 (0 nM)/H2 (25 nM) H1 (6.25 nM)/H2 (18.75 nM) H1 (12.5 nM)/H2 (12.5 nM) H1 (18.75 nM)/H2 (6.25 nM) H1 (25 nM)/H2 (0 nM)

0

0.5 1 1.5 2 Log anti-cRaf ASO concentration (nM) IC50 (nM) Control H1 (0 nM)/H2 (25 nM) H1 (6.25 nM)/H2 (18.75 nM) H1 (12.5 nM)/H2 (12.5 nM) H1 (18.75 nM)/H2 (6.25 nM) H1 (25 nM)/H2 (0 nM)

8.02 8.30 12.42 15.74 17.91 21.48

Figure 2.12 The effects of various ratios of RNase H1 and H2 siRNAs on the potency of anti-cRaf ASO (ISIS 13650). Cells were first transfected with various concentrations of RNase H2 siRNA as indicated for 10 h before the cells were split into 96 well cell culture plates (6000 cells/well) and incubated for 10–14 h. The cells were transfected with various concentrations of ISIS 13650 for 24 h before harvest. Total RNAs were prepared and the cellular cRaf and RNase H2 mRNA levels were determined with RT-PCR in which the reverse transcription and PCR amplification of cRaf and RNase H2 mRNAs were performed in the 96 well format. The total concentration of siRNA was maintained at 25 nM and the ratio of H1 siRNA to H2 siRNA was varied from 0 to 1. The vertical bars represent the standard errors of the mean of three replicates of a representative experiment.

We have shown that in intact cells, RNase H2 appears to play no role in degradation of RNAs targeted by DNA-like ASOs. Nevertheless, IP experiments showed that the presumed RNase H2 protein complex is fully capable of degrading RNA in DNA-like ASO/RNA duplexes and that the pattern of degradation induced by RNase H2 differed from that of RNase H1 (Figure 2.8). Thus, a reasonable hypothesis was that in intact cells human RNase H2 is unavailable to participate in degradation of RNA in DNA-like ASO–RNA duplexes. To evaluate this in more detail, we prepared nuclear extracts and exposed RNA/DNA duplexes to them. In these experiments, we used a different sequence from that used in Figure 2.8 to further confirm the differences in sites of cleavage. Figure 2.13A shows the cleavage of the RNA in the duplex substrate by the nuclear homogenates. The nuclear homogenates immunoprecipitated with the RNase H2 antibody cleaved the heteroduplex at the 11th and 12th ribonucleotide from the 5⬘-terminus of the RNA (Figure 2.13B). Conversely, nuclear homogenates immunoprecipitated with human RNase H1 and H2 antibodies cleaved at ribonucleotides 6 and 7 (Figure 2.13C). When the extract was repeatedly immunoprecipitated with the RNase H2 antibody, the supernatant displayed the human RNase H1 cleavage pattern, predominantly cleaving at ribonucleotides 6 and 7 (Figure 2.13D). The supernatant repeatedly immunoprecipitated with human RNase H1 antibody, displayed the human RNase H2 cleavage pattern, predominantly cleaving at ribonucleotides 11 and 12 (Figure 2.13E). Thus both enzymes are present and active in the nuclear extracts. Again in this experiment using a different substrate, the enzymes displayed different cleavage patterns consistent with the observations shown in Figure 2.8. This result also emphasizes the relative lack of influence of sequence on cleavage with either enzyme. To confirm the results in Figure 2.13 and to quantitate the relative contributions of each of the RNases H in the nuclear homogenates, we preformed two

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(A) Time (min)

RNase H2 specific

RNase H1 specific

0

(B)

5 25 125

0

5 25 125

(C) 0

(D)

5 25 125

0

5 25 125

(E) 0

5 25 125

C U C G A A A C G G A A G A A C G G U A

RNase H1 RNase H2 specific specific

Substrate: Figure 2.13

32 P-AUGGCAAGAAGGCAAAGCUC

TACCGTTCTTCCGTTTCGAG

3’ 5’

Hela cell nuclear extract contains RNase H activity. The 100 nM 20-mer RNA/DNA duplex substrate was prepared and subjected to digestion by Hela cell nuclear extracts (10
g total protein), the H1 or H2 Ab IP samples and the extracts depleted of each RNase H with the Abs for different time periods at 37oC. The digested duplexes were subjected to denaturing polyacrylamide gel analysis. The asterisks indicate the major differences in cleavage sites between RNase H1 and H2. The sequence of the RNA/DNA duplex is shown at the bottom of the figure.

experiments. In the first experiment, (Figure 2.14A), we pretreated cells with the siRNA specific for RNase H1 or RNase H2, and a control siRNA, then prepared cellular homogenates, and determined the initial rate of degradation of RNA in the duplex substrate. Pretreatment of cells with an siRNA to RNase H2 reduced cellular RNase H2 by ⬃60% (data not shown) and reduced the initial rate of cleavage of the substrate by ⬃60%. Treatment of the cells with the siRNA to RNase H1 reduced RNase H1 by ⬃60% (data not shown), but had only a small effect on the initial rate of cleavage in the homogenate (Figure 2.14A). This result is consistent with the results from experiments in which nuclear lysates were immunoprecipitated with antibodies against human RNase H1 and RNase H2 and the extent of reduction of the intact substrate was much lower in the RNase H2-depleted supernatant (Figure 2.13D) than the supernatant in which the RNase H1 was depleted (Figure 2.13E). Taken together, these data suggest that RNase H2 activity is more abundant than RNase H1 activity in cells. In the second experiment (Figure 2.14B), we prepared nuclear extracts, then removed the RNases H with the antibodies, and measured the initial rates of cleavage. Again, depletion of RNase H2 resulted in a much greater reduction of the initial rate than did depletion of RNase H1. Depletion of both RNases H resulted in loss of nearly all of the cleavage activities in the lysate. These data also suggest that in cellular homogenates, the bulk of enzyme activity is a result of RNase H2 (Figure 2.13A). Finally, the fact that IP of both enzymes resulted in almost total loss of activity, suggests that under the conditions of the assay, the two enzymes account for most of the activity. 2.5 THE EFFECTS OF CHIMERIC ASOs ON HUMAN RNase H1 ACTIVITY First-generation phosphorothioate oligodeoxynucleotides have been shown to induce pharmacological effects in vivo and to reduce target RNA in humans [59]. However, they display significant

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION (A)

50

Percent cleavage (%)

45 40

Control siRNA

35 30

H1 siRNA

25 20

H2 siRNA

15 10 5 0 0

I.R. (nM/min) (B)

20 Time (min) Control 1.21 ± 0.05

40 H1 siRNA 1.1±0.09

50 45

Percent cleavage (%)

H2 siRNA 0.46±0.07

IgG Ab strip

40 35

H1 Ab strip

30 25

H2 Ab strip

20 15

H1 Ab &H2 Ab strip

10 5 0 0

20

40

Time (min)

I.R. (nM/min)

IgG strip 1.21±0.05

H1 Ab strip 0.87±0.08

H2 Ab strip H1&H2 strip 0.32±0.05 0.042± 0.021

Figure 2.14 RNase H activity of the cell lysates or supernatants after immunoprecipitation. The enzyme activity was measured with TCA precipitation. 2–5
g proteins were used in the assay with 150 nM RNA/DNA substrate. The experiments were performed in triplicate. The bars show the standard errors of the mean. The initial rates were calculated and presented under the graph. (A) The lysate enzyme activities of Hela cells pretreated with 100 nM RNase H1, H2 or control siRNA for 24 h. (B) The activities of the supernatants after the cell lysates were depleted (immunoprecipitated) with RNase H1 and/or H2 antibody or rabbit IgG. The initial rates are shown as means ⫾ standard error of the mean from at least three separate experiments.

limitations including limited potency and the need to be given intravenously every other day. Second-generation chimeric ASOs consisting of a deoxyribonucleotide region to support RNase H activity flanked by modified nucleotides (e.g., 2⬘-MOE nucleotides) are the products of more than a decade of medicinal chemistry research of antisense oligonucleotides in which more than 1000 modifications were evaluated (for review, see Ref. [3]). 2⬘-MOE chimeric ASOs have been shown to be 10- to 40-fold more potent, safer, and extend elimination half-lives in all species including man from 14 to 30 days (for review, see Ref. [3]). To provide a more precise understanding of the effects of MOE nucleotides on both the overall rate of cleavage and the precise positions of cleavage induced by human RNase H1, we prepared a series of chimeric ASOs in which the deoxyribonucleotides of the ASO were successively substituted

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(A)

2

V0 (nM/min)

with MOE residues at the 3⬘- and 5⬘-poles of the ASO [43]. Successive MOE substitution at the 3⬘-terminus of the ASO resulted in a concomitant ablation of the 5⬘-most cleavage site in the oligoribonucleotide, whereas MOE substitution at the 5⬘-terminus of the ASO resulted in a concomitant ablation of the 3⬘-most cleavage site [43]. As a result, the chimeric substrates produced fewer human RNase H1 cleavage sites and slower overall cleavage rates (Figure 2.15). In fact, a twofold reduction in the initial cleavage rate was observed for chimeric substrates containing as few as two to three MOE substitutions. To examine the effects of MOE substitutions positioned at both the 5⬘- and 3⬘-poles of the ASO on human RNase H1 activity, several ASO sequences were prepared in which the deoxyribonucleotide region was positioned centrally (5-10-5) or toward the 3⬘-pole of the ASO (2-10-8) [43]. All of the unmodified heteroduplex substrates tested exhibited the preferred position for cleavage 7–12 ribonucleotides from the 5⬘-terminus of the RNA. The cleavage sites for the 5-10-5 heteroduplexes were shifted 3⬘ on the RNA compared with the unmodified substrates. A further shift 3⬘ in the RNA was observed for the cleavage sites of the 2-10-8 heteroduplexes. Importantly, the chimeric ASO that shifted the positions of cleavage away from the preferred sites exhibited slower overall cleavage rates [43]. In a control experiment, unmodified substrates were prepared in which the oligodeoxyribonucleotide sequences matched the DNA regions of the 5-10-5 and 2-10-8 chimeric substrates. In all cases the unmodified substrates exhibited significantly faster cleavage rates compared with the chimeric substrates suggesting that the slower cleavage rates observed for the chimeric substrates was due to the MOE substitutions and not the length or sequence of the deoxyribonucleotide region [43]. These results clearly demonstrate that MOE substitutions in the ASO inhibit human RNase H1 activity. Several factors appear to contribute to the reduction in human RNase H1 activity. First,

1

0 U

19-1 18-2 17-3 16-4 15-5 14-6 13-7 12-8 11-9 10-10 9-11 8-12 7-13 6-14

Heteroduplex

V0 (nM/min)

(B) 1.5

1.0

0.5

0.0

U

1-19 2-18 3-17 4-16 5-15 6-14 7-13 8-12 9-11 10-10

Heteroduplex Figure 2.15 Initial cleavage rates for chimeric MOE heteroduplex substrates. (A) Successive substitution of 1–14 MOE-modified nucleotides at the 3⬘-pole of the ASO. (B) Successive substitution of 1–10 MOE-modified nucleotides at the 5⬘-pole of the ASO. Initial cleavage rates (V0) were determined as previously described [26]. The vertical bars represent the standard errors of the mean of three replicates of a representative experiment.

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chimeric heteroduplexes redirected the position of cleavage outside the positional preference for cleavage. Second, the influence of the MOE residues on human RNase H1 activity was directional (Figure 2.15). The observed differences in activity between chimeric heteroduplexes containing MOE residues at the 5⬘-pole versus 3⬘-pole of the ASO is likely the result of the binding directionality of the enzyme, in which the 3⬘-pole MOE substitutions would be positioned at the RNA-binding domain and the 5⬘-pole MOE substitutions would be positioned adjacent to the catalytic domain of the enzyme (Figure 2.16). Given that the RNA-binding domain of human RNase H1 has been shown to be important for properly positioning the enzyme on the heteroduplex for catalysis [36], 3⬘-pole MOE substitutions likely interfere with the binding of the enzyme to this portion of the substrate, effectively redirecting the enzyme to the RNA/DNA region of the chimeric heteroduplex (Figure 2.15A and Figure 2.16). Consistent with this model, 5⬘-pole MOE substitutions would interfere with the catalytic domain of the enzyme, resulting in the observed progressive ablation of human RNase H1 cleavage sites without the concomitant production of new preferred cleavage sites and slower overall cleavage rates (Figure 2.15B and Figure 2.16). Third, the reduction in human RNase H1 activity observed for the chimeric heteroduplexes appeared to be primarily due to the MOE substitutions as comparable cleavage rates were observed for the unmodified heteroduplexes containing the 10 and 20 deoxyribonucleotides without flanking MOE residues. In addition, the MOE residues appeared to effect the cleavage activity of the adjacent deoxyribonucleotide regions as a 4–5 base-pair separation from the 5⬘-most cleavage site on the RNA and the nearest MOE at the 3⬘-pole of the ASO and a 2 base-pair separation between the 3⬘-most cleavage site on the RNA and the nearest MOE at the 5⬘-pole of the ASO was observed. The influence of the MOE substitutions on human RNase H1 activity is consistent with the solution structure of a chimeric heteroduplex containing deoxyribonucleotides flanked on both ends

(A)

3′-MOE

RNA-BD

cat

5′ 3′

(B)

5′-MOE

RNA-BD

cat

5′ 3′ Figure 2.16 Model for the interaction of RNase H1 with the heteroduplex substrate containing MOE modifications at the 3⬘- and 5⬘-poles of the ASO. The light gray and dark gray lines represent, respectively, the sense oligoribonucleotide oriented 5⬘ → 3⬘ and chimeric ASO 3⬘ → 5⬘. The light gray and dark gray boxes represent, respectively, MOE residues at the 3⬘- and 5⬘-poles of the ASO. The binding interaction for human RNase H1 is shown with the RNA-binding domain (RNA-BD) of the enzyme positioned 5⬘ to the catalytic domain (Cat) on the oligoribonucleotide. Each observed cleavage site on the RNA is coupled to a specific binding interaction between the RNA-BD and the 3⬘-DNA/5⬘-RNA pole of the heteroduplex substrate. (A) Alteration in helical geometry or steric interference by the MOE substitutions at the 3⬘-pole of the ASO disrupts the binding interaction between the RNA-BD and the heteroduplex resulting in the observed ablation of catalytic activity at the 5⬘-most cleavage sites on the RNA. (B) MOE substitutions at the 5⬘-pole of the ASO are positioned adjacent to the catalytic domain of enzyme.

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with 2⬘-methoxy residues hybridized to RNA [60]. Specifically, the central deoxyribonucleotides exhibited the eastern-biased sugar conformation preferred by RNase H1, whereas the flanking 2⬘-methoxy residues exhibited a northern sugar conformation. Importantly, the deoxyribonucleotides adjacent to the 2⬘-methoxy residues were also shown to exhibit a northern sugar conformation suggesting the conformational transmission of the northern-biased 2⬘-methoxy residues into adjacent deoxyribonucleotides [60]. Given that heteroduplexes containing northern-biased nucleotides within the DNA strand do not support human RNase H1 activity [35], the attenuation of the human RNase H1 activity observed for the chimeric heteroduplexes is likely due to the conformational transmission of the northern-biased MOE residues into the adjacent deoxyribonucleotides. To better understand the mechanisms of the observed reduction in catalytic efficiency of chimeric substrates containing MOE nucleotides and to begin to identify means to mitigate these effects, we introduced modified nucleotides at the MOE/DNA junction of the chimeric ASO to modulate the transmission of conformation of the MOE substitutions into the area of the duplex in which cleavage occurs (Figure 2.17) [61]. In addition, mismatched base pairs were introduced at various positions in the chimeric substrate and the initial cleavage rates (V0) for the modified heteroduplexes were compared with the wild-type DNA/RNA heteroduplex. (A)

B

O

O

O

B

F

O

O O

SCH 3

O

O

O P O

O P O

O P O

O

O

O

2′-ara-F

2′-S-Me

LNA CH 3

(B)

O

F O

N

O F

O O

F

N

N

O

CH 3

O F

O

O P O

O P O

O

O

N-Me-T

TFI (C)

B

O

O

5’-UCAAAUCCAGAGGCUAGCAG 3’-AGTTTAGGTCTCCGATCGTC 5’-UCAAAUCCAGAGGCUAGCAG 3’-AGTTTAGGTCTCCGATCGTC

Figure 2.17 Structure of the nucleotide modifications. (A) Modified nucleotides containing conformationally biased sugars include the southern-biased 2⬘-methylthiothymidine (2⬘-S-Me-T), the northernbiased locked nucleic acid (LNA) and the eastern-biased 2⬘-ara-fluorothymidine (2⬘-ara-F-T). (B) Structures of the modifications designed to introduce conformational flexibility into the heteroduplex. These modifications include 2⬘-methoxyethoxy-N-methylthymidine (N-Me-T) and tetrafluoroindole (TFI). (C) Sequences of the heteroduplex substrates test with oligoribonucleotide (above) orientated 5⬘→ 3⬘ and the chimeric ASO (below) orientated 3⬘-5⬘. Underlined sequences indicate the position of the MOE residues. The bold sequences indicate the positions of the nucleotide modifications listed in (A) and (B).

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Consistent with the effects of the MOE modifications on human RNase H1 activity, the strong northern-biased locked nucleic acid (LNA) modification positioned at either junction exacerbated the negative effects of the MOE modifications resulting in slower human RNase H1 cleavage rates compared with the MOE chimeric substrates. In contrast, enhanced cleavage rates were observed for the eastern-biased 2⬘-ara-fluorothymidine and bulge-inducing N-methylthymidine modifications positioned at the 5⬘-DNA/3⬘-MOE junction as well as the southern-biased 2⬘-methylthiothymidine positioned at the 5⬘-MOE/3⬘-DNA junction [61]. The greatest improvement in activity was observed for the conformationally flexible tetrafluoroindole (TFI) modifications positioned at either junction. The TFI modification is predicted to -stack with adjacent nucleotides, but not form hydrogen bonds with the opposing RNA. Consistent with the predicted interactions of the TFI, the heterocycle of the ribonucleotide opposing the TFI deoxyribonucleotide had no effect on the human RNase H1 activity whereas nucleotide substitutions adjacent to the TFI significantly affected the cleavage rate [61]. In addition, mismatch base pairs exhibited similar effects on human RNase H1 activity as the TFI modifications [61]. Finally, the cleavage patterns for the modified heteroduplexes suggest that the junction modifications are modulating the conformation transmission of the MOE residues. Specifically, the LNA substitutions ablated the cleavage activity nearest the modifications. In contrast, the rate-enhancing junction modifications enhanced cleavage activity for the cleavage sites nearest these junction modifications [61].

2.6 IMPLICATIONS FOR ANTISENSE THERAPEUTICS The demonstration that human RNase H1 plays a dominant role in the activities of DNA-like ASOs suggests that additional studies that explore the substrate preferences, enzymology, and regulatory processes for RNase H1 should support improved design of antisense agents. In addition, the demonstration that increases in RNase H1 activity correlated with increases in potency suggests that recruitment of RNase H1 to the ASO–RNA duplex and/or cleavage of the RNA by the enzyme is limiting for ASO activity. We have demonstrated that human RNase H1 is comprised of an RNA-BD, a spacer region, and a catalytic domain. Once bound to the heteroduplex substrate, the RNA-binding and the catalytic domains are separated by approximately one helical turn with the RNA-binding domain positioned 3⬘ on the RNA to the catalytic domain. The architecture of the enzyme is used to sense subtle changes in the helical geometry of potential substrates such that the enzyme may bind to many nucleic acid structures, but will only cleave those structures with the appropriate characteristics. To be cleaved by human RNase H1, a substrate must display a minor groove of appropriate dimensions unobstructed by any substituent such as those deriving from 2⬘ modification of the deoxyribose. Further, the intra- and interphosphate distances of the heteroduplex substrate are crucial as is the flexibility of the backbone. Any strategy that would improve these processes should improve ASO potency. Clearly, MOE as well as phosphorothioate modifications inhibit human RNase H1 activity [26]. Given the beneficial properties of these modifications, designing chimeric ASOs for enhanced human RNase H1 activity should improve the potency of these chimeric drugs. For example, optimizing the placement of the DNA-like domains relative to preferred sites of cleavage. In addition, reducing the number of 2⬘-modified nucleotides should improve the enzyme activity. Obviously, as 2⬘-modified nucleotides improve affinity to target RNA, reducing the number of 2⬘-modified nucleotides would reduce the ability of the ASO to invade structured RNA sites resulting in fewer active sites in the mRNA. Identifying modified nucleotides with even greater affinity for the target RNA or more accessible sites should enable the need for fewer modifications. In fact, several “gap-widened” chimeric ASOs containing longer deoxyribonucleotide regions and fewer MOE residues have demonstrated improved potency in cell culture and in vivo (Swikowski, unpublished data).

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Medicinal chemical approaches that optimize the structures of chimeric ASOs should also improve the potency of these drugs. For example, inserting modified nucleotides to modulate the transmission of the 2⬘-modified/RNA geometry into the RNA/DNA portion of the chimeric heteroduplex should also improve human RNase H1 activity. Specifically, modifications that disrupt base-stacking interactions or exhibit an eastern sugar conformation (e.g., N-Me-T and 2⬘-ara-F-T) should be positioned at the DNA/MOE junction adjacent the phosphate-binding pocket in the enzyme (Figure 2.4, Figure 2.16, and Figure 2.17). In contrast, modifications exhibiting conformational flexibility or the opposing southern sugar conformation (e.g., TFI and 2⬘-S-Me-T) should be positioned at the DNA/MOE junction adjacent the ribonucleotides downstream of the scissile phosphate (Figure 2.4, Figure 2.16, and Figure 2.17). The TFI modification can be used to modulate the negative effects of the 2⬘-alkoxy residues both upstream and downstream of the scissile phosphate irrespective of the heterocycle of the opposing nucleotide. However, the TFI modification should be positioned adjacent to the stable base pairs and not at sites that interact directly with the enzyme (Figure 2.4). Mismatch base pairs can also be used to enhance the human RNase H1 activity of the chimeric ASOs, although predicting favorable noncanonical base-pair substitutions may prove more difficult than TFI substitutions. Finally, given that the elimination half-lives and proinflammatory effects of chimeric ASO drugs are determined by the MOE residues, substitution of one or two MOE modifications at the junction should have only modest effects on the properties of these drugs. REFERENCES 1. Crooke, S. T. (1999) Molecular mechanisms of antisense drugs: human RNase H. Antisense Nucl. Acid Drug Dev. 9: 377–379. 2. Crooke, S. T. (2000) Progress in antisense technology: the end of the beginning. Methods Enzymol. 313: 3–45. 3. Crooke, S. T. (2001) In: Antisense Technology: Principles, Strategies and Applications. (Crooke, S. T., ed.) Marcel Dekker, New York, pp. 1–28. 4. Grishok, A., Tabara, H., and Mello, C. C. (2000) Genetic requirements for inheritance of RNAi in C. elegans. Science 287: 2494–2497. 5. Zamore, P. D., Tuschl, T., Sharp, P. A., and Bartel, D. P. (2000) RNAi: double-stranded RNA directs the ATP-dependent cleavage of mRNA at 21 to 23 nucleotide intervals. Cell 101: 25–33. 6. Stein, H. and Hausen, P. (1969) Enzyme from calf thymus degrading the RNA moiety of DNA–RNA hybrids: effect on DNA-dependent RNA polymerase. Science 166: 393–395. 7. Busen, W. (1980) Purification, subunit structure, and serological analysis of calf thymus ribonuclease HI. J. Biol. Chem. 255: 9434–9443. 8. Crouch, R. J. and Dirksen, M. L. (1982) In: Nucleases (Linn, S. M. and Roberts, R. J., eds.) Cold Spring Harbor. Cold Spring Harbor Press, Plainview, New York. 9. Kane, C. M. (1988) Renaturase and ribonuclease H: a novel mechanism that influences transcript displacement by RNA polymerase II in vitro. Biochemistry 27: 3187–3196. 10. Masutani, C., Enomoto, T., Suzuki, M., Hanaoka, F., and Ui, M. (1990) DNA primase stimulatory factor from mouse FM3A cells has an RNase H activity. Purification of the factor and analysis of the stimulation. J. Biol. Chem. 265: 10210–10216. 11. Eder, P. S. and Walder, J. A. (1991) Ribonuclease H from K562 human erythroleukemia cells. J. Biol. Chem. 266: 6472–6479. 12. Frank, P., Albert, S., Cazenave, C., and Toulme, J. J. (1994) Purification and characterization of human ribonuclease HII. Nucl. Acids Res. 22: 5247–5254. 13. Ohtani, N., Haruki, M., Morikawa, M., Crouch, R. J., Itaya, M., and Kanaya, S. (1999) Identification of the genes encoding Mn2⫹-dependent RNase HII and Mg2⫹-dependent RNase HIII from Bacillus subtilis: classification of RNases H into three families. Biochemistry 38: 605–618.

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3

Small RNA Silencing Pathways Alla Sigova and Phillip D. Zamore

CONTENTS 3.1 3.2

Introduction.............................................................................................................................75 Small Interfering RNAs..........................................................................................................76 3.2.1 Genesis of siRNAs......................................................................................................76 3.2.2 siRNA Strand Selection..............................................................................................77 3.3 miRNAs ..................................................................................................................................77 3.3.1 Biogenesis of miRNA Precursors...............................................................................78 3.3.2 Processing of miRNA Precursors into Mature miRNAs............................................79 3.4 The RNAi Enzyme Complex..................................................................................................79 3.4.1 RISC ...........................................................................................................................79 3.4.2 Structure of Argonaute Proteins .................................................................................80 3.5 RISC Assembly.......................................................................................................................81 3.6 Small RNA-Directed mRNA Destabilization and Translational Repression.........................82 3.7 Conclusions.............................................................................................................................83 References ........................................................................................................................................84

3.1 INTRODUCTION RNA silencing pathways use small RNA guides to destroy a complementary mRNA, inhibit its translation, or reduce its rate of transcription. RNA silencing was first discovered by plant scientists in 1990 during an attempt to engineer transgenic petunias overexpressing enzymes that limit flower pigment production [1,2]. Remarkably, the introduction of transgenes expressing either of the flower pigmentation pathway enzymes chalcone synthase or dihydroflavonol-4-reductase failed to produce more intensely pigmented flowers. Instead, the transgenes repressed—that is, “silenced”— expression of both the endogenous and transgenic copies, causing partial or complete loss of flower pigmentation. The phenomenon was therefore dubbed “cosuppression” to reflect the coordinate silencing of the transgene and the endogenous gene. In Neurospora crassa, a similar silencing phenomenon, “quelling,” was also discovered when additional copies of sequences from genes were introduced into the N. crassa genome, again causing an unexpected and dramatic reduction in the expression of the corresponding endogenous genes. Quelling proved to be reversible: the loss of the exogenous DNA from the genome restored expression 75

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of the endogenous gene [3,4], suggesting that silencing required the active production of a silencing signal from the transgene. These earlier studies paved the way for the central breakthrough in understanding RNA silencing—the demonstration by scientists working on the nematode Caenorhabditis elegans that double-stranded RNA (dsRNA) triggers specific repression of genes corresponding to its sequence. RNA silencing was discovered in C. elegans when antisense RNA was used to inhibit the function of par-1 gene in a study reporting the positional cloning of the par-1 locus. While injection of antisense par-1 RNA gave the expected par-1 mutant phenotype, par-1 mRNA itself also disrupted par-1 function [5]. Fire, Mello, and their colleagues revolutionized our understanding of RNA-directed silencing, which came to be know as an RNA interference (RNAi), when they demonstrated that the presence of contaminating dsRNA in both the antisense and sense RNA transcripts was the silencing trigger [6], an insight for which they received the 2006 Nobel Prize in Medicine. They showed that dsRNA, but not purified sense or antisense RNA, induced potent and specific silencing in C. elegans. RNAi has quickly become the reverse genetics tool of choice, even in animals with long established genetic traditions. In plants, RNAi is being used to engineer crops with improved disease resistance and other economically desirable traits. The discovery that long dsRNA is converted to small interfering RNAs (siRNAs) [7], allowed RNAi to be extended to mammalian cells [8]. In fact, siRNA-based therapies for human disease are now being sought in biotechnology companies and academic laboratories, and several siRNA-based drugs are currently in clinical trials. In this chapter, we summarize our current understanding of the biological purpose and biochemical mechanisms underlying RNA silencing pathways.

3.2 SMALL INTERFERING RNAs 3.2.1

Genesis of siRNAs

In 1999, Hamilton and Baulcombe reported finding small (⬃25 nt) RNAs that correlated with posttranscriptional gene silencing (PTGS), the plant cousin of RNAi. They analyzed transgeneinduced PTGS of an endogenous gene, PTGS induced by transgenes without homology to an endogenous gene, systemic PTGS of GFP transgenic plants initiated by infiltration of a single leaf with Agrobacterium tumefaciens expressing GFP sequence, and the antiviral response triggered by infection with potato virus X [7]. In all four cases, ⬃25 nt sense and antisense RNAs complementary to the silenced mRNAs (or to the virus) were detected; these small RNAs were not found in plants in which no silencing response had been triggered. The authors suggested that the novel small RNAs were both the specificity determinants for RNA silencing and the mobile signal by which silencing spreads from the initial site of silencing (such as the Agrobacterium-infiltrated leaf) to the rest of the plant. Biochemical experiments conducted in animal cell extracts that could recapitulate RNAi demonstrated that the small RNAs—now called siRNAs—derive directly from the dsRNA that triggered silencing and function as guides for protein complexes that degrade the targeted mRNA. Cell-free systems recapitulating in vitro the conversion of dsRNA into siRNAs, the assembly of siRNAs into functional protein complexes, and the destruction of target RNAs were developed first in Drosophila, either from syncitial blastoderm embryos or cultured Drosophila S2 cells [9,10] and later from cultured human HeLa cells [11,12]. During the RNAi reaction, both strands of the long dsRNA trigger are processed to RNA segments 21–23 nt long [13] by the ribonuclease (RNase) III–like enzyme Dicer [14]. Consequently, siRNAs bear the hallmarks of RNase III cleavage products: they are double-stranded, have 2-nt overhangs at their 3⬘ ends, a monophosphate at the 5⬘ end, and 2⬘ and 3⬘ hydroxyls at the 3⬘ end [15–17]. Because siRNAs are short, they can be readily synthesized chemically and can be delivered by transfection to mammalian cells without provoking

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undesirable sequence-independent responses. Thus, the discovery of the structure of siRNAs led rapidly to their use as triggers of sequence-specific RNAi in mammalian cultured cells [8,18] and in vivo in both rodents and primates [19–22]. 3.2.2

siRNA Strand Selection

Fulfilling the prediction of Hamilton and Baulcombe, siRNAs are the specificity determinants for RNA silencing, guiding the recognition of mRNA targets and directing their destruction. After their production from long dsRNA by Dicer, siRNAs are incorporated into a functional protein–RNA complex, the RNA-induced silencing complex (RISC) [23], through which they direct the endonucleolytic cleavage of the target 10 nt upstream of the 5⬘-most nucleotide of the siRNA [10,13,15–17,24,25]. For example, introduction of 21-nt siRNA duplexes in Drosophila embryo lysates or cultured mammalian cells leads to sequence-specific mRNA degradation [8,15,17,24]. To bind target mRNAs, the two strands of an siRNA duplex must be separated, and then one or both strands assembled separately into RISC. In vitro in Drosophila embryo lysate, efficient RISC assembly requires ATP [16,26]. Single-stranded siRNA can bypass the requirement for siRNA strand separation in vitro in Drosophila embryo lysate or HeLa cell S100 extracts and in vivo in cultured human cells, but the inherent instability of single-stranded RNA and its inefficient entry into the RNAi pathway limits its practical utility [11,17]. Which of the two siRNA strands assembles into RISC is not random. For convenience, the strand that forms RISC is termed the guide and the strand disfavored for RISC assembly is called the passenger. In flies and mammals, the target RNA does not influence the choice of guide strand. Thus, when the guide strand is complementary to the mRNA target, it directs its destruction. If the siRNA strand complementary to the target is the passenger strand, no cleavage will be observed. This explains why some synthetic siRNA duplexes are not active: they load the wrong strand into RISC. Which of two strands of an siRNA duplex is incorporated into RISC reflects the relative thermodynamic stability of the 5⬘ ends of the two siRNA strands [12,27]. Prediction of the active siRNA strand can be made by a nearest-neighbor analysis of the free energy of the two to four 5⬘ terminal base pairs of each siRNA strand [28,29]. siRNAs in which one strand dominates RISC assembly are said to be “functionally asymmetric.” Such functional asymmetry can be engineered into an siRNA by incorporating a mismatched nucleotide at or near the 5⬘ end of the siRNA strand intended to function as guide [12,30].

3.3 miRNAs A high degree of complementarity is required for a small RNA to direct endonucleolytic cleavage of a target RNA. When the small RNA pairs imperfectly with the target, it cannot cleave the target, but may nonetheless repress the translation and/or destabilize the RNA target. In animals, a large class of endogenous small RNAs, microRNAs (miRNAs), act to repress translation or reduce the stability of their mRNA targets (Fig. 3.1); only a few animal miRNAs pair extensively with their mRNAs targets, allowing them to promote endonucleolytic cleavage [31–33]. In contrast, all plant miRNAs likely direct endonucleolytic cleavage of the mRNAs whose expression they repress [34–36]. miRNAs are close relatives of siRNAs. They are the same length as siRNAs, can function in the same pathways, and differ from siRNAs only in the details of their biogenesis. miRNAs regulate expression of endogenous mRNAs, and some computational studies suggest that from one-third to all the genes in a typical animal are targeted by miRNAs at some developmental time or in some cell lineage [37–42]. miRNAs may even function to restrict the host range and tissue-tropism of viruses infecting mammals [43,44]. The human genome may encode thousands of miRNAs [45].

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miRNA gene

polyA pri-miRNA cap Pasha Drosha pre-miRNA Nucleus Exportin 5

Cytoplasm

pre-miRNA Dicer-1 Loqs miRNA/miRNA* duplex

? Ago1

cap

ORF Ribosome

polyA

Target mRNA

Figure 3.1 The miRNA pathway in Drosophila.

3.3.1

Biogenesis of miRNA Precursors

miRNAs were first discovered as mutations that disrupt the timing of development in C. elegans [46,47]. miRNAs originate as much longer primary miRNAs (pri-miRNAs) [48]. pri-miRNAs are transcribed by RNA polymerase II, and thus contain a 5⬘ cap and a poly(A) tail (Fig. 3.1) [48,49]. A pri-miRNA may contain a single miRNA—with thousands of bases transcribed, apparently to yield a single 21 nt RNA guide—or several miRNAs, a polycistronic pri-miRNA. The majority of worm and human miRNAs reside in their own pri-miRNA transcripts, where as more than half of Drosophila miRNAs are polycistronic [50,51]. Some animal viruses also produce miRNAs [52–55]. Within the pri-miRNA, the mature miRNA resides in one arm of a ⬃70-nt stem-loop structure. In the nucleus, a complex of proteins, including the RNase III enzyme Drosha and its dsRNA-binding protein partner Pasha in flies or DGCR8 in mammals, recognizes the stem-loop, cleaves it about one helical turn up from the base of the stem, liberating a ⬃60-nt pre-miRNA [56–59]. Pasha/DGCR8 binds pri-miRNA, restricting Drosha to pri-miRNA processing [56,60]. Pasha comprises an N-terminal WW domain and two C-terminal dsRNA-binding domains. Similar to all RNase III enzymes, Drosha cleavage produces 2-nt, 3⬘ overhanging ends [56,61]. Exportin 5, a member of karyopherin ␤ family of nucleocytoplasmic transport proteins, recognizes the characteristic terminal structure of the pre-miRNA, exporting it from the nucleus to the

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cytoplasm [62,63]. In the cytoplasm, Dicer binds pre-miRNAs and cleaves off the loop, to generate a ⬃21-nt RNA duplex bearing 2-nt, 3⬘overhangs at each end [64–66]. This duplex therefore resembles an siRNA. Unlike siRNAs, the ⬃19-nt double-stranded region of the miRNA/miRNA* duplex contains strategically placed mismatches, G:U wobbles, and internal loops. Current data suggest that these discontinuities help align Drosha on the pri-miRNA and Dicer on the pre-miRNA [67]. The helical discontinuities also establish which of the two strands of the small RNA duplex becomes the mature miRNA, because the strand with the less stably base-paired 5⬘ end is preferably selected to become the miRNA, whereas the strand with the more stably base-paired 5⬘ end, the miRNA* strand, is degraded [12,27]. 3.3.2

Processing of miRNA Precursors into Mature miRNAs

Just as Drosha requires a dsRNA-binding protein partner to process pri-miRNAs, Drosophila Dicer-1 (Dcr-1) [68] requires its dsRNA-binding partner protein Loquacious (Loqs) to process premiRNAs [69–71]. Loqs contains three dsRBDs (two canonical dsRBDs at the N-terminus, and one noncanonical dsRBD at the C-terminus). Loqs not only stimulates the pre-miRNA processing activity of Dcr-1 in vitro, it also makes it specific. Indeed, Dcr-1 alone can process both long dsRNA and pre-miRNA substrates into ⬃21-nt small RNAs; Loqs restricts Dcr-1 to processing pre-miRNA, inhibiting this nonspecific effect [70]. Mammals appear to use two Loqs homologs, the transactivating response (tar) RNA-binding protein (TRBP) and PKR (protein kinase R) activating protein (PACT) as Dicer partner proteins [72–75]. Similar to Loqs, both TRBP and PACT contain three dsRNA-binding domains and use the third domain to bind Dicer [73,75]. Both proteins are not required for pre-miRNA processing by Dicer. TRBP and PACT may have partially redundant functions. Some studies suggest that TRBP also participates in loading of small RNAs into RISC [76,77], suggesting that TRBP functions like R2D2, the Drosophila partner protein of Dicer-2 (Dcr-2), to transfer the small RNA to Argonaute2 (Ago2). If TRBP functions in mammalian RISC loading, it is unlikely that Dicer also participates (unlike Drosophila Dcr-2), because Dicer mouse knockout cells are competent for siRNA-directed RNAi [78]. In contrast, depletion of PACT in cultured human cells decreases the abundance of mature miRNAs, but has little or no effect on siRNA-directed silencing of a reporter [73].

3.4 THE RNAi ENZYME COMPLEX 3.4.1

RISC

Small RNAs such as miRNAs and siRNAs function bound to members of the Argonaute family of proteins. Argonautes are proteins specialized for small RNA-directed target recognition. Plants and animals typically contain several distinct Argonaute proteins with apparently different functions. In flies and mammals, Ago2 mediates siRNA-directed RNAi [79,80]. (miRNAs can also associate with Ago2.) For historical reasons, any protein complex containing a small RNA and capable of directing posttranscriptional gene silencing is termed an RNA-induced silencing complex (RISC). RISC can be as simple as Ago2 loaded with siRNA [11,81], but because Argonaute proteins associate with auxiliary proteins suspected to enhance or modify their intrinsic activity, RISC may also be a multiprotein complex rivaling the ribosome in size [82]. Both Drosophila and human RISCs are Mg2⫹-dependent endonucleases producing a 3⬘ cleavage product bearing a 5⬘ phosphate and a 5⬘ cleavage product with 2⬘, 3⬘ hydroxyl termini [25,83]. While RISC does not require ATP for target recognition or cleavage [81,84], product release is a greatly facilitated by ATP, suggesting that an ATP-dependent RNA helicase acts to dissociate the cleavage products from the siRNA guide [84]. Alternatively, ATP may promote a conformational change in RISC itself, releasing the cleaved target. RISC is an enzyme, catalyzing multiple rounds of endonucleolytic target cleavage without consuming its guide siRNA [84,85]. Following cleavage by Drosophila Ago2-RISC,

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the target fragments are degraded by the 3⬘-to-5⬘ activity of the exosome and by the 5⬘-to-3⬘ exonuclease XRN1 [86]. Although siRNAs typically pair fully with their target RNAs, the 5⬘, central, and 3⬘ regions of an siRNA make distinct contributions to binding and catalysis. Enzyme kinetics measurements and the structures of archael Argonaute proteins suggest that the first nucleotide of a small RNA guide (G1) does not participate in the binding of RISC with target mRNA [84,87,88]. Nucleotides G2 through G8 provide most of the energy of target binding. The importance of this region of the small RNA, which is called the seed sequence, emerged first from efforts to identify mRNAs regulated by microRNAs [39,41,42,89–91]. While the central and 3⬘ regions of a small RNA guide must also be base-paired with the target for Argonaute to catalyze cleavage, these bases contribute little energy to target binding [84]. In general, target cleavage requires base pairing of nucleotides 2 to12, which corresponds to one turn of A-form helix. In vitro, RISC is remarkably tolerant to mismatches. In vitro, up to nine contiguous mismatches at the 3⬘ end and up to five at the 5⬘ end are tolerated [25,84], although regulation by siRNAs with so little pairing with their target RNAs is unlikely to proceed through an endonucleolytic mechanism in vivo. 3.4.2

Structure of Argonaute Proteins

Argonaute proteins contain two highly conserved domains: the PAZ domain (first identified in the Drosophila protein Piwi and the plant proteins Argonaute and Zwille) and the PIWI domain [92]. The ⬃110 amino acid PAZ domain, found in both Argonaute and Dicer proteins, binds the two single-stranded 3⬘ nucleotides of a small RNA duplex [93–96]. The central domain of PAZ is a 5 or 6 strand, left-handed ␤-barrel with two ␣-helices at the N-terminus. The PAZ domain resembles the oligonucleotide-binding “OB” fold present in some nucleic acid–binding proteins [97]. The sequences of PAZ domains diverge considerably, and the PAZ domain of Pyrococcus furiosus Argonaute was detected only by structural homology [98]. The structure of the PAZ domain is essentially unchanged by RNA binding, even though the siRNA 3⬘ tail is positioned deep inside a cleft lined with aromatic and basic amino acid side chains, which cannot accommodate terminal phosphate or paired nucleotides [93,94,99]. The PIWI domain is a structural homolog of the catalytic domain of RNase H [98], a discovery unanticipated by comparisons of the sequences of the two protein classes. Both RNase H and Argonaute proteins recognize A-form nucleic acid helices and use the sequence of one of the strands (DNA for RNase H, RNA for Argonaute) to direct cleavage of the other strand (RNA for both). Unlike RNase H, the Argonaute proteins remain stably bound to their nucleic acid guide, in part because the PIWI domain contains a 5⬘ phosphate-binding pocket that secures and aligns the siRNA guide strand on the protein. The phosphate-binding pocket is formed by the C-terminal carboxyl group of Argonaute bound to a divalent cation and is the most evolutionarily conserved feature of the PIWI domain [87,88]. Insertion of the 5⬘ phosphate of the siRNA guide strand into the binding pocket precludes pairing of the first base of the guide (G1) with the complementary base in the passenger strand or the RNA target (T1) [88], consistent with the earlier findings that unpairing the first base of the guide within an siRNA duplex promotes its incorporation into RISC [12] and that a mismatch between guide nucleotide G1 and target nucleotide T1 enhances, rather than disrupts, target cleavage [84]. The phosphate groups of guide strand nucleotides 2 through 5 interact extensively with amino acids in the PIWI domain, whereas few contacts are made with the passenger strand, consistent with the need to release the passenger strand and, later, the cleaved target RNA, from Argonaute. Little structural information is available about the geometry of the RNA in the catalytic cleft of the PIWI domain, but model building suggests that the scissile phosphate, which always lies between the target nucleotides paired to guide strand bases G10 and G11, resides within RNase H fold [100]. Biochemical data suggest that the target cleavage site is determined by the placement of the guide strand on Argonaute, not the length of the helix formed between the siRNA and its target

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[10,13,15–17,24,25]. Similar to RNase H, the Argonaute PIWI domain contains a trio of conserved amino acids required for catalysis [101]. In RNase H, Asp-Asp-Glu (DDE) residues coordinate two metal ions at the active site; in the catalytic center of Pyrococcus furiosis Argonaute, the two metal ions are coordinated by two aspartic acids and a histidine (DDH). The metal ions—likely Mg2⫹—are proposed to recruit a water or hydroxide ion as the nucleophile attacking the phosphodiester bond and to stabilize the transition state during catalysis. When these catalytic residues in human Ago2 are mutated, siRNA-directed mRNA cleavage is abolished, supporting the view that the other human Argonaute proteins, Ago1, Ago3, and Ago4, cannot direct endonucleolytic cleavage of their targets because they lack the appropriate catalytic amino acids [79,81].

3.5 RISC ASSEMBLY RISC assembly, the process by which a small RNA is loaded into an Argonaute protein, has been most extensively studied for Drosophila Ago2. Assembly of Drosophila Ago2-RISC in vitro is a highly ordered process (Fig. 3.2). The central step in Drosophila Ago2-RISC assembly is formation of the RISC loading complex (RLC), which requires Dcr-2, its dsRNA-binding partner protein R2D2, and is stimulated by ATP [26,68,102]. Production of the RLC requires binding of a heterodimeric complex of Dcr-2 and R2D2 to the siRNA duplex, but other proteins are likely required, as the RLC is considerably larger than a complex containing only siRNA, Dcr-2, and R2D2 [26,102]. Dcr-2 alone binds poorly to siRNA, requiring R2D2 for stable binding [103,104]. Conversely, recombinant R2D2 does not detectably bind siRNA without Dcr-2 [105]. The central function of the RLC appears to be selecting which strand of an siRNA duplex will be loaded into Ago2—the guide strand—and which will be degraded—the passenger strand. The Dcr-2/R2D2 heterodimer binds an siRNA duplex with a nonrandom orientation. R2D2 binds preferentially to the siRNA 5⬘ end with more double-stranded RNA character, i.e., the more thermodynamically stable end. Dcr-2 therefore lies near the 5⬘ end of the guide strand. The Dcr-2/R2D2 heterodimer is thus primary sensor in Drosophila of siRNA thermodynamic stability [104]. Binding of Dcr-2/R2D2 to the siRNA promotes its loading into RISC, perhaps because Ago2 can bind Dcr-2 directly [106]. Selection of the siRNA guide strand does not appear to be coupled to the polarity of dsRNA processing by Dcr-2, suggesting that newly generated siRNA are released from Dcr-2 and then reenter the Dcr-2-dependent Ago-2-RISC assembly pathway [107]. Surprisingly, separation of the two strands of an siRNA occurs after they are loaded into Ago2. Initially, siRNA unwinding was presumed to precede loading of the guide strand into Ago2 [16]. The first hints that this model was flawed were the findings that siRNA unwinding requires Ago2 [108] and that double-stranded siRNA accumulates in the RLC in lysates prepared from ago2 mutant embryos [104]. Current data suggest that first Dcr-2 and then R2D2 is exchanged for Ago2, placing the 5⬘ phosphate of the guide strand of the double-stranded siRNA in the phosphate-binding pocket of Ago2 [109,110]. The single-stranded RNA-binding PAZ domain of Ago2 is hypothesized to bind the 3⬘ end of the guide strand, displacing R2D2. For an siRNA duplex, the resulting configuration of siRNA on Ago2 is essentially the same as an Ago2:siRNA complex bound to a target RNA. In vitro and cell culture data suggest that endonucleolytic cleavage of the passenger strand initiates its displacement from Ago2, generating a functional RISC in which the guide strand is ready to bind an mRNA target [111–113]. In vitro data also suggest that Ago2-catalyzed cleavage of the passenger strand facilitates loading of siRNA into human Ago2-RISC [111]. Moreover, the same mechanism is likely used for miRNA/miRNA* duplexes that have both a paired central region—that is, one that permits passengerstrand cleavage—and a fully paired seed sequence [111]. Recall that the seed sequence is a specialized region of a small RNA guide—roughly nucleotides 2 through 8—that provides most of the binding energy for target recognition. The seed is thought to be created when a small RNA binds an Argonaute protein. Thus, the special properties of the seed do not exist before the siRNA or

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Dicer-2 R2D2

siRNA duplex

ATP

RLC

ATP

Ago2

ATP? Ago2

polyA

cap

ATP polyA Figure 3.2

cap

Drosophila RISC assembly.

miRNA/miRNA* duplex binds Argonaute. After binding Argonaute, the passenger strand or miRNA* strand is hypothesized to be tethered to its complement mainly through the seed sequence. Small RNA duplexes with disrupted seed sequences (e.g., miRNA/miRNA* sequences with a mismatch or G:U wobble within the seed region) likely release the passenger strand without the need to cleave it, because little holds the two strands of the small RNA together once the duplex binds an Argonaute protein.

3.6 SMALL RNA-DIRECTED mRNA DESTABILIZATION AND TRANSLATIONAL REPRESSION The first examples of miRNA-directed regulation led to the idea that animal miRNAs bind imperfectly to multiple sites in the 3⬘ untranslated region (UTR) of the mRNAs they regulate. This view was reinforced by computational efforts to predict miRNA targets, which relied heavily on the evolutionary conservation of mRNA sequences complementary to the miRNA seed sequence,

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because conservation is simpler to detect in UTRs than in coding sequences, which are constrained to encode a functional protein [41,42,91,114]. Recently, algorithms have been proposed to predict miRNA targets without resort to sequence conservation [40]. These methods suggest that miRNAs also regulate targets by binding in the 5⬘ UTR and coding sequence. The success of this newer computational approach to predict biologically important miRNA–mRNA regulatory relationships remains to be determined, and experimental approaches suggest that 3⬘ UTR sequences are generally more amenable to miRNA-like regulation than other mRNA regions [115–117]. Initial studies of the C. elegans miRNA lin-4, the first discovered miRNA, demonstrated that lin-4 directs repression of lin-14 mRNA posttranscriptionally and suggested that repression occurs through a block to translation of LIN-14 protein [118,119]. Surprisingly, repression of LIN-14 protein synthesis by lin-4 did not appear to alter the association of either lin-14 mRNA with polysomes or the length of the lin-14 poly(A) tail, consistent with repression of LIN-14 translation after the initiation of protein synthesis. Similar conclusions were reached for a second lin-4-regulated gene, lin-28 [120,121]. More recently, a reexamination of the mechanism by which the miRNA let-7 regulates expression of lin-41 and by which lin-4 regulates lin-14 and lin-28 suggests that these miRNAs act mainly by decreasing the stability of their mRNA targets, rather than by altering their rates of translation [122]. Apparently conflicting data have also been reported for human miRNA regulatory mechanisms. In cultured human cells, endogenous let-7 represses translation of a reporter construct by decreasing the rate of translational initiation [123,124]. In contrast, siRNAs engineered to bind imperfectly to multiple sites in the 3⬘ untranslated region of a reporter mRNA appear to repress protein synthesis without altering the rate of translational initiation [125–127]. miRNAs can sequester their mRNA targets in cytoplasmic Processing bodies (P-bodies), and both miRNA-directed mRNA destruction and translational repression may reflect sequestration of the mRNA in P-bodies [123,128–131]. The human Argonaute proteins Ago1 and Ago2 directly associate with the P-body component GW182; depletion of GW182 impairs small RNA-directed silencing of a reporter mRNA [132–134]. Mutations in Ago1 and Ago2 that prevent them from localizing to P-bodies disrupt small RNA-independent translational inhibition of target mRNA when the Argonaute proteins are tethered to the 3⬘ UTR of the target mRNA through a bacteriophage RNAbinding protein [132]. Yet, RCK/p54, a putative DEAD box helicase protein and a general repressor of translation found in P-bodies, interacts with hAgo1 and hAgo2, and this interaction as well as miRNA-directed translational repression of target mRNAs is independent of P-body integrity [135]. Thus it remains possible that miRNA-directed translational repression does not require P-bodies per se, but rather that the relocalization of miRNA-repressed mRNAs to P-bodies is a consequence of translation repression [135]. Adding to the plethora of proposed mechanisms for miRNA-directed regulation of mRNA expression, Drosophila, zebrafish, and mammalian miRNAs can also promote mRNA degradation by triggering poly(A) tail shortening and by promoting removal of the 5⬘ cap [124,134,136,137]. A more detailed molecular understanding of all these proposed mechanisms for miRNA function awaits the development of robust in vitro systems that recapitulate both the loading of miRNAs into RISC and the subsequent repression of target mRNA expression by miRNAprogrammed Argonaute protein complexes.

3.7 CONCLUSIONS The impact of RNAi technology on biology and biotechnology cannot be overestimated. The idea that small RNA-directed regulatory pathways eluded detection decades after the discovery of tRNAs, snRNAs, and ribozymes is stunning. Our molecular understanding of the diversity of small silencing RNAs and the pathways that produce, sort, and use them to regulate gene expression continues to grow at a breathtaking pace. Moreover, fundamental advances in the biochemistry of RNA silencing pathways are rapidly being converted into practical technologies for the silencing of gene

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expression in basal eukaryotes, plants, and animals. Finally, the development of siRNAs as therapy for human disease continues to show great promise. We may soon see small RNA drugs in clinical use to improve human health.

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117. Reynolds, A. et al. Induction of the interferon response by siRNA is cell type- and duplex lengthdependent, RNA, 12, 988, 2006. 118. Wightman, B., Ha, I., and Ruvkun, G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans, Cell, 75, 855, 1993. 119. Olsen, P.H., and Ambros, V. The lin-4 regulatory RNA controls developmental timing in Caenorhabditis elegans by blocking LIN-14 protein synthesis after the initiation of translation, Dev. Biol., 216, 671, 1999. 120. Moss, E.G., Lee, R.C., and Ambros, V. The cold shock domain protein LIN-28 controls developmental timing in C. elegans and is regulated by the lin-4 RNA, Cell, 88, 637, 1997. 121. Seggerson, K., Tang, L., and Moss, E.G. Two genetic circuits repress the Caenorhabditis elegans heterochronic gene lin-28 after translation initiation, Dev. Biol., 243, 215, 2002. 122. Bagga, S. et al. Regulation by let-7 and lin-4 miRNAs results in target mRNA degradation, Cell, 122, 553, 2005. 123. Pillai, R.S. et al. Inhibition of translational initiation by let-7 microRNA in human cells, Science, 309, 1573, 2005. 124. Humphreys, D.T. et al. MicroRNAs control translation initiation by inhibiting eukaryotic initiation factor 4E/cap and poly(A) tail function, Proceedings of the National Academy of Science USA, 102, 16961, 2005. 125. Petersen, C.P. et al. Short RNAs repress translation after initiation in mammalian cells, Mol. Cell, 21, 533, 2006. 126. Doench, J.G., Petersen, C.P., and Sharp, P.A. siRNAs can function as miRNAs, Genes Dev., 17, 438, 2003. 127. Doench, J.G., and Sharp, P.A. Specificity of microRNA target selection in translational repression, Genes Dev., 18, 504, 2004. 128. Liu, J. et al. MicroRNA-dependent localization of targeted mRNAs to mammalian P-bodies, Nat. Cell Biol., 7, 719, 2005. 129. Andrei, M.A. et al. A role for eIF4E and eIF4E-transporter in targeting mRNPs to mammalian processing bodies, RNA, 11, 717, 2005. 130. Sheth, U., and Parker, R. Targeting of aberrant mRNAs to cytoplasmic processing bodies, Cell, 125, 1095, 2006. 131. Teixeira, D. et al. Processing bodies require RNA for assembly and contain nontranslating mRNAs, RNA, 11, 371, 2005. 132. Liu, J. et al. A role for the P-body component GW182 in microRNA function, Nat. Cell Biol., 7, 1261, 2005. 133. Jakymiw, A. et al. Disruption of GW bodies impairs mammalian RNA interference, Nat. Cell Biol., 7, 1267, 2005. 134. Behm-Ansmant, I. et al. mRNA degradation by miRNAs and GW182 requires both CCR4:NOT deadenylase and DCP1:DCP2 decapping complexes, Genes Dev., 20, 1885, 2006. 135. Chu, C.Y., and Rana, T.M. Translation repression in human cells by microRNA-induced gene silencing requires RCK/p54, PLoS Biol., 4, e210, 2006. 136. Wu, L., Fan, J., and Belasco, J.G. MicroRNAs direct rapid deadenylation of mRNA, Proceedings of the National Academy of Science USA, 103, 4034, 2006. 137. Giraldez, A.J. et al. Zebrafish MiR-430 promotes deadenylation and clearance of maternal mRNAs, Science, 312, 75, 2006.

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CHAPTER

4

Splice Switching Oligonucleotides as Potential Therapeutics Peter Sazani, Maria A. Graziewicz, and Ryszard Kole

CONTENTS 4.1 4.2 4.3 4.4 4.5

4.6

Introduction.............................................................................................................................90 Pre-mRNA Splicing................................................................................................................90 Alternative Splicing ................................................................................................................92 Splice Switching Oligonucleotides.........................................................................................93 Therapeutic Targets of SSOs ..................................................................................................94 4.5.1 Restoration of Defective Gene Function by SSOs .....................................................94 4.5.1.1
-Globin ......................................................................................................94 4.5.1.2 Dystrophin ...................................................................................................95 4.5.1.3 Cystic Fibrosis Transmembrane Conductance Regulator ...........................95 4.5.1.4 Tau ...............................................................................................................96 4.5.1.5 Lamin A.......................................................................................................96 4.5.1.6 Survival Motor Neurons..............................................................................97 4.5.1.7 OA1 .............................................................................................................97 4.5.2 Modification of Natural Alternative Splicing as a Potential Therapeutic Tool..........98 4.5.2.1 Bcl-x ............................................................................................................98 4.5.2.2 PSMA ..........................................................................................................98 4.5.2.3 WT1.............................................................................................................99 4.5.2.4 CD40 ...........................................................................................................99 4.5.2.5 MyD88 ........................................................................................................99 4.5.2.6 AChE ...........................................................................................................99 4.5.2.7 Polyadenylation Regions...........................................................................100 Other Mechanisms to Alter RNA Splicing...........................................................................100 4.6.1 Hybridization Strategies ...........................................................................................100 4.6.1.1 ESSENCE..................................................................................................100 4.6.1.2 TOSS .........................................................................................................101 4.6.1.3 Trans-Splicing ...........................................................................................101 4.6.1.4 snRNA .......................................................................................................102 4.6.2 Global Strategies.......................................................................................................103 4.6.2.1 RNAi..........................................................................................................103 4.6.2.2 Small Molecules........................................................................................103 89

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4.7 An Assay for Splice Switching Oligonucleotide Potency....................................................104 References ......................................................................................................................................105

4.1 INTRODUCTION Recent studies found that there are ⬃26,000 human genes, 70% of which undergo alternative splicing [1–3]. Thus, alternative splicing allows increased diversity of the proteins encoded by a limited number of primary transcripts. This tightly regulated process leads to splice variants with unique, sometimes opposing, functions, and is a part of normal organism physiology and development [4–6]. It has also been associated with several genetic diseases (
-thalassemia, muscular dystrophy, cystic fibrosis [CF], and others) as well as with several types of cancer [7]; an estimated 15–50% of genetic disorders result from mutations that alter pre-mRNA splicing [8–11]. This chapter reviews antisense-based technologies to manipulate alternative and correct aberrant splicing. This approach may be critical for the treatment of a number of diseases, especially those where targeting by small molecules is not applicable. Antisense oligonucleotides (AONs) are typically applied as targeted downregulators of mRNA translation. This action is achieved by base pairing of specific 2
-deoxyoligonucleotides with the targeted mRNA, a process that elicits destruction of the mRNA by RNase H, an enzyme that catalyzes RNA breakdown in an RNA/DNA duplex [12]. In recent years, the manipulation of splicing has been accomplished with chemically modified AONs that do not activate RNase H, thereby redirecting aberrant or alternative splicing rather than causing destruction of the targeted pre-mRNA, thus upregulating the synthesis of correct and/or therapeutic gene products. Benefits of such approach include possible use as a method to determine function of gene isoforms, positive readout assay for antisense activity, and most importantly, therapeutic potential for genetic diseases and a variety of other disorders that can be addressed by manipulation of alternative splicing of specific genes. In this chapter we will review pertinent literature since 2001, the time of publication of the previous edition of this volume, to present.

4.2 PRE-mRNA SPLICING Most eukaryotic pre-mRNAs contain coding fragments, or exons, and noncoding fragments, or introns, the latter of which must be removed by the splicing machinery before a mature mRNA is formed (Figure 4.1). For a comprehensive review of pre-mRNA splicing, including its alternative processing, refer to the following reviews [8,13,14]. Here, we present only a brief description of the process, highlighting the topics relevant to the use of AONs to manipulate aberrant and alternative splicing. The splicing machinery, or spliceosome, is a complex system based on multiple interactions among specific sequences in the pre-mRNA, small U-rich nuclear RNAs (snRNAs) forming nucleoprotein complexes (e.g., U1–U6 snRNP), heterogeneous nuclear ribonucleoproteins (e.g., polypyrimidine tract-binding protein [PTB/hnRNPI]), and numerous serine–arginine-rich (SR) family proteins [15–17]. The snRNPs are highly involved in the removal of the introns and splicing of the exons. Proteins from hnRNP family contain RNA-binding domains and auxiliary regions that participate in protein–protein interaction, and play a role as splicing repressors [18,19]. SR proteins have the opposing function, and are responsible for spliceosome stabilization and bridging the 3
and 5
splicing sites. At the C-terminus, SR proteins contain SR domains involved in protein–protein interactions and at the N-terminus region there are one or two RNA-binding domains, shown to interact with purine-rich exonic splicing enhancers (ESEs) in several exons [13,20–27]. Serine–arginine domains have also been found in core splicing regulating factors such as U2AF65 (U2 auxiliary factor 65 kDa) and U2AF35 or alternative splicing regulator TRA in Drosophila melanogaster [28–30].

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+

5′ss

E S KAG GURAGU E

(B)

− ISS

YNCURAY

+ ISE

91

3′ss

−

Y AGG

E S S

A

(C)

A Figure 4.1

Schematic representation of pre-mRNA processing. (A) Schematic exon–intron–exon structure. The conserved pre-mRNA sequences are recognized by the splicesome complex. (B) In the first transesterification reaction, the adenosine nucleotide within conserved branch point motif attacks the 5
-most phosphodiester bond at the 5
splice site, resulting in the upstream exon displacement; the intron assumes lariat structure. (C) In the second trans-esterification reaction, the nucleoside at the 3
end of the released exon attacks the 3
end phosphodiester bond of the intron, completely releasing the intronic lariat and forming a new phosphodiester bond between the two neighboring exons. Boxes, exons; line, intron; ESE, exonic splicing enhancer; ISS, intronic splicing silencer; ISE, intronic splicing enhancer; ESS, exonic splicing silencer; , elements enhancing splicing; , sequences suppressing splicing; KAG GURAGU, Y AGG, conserved 5
and 3
splice site motifs, respectively;YNCURAY, branch point sequence.

Chemically, pre-mRNA splicing takes place at exon–intron junctions (5
and 3
splice sites) via two trans-esterification steps. The spliceosome catalyzing this reaction is comprised of up to 100 proteins and 5 snRNAs [16]. The spliceosome component that interacts with pre-mRNA the earliest is U1 snRNA [31,32], which hybridizes with the 5
splice site sequence via base pairing of its nine 5
-most nucleotides. Next, U2 snRNP is bound, via base pairing of its internal region with the branch point sequence positioned 20–40 nucleotides upstream from the 3
splice site. This binding is stabilized by other splicing factors, including U2AF binding to the polypyrimidine tract and the 3
splice site, and splicing factor 1 binding to the branch point. SR proteins bind to ESEs and interact with U2AF, U1 snRNP, and the branch point [29]. Numerous rearrangements of spliceosome then take place, leading to dissociation of U1 and U4 snRNPs, and association of U5 snRNP simultaneously with the 5
and 3
sites. The first trans-esterification reaction, leads to formation of the intron lariat structure and displacement of the upstream exon. In the second reaction, the nucleoside at the 3
end of the released exon interacts with the phosphodiester bond at the 3
end of the intron. The intron lariat is then released and a phosphodiester bond is formed with the downstream exon resulting in spliced mRNA. Each splicing event requires de novo assembly and disassembly of the spliceosome [33]. In addition to the branch point and 5
and 3
splice sites involved in splicing, which are moderately conserved, other cis-acting RNA sequences are present within introns and exons, which have proven equally necessary for accurate and correct processing of pre-mRNA. Sequences termed exonic and intronic splicing enhancers (ESEs and ISEs, respectively) stimulate, while exonic and intronic splicing silencers (ESSs and ISSs, respectively) block inclusion of certain exons. ESEs and ISEs are recognized by SR proteins, whereas ESSs and ISSs are recognized by a complex often containing hnRNPs. One of the most studied ESEs regulates the inclusion of the female-specific exon 4 in the doublesex (dsx) gene of D. melanogaster; the ESE consists of six repeats of a 13 nucleotide

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consensus sequences and a purine-rich element positioned between repeats 5 and 6 [34]. This element binds an SR protein and two splicing regulators, TRA and TRA2 [28]. In human genes, ESE sequences contain mostly purines, however no sequence consensus has yet been described. However, based on knowledge of sequences known to bind SR proteins, an algorithm to predict ESEs (ESEfinder) in transcripts has been developed [35]. Several ESSs have been described. For example, an ESS was found within exon IIIb of fibroblast growth factor receptor 2 (FGFR2); it consists of crucial UAGG sequence, and has been demonstrated to inhibit splicing of IIIb exon from the FGFR2 pre-mRNA [36]. A systematic screen for ESS sequences has been conducted [37,38], and this body of work has led to the hypothesis that ESSs are responsible for the repression of the vast number of pseudoexons, which have relatively strong 5
and 3
splice sites yet are not spliced into the mRNA and remain within intronic sequences. ESS has thus emerged as an important cis-acting element in the regulation of splicing. ISEs were found to be involved in efficient transcript splicing of genes such as
-tropomyosin (ISE motif (A/T)GGG), human GH-1 gene (ISE motif GGX1–4GGG) or human -globin, which contains motif GGGX0–4 GGG [39–41]. One example of ISS comes from studies on FGFR2 gene splicing, where the specific sequence has been demonstrated to regulate the exclusion of exon IIIb from FGFR2 pre-mRNA. The identified ISS sequence consists of a polypyrimidine-rich region that binds PTB [42].

4.3 ALTERNATIVE SPLICING Sequence elements within pre-mRNA that participate in the selection of splice sites include the relative match of the 3
and 5
splice sites and branch points to their respective consensus sequences, some secondary structures, ESEs (for comprehensive review see Refs. [8,43]), and most likely other enhancer and silencer elements discussed above. Experimental introduction of mutations that increase the match between a branch point or a splice site and their corresponding consensus sequences resulted in inclusion of exons that were otherwise skipped [20,44]. Other studies showed that splice site mutations resulting in an exclusion of adjacent exons could also activate cryptic splicing sites [45]. Likewise, manipulation of polypyrimidine track, and introduction or removal of ESEs/ESSs affected splice site choice [46–49]. This data suggest that splice site selection is the result of competition between splice sites and sequence elements for splicing factors during the assembly of spliceosome [50–52]. Observations that the choice of splicing pathway depends on the availability of constitutive splicing factors and the presence/absence of gene-specific splicing factors further support this notion [51,53]. The levels and ratios between splicing factors in different cell types are key in splicing regulation. For example, hnRNP A1 functionally opposes the SR protein SF2/ASF during 5
splice site selection; SF2/ASF enhances proximal 5
splice site selection, while higher hnRNPA1 to SF2/ASF ratio promotes splicing at the distal 5
splice site. This ratio has been demonstrated to vary 100-fold in different tissues [54]. In general, different interaction patterns between the pre-mRNA sequence elements and splicing factors determine tissue-specific expression of majority of the pre-mRNAs [55,56]. Different outcomes of alternative splicing include (but are not limited to) exon skipping, exon inclusion, splicing out or retention of an intron. Retained introns become part of a coding sequence—an exon—whereas a skipped exon becomes part of the spliced-out intron (Figure 4.2). This suggests that a single mechanism is responsible for both outcomes. In addition, splice site selection can be influenced by alternative polyadenylation sites and vice versa. In some cases, transcripts can be initiated at different transcription start sites, which results in the synthesis of mRNA with different first exons. In some cases, for example, splicing of thalassemic
-globin pre-mRNA, even temperature can affect splice site seletion [57]. More in-depth description of alternative splicing machinery and its regulation can be found in these comprehensive reviews [8,43,58].

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(B)

A

B

(i)

Figure 4.2

C

(ii) A

93

B

C

A

(iii) A

C

(i) A

B

C

A A′

B

A′

(ii) B

A

C

Schematic representation of alternative splicing. (A) Alternative exon or intron skipping/inclusion: (i) exon B inclusion; (ii) exon B skipping; (iii) intron retention. (B) Alternative 5
splice site may induce skipping of fragment of an exon: (i) use of 5
proximal splice allows inclusion of the whole exon A; (ii) use of 5
distal splice site results in inclusion of incomplete exon A. Similarly, alternative 3
splice sites can be utilized (not shown). Boxes, exons; solid line, intron.

The proteomic complexity due to alternative splicing prompted development of methods allowing prediction of alternative gene splicing patterns based on alignment of genomic sequences with transcript sequences [59–62]. The technologies utilize user-supplied sequences as well as gene predictions generated by Ensambl, Acembly, Genscan (ASmodeler) [62] or graph-based gene finder EuGENE [61,62]. Among others, mouse, rat, and human genomes can currently be analyzed for potential alternative splicing events using the aforementioned approaches. Another algorithm, ExonScan, uses splice site, branch point, ESE, and ESS scores to predict exon selection [38]. The incorporation of cis-acting sequences into splicing prediction programs has significantly increased their accuracy.

4.4 SPLICE SWITCHING OLIGONUCLEOTIDES The expanding field of modification of the splicing patterns of genes is based on early work by Dominski and Kole [63] in cell-free extracts. In this work, human
-globin pre-mRNAs containing splicing mutations were targeted with antisense 2
-O-methyl (2
OMe) oligonucleotides to restore correct splicing. The mutations,
-110 (in the first intron), and IVS2-705 and IVS2-654 (in the second intron) had been previously identified as leading to
-thalassemia. AONs targeted against the branch point sequence in the first intron (
-110), or aberrant 5
and cryptic 3
splice sites activated by IVS2-705 and IVS2-654 in the second intron, significantly reversed the aberrant and restored correct splicing. These studies demonstrated that the AONs could compete with splicing factors for splicing elements and therefore alter the manner in which pre-mRNA was spliced, restoring the correct function of the defective gene without downregulating its expression. In general, AONs can serve as splice switching oligonucleotides (SSOs) in three ways: (i) by restoring correct splicing of a specific gene, (ii) by modification of natural alternative splicing, shifting the balance towards one type of splicing product, and (iii) by generating an unnatural splice variant. This approach may be used to study the mechanisms of splice site selection. More importantly, because variations in splicing are clearly associated with disease states, SSO-induced modulation of splicing should produce clinically beneficial splice variants. All three SSO mechanisms require that the oligonucleotides do not activate RNA cleavage by RNase H, which would destroy the pre-mRNA before splicing can occur. Such oligonucleotides include 2
OMe, 2
-O-methoxyethyl (MOE), and 2
-O-aminopropyl, phosphoramidate, and methylphosphonate derivatives, as well as locked nucleic acid (LNA) [64], peptide nucleic acid (PNA), and morpholinobased oligomers [65]. They productively compete with the splicing factors for target sequences in pre-mRNA during splicing and, in addition, these modifications improve oligonucleotide target binding.

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4.5 THERAPEUTIC TARGETS OF SSOs Below we present examples of genes where altered splicing is a cause of disease or where modulation of splicing may translate into a therapeutic effect. These clinically relevant outcomes can be achieved by either restoration of defective gene function or modification of natural alternative splicing. 4.5.1

Restoration of Defective Gene Function by SSOs

4.5.1.1
-Globin
-Thalassemia is a genetic blood disorder resulting from defective
-globin gene expression that leads to partial or complete ablation of
-globin, a hemoglobin subunit [66]. The result is a decreased oxygen-transport capacity of red blood cells, expansion of the bone marrow, and fatal iron overload. There are ⬃200 different mutations in the human
-globin gene but the most common ones disrupt splicing of intron 1 and 2 of
-globin pre-mRNA. In the general population the frequency of the thalassemic gene is about 1 in 300, while in those who are of Mediterranean, South East Asian or African ancestry the risk of carrying it is about 1 in 30 [67]. A series of recent reports from this laboratory demonstrated correction of splicing in cells of thalassemia patients with point mutations at nucleotides 654, 705, and 745 of intron 2 using splice switching morpholino oligonucleotides [68–70]; see also selected reviews [71–74]. The mutations create an additional 5
splice site and activate a cryptic 3
splice site, and lead to inclusion of an aberrant exon-like sequence in the spliced
-globin mRNA. Blocking these splice sites with morpholino oligonucleotides resulted in correction of
-globin pre-mRNA splicing, and subsequently, led to hemoglobin upregulation in erythropoietic progenitor cells from IVS2-654 and IVS2-745 forms of thalassemia, as well as in erythroid cells from the mouse model of IVS2-654 thalassemia. Relatively high doses (45 M) of morpholino oligomer, used for effective splicing correction and
-globin upregulation in human erythropoietic progenitor cells, did not affect sequence specificity and seemed to have little toxicity. Another
-globin mutation, HbE, was also successfully targeted by this approach. Note that only morpholino oligonucleotides were taken up and effective in splicing modulation in erythroid progenitor cells; the other chemistries discussed in the previous section were not. Oligonucleotide delivery with cationic lipids was unsuccessful; these cells seem particularly sensitive to cationic lipid toxicity (data not shown). In vivo experiments in IVS2-654 thalassemia mouse model showed that morpholino oligonucleotides conjugated with Tat-like peptides were effective in systemic delivery by i.v. injection. Thirteen-day treatment restored ⬃10% of human hemoglobin expression. This resulted in visible improvement of red blood cell morphology (Figure 4.3) (Svasti and Kole, unpublished). This is a promising result since no more than 20% levels of hemoglobin are needed to eliminate or drastically reduce blood transfusions, the current treatment for thalassemia patients.

M-tat D-0

M-tat D-13

Normal

Figure 4.3 Red blood cell morphology is improved in thalassemic IVS2-654 mice treated with M-tat oligonucleotide inducing splicing repair of IVS2-654
-globin pre-mRNA. Wright–Giemsa staining of blood smears.

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4.5.1.2 Dystrophin Mutations that result in the formation of stop codons in the coding exons of dystrophin gene are the main causes of Duchenne muscular dystrophy (DMD) [75]. A similar, but milder form of the disease, Becker muscular dystrophy (BMD), is associated with deletions that maintain the reading frame and as a result produce shorter, partially functional variants of dystrophin protein. It was hypothesized that skipping the mutated exon would remove the stop codon mutation and restore the reading frame, allowing for at least partial recovery of dystrophin function. This hypothesis was proven correct, in a series of papers showing that in cultured cells derived from the mdx dystrophic mouse, SSO targeted to the splice sites of the mutated exon 23 caused exon skipping and restoration of the reading frame [76–80]. Similar results were shown in cells derived from a canine model of muscular dystrophy [81]. Local administration of SSOs in vivo was also able to restore the mdx reading frame in mice. Mann et al. [78] demonstrated that 2
OMe oligonucleotides complexed with cationic lipids targeted to the 5
splice site of the exon 23 of mdx caused exon skipping and the production of revertantmuscle fibers when locally injected into the muscle tissue mouse. Similar results were obtained with 2
-O-methyl oligonucleotides delivered to muscle tissue by electroporation [82]. Recently, morpholino oligomers were delivered locally into muscle by hybridizing to a DNA oligonucleotide and complexing with a cationic lipid. This treatment led to significant increases in functionaldystrophin, as detected by Western blot and immunostaining [83]. Impressively, the delivery of 2
-O-methyl oligonucleotides with the aid of the F127 block copolymer by local injection into the muscle induced a shift in splicing that produced functional amounts of dystrophin [84]. Treated mice exhibited the ability to exert more force with their contralateral muscles. The combination of the oligonucleotide and F127 was nonimmunogenic. More recently, systemic delivery of SSOs in the mdx mouse has been accomplished, using the same F127/2
-O-methyl formulation injected intravenously, as either a single or three weekly very high doses of ⬃100 mg/kg of the oligonucleotide [85]. Following the treatment, mdx mice showed evidence of dystrophin-containing muscle fibers at multiple sites, including the quadriceps, biceps, diaphragm, abdomen but not heart. These fibers were still detectable 6 weeks after a single administration. No toxicity was detected as a result of treatment. Similar impressive restoration of function in the quadriceps and other muscles was obtained using systemically delivered morpholino dystrophin SSOs at 100 mg/kg [86]. Cultures from different human muscular dystrophy patients, containing several known diseasecausing mutations, were also successfully targeted with SSOs to restore gene function [87–93]. A “humanized” mouse model of muscular dystrophy has also been developed and has been used to confirm the activity of human-targeted SSOs in vivo [94]. Together, this body of preclinical work demonstrates the power and feasibility of SSOs to treat muscular dystrophy. Both the toxicity profile and the delivery method are compatible with human drug treatment. Clinical trials will determine the extent of the benefit of SSOs in human muscular dystrophy patients. Peculiarly, Takeshima et al. [95,96] reported that an oligodeoxynucleotide, capable of activating RNaseH, induced skipping of dystrophin exon 20 from tissues of a patient with a mutation in exon 19. They also showed efficacy after i.v. infusion of the oligonucleotide into the patient, the first clinical demonstration of SSOs. The efficacy of the oligodeoxynucleotide is puzzling, since RNaseH should destroy the premRNA before splicing. In fact, dystrophin does appear to be downregulated in treated samples.

4.5.1.3 Cystic Fibrosis Transmembrane Conductance Regulator CF is a disorder resulting from loss of function of the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes a cyclic adenosine monophosphate (cAMP)-regulated chloride channel. CF is characterized by severe pulmonary and pancreatic disease, male infertility associated with congenital bilateral absence of vas deferens (CBAVD), and high electrolyte concentration in sweat [97].

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The most common and severe mutation,
F508, prevents the protein from being localized in apical plasma membrane. Spliceosome-mediated RNA trans-splicing (SMaRT) with recombinant adenoviral vectors that expressed pretrans-splicing molecules (PTMs) was demonstrated to partially restore CFTR chloride channel activity in polarized human
F508 CF airway epithelial cells, but the application of the adenovirus proved to be of limited efficiency [98]. More recently, recombinant adenoassociated vectors carrying PTM that would bind to intron 9 of CFTR pre-mRNA and trans-splice the correct sequence of human CFTR exons 10–24 into the endogenous pre-mRNA were tested, providing conductance restoration to ⬃14% of that of normal human airway epithelia [99]. The SMaRT technology will be discussed in more detail further in this chapter. Milder cases of CF are characterized by late onset and/or less severe pulmonary complications, and CBVAD. Less than 2% of all CF cases are associated with a mutation 3849  10 kb C to T in CFTR intron 19. The mutation has an increased prevalence in patients with mild form of CF as well as in Ashkenazi Jews [100]. It generates an aberrant 5
splice site and activates a cryptic 3
splice site. Friedman et al. [101] reported that three cell lines, 3T3 mouse fibroblasts, C127 mouse epithelial cells, and CFT human tracheal cells, all carrying CFTR with this mutation, when treated with antisense 2
-O-methyl oligonucleotides that target either aberrant donor or acceptor splice site or to the included exon-like fragment, showed dosedependent increase in CFTR protein production. Delivery of both oligonucleotides had additive effect. More recent approach demonstrated that increase in expression of two splicing factors, Htra2-beta1 and SC35, increased the level of correct CFTR mRNA and resulted in the production of functionally correct CFTR channel in CF-derived epithelial cells carrying the 3849  10 kb C to T mutation [102].

4.5.1.4 Tau Tau is a neuron-specific microtubule-associated protein and a key element of neuron integrity and axonal transport, regulating microtubule stability and dynamics [103–105]. The human tau gene contains 16 exons and encodes six splice variants. Alternative splicing of exons 2, 3, and 10 results in tau protein containing 3 (Tau3R) or 4 (Tau4R) microtubule-binding repeats [105–107]. In fetal brain, Tau3R is a dominant form while later in the postnatal period Tau4R levels increase to balance Tau3R; in normal adult human brain Tau3R/Tau4R ratio is ⬃1 [107]. Several neurodegenerative diseases, termed tauopathies, which result from imbalance in the Tau3R/Tau4R ratio, are associated with the presence of profuse filamentous deposits containing hyperphosphorylated tau in neurons and glia of affected individuals. Tauopathies include Down’s syndrome (DS), Pick’s disease (PiD), some variants of prion diseases, Alzheimer’s disease (AD), progressive supranuclear palsy (PSP), and frontotemporal dementias. Point mutations in exon 10 and the following intron of the tau gene are associated with tauopathy described as frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17), with symptoms similar to those of AD [108]. In normal human neurons exon 10 of the tau gene is skipped, due to the fact that a hairpin structure within pre-mRNA masks its 5
splice site and prevents the efficient binding of U1 snRNP. The mutations disrupt the hairpin, leading to inclusion of exon 10 into mature tau mRNA. Kalbfuss et al. [109] used two rat cell lines, neuronal phoechromocytoma PC12 cells and AR42J pancreatic acinar cells, expressing tau protein that included exon 10, as models for oligonucleotide treatment of FTDP-17. 2
-O-methyl-phosphorothioate oligonucleotides, targeted to either the 3
or 5
splice site of tau exon 10, restored exon 10 skipping in a dose-dependent manner. In addition, in AR42J cells, the decrease in the level of exon 10-containing variant, which includes a microtubule-binding element, led to rearrangement of the cytoskeletal structure, a phenomenon that can reflect a potentially clinically beneficial outcome.

4.5.1.5 Lamin A Lamins are proteins that polymerize to form a meshwork of filaments localized predominantly on the inner surface of the inner nuclear membrane. In humans, two genes encoding lamins B1 and B2 are expressed constitutively in somatic cells; in embryonic cells alternative splicing of B2 gene

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leads to synthesis of B3 [110]. One gene (LMNA) [110,111] codes for A-type lamin splice variants, lamin C, A, and A
10 that differ in the splicing of exon 10; their expression is regulated during the development process [112–117]. Lamin C contains the complete 111 nucleotide sequence of exon 10, whereas lamin A contains only the 5
90 bases of this exon through the use of alternative 5
splice site, and lamin A
10 lacks exon 10 altogether [118]. Mutations in LMNA are associated with a number of genetic diseases, such as cardiomyopathies with involvement of variable skeletal muscles, peripheral neuropathy, premature ageing syndromes or partial lipodystrophy disorders. The majority of patients suffering from Hutchinson-Gilford progeria syndrome (HGPS), a form of the premature ageing, are carriers of heterozygous silent substitution at codon 608 localized in exon 11 of the LMNA gene [119,120]. The mutation activates an exonic cryptic donor splice site positioned four nucleotides upstream. The resulting pre-mRNA is spliced at this site, producing LMNA mRNA that lacks 150 nucleotides at the end of exon 11. Thus the mutant lamin A protein carries an internal deletion of 50 amino acids. Observed consequences involve morphological changes of nuclei in HGPS cell lines [119–122], up to sixfold reduction in cellular levels of lamin B, another major element of the lamina meshwork, as well as other lamina-associated polypeptides (LAP2s) [122]. Removal of the truncated lamin A from the cells is a key to successful rescuing of the ageing phenotype in HGPS fibroblasts. Scaffidi and Misteli [122] have reported that while introduction of a wild-type lamin A did not correct cellular symptoms, premature ageing phenotypes of HGPS fibroblasts were efficiently suppressed by splicing correction with a morpholino oligonucleotide targeted to the aberrant 5
splice site. This approach not only allows for synthesis of normal lamin A, but also eliminates or significantly diminishes synthesis of its mutated form.

4.5.1.6 Survival Motor Neurons Spinal muscular atrophy (SMA), an autosomal recessive neurodegenerative disorder associated with the selective destruction of spinal cord -motor neurons, is a leading cause of infant mortality from genetic disorders. SMA results from mutation or deletion of the survival-of-motor-neuron gene, SMN1. A nearly identical SMN1 paralog, SMN2, expresses only limited amounts of functional protein. It retains a correct reading frame but carries translationally silent, single oligonucleotide transition at position 6 of exon 7, which leads to exon 7 skipping [123–125] and production of truncated, unstable protein [124]. In their recent report, Cartegni et al. [126] suggest that skipping of exon 7 in SMN2 pre-mRNA splicing occurs via the loss of the SF2/ASF-dependent ESE. It has been reported that both, in patients suffering from SMA and in mouse models for the disease, increase in expression of SMN2, observed when multiple copies of SMN2 are present, can diminish SMA symptoms [127–130]. Some of the efforts to restore synthesis of the full-length active isoform of survival motor neuron (SMN) protein have been directed towards developing drugs that would encourage SMN2 exon 7 inclusion into its mature mRNA. It was suggested that blocking of exon 8 3
splice site should shift the balance towards exon 7 retention. This was achieved by designing 2
-O-methyl oligonucleotides that targeted the 3
splice site in cell lines constitutively expressing SMN2 minigene constructs [131]. Another group was successful in achieving the same goal by utilizing 2
-O-methyl oligonucleotides complementary to ISSs and ISEs positioned, respectively, upstream and downstream from the exon 7 [132–134].

4.5.1.7 OA1 Ocular albinism type 1 is the most common cause of ocular albinism. It is associated with mutations of the OA1 gene located on chromosome X. Recently, in a family with the X-linked type of ocular albinism, a novel point mutation has been identified within intron 7 of the gene that creates a new acceptor splice site [135]. In affected patients, levels of correct OA1 mRNA were low and the alternatively spliced variant of mRNA, containing a 165 bp fragment of intron 7 retained between exons 7 and 8, was also detected. The new transcript contained a premature stop codon and

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was unstable [135]. The identified mutation was a G  A substitution which created a consensusbinding motif for ASF/SF2. In addition, it activated a cryptic donor splice site leading to the inclusion of intron 7 fragment between exons 7 and 8. The authors demonstrated that treatment of the patient’s melanocytes with antisense morpholino oligonucleotide complementary to the mutationcontaining gene sequence induced skipping of the intronic inclusion, rescuing expression of OA1 gene and protein [135]. 4.5.2

Modification of Natural Alternative Splicing as a Potential Therapeutic Tool

4.5.2.1 Bcl-x Bcl-x is a member of the bcl-2 family of genes that regulates apoptosis. Utilizing two alternative 5
splice sites, the gene undergoes alternative splicing that generates transcripts encoding two protein isoforms characterized by antagonistic functions: the longer, antiapoptotic bcl-xL and shorter, proapoptotic bcl-xS [136]. Several cancers are associated with bcl-xL overexpression, including hematomas, neuroblastomas, myelomas, lymphomas, prostate, and breast cancers [137,138]. This phenomenon is linked to resistance to chemotherapeutics, apoptosis inhibition, and poor clinical prognosis. Overexpression of bcl-xS, in contrast, was demonstrated to induce apoptosis in cancer cell lines. Therefore, blocking the xL (proximal) 5
splice can lead to an increase in bcl-xS/bcl-xL ratio, thereby inhibiting antiapoptotic signals while at the same time enhancing proapoptotic message; the result is potential growth inhibition or death of tumor cells. Several reports show the anticancer value of bcl-xL mRNA downregulation, achieved via AONs that promote its RNase H-mediated cleavage [139–144]. However, simultaneous upregulation of the proapoptotic bcl-xS variant by bcl-x SSO was found more potent, particularly in PC3 cells, than standard AONs at inducing apoptosis [145]. In other cell lines, including A549 and MCF7, oligonucleotides targeted to the downstream alternative 5
splice site in bcl-x exon 2 led to decreased bcl-xL expression, increased bcl-xS expression, and sensitized cell to death by chemotherapeutic agents or UV radiation [139,145,146]. Therefore, upon successful delivery to tumor cells, bcl-x SSOs may be a powerful drug against cancers that are normally refractory to treatment. Overexpression of the bcl-xL mRNA and protein does not only apply to cancer. It has been reported in psoriasis, where it has been linked to lack of apoptosis in psoriasis plaques [147–149]. Shifting splicing of bcl-xL pre-mRNA in psoriasis-affected individuals can be beneficial, especially considering that it would not be necessary to eradicate all affected cells to achieve significant improvement in patients. It may also potentially increase patient response to standard apoptosisinducing treatments, such as methotrexate or UV radiation.

4.5.2.2 PSMA Prostate-specific membrane antigen (PSMA), encoded by the folate hydrolase (FOLH1) gene, is a largely extracellular membrane-anchored protein that is highly expressed in cancerous prostate tissue and nonprostatic tumor neovasculature. Recently, the treatment of LNCap cells with SSOs shifted splicing of FOLH1 pre-mRNA from the full-length PSMA to three different splice variants: the cytoplasmic PSM
alternatively spliced at exon 1 and two other isoforms, PSMA6 and PSMA18, which lack exons 6 and 18, respectively [150]. Decrease in membrane PSMA levels was observed, accompanied by increased levels of PSM
, PSM6, and PSM18 mRNAs. While the PSM
isoform was demonstrated to retain enzymatic activity, two other splice variants were inactive. The function of PSMA in prostate cancer is currently unclear, but it may act as a transport or signaling protein, due to the presence of cytoplasmic MXXXL internalization motif [151,152], and demonstrated the ability to internalize in response to ligand binding; it is possible that its splice variants, PSMA6 and PSMA18, expected to retain that motif, could compete with and disrupt normal PSMA signaling/transporting pathway.

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4.5.2.3 WT1 Wilms’ tumor suppressor (WT1) gene plays key role in mammalian urogenital development. Dysregulation of this gene has been associated with many human cancers, including kidney malignancy in children (Wilms’ tumor) [153], leukemias [154], and breast cancer [155]. WT1 is a transcription factor that activates transcription of specific genes and inhibits transcription of certain genes that are upregulated in patients with Wilms’ tumor. WT1 pre-mRNA can be alternatively spliced at two regions, so that mature mRNA can include/exclude either a region coding for three amino acids—KTS—at the end of exon 9 (WT1  KTS) [156,157] or exon 5 [158]. The splice variant containing exon 5 is elevated in Wilms’ tumor and leukemia [158]. Specific targeting of WT1 transcripts containing exon 5 with antisense oligonucleotide led to the death of HL60 leukemia cells, which suggests that disruption of exon 5 splicing may serve as a proapoptotic signal [159]. In addition, in HL60 cells gene expression profiles, analyzed after WT1 exon 5-targeted antisense oligonucleotide treatment, revealed upregulation of thrombospondin 1, a known WT1 target, which correlated well with cell death [159].

4.5.2.4 CD40 CD40 is a cell membrane protein that together with its ligand, CD154, plays a key role in the initiation and propagation of immune responses. The CD40–CD154 signaling pathway affects immune responses in cellular and animal model systems and inhibition of CD40 function may lead to some therapeutic benefits in inflammatory and autoimmune diseases [160]. Expression of cell surface-associated CD40 protein can be downregulated by a PNA oligomer that binds to the 5
splice site of exon 6 in CD40 pre-mRNA [161]. The PNA alters constitutive splicing which results in accumulation of CD40 transcript lacking exon 6 and, as a consequence CD40 protein without the transmembrane domain. Decrease in membrane-bound CD40 led to the inhibition of CD40-dependent induction of IL-12 in murine macrophages [161]. These data suggest that PNA oligomers may act as efficient inhibitors of membrane-bound CD40, therefore allowing for modulation of immune response.

4.5.2.5 MyD88 Another example of potential modulation of proinflammatory stimuli by splicing alteration comes from studies on systemic delivery in mice of MOE SSO targeted to MyD88 pre-mRNA. [162]. MyD88 is an adaptor protein involved in proinflammatory cytokines signaling pathway leading to phosphorylation of IL-1R-associated kinase (IRAK-1) and activation of the NF-B transcription factor [163,164]. SSO targeted to the 5
splice site of exon 2 increased the ratio of MyD88 to MyD88(S)—a variant that lacks the ability to couple IRAK-1 to NF-B in cell culture and in livers of treated mice. Reduction of proinflammatory signaling via IL-1R pathway was detected [162].

4.5.2.6 AChE Alternatively spliced variants of the acetylcholine-hydrolyzing enzyme acetylcholinesterase (AChE) have been implicated in the progression of such neurodegenerative diseases such as Parkinson’s, Alzheimer’s, and neuromuscular disorders such as myasthenia gravis (recently reviewed in Ref. [165]). Alternative splicing of the AChE gene results in three species of AChE mRNA: synaptic AChE-S, joining exon 4 and 6, the principal multimeric enzyme in normal brain and muscle; soluble, monomeric readthrough AChE-R that retains intron 4 and exon 5, present in embryonic and tumor cells and induced under psychological, chemical, and physical stress; and glypiated dimers of erythrocytic AChE-E, associated with red blood cell membranes, in which exons 4 and 5 are joined directly (reviewed in Ref. [166]). Antisense-mediated degradation of AChE

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mRNA has been demonstrated to prevent AChE-R overexpression and to lessen the symptoms in rat model of myasthenia gravis [167], a closed-head injury model in mice [168], and in mice with enhanced stress-induced memory impairments and fear [169].

4.5.2.7 Polyadenylation Regions It has been demonstrated that AONs can be used to increase abundance of certain mRNAs, and therefore levels of certain proteins, via modifying polyadenylation patterns and thus limiting message degradation [170]. Alternative polyadenylation sequences, often more than two, have been discovered at the 3
end of pre-mRNAs of multiple genes [171]. These pre-mRNAs often contain destabilization sequences that are responsible for mRNA degradation [172]. Hybridization of specific 2
-O-MOE phosphorothioate oligonucleotides to the 3
-most polyadenylation region of E-selectin blocked polyadenylation at this site and redirected it to upstream cryptic sites. The effect was that shorter transcripts contained fewer destabilization regions, which led to increased mRNA stability and altered protein expression [170].

4.6 OTHER MECHANISMS TO ALTER RNA SPLICING As highlighted above, one can potentially manipulate alternative splicing through the use of a target-optimized, hybridizing, and RNase H-inactive oligonucleotide. This strategy is extremely useful for inducing the skipping of one or several coding exons, for controlling the preferential use of alternative 3
or 5
splice sites, and, as will be discussed below, for enhancing exon inclusion. These alternatives comprise the majority of observed splicing events. SSOs therefore represent the simplest and generally most effective splice modulation approach. However, other hybridizationbased alternative splicing techniques have also been developed. These technologies further expand the functionality of SSOs, adapt the technology as gene therapy, and suggest the possibility for global control of alternative splicing with small molecules. 4.6.1

Hybridization Strategies

4.6.1.1 ESSENCE Exon-specific splicing enhancement by small chimeric effectors (ESSENCE) is a PNA oligonucleotide-based approach designed to enhance the inclusion of the targeted, alternatively or aberrantly, spliced exon into the spliced mRNA. The technology is based on manipulating the function of ESEs [173]. An ESSENCE PNA oligonucleotide contains a covalently bound peptide SR domain at the 3
end, designed to mimic SR proteins known to bind ESEs. This oligonucleotide bound internally to the exon targeted for inclusion is expected to promote SR-protein-mediated exon definition and enhance exon inclusion into mature mRNA. Cartegni and Krainer [174] demonstrated the effectiveness of this approach on both BRCA1 and SMN2 pre-mRNA. Correct splicing of BRCA1 exon 18 requires recognition of an intact ESE by the SR protein ASF/SF2. In a mutation found to be prevalent among patients with ovarian cancer [175], the ESE is inactivated by a point mutation and exon 18 is skipped, resulting in improper translation and inactivation of the gene product. ESSENCE PNA oligonucleotides targeted near the mutated ESE restored exon 18 inclusion in mRNA in a sequence- and dose-dependent fashion. Likewise, targeting exon 7 in nonfunctional SMN2 pre-mRNA with ESSENCE oligonucleotides led to enhancement of its inclusion in mRNA. As mentioned above, activation of SMN2 is a potential therapy for SMA. Although in these examples the SR domain enhanced the ability of the oligonucleotide to force inclusion of the targeted exon, it did not appear absolutely essential for activity [174]. For example, a PNA oligomer of identical sequence targeted to BRCA1 but lacking a 3
SR tail was able to induce

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50–55% exon 18 inclusion compared to 25% for the scrambled sequence control and 75% for the optimized ESSENCE oligonucleotide. It seems that an oligonucleotide alone targeted to a putative exon may increase its inclusion, though the mechanism of this action is unclear. It is possible that the oligonucleotide is competing with splicing factors such as hnRNP A1 [176] for binding of other elements, such as ESSs, that serve to decrease exon inclusion. If so, this may indicate that simple oligonucleotides, depending on whether targeted to enhancer or silencer sequences within the exon, may achieve opposite effects, exon skipping or exon inclusion. ESSENCE was also applied to alternative splicing of bcl-x. An antisense 12-mer PNA with an RS domain tail at its “3
” end, was targeted to a region upstream of the distal (xS) 5
splice site of exon 2 [177]. The antisense portion was targeted such that it did not bind at any ESE or ESS sites. Therefore, in contrast to the previous ESSENCE examples, the PNA alone did not influence alternative splicing, but addition of the RS tail enabled induction of splice switching to the xS form, where the ratio of xLxS fell from 11.4 to 4.9 in HeLa cells. These data definitively demonstrate that RS conjugates can influence alternative splicing by recruiting splicing factors to underused splice sites. Interestingly, the cell culture results were obtained by delivery of the PNAs essentially by free uptake at high concentrations (up to 50 M). The use of a better cell culture delivery method for neutral or positively charged oligomers such as scrape loading [178] may result in more efficient splice shifting. In a similar approach, Skordis and colleagues [179] used a tail containing an ESE to increase the inclusion of SMN2 exon 7. Briefly, an oligonucleotide targeted internally to exon 7 was extended with a 5
tail containing an ASFSF2-binding site. The tail recruited the SR protein andenhanced exon 7 inclusion in a sequence- and dose-dependent manner. This approach differs from ESSENCE in that the SR protein functionality is built directly into the oligonucleotide in the latter.

4.6.1.2 TOSS Targeted oligonucleotide silencers of splicing (TOSS) are AONs with overhanging 3
or 5
tails designed to recruit factors that inhibit selection of a targeted splice site. These oligonucleotides repress splicing of a targeted exon either by recruiting hnRNP A1, which represses splice site recognition, or by competing for binding of other critical splicing factors at the target site. In the first iteration of TOSS [180], AONs were designed upstream of the preferentially used proximal, -xL 5
splice site of bcl-x such that, alone, it did not cause an increase in distal 5
splice site utilization. The antisense oligonucleotide induced splice switching when a 20-mer 5
tail consisting of the consensus-binding sequence for hnRNP A1, which recognizes and binds to ESSs [37,38], was added. Cationic lipid-mediated delivery of this TOSS resulted in a dose-dependent increase in the bcl-xS/bcl-xL isoform ratio, while the same oligonucleotide without the 5
tail did not. Furthermore, depletion of hnRNP A1 by siRNA reduced the efficacy of the TOSS, suggesting that it acts by recruiting hnRNP A1, repressing the proximal splice site, and forcing selection of the distal site. The action of TOSS targeted to bcl-x was increased through the use of other linear and branched 5
and 3
tails [181]. A 5
tail containing the
-globin branch site had splice switching activity greater than tailless or poly U-tail-containing oligonucleotides, though only a fraction as potent as the hnRNP TOSS. Furthermore, placement of the same tail at the 3
end, or with the tail extended to include the 5
splice site did not increase activity relative to the hnRNP TOSS. Interestingly, the use of TOSS with 3
branched tails that mimic the intron lariat intermediate had splice switching activity similar to the hnRNP TOSS. Functional studies demonstrated that the branched tail TOSS acts by recruiting U1 snRNP.

4.6.1.3 Trans-Splicing The oligonucleotide- or vector-based use of trans-splicing mechanisms for gene modulation/ repair is also referred to as SMaRT. This technology exploits the ability of the spliceosome to splice

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together two exons from different transcripts. The phenomenon of trans-splicing was originally described in lower organisms, such as trypanosomes, but was found to be present in higher organisms including mammals (see Ref. [182] for a thorough review). Trans-splicing is very similar to cissplicing (see above), in that a branched intermediate is formed as a result of the first transesterification reaction. However, trans-splicing produces a Y-shaped intermediate instead of a lariat, because each exon to be ligated is free and not connected by a single intron. The second transesterification proceeds in a manner very similar to cis-splicing, with the corresponding release of the Y-shaped intermediate. Thus exons from related or unrelated primary transcripts can be joined to form a hybrid mature mRNA. To induce trans-splicing for the purpose of gene modulation/repair [183], an RNA called PTM [184] is introduced into the site of pre-mRNA splicing (e.g., nuclear extracts, the nucleus of live cells, etc.). The PTM consists of the 3
portion of an intron—with an intact branch point, poly-Y tract, and 3
splice site—and a single “exon” immediately downstream. The “exon” may be a natural exon, or a fragment of cDNA comprising of several exons spliced together, as long as the 3
splice site consensus sequence is maintained. Additionally, the PTM contains at its 5
end a sequence complementary to the branch point of the intron targeted for trans-splicing. Thus, for a given target pre-mRNA, the PTM masks the natural branch point of a targeted intron, and increases the probability of a trans-esterification reaction between the 5
ss of the targeted intron and the branch point on the PTM. In the next step, the second trans-esterification ligates the upstream exon of the target pre-mRNA to the exon of the PTM. The result is a hybrid mRNA, with native coding sequence at the 5
end and coding sequence from the PTM at the 3
end. There are several situations, where invoking trans-splicing may be the most practical means of gene modulation/repair. A striking example is the CFTR gene, whose gene spans ⬃200 kb, with a cDNA of ⬃4 kb. The size of CFTR gene or even cDNA makes its replacement via viral vectors difficult. Mansfield et al. [185] transfected 293T cells with a CFTR
F508 minigene containing the mutant codon in exon 10 and the natural intron 9. These cells produced aberrant CFTR. When cotransfected with a PTM targeted to intron 9 and containing the remaining correct CFTR exons 10–24, a detectable level of trans-splicing occurred at an estimated rate of 12%, and normal CFTR protein was produced. In a follow-up study, endogenous
F508 CFTR was corrected by transsplicing in CF bronchial xenografts in mice, at a rate of 16% [98]. More recently, vectors (rAAV) were used to deliver CFTR
F508 PTMs to polarized airway epithelia [99], the goal of providing a permanent therapy for CF. Further studies are needed to determine the feasibility of this approach. Other genes targeted with PTMs include collagen XVII [186,187], tau [188], CD40 Ligand [189], and most notably, coagulation factor VIII [190]. In the latter, a mouse model of FVIII deficiency was used in which exon 16 contained an insertion of neomycin resistance, preventing proper translation of the protein. Repair of this defective FVIII required a PTM targeted to intron 15 and contained FVIII exons 16–26. Delivery of the PTM construct, either delivered and expressed directly from a plasmid or expressed via an adenoviral vector, induced FVIII production by the liver. Though the effects were transient, treatment resulted in increased FVIII activity and coagulation. These results are striking, considering that the investigators estimate that only 2–6% of mutant transcripts are being corrected by trans-splicing. Thus, FVIII represents a potentially excellent target for SMaRT technology.

4.6.1.4 snRNA U7 snRNA is a nonspliceosomal snRNA, which participates exclusively in histone mRNA processing (see Ref. [191] for review) via hybridization to the 3
region of histone pre-mRNA region. As an expressed nuclear RNA with a natural antisense binding sequence, U7 snRNA was altered for inducing splice modulation of targeted pre-mRNA by removing the antihistone pre-mRNA sequence and replacing it with the sequence of interest. This has been done successfully with several genes. Furthermore, since the modified U7 snRNAs can be expressed, driven by an RNA polymerase III promoter, they offer a potentially stable source of splice switching molecules.

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This approach was investigated as a potential treatment for thalassemia. The same antisense sequence used in SSOs [192] was substituted into U7 at the antihistone pre-mRNA-binding site [193]. Stable transfection of the modified U7 snRNA into HeLa cells expressing the mutant
-globin gene partially but permanently restored correct splicing and protein expression. Similar results were also obtained using combinations of modified U7 [194] or a modified U1 snRNA [195]. More recently, modified U7 snRNAs in a lentiviral vector were delivered to human thalassemic bone marrow ex vivo, and restoration of
-globin expression was detected. These studies point the way to a possible cure for
-thalassemia, if a vector targeting blood progenitor cells can be isolated [196]. Dystrophin was also targeted with modified U7 constructs. In C2C12 cells and immortalized muscle cells from an mdx muscular dystrophy mouse model, modified U7 snRNAs were shown to induce skipping of mutant exons that harbor premature stop codons, thus restoring the reading frame and generating a shorter but more functional dystrophin protein. Impressively, modified U7 snRNAs delivered to the mdx mouse via an AAV vector showed long-term upregulation of functional dystrophin after a single administration [197]. Modified U1 constructs are now being evaluated for the same purpose [198]. Other targets have been targeted with moderate success including SMN [199] and human immunodeficiency virus (HIV) [200]. 4.6.2

Global Strategies

4.6.2.1 RNAi Small interfering RNA (siRNA) can be used to target splicing factors and thereby affect alternative splicing. For example, Kashima and Manley [201] show that RNAi targeted to hnRNP can alter splicing of exon 7 of SMN2. According to their model, hnRNP binds to a novel ESS that prevents Tra2 from binding its cognate ESE, and prevents U2AF interaction with the 3
splice site adjacent to exon 7. By reducing hnRNP with the appropriate siRNA, Tra2 binding can take place and exon 7 inclusion is increased. This approach may have therapeutic implications in the treatment of SMA [202]. Massiello and colleagues [203,204] investigated a cis-acting ceramide response element (CRCE1) located in the alternatively spliced region of exon 2 of bcl-x and which binds the splicing factor SAP155. In its normal phosphorylated state, SAP155 promotes usage of the xL splice site, while the addition of ceramide to cell-free splicing extracts causes SAP155 to be altered by an unknown mechanism, and the xS splice site becomes more favored. Downregulation of SAP155 by RNAi in A549 cells had the same, albeit modest effect. siRNA targeted towards splicing factor SC35 was recently used to restore correct splicing in human fibroblasts derived from a patient with pyruvate dehydrogenase (PDH) complex deficiency, which causes mental retardation [205]. The patient had a mutation that increased SC35 binding to an intron of PDH pre-mRNA, causing activation of a cryptic 5
splice site. Titration of SC35 by siRNA almost completely abrogated the use of the cryptic site and significantly increased the quantity of properly spliced PDH mRNA and protein. As with other global strategies, extensive evaluation of possible off-target effects needs to be examined. It will be extremely important in the development of RNAi or small molecules as splice modulators to establish the consequences of altering a general splicing factor such as ASF/SF2, hnRNP A1, SAP155 or SC35 on the expression of other genes.

4.6.2.2 Small Molecules The spliceosome is a large conglomerate of proteins and RNAs that must assemble uniquely to each site of pre-mRNA splicing. It is reasonable to assume, therefore, that small molecules can alter the assembly such that alternative splice site selection is altered. This was found to be the case for dimethyl sulfoxide (DMSO), which is often used to induce differentiation in certain tumor cell

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lines, or simply as a diluent for compounds not easily dissolved in water. In the study, DMSO was shown to modulate alternative splicing of the neurospecific, NCAM, via an effect on the activity of hnRNP A1 [206]. Small molecules can be used to modulate other proteins involved in alternative splicing. Muraki and colleagues recently screened for and found a small molecule inhibitor of Clk (cdc2-like kinase), a protein kinase that phosphorylates SR proteins, including ASFSF2. As mentioned above, the phosphorylation state of SR proteins regulates its influence on alternative splicing. In cell-free extracts, TG003 (a benzothiozole compound) inhibited kinase activity of Clk1 and prevented splicing of
-globin pre-mRNA, with a Ki of 10 nM. In vivo, in Xenopus embryos, TG003 prevented developmental errors that arise from overexpression of Clk. This data suggest that small molecules targeting SR proteins may be used to treat disease. However, the data also show that alternative splicing of Clk1 and SC35 are also altered by TG003. Since Clk1 and SC35 themselves can influence splicing, it is unclear if this has a feedback effect, or if it is simply a side effect. More studies are required to determine the global impact of this inhibitor on splicing. Soret and colleagues [207] discovered a group of indole compounds that specifically bind to and inhibit ASFSF2 in cell-free splicing reactions. In addition to having good affinity for intact but not denatured or truncated ASFSF2 (Kd  200–400 nM), some compounds inhibited ASFSF2-dependent splicing in a dose-dependent manner, while affecting ASFSF2-independent splicing constructs only at very high concentrations. These compounds also abrogated aberrant and restored correct splicing of chimeric
-globin pre-mRNA constructs. Interestingly, splicing steps required for HIV disease progression are also inhibited by the indoles, together indicating possible therapeutic drug targets. Tazi and colleagues [208] have also found diospyrin derivatives that inhibit topoisomerase I and alter pre-mRNA splicing in cell-free extracts, and have discovered indole compounds that selectively inhibit topoisomerase I over ASFSF2 [207]. Determining the therapeutic utility of the indole and diospyrin molecules requires studies in live cells and living organisms.

4.7 AN ASSAY FOR SPLICE SWITCHING OLIGONUCLEOTIDE POTENCY Simultaneous downregulation of aberrant splicing with upregulation of the correct splice variant is a unique feature of splicing manipulation by SSO. In addition to its therapeutic applications, it also has utility as an assay for antisense oligonucleotide potency. This laboratory has developed such an assay, based on the aberrant splicing of human
-globin intron 2 containing the thalassemic IVS2-654 mutation. The intron was inserted into the coding sequence for enhanced green fluorescence protein (EGFP), and was stably expressed in both HeLa cells [209], and in a transgenic mouse line, which ubiquitously expresses the construct [210]. When RNase H-inactive oligonucleotides targeted to the 654 mutation (SSO-654) are successfully taken-up by the nucleus of cells, mRNA containing the correct EGFP reading frame is generated, and the cells become fluorescent. In the HeLa cell culture system, the ability of oligomers to be taken into the cell, reach the nucleus and shift splicing of the reporter gene was examined. It was found that, in the absence of a delivery agent, uncharged or positively charged compounds (e.g., morpholino, PNA) were more apt to reach the nucleus from the extracellular media than negatively charged oligonucleotides (e.g., 2
OMe, 2-O-MOE) [209]. Furthermore, the addition of four positively charged lysines increased the free uptake activity of PNA. Follow-up studies by Thierry et al. [211] show that the addition of up to eight lysines also imparted increased activity of PNA SSOs. Moulton et al. [212,213] also showed that positively charged HIV Tat- and arginine-rich peptides and derivatives significantly increased uptake and activity of morpholino SSOs in cell culture. In another study, base modifications for MOE and PNA were also tested in the EGFP assay in the Kole lab [214]. We found that guanine derivative such as phenoxazine and G-clamp, which is tricyclic and is capable of Hoogstein basepairing, modestly improved the activity of oligomers delivered by cationic lipids.

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1.5

3

6

12

25

105 0 mg/kg Ab Co

Figure 4.4

Splice switching of EGFP-654 pre-mRNA by LNA oligonucleotides in transgenic mice. Increasing concentrations of LNA SSO-654 were administered i.p. once daily for 4 days and whole liver was analyzed by RT-PCR the following day for the ratio of abberantly (Ab) or correctly (Co) spliced EGFP mRNA. The data indicate that as little as 0.75 mg/kg/day induced correction of aberrantly spliced EGFP-654, and that 25 mg/kg induced almost a complete shift from Ab to Co.

Using the EGFP-654 transgenic mouse, we compared the in vivo efficacy of SSO-654 of different oligonucleotide chemistry: 2
OMe, MOE, morpholino, PNA, and LNA. In our original screen, we found that MOE and PNA had good activity in vivo in several tissues, and particularly high activity in the liver and small intestine. In particular, the addition of a 4-lysine tail to PNA significantly increased its efficacy, with a 50% shift in splicing detected in liver after daily i.p. injections of 50 mg/kg for 4 days [210]. The data regarding the PNA–peptide conjugates was applied to improve the antisense activity of oligomers directed towards CD40 [161,215,216]. More recently, we tested the efficacy of LNA and found them to be at least 10-fold more potent than other chemistries in the liver, small intestine, and colon [217]. For example, 50% shift in splicing was achieved with only 6 mg/kg, and achieved ⬃90% shift in splicing with only 25 mg/kg, injected i.p. as above (Figure 4.4). Interestingly, in contrast to MOE and PNA, LNA functional distribution was limited to these organs. Furthermore, LNA showed limited but detectable SSO activity when delivered orally by gavage in PBS, with no other delivery adjuvant. These data suggest that LNA are potent SSOs suitable for clinical development. The data discussed above suggest that cell culture activity studies may or may not be predictive of efficacy in vivo (see also Refs. [71,72,196]). MOE oligonucleotides are clearly potent SSOs in the liver and small intestine in mice, yet in cell culture they require mechanical or chemical delivery agents for nuclear activity. In contrast, peptide conjugates appear to be effective in both systems. The variations in the efficacy of different modified oligomers in in vivo versus in vitro is likely due to their interactions with serum proteins in the blood, which may aid in the delivery of negatively charged oligonucleotides by binding them and extending their half-life in the circulation.

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II

The Basics of Oligonucleotide-Based Therapeutics

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CHAPTER

5

Basic Principles of Antisense Drug Discovery Susan M. Freier and Andrew T. Watt

CONTENTS 5.1 5.2

Introduction .........................................................................................................................118 Design of Antisense Oligonucleotids ..................................................................................118 5.2.1 Gene Sequence Alignment ......................................................................................118 5.2.2 Identification of Cross Reactors ..............................................................................119 5.2.3 Selection of ASO Target Sites .................................................................................119 5.2.3.1 Correlation of Activity with Gene Feature ..............................................119 5.2.3.2 Correlation of Activity with Calculated Secondary Structure in Target or ASO ..........................................................................................120 5.2.3.3 Correlation of Activity with Sequence Motifs in the ASO ......................122 5.2.3.4 Correlation of Activity with Cell-Free Properties ....................................122 5.2.3.5 Correlation of Activity with Multiple Properties .....................................122 5.2.4 Exclusion of ASOs ..................................................................................................123 5.2.4.1 Sequence Motifs Associated with Non-Antisense Activities ...................123 5.2.4.2 Reduction of Nontarget Genes .................................................................123 5.2.5 Sequence Variants Due to Polymorphism ...............................................................123 5.2.6 Cross-Species ASOs ................................................................................................124 5.2.7 Summary of Considerations for ASO Design .........................................................127 5.2.8 How Many ASOs to Design and Test? ....................................................................127 5.3 Primary Screen for Active ASOs .........................................................................................128 5.3.1 Activity Assay ..........................................................................................................128 5.3.1.1 Kinetic RT-PCR ........................................................................................129 5.3.1.2 Activity Assays Other Than Kinetic RT-PCR ..........................................130 5.3.1.3 Other Considerations for Choice of Screening Assay .............................131 5.3.1.4 Positive and Negative Controls for Screening Assays .............................134 5.4 Follow-Up Assays ................................................................................................................134 5.4.1 In Vitro .....................................................................................................................134 5.4.2 In Vivo ......................................................................................................................135 5.5 Comparison of in Vitro Activity to in Vivo Activity ............................................................135 5.6 Summary ..............................................................................................................................138 References .....................................................................................................................................138

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5.1 INTRODUCTION It has been nearly 30 years since the first reports of inhibition of gene expression using an antisense oligonucleotide (ASO) [1]. Although many technological hurdles remained to be solved, the potential of antisense inhibitors for therapeutic applications was clearly apparent at that time. Antisense oligonucleotides offered specificity rarely attainable with small molecule inhibitors. Specific isoforms of proteins could be targeted, allowing direct inhibition of one part of a pathway without affecting related pathways. The approach was universal. With only partial sequence of any RNA, that RNA and its products could be inhibited. Because the mechanism is universal and the chemical structures of ASOs are so similar, lessons learned for ASOs against one molecular target transferred to ASOs for other molecular targets. The result is several ASOs currently in development for a broad range of indications (Chapters 21–28). In addition to the therapeutic utility of ASOs, their value as a tool for functional genomics has been huge. With the availability of the complete genome sequence for human and many model organisms, it has become straightforward in many cases to identify a gene-specific inhibitor and evaluate its function in vitro and in vivo. ASOs have become a fast, effective mechanism for target validation. A gene-specific inhibitor can be quickly identified and tested in a disease model in vivo. The results of that study provide a rapid validation of the target before embarking on expensive screens for a small molecule inhibitor or development of a human antisense therapeutic. Since completion of the human genome sequence, Isis has identified and characterized antisense inhibitors for more than 3000 molecular targets. We will summarize below what has been learned about efficient and effective discovery of antisense oligonucleotides for purposes of functional genomics and direct drug discovery. We will address design of ASOs, screening of ASOs, and follow-up confirmation of active ASOs. We will end with a discussion of the correlation between in vitro target reduction and in vivo target reduction. Later stages of antisense drug discovery including pharmacokinetics, pharmacodynamics, and pharmacology of ASOs in vivo are thoroughly covered elsewhere in this volume.

5.2 DESIGN OF ANTISENSE OLIGONUCLEOTIDS As mentioned above, one attraction of antisense technology is the universality of the approach and the simplicity of drug design. If RNA sequence is known, antisense sequence is easy to work out. The process, however, is not that simple. Decisions must be made with respect to length, chemical modifications, and target site. As discussed in Chapters 1, 2, and 10–14, 2⬘–O–methoxyethyl chimeric ASOs possess desirable properties for antisense therapeutics. These ASOs support RNase H and directly cause decay of the RNA target. Many alternative chemistries and structures continue to be investigated (Chapters 6 and 17–20). The bulk of our experience, however, has been with 5-10-5 MOE gapmers, 20-mer fully phosphorothioate ASOs with five 2⬘–O–methoxyethyl sugars on each end and ten deoxyribose sugars in the gap. Thus, this chapter will focus on finding the optimal 5-10-5 MOE gapmer; Chapter 17 and others mentioned above will discuss effects of varying this basic structure. Although most of the data described are for 5-10-5 MOE gapmers, the processes described below can be applied to any ASO structure and modification including siRNAs and single-stranded ASOs that use double-stranded RNASes for target cleavage. ASOs that work by blocking translation or altering RNA processing may have additional restrictions, both for ASO design and for measurement of ASO activity. 5.2.1

Gene Sequence Alignment

The first step in ASO design is to identify the molecular target(s). The gene of interest may have been selected because it was known to be important in specific biological pathways or diseases. Alternatively, it may have been selected because little was known and the ASO will be used for

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functional genomics studies. In either case, one begins with a gene. The purpose of the sequence alignment is to identify the primary transcript(s) for this gene and all mRNA products. Although I refer to the final products after splicing and polyadenylation as mRNAs, they can equally well be processed noncoding RNAs. We have been successful in targeting spliced and unspliced noncoding RNA as well as coding RNAs using gapmer ASOs. Alternative starts of transcription and alternative polyadenylation will lead to alternative primary transcripts. The reason for identifying these alternative transcripts is to ensure that ASOs are complementary to the pre-mRNA for all variant mRNAs. If one wants to specifically target only one transcription variant, ASOs can be designed to unique regions of primary transcripts [2]. If, however, one wants to inhibit all variants, it is important to ensure that ASOs are complementary to the primary transcripts for all variants. Similarly, identification of mRNA variants is important when designing reverse transcriptase polymerase chain reaction (RT-PCR) primers or other detection methods to be used in the screening assay. If multiple mRNA variants are present, it is important that the assay detect and, if possible, distinguish the variants. ASOs that cross exon:exon junctions can be active [3]; so identification of splice variants is necessary to recognize which processed transcripts will be targeted by these exon:exon ASOs. For many genes, the literature can provide information on known primary and processed RNA variants. For others, the best sources of information are the public sequence databases such as Genbank. Complementary DNAs (cDNAs) and expressed sequence tags (ESTs) can be aligned on the genome using BLAST [4] and common variants can be identified. Possible variants can also be identified by comparison to other species. If, for example, there are three alternate starts of transcription in human resulting in three alternate exons 1 and if three similar exons can be found upstream of the same gene in the rat genome, one could speculate the three alternate exons 1 are also used in rat. 5.2.2

Identification of Cross Reactors

One advantage of antisense technology is that it can be used to specifically target one member of a family of similar gene products. To recognize this specificity, one must avoid ASO binding to other members of the family. Cross-reacting RNAs can be identified by a homology search versus databases of RNAs in the same species. Any gene with even weak homology to the target gene is classified as a potential cross reactor for both ASO design and RT-PCR primer selection. 5.2.3

Selection of ASO Target Sites

There are typically thousands of sites on an mRNA and tens of thousands of sites on a premRNA that can be targeted by ASOs. Thus, any method that can select active ASOs from these thousands of candidates is of value. Many computational and experimental methods have been used and are reviewed elsewhere [5]. Here, I will focus on those that have been tested in the drug discovery process at Isis.

5.2.3.1 Correlation of Activity with Gene Feature Figure 5.1 shows screening data for ASOs designed to different regions of a pre-mRNA target. Active ASOs occurred in all regions of the target. These data were combined with similar data for hundreds of other targets in an attempt to correlate gene feature with ASO activity. Overall, correlation was weak. Active ASOs were found in the untranslated regions (UTRs), coding sequence (CDS), introns, splice sites (intron:exon and exon:intron junctions), and exon:exon junctions. When the correlation was tested using only lead ASOs with confirmed in vivo activity, similar results were obtained; all regions were represented. Activity of oligos complementary to introns [6] provides strong evidence that these ASOs act on RNA in the nucleus before splicing occurs. Activity of ASOs that target an exon–exon junction

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[3] suggests that these ASOs also act on the spliced product. This activity may be in the nucleus, after splicing and before export, or it may occur on the mRNA in the cytoplasm.

5.2.3.2 Correlation of Activity with Calculated Secondary Structure in Target or ASO Figure 5.2 depicts the hybridization steps required for an ASO to bind to its RNA target site. If only hybridization were important, activity would correlate with the free energy required to open up binding sites in the ASO and RNA target and the free energy of duplex formation between the ASO and the target RNA. Researchers at the Mathew’s Lab, University of Rochester Medical Center, have provided programs to calculate free energies for each of the steps in Figure 5.2 [7].

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Intra- and intermolecular oligo self structure

Total binding of an ASO to an RNA target consists of three steps. Self-structure in the ASO must be removed and secondary structure in the RNA target must be opened up. Finally, the ASO binds to the denatured target site with favorable free energy.

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Mathews and colleagues (http://rna.urmc.rochester.edu/rnastructure.html) and many others have reported similar strategies [8–20]. Good correlation between calculated binding free energies and observed antisense activity has been reported for data in a single experiment [7,15]. When data were combined for screens against multiple targets performed under similar conditions, correlations became weaker. Figure 5.3a shows data for 349 20-mers comparing antisense activity to free energy of duplex formation. Although the correlation was weak, it was in the expected direction and, due to the large number of samples, was significant. For this set of ASOs, a filter of ⌬G° ⬍ ⫺35 kcal/mol increased the hit rate from 14% to 24% (Figure 5.3b). The number of active ASOs, however, was reduced from 49 to 5 and the number of candidates was reduced from 349 to 21. A receiver operator curve (ROC) for these data is shown in Figure 5.3c. Good specificity can be achieved but not without a big loss in sensitivity. Similar results were reported by Matveeva et al.; restrictive filtering of candidate ASOs based on free energy calculations improved the fraction (a)

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(a) Correlation of antisense activity with the free energy of duplex formation for 349 ASOs from several screens against several targets. (b) Effect of a free-energy prefilter on activity profile of ASOs. (c) Receiver–operator curve for data in Figure 5.3a. Active ASOs were defined as those with RNA levels ⬍25% control. The free-energy cutoff was varied from ⫺40 kcal/mol to ⫺10 kcal/mol and the effect on sensitivity ([active ASOs predicted to be active]/[all active ASOs]) and specificity ([inactive ASOs predicted to be active]/[all inactive ASOs]) was plotted. Area under this curve was 0.72.

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active and greatly reduced the number of active ASOs identified [14]. Thus, if there are sufficient candidate ASOs and sufficient active ASOs, a strategy using thermodynamic cutoffs can improve hit rates but not to 100%. The weak correlation of ASO activity with calculated free energies for the processes in Figure 5.2 is likely due to two factors. First, the thermodynamic calculations are not perfect. Few thermodynamic parameters are available for modified ASOs [21] and predictions of free energy for unfolding a region of an RNA are still being improved [22,23]. The main source of the discrepancy, however, is that the process in Figure 5.2 is too simple. The ASO is not binding to a folded mRNA at equilibrium but to a transcript that is undergoing refolding as it is processed. Proteins bind both to the transcript and to the ASO. The process is kinetic, not equilibrium. Enzyme kinetics of RNAse H or other terminating mechanisms must be considered. Mismatched sites on other RNAs may compete for binding of the ASO. Given the complexity of the process, it is not surprising that thermodynamic predictions of ASO binding are not sufficient to fully explain ASO activity. Despite the relatively weak correlations reported above, target structure has been shown to play a role in antisense activity. Vickers et al. prepared several constructs containing a complementary site for a single ASO [24]. The RNA sequence surrounding the site was varied to produce transcripts in which the ASO binding site was in a region with more or less secondary structure. By using the same ASO for each experiment, they were able to show that potency of a single ASO was reduced when that ASO was complementary to a structured site in the target RNA.

5.2.3.3 Correlation of Activity with Sequence Motifs in the ASO Experimental correlation of antisense activity with sequence motif has led to identification of motifs positively or negatively correlated with antisense activity [13,25,26]. As for the thermodynamic considerations presented above, the correlation is not strong, so selection of ASOs based on motif rules can improve specificity but not without a big loss in sensitivity.

5.2.3.4 Correlation of Activity with Cell-Free Properties Several investigators have used in vitro–transcribed mRNA or cell extracts to experimentally evaluate ASO activity before performing the cellular assay [16,17,27–35]. Although correlations between activity in the cell-free assay and cellular antisense activity have been reported, there seems to be little advantage to these cell-free assays. They are often more difficult than the direct cellular screen and the lack of strong correlation does not preclude the need for cellular assays. The cell-free assays that could be advantageous are those that sample the full set of ASO binding sites. These include arrays [27,34,36] and combinatorial library approaches [28,29,37,38]. In principle, these strategies could sample more sequence space than a typical screening experiment in cell culture. In practice, however, technical limitations and the lack of strong correlation between the cell-free results and antisense activity in cell culture have limited the utility of these approaches.

5.2.3.5 Correlation of Activity with Multiple Properties The lack of strong correlation for any of the above properties with antisense activity led to an attempt to simultaneously correlate multiple properties with antisense activity. We began with antisense activity data for about 30,000 ASOs and filtered the set to create a training set of ⬃10,000 ASOs with reproducible activity, which were tested under similar conditions. Thermodynamic properties, sequence motifs, and gene feature properties discussed above were included. Both decision tree and neural net models were used to mine the data. The resulting hybrid model for prediction of antisense activity was used to design 39 ASOs to each of 25 target genes. A second set of 39 ASOs was designed using a more traditional approach without use of the

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computational models. When activity was measured for these ASOs, the percentage of active ASOs increased slightly when the models were used, but overall, activity profiles were very similar using the two methods. To date, results from computational methods for prediction of antisense activity have been disappointing. Hit rates have not improved enough to substantially reduce the number of ASOs to be screened. The methods have also not resulted in identification of leads with greater potency than those identified by more traditional screens. 5.2.4

Exclusion of ASOs

5.2.4.1 Sequence Motifs Associated with Non-Antisense Activities Although methods to predict active ASOs have met with limited success, methods to exclude ASOs have been successful. Some sequence motifs have been associated with non-antisense-based pharmacology and exclusion of ASOs with these motifs can reduce the likelihood of confusing pharmacological results later during the discovery process. Immunostimulation in rodents or primates can result from treatment with ASOs containing motifs with unmodified CpG [39,40]. More recently, it has been observed that certain pyrimidine-rich motifs were overrepresented in a small set of ASOs that were hepatotoxic in mice (S. Burel and S. Henry, Isis Pharmaceuticals, unpublished results). A thorough definition of the motif is still in progress. It has long been recognized that strings of guanosine can result in nonantisense activity [28,41]. Avoidance of ASOs containing these motifs may help avoid pharmacological results complicated by ASO activity beyond the pharmacological effect of target reduction.

5.2.4.2 Reduction of Nontarget Genes Studies such as those in Figure 5.4 have demonstrated that even a single mismatch can reduce ASO activity and three mismatches essentially obliterate activity in vitro and in vivo. Candidate ASOs are filtered to have more than three mismatches to potential cross-reactive targets. Potential cross-reacting transcripts are identified as described above. Each ASO is compared to each site on this list of cross reactors, and no ASO with three or fewer mismatches is considered for screening. The most common sequence alignment tool, BLAST [4], cannot be used for this process as the smallest window size is seven and it is possible for a 20-mer ASO to bind to a target site with three mismatches and no region of seven pairs in a row. The alignment tool FASTA [42] allows a window size as small as one and could be used for this comparison. The strategy described avoids ASOs that would hybridize to identified “cross reactors.” It would not, however, identify a single mismatched 20-mer site in an otherwise nonhomologous gene. To identify all potential mismatched sites, one would need to test each candidate ASO versus the entire transcripome. At Isis, we have developed a rapid string-searching algorithm optimized for this purpose. Even with our optimized algorithm, that process is too slow for testing tens of thousands of candidates. Thus we postpone the transcriptome search until after the initial in vitro screen, when there are only about 10 candidate ASOs (see Figure 5.9). Another strategy for avoiding cross-reactive oligonucleotides is to avoid repeat features. These are sequences that appear frequently in the genome and ASOs complementary to them are likely to hit several other genes. Simple repeat features can be identified by DUSTMAPPER [43] and complex repeats can be identified using Repeatmasker [44]. 5.2.5

Sequence Variants Due to Polymorphism

In general, it is desirable for a human ASO therapeutic to inhibit all genetic variants of a gene. Thus known sites of polymorphism should be avoided.

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Cross-Species ASOs

The lead antisense compound in one species is often not the lead compound in another species. This can increase the work necessary for development. For example, the human lead must be tested in rats for “chemical” toxicity and the rat lead must be tested in rats for toxic effects of target knockdown. If the human and rat leads were the same compound, the work and cost would be reduced. There is, therefore, incentive to design “cross-species” ASOs. Fortunately, in 2006, extensive genomic sequence is available for human and many organisms used in disease models (e.g., mouse, rat, dog, pig, chimp, rhesus monkey, rabbit). For most genes, sequence can be identified for several species of interest. If homology is sufficient, cross-species ASOs can be designed.

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Even if cross-species ASOs can be identified, they are frequently concentrated in the CDS or maybe only one region of the CDS. Data in Figures 5.1 and 5.5 combined with results from thousands of screens performed at Isis have demonstrated that lead ASOs can come from anywhere on the transcript. Thus, it may be prudent to screen ASOs from all regions of the transcript even if they are not all cross-species. It is also critical to note that just because an ASO is complementary to two species, it may not be sufficiently active in both species. For example, a lead in mouse may not be a lead in rat, even though it is complementary to rat. The final decision to use a cross-species ASO

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depends on the cost savings and convenience of having only one ASO for studies in multiple species weighed against any reduced potency of a cross-species lead compared with a species-specific lead. Experience at Isis suggests that if a gene is well enough conserved so that at least a hundred wellseparated cross-species ASOs can be designed throughout the mRNA, cross-species leads can be identified that are nearly as active and as potent as non-cross-species leads. In practice, success rates have been good for identification of lead mouse-rat or human-monkey ASOs; but success rates have been lower for identification of lead ASOs with good activity in both rodents and primates. It is possible to use the same strategy to design ASOs complementary to two genes from related families such as AKT1 and AKT2. The disadvantage of such an approach is that potency versus the two targets is usually not identical; so, at any dose, the degree of target knockdown is not equal. If an isoform-specific ASO is designed for each target, then they can be dosed simultaneously and the degree of target reduction can be controlled independently for each target. 5.2.7

Summary of Considerations for ASO Design

The selection of ASOs for screening is a balance of several factors and, depending on the planned use for an ASO, priority of each factor may change. Typically, however, the primary filter as been to avoid cross-reactive ASOs and those with potential aptameric motifs. The second priority has been cross-species design. The number of cross-species ASOs selected depends on the degree of sequence homology and the importance of cross-species homology to the planned studies for that oligo. The final criterion has been calculated prediction of activity using the hybrid decision tree/neural net model described above. 5.2.8

How Many ASOs to Design and Test?

Figure 5.5a shows screening results for 57 ASOs targeted to a single gene. Although “hit rates” vary by target, these results are quite typical. About one third of the ASOs tested reduced target mRNA below 40% control; 12% reduced it below 25% control; and only 7% reduced it below 20% control. Thus, an “active” ASO that reduced target by 60% or more could likely have been identified by testing three to six ASOs at random from this set. Screening 10 ASOs would have identified, on average, about one compound that reduced target better than 25% control. In this example, screening of 57 candidate ASOs identified four candidates with activity below 20% control. It is clear that screening 57 ASOs identified more candidates and more active candidates than would have been obtained testing only three or ten ASOs. The question becomes, “Was it worth it?” The answer is almost always yes. First, target validation is easier with the most potent and most active ASO. It is usually unknown what degree of target inhibition will be required to produce the desired pharmacology. With a potent, active, dose-responsive ASO, target levels can be “dialed down” as low as necessary to evaluate the pharmacological effect. Second, if one is screening for a human therapeutic, it is advantageous to find the most potent drug because therapeutic doses will be smaller, ultimately improving therapeutic index and reducing manufacturing costs. Screening more oligonucleotides may also offer broader intellectual property protection and will provide the drug development organization with potential backup compounds, should they be needed. In practice, for human development candidates, we have often screened several hundred ASOs to a single target. Rarely, however, has continued screening identified an ASO that was clearly more active than any among the first 200. There are also economies of scale when more compounds are tested. Eighteen control samples, including 13 untreated controls, are run on each screening plate, regardless of the number of test compounds. Development of a screening assay often costs about as much as synthesis and testing of 50–75 ASOs. With these relatively high fixed costs, the incremental cost of screening more compounds is small. We have found robotic sample handlers to be efficient and effective for ASO

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synthesis and sample handling, processing, and analysis, supporting the use of microtiter plates for these processes. The combination of economy of scale, the efficiency of using microtiter plates and robotic sample handling, and the desire for at least a few very active ASO inhibitors has led us to typically screen 78 candidate ASOs for one target or 37 ASOs for each of two targets on a single 96-well plate. This screening strategy has typically resulted in two to eight lead compounds for subsequent evaluation.

5.3 PRIMARY SCREEN FOR ACTIVE ASOs Although algorithms for ASO design can improve screening hit rates to some extent and help avoid oligonucleotides with undesirable properties that may show up later in the discovery process, it is still necessary and cost-effective to synthesize candidate ASOs and screen them for antisense activity before evaluating pharmacological effects of ASO treatment. The efficiency and efficacy of the entire antisense drug discovery process depends on the efficiency and efficacy of the initial process of lead identification. In this section, we will discuss options for initial ASO screening and include technical advice to improve reliability of screening results. 5.3.1

Activity Assay

As is the case for most screening exercises, the selection of an assay that is easy to perform and gives accurate results for ASO activity is critical. Assay development and validation can require more resources than the ASO screen itself but investment in a reliable screening assay is always worthwhile. Initial screens of ASOs have usually been performed in cell culture in vitro. If screening data are to be useful, they must be robust and reproducible. There should be good correlation between activity in the screening assay and activity in subsequent experiments. The ideal screen should avoid both false negative and false positive leads. One important criterion for the screening endpoint is that it should directly measure the molecular target of the ASO. If downstream events are followed, both false positive and false negative results can be obtained. If a pharmacological endpoint is used, slow kinetics can result in a false negative. Typically, ASO treatment results in reduction of mRNA levels, which in turn results in reduction of target protein levels followed by changes along a signaling pathway that ultimately results in a pharmacological phenotype. Depending on protein half-life and kinetics of downstream processes, it can be several days between ASO treatment and the pharmacological endpoint. For example, Dean et al. [45] reported that treatment of A549 cells with an anti-PKC
ASO resulted in near-maximal reduction of mRNA as early as 4 h after treatment. After 48 h, mRNA levels began to recover, probably due to cell division and resulting dilution of ASO. A second ASO treatment at 48 h was required to maintain mRNA knockdown until 72 h, at which time protein reduction and pharmacalogical effects of pharbol 12-mystirate 13-acetate (PMA) stimulation on ICAM1 mRNA were observed. In another report, effects of ASO treatment on adipocyte differentiation were not observed until 10 days in cell culture [46]. If assays such as these were used for screening and the treatment regimen and time allowed were not sufficient for the pharmacological endpoint to be observed, an active ASO would appear as a false-negative. Pharmacological assays can also result in false-positives. Non-hybridization-based pharmacological effects of ASOs have been reported [28,41,47]. Often, these effects are observed in the absence of target RNA reduction. Thus, if reduction of the target RNA were used as the primary endpoint, these false-positives would not be reported. Oligonucleotides with robust activities that do not work via an antisense mechanism can become useful therapeutics [39,48,49]. In this chapter, however, we will treat oligonucleotides with non-antisense activities as undesirable false-positives.

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5.3.1.1 Kinetic RT-PCR For ASOs that support RNase H, the primary event is cleavage of the pre-mRNA or mRNA at the ASO binding site. Cleavage is usually followed by rapid degradation of the RNA. The earliest measurable effect is disappearance of the mRNA and measurement of mRNA loss is the assay of choice. Although there are many quantitative assays for mRNA, we have found real-time reverse transcriptase polymerase chain reaction (RT-PCR) to have several advantages. First, it is sensitive. Typically, cells are treated with ASO in a 96-well plate from which 50–200 ng of total RNA can be recovered. For most target mRNAs, as little as 10–20 ng of total RNA will give a strong real-time RT-PCR signal. Thus, multiple measurements can be made from a single treatment well. Second, RT-PCR assays for new targets are relatively easy to design; all that is needed is mRNA sequence. Much has been written about the design of primers and probes for real-time RT-PCR and we will not repeat that here. We will, however, mention a few guidelines that have increased our success rates. These guidelines are most important for genes that are very weakly expressed because they help avoid a false signal from nontarget genes. ●

●

●

As much as possible, use an amplicon that crosses an exon–exon junction. This will prevent the signal from contaminating DNA or pre-mRNA. Avoid all repeat motifs. This can be easily done by using Repeatmasker before design of the primer probes [44]. Identify related transcripts in the same gene “family” (e.g., JNK1, JNK2, and JNK3 or proteins with zinc-finger domains). Mask out any regions in the target mRNA that have even weak homology to these potential cross reactors.

Additional advantages of kinetic RT-PCR for mRNA quantitation are reviewed by Bustin [50]. This review addresses many of the issues to be considered when developing a real-time RT-PCR assay for ASO screening, including options for fluorescent probes and issues such as one-step versus twostep RT-PCR, standard curves, and normalization. One drawback of PCR for ASO screening is that it measures levels of intact amplicon RNA only. Thus, if RNA cleavage occurs at the ASO binding site but cleavage products are long lived, then the RT-PCR signal would not detect cleavage outside the amplicon and false-negatives could result. It is tempting, therefore, to design RT-PCR probes to span the ASO binding site. This, however, can result in false-positive results. ASOs complementary to the amplicon of the RT-PCR reaction have been found to inhibit the RT-PCR signal (S. Davis, Isis Pharmaceuticals, unpublished results). The presumed mechanism of inhibition is the binding of the ASO to the primers, probe, RNA, or amplification products, resulting in reduced amplification. Although RNA purification methods remove most of the ASO from a treated sample, we have encountered examples where enough ASO was carried over into the RNA such that RT-PCR was inhibited. Figure 5.6a shows the effect of five different ASOs on mRNA levels after treatment in cultured cells. Two different real-time RT-PCR primer-probe sets were used to measure mRNA levels. ASOs 1–3 did not overlap the amplicon of either primer set and measured mRNA levels were similar for both sets. In contrast, each of the other two ASOs overlapped the amplicon of one of the primer sets. For both these ASOs, the signal from the amplicon overlapping the ASO was reduced compared with that from the amplicon away from the ASO, consistent with the observation that ASO “carry-over” can inhibit RT-PCR when the ASO is within the PCR amplicon. Figure 5.6b shows results of a screen in which two of the ASOs overlapped the amplicon completely and two others partially overlapped the amplicon. The two ASOs contained within the amplicon appeared more active than any others in the screen. Screening data with active “amplicon oligos” needs to be confirmed using a PP-set away from the ASO binding site. Although uncommon, ASOs that cleave target RNA and result in stable products have been observed [51]. Depending on placement of the RT-PCR probes, such ASOs could result in falsenegatives. The best way to avoid these false-negatives is to use a Northern blot assay with a full length cDNA probe so that all products can be detected and their lengths measured.

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PP set A

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0

ASO position on mRNA

Figure 5.6

Demonstration of “amplicon effect”. (a) Human HepG2 cells were transfected with five ASOs complementary to different regions of a target mRNA. RNA was isolated and analyzed by TAQ-man RT-PCR using two different primer-probe sets. Positions of the ASOs and the primer-probes are shown in the inset. (b) Human A549 cells were transfected with ASOs complementary to different regions of a target mRNA. The two ASOs that completely overlap the PCR amplicon are highlighted.

5.3.1.2 Activity Assays Other Than Kinetic RT-PCR Screening of ASOs that do not result directly in cleavage of target RNA requires a screening assay other than quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). If the primary event is inhibition of translation, then a protein assay is required. An assay for protein activity can be used but can be susceptible to direct inhibition of protein activity by an oligonucleotide [41]. If the mechanism of action is redirection of splicing, variant-specific RT-PCR can be used [52]. Northern blots and ribonucleoase protection assays can be used to measure loss of mRNA but usually require more RNA and are lower throughput than RT-PCR. Other hybridizationbased assays for mRNA that do not rely on target amplification such as b-DNA probes have also been used successfully for evaluation of mRNA target reduction by ASOs [53].

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Finally, one can design a reporter assay with a simple readout such as luciferase activity. The disadvantage of such an assay is that it is indirect and thus subject to false-positive and false-negative results as discussed above for indirect assays. In addition, if the target RNA is expressed as part of the reporter system, it is not the natural target and accessibility of target sites to ASOs may not mimic the endogenous target.

5.3.1.3 Other Considerations for Choice of Screening Assay In addition to the choice of assay for RNA, there are other decisions to be made when designing a screening assay. Criteria for selection of cell lines are straightforward; the cell line must express the target at a quantitatively reproducible level and it should be possible for the cells to be transfected with oligonucleotide. There are no data indicating any advantage of selecting a cell line related to the ultimate target cell in vivo. Activity profiles appear to be independent of cell line as long as the same transcript is expressed in both lines. Precision of the screening data is more important than the tissue source of the cell line. When no cell line can be found that expresses the target of interest, a few options are still available. The first is primary cells. Primary cells such as hepatocytes, endothelial cells, neurons, and adipocytes have been used successfully to screen for active ASOs [54]. A second possibility is to clone the transcript and screen in transiently or stably transfected cells. Figure 5.7(a) shows a screen of ASOs targeting a message expressed off a cDNA clone transiently transfected into COS-7 cells. The most obvious features of this screen are the lack of strong inhibition by any ASO and the relatively high variability observed for the less active ASOs. The relatively poor activity is partly due to the relatively high level of plasmid DNA in these transfected cells compared to expressed mRNA levels. Despite pretreatment of the RNA with DNase, there was sufficient plasmid DNA in the screening samples to result in a high background signal. Inhibition of an endogenous target using a monkey positive control also suggested that ASO transfection efficiency was not optimal. Despite the noisy screen, retest of the most active nonamplicon ASOs (marked with arrows in Figure 5.7a) demonstrated clear, reproducible, dose-responsive activity of the leads (Figure 5.7b). Assays using stable transfectants have typically resulted in greater activity and more reproducible results (Figure 5.7c). The most plausible explanation for this observation is that integration of the cDNA into the host genome reduced the amount of DNA background in the RNA fraction. Although both transiently and stably transfected expression vectors have been successfully used to screen for active antisense inhibitors, one must consider the fact the primary transcript is mRNA, often with shortened UTR sequence, and is thus a different target than the endogenous pre-mRNA. Limited data available on the correlation of ASO activity between endogenous target and cDNA target suggest that leads identified using cloned cDNAs can provide ASOs that are active in vivo [55]. This correlation, however, would be expected to vary on a target-by-target basis. A third option for targets that cannot be screened in cell culture is to screen directly in vivo. If the target is expressed in a tissue where an ASO is known to distribute and be active, a screen of 10–20 ASOs in vivo can find compounds with good activity. Figure 5.8 shows data for 21 ASOs targeting a single mRNA in mouse kidney. Four active ASOs were easily identified. In vivo screening is costly and slow, so fewer compounds can be tested but it may be the best option for high-value targets that cannot be screened in cells. As mentioned above, cells lines for in vitro screens must be reproducibly transfectable with ASOs. For many cultured cells, commercially available cationic lipid preparations can be used to transfect oligonucleotides. Transfection conditions should, however, be optimized for each cell line and ASO chemistry. Transfection conditions reported to be effective for plasmids may not work for oligonucleotides. Transfection of fluorescently labeled oligonucleotides can result in fluorescent cells but those conditions have not always resulted in ASO activity. To assess ASO transfection, it is essential to observe target knockdown using a positive-control ASO targeting a gene expressed in the selected cell line. PTEN is a broadly expressed target and ISIS 116847 has been shown to effectively

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12 31 92 189 236 326 350 361 367 382 389 395 415 435 442 449 463 471 521 545 551 569 575 580 604 619 626 635 640 677 683 698 708 717 722 750 757 775 784 794 828 853 883 935 941 946 951 985 993 1027 1032 1040 1060 1080 1085 1103 1119 1203 1211 1217 1247 1254 1259 1265 1270 1278 1288 1341 1367 1402 1430 1449

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ISIS # (b) No treatment 13 nM

40 nM 120 nM

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ASO 442 Figure 5.7

Control ASO

Screening data for ASOs targeting an mRNA expression vector. (a) Antisense activity for ASOs complementary to a cDNA expression vector transiently transfected into COS-7 cells. Only ASOs within the CDS were complementary to the expressed mRNA. (b) Dose-response curve for the most active compound from panel a. (c) Screen of ASOs to a different cDNA target expressed in stably transfected CHO cells.

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Amplicon 428

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Figure 5.7

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inhibit PTEN in primate and rodent cells. Thus, it is a useful control for optimization of transfection conditions. For lipid transfection, conditions that should be optimized are the composition of the lipid cocktail, concentration of lipid, and ratio of lipid concentration to ASO concentration. Cell density and culture medium also affect transfection efficiency. When optimizing transfection conditions, one optimizes for the greatest potency and activity without nonspecific activity or toxicity. Nonspecific activity can be monitored using nontarget ASOs. Total RNA can be measured using Ribogreen RNA quantitation reagent. A significant and dose-responsive decrease in the ribogreen

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signal is typically correlated with cytotoxicity and suggests that the transfection conditions are too harsh and may result in nonantisense effects of the ASOs. Electroporation provides an alternative to lipid transfection agents. Optimization is even more important for electroporation because the window between effective transfection conditions and cell death can be small. The most important parameters to be varied have been voltage and ASO concentration. Less frequently, optimization of cell volume and path length has further improved transfection efficiency.

5.3.1.4 Positive and Negative Controls for Screening Assays Three kinds of controls are important for an ASO screen. The first is untreated control (UTC) samples. These are cells treated with culture medium, transfection reagent, and no oligonucleotide. Levels of mRNA for all ASO treated samples are normalized to UTC. Thus, more UTC samples will reduce the standard error for all data points. Pooled UTC RNA is also used to prepare the standard curve for RT-PCR. Pooling wells ensures that the sample is representative and the use of UTC samples rather than RNA prepared in a separate experiment ensures that the amplification per cycle will be as identical as possible between standards and test samples. The second type of control is a negative-control oligonucleotide of the same chemistry. Seventy-eight ASOs are typically screened in each experiment; so it is unreasonable to include mismatched or scrambled controls for each test oligonucleotide at this stage. We usually include two ASOs complementary to no target as negative controls in every experiment and usually observe no significant target reduction compared with untreated controls. Finally, a positive-control ASO should be included. This should be an ASO such as ISIS 116847 that has been reproducibly active against its PTEN target in all cell lines. The positive-control ASO provides confirmation of adequate transfection and provides an estimate of the amount of target reduction expected from an active ASO in this particular experiment.

5.4 FOLLOW-UP ASSAYS 5.4.1

In Vitro

Once leads have been identified in the screening assay, follow-up assays should be performed. The purpose of these assays is to compare the leads for activity and potency in multiple assays and to identify, as quickly as possible, any false-positive ASOs that passed through the screen. First, the screening assay should be repeated on the most active compounds over a range of concentrations to determine relative potency and efficacy of the lead candidates. Figure 5.5b shows a repeat doseresponse experiment for six of the most active compounds from Figure 5.5a. This experiment helps distinguish small differences in potency and efficacy between the most active compounds from the initial screen. Additional assays of mRNA target reduction can also be employed. These include northern blots, ribonucleoase protection assays, RT-PCR with alternate primer sets, and alternate detection probes. The purpose of these alternate mRNA assays is to confirm that the reduction in the RT-PCR signal observed with the primary assay is truly representing a reduction in cellular mRNA. This second RNA assay is particularly important if one of the leads lies within the amplicon of the initial primer set (see above). Although apparent activity of an “amplicon ASO” can be artifactual, frequently the activity has been reproduced using a northern blot or a primer set away from the ASO binding site, indicating that the result was not an artifact. Another possible follow-up assay is to repeat the in vitro dose response in an additional cell line. If the primary transcript differed between cell lines, it would be possible for an ASO to be active in one cell line but not another. For example, different sites of initiation of transcription would result in a region of pre-mRNA being transcribed in one cell line but not another and ASOs to that region being active on one cell line but not another. This scenario has been avoided by using expression

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databases such as dbEST to identify alternative sites for transcription initiation and termination and, if ASOs are targeted to these regions, to use transcript-specific probes to measure the effect on each transcript. In practice, the only time we have found ASO activity not to reproduce between cell lines was when transfection was ineffective in the second cell line. This was immediately recognized by lack of activity with the positive-control ASO. Inhibition of protein also provides confirmation that the ASO is reducing the target of choice. As mentioned above, the major concern when assaying for protein reduction is to make sure that enough time has elapsed between ASO treatment and protein assay for preexisting protein to decay. It is not necessary to perform all the secondary in vitro assays described. They provide confirmation of antisense activity and multiple comparisons of the most active oligonucleotides to ensure that the most active and potent are carried to the next step. Depending on cost and resources, it may be prudent to postpone or omit some of the in vitro follow-up assays and move rapidly to in vivo confirmation. 5.4.2

In Vivo

For nonhuman targets, the final step in lead identification usually is screening for target reduction in vivo. In general, we have compared the top four ASOs identified in the in vitro dose-response experiment in an in vivo study at a single dose with reduction of target RNA as the primary endpoint. Secondary endpoints such as serum levels of transaminases, creatinine, urea, body weight, and organ weight were used to identify ASOs with non-target-related toxicity. Often, the top two compounds have been repeated in dose response in vivo before initiation of pharmacological assays. Identification of two leads that reduce target in vivo allowed pharmacological results to be confirmed with a second compound that effectively reduced the target gene.

5.5 COMPARISON OF IN VITRO ACTIVITY TO IN VIVO ACTIVITY Figure 5.9 summarizes the process described above. One begins with a list of all possible ASOs to an RNA transcript and ends with about two ASOs to be used in pharmacology models in vivo. If the ASO will be used only for in vitro functional genomics, the last two steps are, of course, unnecessary. The top two to four ASOs from the in vitro confirmation and dose response can be used to test pharmacology in vitro. However, if one is screening for a human antisense therapeutic or doing functional genomics in a model organism, the critical point in this flow scheme is the transition from in vitro to in vivo. If this process is to be effective, the best ASOs from the cellular screens must be the best ASOs in vivo. Years of experience of in vitro screening followed by testing in vivo has provided substantial evidence that relative efficacy and potency measured in vitro correlates reasonably well with efficacy and potency in vivo. There are two lines of evidence to support this correlation. Affinity of an ASO for its RNA target can be reduced in a systematic and controlled fashion by introducing mismatches between the ASO and the target. Table 5.1 shows Isis 22023 and a series of mismatched ASOs for mouse Fas. Figure 5.4 reports the effect of these ASOs on FAS expression in tissue culture (Figure 5.4a) and in mouse liver (Figure 5.4b). Activities of the ASOs were similar in vitro and in vivo [56]. Table 5.1 also describes Isis 11061, which targets mouse C-raf kinase and a series of ASOs with 1–6 mismatches to the target. Figure 5.4c presents in vitro data for these ASOs, measuring inhibitions of mRNA levels as a function of oligonucleotide concentration. ASO potency clearly decreased with increasing number of mismatches. When these same ASOs were tested for target reduction in mouse liver, the same relative activity was observed (Figure 5.4d). ASO affinity can also be increased by incorporation of high-affinity sugar modifications into the sequence. Figure 5.4e demonstrates the increase in potency of 11061 in vitro when the five residues in each “wing” were modified with high-affinity 2⬘–O–methoxyethyl sugars. The same increase in activity was observed in vivo (Figure 5.4f).

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Generate all ASOs complementary to pre-mRNA and variant mRNAs

~10,000

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~100

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~10

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~2

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Pharmacology in vivo

Figure 5.9 Flow scheme for selection of leads ASOs for any gene target. All possible ASOs to the unprocessed RNA are generated and filtered in silico to generate a set of about 100 oligonucleotides for synthesis. A primary screen of these 100 oligonucleotides in cells reduces the number of leads about 10-fold. Further cellular assays allow ranking of the screening leads and selection of a few ASOs for evaluation in a screen in vivo followed by a dose-response experiment in vivo. The end result is typically two ASOs with good activity and potency in vivo for use in pharmacology assays.

Table 5.1 ASOs to Fas and c-RAF Kinase and Mismatched ASOs. All ASOs to FAS Were 2⬘⬘ MOE Chimeras. ASOs to c-RAF Were Oligodeoxynucleotides, Except ISIS 15570 Which Was a 2⬘⬘ MOE Chimera. Isis #

Sequence

Mismatches

Target

TM(°C)

22023 29834 29835 29836 29837 11061 13490 13491 13492 13493 13494 13495 15770

TCCAG CACTTTCTTT TCCGG TCCAG CACCTTTTTT TCCGG TCCAG CTCCTTTTAT TCCGG TCCAT CTCCTTTTAT GCCGG TCGAT CTCCTTTTAT GCCCG ATGCATTCTGCCCCCAAGGA ATGCATTCTCCCCCCAAGGA ATGCATTCTCGCCCCAAGGA ATGCATTCCCGCCCCAAGGA ATGCATTCCCGTCCCAAGGA ATGCATTACCGTCCCAAGGA ATGCATTACCGTACCAAGGA ATGCA TTCTGCCCCC AAGGA

0 2 4 6 8 0 1 2 3 4 5 6 0

Fas Fas Fas Fas Fas C-raf C-raf C-raf C-raf C-raf C-raf C-raf C-raf

ND ND ND ND ND 60.5 45.8 42.2 33.8 25.0 – – –

A second line of evidence supporting the correlation between activity in vitro and activity in vivo comes from an unpublished study by N. Dean and colleagues at Isis. They compared rankorder potency in vitro and rank-order efficacy in vivo for a series of 44 ASOs to 13 targets expressed in mouse liver. For each of these targets, in vitro screening was performed as described above. In vitro activity was confirmed by repeating the screening assay at several doses for the most active

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ASOs. In each case, 3–4 ASOs from the in vitro study were tested in vivo for their ability to reduce target mRNA in mouse liver. Using a Pearson’s product-moment correlation test, the correlation coefficient between in vitro and in vivo was 0.61 with p ⬍ 0.0001 for a highly significant positive correlation between the activity of the ASO in vivo compared to potency in vitro. In every case (13 genes), if both the rank 1 and rank 2 ASO (in vitro) were evaluated in vivo, at least one of these gave target down-regulation in liver more than 50%. None of the rank 1 or rank 2 ASOs were inactive in vivo (⬍25% reduction). Four examples of this correlation are shown in Figure 5.10. ASOs with good potency in vitro demonstrated good efficacy in vivo. The four targets in Figure 5.10 and the other nine targets used for the correlation study described above were selected because the target was expressed primarily in liver hepatocytes and ASO

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Figure 5.10 Comparison of in vitro potency to in vivo activity for ASOs to four targets. The most potent ASOs from the in vitro assay were tested in vivo for target reduction in mouse liver.

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distribution to hepatocytes is high. In this case where target expression was the highest in cell types to which ASO distributes, the correlation between in vitro and in vivo was excellent. The correlation between good activity in vitro and in vivo can be less good where other tissues and cell types are considered. There are some tissues and cell types where standard unformulated ASOs distribute poorly. For targets expressed in those cell types in those tissues, in vivo activity will be poor regardless of in vitro activity. When an ASO has shown good potency in vitro and does not demonstrate good efficacy in vivo, we have usually been able to explain the result by multiple cell types in the target tissue and differential expression and differential ASO distribution to the multiple cell types. For example, ASO activity is better in hepatocytes than in nonparenchymal cells. If a liver target is expressed at higher levels in nonparenchymal cells compared to parenchymal cells, an ASO that shows good activity in primary hepatocytes in vitro would appear less active in vivo because the bulk of the target is in a cell type where ASOs are less active. Another explanation for in vitro results not correlating with in vivo results is the proinflammatory effects of the oligonucleotide. As discussed elsewhere in this volume, some oligonucleotide sequences can produce profound proinflammatory effects including cell infiltrates and induction of a target gene. In some cases, the antisense effects in a tissue can be masked by an infux of inflammatory cells that express the target gene at higher levels than normal parenchymal cells. Alternatively, the proinflammatory effect of the oligonucleotide could lead to induction of the target gene in parenchymal cells, attenuating specific antisense effects. 5.6 SUMMARY The process described above for antisense lead identification is designed to move from a large number of candidates to the most active and potent compounds as quickly and efficiently as possible. It is designed to find the best compounds and avoid any with activity other than direct reduction of the intended target. Figure 5.9 illustrates the process by which tens of thousands of candidate ASOs are filtered down to two compounds for evaluation in pharmacology models. With robotic synthesis and sample handling, the time required for the in vitro portion of this process can be as short as a week, although 3–4 weeks has been more common. Scale-up synthesis and in vivo testing requires another 4–10 weeks. The bottom line is that one can select a target and have two specific inhibitors with confirmed in vivo activity within a few months. Not only is the process fast, but also the success rate has been high. Some targets have taken longer due to some of the technical issues discussed above but, in the end, active compounds have been identified to almost every target attempted. The success of this approach can be attributed to the universality of the principles of antisense, coupled with lessons learned over the past 15–20 years of antisense research. REFERENCES 1. M. L. Stephenson and P. C. Zamecnik; Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide; Proceedings of the National Academy of Science USA; 75; 285–258; 1978. 2. K. V. Prasanth, S. G. Prasanth, Z. Xuan, S. Hearn, S. M. Freier, C. F. Bennett, M. Q. Zhang, and D. L. Spector; Regulating gene expression through RNA nuclear retention; Cell; 123; 249–263; 2005. 3. B. A. Zinker, C. M. Rondinone, J. M. Trevillyan, R. J. Gum, J. E. Clampit, J. F. Waring, N. Xie, D. Wilcox, P. Jacobson, L. Frost, P. E. Kroeger, R. M. Reilly, S. Koterski, T. J. Opgenorth, R. G. Ulrich, S. Crosby, M. Butler, S. F. Murray, R. A. McKay, S. Bhanot, B. P. Monia, and M. R. Jirousek; PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice; Proceedings of the National Academy of Science USA; 99; 11,357–11,362; 2002. 4. S. F. Altschul, W. Gish, W. Miller, E. W. Myers, and D. J. Lipman; Basic local alignment search tool; J. Mol. Biol.; 215; 403–410; 1990.

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5. M. Lutzelberger and J. Kjems; Strategies to identify potential therapeutic target sites in RNA; in: Handbook of Experimental Pharmacology; V. A. Erdmann, J. Brosius, and J. Barciszewski, Eds; Vol. 173; Springer, Berlin, 2006; pp. 243–259. 6. T. A. Vickers, S. Koo, C. F. Bennett, S. T. Crooke, N. M. Dean, and B. F. Baker; Efficient reduction of target RNAs by small interfering RNA and RNase H-dependent antisense agents. A comparative analysis; J. Biol. Chem.; 278; 7108–7118; 2003. 7. D. H. Mathews, M. E. Burkard, S. M. Freier, J. R. Wyatt, and D. H. Turner; Predicting oligonucleotide affinity to nucleic acid targets; RNA; 5; 1458–1469; 1999. 8. P. Arrigo, G. Ivaldi, and P. P. Cardo; In silico determination of potential antisense targets for human beta-globin variants; In Silico Biol.; 2; 143–150; 2002. 9. X. Bo, S. Lou, D. Sun, W. Shu, J. Yang, and S. Wang; Selection of antisense oligonucleotides based on multiple predicted target mRNA structures; BMC Bioinformatics; 7; 122; 2006. 10. X. Bo and S. Wang; TargetFinder: a software for antisense oligonucleotide target site selection based on MAST and secondary structures of target mRNA; Bioinformatics; 21; 1401–1402; 2005. 11. A. M. Chalk and E. L. Sonnhammer; Computational antisense oligo prediction with a neural network model; Bioinformatics; 18; 1567–1575; 2002. 12. R. K. Far, J. Leppert, K. Frank, and G. Sczakiel; Technical improvements in the computational target search for antisense oligonucleotides; Oligonucleotides; 15; 223–233; 2005. 13. M. C. Giddings, A. A. Shah, S. Freier, J. F. Atkins, R. F. Gesteland, and O. V. Matveeva; Artificial neural network prediction of antisense oligodeoxynucleotide activity; Nucleic Acids Res.; 30; 4295–4304; 2002. 14. O. V. Matveeva, D. H. Mathews, A. D. Tsodikov, S. A. Shabalina, R. F. Gesteland, J. F. Atkins, and S. M. Freier; Thermodynamic criteria for high hit rate antisense oligonucleotide design; Nucleic Acids Res.; 31; 4989–4994; 2003. 15. V. Patzel, U. Steidl, R. Kronenwett, R. Haas, and G. Sczakiel; A theoretical approach to select effective antisense oligodeoxyribonucleotides at high statistical probability; Nucleic Acids Res.; 27; 4328–4334; 1999. 16. M. Scherr, J. J. Rossi, G. Sczakiel, and V. Patzel; RNA accessibility prediction: a theoretical approach is consistent with experimental studies in cell extracts; Nucleic Acids Res.; 28; 2455–2461; 2000. 17. G. Sczakiel; Theoretical and experimental approaches to design effective antisense oligonucleotides; Front. Biosci.; 5; D194–201; 2000. 18. S. A. Shabalina, A. N. Spiridonov, and A. Y. Ogurtsov; Computational models with thermodynamic and composition features improve siRNA design; BMC Bioinformatics; 7; 65; 2006. 19. R. A. Stull, L. A. Taylor, and F. C. Szoka, Jr.; Predicting antisense oligonucleotide inhibitory efficacy: a computational approach using histograms and thermodynamic indices; Nucleic Acids Res.; 20; 3501–3508; 1992. 20. N. Toschi; Influence of mRNA self-structure on hybridization: computational tools for antisense sequence selection; Methods; 22; 261–269; 2000. 21. E. Kierzek, D. H. Mathews, A. Ciesielska, D. H. Turner, and R. Kierzek; Nearest neighbor parameters for Watson-Crick complementary heteroduplexes formed between 2⬘-O-methyl RNA and RNA oligonucleotides; Nucleic Acids Res.; 34; 3609–3614; 2006. 22. D. H. Mathews; Revolutions in RNA secondary structure prediction; J. Mol. Biol.; 359; 526–532; 2006. 23. D. H. Mathews and D. H. Turner; Prediction of RNA secondary structure by free energy minimization; Curr. Opin. Struct. Biol.; 16; 270–278; 2006. 24. T. A. Vickers, J. R. Wyatt, and S. M. Freier; Effects of RNA secondary structure on cellular antisense activity; Nucleic Acids Res.; 28; 1340–1347; 2000. 25. G. C. Tu, Q. N. Cao, F. Zhou, and Y. Israel; Tetranucleotide GGGA motif in primary RNA transcripts. Novel target site for antisense design; J. Biol. Chem.; 273; 25,125–25,131; 1998. 26. O. V. Matveeva, A. D. Tsodikov, M. Giddings, S. M. Freier, J. R. Wyatt, A. N. Spiridonov, S. A. Shabalina, R. F. Gesteland, and J. F. Atkins; Identification of sequence motifs in oligonucleotides whose presence is correlated with antisense activity; Nucleic Acids Res.; 28; 2862–2865; 2000. 27. R. Bakalova, Z. Zhelev, H. Ohba, and Y. Baba; Quantum dot-conjugated hybridization probes for preliminary screening of siRNA sequences; J. Am. Chem. Soc.; 127; 11,328–11,335; 2005. 28. D. J. Ecker, T. A. Vickers, R. Hanecak, V. Driver, and K. Anderson; Rational screening of oligonucleotide combinatorial libraries for drug discovery; Nucleic Acids Res.; 21; 1853–1856; 1993.

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29. S. P. Ho, Y. Bao, T. Lesher, R. Malhotra, L. Y. Ma, S. J. Fluharty, and R. R. Sakai; Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries; Nat. Biotechnol.; 16; 59–63; 1998. 30. S. P. Ho, D. H. Britton, Y. Bao, and M. S. Scully; RNA mapping: selection of potent oligonucleotide sequences for antisense experiments; Methods Enzymol.; 314; 168–83; 2000. 31. O. Matveeva, B. Felden, A. Tsodikov, J. Johnston, B. P. Monia, J. F. Atkins, R. F. Gesteland, and S. M. Freier; Prediction of antisense oligonucleotide efficacy by in vitro methods; Nat. Biotechnol.; 16; 1374–1375; 1998. 32. M. Ooms, K. Verhoef, E. Southern, H. Huthoff, and B. Berkhout; Probing alternative foldings of the HIV-1 leader RNA by antisense oligonucleotide scanning arrays; Nucleic Acids Res.; 32; 819–827; 2004. 33. M. Sohail, G. Doran, S. Kang, S. Akhtar, and E. M. Southern; Structural rearrangements in RNA on the binding of an antisense oligonucleotide: implications for the study of intra-molecular RNA interactions and the design of cooperatively acting antisense reagents with enhanced efficacy; J. Drug Target; 13; 61–70; 2005. 34. M. Sohail, H. Hochegger, A. Klotzbucher, R. L. Guellec, T. Hunt, and E. M. Southern; Antisense oligonucleotides selected by hybridisation to scanning arrays are effective reagents in vivo; Nucleic Acids Res.; 29; 2041–2051; 2001. 35. S. P. Walton, G. N. Stephanopoulos, M. L. Yarmush, and C. M. Roth; Thermodynamic and kinetic characterization of antisense oligodeoxynucleotide binding to a structured mRNA; Biophys. J.; 82; 366–377; 2002. 36. S. Duan, D. H. Mathews, and D. H. Turner; Interpreting oligonucleotide microarray data to determine RNA secondary structure: application to the 3⬘ end of Bombyx mori R2 RNA; Biochemistry; 45; 9819–9832; 2006. 37. W. F. Lima, V. Brown-Driver, M. Fox, R. Hanecak, and T. W. Bruice; Combinatorial screening and rational optimization for hybridization to folded hepatitis C virus RNA of oligonucleotides with biological antisense activity; J. Biol. Chem.; 272; 626–638; 1997. 38. O. Matveeva, B. Felden, S. Audlin, R. F. Gesteland, and J. F. Atkins; A rapid in vitro method for obtaining RNA accessibility patterns for complementary DNA probes: correlation with an intracellular pattern and known RNA structures; Nucleic Acids Res.; 25; 5010–5016; 1997. 39. A. M. Krieg; Therapeutic potential of Toll-like receptor 9 activation; Nat. Rev. Drug Discov.; 5; 471–484; 2006. 40. A. M. Krieg, A. K. Yi, S. Matson, T. J. Waldschmidt, G. A. Bishop, R. Teasdale, G. A. Koretzky, and D. M. Klinman; CpG motifs in bacterial DNA trigger direct B-cell activation; Nature; 374; 546–549; 1995. 41. C. F. Bennett, M. Y. Chiang, L. Wilson-Lingardo, and J. R. Wyatt; Sequence specific inhibition of human type II phospholipase A2 enzyme activity by phosphorothioate oligonucleotides; Nucleic Acids Res.; 22; 3202–3209; 1994. 42. W. R. Pearson and D. J. Lipman; Improved tools for biological sequence comparison; Proceedings of the National Academy of Science USA; 85; 2444–2448; 1988. 43. J. C. Wootton and S. Federhen; Analysis of compositionally biased regions in sequence databases; Methods Enzymol.; 266; 554–571; 1996. 44. A. Smit, R. Hubley, and P. Green; RepeatMasker; http://repeatmasker.org; 2006. 45. N. M. Dean, R. McKay, T. P. Condon, and C. F. Bennett; Inhibition of protein kinase C-alpha expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters; J. Biol. Chem.; 269; 16,416–16,424; 1994. 46. C. Esau, X. Kang, E. Peralta, E. Hanson, E. G. Marcusson, L. V. Ravichandran, Y. Sun, S. Koo, R. J. Perera, R. Jain, N. M. Dean, S. M. Freier, C. F. Bennett, B. Lollo, and R. Griffey; MicroRNA143 regulates adipocyte differentiation; J. Biol. Chem.; 279; 52,361–52,365; 2004. 47. W. Wang, H. J. Chen, A. Schwartz, P. J. Cannon, C. A. Stein, and L. E. Rabbani; Sequence-independent inhibition of in vitro vascular smooth muscle cell proliferation, migration, and in vivo neointimal formation by phosphorothioate oligodeoxynucleotides; J. Clin. Invest.; 98; 443–450; 1996. 48. D. J. Ecker, J. R. Wyatt, T. A. Vickers, R. Buckheit, J. Roberson and J.-L. Imbach; Novel guanosine quartet structure binds to the HIV envelope and inhibits envelope mediated cell fusion; Nucleos. Nucleot.; 14; 1117–1127; 1995.

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49. E. W. Ng, D. T. Shima, P. Calias, E. T. Cunningham, Jr., D. R. Guyer, and A. P. Adamis; Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease; Nat. Rev. Drug. Discov.; 5; 123–132; 2006. 50. S. A. Bustin; Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays; J. Mol. Endocrinol.; 25; 169–193; 2000. 51. T. P. Condon and C. F. Bennett; Altered mRNA splicing and inhibition of human E-selectin expression by an antisense oligonucleotide in human umbilical vein endothelial cells; J. Biol. Chem.; 271; 30,398–30,403; 1996. 52. T. A. Vickers, H. Zhang, M. J. Graham, K. M. Lemonidis, C. Zhao, and N. M. Dean; Modification of MyD88 mRNA splicing and inhibition of IL-1beta signaling in cell culture and in mice with a 2⬘-O-methoxyethyl-modified oligonucleotide; J. Immunol.; 176; 3652–3661; 2006. 53. J. Soutschek, A. Akinc, B. Bramlage, K. Charisse, R. Constien, M. Donoghue, S. Elbashir, A. Geick, P. Hadwiger, J. Harborth, M. John, V. Kesavan, G. Lavine, R. K. Pandey, T. Racie, K. G. Rajeev, I. Rohl, I. Toudjarska, G. Wang, S. Wuschko, D. Bumcrot, V. Koteliansky, S. Limmer, M. Manoharan, and H. P. Vornlocher; Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs; Nature; 432; 173–178; 2004. 54. L. M. Watts, V. P. Manchem, T. A. Leedom, A. L. Rivard, R. A. McKay, D. Bao, T. Neroladakis, B. P. Monia, D. M. Bodenmiller, J. X. Cao, H. Y. Zhang, A. L. Cox, S. J. Jacobs, M. D. Michael, K. W. Sloop, and S. Bhanot; Reduction of hepatic and adipose tissue glucocorticoid receptor expression with antisense oligonucleotides improves hyperglycemia and hyperlipidemia in diabetic rodents without causing systemic glucocorticoid antagonism; Diabetes; 54; 1846–1853; 2005. 55. S. P. Ho, L. K. Takahashi, V. Livanov, K. Spencer, T. Lesher, C. Maciag, M. A. Smith, K. W. Rohrbach, P. R. Hartig, and S. P. Arneric; Attenuation of fear conditioning by antisense inhibition of brain corticotropin releasing factor-2 receptor; Brain Res. Mol. Brain Res.; 89; 29–40; 2001. 55. H. Zhang, J. Cook, J. Nickel, R. Yu, K. Stecker, K. Myers, and N. M. Dean; Reduction of liver Fas expression by an antisense oligonucleotide protects mice from fulminant hepatitis; Nat. Biotechnol.; 18; 862–867; 2000.

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CHAPTER

6

The Medicinal Chemistry of Oligonucleotides Eric E. Swayze and Balkrishen Bhat

CONTENTS 6.1

6.2

6.3

Introduction .........................................................................................................................144 6.1.1 Scope .......................................................................................................................144 6.1.2 Why Oligonucleotide Medicinal Chemistry Is Needed ...........................................144 6.1.2.1 Stability Is Insufficient ..............................................................................144 6.1.2.2 Pharmacokinetics Are Insufficient ............................................................144 6.1.2.3 Affinity Is Insufficient...............................................................................145 The Medicinal Chemistry Challenge....................................................................................145 6.2.1 Properties of Oligonucleotides .................................................................................145 6.2.2 Desired Properties of Oligonucleotide Drugs ..........................................................145 6.2.2.1 High Affinity and Specificity for the Receptor.........................................146 6.2.2.2 Ability to Effect the Desired Pharmacology.............................................146 6.2.2.3 Favorable Pharmacokinetics .....................................................................146 6.2.2.4 An Acceptable Therapeutic Index ............................................................147 6.2.2.5 Cost-Competitive Manufacture ................................................................147 Oligonucleotide Modifications ............................................................................................147 6.3.1 Potential Sites of Modification ................................................................................147 6.3.2 Backbone Modifications ..........................................................................................148 6.3.2.1 Furanose-Containing Phosphate Backbones ............................................148 6.3.2.2 Furanose-Containing Nonphosphate Backbones .....................................151 6.3.2.3 Sugar and Backbone Replacements .........................................................153 6.3.3 Sugar Modifications ................................................................................................154 6.3.3.1 2⬘-Modifications .......................................................................................154 6.3.3.2 Other Furnanose Substitution Positions ...................................................158 6.3.3.3 Bicyclic Sugars .........................................................................................158 6.3.3.4 Substitutions for the Ribofuranose Sugar ...............................................161 6.3.4 Heterocyclic Modifications .....................................................................................163 6.3.4.1 Pyrimidine 5⬘-Position Modifications ......................................................163 6.3.4.2 Tricyclic Cytosine Analogs ......................................................................165 6.3.4.3 Other Pyrimidine Modifications ..............................................................166 6.3.4.4 Purine Modifications ................................................................................166

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6.3.5

Conjugates ...............................................................................................................167 6.3.5.1 Cholesterol Conjugates ............................................................................167 6.3.5.2 Fatty Acid Conjugates ..............................................................................168 6.4 Optimizing Oligonucleotide Drugs .....................................................................................169 6.4.1 General Strategies ....................................................................................................169 6.4.2 Oligonucleotide Design Strategies ..........................................................................169 6.4.2.1 Gapmer Designs ......................................................................................169 6.4.2.2 siRNA Designs .........................................................................................170 6.4.2.3 Occupancy Only Designs .........................................................................170 6.4.2.4 Aptamer Designs ......................................................................................171 6.5 Outlook ................................................................................................................................171 References .....................................................................................................................................172

6.1 INTRODUCTION 6.1.1

Scope

This review will focus on the properties that an oligonucleotide drug must have, the many modifications available to the medicinal chemist to engineer those properties into the oligonucleotide, and finally on motifs that have been utilized in oligonucleotide drugs and drug candidates. No attempt has been made to be exhaustively complete in the coverage. We have instead highlighted properties of modifications, and strategies that we feel are likely to be successful in drug development, of value to the field, as well as those that add to the understanding of oligonucleotide medicinal chemistry. Much of the discussion centers around RNase H–based antisense therapeutics, since most of the efforts to optimize oligonucleotide drugs has focused on that application. However, the chemical modifications and principles apply equally to other classes of oligonucleotide drugs, including immunostimulatory motifs, aptamers, microRNA inhibitors, and duplex small interfering RNAs (siRNAs). 6.1.2

Why Oligonucleotide Medicinal Chemistry Is Needed

6.1.2.1 Stability Is Insufficient Unmodified DNA and RNA are inherently unstable molecules in biological systems. This instability is primarily due to ubiquitous nucleases, which cleave the phosphodiester linkage, but can also be chemical in nature, particularly for RNA. This instability precludes the use of unstabilized nucleic acids as drugs, since they are destroyed before they have a chance to reach their target receptor after being administered to an animal. A chemical modification strategy is an obvious way to improve the stability of oligonucleotide drugs, and has been highly successful. In fact, this strategy is used by living cells to stabilize endogenous nucleic acids such as ribosomal and transfer RNAs, and cues for the medicinal chemist can be taken from the myriad of naturally occurring nucleoside modifications [1].

6.1.2.2 Pharmacokinetics Are Insufficient In addition to their susceptibility to attack by nucleases, the pharmacokinetics of RNA and DNA are still insufficient to make acceptable systemic therapeutics. Because they are small enough to be filtered by the glomerulus and are poorly protein-bound in plasma, unmodified oligonucleotides are rapidly filtered and excreted. Therefore, beyond the requirement for improved nuclease stability, the plasma half-life must be improved such that distribution to tissues can occur. Moreover, unmodified

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oligonucleotides distribute poorly to tissues. This process is poorly understood, but appears to involve binding to acceptor sites on a tissue surface, which facilitates uptake.

6.1.2.3 Affinity Is Insufficient Because RNA is highly structured within a cell, any antisense drug must be able to compete with those structures. DNA, the initial antisense “lead” drug motif, has a lower affinity for RNA than RNA does for itself, which presents a thermodynamic problem that must be overcome. Increasing the intrinsic affinity for a complementary nucleic acid target is the most straightforward way to address this problem. Improving affinity should correlate with improvements in potency, until binding no longer becomes limiting.

6.2 THE MEDICINAL CHEMISTRY CHALLENGE 6.2.1

Properties of Oligonucleotides

Oligonucleotide drugs are intermediate in size between protein-based biological drugs and conventional small-molecule drugs. They range in length from about 13 nucleotides (the telomerase inhibitor GRN163L) to over 27 nucleotides (the aptamer Macugen, pegaptanib). Duplex drugs consisting of two strands (such as the antiviral siRNA ALN-RSV01) contain about 40 total nucleotides, in two distinct molecules held together by Watson–Crick base pairing. Most antisense oligonucleotides (ASOs) have a length of around 20 bases, which translates to a molecular weight of around 7000 atomic mass units. Due to the phosphate backbone, the same 20mer oligonucleotide has a formal negative charge of ⫺19, though not all of those residues are anionic at any given time. Electrospray ionization mass spectrometry experiments have indicated that at physiologic salt concentrations, 20–30% of the phosphates are charged, putting the likely actual negative charge of a 20mer oligonucleotide drug in the range ⫺4 to ⫺6 [2]. Because of their size and charge, the prevailing perception among medicinal chemists has been that oligonucleotides are not “drug-like” and would therefore not likely succeed as drugs. However, this line of thinking is somewhat shortsighted, as many successful small-molecule drugs are not very “drug-like,” nor are the highly successful protein-based therapeutics. Oligonucleotide drugs that utilize Watson–Crick base pairing as the mode of binding to the receptor (usually a target RNA) possess the unique property of having information encoded within their pharmacophore, which is essentially the nucleotide sequence. Molecules of this nature have been termed “informational drugs” [3]. This property allows a very high level of specificity to be easily achieved for the intended receptor, and furthermore allows for the rapid discovery of leads for diverse targets. Although the sequence is changed for each target, and results in changes to the drug properties at the microscopic level, the overall pharmacokinetic and toxicological profiles are similar within each class of oligonucleotides. Furthermore, the synthesis and manufacturing process is almost identical. These generic properties resulting from the nature of oligonucleotide receptor binding allow for large gains in the efficiency of lead discovery, optimization, and development. 6.2.2

Desired Properties of Oligonucleotide Drugs

Similar to all drugs, oligonucleotide drugs should have the following properties: (1) high affinity and specificity for the target receptor, (2) ability to effect the desired pharmacology (activate RNase H cleavage of mRNA, block VEGF action, etc.), (3) favorable pharmacokinetics (a component of which is stability), (4) an acceptable therapeutic index, and (5) manufacturing costs that make the product competitive in the marketplace.

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6.2.2.1 High Affinity and Specificity for the Receptor It is obvious that one must not make an exceedingly stable drug at the expense of large reductions in potency and specificity. Therefore, the ability to maintain binding to the receptor has been carefully balanced with the optimization of other properties. For traditional antisense approaches involving activation of RNase H, and especially steric blocking mechanisms, this means that modifications must retain, and preferably enhance, the ability to recognize their target RNA by Watson–Crick base pairing. This property is commonly measured by the melting temperature (Tm) of an oligonucleotide containing the modification of interest duplexed with a complementary strand of RNA. Specificity can be measured by studying the effects of mismatches. This approach has been well validated via correlations of activity in cell culture to affinity [4], and detailed structure–stability studies of many modifications have been undertaken [5]. For drugs that utilize an RNA-induced silencing complex (RISC)-based mechanism such as siRNAs, the interactions that govern binding to the mRNA receptor are not as clear, nor is it obvious whether high affinity for complementary RNA is a desired property. However, it is reasonably well established that Watson–Crick base-pairing rules determine the specificity of the drug, and they must therefore be maintained. For aptamer-derived drugs, affinity and specificity are likely key determinants of potency and efficacy. Unlike antisense approaches, the rules for specificity and affinity are no longer decipherable via the informational code of base-pairing rules, and as such optimization of a selected aptamer becomes much more akin to traditional drug optimization projects.

6.2.2.2 Ability to Effect the Desired Pharmacology For many drugs, binding to the receptor is sufficient to effect the desired pharmacology. For aptamers, this is likely to be the case as sterically blocking a receptor or binding a ligand will generally inhibit its function. However, for antisense approaches this is not always the case. The most widely used antisense drugs to date utilize RNase H cleavage of the target RNA to achieve their desired effects. Many studies have shown that modifications that are not substrates for RNase H activity are poor antisense drugs, despite being tight binders to their RNA complement, and presumably their receptor. siRNA drugs face a similar requirement in the ability to be loaded into the RISC complex and activate cleavage of the target. It is therefore crucial to examine modifications for their effects on these terminating events, and to understand these effects when designing oligonucleotide structure–activity relationship (SAR) series in optimization efforts.

6.2.2.3 Favorable Pharmacokinetics As discussed above, resistance to nucleases must be engineered into oligonucleotides for them to be useful as drugs. Nuclease stability is required but not sufficient for achieving a favorable pharmacokinetic profile. The drug must also distribute to the site of action, and persist at the site of action for a reasonable period of time. Unmodified nucleic acids are poorly bound to plasma proteins in vivo, and are therefore rapidly excreted to urine. For drugs such as aptamers that have their site of action in the blood compartment, modifications such as pegylation, which minimize or avoid renal filtration, are sufficient. However, for drugs that must distribute to tissues and penetrate cells to reach their target receptors, binding to other acceptor sites within the animal presumably mediates drug distribution. Thus, modifications that generally improve protein binding are often beneficial. However, strong protein-binding interactions can decrease the concentration of drug at the receptor site, and the effects are complex and difficult to predict. This underscores the necessity of early in vivo pharmacokinetic studies with new classes of oligonucleotide drugs, just as is commonplace with small-molecule drugs.

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6.2.2.4 An Acceptable Therapeutic Index As with all drugs, it should be obvious that known toxic modifications should be avoided. This is an especially important consideration for oligonucleotide drugs, as many nucleoside monomers have wide-ranging biological activities of their own. Known toxic sequence motifs should also be avoided. This can be achieved by building a database of toxic motifs, then filtering them out in a sequence selection process (see Chapter 5 of this volume). However, just as with small-molecule drug discovery, toxicological surprises occur, and detailed toxicological evaluation of new modifications, new usage contexts of these modifications, and new sequences must be conducted.

6.2.2.5 Cost-Competitive Manufacture Because oligonucleotide drugs are fairly large (molecular weight around 7000) molecules composed of monomeric building blocks, the cost of the monomers is a key driver in the ultimate cost of the active pharmaceutical ingredient. Therefore, monomers with intractably complex syntheses, or those for which there is no cheap source of starting materials, are likely doomed to fail for use in oligonucleotide drugs. This should not be overemphasized, as process chemists can work magic on even the most challenging syntheses, but it should not be ignored when choosing modifications to invest in for potential therapeutic use.

6.3 OLIGONUCLEOTIDE MODIFICATIONS 6.3.1

Potential Sites of Modification

A dinucleotide depicting subunits that may be modified to enhance oligonucleotide drug properties is shown in Figure 6.1. Modifications may be made to the backbone (including complete replacement of the sugar phosphate backbone), sugar, and heterocycle portion of the nucleic acid monomer. Additionally, many diverse moieties have been conjugated to various positions in the subunits, mainly in an attempt to alter the pharmacokinetic properties of the oligomer. There are approximately 25 positions for each dinucleotide that do not directly interfere with Watson–Crick base-pair hydrogen bonding. Most of these positions have been modified in at least one way, and studied for their effects on the properties of the resulting oligonucleotides. We highlight certain modifications and review their important characteristics in the following sections, divided according to structural class.

5′ end

4′

X

1′

Base (A, C, G, T)

5′

Linkage

3′

2′

O S P O O

R

Sugar

O O

3′ end

Conjugates

Base

Phosphorothioate

Figure 6.1

Base

R 2′-Substitution

Positions for modification on an oligonucleotide dimer subunit.

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Backbone Modifications

Because the phosphodiester backbone of DNA and RNA is extremely sensitive to ubiquitous nucleases, it is poorly suited for therapeutic utility. The backbone was therefore a necessary and obvious first target for improvement with chemical modification. As a result, extensive medicinal chemistry research focused on the oligonucleotide backbone in order to find modifications that increased nuclease resistance and maintained or improved affinity and specificity to target RNA. Secondary goals included retaining the ability to efficiently recruit RNase H and improve pharmacokinetic properties in animals. The following sections highlight some of the more important backbone modifications that have found use in antisense technology.

6.3.2.1 Furanose-Containing Phosphate Backbones Phosphorothioate (PS) Phosphorothioate-containing oligonucleotides (Figure 6.2) represent by far the most widely used backbone modification for antisense drugs. The properties of the first-generation PS oligodeoxynucleotide antisense drugs have been extensively reviewed in a previous volume [6]. These differ from natural nucleic acids in that one of the nonbridging phosphate oxygen atoms is replaced with a sulfur atom. This substitution of a sulfur for oxygen in the phosphate ester confers several properties onto oligonucleotides, which are crucial for their use as systemic antisense drugs [7]. Foremost, the PS linkage greatly increases stability to nucleolytic degradation [8], such that PS oligonucleotides possess sufficient stability in plasma, tissues, and cells to avoid metabolism prior to reaching the target RNA after systemic administration to an animal. Secondly, but of importance for antisense applications,

O S P O O Phosphorothioate (PS) O H3C P O O

O S P O S O O

Methylphosphonate

O O P O O O P O O

O

B

O H3B P O O

O

3′-Methylene phosphonate

O

DNA phosphodiester (PO)

O P O O

O Phosphonoacetate

Figure 6.2

Phosphorodithioate

B

Furanose-containing phosphate backbones.

Boranophosphate

HN O P O O N3′→ P5′ phosphoramidate

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PS oligodeoxynucleotides are able to efficiently elicit RNase H cleavage of the target RNA, which is critical for the success of most antisense drugs (see Chapter 2 of this volume). Additionally, the PS modification confers a substantial pharmacokinetic benefit by increasing the binding to plasma proteins, which prevents rapid renal excretion and facilitates binding to other acceptor sites that facilitate uptake to tissues [9]. While greatly increasing the stability of ASOs, PS-modified drugs are still subject to metabolism, and have tissue half-lives of 1–3 days [9], which is suboptimal for a parenterally administered drug. Furthermore, the PS modification reduces affinity for the target RNA (the ultimate biological receptor for ASO drugs) [8], which adversely effects potency. These limits can be effectively addressed with chimeric “gapmer” oligonucleotide designs, which will be discussed in this section. Introduction of each sulfur atom in the phosphate backbone generates a chiral center containing a mixture of diastereomers [10]. It has been reported that the stereoisomers have different and potentially complementary physiochemical properties (one is more stable, the other a better RNase H substrate), thereby prompting research efforts toward the stereo-controlled synthesis of chiral PS oligonucleotides [11]. Progress toward this goal has been reviewed recently [12]. However, thus far it has proven difficult to achieve a stereo-controlled synthesis via cost-competitive methods compatible with the current manufacturing process, which is able to provide antisense drugs at a reasonable cost (see Chapter 14 of this volume). Though often maligned, PS still remains the most successful modification to date in oligonucleotide therapeutics. It is difficult to envision a modification that will be able to completely displace PS from current antisense drugs, and other classes of oligonucleotides often employ PS to achieve the improved properties required for use as drugs.

Phosphorodithioate The phosphorodithioate backbone (Figure 6.2) modification was introduced by Caruthers and coworkers [13–15]. In this case, both nonbridging oxygen atoms of the phosphate linkage are replaced with sulfur. The phosphorus atom is therefore achiral. Though this modification rendered oligonucleotides nuclease-resistant and was found to support the RNase H mechanism of action, it suffered from loss of affinity and difficulty in scalable synthesis. Phosphorodithioate analogs were found to lead to stronger nonspecific protein binding than the PSs [16], which raised toxicity concerns. Recently, an improved synthesis was reported [17]; however, the disadvantages in affinity and protein binding properties have made this modification less desirable.

Boranophosphate In boranophosphate oligodeoxynucleotides, the nonbridging phosphodiester oxygen is replaced with a borane (BH3–) group (Figure 6.2). This DNA modification was introduced by Shaw and coworkers [18–20], and has been recently reviewed [21,22]. The boranophosphate is isoelectronic with phosphodiester, and isosteric with methylphosphonate group. It is considered to be more lipophilic than DNA, but due to the presence of negative charge is still highly water-soluble. There are reports suggesting that the boranophosphate-modified ASOs are considerably more stable to nucleases than natural DNA and slightly more stable than PS oligodeoxynucleotides [20]. Boranophosphate oligodeoxynucleotides have also been found to activate Escherichia coli RNase H and induce cleavage of target mRNA [23,24]. More recently, boranophosphate-modified siRNAs were tested in vitro and were found to be slightly more active than unmodified siRNAs. The level of activity and efficacy depended on the number and the position of boranophosphate residues in the molecule [25]. The lack of improved affinity for RNA targets coupled with the difficulty in chemical synthesis to produce sufficient quantities of boranophosphate-modified ASOs for studies in animals is likely to hinder further development of this modification for oligonucleotide drugs.

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N3 ⬘→P5 ⬘ Phosphoramidates and Thiophosphoroamidates In the phosphoramidate oligodeoxynucleotides, the 3⬘-amino group is substituted for 3⬘-oxygen in the deoxyribose ring and the nucleosides are linked via phosphoramidate monoester linkages (Figure 6.2). Synthesis of these compounds was first reported by Gryaznov and coworkers [26]. Phosphoramidates exhibit both high affinity toward complementary RNA (⌬Tm⬃2.5°C per modification) and high nuclease resistance [27,28]. Although these molecules do not activate RNase H, they have shown encouraging in vivo results. For example, a phosphoramidate-modified ASO was specifically used to down-regulate the expression of c-myc gene [29]. Moreover, in a leukemia model, the phosphoroamidates were found to outperform PS oligodeoxynucleotides via steric blocking of translation initiation [30]. Although possessing some promising properties, this modification was found to have low affinity to intra- and extracellular proteins and decreased acid stability relative to phosphodiester counterparts. To address these issues, thiophosphoramidate linkages were prepared. They were found to be more acid-stable when incorporated into oligonucleotides, and introduction of sulfur in place of one of the nonbridging oxygen atoms did not alter the RNA-binding properties of these compounds [31]. Recently, Wang et al. [32] used the N3⬘→P5⬘ thiophosphoramidate (GRN163) to study telomerase inhibition in vitro and in vivo in multiple myeloma and lymphoma with an oligonucleotide telomerase template antagonist. Oligonucleotide phosphoramidates, especially thiophosphoroamidates modified with various lipid groups, showed significant promise as antitelomerase agents [33]. The relatively well-established synthesis and attractive chemical and biophysical properties, including increased affinity and stability, makes them a promising backbone modification.

Phosphonoacetate, Thiophosphonoacetate, and Phosphonoformate These relatively new analogs discovered by Caruthers and his coworkers [34] possess an acetic acid functional group instead of a nonbridging oxygen atom at the internucleotide linkage. They are chiral at phosphorus and isoelectronic with the DNA at neutral pH [35]. Under physiological pH conditions, these analogs possess a negative charge and form stable, complementary A-like DNA/RNA duplexes. Phosphonoacetate (Figure 6.2), thiophosphonoacetate, and phosphonoformate oligodeoxynucleotides were found to support robust RNase H activity only when incorporated in the wings of gapmer (see Section 6.4, Figure 6.11) designs [36]. Appropriate placement of phosphonoformate within an oligonucleotide leads to improved affinity when paired with complementary RNA. They were highly resistant to snake venom phosphodiesterase, DNase 1, and the nucleases present in HeLa cell extracts. These analogs have been reported to enhance uptake in cell culture as well [36]. The progress in solid-phase synthesis as well as favorable biophysical and biochemical properties warrant further investigation of this modification in animal studies.

3⬘-Methylene Phosphonate 3⬘-Methylene-modified oligonucleotides have the 3⬘-hydroxyl of deoxy ribose replaced with a carbon atom. They are achiral, negatively charged, and isoelectronic with phosphodiesters. The incorporation of this modification in a DNA has been shown to lead to C3⬘-endo sugar pucker [37], which is expected to improve target RNA binding affinity. The synthesis of 3⬘-methylene H-phosphonate (Figure 6.2) nucleotides using modified Arbuzov reaction conditions allowed the synthesis of a number of oligonucleotides [38]. The 2⬘-modified-3⬘-methylene phosphonate–modified oligonucleotides showed excellent binding affinity to the complementary RNA with a ⌬Tm of up to 3.6°C per modification, relative to a PS DNA/RNA duplex. Substitution of this modification at the 3⬘-end rendered the oligonucleotide highly resistant to snake venom phosphodiesterase. Although some promising results have been observed with 3⬘-methylene-modified oligonucleotides, the lack of a robust synthetic method for nucleotide monomers and oligonucleotides has prevented the further advancement of this modification.

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Methylphosphonate Methylphosphonates (Figure 6.2) represent one of the earliest examples of neutral oligonucleotide analogs, and have one of the nonbridging oxygen atoms replaced by a methyl group. Early progress in the standard solid-phase synthesis of these molecules made possible a rigorous evaluation of the biophysical and biochemical properties of methylphosphonate-modified oligonucleotides. While providing high nuclease resistance, this modification does not support RNase H. Additionally, high methylphosphonate content in an oligomer leads to loss in affinity toward its complementary RNA and poor solubility. These shortcomings have made this modification less desirable. However, employing a design of methylphosphonates in the wings and PS DNA in the gap, oligonucleotides were utilized ex vivo for purging autografts in chronic myeloid leukemia [39]. Recently, Miller and his coworker [40] used 2⬘-O-methyl oligonucleotide hairpins containing methylphosphonate linkages against TAR RNA. Placement of the linkage showed a strong positional preference, and the configurations of the methylphosphonate linkages also affected binding affinity, with the Rp,Rp isomer showing significantly higher binding than the Sp,Sp isomer. These studies highlight a potential utility as site-specific modifications to improve nuclease resistance. However, many other modifications will accomplish this task, and the value of methylphosphonates to oligonucleotide drug applications remains unclear.

6.3.2.2 Furanose-Containing Nonphosphate Backbones Amides Amide-based backbones were prepared by De Mesmaeker and colleagues in an attempt to control the charge and chirality of the backbone. The design of these backbones consisted of dimeric nucleosides connected by various amide linkages, such that the resulting oligonucleotide consisted of alternating stretches of phosphodiester and amide linkages. A modest improvement in duplex stability with complementary RNA (⌬Tm of 0.5°C per modification) was observed with two positional isomers of the amide linkage [41,42]. The affinity for complementary DNA was somewhat lower for these amide dimers. It is argued that restricted rotation about the amide bond, with a preference for the trans isomer, preorganizes these backbones into a conformation that gives the oligomer a more favorable A-form-like geometry, which favors base pairing with RNA. Substituting the 2⬘-position in both units of the dimer with 2⬘-O-methyl further biased the conformation of the ribofuranoses to C3⬘-endo, which improved the binding affinity to ⌬Tm of up to 2–3°C per dimer [43]. Because two nucleosides are part of this “modification,” it should be noted that the affinity increase observed is just slightly improved relative to that seen with 2⬘-O-methyl RNA. Additional chemical SAR around the amide backbone was done and a 5⬘-(S )-C-methyl group further rigidified the backbone, which when combined with 2⬘-O-methyl-modified sugars (Figure 6.3) resulted in a

O

O O

O O

B OMe

O

NH

O

5′-(S)-methyl amide-3 Figure 6.3

O

O O

B OMe

B

S

OMe N

O

O

B

O MMI

Furanose-containing nonphosphate backbones.

O

B OMe

O Thioformacetal

B

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⌬Tm of up to 4.4°C per dimer incorporation versus RNA complements [44]. The amide linkages are highly resistant to nucleases, but the stability of the amide linkage is unknown in vivo. Recently, a number of structural studies have been reported for a number of amide backbone analogs using NMR, crystallography, and molecular simulations to understand the SAR between nature, length, and steric properties of these molecules and possible reasons for their contribution to DNA/RNA duplex stability [45,46]. Despite these promising results, no significant biological data have been reported for these modified oligonucleotides. This perhaps is due to the fact that large-scale synthesis has not been achieved. Because of the ability to control charge and completely eliminate degradation due to nucleases while increasing affinity, modifications of this type merit further investigation, particularly in animal studies.

Methylene(methylimino) (MMI) Several nitrogen-containing backbone modifications similar to the amides that are achiral and have five-atom spatial separation between the two sugar residues were evaluated as dimeric nucleosides [47]. The best compounds were identified from biophysical studies as methylene(methylimino) (MMI, Figure 6.3) linkages with a 5⬘–O–NCH3–CH2– linkage and 2⬘–O–methyl or 2⬘–fluoro substitutions on each sugar [48]. It has been suggested that the presence of a 3⬘–C–C bond in the MMI linkage reduces the O–4⬘ and C–3⬘ gauche interaction compared to the natural phosphodiester backbone linkage. The data unambiguously reveal that combination of an appropriate 2⬘-modification with a neutral and achiral linkage provides oligomers with high affinity for an RNA target. For example, 2⬘–O–methyl or 2⬘–fluoro substituted MMI linkages increased the affinity for complementary RNA as much as 3.2 to 3.7°C ⌬Tm per modification. Because these modifications contain no phosphodiester, it is obvious that they will not be degraded at the modified backbone by nucleases. However, it was found that the dimer structures also stabilized the internal phosphodiester such that it was not degraded as well. This allows a molecule free of chirality and PS to be prepared, which carries only 50% of the charge of the parent. Unfortunately, none of the backbones studied to date support RNase H, relating their use for antisense applications to gapmer designs or stericblocking-type mechanisms. MMI-modified gapmers were found to be very potent in an RNase H enzymatic assay as well as reducing target mRNA of PKC-α and H-Ras in vitro. However, their in vivo activity was similar to 2⬘-methoxyethylribose (MOE) gapmers (unpublished results). Moreover, more rapid clearance and lower tissue levels of drug were observed for MMI ASOs with more than 50% MMI content. This can be attributed to a reduced number of PS linkages, which leads to decreased plasma protein binding and increased renal excretion.

Formacetal and Thioformacetal A very simple yet interesting backbone substitution is the formacetal, which replaces the phosphate diester with a methylene unit, and the related thioformacetal (Figure 6.3), which also replaces the 3⬘-side oxygen with a sulfur atom [49]. The formacetal-linked oligonucleotides bind in a sequence-specific manner, but with less affinity compared to unmodified oligonucleotides, but the thioformacetal linkage increases affinity slightly with dimeric incorporations and is neutral when incorporated as a thioformacetal pentamer [50]. The NMR structure of these duplexes has been studied and the stabilities rationalized based on the structure, which indicated that the thioformacetal adopted a northern sugar pucker [51]. An in vivo therapeutic application of a formacetal-modified oligonucleotide aptamer has been reported [52]. A systematic positional SAR was conducted that led to the selection of a modified aptamer with four formacetal groups that increased anticoagulant effect combined with extended half-life in monkeys, when compared to the parent aptamer. This result suggests that other rationally designed neutral backbones that are known to increase binding affinity and in vivo nuclease

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stability might be useful for other aptamers selected for binding to therapeutically relevant protein targets.

Other Nonphosphate Backbones There are numerous other backbone modifications that contain guanidine, silyl, sulfone, sulfoxide, urea, carbamate, sulfide, etc. that are not discussed in this chapter, either because of lack of recent progress or perceived value for use in oligonucleotide drugs. Most have been covered to some extent in one or more of several reviews, and the interested reader is referred to these reviews [22,53–55].

6.3.2.3 Sugar and Backbone Replacements Morpholino Phosphorodiamidate Phosphorodiamidate morpholino oligonucleotides (Figure 6.4) are a class of backbone modification that has a morpholine ring as a replacement for the furanose, with a phosphorodiamidate linkage connecting the morpholine nitrogen atom with the hydroxyl group of the 3⬘-side residue. Because of the phosphorodiamidate linkage, morpholino oligonucleotides are neutral. These modifications are similar in affinity to DNA/DNA duplexes, and are nuclease-stable. However, they do not activate RNase H, and are primarily used in translation arrest or other steric blocking mechanisms such as alteration of splicing, where they have shown activity in animal models [56,57]. Morpholinos have advanced to human clinical trials and pharmacokinetic data were reported [58]. Morpholino oligonucleotides have been reviewed [59–61], and are covered in depth in Chapter 20 of this volume.

Hydroxyproline Backbones Use of a hydroxyproline sugar replacement in two different contexts has resulted in the trans-4-hydroxy-pyrrolidine-2-phosphonic acid (pHypNA) and HypNA-pPNA (Figure 6.4), which consists of a hydroxyproline-based monomer alternating with a phosphono-peptide nucleic acid monomer [62,63]. These analogs are stable to nucleases and maintain affinity for target nucleic acids; however, they are not strongly stabilizing and do not activate RNase H. They have been used in steric blocking mechanisms, where interesting results have been reported in cell culture [64,65].

(5′)

(5′)

O O N N P O O

(5′)

O B

N

N

O O

O

NH B

O

B

N

Morpholino

Sugar and backbone replacements.

O

NH B

N O O

Figure 6.4

NH B

B

N

O

O

P O

PNA (peptide nucleic acid)

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Peptide Nucleic Acid (PNA) Peptide nucleic acids (Figure 6.4) are a radically different class of oligonucleotide analogs that contain a peptide replacement for the sugar phosphate backbone, yet maintain the ability to Watson–Crick base pair with complementary RNA and DNA [66]. First introduced by Nielsen and coworkers [67], PNAs are highly resistant to degradation by nucleases and proteases and are very stabilizing when paired with RNA and DNA. PNAs do not activate RNase H, and as such have been used in translation inhibition [68,69] and splicing modulation antisense mechanisms [57,70]. Recently, PNAs have been utilized to both activate [71] and inhibit transcription [72]. PNA and its properties are covered in depth in Chapter 18 of this volume. Despite their uncharged nature, PNAs do not cross cell membranes readily, and the cell culture data generated with PNAs have required methods to deliver them to cells. PNA oligonucleotides also have generally poor drug properties for in vivo applications. They have very low solubility and are very poorly protein-bound. These limitations have been addressed by conjugation of short peptides [73,74] and use of charged amino acids [75] in the PNA backbone, which serves to increase solubility. Importantly, the short peptides employed were able to dramatically improve tissue distribution. In a follow-up study from these advances [76], PNAs targeting a splice junction of PTEN, which alter PTEN splicing and reduce protein, were conjugated to 8mer lysine peptides. Compound administered at 40 mg/kg twice weekly for 3 weeks showed little activity in liver and kidney, which was surprising given the high drug concentrations achieved (108 and 408 ␮M in liver and kidney, respectively). In contrast, robust activity was observed in adipose tissue, with an alteration of splice products observed concomitant with a dose-dependent down-regulation of PTEN protein. As in liver and kidney, a relatively high drug concentration of 79 ␮M was achieved. A comparison of estimated EC50 values in adipose tissue between PNA and MOE ASOs shows that the MOE ASO is far superior, with an EC50 of around 1 ␮M, as opposed to the 70–80 ␮M value for the PNA. This ⬎50-fold difference in potency suggests that many hurdles remain for PNA oligonucleotide drugs. 6.3.3

Sugar Modifications

6.3.3.1 2 ⬘-Modifications To date, modifications to the 2⬘-position of the sugar moiety have provided the most value in the quest to enhance oligonucleotide drug properties. Preorganization of the sugar into an RNA-like 3′-endo pucker, or northern conformation in the furanose pseudorotation cycle [77], is responsible for the increased binding affinity. Furthermore, the proximity of the 2⬘-substituent to the 3⬘-phosphate in an oligonucleotide causes 2⬘-modified oligonucleotides to generally increase nuclease resistance. As such, 2⬘-modification generally adds two desirable properties to oligonucleotides with a single modification. Unfortunately, essentially all 2⬘-modifications greatly reduce or completely inhibit the ability of RNase H to cleave the RNA strand opposite the modification, which restricts the use of 2⬘-modifications for antisense purposes. This limitation has been minimized by use of a gapmer strategy, where regions of 2⬘-modified residues flank a central DNA region of the oligonucleotide. The 2⬘-modified “wings” thus serve to increase affinity and nuclease resistance, while the central gap region allows RNase H–mediated cleavage of the target RNA. Similar limitations are currently in the process of being elucidated for the use of 2⬘-modified nucleosides in siRNA duplexes, which must utilize the RNAi machinery. Considerably more flexibility is afforded for use in oligonucleotide drugs that do not require a terminating mechanism.

2⬘-Fluoro Modifications The increase in affinity observed with 2⬘-modifications is energetically driven by the electronegative substituent at the 2⬘-position [78]. As such, the 2⬘-fluoro (Figure 6.5) modification

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B

O O

O

B

O O

F

155 O

B

O O

O

O O

O

2′-Fluoro O

B

O O

MOE (2′-O-methoxyethyl)

O

FANA

B

B

O

O O

HN O N

DMAOE

O O

O

OCH3

2′-O-methyl

B

O

O

O

Figure 6.5

NMAc

F O

O

HN

N DMAEAc

Sugar 2⬘-modifications.

imparts the highest binding affinity (⌬Tm ⬃ 2°C per modification, relative to DNA) for target RNA of the 2⬘-class of modifications [5]. The 2⬘-fluoro substitution also improves stability relative to RNA. These favorable properties have led to substitution of 2⬘-fluoro for RNA nucleosides in the design of the aptamer Macugen, currently marketed for wet age-related macular degeneration [79]. The 2⬘-fluoro modification has also been employed in the design of duplex siRNA oligonucleotides [80–82]. This substitution has allowed for the complete elimination of RNA from siRNAs, providing duplexes with increased stability and potency that act via activation of the RNAi pathway [83,84]. For antisense applications, since 2⬘-fluoro does not activate RNase H or improve nuclease stability beyond DNA, its use has been limited predominantly to cell culture [85,86]. The 2⬘-fluoro substituent has also been evaluated in the arabino configuration, which places the fluorine atom on the same face of the sugar as the base moiety to provide 2⬘-deoxy2⬘-fluoro-D-arabinonucleic acid (FANA, Figure 6.5) [87]. The arabino 2⬘-fluoro configuration also displays an enhancement of affinity toward complementary RNA, but is about half that observed with the ribo-2⬘-fluoro configuration. Structural studies have shown that FANA residues assume an O4⬘-endo pucker (eastern conformation) in B-DNA and a predominantly northern conformation in the context of an A-form duplex, and have rationalized the affinity enhancement based upon stereoelectronic conformational preorganization [88,89]. Uniformly modified FANA oligonucleotides have been shown to support RNase H–mediated cleavage of an RNA complement; however, the cleavage rates are reduced and antisense activity in mammalian cells was not observed for the uniformly modified FANA oligonucleotides [90]. However, a gapmer strategy using FANA wings surrounding a DNA gap increased both the affinity for an RNA target and the RNase H cleavage rates relative to a uniform DNA oligonucleotide, and the antisense potency in a luciferase reporter system was substantially improved [90]. These results have been extended to an endogenous target for both FANA gapmers and designs that employ alternating blocks of 3 FANA and DNA residues [91]. The utility of the FANA modification in siRNA duplexes has also been investigated. The optimal design consisting of a uniformly modified FANA sense strand and FANA overhangs on the antisense strand maintained potency and improved serum stability [92]. No in vivo studies employing FANA designs of this nature have been reported to date; so although promising, the ultimate value of FANA is still unknown.

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2⬘-O-Methyl and Methoxyethyl (MOE) 2⬘-O-Alkyl groups improve binding affinity to a lesser degree than the 2⬘-fluoro nucleosides, but impart a substantial degree of nuclease resistance to the resulting oligonucleotide. The use of 2⬘-O-methyl nucleosides (Figure 6.5) in siRNA holds promise in both the sense [93] and antisense strands. The steric requirements of the RNAi machinery for recognition of the antisense strand appear to restrict the use of bulkier modifications, while 2⬘-O-methyl is often well tolerated [94]. Minimal use of 2⬘-O-methyl nucleosides has been employed to stabilize siRNAs for successful in vivo proof of concept experiments [95,96], and to minimize off-target effects [97,98]. Gapmer designs employing 2⬘-O-methyl ASOs have been well investigated [85], and these designs have advanced to human clinical trials [99]. However, the overall profile of 2⬘-O-methyl gapmer ASOs is less appealing than that of other modifications currently in development. The 2⬘-O-methoxyethyl (MOE, Figure 6.5) modification is currently the most advanced of the 2⬘-modified series, and has entered clinical trials for multiple indications. MOE increases Tm by about ⫹2°C per modification versus RNA, relative to DNA, and greatly increases resistance to nucleases. It also appears to reduce certain nonspecific protein binding, which can reduce toxicities. MOE oligonucleotides have unique structural features evident from structural studies, which help explain the properties of MOE oligonucleotides [100]. MOE substitution at the 2⬘-position induces a C3⬘-endo (northern) conformation of the sugar, and the methoxyethyl substituent assumes a gauche orientation that traps water in a shell of hydration that includes the adjacent phosphate residue. This further increases rigidity of the C3⬘-endo sugar conformation, and the preorganization of the oligonucleotide into an A-form geometry is thought to largely account for the increased affinity for RNA. The increased nuclease resistance is most likely due to steric hindrance imparted by the methoxyethyl substituent combined with the shell of hydration created by the bound water. These fundamental properties of MOE have profound beneficial effects on the potency, pharmacokinetics, and toxicology of MOE containing antisense oligonucleotides, which are extensively reviewed in Chapters 10–14 of this volume. Systematic optimization of the design of MOE ASOs can further improve potency, which is reviewed in Chapter 17 of this volume, and optimization of other oligonucleotide drug classes such as aptamers and siRNA duplexes using MOE chemistry is expected to have similarly beneficial effects.

Lipophilic 2⬘-O-Alkyl Modifications A range of lipophilic 2⬘-O-alkyl groups have been investigated to varying extents in attempts to improve the pharmacokinetic properties of oligonucleotides. While the nuclease resistance of a given 2⬘-modification generally increases with increasing steric bulk, the affinity for target RNA decreases. The 2⬘-O-propyl modification carved out somewhat of a middle ground, in that it modestly increased both affinity and nuclease resistance, and the increased lipophilicity is presumably responsible for a demonstrated improvement in the plasma protein binding of fully phosphodiester 2⬘-O-propyl oligonucleotide [101]. However, the ultimate potency and nuclease resistance achieved were not sufficient to outperform 2⬘-O-methyl or MOE as ASO drugs. In an attempt to improve the protein binding, while maintaining the increased affinity and nuclease resistance of MOE, a methylthio ether version of MOE was tested. This compound improved protein binding as expected, maintained affinity, but showed greatly reduced nuclease resistance when compared to MOE. This was attributed to loss of organization of the side chain as demonstrated by structural analysis [102]. In a study comparing the effects of various lipophilic 2⬘-substituents, the affinity and nuclease resistance of propyl, butyl, 2-(fluoro)ethyl, 2-(trifluoro)ethyl, allyl, propargyl, and 2-(benzyloxy)ethyl 2⬘-O-substituents were compared and rationalized by crystallographic studies [103]. All groups improved affinity, with the largest gain due to the 2-(fluoro)ethyl side chain, which adopts

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a conformational preorganization due to the gauche effect of the electronegative fluorine atom. The lowest level of nuclease resistance was found in the propargyl analog, followed by the bulky 2-(benzyloxy)ethyl derivative. The structural data are consistent with a stacking interaction of the aromatic benzyl group with the furanose ring of the adjacent 3⬘-side residue. This moves the side chain away from the phosphate linkage, and presumably reduces nuclease resistance due to decreased steric interactions. A 2⬘-O-2,4-dinitrophenyl substitution has been reported to provide enhanced affinity for target, increased nuclease resistance, and result in remarkable activities both in cell culture and in animals [104–107]. The nature of the way the modification has been prepared makes confirmation of these findings difficult, as the composition is prepared via reaction of an enzymatically prepared RNA with 1-fluoro-2,4-dinitrobenzene to generate a mixture of compounds with approximately 70% derivatization of the RNA 2⬘-position. Furthermore, the reported effects have been primarily against oncology targets and endpoints, no data against endogenous gene targets in normal animals has been reported, and no pharmacokinetic properties of the 2,4-dinitrophenyl RNA have been described. Thus, while the early data is encouraging and substitutions of this type are interesting, rigorous characterization of the drug substance and thorough pharmacokinetic/pharmacodynamic (PK/PD) relationships must be obtained for this modification to advance toward use in oligonucleotide therapeutics.

Cationic 2⬘-O-Alkyl Modifications The cationic 2′-O-aminopropyl [108] and corresponding dimethylaminopropyl version [109] have shown favorable binding affinity, with dramatically improved nuclease resistance. In an attempt to extend the increased nuclease resistance of these cationic modifications to the higher affinity seen with MOE, the terminal methoxy group of MOE was replaced with a guanidinium [110], and imidazole [103], or a (dimethylamino)ethoxy group to give a 2⬘-O-[2-[N,N-(dimethyl)aminoethoxy]ethyl] (DMAEOE) functionality [111]. These modifications display hybridization properties equal or superior to those of MOE, and nuclease resistance equal to that of the 2⬘-O-aminopropyl modification. However, in unpublished work from our laboratory, we have found that incorporation of DMAEOE nucleosides into the 5⬘ and 3⬘ wings of an active MOE gapmer did not translate to improved activity in animals. Despite increasing the concentration delivered to kidney, we found little to no inhibition of gene expression in kidney, whereas the corresponding MOE ASOs reduced target as expected. Whether this translates to other cationic modifications in other tissues remains to be determined. The less basic (dimethylaminooxy)ethyl (DMAOE, Figure 6.5) [112] substituent, which contains a hydroxylamine functionality, increased both nuclease resistance and affinity for RNA, yet maintained activity both in vitro and in vivo [113]. This suggests that cationic modifications inhibit antisense activity in animals, in spite of their improved affinity and nuclease resistance profiles. A conformation similar to that assumed by MOE can also be achieved by utilizing a carbamoylmethoxy or acetamide functionality [114]. These modifications have been prepared in both unsubstituted, methylated (NMAc, Figure 6.5), dimethylated, and the cationic dimethylaminoethylated (DMAEAc, Figure 6.5) versions [115]. As expected, the cationic version increased nuclease resistance the most. All substitution patterns increased affinity for RNA, with the NMAc having the best overall profile. In unpublished work from our laboratory, a gapmer substituting NMAc for MOE was found to be equipotent in vitro and in vivo to MOE in reducing PTEN mRNA in mouse liver. Additionally, NMAc has been found to substitute for MOE on the sense strand of siRNA designs (unpublished results). These results suggest that the acetamido 2⬘-modification has intriguing possibilities in both antisense and other oligonucleotide drug discovery efforts. As any amine can be used to form the amide on a nucleoside precursor, a diverse array of functionality can be introduced onto 2⬘-O-acetamido nucleosides, further illustrating the potential versatility of this substitution.

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6.3.3.2 Other Furnanose Substitution Positions In comparison to the 2⬘-modifications, comparatively little success has been had with other furanose modifications for antisense applications. Substitutions at the various carbon positions on the sugar ring have been investigated. 4⬘-C-methyl [116], hydroxymethyl [117,118], methoxymethyl, and aminomethyl [119,120] substitutions in general decrease affinity for target RNA, increase nuclease resistance substantially, favor a southern sugar conformation, and inhibit RNase H activity. Combination of 4⬘-C-aminomethyl with a 2⬘-fluoro substituent (Figure 6.6) provided an analog with increased affinity for both RNA and DNA targets, while maintaining the high level of nuclease resistance [121]. Substitutions at the 3⬘-C-position increase nuclease resistance somewhat, decrease binding affinity, and are inhibitory to RNase H activity [116,122,123]. Modification of the 5⬘-C-position with a methyl group has been shown to increase nuclease resistance and be modestly destabilizing; however, the compounds tested were diastereoisomeric mixtures [124], so the effects of the individual methyl isomers cannot be judged. Groups larger than methyl such as 5⬘-allyl have been prepared in pure form, and it was found that both isomers greatly improved nuclease stability relative to deoxy, and were about equally destabilizing [119]. 5⬘-C-aminomethyl, hydroxymethyl, and methoxymethyl analogs were also prepared in the (S) configuration, and found to be only slightly destabilizing in a mixed sequence having eight modifications spaced relatively evenly in a 19mer DNA parent sequence. The resulting sequences were highly stable to nucleases, and the 5⬘(S)-C-hydroxymethyl analog (Figure 6.6) supported cleavage of a duplex by RNase H [119]. The greatly increased nuclease stability of the 5⬘-C-substituted analogs, combined with the minimal disruption to binding affinity and overall structure of the nucleic acid underscores the potential of this class of modification for improving oligonucleotide drug properties.

6.3.3.3 Bicyclic Sugars Much effort has been invested in restricting the conformation of nucleosides via the conformational restraint introduced in making bicyclic nucleosides. As with traditional small-molecule drug optimization efforts, the rewards for correctly constraining a ligand are large entropic gains in binding affinity. This is most evident with the 2⬘,4⬘-bicyclic nucleic acids (2⬘,4⬘-BNAs) such as locked nucleic acid (LNA) and its analogs.

Locked Nucleic Acid (LNA, 2⬘,4⬘-BNA) The sugar modification showing the largest known improvement in binding affinity is a bicyclic system having the 4⬘-carbon tethered to the 2⬘-hydroxyl group, known as 2⬘,4⬘-BNA by Imanishi [125,126], and LNA by Wengel [127–129], who published shortly thereafter (Figure 6.7). This modification can also be thought of as a constrained analog of 2⬘-O-methyl RNA, where the 2⬘substituent is tethered to the 4⬘-C atom. This enforces a northern sugar pucker, which is essentially O

O O

B

O

HO

B

H2N O

F

4′-C-aminomethyl (with 2′-Fluoro)

Figure 6.6

Sugar modifications.

O 5′-(S)-hydroxymethyl

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O

O B

O

B

O

LNA (2′,4′-BNA) O

O

-L- LNA

O O

2′,4′-BNANC

B

O

ENA

O B

O NO

Figure 6.7

O O

O

O

O

159

O H O

B

O Oxetane 2′,3′-BNA

H

B

O

Tricyclo-DNA

Bicyclic sugars (BNAs).

identical to that adopted by A-form RNA and has been confirmed by structural analysis [130–132]. LNA shows dramatically improved hybridization properties relative to a DNA/RNA duplex, and improves nuclease resistance. The use of LNA in siRNAs has resulted in improved stability [133,134], and decreased immune stimulation [135], and LNA ASOs have successfully inhibited microRNA function in cell culture [136]. LNA has also been investigated for use in aptamer applications, as LNA/DNA hybrids were able to replace RNA in a TAR aptamer [137]. Replacement of specific nucleotides in the Tenascin-C binding aptamer TTA1 with LNA provided a molecule with improved target binding, stability in plasma, in vivo pharmacokinetics, and uptake to tumor [138], clearly demonstrating the potential of LNA to improve the in vivo properties of oligonucleotide aptamers. LNA has been reviewed in 2004, and many applications are highlighted [139]. Since uniform LNA oligonucleotides do not support RNase H [140], the most promising antisense applications of LNA employ a gapmer strategy. In a recent study, LNA gapmers targeted to H-Ras were able to reduce tumor growth with dosage levels of 1 mg/kg per day for 11 days of treatment, in the absence of toxicity as measured by plasma transaminase levels [141]. However, in an earlier study employing uniform LNA ASOs, transaminase levels were increased at a 5 mg/kg daily dose after 14 days [142]. In order to examine the effects on endogenous target genes, we examined the effects of LNA gapmers targeting mouse TRADD, PTEN, and ApoB in liver [143], and compared their potency and therapeutic index with the corresponding MOE gapmers. In some, but not all cases, the LNA gapmers increased potency, up to five- to tenfold over the MOE ASOs. However, they also showed profound hepatotoxicity as measured by serum transaminases, organ weights, and body weights. This toxicity was evident for multiple LNA sequences targeting each of the different biological targets, as well as in mismatch control sequences having no known mRNA targets. For example, the best LNA ASO targeting TRADD showed an ED50 of about 2 mg/kg when dosed twice weekly for 3 weeks. However, this ASO caused about fivefold and ⬎50-fold increases in liver transaminases at doses of about 5 and10 mg/kg, respectively. In contrast, a MOE ASO showed an ED50 of 13 mg/kg, and no toxicity at doses of over 40 mg/kg, which gave a ⬎86% reduction in target. These studies suggest that while LNA ASOs have the potential to improve antisense potency, they impose a significant risk of hepatotoxicity, which must be considered when designing LNAcontaining antisense therapeutics. The mechanism of the hepatotoxicity, as well as the sequence dependence of this toxicity, is unknown. LNA oligonucleotides are nearing human clinical trials, and will be reviewed in greater detail in Chapter 19 of this volume.

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␣-L-LNA After the discovery of LNA, the Wengel group investigated the duplex stability of the eight possible stereoisomers of LNA by preparing four diastereomeric isomers, and inferring the properties of the other four enantiomers by comparing their hybridization properties to both D-RNA and L-RNA [144]. This work resulted in the surprising observation that ␣-L-LNA (Figure 6.7), which contains the 2⬘,4⬘-bridge on the same side of the sugar as the nucleobases, along with an inversion of the 3⬘-hydroxy, has nearly the same affinity for target RNA as does LNA. All other isomers either did not hybridize, or were significantly reduced in affinity, relative to LNA. Subsequent structural studies showed ␣-L-LNA to assume a DNA-like southern conformation, and form H-form duplexes with target RNA [145,146]. These observations suggest that the dogma that a northern sugar pucker is the major determinant of high binding affinity is not necessarily correct, and that conformational preorganization is a major factor. Despite being locked into a southern conformation, ␣-L-LNA does not support RNase H, and has been utilized successfully in gapmer ASO designs [147]. ␣-L-LNA shows increased nuclease resistance over LNA, and evaluation of an ␣-L-LNA version of the H-Ras sequence for reduction of tumor growth in a xenograft model found it to be slightly more effective than LNA [141]. To date, the toxicological properties of ␣-L-LNA are unknown, and the potential of this interesting modification will only be understood with continued rigorous in vivo studies.

Other 2⬘,4⬘-Bicyclic Sugars Related to LNA are other classes of 2⬘,4⬘ linked BNAs such as 2⬘-amino and 2⬘-thio LNA [148]. These analogs were also studied in the H-Ras sequence, and found to be slightly less effective than LNA [141]. The tissue distribution was different, highlighting the ability to alter distribution by modulating charge and lipophilicity. Ethylene nucleic acid (ENA, Figure 6.7) contains a two-carbon-atom bridge as opposed to a single atom bridge, has similar affinity, and improved nuclease resistance relative to LNA [149]. ENA has shown antisense activity in cell culture in gapmer designs [150,151] and as uniformly modified ASOs working via a steric blocking mechanism [152]. Several other 2⬘,4⬘ BNA analogs have been reported, which include additional atoms [153] or rearrangement of the oxygen and carbon atoms [154], both of which reduce hybridization relative to LNA and ENA, but are still stabilizing. Substitution of a hydroxylamino moiety to give a 2⬘,4⬘-BNA having a 2⬘-O–N–C bridged system (2⬘,4⬘-BNANC, Figure 6.7) increased affinity slightly over LNA, and increased nuclease resistance [155]. As such, this modification appears superior to LNA in its biophysical properties.

1⬘,4⬘-Bicyclic Sugars A bicyclo[3.2.1]octane system employing a 1⬘-C,4⬘ bridge adopted an O4⬘-endo furanose conformation. The 1⬘-CH2–O–CH2-4⬘ bridging group adopted a chair-like conformation of the resulting 1,4-dioxane ring. It was modestly destabilizing to RNA, having a ⌬Tm of –1.5°C per modification [156]. This was ascribed in part to steric clashes involving the dioxane bridging ring, and highlights the unique properties of the 2⬘,4⬘-bicyclic structures in improving hybridization properties to DNA and RNA targets.

1⬘,2⬘-Bicyclic Sugars Oxetanes formed by 1⬘-C,2⬘-O bridges are highly destabilizing when paired to RNA, having a ⌬Tm of around ⫺5°C per modification for the pyrimidines, and do not support RNase H cleavage near the modification site. Interestingly, the purines were essentially neutral when paired to RNA complements, relative to the DNA. This is despite all nucleosides having a north-eastern conformational bias [157]. Not surprisingly, these modifications did not improve potency over PS oligodeoxynucleotides [158].

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2⬘,3⬘-Bicyclic Sugars Both oxetane and tetrahydrofuran 2⬘-O,3⬘-C bridged arabinose derivatives, which have the bridging group on the alpha-face of the nucleoside, have been reported [159]. The oxetane analog (Figure 6.7) adopts an eastern-like O4⬘-endo sugar conformation in B-DNA [160], and showed an increase in affinity for target RNA, with a ⌬Tm of ⬃1°C per modification. It was destabilizing by about ⫺1°C versus DNA targets. The tetrahydrofuran, predicted to assume a C1⬘-exo conformation was highly destabilizing.

3⬘,5⬘-Bicyclic Sugars Analogs of DNA utilizing 5⬘-amino nucleosides and a P3⬘→N5⬘ phosphoramidite linkage are known to be destabilizing. This was corrected by constraint via a 3⬘-C,5⬘-N methylene bridge to provide a 5⬘-amino 3⬘,5⬘-BNA, which was neutral when paired to DNA and moderately stabilizing when paired to RNA [161]. A bicyclic DNA analog having a 3⬘-C,5⬘-C bridged bicyclo[3.3.0] octane system (bicycloDNA) [162] has been elaborated to a tricyclic analog having a spirocyclopropyl ring at the 5⬘-C position (tricyclo-DNA, Figure 6.7) [163]. This system has been shown to adopt a DNAlike southern conformation, increase affinity for complementary DNA and RNA, and increase nuclease resistance. Use in a mixed sequence gave an increase in Tm of over 2°C per modification versus an RNA target, and this same sequence improved potency over a 2⬘-O-methyl ASO in a splicing application in cell culture [164]. Uniformly modified tricyclo-DNA oligonucleotides from 11 to 15 bases in length were four- to fivefold more potent than the corresponding LNA oligonucleotides, despite having lower affinity for the complementary RNA by about 25°C [165]. This result was rationalized by the observation of differential distribution within cells by fluorescence microscopy after transfection. Because of these observations, tricycloDNA is an interesting modification that merits further investigation in animal studies for its antisense effects.

Other Bicyclic Sugar Modifications Early efforts at constraint of oligonucleotide monomers focused on the use of small rings to enforce a predominant sugar conformation. Carbocyclic nucleosides having cyclopropyl bridges on either side of the methylene substituted for the furanose oxygen atom were found to be slightly stabilizing with a 1⬘,6⬘ bridge, and slightly stabilizing with a 4⬘,6⬘ bridge [166–168]. Oxetane constrained bicyclic nucleosides having a 3⬘-O,4⬘-C bridging group involving the C3⬘ hydroxyl adopt a southern-type conformation [169]. When incorporated into oligonucleotides using a 2⬘,5⬘-linkage, they are modestly destabilizing to RNA with multiple incorporations, but can be stabilizing with single incorporations. The modification was highly destabilizing when paired with DNA, and increased nuclease resistance. However, somewhat surprisingly, the increase was not dramatic [170]. An isomeric alpha-L version was also highly destabilizing [171]. A trans-3⬘-C,4⬘-C bridged structure, which is restricted to a southern conformation, was strongly destabilizing when paired with RNA or DNA [172].

6.3.3.4 Substitutions for the Ribofuranose Sugar 4⬘-Thioribose A simple substitution of sulfur for the furanose oxygen in RNA or DNA provides 4⬘-thio nucleic acids. 2⬘-Deoxy-4⬘-thioribose was modestly stabilizing, and improved resistance to endo, but not 3⬘-exo nucleases [173]. Regardless of the properties conferred upon an oligonucleotide, the use of

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2⬘-deoxy-4⬘-thioribose for oligonucleotide therapeutics would be unwise given the cellular toxicity of the nucleoside monomers. In contrast, the 4⬘-thioribose modification (4⬘-thio-RNA, Figure 6.8) is not known to suffer these limits. It has been prepared originally by Imbach. They showed that phosphodiester 4⬘-thio-RNA oligomers improved hybridization to RNA targets, and were significantly more resistant to cleavage by serum nucleases than unmodified phosphodiester RNA [174,175]. Crystallography studies have shown that 4⬘-thio-RNA hybridizes with complementary RNA sequences to form stable duplexes with standard A-form helical geometry [176]. As other nucleoside modifications incorporating sulfur have shown improved binding to plasma proteins, it is also possible that 4⬘-thio-RNA incorporated into oligonucleotides could alter pharmacokinetic properties, but to date, no in vivo studies have been reported. These properties make 4⬘-thio-RNA a structurally similar yet nuclease-resistant analog of wildtype RNA and hence attractive for applications in oligonucleotide drug optimization efforts. Accordingly, 4⬘-thio-RNA has been examined for applicability to siRNA applications, and shown to maintain [177] or slightly improve activity and stability [178] when utilized in certain contexts. 4⬘-Thio-RNA forms exceptionally stable duplexes when paired with a complementary 4⬘-thio-RNA strand [179]. This property, combined with the nuclease resistance profile, was exploited to optimize the stability of an aptamer [180].

Other Sugar Analogs Having Five-Membered Rings Substitution of the furanose oxygen atom with nitrogen and carbon has been explored to a lesser extent than 4⬘-thio-RNA. In general, carbocyclic neucleosides either reduce or do not substantially change affinity, depending upon the nature and location of other substituents [181,182]. Substitution of the methylene replacing the furanose oxygen with acylated aminosugars was strongly destabilizing [183]. Nuclease resistance was generally increased for both azasugars and carbocycles, but the profile of these modifications, especially given the difficulty of synthesis, did not merit continued investment. A threofuranosyl-(3→2⬘)-linked nucleic acid analog (TNA) has been prepared [184], which is stabilizing when paired with RNA. The structure of this duplex has been studied and provides a rationale for the affinity [185]. Additional phosphoramidite analogs of the TNA system are essentially neutral when paired with RNA, relative to an RNA/DNA duplex [185]. None of the TNA analogs have been investigated for antisense applications to date.

Sugar Analogs Having Six-Membered Rings Due to their increased conformational rigidity relative to furanoses, hexoses have the potential to have superior hybridization properties, and would be expected to be highly nuclease-resistant. Pyranosyl sugar–derived nucleic acids have been studied and reviewed extensively by Eschenmoser [186]. They often display excellent Watson–Crick base-pairing properties that have been rationalized by structural studies [187], but do not hybridize well to DNA or RNA. As such, they have not been explored for antisense applications of oligonucleotide drugs. However, they could have potential utility in aptamer-derived oligonucleotide drugs.

O

O S

O

B OH

4′-Thioribose

Figure 6.8

Sugar substitutions.

O O

O

B HNA

O O

O

B OCH3

2′-O-methyl ANA

O

B CeNA

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In contrast, a family of hexitol-derived nucleic acids, pioneered and reviewed by Van Aerschot [188] and Herdewijn [189], hybridizes well to RNA. The lead modification in this family, termed hexitol nucleic acid (HNA, Figure 6.8) forms very stable duplexes with RNA (⌬Tm ⬃ ⫹3°C per modification), and is selective for RNA over DNA. HNA also has clear mismatch discrimination, and shows a high level of resistance to nucleases. HNA/RNA duplexes are poor substrates for RNase H, but antisense activity has been shown both in translation arrest or steric blocking mechanisms [190], and in gapmer motifs [191]. HNA has also been used to improve the properties of an anti-TAR aptamer [192]. An NMR structure of an HNA duplex has been solved, and its surprising similarity to A-form RNA structures provides insights into the mode of binding enhancement [193,194]. Analogs of HNA that have an additional hydroxyl in an axial (altritol derived sugar, ANA) conformation display improved hybridization to RNA, but those with the hydroxyl in an equatorial position (mannitol-derived sugar) show very poor affinity. Methylation of the hydroxyl group to provide 2⬘-O-methyl-ANA (Figure 6.8) further improved affinity (⌬Tm ⬃ ⫹4°C per modification), and simplified synthesis, providing the most promising hexitol nucleoside to date based on hybridization properties versus RNA targets [195]. Cyclohexenyl analogs of HNA (termed CeNA, Figure 6.8) contain an ethylene unit substituted for the furanose oxygen atom in DNA. They have been found to be modestly destabilizing toward DNA, and stabilizing when paired with RNA [196]. This has been attributed to flexibility introduced by the cyclohexenyl system as opposed to a more rigid cyclohexane system, and the flexibility demonstrated in an NMR study [197]. CeNA has been shown to support RNase H cleavage of an RNA complement, though at a high enzyme concentration and low efficiency. It is therefore likely that CeNA would have to be utilized in a gapmer motif for antisense applications. Therefore, because of lower affinity and complex synthesis relative to other sugar modifications, its potential utility is limited. 6.3.4

Heterocyclic Modifications

Historically, very few heterocycle modifications that increase nuclease resistance have been found. As such, modification of the heterocycle has focused mainly on increasing binding affinity for complementary nucleic acids. This may be accomplished through increasing stacking interactions, or by increasing the strength of the Watson–Crick base pairs. Preserving Watson–Crick hydrogen-bonding interactions is of critical importance for the recognition of the receptor by antisense drugs, and for maintaining the proper structure of aptamer-based drugs. This prohibits alteration of the hydrogen bond donor–acceptor footprint of the bases. Furthermore, substitution near the glycoside bond often disrupts the glycosidic torsion angle, which usually results in poor hybridization. These considerations place severe design constraints on modification to the heterocycles, such that there are very few positions on the heterocyclic bases that can be productively modified. It is therefore not surprising that in spite of more than two decades of significant research effort, there has been only modest progress in identifying a base-modified chemistry that demonstrated meaningful and safe in vivo pharmacology in animal models. In the following sections, we highlight some key heterocyclic modifications to highlight their unique properties, potential utilities, and known liabilities. A number of comprehensive review articles regarding heterocyclic modified oligonucleotides have been published, and we refer the interested reader to these for more in-depth coverage [198–200].

6.3.4.1 Pyrimidine 5⬘⬘-Position Modifications C5 Alkyl and Halogen Substitution The C5 position has been one of the most common places for chemical manipulations on pyrimidine heterocycles, as modification of this site has little effect on Watson–Crick base pairing. Furthermore, substitution at the 5-position places the modification directly above the 5⬘-side

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heterocyclic base. This suggests substitutions that would be expected to enhance the base stacking properties should provide increases in affinity. For example, a simple modification such as substituting C5 hydrogen of deoxycytidine with a methyl group (Figure 6.9) improves the Tm of the DNA/RNA duplex by about 0.5°C per substitution [5] while a slightly larger group such as 5-ethyl was destabilizing [201]. Because of the affinity increase of the 5-methyl group, combined with reduction in immunostimulatory toxicities [202], 5-methylcytosine is broadly used in place of cytosine for most modified “C” nucleosides. The effect on affinity of a halogen substitution at C-5 position in pyrimidines is similar to methyl. For use in oligonucleotide therapeutics, however, 5-halopyrimidines should not be considered due to the chemical reactivity and biological activities of the bases and nucleosides.

Propynes Since the stacking interactions between the planar heterocycles of nucleic acids are in large part responsible for the stability of DNA and RNA duplexes [203], enhancing them with additional aromatic or ␲-rich surfaces is a logical way to increase duplex stability, and presumably affinity for a target RNA. This prompted substituting the C5 position with a propynyl group (Figure 6.9) to enhance the stacking interactions in duplex helices [204]. Enhanced duplex stability (⌬Tm ⬃ ⫹2°C per modification) and potent gene-specific inhibition was achieved in vitro using T- and C-rich PS

O

O

NH

NH N

N

O

5-Propynyluracil NH2

N

N

N

O

N

5-Methylcytosine

N

N

N

N

N

N

NH2

N N

NH2

N N

NH2

NH

O

NH N

O

5-Thiazolylcytosine

O

NH

Guanine NH2 N2-Aminopropylguanine Figure 6.9

N

NH O

O NH

N

O

Phenoxazine

O

NH2

O

N N

O

5-Thiazolyluracil S

5-Propynylcytosine

Diaminopurine

Adenine

N

N

NH2

NH2 N

N

O

NH

O

NH2

Cytosine

N

N

NH2

N

N

S

O

N

NH

2-Thiothymine

Thymine

S

O

The DNA bases and selected heterocyclic modifications.

N N

O

G-Clamp

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oligodeoxynucleotides poly-substituted with 5-propynyl nucleosides [205,206]. Mechanistic studies implicated RNase H cleavage and intracellular dissociation rates as the limiting factors in activity [207]. A length SAR study led to the discovery that poly propynylated ASOs as short as seven nucleotides in length could exert potent antisense effects [208]. Though this extremely short length has potential negative implications in terms of specificity for the target gene, there are many benefits including cost of manufacture. The presence of C-5 propynyl pyrimidine bases did not interfere with the RNase H cleavage kinetics but were found to be more toxic than unmodified counterparts when tested in vitro [209]. Monia and coworkers evaluated the effect of the propyne modification both in vitro and in vivo [210]. They found that 5-propynyl pyrimidine modification, whether incorporated as a deoxy nucleoside or as a MOE nucleoside, showed substantial increase in affinity toward complementary RNA target. However, the in vitro data show only a moderate increase in potency for a propyne PS oligodeoxynucleotide ASO targeting murine PTEN. No such improvement was observed with MOE gapmer ASOs containing 5-propynyl substitution, and all propynyl-modified ASOs were found to have similar or reduced potency as compared to the parent MOE gapmer. in vivo studies paralleled this result, where the MOE gapmer ASO was the most potent compound, and was not outperformed by any configuration of propyne ASO. Furthermore, the propynyl pyrimidine containing PS oligodeoxynucleotide induced severe liver toxicity after a single administration at 20 mg/kg and was lethal at 50 mg/kg. The hepatotoxicity was reduced by using a gap design ASO in which the C-propynyl group was placed only in MOE-modified nucleotides; however, potency was not increased, and toxicity was still evident. Given the lack of improvement in in vivo activity coupled with the severe in vivo toxicity, it is likely that C5 propynyl-modified oligonucleotides will not be useful in oligonucleotide drugs.

Thiazoles Several 5-heteroaryl-substituents that are coplanar with the pyrimidine ring have been shown to possess duplex-stabilizing properties due to increased stacking interactions [211]. Similar to the propynes, 5-thiazolo-substituted oligonucleotides (Figure 6.9) showed high affinity (⌬Tm about ⫹1.7°C) for complementary RNA, compared to DNA. The nature of the heterocycle is important, as protons adjacent to the biaryl linkage cause twisting of the C5 aromatic group out of plane, which decreases the stacking interaction and reduces the gain in affinity. Whether these modifications share the same toxicity profile with propynes is unknown, but it is possible they would have a different profile due to the differences in electronic effects between the alkyne and the thiazole heterocycle. However, unlike propynes and phenoxazines described below, they do not support RNase H or show activity in cell culture when incorporated throughout the oligonucleotide [207]. This discrepancy is as yet unexplained, but limits the use of thiazoles to gapmer designs where other modifications such as 2⬘- and bicyclic modifications adequately increase affinity. The utility of the thiazoles is therefore likely to be limited, regardless of their toxicity profile, unless they are additive in Tm with 2⬘-modifications.

6.3.4.2 Tricyclic Cytosine Analogs Phenoxazine Cytosine analogs containing a lipophilic phenothiazine or phenoxazine (Figure 6.9) substitution were synthesized and incorporated into ASOs by Matteucci and coworkers [212]. These tricyclic compounds were shown to specifically pair with guanosine, and increased affinity dramatically (⌬Tm ⫹6°C). Incorporation of 4-phenoxazine bases in a 7mer PS oligonucleotide targeting Tag expression showed a five-fold increase in relative binding affinity for its RNA target [213]. RNase H was found to cleave the tag RNA opposite to phenoxazine dimers. Most importantly, the

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phenoxazine containing ASO had activity in cell culture without the aid of delivery methods, giving an IC50 of 5 ␮M. Fluorescence labeling experiments found the uptake to be length-dependent, and showed nuclear staining only with phenoxazine-containing ASOs. These experiments suggest that lipophilicity and size of the oligonucleotide are important for uptake into cells, concepts well established among small-molecule medicinal chemists. Whether these observations translate to improved activity in vivo is unknown, and no animal results have been described in the intervening years since publication of the initial reports.

G-Clamp The success with phenoxazine led to the addition of an aminopropyl-substituted phenoxazine termed G-Clamp (Figure 6.9), which as a single incorporation in a 10mer sequence increased Tm by an astounding 18°C [214]. This affinity did not come at the expense of selectivity, as shown with mismatch studies. A model where the aminopropyl group forms an additional hydrogen bond to the major groove side of guanine in a C:G base pair was proposed to rationalize the results. A single substitution of a G-clamp into a 15mer PS oligonucleotide was found to enhance antisense activity compared to propynyl-modified ASO when tested in CV1 cells [215]. Furthermore, the G-clamp increased potency 25-fold when substituted into a 20mer PS oligodeoxynucleotide. The G-clamp modification, like the propynes and phenoxazines, is reported to support RNase H. The synthesis and subsequent structural studies on guanidinium-modified G-clamp has been reported [216]. The guanidinium G-clamp analog increased affinity in a similar manner as the original version, and structural data supported the major groove side hydrogen-bonding model originally proposed by Matteucci. The synthesis of 2⬘-O-methyl and MOE modified G-clamps along with their in vitro activity has been reported [217,218]. The analogs both improved potency in cellculture applications. As with the phenoxazine, whether this improved activity in cell culture will translate to improved in vivo activity remains unknown.

6.3.4.3 Other Pyrimidine Modifications The replacement of oxygen at the C2 and C4 positions of thymine have been investigated [219,220]. Sulfur is bulkier, more polarizable, and is a weaker hydrogen-bond acceptor compared to oxygen. As a result, it increases stacking interactions. Substitution of sulfur for oxygen at C4 of thymine gave a modest improvement in duplex stability, which is surprising, given the expected loss of a hydrogen-bond acceptor. Incorporation of 2-thiothymidine (Figure 6.9) leads to sequencedependent stabilization of the duplex. This is probably a combination of enhancement of stacking interactions, and an effect on sugar conformation, as the 2⬘-thio substitution imposes a C-3⬘-endo (northern) pucker. Combination of the 2-thio and a sugar-2⬘-modification such as 2⬘-fluoro or MOE improved thermodynamic stability and nuclease resistance, and the effects rationalized by crystallography studies [221]. No in vitro or in vivo activity has been reported to date.

6.3.4.4 Purine Modifications Diaminopurine The 2-amino-2⬘-deoxyadenosine modification employs a diaminopurine base (Figure 6.9), which donates a third hydrogen bond and is therefore analogous to a GC base pair [222,223]. The resulting increase DNA/RNA duplex stability is reflected in increases in Tm of between 1.4°C and 1.8°C per incorporation over the unmodified wild-type duplex [224]. Diaminopurine unfortunately increases the rate of depurination under acidic conditions, which limits the practical utility of this otherwise interesting modification.

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N2-Substituted Guanine Both an N2-imidazolylpropyl [225] and N2-aminopropyl [226] guanine analogs (Figure 6.9) showed a duplex stabilization (⌬Tm⫹1°C to⫹3.3°C) with a single substitution. The cationic group is believed to lie in the minor groove, making key new contacts with the phosphate backbone.

Deazapurines Several heterocyclic modifications have been reported in deaza series where a nitrogen atom was replaced with a carbon atom. These modifications did not improve affinity to any significant degree. However, the propynyl substitution on 2-amino-7-deaza-adenosine stabilizes duplexes considerably [227]. Increased RNA-binding affinity has also been reported for oligonucleotides incorporating 7-propynyl-7-deazadeoxy adenosine or 7-propynyl-7-deazadeoxyguanosine [228]. Despite these improvements, the use of 7-deazapurines carries a large toxicity risk, due to the known toxicities of this class of nucleosides. Furthermore, the synthesis of these compounds is laborious, making the preparation of quantities suitable for evaluation in animals difficult. 6.3.5

Conjugates

In attempts to alter and improve the pharmacokinetics of oligonucleotide drugs, many conjugation strategies have been investigated. In the simplest form, pegylation of an oligonucleotide to increase the size sufficiently to prevent renal filtration can dramatically improve plasma half-life. This is likely to be broadly useful for applications (such as aptamers) where the active compartment is in the plasma, but does not improve distribution to tissues. Conjugation of oligonucleotides to ligands that can bind to various acceptor sites would be expected to alter the pharmacokinetic properties of the oligonucleotide, and many attempts have been made to increase tissue and cellular uptake of oligonucleotide drugs via conjugation of various ligands. We will briefly address some recent applications of conjugated oligonucleotides, and refer the interested reader to comprehensive review articles for more in-depth coverage [229,230].

6.3.5.1 Cholesterol Conjugates Cholesterol conjugates of oligonucleotide are one of the most studied classes of conjugates. Cholesterol conjugation has been clearly shown to increase the exposure of an ICAM-1 targeted PS oligonucleotides to liver, with a concomitant reduction in exposure to kidney [101]. The cholesterol conjugate maintained activity in cell culture, and showed activity in vivo at doses where the unconjugated oligonucleotide had no effect [229]. This is consistent with improved distribution to liver. In subsequent pharmacokinetic studies, the uptake to liver endothelial cells was shown to be greatly increased, presumably due to increased binding to LDL for the cholesterol-conjugated oligonucleotide [231,232]. A bis-cholesterol conjugated oligonucleotide was found to lead to almost complete hepatic uptake of the oligonucleotide [233]. More recently, intravenously administered cholesterol-conjugated anti-microRNA oligonucleotides (Figure 6.10) resulted in inhibition of mirRNA function for the liver expressed miR-122. In this case, the cholesterol conjugation allowed use of a mostly phosphodiester oligonucleotide (2 or 3 PSs were used at each end for stability), which would be expected to have poor tissue uptake in the absence of the cholesterol conjugate [234]. Also described were broad systemic effects of the oligonucleotide in tissues other than liver, which is somewhat inconsistent with previous studies using fully PS cholesterol–conjugated oligonucleotides. The observed distribution of cholesterol-conjugated oligonucleotide to tissues other than liver is likely due to a combination of the phosphodiester backbone with the high (80 mg/kg) intravenous dose. Since the presence of high tissue concentrations of

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O O O

5′

H (CH2)5 N

O

O

O P

S

N

O

O OH

3′-Cholesterol-conjugated microRNA inhibitor O

O N H

O OH

S P

O O HN S P O O

B

O

HN

B 12

H

GRN163L Figure 6.10 Design of lipophilic conjugates.

antisense oligonucleotide makes northern blot detection of the target microRNA technically problematic, the broad reduction of microRNA observed in many tissues should be interpreted with caution until verification of microRNA reduction can be achieved by another method. In a similar study, cholesterol conjugation was used to demonstrate activity of a duplex siRNA drug in mice. Conjugation of cholesterol at the 3⬘ end of sense strand of a duplex siRNA and intravenous administration gave reduction of apolipoprotein B (ApoB) mRNA in liver and jejunum, as well as the expected decrease in plasma levels of ApoB protein and cholesterol [96]. The nonconjugated ApoB siRNA was inactive. While clearly important for establishing proof of concept for the utility of chemical modifications to enable siRNA activity in vivo, the generality of this approach is unknown. Furthermore, cholesterol conjugation raises concerns regarding toxicity, and increased toxicity of cholesterol-conjugated oligonucleotides has been observed, although it is possible that this is due to increased tissue exposure [235]. While cholesterol conjugation may have utility as research tools, it is our opinion that cholesterol conjugation is unlikely to be broadly useful for oligonucleotide drugs.

6.3.5.2 Fatty Acid Conjugates In addition to cholesterol, other lipid groups can alter distribution of oligonucleotides in animals. Similar to cholesterol, but to a lesser degree, modification with fatty acid chains (C18 in this study) increased exposure to liver [101]. Lipid modification with a palmitoyl (C16) functionality has been shown to enhance the potency of a 13mer telomerase inhibitor (Figure 6.10) in cell culture and in vivo [236]. Increased levels of telomerase inhibition in xenograft tumor were demonstrated for the lipid conjugate, which correlated with increased drug levels relative to the unconjugated analog. This suggests that the appropriate lipophilic groups can influence uptake, distribution, and potency of oligonucleotides in vivo. Although detailed structure–activity relationships of various lipid conjugates have not been reported, nor have toxicology studies, we expect simple fatty acid conjugation to be useful for oligonucleotide drugs, especially for improving the pharmacokinetics of shorter oligonucleotides, or those with reduced PS content.

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6.4 OPTIMIZING OLIGONUCLEOTIDE DRUGS 6.4.1

General Strategies

It is extremely likely that no one modification will be found that confers optimal properties upon an oligonucleotide. By analogy, it would be unreasonable to expect one chemical substitution (a chlorine atom, for example) to confer increased potency, and improved pharmacokinetic properties upon a range of small molecules. Just as with traditional medicinal chemistry applied to small molecules, a successful oligonucleotide medicinal chemistry optimization must utilize various chemical modifications together, at the appropriate position in the oligonucleotide to achieve the desired outcome. For antisense oligonucleotide modifications, a heavy emphasis has often been given to biophysical properties such as affinity and stability to 3⬘-exonucleases such as snake venom phosphodiesterase. A modification that “passes” these tests was then substituted into a gapmer and a uniform design, and tested in cell culture. Seldom was the oligonucleotide optimized to suit the modification, which may help explain the often disappointing results and lack of a clear SAR. Furthermore, the endpoints must be sufficiently robust and relevant to provide a meaningful SAR set to guide the optimization process. The traditional path has been biophysical studies, followed by cell culture experiments, followed by in vivo studies with a few limited motifs. In general, we feel that time and effort spent studying model systems (such as complex reporter systems, cell free assays, cell uptake systems, etc.) does not get one closer to understanding the structural features responsible for activity in a system more relevant to improving drug properties (potency, pharmacokinetics, toxicity). Once a modification has demonstrated acceptable basic physiochemical properties (reasonable hybridization properties, for example), in vivo studies should be carried out with as many motifs as possible as soon as possible. Preferred endpoints for this optimization are drug levels and target reduction in liver, and measures of toxicity such as organ weights, body weights, and plasma transaminases. This type of approach is increasingly more tractable due to the availability of medium-scale synthesizers (like the Pharmacia AKTA) that consume low levels of amidite excess, such that less than about a gram of amidite can supply enough oligonucleotide for a first-pass mouse experiment. Murine TRADD, PTEN, and ApoB have been excellent systems for studying ASO designs using this paradigm. 6.4.2

Oligonucleotide Design Strategies

6.4.2.1 Gapmer Designs Current second-generation antisense drugs are combinations of three different monomer types: a PS backbone, a deoxyribose gap, and MOE wings (Figure 6.11). Each modification serves a purpose, and the placement is appropriate for that purpose. The 2⬘-deoxy gap region allows for the recruitment of RNase H by the oligonucleotide/target RNA duplex. The PS stabilizes the molecule to degradation by nucleases, and confers a fairly broad protein-binding character onto the molecule. This, among other things, prevents filtration by greatly increasing binding to plasma proteins. The MOE further increases nuclease resistance and stability in tissues, decreases toxicities, and increases binding affinity for the target RNA. These combined properties greatly improve potency and make the second-generation ASO platform very promising. This is exemplified by the performance of ISIS-301012 in early clinical trials, where it showed a dose dependent reduction of ApoB protein concurrent with lowering of LDL cholesterol. Doses as low as 100 mg/week produced statistically significant reductions in ApoB protein, and a dose of 200 mg/week reduced serum ApoB protein by 50% [237]. Second-generation MOE oligonucleotides are discussed in detail in Chapters 10–14 of this volume. This fairly simple combination of modifications can be further optimized for a specific utility by varying the size of the gap region, and/or the length of the molecule to improve activity, as discussed in Chapter 17 of this volume.

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3′ Wing

Gap

GCCTC AGTCTGCTTC GCACC HO

O S P O O

O

O

O

O

B O OCH3 O

O

B O

O S P O O

O

B

O O

B

O S P O O

O OCH3 O

B HO

OCH3 MOE

B O OCH3

DNA

MOE

Figure 6.11 Gap design of ISIS-301012, a second-generation antisense oligonucleotide.

6.4.2.2 siRNA Designs To date, two general strategies of chemical modification have been employed when optimizing siRNA for systemic in vivo activity. The first approach aspires to remove as much RNA from the molecule as possible to maximize stability. This has been employed in a viral hepatitis model to show activity in reducing viral titer [81,82]. The design consisted of all 2⬘-fluoro and 2⬘-deoxy residues in the sense strand, which was further stabilized with inverted deoxy abasic residues at each end. This was combined with a heavily 2⬘-fluoro- and 2⬘-O-methyl-modified antisense strand that contained only a few RNA residues at the 5⬘ end. To improve exonuclease stability, PS linkages were employed at the 3⬘-end of the antisense strand. In a slightly different approach, the RNA has also been completely eliminated from the duplex siRNA by using alternating 2⬘-fluoro/ 2⬘-O-methyl motifs [83]. These designs improved potency in cell culture and stability, but no in vivo results have yet been reported. The second general approach is a more minimal modification strategy, where an siRNA lead is modified with 2⬘-O-methyl phosphorothioates at the 3⬘-ends to protect against exonucleases, and with 2⬘-O-methyl groups at precise sites within the duplex to protect against endonucleases. This strategy, when combined with a conjugation approach, gave activity in vivo [96]. Minimal 2⬘-Omethyl modification to specific locations in the sense and antisense strand have also been used to decrease the off-target effects of siRNAs [97]. Determining whether chemically modified siRNA designs will provide an acceptable therapeutic index with systemic administration at pharmacologically relevant doses requires designs that provide robust activity in animals. None of the current siRNA designs achieve this goal without the use of lipid formulations, and poor pharmacokinetic properties appear to be responsible for the observed discrepancy between potencies in cell culture and in animals. Detailed pharmacokinetic studies with modified siRNAs will be required to understand how to best optimize siRNA designs for in vivo activity. The properties of duplex RNA drugs such as siRNAs will be reviewed in detail in Chapters 16 and 17 of this volume.

6.4.2.3 Occupancy Only Designs Mechanisms that rely primarily on binding to target RNA include translation arrest, alteration of splicing, and inhibition of microRNA function. These mechanisms do not require activation of a termination event, and as such, the many high-affinity modifications that do not support terminating mechanisms can be applied in the optimization effort. This opens up design possibilities employing multiple modifications to improve potency and pharmacokinetics. In practice, however, most reports have focused on oligonucleotides uniformly substituted with a single modification.

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As PNA and morpholino oligonucleotides do not induce a terminating event, they have been extensively used to both modulate splicing and inhibit translation. PNA and morpholino ASOs are reviewed in Chapters 18 and 20 of this volume, respectively. Uniform MOE ASOs have been shown to modulate splicing [238], and inhibit translation [239]. Additionally, a uniform MOE ASO was shown to redirect polyadenylation to form a more stable mRNA that altered protein expression [240]. It is also possible to use antisense strategies to inhibit microRNAs, which are negative regulators of gene. In a recent application, a uniformly modified MOE PS inhibited the function of a microRNA in liver, causing reductions in cholesterol as well as expected changes in gene expression [241]. In a related cell culture study, multiple uniform, and some motifs containing multiple modifications were examined for their ability to inhibit a microRNA, and a clear SAR emerged suggesting that affinity as well as steric bulk were important factors [242]. It seems unlikely that any uniform modification strategy is optimal for all occupancyonly mechanisms, but detailed SAR studies employing multiple modifications have not been reported.

6.4.2.4 Aptamer Designs Aptamers have different design criteria than other oligonucleotides, as they must maintain binding to a target driven by three-dimensional structure, not Watson–Crick base pairing. Therefore, maintaining the three-dimensional structure is crucial during the optimization process. A common strategy is substitution of nucleosides in the stem structure with higher affinity and stabilizing modifications such as 2⬘-fluoro, 2⬘-O-methyl, LNA, or 4⬘-thioribose. To address pharmacokinetics, pegylation has been employed to prevent clearance of the aptamer from the desired compartment. Macugen (pegaptanib) is a result of this strategy, as it is pegylated, heavily modified with 2⬘-fluoro and 2⬘-O-methyl nucleosides, and contains only 2 unmodified RNA residues [79]. The use of the many modifications known to modulate the protein binding and stability of oligonucleotides holds promise for the development of aptamers having improved properties, including the ability to penetrate tissues and access intracellular targets. Aptamers are discussed in detail in Chapter 28 of this volume.

6.5 OUTLOOK Which modifications and designs hold the most promise for use in oligonucleotide drugs? Despite the common misperception that PS is something that should be engineered out of an oligonucleotide drug, it remains the backbone modification that has done the most to advance antisense technology. PS linkages improve stability while maintaining the ability to elicit RNase H activity, and contribute protein-binding properties that prevent rapid excretion and facilitate uptake to tissues. The most advanced sugar modification is MOE, which improves nuclease stability, affinity for target RNA, and can decrease toxicities. When combined with PS DNA using a gapmer design strategy, these properties produce molecules that display robust pharmacodynamic and pharmacological effects in animals, a goal that remains elusive for most other oligonucleotide drug classes. The success of this second-generation antisense design suggests that both MOE and PS will add substantial value to other oligonucleotide drug classes. Although less valuable for traditional antisense applications than MOE, 2⬘-fluoro and 2⬘-Omethyl have already been employed in the aptamer Macugen (pegaptanib), and are also likely to be useful for siRNA designs. Another sugar modification class that holds much promise is the BNAs, and LNA in particular. The large increases in affinity of the 2⬘,4⬘-BNAs such as LNA, combined with their increased stability to nucleases, makes them potentially important in many oligonucleotide drug applications. Although LNA oligonucleotides have entered clinical trials, some early experiments on the safety of LNA oligonucleotides in rodents raise concerns. It remains to be seen

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whether the toxicity profile of LNA is ultimately determined to be acceptable for broad use in oligonucleotide drugs. It should also be noted that there are many analogs of LNA in the 2⬘,4⬘-BNA class that have not been extensively investigated, and may not share this toxicity risk if it is found to be a liability. Less likely to be valuable are the heterocyclic modifications such as propynes, which have very significant toxicity liabilities, and G-clamp and other modifications that have unknown properties in vivo. Sugar replacements such as 4⬘-thioribose and HNA have interesting physiochemical properties, but appear to add little advantage over modifications such as MOE and the 2⬘,4⬘-BNAs for traditional antisense designs. Whether they will have utility in siRNAs, aptamers, or other drug classes remains to be seen. Neutral backbone modifications such as MMI have not progressed in recent years as in vivo studies on a few analogs have shown that they do not increase potency over simpler 2⬘-modifications such as MOE. Sugar-phosphate backbone replacements such as PNA and morpholino, continue to attract high levels of interest, but appear to be less potent than second-generation designs in vivo. This could be a result of less favorable pharmacokinetics, or not having an efficient terminating mechanism, or a combination of both. Lastly, modulating the protein-binding properties of oligonucleotides with simple lipophilic conjugates is likely to find utility; however, conjugate-directed cell and tissue targeting of oligonucleotides is not likely to succeed in therapeutic applications due to the complexity of designs and limitations of receptor-mediated uptake. Regardless of which modifications and designs ultimately prove useful, there is a promising future for oligonucleotide drugs, and therefore a need for oligonucleotide medicinal chemistry. It is clear that many good nucleic acid drug targets exist. In addition to traditional oligonucleotide drug targets such as coding RNAs, which continue to be exploited, new RNA targets are emerging. This is exemplified by the discovery of microRNAs, an entirely new class of RNAs, which are endogenous regulators of gene expression. Currently, the only practical way to “drug” these targets in a specific manner is using oligonucleotides. Thus, the application of sound medicinal chemistry principles to improve the potency and profile of oligonucleotide drugs holds much promise.

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189. Herdewijn, P., Conformationally restricted carbohydrate-modified nucleic acids and antisense technology, Biochim. Biophys. Acta, 1489, 167–179, 1999. 190. Vandermeeren, M. et al., Biological activity of hexitol nucleic acids targeted at Ha-ras and intracellular adhesion molecule-1 mRNA, Biochem. Pharmacol., 59, 655–663, 2000. 191. Kang, H. et al., Inhibition of MDR1 gene expression by chimeric HNA antisense oligonucleotides, Nucleic Acids Res., 32, 4411–4419, 2004. 192. Kolb, G. et al., Hexitol nucleic acid–containing aptamers are efficient ligands of HIV-1 TAR RNA, Biochemistry, 44, 2926–2933, 2005. 193. Lescrinier, E. et al., Solution structure of a HNA-RNA hybrid, Chem. Biol., 7, 719–731, 2000. 194. Maier, T. et al., Reinforced HNA backbone hydration in the crystal structure of a decameric HNA/RNA hybrid, J. Am. Chem. Soc., 127, 2937–2943, 2005. 195. Van Aerschot, A. et al., Improved hybridisation potential of oligonucleotides comprising O-methylated anhydrohexitol nucleoside congeners, Nucleic Acids Res., 29, 4187–4194, 2001. 196. Wang, J. et al., Cyclohexene nucleic acids (CeNA): serum stable oligonucleotides that activate RNase H and increase duplex stability with complementary RNA, J. Am. Chem. Soc., 122, 8595–8602, 2000. 197. Nauwelaerts, K. et al., Cyclohexenyl nucleic acids: conformationally flexible oligonucleotides, Nucl. Acids Res., 33, 2452–2463, 2005. 198. Herdewijn, P., Heterocyclic modifications of oligonucleotides and antisense technology, Antisense Nucleic Acid Drug Dev., 10, 297–310, 2000. 199. Revenkar, G. R. and Rao, T. S., DNA with Altered Bases, in Comprehensive Natural Products Chemistry, Barton, D. H. R., Nakanishi, K., and Kool, E. T., eds., Pergamon Press, Oxford, 1999, pp. 313–339. 200. Sanghvi, Y. S. et al., Antisense oligodeoxynucleotides: synthesis, biophysical and biological evaluation of oligodeoxynucleotides containing modified pyrimidines, Nucleic Acids Res., 21, 3197–3203, 1993. 201. Sagi, J. et al., Destabilization of the duplex and the high-salt Z-form of poly(dG-methyl5dC) by substitution of ethyl for the 5-methyl group, Int. J. Biol. Macromol., 13, 329–336, 1991. 202. Henry, S. et al., Chemically modified oligonucleotides exhibit decreased immune stimulation in mice, J. Pharmacol. Exp. Ther., 292, 468–479, 2000. 203. Devoe, H. and Tinoco, I., Jr., The stability of helical polynucleotides: base contributions, J. Mol. Biol., 4, 500–517, 1962. 204. Froehler, Brian C. et al., Oligodeoxynucleotides containing C-5 propyne analogs of 2⬘-deoxyuridine and 2⬘-deoxycytidine, Tetrahedron Lett., 33, 5307–5310, 1992. 205. Wagner, R. W. et al., Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines, Science, 260, 1510–1513, 1993. 206. Moulds, C. et al., Site and mechanism of antisense inhibition by C-5 propyne oligonucleotides, Biochemistry, 34, 5044–5053, 1995. 207. Gutierrez, A. J. et al., Antisense gene inhibition by C-5-substituted deoxyuridine-containing oligodeoxynucleotides, Biochemistry, 36, 743–748, 1997. 208. Wagner, R. W. et al., Potent and selective inhibition of gene expression by an antisense heptanucleotide, Nat. Biotechnol., 14, 840–844, 1996. 209. Wagner, R. W., Structure-activity relationships in cell culture, Ciba Found. Symp., 209, 142–154; discussion 154–157, 1997. 210. Shen, L. et al., Evaluation of C-5 propynyl pyrimidine-containing oligonucleotides in vitro and in vivo, Antisense Nucleic Acid Drug Dev., 13, 129–142, 2003. 211. Gutierrez, A. J. et al., 5-Heteroaryl-2⬘-deoxyuridine analogs. Synthesis and incorporation into highaffinity oligonucleotides, J. Am. Chem. Soc., 116, 5540–5544, 1994. 212. Lin, K.-Y., Jones, R. J., and Matteucci, M., Tricyclic 2⬘-deoxycytidine analogs: syntheses and incorporation into oligodeoxynucleotides which have enhanced binding to complementary RNA, J. Am. Chem. Soc., 117, 3873–3874, 1995. 213. Flanagan, W. M. et al., Cellular penetration and antisense activity by a phenoxazine-substituted heptanucleotide, Nat. Biotechnol., 17, 48–52, 1999. 214. Lin, K.-Y. and Matteucci, M. D., A cytosine analog capable of clamp-like binding to a guanine in helical nucleic acids, J. Am. Chem. Soc., 120, 8531–8532, 1998. 215. Flanagan, W. M. et al., A cytosine analog that confers enhanced potency to antisense oligonucleotides, Proc. Natl. Acad. Sci. USA., 96, 3513–3518, 1999.

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216. Wilds, C. J. et al., Structural basis for recognition of guanosine by a synthetic tricyclic cytosine analogue: Guanidinium G-clamp, Helv. Chim. Acta, 86, 966–978, 2003. 217. Sazani, P., Astriab-Fischer, A., and Kole, R., Effects of base modifications on antisense properties of 2⬘-O-methoxyethyl and PNA oligonucleotides, Antisense Nucleic Acid Drug Dev., 13, 119–128, 2003. 218. Holmes, S. C. and Gait, M. J., The synthesis of 2⬘-O-methyl G-clamp containing oligonucleotides and their inhibition of the HIV-1 Tat-TAR interaction, Nucleos. Nucleot. Nucleic Acids, 22, 1259–1262, 2003. 219. Connolly, B. A. and Newman, P. C., Synthesis and properties of oligonucleotides containing 4-thiothymidine, 5-methyl-2-pyrimidinone-1-beta-D(2⬘-deoxyriboside) and 2-thiothymidine, Nucleic Acids Res., 17, 4957–4974, 1989. 220. Newman, P. C. et al., Incorporation of a complete set of deoxyadenosine and thymidine analogues suitable for the study of protein nucleic acid interactions into oligodeoxynucleotides. Application to the EcoRV restriction endonuclease and modification methylase, Biochemistry, 29, 9891–9901, 1990. 221. Diop-Frimpong, B. et al., Stabilizing contributions of sulfur-modified nucleotides: crystal structure of a DNA duplex with 2⬘-O-[2-(methoxy)ethyl]-2-thiothymidines, Nucleic Acids Res., 33, 5297–5307, 2005. 222. Howard, F. B. and Miles, H. T., 2NH2A X T helices in the ribo- and deoxypolynucleotide series. Structural and energetic consequences of 2NH2A substitution, Biochemistry, 23, 6723–6732, 1984. 223. Lamm, G. M. et al., Antisense probes containing 2-aminoadenosine allow efficient depletion of U5 snRNP from HeLa splicing extracts, Nucleic Acids Res., 19, 3193–3198, 1991. 224. Gryaznov, S. M. and Schultz, R. G., Stabilization of DNA:DNA and DNA:RNA duplex by substitutionof 2⬘-deoxyadenosine with 2⬘-deoxy-2-aminoadenosine, Terahedron Lett., 36, 2489–2492, 1994. 225. Ramasamy, K. S. et al., Remarkable enhancement of binding affinity of heterocycle-modified DNA to DNA and RNA. Synthesis, characterization and biophysical evaluation of N2-imidazolylpropylguanine and N2-imidazolylpropyl-2-aminoadenine modified oligonucleotides, Tetrahedron Lett., 35, 215–218, 1994. 226. Manoharan, M. et al., Oligonucleotides bearing cationic groups: N2-(3-aminopropyl)deoxyguanosine. Synthesis, enhanced binding properties and conjugation chemistry, Tetrahedron Lett., 37, 7675–7678, 1996. 227. Balow, G. et al., Biophysical and antisense properties of oligodeoxynucleotides containing 7-propynyl-, 7-iodo- and 7-cyano-7-deaza-2-amino-2⬘-deoxyadenosines, Nucleic Acids Res., 26, 3350–3357, 1998. 228. Buhr, C. A. et al., Oligodeoxynucleotides containing C-7 propyne analogs of 7-deaza2⬘-deoxyguanosine and 7-deaza-2⬘-deoxyadenosine, Nucleic Acids Res., 24, 2974–2980, 1996. 229. Manoharan, M., Oligonucleotide conjugates in antisense technology, in Antisense drug technology: Principles, strategies, and applications, Crooke, S. T., ed., Marcel Dekker, New York, 2001, pp. 391–470. 230. Manoharan, M., Oligonucleotide conjugates as potential antisense drugs with improved uptake, biodistribution, targeted delivery, and mechanism of action, Antisense Nucleic Acid Drug Dev., 12, 103–28, 2002. 231. Bijsterbosch, M. K. et al., Delivery of cholesteryl-conjugated phosphorothioate oligodeoxynucleotides to Kupffer cells by lactosylated low-density lipoprotein, Biochem. Pharmacol., 62, 627–633, 2001. 232. Bijsterbosch, M. K. et al., Modulation of plasma protein binding and in vivo liver cell uptake of phosphorothioate oligodeoxynucleotides by cholesterol conjugation, Nucleic Acids Res., 28, 2717–2725, 2000. 233. Bijsterbosch, M. K. et al., bis-Cholesteryl-conjugated phosphorothioate oligodeoxynucleotides are highly selectively taken up by the liver, J. Pharmacol. Exp. Ther., 302, 619–626, 2002. 234. Krutzfeldt, J. et al., Silencing of microRNAs in vivo with “antagomirs,” Nature, 43, 685–689, 2005. 235. Henry, S. P. et al., Toxicological properties of several novel oligonucleotide analogs in mice, Anticancer. Drug Des., 12, 1–14, 1997. 236. Herbert, B.-S. et al., Lipid modification of GRN163, an N3⬘-P5⬘ thio-phosphoramidate oligonucleotide, enhances the potency of telomerase inhibition, Oncogene, 24, 5262–5268, 2005. 237. Kastelein, J. J. P. et al., Potent reduction of apolipoprotein B and LDL cholesterol by short-term administration of an antisense inhibitor of apolipoprotein B, Circulation, in press, 2006. 238. Karras, J. G. et al., Peptide nucleic acids are potent modulators of endogenous pre-mRNA splicing of the murine interleukin-5 receptor-a chain, Biochemistry, 40, 7853–7859, 2001.

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239. Baker, B. F. et al., 2⬘-O-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells, J. Biol. Chem., 272, 11,994–12,000, 1997. 240. Vickers, T. A. et al., Fully modified 2⬘ MOE oligonucleotides redirect polyadenylation, Nucleic Acids Res., 29, 1293–1299, 2001. 241. Esau, C. et al., miR-122 regulation of lipid metabolism revealed by in vivo antisense targeting, Cell Metabol., 3, 87–98, 2006. 242. Davis, S. et al., Improved targeting of miRNA with antisense oligonucleotides, Nucl. Acids Res., 34, 2294–2304, 2006.

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CHAPTER

7

Basic Principles of the Pharmacokinetics of Antisense Oligonucleotide Drugs Arthur A. Levin, Rosie Z. Yu, and Richard S. Geary

CONTENTS 7.1 7.2

7.3

7.4

Introduction...........................................................................................................................184 Basic Chemistry....................................................................................................................185 7.2.1 Phosphorothioate Backbone .....................................................................................185 7.2.1.1 Phosphorothioate Chirality........................................................................185 7.2.1.2 Protein Binding .........................................................................................187 7.2.2 Methoxyethyl Modifications at the 2⬘ Position of Ribose .......................................188 7.2.2.1 Nuclease Resistance ..................................................................................188 7.2.2.2 Protein Binding .........................................................................................189 Absorption, Distribution, Metabolism, and Excretion .........................................................191 7.3.1 Absorption ................................................................................................................191 7.3.1.1 Parenteral ..................................................................................................191 7.3.1.2 Local Delivery...........................................................................................191 7.3.1.3 Enteral .......................................................................................................192 7.3.2 Distribution ...............................................................................................................192 7.3.2.1 Roles of Perfusion and Tissue Affinity .....................................................192 7.3.2.2 Plasma Protein Binding in Distribution ....................................................195 7.3.2.3 Distribution to Other Organs.....................................................................196 7.3.3 Metabolism ...............................................................................................................198 7.3.3.1 Enzymes Responsible for Metabolism......................................................199 7.3.3.2 Metabolites ................................................................................................200 7.3.3.3 Metabolism and Clearance........................................................................201 7.3.4 Excretion...................................................................................................................202 7.3.4.1 Overview of Excretion ..............................................................................202 7.3.4.2 Early Excretion and Protein Binding ........................................................203 7.3.4.3 Excretion of Metabolites ...........................................................................204 7.3.5 Summary of Absorption, Distribution, Metabolism, and Excretion ........................206 Application of Pharmacokinetics to the Design of Treatment Regimens ............................206 7.4.1 Steady-State Kinetics................................................................................................208 7.4.2 Dose and Schedule ...................................................................................................209

183

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7.4.3 Relating Exposure and Effect...................................................................................211 7.4.4 Drug–Drug Interactions............................................................................................211 7.4.5 Remaining Questions................................................................................................211 References ......................................................................................................................................211

7.1 INTRODUCTION The pharmacokinetic properties of antisense oligonucleotides (ASOs) are best understood in the context of their physical and chemical properties. Charge, molecular weight, and amphipathic nature of the ASOs all influence the pharmacokinetics of phosphorothioate ASOs. This review will concentrate on how chemical modifications, particularly, the phosphorothioate modification and the 2⬘-methoxyethyl (MOE) modification, of second-generation ASOs improve the pharmacokinetic properties. The absorption, distribution, metabolism, and excretion of first-generation phosphorothioate oligodeoxynucleotides (ODNs) have been thoroughly characterized in laboratory animals and in human clinical trials [1–10]. Our understanding of the pharmacokinetics of phosphorothioate ASO drugs can be summarized as follows: phosphorothioate ODNs administered parenterally appear rapidly in plasma where they are bound to hydrophilic sites on plasma proteins and are thus protected from glomerular filtration. These hydrophilic sites are distinct from the hydrophilic sites where lipophilic drugs bind, and thus there is little competition for plasma protein binding by ASOs. Plasma kinetics are multiphasic with a short distribution phase that is on the order of hours, and then a terminal elimination phase that has half-lives on the order of days or weeks. The initial distribution phase is primarily related to binding and distribution to tissues with liver, kidney, lymph nodes, and spleen being the sites of highest binding and uptake. Phase clearance due to this initial clearance is dependent on protein binding. It is saturable, and therefore, the distribution kinetics are dose-dependent with clearance rates slowing and area under the plasma curve (AUC) increasing with increasing doses. Once bound to the cells, ASOs transit into cells by moving down concentration gradients from extracellular compartments to intracellular compartments, probably by shuttling from one protein-binding site to another across cell membranes. ASOs in cells bind to available targets, but the majority of the ASO in cells is likely bound to intracellular proteins. Within the cell, ubiquitous nucleases metabolize ASO drugs, not the cytochrome P450 enzymes that typically metabolize low-molecular-weight drugs. ASOs, therefore, do not compete with traditional small molecular weight drugs for metabolic processes, reducing the potential for drug–drug interactions. Whole body elimination is the result of metabolism in tissues, and a reequilibration of metabolites and parent drug out of tissues and into circulation, where they are ultimately excreted in the urine. These processes are very similar in laboratory animals and man. In fact the processes are similar enough that doses scale from species to species on the basis of body weight not surface area, allowing for good extrapolations from laboratory animals to man. The focus of the review is the “second generation” of ASOs that feature MOE groups on the 2⬘ position of nucleotides in the 3⬘ and 5⬘ termini (see Chapter 1, this volume). This configuration is the most widely used in clinical studies at present time and represents an advance over the unmodified phosphorothioate ODNs that constituted the first generation of antisense drugs, because of increased potency, reduced toxicity, and increased half-life. Many of the advantages associated with moving from the first-generation to the second-generation ASOs are directly related to improved pharmacokinetics and this review will describe our understanding of the fundamental biology and chemistry that modulate the pharmacokinetics of this new class of ASOs. (A list of sequences discussed in this chapter can be found in Table 7.1.)

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Table 7.1 List of Sequences Discussed in This Chapter Isis Number or Other 2302

Sequence

Generation

GCCCAAGCTGGCATCCGTCA

5132

TCCCGCCTGTGACATGCATT

13650

TC C C GC CTGTGACATGC ATT

m

m

m

m

m

m

m

m

m

104838

GC TGATTAGAGAGAGGTC C C

107248 ATL1102

C TGAGTC TGTTTTC C ATTC T

m

m

m

m

m

m

m

m

112989 OGX-011

C AGC AGCAGAGTCTTCATC AT

113715

GC TC C TTC C AC TGATC C TGC

301012

m m

m

m

m

m

m

m

m

m

m

m

m

m

m

m

m

m

GC C C AGTC TGC TTC GC AC C

Human Data

1

Y

1

Y

2

N

2

Y

2

Y

2

Y

2

Y

2

Y

Note: Underlined letters represent 2⬘-MOE-modified nucleotides; C ⫽5 Methyl C. m

7.2 BASIC CHEMISTRY 7.2.1

Phosphorothioate Backbone

The earliest attempts to produce antisense activity used unmodified DNA, which unfortunately was highly susceptible to the nuclease degradation. Ubiquitous nucleases cleave the phosphodiester bonds of native DNA, and as a result, circulating half-lives for unmodified ASOs are on the order of minutes. This rapid degradation was the predominate factor driving the pharmacokinetic profile of unmodified antisense DNA. Modifying the phosphodiester backbone of DNA to a phosphorothioate backbone dramatically alters the pharmacokinetic profile. The phosphorothioate modification substitutes one sulfur for one of the nonbridging oxygen atoms in the phosphodiester linkages. This modification yields ASOs with much greater utility as a result of the improved resistance to nucleases. The improvements in the utility go much further than the increase in nuclease resistance [11].

7.2.1.1 Phosphorothioate Chirality Thiation of the diester backbone of the ASO not only increases the resistance of the backbone to nucleases but, the substitution of sulfur changes the nature of the backbone and imparts chirality on each of the phosphorothioate linkages such that in any 20-mer ASO there are 219 Sp or Rp stereoisomers [11]. Combined, nuclease resistance and increased protein binding have profound influences on the pharmacokinetics of ASOs. Increased nuclease stability of phosphorothioate ASOs results in plasma half-lives that are 30–60 min compared to 1–2 min of phosphodiester ASOs. The nuclease resistance and the chirality produced by the substitution of the phosphorothioate for the phosphodiester linkages merit discussion, because the physical properties associated with this modification are important to both first- and second-generation ASOs. Nuclease resistance in phosphorothioate ASOs derives in part from the proximity of the sulfur on the nonbridging oxygen to metal ions in the active site of exonucleases. This proximity may cause the displacement of the metal ion from the active site. This effect was demonstrated in a model 3⬘–5⬘ exonuclease, DNA polymerase. X-ray crystallographic data support this hypothesis and apparent differences in sensitivity of the stereoisomers to exonuclease activity by this enzyme are consistent with the proposed conformation of the phosphorothioate ODN stereoisomers within the active site. The Sp stereoisomer apparently rests in the enzyme with the sulfur displacing the metal ions in the active site, while the Rp, the more rapidly degraded isomer, does not interfere with the active site metal ions

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(reviewed in Ref. [11]). It is likely that the chirality of the phosphorothioate linkages may interfere with other nucleases in a similar manner, though different exonucleases may show differing preferences for Rp and Sp linkages depending on the nature of the active site of the enzyme. Alternatively, the sulfur may simply take the place of the oxygen in the diester linkage that would normally complex the Mg2⫹ ion at the active site. The differences in the ability of the sulfur to take on a negative charge versus that of oxygen could be expected to change the properties at the active site. This change would most likely occur because the hydrolysis of the phosphodiester linkage may proceed through the formation of a transient pentacoordinate phosphorous with two negatively charged oxygens (Figure 7.1). Substitution of one of the equatorial oxygens with sulfur would have negative consequences on the enzyme activity: (1) reduced binding of water which stabilizes the local negative charge on the atoms, making it more difficult to generate a second negative charge on the sulfur; and (2) reduction in Mg2⫹ binding to the location with a sulfur substitution. There is evidence for the latter effect in studies of ribozyme cleavage rates following diastereomeric phosphorothioate incorporation, where addition of Mn2⫹ rescues the inhibited cleavage activity of a phosphorothioate ribozyme [12]. It has also been shown that there is some reduction in nuclease activity at a distance from the phosphorothioate linkages, suggesting some transmission of the chirality effect [13]. This phenomenon would be best explained by changes in the packing of water molecules around the phosphorothioate backbone compared to the phosphodiester backbone [14]. The stereospecific effects on nuclease activity markedly affect the pharmacokinetics of phosphorothioate ASOs and this was most apparent in the rates of metabolism of first-generation phosphorothioate ASOs in plasma. The rates of disappearance of the full-length ASOs from plasma decrease overtime, suggesting that there was a slowing of metabolism (reviewed in Ref. [8]). These changes in rates could not be explained on the basis of first-order kinetics alone, but could be explained by the differences in susceptibility of the Rp and Sp linkages. Metabolism (or 3⬘ exonuclease activity) would proceed until a resistant (Sp) linkage was encountered, then metabolism would slow. Stereospecific differences in exonuclease activities are less important for second-generation ASOs. Because second-generation ASOs have 2⬘ modifications at the 3⬘ and 5⬘ termini that prevent exonuclease cleavage, the rate-limiting step in the metabolism of second-generation ASOs is not exonuclease-mediated, but rather endonuclease-mediated. The effects of chirality of phosphorothioate linkages have been explored using a model endonuclease from Serratia marcescens [15]. R −

O

R

R O P

O

O O

OH2

−

OH O

P

Mg 2+ O

−

O

O− Mg2+

OH

O R

O

P

R

HO R

R −

O

R

R O P

O

O S

OH2

−

OH O

P

Mg 2+ O

−

S− Mg 2+

O

OH

O R

R

HO Disfavored

Figure 7.1

S

P

R

Scheme comparing the interaction between a diester linkage and the metal ion of a nuclease and a phosphorothioate linkage and the same metal ion at the active site of a nuclease. The phosphorous of the diester linkage can form a pentavalent oxygen complex while the analogous complex with the phosphorothioate linkage is disfavored because of the less electronegativity of the sulfur (as indicated).

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The endonuclease much like the exonuclease requires one of the nonbridging oxygens for the hydrolysis of the phosphodiester linkage. In a similar fashion, Sp diastereomer puts the sulfur in proximity to the Mg2⫹ and diminishes the activity of the enzyme while the Rp diastereomer does not. The Serratia endonuclease has significant homology to some mammalian endonucleases, suggesting that this mechanism might be broadly applicable. How the stereochemistry of the phosphorothioate linkage affects nuclease activity for other endonucleases has not been characterized. However, assuming that the reaction follows a similar scheme as the Serratia endonuclease, the active site will have a metal ion (probably Mg2⫹) and that sulfur substitution will interfere with the activity when the sulfur is oriented towards the metal ion. If endonucleases are sensitive to chirality, it might have important consequences for the disposition of second-generation drugs. Like the chirality effect for first-generation drugs, a chiral effect on the metabolism of second-generation drugs might result in a slowing of metabolism as the rapidly metabolized stereoisomers are degraded selectively, leaving only the slowly metabolized isomers. This type of effect would tend to result in longer half-lives with prolonged dosing.

7.2.1.2 Protein Binding The phosphorothioate modifications also alter the pharmacokinetic profile of ASOs as a result of their enhanced protein binding compared to ASOs with a phosphodiester backbone. The chemical basis for the increase in protein binding stems from the increased lipophilicity of the phosphorothioate linkage over the phosphodiester linkage as well as the increased “stickiness” of the thioate compared to the diester. Molecular interactions with water determine the form and function of macromolecules in solution [14]. In this regard, even slight changes in the lipophilicity of the backbone over the entire length of the ASOs can have marked consequences for the interactions of the oligomer with water and by extension with macromolecules, like proteins. At the gross level, phosphorothioate ODNs bind more promiscuously to cellular proteins than do phosphodiester ODNs [16]. Specific interactions with proteins are also enhanced by the addition of phosphorothioate linkages as was demonstrated with circular dichroism [17]. Why phosphorothioate ASOs bind to proteins with greater affinity has not been completely elucidated. Two possible explanations include increased lipophilicity and metal ion complexation. Interactions of the sulfurs in the phosphorothioate with metal ions (e.g., Mg2⫹) associated with specific proteins have been proposed for the affinity of phosphorothioate ASOs for proteins [11]. That the phosphorothioate linkages are a driving force in the binding of phosphorothioate ASOs was demonstrated using abasic phosphorothioate oligodeoxyriboses of various lengths. Binding was length-dependent, and like the binding of phosphorothioate ASOs, the binding of the phosphorothioate oligodeoxyribose was highly dependent on the species (H. Gaus, Isis unpublished observations). Unlike the inhibitory effects on nucleases, chirality does not appear to be a factor modulating the binding of phosphorothioate ASOs to human serum albumin, fibrinogen, or
-globulin [18]. The importance of the plasma protein binding to the pharmacology, pharmacokinetics, and toxicity of phosphorothioate ASOs (both first and second generation) cannot be overstated. At concentrations in the micromolar range more than 90% of phosphorothioate ODNs are bound in plasma. There are sequence-dependent differences in protein binding at micromolar concentrations and binding can vary from 90 to 98% yielding varying amounts of free ASO (see below). And there are species differences in protein-binding affinities and capacity (Figure 7.2), as well as qualitative differences in ASO binding to specific protein in some species. Binding of phosphorothioate ASOs to circulating proteins spares these compounds from glomerular filtration and urinary excretion. As protein binding becomes saturated, for example, when a large bolus dose is administered, urinary excretion of full-length ASOs is increased. Urinary excretion increases as the degree of protein binding decreases and the amount of free or unbound ASO increases [9]. Because of species

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Mouse

Monkey

Rat

Human

100

% Bound

95

90

85

80 ISIS 104838 Figure 7.2

ISIS 113715

ISIS 301012

Protein binding was determined by ultrafiltration [68] in whole plasma from mice, rats, monkey, and man at the concentration of 1 ␮M (⬃7 ␮g/mL). Data shown are means ⫾ SD of triplicate determinations.

differences, saturation will occur in different species at differing concentrations. For example, plasma proteins in the mouse have less ASO-binding capacity than rat or human (see Figure 7.2), and therefore, plasma protein binding in mice is saturated at lower concentrations than other species. Species differences in plasma protein binding are a significant contributing factor to species differences in pharmacokinetics. The enhanced nuclease resistance and protein binding that are imparted to ASOs by phosphorothioate linkages modulate the pharmacokinetics of ASO therapeutics. The additional chemical modifications that are made in the second-generation antisense compounds have even more profound effects. 7.2.2

Methoxyethyl Modifications at the 2⬘⬘ Position of Ribose

Second-generation ASOs were developed to improve on some of the characteristics of the ODNs of the first generation. Like the phosphorothioate modification, the addition of MOE groups to the 2⬘ position increases nuclease resistance and modifies protein binding.

7.2.2.1 Nuclease Resistance The MOE modification results in a remarkable increase in nuclease stability. While the phosphorothioate modification reduced the activity of exonucleases, the modification of the 2⬘ position nearly eliminates exonuclease activity. Nuclease resistance may result from (1) the replacement of a critical 2⬘ hydrogen with MOE, (2) steric effects of the MOE group or (3) tighter organization of water shell [19]. Whatever the exact structural basis for the nuclease resistance, the presence of the MOE groups makes the 2⬘-modified ASOs virtually impervious to degradation by circulating nucleases and most cellular nucleases as well. For therapeutic applications that depend on RNase H activity, MOE modifications are generally limited to the terminal nucleotides so as to protect the ends of the molecules from exonuclease degradation. The central region of the molecule is unmodified phosphorothioate ODN to support RNase H activity (see Lima et al., Chapter 2 in this volume). This arrangement in the so-called gapmer (or chimeric RNA–DNA–RNA-like configuration) takes advantage of the higher affinity and nuclease resistance of the MOE modifications and still supports the enzymatic activity of RNase H cleavage of the targeted mRNA. Without metabolic degradation due to exonuclease-mediated trimming of the termini, the slower (and presumably) more sequence-selective endonuclease-mediated

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Absorbance (AU, x1E-03)

MOE gapmer 16 days

PS unmodified 10.0

A

6 days

B T27

ISIS 13650

3.0

ISIS 3521

5.0 Metabolites

13866

1.0 0

0 2.00

4.00 6.00 Time (min)

8.00

2.00

4.00 6.00 Time (min)

8.00

Figure 7.3 Capillary gel electrophoresis of renal extracts from monkeys treated with on the left: a secondgeneration oligodeoxynucleotide (ISIS 13650); right panel: a first-generation oligodeoxynucleotide (ISIS 3521). Monkeys were treated on alternate days for the duration shown.

cleavage of the central deoxy region becomes the dominant force in metabolism. The central region consisting of deoxy phosphorothioates is not nearly as resistant to nuclease-mediated cleavage and it is the cleavage of this central portion that appears to be the rate-limiting step of metabolism. The differences in the sites of metabolism of first- and second-generation ASOs yield different metabolic patterns (see this chapter, Section 7.3.3.2). When the metabolite patterns of gapmers are assessed by capillary gel electrophoresis (CGE) (Figure 7.3) or by liquid chromatography/mass spectroscopy (LC/MS), rather than seeing processive metabolite shortening a single nucleotide at a time, the major pattern consists of metabolites that have apparently been cleaved centrally in the deoxy region (presumably by endonucleases) followed by processive shortening of the cleavage products. The resistance to exonucleases and the slow metabolism by endonucleases yield compounds with long biological half-lives.

7.2.2.2 Protein Binding Phosphorothioate ASOs with 2⬘-MOE modifications have slightly lower affinity for plasma proteins compared with unmodified phosphorothioate ASOs. For example, with the addition of five MOE modifications to the 3⬘ end of a first-generation phosphorothioate ODN, the dissociation constants for albumen increased from ⬃18 to ⬃40 ␮M. Interestingly, full MOE modification of the ASO only slightly diminished the affinity further [20]. Why MOE modifications of all riboses in an oligonucleotide did not further reduce the affinity is not understood, but might be related to secondary structure. The contribution of the phosphorothioate linkages in protein binding of second-generation ASOs is demonstrated in a compound with MOE modifications and only diester linkages. This compound had the weakest affinities for albumen with values in the range of 600–700 ␮M [20]. The reduction in protein-binding affinity associated with the MOE modification can be attributed to some of the physical properties that have already been alluded to in this chapter. Probably, the most significant physical factor that distinguishes the MOE-modified ASOs from unmodified ASOs is the organization of a molecular shell of water that appears to be induced by the presence of the MOE side chain [19]. The MOE substituent in the context of the sugar backbone “chelates” water molecules (and possibly metal ions) between it and the backbone producing a structure that is encased in shells of water molecules, diminishing the potential contact between the “sticky” sulfur molecules and plasma or cellular proteins. (Note that this is the same phenomenon that reduces the accessibility of nucleases to the phosphate or phosphorothioate internucleotide bridges and enhances the nuclease stability.) Arrangement of the modifications within an ASO can also affect protein binding. ASOs with varying numbers of nucleotides modified with MOE groups have varying degrees of (plasma clearance and)

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protein binding compared with a PS ODN with five MOE-modified nucleotides at the 5⬘ end (hemi) and PS full MOE (Figure 7.4a and Figure 7.4b). The degree of protein binding is inversely correlated with urinary excretion (Table 7.2, abstracted from Ref. [21]). The addition of phosphorothioate linkages and 2⬘-MOE substitutions modify protein binding and as a result alter ASO disposition.

Plasma clearance (mL/h)

(a) PO full MOE

160

Altern. PS/PO full MOE 120

80 PS hemi-MOE PS ODN

40 PS full MOE 0

(b)

50

% Dose excreted in urine

PO full MOE 40 30 Altern. PS/PO full MOE

20

PS hemi-MOE 10 0

PS ODN

PS full MOE 75

80

85

90

95

100

Percent bound Figure 7.4

Plasma protein binding predicts clearance (a) and urinary excretion (b). The 20-mer oligonucleotides were modified as follows: PO full MOE, 20-mer oligonucleotide with all sugars modified with 2⬘ MOE and a full diester backbone. Altern. PS/PO full MOE, internucleotide linkages alternated between phosphorothioate and phosphodiester, with every nucleotide modified with MOE. PS full MOE—fully phosphorothioate oligonucleotide with 5 terminal MOE groups on each of the 5⬘ and 3⬘ termini (aka second generation). PS hemi-MOE, a phosphorothioate backbone with 5 terminal nucleotides on the 3⬘ end modified at the 2⬘ position of the ribose with MOE. PS ODN, a full phosphorothioate backbone with 20 deoxynucleotides (first generation).

Table 7.2 Relationships between Extent of Protein Binding and Urinary Excretion

a b

ASO

Modification

Percent Bindinga

Urinary Excretionb

13650 15839 16592

5 MOE-10 deoxy-5 MOE 10 deoxy-7 MOE 20 MOE full diester

93.5–94.0 96.0–98.0 77.0–80.0

7.7 ⫾ 2.3 4.6 ⫾ 2.4 32.3 ⫾ 15.6

Plasma protein binding over the concentration ranges of 0.7–140 ␮g/mL measure by ultrafiltrations. Urinary excretion after treatment of cynomolgus monkeys (n ⫽ 6–8) with 2 h infusions of 10 mg/kg of each ASO.

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7.3 ABSORPTION, DISTRIBUTION, METABOLISM, AND EXCRETION 7.3.1

Absorption

7.3.1.1 Parenteral Because of the molecular weight (⬃7000 Da) and negative charges of ASOs, gastrointestinal uptake is limited and parenteral routes of administration are typically used. Subcutaneous, intravenous infusions or intravitreal injections have been used to administer first-generation drugs. The reduced proinflammatory effects of second-generation ASOs (for review see Chapter 12, this volume) make them more suitable for subcutaneous injections. The plasma pharmacokinetics of subcutaneous injections and intravenous infusions are comparable, and studies in laboratory animals demonstrate that ultimately the distribution of ASO to various organs is also comparable with the exception of the local injection site and the draining lymph nodes. Following subcutaneous administration, both first- and second-generation ASOs are absorbed rapidly from the injection sites into the circulation. In clinical trials, after subcutaneous injection of a second-generation ASO, the time to maximum plasma levels, Tmax, ranged from 1.5 to 4.7 h as dose was increased from 25 to 200 mg (in a constant volume of 1 mL) [22]. The bioavailability of subcutaneous doses ranged from 36 to 82% over this dose range. Preclinical studies [23] and a clinical trial [22] suggest that bioavailability increases with concentration, but additional unpublished clinical studies have yielded less consistent concentration effects, such that it is not appropriate to generalize at this point. The kinetics at the local injection sites can affect local responses, as well as broader systemic pharmacokinetics and distribution. Reducing the concentration of ASO at the injection site is hypothesized to reduce local responses to injection. One approach to reducing concentrations of ASO at the active site is to dilute the formulation, and therefore, increase the surface area available for absorption from a subcutaneous depot. In theory, dilute solutions and greater surface area for absorption should reduce local concentrations and diminish local responses. The same phenomena would be expected to increase the rate of systemic absorption, and alterations of dose and volume can be considered as potential variables that can be manipulated. However, even with the present subcutaneous injection regimens of low volumes and relatively high concentrations, it is possible to demonstrate pharmacology, good tolerability, and high bioavailability.

7.3.1.2 Local Delivery Aerosol formulations, enema, and intravitreal injections have been used as local treatments. To treat lung disease, it is possible to achieve relatively high concentrations of ASO in the lung using aerosolized ASO [24]. The kinetics for a first-generation ODN have been characterized after inhalation exposure in mice. The kinetics of a first-generation ODN are dose-dependent with the lung clearing with a terminal elimination half-life of ⬃2 h. In contrast, studies with MOE-modified second-generation ASOs show that the terminal elimination half-life is greater than 4 days (unpublished observations). Like other local treatment therapies, the advantage to inhalation is that it is possible to achieve high local concentrations in a tissue that is not normally a site of significant accumulation. The lung does not normally accumulate ASOs after parenteral administration. Local treatments also have the added advantage of minimizing systemic exposure. For example, treatment of a monkey with 0.1 mg/kg via inhalation (estimated delivered dose) for three doses over 1 week resulted in lung concentrations of ⬃1 ␮g/g. Yet in these same monkeys, concentrations in the liver and kidney were ⬍5% of the concentrations in lung. As drug is cleared from the lung, it will ultimately deposit in the kidney, but the amounts of drug administered via inhalation is far less than for systemic treatment, so it follows that total exposure will be less.

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Similarly, enema formulations have been successfully used to treat ulcerative colitis. In contrast to inhalation, where small amounts of drug could be used to treat local disease, in ulcerative colitis an enema formulation containing 240 mg of the first-generation drug alicaforsen was administered in clinical trials. In this case, the epithelium of the colon is exposed, but because of the limited permeability of the gut to highly charged ODNs, absorption is minimal and the total systemic exposure is minimal. Colonic epithelial biopsy samples taken within 12 h of administration of enemas in a phase 2 trial had concentrations up to 20 ␮g/g. Plasma bioavailability in human subjects with ulcerative colitis ranged from 0.03 to 2.14% and the maximum concentration observed in plasma was only 0.126 ␮g/mL [25]. The lack of significant absorption systemic absorption and the locally high concentrations work in favor of the efficacy of this local therapy. Intravitreal injections have been used to deliver both first- and second-generation drugs. With this form of local therapy the total dose administered into the eye is quite small (micrograms). Local sequestration seems to limit systemic exposure even further. Within the eye, the vitreous and the retina clear ASOs at different rates: vitreous faster than retina. For fomivirsen, the first marketed antisense drug, vitreous appeared to clear drug (over the course of days) as a result of distribution to tissue; in this case retina. For a second-generation version of fomivirsen, the tissue half-life was estimated as ⬎30 days. Both local metabolism and diffusion out of the eye contribute to the elimination of half-life from the eye (see Chapter 21 in this volume for a complete review of ocular use of ASOs). Again because of the small quantities of drug delivered to the eye, systemic exposure is minimized. However, that pattern of systemic distribution is similar, with liver and kidney accumulating most drugs.

7.3.1.3 Enteral The marked nuclease stability that is afforded by 2⬘-MOE modifications of second-generation ASOs provides the longevity in the gut that can be exploited for parenteral absorption. However, molecular size and charges on the ASOs limits transcellular absorption and researchers have relied on paracellular approaches to enhance uptake from the gut. Oral bioavailability of second-generation drugs (ISIS 104838 or ISIS 301012) is as high as 6–12% and pharmacology by the oral route has been demonstrated after administration of enterically coated solid dosage forms of ASOs combined with permeation enhancers (see Chapter 8 in this volume for a discussion of oral formulations and bioavailability). Once absorbed from the gut, the fate of the ASOis generally similar to intravenous and subcutaneous injections. Although liver is one of the sites of the greatest accumulation of ASO, first-pass effects are not important factors in determining the fate of ASO absorbed from the gut [26,27]. 7.3.2

Distribution

7.3.2.1 Roles of Perfusion and Tissue Affinity Distribution of ASOs like all other drugs is dependent upon permeability, blood flow, plasma protein binding, tissue, and cellular binding. Phosphorothioate ASOs (first and second generation) with their multiple negative charges and their protein-binding characteristics display disposition characteristics that are less dependent on blood flow and more dependent on factors that control the uptake of ASOs into cells. The initial distribution from plasma to tissues is relatively rapid with dose-dependent half-lives that range from 30 to 90 min after intravenous administration. Distribution half-lives are longer after the subcutaneous administration than after intravenous injection. The difference is related to prolonged absorption from the site of injection rather than a true difference in movement from plasma into tissues [7,23,28].

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There are small differences in the distribution kinetics between first- and second-generation drugs. The greater nuclease stability of second-generation ASOs is offset by slightly less protein binding. The plasma profiles of the same sequence in a first- and second-generation configuration are similar. They are nearly superimposable if the sum of parent and ASO metabolites (total ASO) of the first-generation drug is compared with the intact second-generation drug (ISIS 13650) (Figure 7.5). Otherwise, the more rapid degradation by exonucleases shortens the half-life of the first-generation drug compared to the second. Both distribute to tissues in a dose-related manner. At higher doses, the distribution changes and more ASO goes to lower-avidity tissues, like spleen and lymph nodes due to saturation of uptake in liver and kidney [23]. Once absorbed from the site of injection or the gut, ASOs circulate bound to plasma proteins. Two plasma proteins that are known to bind phosphorothioate ASOs are albumen and alpha 2 macroglobulin. Another abundant protein, acid glycoprotein neither binds ASOs very well [29] nor was significant binding observed with purified LDL or HDL [30]. Protein binding is critical to the distribution of the ASO to target tissues. Chemical modifications that reduce binding result in a marked reduction in tissue concentrations and concomitant increases in ASO filtered by the glomeruli and excreted in urine (Table 7.2). This phenomenon can be observed when diester linkages are included in the ASO backbone or when MOE modifications are present or both or in shorter ASOs. The reduced protein-binding affinity of diester and MOE-modified compounds (or both modifications) allows more free ASO to circulate, resulting in increased renal uptake and greater urinary excretion (see Figure 7.4) [28]. In all our studies with parenterally administered first- and second-generation ASOs, we have observed that the initial clearance from plasma is nonlinear. Clearance decreases with increasing doses, and consequently, plasma AUC increases more than that would be predicted on the basis of dose alone [6,22,23,31–33]. Because the initial clearance is driven by distribution and tissue uptake the observation of dose-dependent kinetics implies that tissue uptake has some saturable component. ASOs bound to plasma proteins distribute rapidly to tissues from plasma as a result of binding to cell surface proteins. While the exact mechanisms for cell binding and uptake are not known, one can envision that ASOs would continually bind to available protein-binding sites in extracellular matrix or on cell surfaces. From there, bound ASOs continue traveling down the concentration gradients even crossing the cell membranes by exchange from cell surface protein to membrane or intracellular protein. In that way, the driving force for the movement into cells is the gradient from high concentrations to low. Shuttling has been described for ASO movement between cytoplasm and nucleus [34]. Protein binding

Plasma concentration (µg/mL)

1000 ISIS 13650 ISIS 5132 (Parent) ISIS 5132 (Total)

100

10

1 0

2

4

6

8

Time (h) Figure 7.5

Plasma concentrations of ISIS 5132 shown in dark triangles and total oligodeoxynucleotide shown in light triangles, compared with the plasma concentration of ISIS 13650 a second-generation oligonucleotide.

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facilitates the movement of ASOs across barriers that would normally inhibit the movement of highly charged hydrophilic molecules. This mechanism of shuttling across membranes remains a hypothesis for this class of compounds, but has been previously described for organometallic compounds [35]. The transit of ASO from extracellular matrix to intracellular compartment has been visualized in livers of treated mice using immunohistochemistry [36] and in the kidneys of rats by vital microscopy with fluorescently tagged second-generation ASOs (S. Henry and B. Molitoris, unpublished observations). The identity of the proteins that are responsible for the transit of ASO from the extracellular to the intracellular compartments is not known, but the scavenger receptor has been implicated [37]. However, cellular distribution and pharmacology of ASOs in transgenic mice lacking the scavenger SR-A I/II did not differ appreciably from syngeneic controls [38]. Thus, this specific receptor is not responsible for the majority of uptake into liver and kidney. Other protein acceptors that are capable of binding and translocating ASOs across cell membranes must be responsible for cellular uptake. Membrane proteins that function as transporters are present in all organisms [39]. In considering the transport of drugs, two major superfamilies are generally considered: ABC (ATP binding cassette—requiring energy) and SLC (solute carrier—not requiring energy) transporters. It is known that the rate of transport across a biological membrane via transporter-mediated processes is characterized by saturability. There are multiple members of the SLC superfamily known to function primarily as nucleoside, nucleoside-sugar, and phosphate-sugar transporters. We believe that future work is likely to implicate membrane protein transporters in the facilitated uptake of ASOs as well. Whether they are known receptors or transporters, or whether proteins are acting as nonspecific transport vectors remains to be elucidated. What is clear is that there are differences in the level of avidity and uptake in different organs and tissues and that uptake is saturable at higher doses leading to dose-related plasma clearance and distribution. This distribution of ASO to tissues is determined by a combination of delivery of ASO, blood flow, possible residence time, and finally by the inherent avidity of the tissue for the ASO. The initial binding of ASO to the cell surface appears to be one of the events determining the organ distribution of ASOs. This hypothesis is based upon the observation that liver and kidney are the initial sites of distribution for ASOs with some additional accumulation occurring in the first 24 h after dosing [40]. There is a fractional increase in liver and kidney concentrations over the first 24 h that occurs during a period of marked redistribution at the cellular and subcellular level [41], but the initial organ distribution seems to predict for the sites of more permanent accumulation over time [23,33], suggesting that these organs have greater avidity for ASOs. The proteins responsible for this avidity may include heparin-like binding sites on laminin and fibronectin, and FGF [42–45]. Binding of first-generation ASOs to extracellular matrix was followed by internalization into cells particularly phagocytically active cells [36]. The early binding of ASOs to cells may be related to binding to these proteins, but the nature of the proteins that are ultimately associated with the internalization of the ASOs is not known. Phagocytically active cells like Kupffer cells, tissue macrophages, and proximal tubular cells have high avidity for ASO uptake suggesting that there are receptors on their cell surfaces. Preadministration of an ASO drug to mice can be used to saturate uptake by phagocytically active cells in the liver. When the binding sites on Kupffer cells approach saturation, lower affinity sites become accessible. Subsequent administration of a pharmacologically active ASO to mice that have been pretreated in this way improves the distribution of ASO to nonphagocytic cells in the liver and as a result improves pharmacologic activity in hepatocytes compared to nonpretreated mice (Geary and Sinkowski, unpublished data). Conversely, at plasma concentrations below 1 ␮g/mL ASO tends to favor the binding sites with greater avidity in the liver like Kupffer cells, and it accumulates less in hepatocytes. The combination of the specific cellular binding sites and blood flow results in a distinctive pattern of organ and cell distribution for phosphorothioate ASOs. Experience with dozens of sequences, both first- and second-generation chemistries, demonstrates that the pattern of distribution is generally reproducible from sequence to sequence, and appears to be qualitatively similar from species to species. Kidney, liver, lymph node, spleen, and bone marrow are the organs that accumulate the highest concentrations of ASO and ASO metabolites (Figure 7.6). That lung and

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Oligonucleotide concentration in tissue (µg/g)

300

Parent Total oligo 250

200

150

100

50

Ax

illa

ry

LN Br ai n Fa Ki t dn He Ki ey art dn co ey rte x m ed ul la Li ve O va r Sk Pa rie el n c s et al rea m s us c Sp le le e Te n st e U s te ru s

0

Figure 7.6

Bar graph distribution. Biodistribution of 2⬘ MOE ASO in monkeys; concentrations presented as average microgram oligo per gram tissue for a 20-mer partially modified 2⬘ MOE PS ASO, ISIS 113715 (n ⫽ 2); error bar represents range. Tissues assayed 24 h after single 1 h i.v. infusion of 3 mg/kg ISIS 113715. Assayed by capillary gel electrophoresis (CGE-UV).

heart are not sites of accumulation suggest that blood flow alone is not sufficient to explain the high levels of uptake by kidney and liver. In fact, the site with the highest blood flow in kidney is presumably the glomerulus and that is not a site that accumulates significant quantities of ASOs. In contrast, the renal proximal tubules may represent an extreme situation where there is ample delivery from both the ultrafiltrate and interstitial blood supply. Proximal tubular cells are phagocytically active cells that may be capable of taking up both free ASO as well as ASO bound by proteins that are normally reabsorbed in the proximal tubules [46]. As a result, the renal cortex and in particular the proximal tubular epithelium contains the highest concentrations of phosphorothioate ASOs, both first- and second-generation ASOs (reviewed in Ref. [47]). Blood flow (mL/g of tissue) to the liver is only 苲20% of that to the kidney. The four- to fivefold difference in perfusion between liver and kidney does not translate into four- to fivefold difference in tissue concentrations. Fenestrated capillaries expand the surface area available to the ASO and therefore an abundance of binding sites in the liver may compensate for the lower perfusion rates between liver and kidney.

7.3.2.2 Plasma Protein Binding in Distribution Although cellular avidity may ultimately dictate uptake into target organs, plasma protein binding can alter the relative distribution to kidney and liver of different sequences. There are sequence differences in protein binding. Sequences that bind less to plasma proteins have reduced uptake into liver and increased accumulation in kidney. There is an inverse relationship between extent of protein binding and the accumulation in rat and monkey kidneys after repeated intravenous or subcutaneous injections. In fact, for second-generation MOE-modified ASO sequences with the typical 5 MOE-10 ODNs-5 MOE nucleotides, differences in renal accumulation can be as high as twofold from one sequence to another (Figure 7.7).

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Total oligo concentration (µg/g) in renal cortex

196 7000 r ² = 0.869

6000 5000 ISIS 113715

4000 ISIS 104838

3000 ISIS 107248

2000

ISIS 301012

1000

ISIS 112989

0 2

Figure 7.7

3

4 5 6 7 % Unbound to plasma protein

8

Renal cortex concentrations as a function of the percent oligonucleotide unbound at 1 ␮M in monkey plasma using the method described in Ref. [28]. The renal cortex concentrations were determined from six subjects treated with each drug for 4–13 weeks at ⬃10 mg/kg/week.

Sequences with higher binding affinities with less circulation as free drug will have less accumulation in kidney and more accumulation in other organs. Protein binding can be used as a selection criterion for choosing ASOs for clinical development. Determination of protein binding is an empirical process at this time with no way to accurately predict which sequences will bind plasma proteins with greater or lesser affinity.

7.3.2.3 Distribution to Other Organs Independent of sequence, there is characteristic pattern of distribution of first- and secondgeneration phosphorothioate ASOs with significant distribution to the kidney and liver, as well as spleen, and lymph nodes, bone, and the cytoplasm of adipocytes [48–50]. Lymphoid tissue uptake can be explained partly by the presence of phagocytically active histiocytes and other mononuclear cells. It is possible to visualize ASO in phagolysosomes of histiocytes and tissue macrophages with hematoxylin staining. ASO is taken up by endocytosis, compartmentalized in endosomes, and the ASO is retained within these membrane-bound structures. The local concentrations of ASO in phagolysosomes are often sufficient such that they stain with hematoxylin (much like nuclear DNA). ASO in phagolysosomes probably accounts for much of the ASO accumulated in spleen and lymphoid organs. Similarly, the phagocytically active cells in bone and bone marrow may also account for the tissue concentrations observed in these tissues and that appears to be true based on the presence of basophilic granules in these cells and tissues. Muscles (either skeletal or cardiac) do not take up significant quantities of ASO, although low concentrations have been measured in this tissue. Close examination of muscle using immunohistochemistry or autoradiography demonstrates that resident macrophages in the stroma of a muscle contain ASOs in phagolysosomes and this accumulation may account in part for the ASO measured in muscles and heart. The accumulation of ASO in the cytoplasm of adipocytes is interesting and of therapeutic utility. It is interesting because unlike most compounds that accumulate in adipocytes, ASOs are hydrophilic not hydrophobic. Immunohistochemistry demonstrates clearly that there is ASO in the cytoplasmic fraction of the adipocyte, with little or no ASO in the lipid storage vacuole. The intensity of staining indicates that there is significant accumulation within the cytoplasm. Correcting for the fact that greater than 90%

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of the cell mass is composed of the lipid droplet from which ASO is excluded, suggests that concentrations in the cytoplasm of adipocytes may rival the concentrations in hepatocytes. For example, in cynomolgus monkeys after 13 weeks of treatment with ISIS 113715 at 10 mg/kg/week concentrations in liver are 301 ⫾ 88.1 ␮g/g, but only 37.3 ⫾ 14.4 ␮g/g in adipose tissue from the same animals. Assuming that the lipid-free weight of the tissue is 10% of the total weight, then the corrected concentrations would be 10 times the observed concentration, and therefore, in the range of concentrations in liver. Significant concentrations in adipocytes predict that it should be possible to modulate the expression of disease-related genes in this cell type: a source of hormones and cytokines. Pharmacologically active concentrations of ASOs reach adipocytes and treatment of ob/ob mice with ISIS 113715 at 25 mg/kg twice weekly lowers the expression of its target gene PTP-1B in liver as well as in adipocytes [51]. Similar observations were made in monkeys treated with ISIS 113715. Because subcutaneous fat biopsies can be obtained in clinical studies, and ASOs are active and accumulate in fat, it may be possible to use fat biopsies for measuring pharmacologic effects (reductions in mRNA) and tissue concentrations of drug to directly measure exposure to human tissues (Figure 7.8). Phosphorothioate ASOs distribute to other organs and cell types, and for some tissues and targets cells, concentrations of ASO are not high enough to produce pharmacologic activity. For example, mature lymphocytes do not accumulate ASOs and antisense activity appears to be limited in T-cells. Immunohistochemistry studies demonstrate that endothelial cells accumulate ASO [36,37] at concentrations sufficient to reduce target mRNA levels, making them a potential site for pharmacologic intervention. Bone cells, particularly the phagocytically active osteoblasts, can take up ASO and this propensity for uptake may be exploitable for therapeutic use. Likewise, pancreatic islet cells accumulate ASOs to a degree that might be exploitable (ISIS, unpublished observations). Both first- and second-generation ASOs are highly charged hydrophilic molecules that do not appear to penetrate the blood–brain barrier, or the blood–testes barrier. However, like muscle, in the interstitial regions of the testes, resident macrophages contain ASO and this phagocytized ASO may account for low levels of antisense drugs observed in the testes as well (Figure 7.6). Ovaries are not afforded the same barrier. ASO can be measured in ovaries and can be visualized by immunohistochemistry in the stroma (depicted in Ref. [47]).

500 Intercept = 54.6

Liver concentration (µg/g)

Slope = 8.11 r ² = 0.923

400

300

200

100

0 0

10 20 30 40 Subcutaneous fat concentration (µg/g)

50

Figure 7.8 The relationship between concentrations of ISIS 113715 in liver of monkeys treated for 13 weeks with doses from 1 to 30 mg/kg/week versus concentrations in subcutaneous adipose tissue. Each animal on study is represented by single liver and fat values. The slope and the correlation coefficients are shown.

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The limited distribution from plasma to brain cells and cardiac myocytes makes these tissues unlikely targets for antisense intervention, despite the fact that selective inhibition or expression of some ion channel proteins in brain and muscle might be therapeutically useful. However, the limited distribution to these tissues can be considered as an advantage because the absence of significant concentrations of ASOs in brain and heart diminishes the risk for central nervous system (CNS) and cardiac effects: two common sites for adverse effects. That being said, direct administration to the CNS via intrathecal or intraventricular infusion or injections distributes within the CNS and may be useful therapeutically [52,53]. Another site that appears to be shielded from antisense distribution is the fetus. Though there has been a single report of transplacental antisense activity in early rodent embryos [54], studies of the transplacental kinetics of ASOs indicated that there is little or no fetal accumulation of ASO and relatively low levels of ASO taken up by the rodent placenta [55,56]. Even with constant infusions of ASO throughout gestation, little and in many cases no, ASO could be detected in fetal tissues [55]. These results have been consistently reproduced in teratogenicity studies in mice, rats, and rabbits with various second-generation ASOs (Henry, unpublished observations). In these studies only minimal exposure to the fetal liver and kidney can occasionally be measured. The placenta, despite high perfusion, is not a site of high accumulation of ASOs [55,56]. Distribution is one of the key factors that influences the activity of ASOs. The distinctive pattern of ASO distribution appears to be related to the avidity of tissues for ASO and to a lesser extent perfusion. The conclusion from this section of the review is that distribution is much more a function of the affinity of cells and tissues and less a function of delivery. 7.3.3

Metabolism

Metabolism is the major mechanism of whole body clearance of ASO drugs. ASOs like first- and second-generation antisense drugs are metabolized by nucleases, not the typical mixed function oxidases like the cytochromes P450 that metabolize traditional lipophilic low-molecular-weight drugs. ASOs are neither thought to be substrates for the P450 enzyme family, nor do they induce or inhibit the activity of cytochrome P450 at clinically relevant concentrations (Isis, unpublished observations). ISIS 301012 5′ - G C C T C a g t c t g c t t c G C A C C -3′ 5′-

-3′

2′-MOE

2′-MOE

deoxy

endonuclease

exonuclease

urine

+ + Figure 7.9

A scheme depicting the metabolism of a 20-mer second-generation oligonucleotide. The MOEmodified termini are depicted as capital letters or the ellipse-shaped ends of the molecule.

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Rather than cytochromes P450—mediated metabolism, ASOs are metabolized by nucleases. Exonuclease-mediated shortening of the ASO, which was a prominent mechanism of metabolism and clearance of first-generation phosphorothioate ODNs, has only a secondary role in the clearance of second-generation ASOs. The rate-limiting step is endonuclease cleavage yielding two fragments of the original compound. One fragment has a 3⬘ MOE terminus and the other a 5⬘ MOE terminus (depicted in Figure 7.9).

7.3.3.1 Enzymes Responsible for Metabolism The specific enzymes that metabolize second-generation ASOs are not known. Nucleases are ubiquitously distributed and it is possible to identify cleaved ASO metabolites in most tissues. Hepatic and renal homogenates are both capable of metabolizing second-generation ASOs in vitro (without the addition of exogenous energy sources such as nicotinamide–adenine dinucleotide phosphate [NADPH] regenerating systems or adenosine 5⬘-triphosphate [ATP]). The initial cleavage event results in two products each with a protected MOE terminus 5⬘ and 3⬘. These metabolites no longer bind to proteins with significant affinity, and are therefore, cleared from tissue. Because metabolites are cleared more quickly than they are formed, there is little accumulation of metabolites in tissues (in Figure 7.6, compare parent versus total ASO). Thus, it is not possible to use the amount of metabolites in a tissue as an index of metabolic capability of tissues. Clearance from tissues is dependent on metabolism so that differences in rates in metabolism or different tissues can be estimated based on elimination of half-lives for these tissues. On the basis of data for four second-generation ASOs, the behaviors of members of this class are similar, but there are slight differences in the half-lives in liver and kidney of monkeys treated for 4–13 weeks (Table 7.3, compare down the columns). For ISIS 104838 and 112989 the tissue half-lives are very similar in liver and kidney. These data are suggestive that for some sequences there may not be significant differences in the rates of metabolism in different organs. For ISIS 107248, 113715, and 301012 there are differences in tissue half-lives. The differences in metabolic rates between liver and kidney indicate that there could be differences in metabolic rates in different tissues or that there are more complex kinetics for some sequences (or they could simply reflect the small numbers of samples taken over a limited sampling time). We have already determined that for second-generation ASOs there are different rates of clearance in different cell types in the liver [57], and that some cell types within organs like the proximal tubular cells in kidney cortex tend to sequester ASO in phagolysosomes. Because of this sequestration, it is very likely that there are differences in metabolic rates in different tissues. In addition, it is likely that specific sequences may have different sensitivity to the endonucleolytic cleavage. Bacterial exonucleases exhibit a high degree of sequence specificity presumably for defense against viruses. Much of the nuclease activity in mammalian cells has been associated with the transcription machinery and DNA repair, and thus, the sequence specificity may differ from bacterial endonucleases. It is not known if the replication and repair-associated enzymes are the same ones responsible for catabolism of these synthetic ASOs. At high concentrations ssDNA [58], and by extension ASOs, may be able to compete effectively with the favored natural substrate. Multiple enzyme families have nuclease activity capable of catabolizing ASOs. The identification of the Table 7.3 Tissue Half-Lives in Days (SE) in Monkey’s Organsa Organ Liver Kidney cortex a

104838

107248

113715

301012

112989

13 (5) 15 (6)

22 (4) 14 (3)

8 (4) 16 (1)

37 (7) 17 (11)b

10 (2) 10 (2)

Organ samples collected at various times after the administration of loading regimens of four alternate day doses ⬃3mg/kg. b Appears to be a biphasic elimination for this sequence in renal cortex, elimination half-life was poorly estimated.

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specific enzymes involved in ASOs is not critical for the development of antisense drugs, but having a more thorough understanding of the metabolic pathways will be useful for the technology as we refine it.

7.3.3.2 Metabolites Differences in metabolism from first- to second-generation ASOs are very apparent when comparing metabolic profile with CGE (Figure 7.3). When tissue samples or urine are analyzed by LC/MS, the nature of the metabolic process becomes more clear [28]. Typically a first-generation phosphorothioate ODN is metabolized into a ladder of ASO metabolites each differing by a single nucleotide as a result of single-nucleotide excision by exonucleases. Thus after administering a 20mer first-generation ODN, plasma and tissue contain a family of processively shortened ASOs starting at 19-mers and working down to shorter ASOs (Figure 3[b]). In contrast, administration of a second-generation ASO yields a very different pattern. The initial cleavage event in the metabolism of second-generation ASOs is mediated by an endonuclease, yielding two ASOs one with the 3⬘ MOE terminus and the other with the 5⬘ MOE terminus (see the metabolic scheme in Figure 7.9). The cleavage products have unprotected termini that can be substrates for either 5⬘ exonucleases or 3⬘ exonucleases. In tissues, ASO metabolites consistent with both 3⬘ and 5⬘ exonuclease metabolism have been identified [33,59]. The results of the combined endo- and exonuclease activities are ladders of chain-shortened cleavage products, many if not all of which can be identified in urine collected from treated animals or humans. Urine of treated animals or human subjects treated with a 5-10-5 type of second-generation ASO can have metabolites that range from the two possible 15-mers to the two possible MOE 5-mers and multiple combinations of each of the intermediate length metabolites [28]. Metabolites shorter than the length of the MOE termini would only be present if the MOE termini themselves were substrates for nucleases. That metabolites shorter than the MOE termini are generally undetectable in mass spectral analyses is suggestive that MOE mononucleotides are not generally liberated in the metabolic process. However, some exceptions to this generalization have been observed. For a limited number of sequences, we have observed single-nucleotide trimming of second-generation ASOs in tissues and plasma by either CGE or LC/MS: metabolites apparently the result of exonuclease cleavage. These metabolites can be observed in the initial distribution phase, suggesting that they are generated rapidly in plasma. The MOE mononucleotides that are released are not substrates for phosphorylation, and are therefore, unlikely to be incorporated into nucleotide triphosphates and ultimately into endogenous nucleotides. MOE mononucleotides are not inhibitors of DNA synthetic enzymes. Thus, MOE mononucleotides, if they are released should not interfere with endogenous nucleotide pools. The central deoxynucleotide portion of second-generation ASOs is subject to exonuclease cleavage, which results in the release of single nucleotides. The monodeoxynucleotides that are produced by exonuclease cleavage are identical to endogenous nucleotides with the exception of the thiophosphate group that theoretically might be present. However, the thiophosphate group is labile to oxidation and loss of the sulfur, making the terminal phosphate group identical to an endogenous nucleotide. Therefore, in contrast to MOE mononucleotides, any liberated deoxynucleotide would naturally be incorporated into the intracellular pool of endogenous nucleotides. Nucleotides that retain the thiophosphate group would be substrates for addition of one or two phosphate groups yielding a mixed phosphate thiophosphate di- or triphosphate nucleotide. The phosphorylation is possible, but the presence of the alpha thiophosphate would make it a thermodynamically disfavored reaction (discussed in Ref. [47]). Even if the alpha thiophosphate were phosphorylated and incorporated, base pairing and replication fidelity would be retained. In the metabolic scheme presented (Figure 7.9), other than the deoxymononucleotides, the metabolites that are characteristic of second-generation drugs are the shortened fragments with MOE termini and these appear to be excreted in urine.

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7.3.3.3 Metabolism and Clearance The rapid clearance of metabolites from the organs in which they are formed is demonstrated in two ways. First, there is no significant buildup of metabolic products. The concentration of parent ASO represents a very high fraction of the total ASO present in an organ or tissue (Table 7.4). With increasing time after injection, the fraction of ASO that is comprised of metabolites only increases slightly with the exception of spleen in which the ASO concentration diminished 90% over the 3-month period and may not be representative. With the exception of spleen, there was no meaningful buildup of metabolites. Thus, it appears that formation of metabolites is generally slower than clearance. Second, that metabolites are cleared as rapidly as they are formed is supported by the similarities in the half-life of the parent ASO and the half-life of total ASO (Table 7.5). There is a close correlation between the tissue half-lives observed in monkeys and the plasma terminal elimination half-lives observed in human subjects (Table 7.6). Comparing mean terminal elimination rates in human subjects treated with different second-generation ASOs, the terminal elimination half-lives in human subjects differed by as much as twofold depending on the sequence (Table 7.6). That these are sequence-related differences is confirmed by the similarities between the tissue half-lives determined in monkeys and the terminal elimination half-lives in man. Table 7.4 ISIS 301012 in Tissues from Monkeys Either 2 or 90 Days after the Last Dose 2 Days Sample

Concentration of ISIS 301012

Liver Kidney cortex Spleen Lymph node

1129 ⫾ 242 1593 ⫾ 518 291 ⫾ 125 767 ⫾ 255

90 Days Percent Intact Drug 99 ⫾ 25 94.7 ⫾ 6.2 96.7 ⫾ 5.3 96.1 ⫾ 3.4

Concentration of ISIS 301012

Percent Intact Drug

154 245 31.1 107

74 91.7 50.4 100

Note: Tissues from monkeys treated for 13 weeks with 20 mg/kg/week of ISIS 301012. N ⫽ 6 for 2 days after the last dose group and N ⫽ 2 for 90 days after the last dose group. Table 7.5 Half-Lives of ISIS 113715 and Total ASO in Tissues from Monkeysa Tissue

Parent t1/2 (day)

Axillary lymph Inguinal lymph Kidney cortex Kidney medulla Liver Spleen a

Total Oligo t1/2 (day)

9.8 11.3 15.6 23.2 7.9 11.8

9.5 11.9 16.1 26.1 7.7 10.8

ISIS 113715 tissue exposure parameters in the major tissues of distribution of the pharmacokinetic animals (3 mg/kg i.v. administered on days 1, 4, and 7 only). Tissue samples collected on study days 2, 8, 14, 21, 35, and 63 from two monkeys at each time point.

Table 7.6 Table of Terminal Elimination Half-Lives for 104838, 113715, and 301012

a

ASO

Sequence

t1/2 Livera

104838

GCTGATTAGAGAGAGGTCCC

13

t1/2 Kidneya 15

113715

GCTCCTTCCACTGATCCTGC

8

16

107248

CTGAGTCTGTTTTCCATTCT

22

14

301012

GCCTCAGTCTGCTTCGCACC

37

17

Tissue half-life (days) calculated from data obtained in studies in monkeys (see Table 7.3) b Terminal elimination half-life data from phase 1 clinical trials.

Terminal Elimination (day)b 12 (3)

⬃15 ⬃24 31 (11)

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If endonuclease cleavage is truly the rate-limiting step in the degradation and clearance of the ASO, then it appears from the wide range of terminal elimination half-lives that there may be differences in the susceptibility of these compounds to degradation. The longer terminal elimination of ISIS 301012 suggests that it may be less prone to metabolism and clearance than the other sequences listed. The factors that control the susceptibility of the deoxy portion of the ASO need to be further characterized, as do the enzymes responsible for the catabolism. The nucleases that metabolize ASOs are thought to be ubiquitous and there is no induction of metabolism with repeated administration. The pattern and extent of metabolism are similar after single or multiple injections. However, there may be changes in terminal elimination half-lives that are dependent on dose or tissue exposure. For ISIS 104838 in a phase 1 study [22], half-life increased as dose was increased, but the number of subjects was only three per group. For ISIS 301012, the terminal elimination half-life ranged from 23 ⫾ 1 to 31 ⫾ 11 days in a phase 1 study in which subjects (n ⫽ six/group) treated with 50 or 200 mg/week [67]. The potential inhibitory effects of phosphorothioate ASOs on nucleases might explain the dose-related phenomenon. As studies are performed with larger cohorts with additional sequences, it will become more clear as to whether terminal elimination kinetics are dose-related for more sequences. Similarly, because ASO therapeutics have not yet been tested in large populations, we have not yet determined if there are polymorphisms in the human population that may affect the rates of nuclease-mediated metabolism and clearance of these compounds like those that have been seen with traditional drugs. 7.3.4

Excretion

The excretion of ASOs and ASO metabolites changes depending on the proximity to the last dose. Initial excretion rates are a function of protein binding while excretion rates at later times are a function of metabolism and the disposition of those metabolites.

7.3.4.1 Overview of Excretion The distinctly multiphasic behavior of ASOs in plasma influences their excretion. During the initial plasma clearance phase while ASOs are being distributed to tissues, excretion plays a minor role in plasma clearance. During this period the small amount of urinary excretion is the result of spilling 100 Total excreted

Percent radiolabel excreted

80 Urine

60 40

Carcass

20

Feces

0 −20

0

20

40 60 Time (days)

80

100

Figure 7.10 Excretion of radiolabel from rats treated with 5 mg/kg ISIS 113715 labeled with tritiums at nonexchangeable position of thymidines in MOE-modified wings. Each of the radiolabeled thymidines was 3 positions inward from the termini. The data are from three to six rats per time point.

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of the parent ASO (for second-generation drugs) in urine. The extent of excretion in this phase is dependent on the extent and capacity of plasma protein binding. Later during the equilibrium phase, the nature of the excreted products changes primarily from the excretion of the parent to the excretion of metabolites. The ultimate fate of second-generation ASOs is metabolism and urinary excretion, and to a much lesser extent fecal excretion. In a mass balance experiment, typical of secondgeneration ASOs, the urinary metabolites account for nearly 80% of the total dose, 90 days after administration of a single dose. A few percent of the administered dose was excreted with feces and the remainder had not yet been cleared from the carcass (Figure 7.10). This illustrates the importance of urinary excretion in the whole body clearance of the long-lived second-generation ASOs.

7.3.4.2 Early Excretion and Protein Binding After initial parenteral administration of ASOs, any fraction of drug that is not protein bound in plasma can be filtered by the glomerulus and potentially excreted in urine. There are some sequence-related differences in plasma protein binding and the differences translate into differences in degree of urinary excretion. The amount of free ASO correlates directly with the fraction of the dose excreted in urine in rats (Figure 7.11). Saturation of plasma protein binding often occurs at high doses in toxicology studies, resulting in a sharp increase in the fraction of drug excreted in urine of mice as dose is increased [10,31]. In primates the same holds true: though the capacity for plasma protein binding is greater. The fraction of the dose excreted as the parent compound in the urine is also dose dependent, increasing from 0.2 to 2.9% in monkeys treated with ISIS 301012 after single doses of 2 to 12 mg/kg. Similarly in clinical trials the fraction excreted in urine tends to increase with increasing dose. For ISIS 113715 a compound that binds to plasma proteins to a lesser degree than ISIS 301012 (see Figure 7.11), the fraction excreted in urine as parent compound increased from 0.2 to 7.4% over the dose range of 0.1 to 7.5 mg/kg. The percent of the drug excreted as intact drug (versus total ASO) also increased as dose increased. At 0.5 mg/kg, only 15% of the compound was excreted in urine intact compared to 81% intact after an intravenous infusion of 7.5 mg/kg, consistent with the concept that at high doses when protein binding is saturated there is a spilling of free parent drug into glomerular filtrate and ultimately urine (Table 7.7). However, the spillage of parent drug into the urine at 16 r ² = 0.808

% Dose excreted in urine

14 12

ISIS113715

10 8

ISIS 301012

6 ISIS 104838 4 2 ISIS112989

0 −2

2

3

4

5

6

7

8

% Unbound to plasma protein Figure 7.11 Amount of free oligonucleotide in whole plasma incubated with 1 ␮M of the indicated secondgeneration oligonucleotides versus the percent of the dose excreted in urine 24 h after administration of the indicated oligonucleotides. The dose administered was ⬃10 mg/kg/week.

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Table 7.7 Urinary Excretion of ISIS 113715 in a Phase 1 Clinical Study in Normal Human Subjects % Dose Excreted in Urinea Dose (mg/kg) 0.5 1 2.5 5 7.5

Intact 0.2 0.2 0.6 3.2 7.4

(n ⫽ 2) ⫾ 0.1 ⫾ 0.6 ⫾ 2.7 ⫾ 4.4

Totalb 1.3 1.7 1.6 5.2 9.1

(n ⫽ 2) ⫾ 0.5 ⫾ 1.8 ⫾ 3.2 ⫾ 3.7

% Intact 15 13 38 62 81

Note : Urinary excretion of ISIS 113715 and total ASO over 24 h in humans (n ⫽ 3, except where noted; data presented as mean ⫾ standard deviation). a Urine samples (n ⫽ 3/study day) were collected over 24 h after a single dose. b Total ASO ⫽ sum of parent drug and all measurable ASO metabolites.

early times after injection is only a small contribution to total elimination compared to the total amount of drug administered. Excretion of ASO metabolites in urine contributes much more to the ultimate elimination of the drug.

7.3.4.3 Excretion of Metabolites Once the initial distribution phase is complete, there is equilibrium between plasma and tissues. At equilibrium, the levels of drug in plasma will be proportional to the concentration of drug in tissue. This concept is described in more detail in Chapter 11 (Geary et al., this volume). In addition to the equilibrium between tissue and plasma for the parent drug, there is also an exchange between tissue and plasma of the chain-shortened ASO metabolites. The metabolites formed in tissues (as a result of endo- and subsequent exonuclease) metabolism equilibrate down the concentration gradient from the intracellular compartment into extracellular fluid and then into plasma. Ultimately they are filtered in the kidney and excreted. The concentrations of parent drug at equilibrium are very low compared to the distribution phase. In the distribution phase, concentrations are in the microgram per milliliter range while in the equilibrium phase they are in the range of nanograms per milliliter and the metabolite concentrations at equilibrium are a fraction of that. Despite the low circulating concentrations of parent drug and metabolites at equilibrium, this redistribution of parent drug and ASO metabolites represents the major route of excretion for second-generation ASO drugs and these can be both found in the urine of laboratory animals and man (Figure 7.12, [28,68]). Because metabolites bind to proteins more weakly than the parent drugs, one question that we wanted to address is whether these metabolites are simply filtered in the kidney, or whether these shortened metabolites are filtered and then reabsorbed by the kidney. In light of what is known about the role of protein binding in cell uptake, the reduced protein binding of these shortmers could limit the efficiency of renal uptake. To test that hypothesis, studies on the disposition of the two 10-mer metabolites of ISIS 104838 was characterized in rats. One of the cleavage products had 5⬘ MOE groups and the other the MOE group on the 3⬘ end. Because of reduced plasma protein binding of the metabolites (Figure 7.13), the urinary excretion of the metabolites was considerably faster than that of the parent (Table 7.8), but in the first 24 h, the urinary excretion of the metabolites still only represented a small fraction of the administered metabolites, with the remainder appearing in the kidney (data not shown). Because these metabolites are reabsorbed readily by the kidney, one might expect that metabolites would accumulate in kidney, but they do not. The metabolic products apparently clear three to fivefold faster than the parent ASO itself. The metabolites themselves are also subjected to further metabolism, in particular the cleavage product with an unprotected 3⬘ end was subjected to further

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205

Metabolites

I.S. (0.25 µM)

5 mg/kg

Mouse, 0−24 h

Metabolites

ISIS 104838

I.S. (0.25 µM)

3 mg/kg

ISIS 104838

Metabolites

Monkey, 0−24 h

2 mg/kg

I.S. (0.25 µM)

Human, 2−4 h

Relative retention time

Figure 7.12 Capillary gel electrophoresis of urine collected from mice, monkeys, and humans treated with ISIS 104838 at the indicated doses.

100

% Bound

90

80

70 ISIS 104838 n-9 n-10

60

n-11 n-12

50 0.1

1

10

15

20

50

[ISIS 104838] in Plasma (µM)

Figure 7.13 Binding of ISIS 104838 and representative metabolites in whole human plasma. The oligonucleotides were incubated at the concentrations indicated and the percent bound determined by ultrafiltration.

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Table 7.8 Percent of Drug Excreted by Rats in Urine 7 Days after the Administration of ISIS 104838 or Two Metabolites Compound ISIS 104838 5⬘ Endo metabolite 10-mer 3⬘ Endo metabolite 10-mer

Parent (%) 0.2 2.5 4.1

Total Oligo (%) 1.1 3.6 5.2

metabolism before being excreted in urine. Taken together these data suggest that the fate of metabolites is more complex than simple filtration in the glomerulus and excretion in urine. 7.3.5

Summary of Absorption, Distribution, Metabolism, and Excretion

In summary, parenterally administered phosphorothioate ASOs circulate bound to plasma proteins. Unbound or free ASO is subjected to glomerular filtration and is excreted in urine reabsorbed into renal tubular cells. In the distribution phase (immediately after administration), ASO moves down a concentration gradient from plasma protein bound, to cell surface bound, and then to intracellular protein bound (Figure 7.14, top insert). Movement down the concentration gradients continues until equilibrium between plasma and tissue is obtained. At high enough concentrations, the steps in this process can be saturated, explaining in part some of the nonlinear kinetics observed. Tissue clearance is dependent on the loss of ASO from the system as a result of the urinary excretion of endonuclease-cleaved metabolites and intact ASO (Figure 7.14, top panel). Metabolism may be influenced by concentration of the substrate and by the chirality of the phosphorothioate linkages. Because of diminished protein binding compared to parent ASO, shortened ASO metabolites that are formed intracellularly move down a concentration gradient from the cellular compartment where they are formed to the circulation, and then to the kidney where the cleavage products are filtered and ultimately excreted (Figure 7.14, bottom panel). The nuclease resistance of secondgeneration drugs effectively slows catabolism, increasing the terminal elimination half-life and prolonging the pharmacologic activity.

7.4 APPLICATION OF PHARMACOKINETICS TO THE DESIGN OF TREATMENT REGIMENS One of the advantages in having a platform technology in which different drugs share so many similarities from sequence to sequence is that the information obtained from studies with one sequence is applicable, in part, to the understanding of all sequences with similar chemistries. Though there are some quantitative differences between sequences, qualitatively different sequences behave similarly. These similarities have been most useful in the application of our understanding of the basic principles of the pharmacokinetics of antisense drugs to the design of clinical dose regimens. Another advantage of the technology is that there is significant predictability of the pharmacokinetics in laboratory animals to humans. Initial plasma clearance rates scale linearly with body weight [28], and plasma concentrations in monkeys and man are similar after equivalent doses are administered on the basis of body weight, and not surface area. The predictability from sequence to sequence and the ability to extrapolate from animals to man together provide distinct advantages for understanding the pharmacokinetics of the class and for the design of clinical dose regimens. In addition to the cumulative information available for the second-generation platform, another key characteristic of the class that allows for significant flexibility in the design of therapeutic regimens is the long terminal elimination half-life. This improvement over the first-generation

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Antisense drug travel ASOs travel from high concentration to low concentration and from proteins of low affinity to high affinity. low

high

highest

higher

Antisense drug disposition, activity and metabolism Cell surface protein Membrane protein Intracellular protein Plasma protein

mRNA

Cleaved mRNA

Endonuclease

Cleaved ASO Free ASO

Cell

Metabolites

Capillary

Antisense metabolite disposition

Glomerulus Excreted

Only unbound drug or metabolites can be filtered

Metabolites

Free ASO

Kidney

Capillary

Figure 7.14 Cartoon of oligonucleotide distribution, metabolism, and excretion. Top insert: ASOs move down concentration gradients and move from protein to protein based on mass flow and protein-binding affinity. Top panel: ASO circulating in plasma is primarily bound to plasma proteins. Bound or free ASO can interact with cell surface proteins and move down the concentration gradients and to proteins of higher affinity as it makes its way across cell membranes and into the cell where it can hybridize to its cognate mRNA, activate RNase H and/or be cleaved by endonucleases. Cleavage products bind weakly to protein and can be lost from the cell. Bottom panel: ASO or metabolites in circulation that are unbound can be filtered in the kidney and excreted in the urine or bound drug is not readily filtered.

ODNs allows for infrequent dosing. Whereas first-generation ASOs had 20–40-h terminal elimination half-lives, second-generation drugs have 14- to 30-day terminal elimination half-lives. Maintenance of therapeutic concentrations of first-generation drugs required dosing on alternate days. For secondgeneration drugs, therapeutic concentrations can be maintained with doses as infrequent as weekly or

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monthly, depending on dose and the terminal elimination half-life of the specific sequence. By adjusting dose and dose frequency, it is possible to design regimens that achieve specific concentrations of drug in target organs. 7.4.1

Steady-State Kinetics

Repeated administration of an ASO with a long terminal elimination half-life can result in accumulation in organs and tissues. First-order kinetics dictate that depending on the dose and dose frequency, there will be accumulation of ASO in tissues until a steady state is reached. Animal studies with a second-generation ASO demonstrate that a steady state is obtained in tissues such that no additional accumulation of ASO occurs in kidney, liver, and spleen as dosing is extended from 3 to 9 months (Figure 7.15). For most ASOs, steady-state concentrations of ASO can be predicted on the basis of half-life only, but for some sequences the data are better-fitted assuming first-order kinetics with a saturable or dose-related factor. Drugs with long half-lives require long dosing periods to achieve steady state. Loading regimens have been used to shorten the time to steady state in both clinical trials and in animal studies. Attaining steady state or near steady-state concentrations more quickly than the 3–5 half-lives are useful when exposure needs to be maximized in a short period of time, for example, in a toxicology study or in clinical trials when duration of treatment is limited. In one loading strategy, ASO is given on alternate days for the first week followed by once weekly dosing. In the study results depicted in Figure 7.16, the loading regimen produced renal cortex concentrations of ⬃400 ␮g/g at the end of 8 days. At the end of 13 weeks of treatment the concentration data point attained for renal cortex was ⬃500 ␮g/g. Thus, the loading regimen achieved ⬃80% of the observed steady-state concentrations in cynomolgus monkeys treated with 10 mg/kg/week of ISIS 113715. In this example, the steady-state concentration in the renal cortex could be predicted on the basis of the half-life, the dose, and the frequency. Note that the steady state was attained even though the weekly dose regimen was twice as frequent as would be required by the 14-day half-life. With a half-life of ⬃14 days, accumulation should reach approximately threefold compared to the concentration obtained after a

Tissue concentrations (µg/g)

2000

1500

1000

500

0

3

6 Time

9

Figure 7.15 Concentrations of ISIS 104838 in renal cortex (open cicles), liver (open squares) or spleen (filled diamonds) in monkeys treated with ⬃20 mg/kg/week for 3, 6 or 9 months. Each data point represents the mean of at least four subjects (⫾SD).

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3 mg/kg Simulation Actual measured 113715 concentration

Kidney cortex (µg/g)

600 500 400 300 200 100 0 0

20

40

60 80 Time (days)

100

120

Figure 7.16 Renal accumulations of ISIS 113715 in monkeys treated with 3 mg/kg/week following a loading regiment of alternate day dosing for the first week. One group of monkeys received only the loading regimen and then tissues were assayed by CGE for two monkeys at each of the indicated time points. The data were fitted assuming first-order kinetics and then that model was used to simulate repeated administration at weekly dosing. The data point at 90 days represents the mean (⫾SD) concentration in renal cortex (n ⫽ 6).

single dose.* Comparing the concentration in kidney after day 1 to the concentration after 90 days demonstrates that ISIS 113715 follows first-order kinetics and steady state is attained (Figure 7.16). Because both pharmacology and toxicity are related to tissue concentrations, confirming that second-generation ASOs achieve steady state in predictable ways is useful for setting dose and dose intervals as well as understanding drug safety. It is particularly important for safety because there will not be continuous accumulation of ASO in target tissues like renal cortex with long-term administration of second-generation drugs. 7.4.2

Dose and Schedule

Because the pharmacology with ASOs is dependent on tissue concentrations and it is nearly impossible to collect tissue samples in most clinical trials, we have had to develop surrogates for tissue concentrations. Under equilibrium conditions (defined as ⬎48 h after the last dose), plasma concentrations reflect the exchange between the major organs of accumulation and plasma. Being able to assess equilibrium plasma concentrations has only become possible because of the introduction of a high-sensitivity hybridization-dependent enzyme-linked specific immunoassay [60]. In studies in monkeys, we have demonstrated that equilibrium plasma concentrations (troughs) have a linear relationship to liver and kidney concentrations (see Geary et al., Chapter 11). Like tissue concentrations, with continuous dosing, trough plasma concentrations eventually reach a steady state and remain there (Figure 7.17). The trough (or equilibrium) plasma concentrations obtained at steady state increase linearly with increasing dose, confirming that trough plasma concentrations reflect exposure (Figure 7.18). The use of trough plasma concentrations as a surrogate for tissue concentrations is providing insight into dose and schedule and more informed design of clinical dose regimens. First-order kinetics predict that changing dose interval, for example by administering twice the dose half as frequently should result in similar steady-state concentrations albeit with more variation about the mean. Greater peak to trough variations are acceptable as long as the antisense

*

Assuming first-order kinetics: (concentration ⫽ e⫺
t,
⫽ 0.0495/day) and accumulation factor ⫽ 1/e⫺
⫻ 7 days ⫽ ⬃3.

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Plasma trough concentration ISIS 301012 (ng/mL)

210 20

15

10

5

0 20

30

40

50

60

70

80

90

100

Study days Figure 7.17 Mean plasma trough concentrations (ng/mL) of ISIS 301012 in human subjects (7 days after dose) following 200 mg dose q2w (100 mg/week) for 13 weeks. Plasma samples were analyzed using a hybridization-dependent enzyme-linked immune assay that is specific for the parent drug.

Plasma trough concentration ISIS 301012 (ng/mL)

40

y = 0.147x − 1.2659 R 2= 0.9971

35 30 25 20 15 10 5 0 0

50

100 150 Weekly dose (mg)

200

250

Figure 7.18 Plasma trough concentration (ng/mL) of ISIS 301012 as a function of equivalent weekly dose at the end of 13 weeks treatment in human subjects. Plasma samples collected in ⬃7 days after the last dose were analyzed as described above for Figure 7.16 (mean ⫾ SE, n ⫽ 7 or 8).

activity is maintained at the trough concentration and the peak concentrations are below toxic levels. Clinical trials have been designed to explore the effects of dose and dose frequency. The results obtained with ISIS 301012 in phase 2 clinical trials confirm predictable kinetics and the concentrations in plasma approach steady state (Figure 7.17). Among the variables available in study design is the loading regimen. Whether or not a clinical study uses a loading regimen is dependent on how quickly steady state needs to be achieved which is in turn dependent on how quickly a therapeutic effect is needed. Those variables will vary with the study duration and with the disease indication. Clearly chronic and acute indications will require very different loading regimens. In chronic indications it may be acceptable for it to take weeks or months to achieve steady state, however in acute indications aggressive loading would be more likely used to achieve steady state quickly. Thus, the use of loading or induction regimens will be established for each disease indication or clinical trial.

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Relating Exposure and Effect

One of the most important advances that has been made with second-generation drugs comes from combining pharmacokinetic data (either trough plasma concentrations or tissue concentrations) with direct measurements of the molecular target for antisense activity. Using these types of analyses it has been possible to demonstrate concentration response relationships for the reduction in expression of TNF in the synovium of rheumatoid arthritis patients (unpublished observation), reduction in clusterin in the prostates in hormone refractory prostate cancer [61–63] and reductions in circulating apolipoprotein B levels in subjects with elevated cholesterol (Kastalein, in press circulation). These concentration response relationships are relatively consistent from laboratory animals to man. Having plasma as a surrogate for tissue concentrations or by knowing concentrations of drug required at sites of activity is allowing us to design improved dose regimens for critical studies in larger populations (see Chapter 11, this volume for a more complete discussion of pharmacokinetic and pharmacodynamic relationships). 7.4.4

Drug–Drug Interactions

In a typical clinical setting patients are exposed to a multitude of drugs and bioactive foodstuffs. Drug–drug interactions are often the result of competition for binding sites in plasma proteins or competition for drug-metabolizing enzymes. ODNs do not compete with coumadin, valproic acid, asprin, and other commonly used drugs known to be highly protein bound [29] and similar results have been found for second-generation antisense drugs (Isis, unpublished observations). In clinical studies no drug–drug interactions have been observed with first- or second-generation ASOs with cytotoxic oncolytics [64,65], statins (unpublished observations), and orally active antidiabetes drugs [66]. Thus, nonspecific drug–drug interactions do not appear to be common. Drug interactions related to additive or synergistic pharmacology will have to be assessed on a drug by drug basis for each indication, but to date drug interactions based on competition for distribution or metabolism have not been observed in studies designed to detect pharmacokinetic differences. 7.4.5

Remaining Questions

The last remaining question for the use of second-generation ASOs relates to chronic administration and what dose and schedule will be required to maintain activity. First-order kinetics would suggest that maintenance doses would require replenishment of the small fraction of parent drug metabolized and cleared each dosing interval. The amount may vary depending on the sequence and the half-life for the sequence. Preliminary evidence exists that with longer exposure periods there may be deviations from simple first-order kinetics. As we expand our experience to include more chronic exposure studies in laboratory animals and in clinical trials, we will monitor for changes in kinetics with the tools and understanding that we have developed for the platform of secondgeneration antisense drugs. REFERENCES 1. Geary, R. S., Current assessment of PK/PD relationships for antisense therapeutics, World Congress of Pharmacy and Pharmaceutical Sciences, Nice, France, 2002. 2. Geary, R. S., Leeds, J. M., Fitchett, J., Burckin, T., Troung, L., Spainhour, C., Creek, M., and Levin, A. A., Pharmacokinetics and metabolism in mice of a phosphorothioate ASO antisense inhibitor of C-raf-1 kinase expression, Drug Metab. Dispos. 25 (11), 1272, 1997. 3. Geary, R. S., Leeds, J. M., Henry, S. P., Monteith, D. K., and Levin, A. A., Antisense oligonucleotide inhibitors for the treatment of cancer: 1. Pharmacokinetic properties of phosphorothioate ODNs, Anticancer Drug Des. 12 (5), 383, 1997.

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4. Geary, R. S., Leeds, J. M., Shanahan, W., Glover, J., Pribble, J., Truong, L., Fitchett, J., Burckin, R., Nicklin, P., Philips, J., and Levin, A. A., Sequence independent plasma and tissue pharmacokinetics for 3 antisense phosphorothioate ASOs: mouse to man, in American Association of Pharmaceutical Scientists, Pharm. Research, Plenum Press, Seattle, Washington, 1996, p. S. 5. Geary, R. S., Yu, R. Z., and Levin, A. A., Pharmacokinetics of phosphorothioate antisense ODNs, Curr. Opin. Invest. Drugs 2 (4), 562, 2001. 6. Glover, J. M., Leeds, J. M., Mant, T. G., Amin, D., Kisner, D. L., Zuckerman, J. E., Geary, R. S., Levin, A. A., and Shanahan, W. R., Jr., Phase I safety and pharmacokinetic profile of an intercellular adhesion molecule-1 antisense ODN (ISIS 2302), J. Pharmacol. Exp. Ther. 282 (3), 1173, 1997. 7. Leeds, J. M., Henry, S. P., Geary, R. S., Burckin, T. A., and Levin, A. A., Comparison of the pharmacokinetics of subcutaneous and intravenous administration of a phosphorothioate oligodeoxynucleotide in cynomolgus monkeys, Antisense Nucl. Acid Drug Dev. 10 (6), 435, 2000. 8. Levin, A. A., A review of issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides, Biochim. Biophys. Acta 1489 (1), 69, 1999. 9. Levin, A. A., Henry, S. P., Bennett, C. F., Cole, D. L., Hardee, G. E., and Srivatsa, G. S., Preclinical development of antisense therapeutics, in Novel Therapeutics from Modern Biotechnology: From Laboratory to Human Testing, 1st ed., Oxender, D. L. and Post, L. E., eds., Springer-Verlag, Heidelberg, Germany, 1998, p. 131. 10. Levin, A. A., Geary, R. S., Leeds, J. M., Monteith, D. K., Yu, R. Z., Templin, M. V., and Henry, S. P., The pharmacokinetics and toxicity of phosphorothioate ASOs, in Biotechnology and Safety Assessment, 2nd ed., Thomas, J. A., ed., Taylor & Francis, Philadelphia, PA, 1998, p. 151. 11. Eckstein, F., Phosphorothioate ODNs: what is their origin and what is unique about them? Antisense Nucl. Acid Drug Dev. 10 (2), 117, 2000. 12. Slim, G. and Gait, M. J., Configurationally defined phosphorothioate-containing oligoribonucleotides in the study of the mechanism of cleavage of hammerhead ribozymes, Nucl. Acids Res. 19 (6), 1183, 1991. 13. Spitzer, S. and Eckstein, F., Inhibition of deoxyribonucleases by phosphorothioate groups in oligodeoxyribonucleotides, Nucl. Acids Res. 16 (24), 11691, 1988. 14. White, A. P., Reeves, K. K., Snyder, E., Farrell, J., Powell, J. W., Mohan, V., and Griffey, R. H., Hydration of single-stranded phosphodiester and phosphorothioate oligodeoxyribonucleotides, Nucl. Acids Res. 24 (16), 3261, 1996. 15. Koziolkiewicz, M., Krakoviak, A., Kwinkowski, M., Boczkowska, M., and Stec, W. J., Stereodifferntiation—the effect of P chirality of oligo(nucleoside phosphorothioates) on the activity of bacterial RNase H, Nucl. Acids Res. 23 (24), 5000, 1995. 16. Brown, D. A., Kang, S. H., Gryaznov, S. M., DeDionisio, L., Heidenreich, O., Sullivan, S., Xu, X., and Nerenberg, M. I., Effect of phosphorothioate modification of ODNs on specific protein binding, J. Biol. Chem. 269 (43), 26801, 1994. 17. Mou, T. C. and Gray, D. M., The high binding affinity of phosphorothioate-modified oligomers for Ff gene 5 protein is moderated by the addition of C-5 propyne or 2⬘-O-methyl modifications, Nucl. Acids Res. 30 (3), 749, 2002. 18. Yu, D., Kandimalla, E. R., Roskey, A., Zhao, Q., Chen, L., Chen, J., and Agrawal, S., Stereo-enriched phosphorothioate ODNs: synthesis, biophysical and biological properties, Bioorg. Med. Chem. 8 (1), 275, 2000. 19. Teplova, M., Minasov, G., Tereshko, V., Inamati, G. B., Cook, P. D., Manoharan, M., and Egli, M., Crystal structure and improved antisense properties of 2⬘-O-(2-methoxyethyl)-RNA, Nat. Struct. Biol. 6 (6), 535, 1999. 20. Geary, R. S., Ushiro-Watanabe, T., Truong, L., Freier, S. M., Lesnik, E. A., Sioufi, N. B., Sasmor, H., Manoharan, M., and Levin, A. A., Pharmacokinetic properties of 2⬘-O-(2-methoxyethyl)-modified ASO analogs in rats, J. Pharmacol. Exp. Ther. 296 (3), 890, 2001. 21. Yu, R. Z., Geary, R. S., and Levin, A. A., Application of novel quantitative bioanalytical methods for pharmacokinetic and pharmacokinetic/pharmacodynamic assessments of antisense oligonucleotides, Curr. Opin. Drug Discov. Dev. 7 (2), 195, 2004. 22. Sewell, L. K., Geary, R. S., Baker, B. F., Glover, J. M., Mant, T. G. K., Yu, R. Z., Tami, J. A., and Dorr, A. F., Phase I trial of ISIS 104838, a 2⬘-methoxyethyl modified antisense oligonucleotides targeting tumor necrosis factor-alpha, J. Pharmacol. Exp. Ther. 303 (3), 1334, 2002.

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23. Phillips, J. A., Craig, S. J., Bayley, D., Christian, R. A., Geary, R. S., and Nicklin, P. L., Pharmacokinetics, metabolism and elimination of a 20-mer phosphorothioate ODN (CGP 69846A) after intravenous and subcutaneous administration, Biochem. Pharmacol. 54 (6), 657, 1997. 24. Templin, M. V., Levin, A. A., Graham, M. J., Aberg, P. M., Axelsson, B. I., Butler, M., Geary, R. S., and Bennett, C. F., Pharmacokinetic and toxicity profile of a phosphorothioate ASO following inhalation delivery to lung in mice, Antisense Nucl. Acid Drug Dev. 10 (5), 359, 2000. 25. Miner, P. B., Jr., Geary, R. S., Matson, J., Chuang, E., Xia, S., Baker, B. F., and Wedel, M. K., Bioavailability and therapeutic activity of alicaforsen (ISIS 2302) administered as a rectal retention enema to subjects with active ulcerative colitis, Ailment Pharmacol. Ther. 23 (10), 1427, 2006. 26. Geary, R. S., Teng, C. L., Truong, L., Perry, B., Sinko, P., and Hardee, G. E., First pass hepatic extraction of a partially modified chimeric antisense oligonucleotides in Beagle dogs, in Annual Meeting of the American Association of Pharmaceutical Scientists, Indianapolis, IN, 2000, p. 216. 27. Stavchansky, S., Geary, R. S., and Cho, M., Pharmacokinetics and hepatic first pass effect of an antisense oligonucleotide (ISIS 2302) in rats, in Annual Meeting of the American Association of Pharmaceutical Scientists, Indianapolis, IN, 2000, p. 216. 28. Geary, R. S., Yu, R. Z., Watanabe, T., Henry, S. P., Hardee, G. E., Chappell, A., Matson, J., Sasmor, H., Cummins, L., and Levin, A. A., Pharmacokinetics of a tumor necrosis factor-alpha phosphorothioate 2⬘-O-(2-methoxyethyl) modified antisense oligonucleotides: comparison across species, Drug Metab. Dispos. 31 (11), 1419, 2003. 29. Watanabe, T. A., Geary, R. S., and Levin, A. A., Plasma protein binding of an antisense oligonucleotide targeting human ICAM-1 (ISIS 2302), ASOs 16 (2), 169, 2006. 30. Bijsterbosch, M. K., Rump, E. T., De Vrueh, R. L., Dorland, R., van Veghel, R., Tivel, K. L., Biessen, E. A., van Berkel, T. J., and Manoharan, M., Modulation of plasma protein binding and in vitro liver cell uptake of phosphorothioate ODNs by cholesterol conjugation, Nucl. Acids Res. 28 (14), 2717, 2000. 31. Yu, R. Z., Geary, R. S., Leeds, J. M., Ushiro-Watanabe, T., Moore, M., Fitchett, J., Matson, J., Burckin, T., Templin, M. V., and Levin, A. A., Comparison of pharmacokinetics and tissue disposition of an antisense phosphorothioate ASO targeting human Ha-ras mRNA in mouse and monkey, J. Pharm. Sci. 90 (2), 182, 2001. 32. Leeds, J. M. and Geary, R. S., Pharmacokinetic properties of phosphorothioate ASOs in humans, in Antisense Research and Applications, 1st ed., Crooke, S. T., ed., Springer, Heidelberg, 1998, p. 217. 33. Nicklin, P. L., Craig, S. J., and Phillips, J. A., Pharmacokinetic properties of phosphorothioates in animals—absorption, distribution, metabolism and elimination, in Antisense Research and Applications, 1st ed., Crooke, S. T., ed., Springer, Berlin, 1998, p. 141. 34. Lorenz, P., Misteli, T., Baker, B. F., Bennett, C. F., and Spector, D. L., Nucleocytoplasmic shuttling: a novel in vitro property of antisense phosphorothioate ODNs, Nucl. Acids Res. 28 (2), 582, 2000. 35. Snyder, R. M., Mirabelli, C. K., and Crooke, S. T., Cellular association, intracellular distribution, and efflux of auranofin via sequential ligand exchange reactions, Biochem. Pharmacol. 35 (6), 923, 1986. 36. Butler, M., Stecker, K., and Bennett, C. F., Cellular distribution of phosphorothioate ODNs in normal rodent tissues, Lab. Invest. 77 (4), 379, 1997. 37. Bijsterbosch, M. K., Manoharan, M., Rump, E. T., De Vrueh, R. L. A., van Veghel, R., Tival, K. L., Biessen, E. A. L., Bennett, F. C., Cook, D. P., and van Berkel, T. J. C., In vitro fate of phosphorothioate antisense ODNs: predominant uptake by scavenger receptors on endothelial cells, Nucl. Acids Res. 25 (16), 3290, 1997. 38. Butler, M., Crooke, R. M., Graham, M. J., Lemonidis, K. M., Lougheed, M., Murray, S. F., Witchell, D., Steinbrecher, U., and Bennett, C. F., Phosphorothioate ODNs distribute similarly in class A scavenger receptor knockout and wild-type mice, J. Pharmacol. Exp. Ther. 292 (2), 489, 2000. 39. Giacomini, K. M. and Sugiyama, Y., Membrane transporters and drug response, in Goodman & Gilman’s The Pharmacological Basis of Therapeutics, 11th ed., Brunton, L. L., ed., McGraw-Hill, New York, 2006, pp. 41. 40. Peng, B., Andrews, J., Nestorov, I., Brennan, B., Nicklin, P., and Rowland, M., Tissue distribution and physiologically based pharmacokinetics of antisense phosphorothioate ASO ISIS 1082 in rat, Antisense Nucl. Acid Drug Dev. 11 (1), 15, 2001.

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41. Graham, M. J., Crooke, S. T., Monteith, D. K., Cooper, S. R., Lemonidis, K. M., Stecker, K. K., Martin, M. J., and Crooke, R. M., In vitro distribution and metabolism of a phosphorothioate ASO within rat liver after intravenous administration, J. Pharmacol. Exp. Ther. 286 (1), 447, 1998. 42. Benimetskaya, L., Tonkinson, J. L., Koziolkiewicz, M., Karwowski, B., Guga, P., Zeltser, R., Stec, W., and Stein, C. A., Binding of phosphorothioate ODNs to basic fibroblast growth factor, recombinant soluble CD4, laminin and fibronectin in P-chirality independent, Nucl. Acids Res. 23 (21), 4239, 1995. 43. Benimetskaya, L., Loike, J. D., Loike, G., Siverstein, S. C., Long, C., Khoury, J. E., Cai, T.-Q., and Stein, C. A., Mac-1 (CD11b/CD18) is an ODN-binding protein, Nat. Med. 3 (4), 414, 1997. 44. Loke, S. L., Stein, C. A., Zhang, X. H., Mori, K., Nakanishi, M., Subasinghe, C., Cohen, J. S., and Neckers, L. M., Characterization of ASO transport into living cells, Proc. Natl. Acad. Sci. USA 86, 3474, 1989. 45. Guvakova, M. A., Yakubov, L. A., Vlodavsky, I., Tonkinson, J. L., and Stein, C. A., Phosphorothioate ODNs bind to basic fibroblast growth factor, inhibit its binding to cell surface receptors, and remove it from low affinity binding sites on extracellular matrix, J. Biol. Chem. 270, 2620, 1995. 46. Sawai, K., Mahato, R. I., Oka, Y., Takakura, Y., and Hashida, M., Disposition of ASOs in isolated perfused rat kidney: involvement of scavenger receptors in their renal uptake, J. Pharmacol. Exp. Ther. 279 (1), 284, 1996. 47. Levin, A. A., Henry, S. P., Monteith, D., and Templin, M., Toxicity of antisense oligonucleotides, in Antisense Drug Technology, Crooke, S. T., ed., Marcel Dekker, New York, 2001, p. 201. 48. Cossum, P. A., Sasmor, H., Dellinger, D., Truong, L., Cummins, L., Owens, S. R., Markham, P. M., Shea, J. P., and Crooke, S. T., Disposition of the 14C-labeled phosphorothioate ASO ISIS 2105 after intravenous administration to rats, J. Pharmacol. Exp. Ther. 267 (3), 1181, 1993. 49. Cossum, P. A., Troung, L., Owens, S. R., Markham, P. M., Shea, J. P., and Crooke, S. T., Pharmacokinetics of a 14C-labeled phosphorothioate ASO, ISIS 2105, after intradermal administration to rats, J. Pharmacol. Exp. Ther. 269 (1), 89, 1994. 50. Crooke, S. T., Graham, M. J., Zuckerman, J. E., Brooks, D., Conklin, B. S., Cummins, L. L., Greig, M. J., Guinosso, C. J., Kornbrust, D., Manoharan, M., Sasmor, H. M., Schleich, T., Tivel, K. L., and Griffey, R. H., Pharmacokinetic properties of several novel ASO analogs in mice, J. Pharmacol. Exp. Ther. 277 (2), 923, 1996. 51. Zinker, B. A., Rondinone, C. M., Trevillyan, J. M., Gum, R. J., Clampit, J. E., Waring, J. F., Xie, N., Wilcox, D., Jacobson, P., Frost, L., Kroeger, P. E., Reilly, R. M., Koterski, S., Opgenorth, T. J., Ulrich, R. G., Crosby, S., Butler, M., Murray, S. F., McKay, R. A., Bhanot, S., Monia, B. P., and Jirousek, M. R., PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice, Proc. Natl. Acad. Sci. USA 99 (17), 1137, 2002. 52. Hua, X. Y., Moore, A., Malkmus, S., Murray, S. F., Dean, N., Yaksh, T. L., and Butler, M., Inhibition of spinal protein kinase C alpha expression by an antisense oligonucleotide attenuates morphine infusion-induced tolerance, Neuroscience 113 (1), 99, 2002. 53. Butler, M., Hayes, C. S., Chappell, A., Murray, S. F., Yaksh, T. L., and Hua, X. Y., Spinal distribution and metabolism of 2⬘-O-(2-methoxyethyl)-modified ASOs after intrathecal administration in rats, Neuroscience 131 (3), 705, 2005. 54. Driver, S. E., Robinson, G. S., Flanagan, J., Shen, W., Smith, L. E. H., Thomas, D. W., and Roberts, P. C., ASO-based inhibition of embryonic gene expression, Nat. Biotechnol. 17, 1184, 1999. 55. Soucy, N. V., Riley, J. P., Templin, M. V., Geary, R., de Peyster, A., and Levin, A. A., Maternal and fetal distribution of a phosphorothioate ASO in rats after intravenous infusion, Birth Defects Res. B Dev. Reprod. Toxicol. 77 (1), 22, 2006. 56. Henry, S. P., Denny, K. H., Templin, M. V., Yu, R. Z., and Levin, A. A., Effects of human and murine antisense oligonucleotide inhibitors of ICAM-1 on reproductive performance, fetal development, and post-natal development in mice, Birth Defects Res. B Dev. Reprod. Toxicol. 71 (6), 359, 2004. 57. Yu, R. Z., Zhang, H., Geary, R. S., Graham, M., Masarjian, L., Lemonidis, K., Crooke, R., Dean, N. M., and Levin, A. A., Pharmacokinetics and pharmacodynamics of an antisense phosphorothioate ASO targeting Fas mRNA in mice, J. Pharmacol. Exp. Ther. 296 (2), 388, 2001. 58. Wilson, D. M., 3rd, Ape1 abasic endonuclease activity is regulated by magnesium and potassium concentrations and is robust on alternative DNA structures, J. Mol. Biol. 345 (5), 1003, 2005. 59. Gaus, H. J., Owens, S. R., Winniman, M., Cooper, S., and Cummins, L. L., On-line HPLC electrospray mass spectrometry of phosphorothioate ASO metabolites, Anal. Chem. 69 (3), 313, 1997.

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60. Yu, R. Z., Baer, B., Chappel, A., Geary, R. S., Chueng, E., and Levin, A. A., Development of an ultrasensitive noncompetitive hybridization-ligation enzyme-linked immunosorbent assay for the determination of phosphorothioate ODN in plasma, Anal. Biochem. 304 (1), 19, 2002. 61. Jackson, J. K., Gleave, M. E., Gleave, J., and Burt, H. M., The inhibition of angiogenesis by antisense oligonucleotides to clusterin, Angiogenesis 8 (3), 229, 2005. 62. Gleave, M. and Chi, K. N., Knock-down of the cytoprotective gene, clusterin, to enhance hormone and chemosensitivity in prostate and other cancers, Ann. NY Acad. Sci. 1058, 1, 2005. 63. Gleave, M. and Miyake, H., Use of antisense oligonucleotides targeting the cytoprotective gene, clusterin, to enhance androgen- and chemo-sensitivity in prostate cancer, World J. Urol. 23 (1), 38, 2005. 64. Adjei, A. A., Dy, G. K., Erlichman, C., Reid, J. M., Sloan, J. A., Pitot, H. C., Alberts, S. R., Goldberg, R. M., Hanson, L. J., Atherton, P. J., Watanabe, T., Geary, R. S., Holmlund, J., and Dorr, F. A., A phase I trial of ISIS 2503, an antisense inhibitor of H-ras, in combination with gemcitabine in patients with advanced cancer, Clin. Cancer Res. 9 (1), 115, 2003. 65. Villalona-Calero, M. A., Ritch, P., Figueroa, J. A., Otterson, G. A., Belt, R., Dow, E., George, S., Leonardo, J., McCachren, S., Miller, G. L., Modiano, M., Valdivieso, M., Geary, R. S., Oliver, J. W., and Holmlund, J., A phase I/II study of LY900003, an antisense inhibitor of protein kinase C-alpha, in combination with cisplatin and gemcitabine in patients with advanced non-small cell lung cancer, Clin. Cancer Res. 10 (18 Pt 1), 6086, 2004. 66. Geary, R. S., Bradley, J. D., Watanabe, T., Kwon, Y., Wedel, M., van Lier, J. J., and Vanvliet, A. A., Lack of pharmacokinetic interaction for ISIS 113715, a 2⬘-O-methoxyethyl modified antisense oligonucleotide targeting protein tyrosine phosphatase 1B messenger RNA, with oral antidiabetic compounds metformin, glipizide or rosiglitazone, Clin. Pharmacokinet. 45 (8), 789, 2006. 67. Kastelein, J. J. P., Wedel, M. K., Baker, B. F., Su, J., Bradley, J. O., Yu, R. Z., Chuang, E., Graham, M. J., and Crooke, R. M. Potent reduction of apolipoprotein B and Low-density lipoprotein cholesterol by short-term administration of an antisense inhibitor of apolipoprotein B. Circulation 114 (16), 1729, 2006. 68. Yu. R. Z., Kim, T. W., Hong, A., Watanabe, T. A., Gaus, H. J., and Geary, R. S. Cross species, Pharmacokinetic comparison from mouse to man of a second generation antisense oligonucleotide ISIS 301012, targeting human apolipoprotein 8-100. Drug Metab. Dispos. 35, 460, 2007.

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8

Routes and Formulations for Delivery of Antisense Oligonucleotides Gregory E. Hardee, Lloyd G. Tillman, and Richard S. Geary

CONTENTS 8.1 8.2

Introduction .........................................................................................................................217 Systemic Administration .....................................................................................................218 8.2.1 Physical and Chemical Properties ...........................................................................218 8.2.2 Intravenous Infusion ................................................................................................219 8.2.3 Subcutaneous Injection ............................................................................................219 8.3 Local Administration ...........................................................................................................221 8.3.1 Brain ........................................................................................................................221 8.3.2 Ocular ......................................................................................................................221 8.3.3 Topical Delivery ......................................................................................................222 8.3.4 Pulmonary Delivery .................................................................................................223 8.3.4.1 Formulation Considerations .....................................................................223 8.3.4.2 Deposition and Uptake .............................................................................223 8.3.4.3 Demonstration of Pharmacology by the Pulmonary Route .....................224 8.4 Oral and Gastrointestinal Delivery ......................................................................................225 8.4.1 Presystemic Metabolism ..........................................................................................225 8.4.2 Permeability .............................................................................................................227 8.4.3 Systemic Bioavailability ..........................................................................................228 8.4.4 Local GI Uptake ......................................................................................................231 8.5 Conclusions .........................................................................................................................233 References .....................................................................................................................................234

8.1 INTRODUCTION Initial efforts in the creation of antisense therapeutics have focused on fundamental questions such as safety, efficacy, and technology costs, as would be expected for any emerging technology with a potential to create a new therapeutic paradigm. Clinical proof of efficacy has been established with the approval of the first antisense oligonucleotide therapeutic, Vitravene™ (fomivirsen), indicated for the local treatment of cytomegalovirus (CMV) retinitis in patients with AIDs [1].

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Following this approval, we have seen a dramatic and continuing fall in the cost of goods; it is appropriate that we now address the area of delivery routes and formulations. Advances in this area have the potential to impact oligonucleotide biopharmaceutics and expand the utility of antisense technology. Early research in drug delivery systems focused on a perceived weakness of the technology: cellular uptake. In vitro observations made with phosphorothioate oligodeoxynucleotides led many working in the area to conclude that because of metabolic instability, size and charge, oligonucleotides would not be adequately taken up into the targeted tissues to the extent necessary to exert pharmacological activity. However, an ever expanding body of in vivo animal and clinical data has shown that even simple saline solutions delivered parenterally can lead to significant oligonucleotide uptake and effect. While such simple formulations are efficacious for some therapeutic applications, the development of novel formulations and delivery routes should enable a wider variety of target tissues to be treated through increased efficiency of target-tissue uptake and accumulation. In this chapter, we will address a variety of challenges and opportunities in the delivery of antisense oligonucleotides for a wide range of diseases.

8.2 SYSTEMIC ADMINISTRATION 8.2.1

Physical and Chemical Properties

In the development of any drug product, it is essential that the fundamental physical and chemical properties of the drug molecule and of prototype drug formulations be determined. Such data often dictate the choices considered for dosage form design and subsequent development events. With respect to the inherent properties of the unformulated oligonucleotide, there is sufficient evidence that we may assign common properties for all oligonucleotides for a given class of oligonucleotide chemistry. That is, these data consistently demonstrate that all “firstgeneration” or phosphorothioate oligodeoxynucleotides exhibit similar physiochemical properties. Likewise, newer chemistries appear to have conformity within their defined chemistry, such as for sugar-modified chemistries (e.g., 2
-O-methoxyethyl [2
-MOE]) or for backbone-modified chemistries (e.g., methylphosphonates). This important feature of class conformity allows estimation of biopharmaceutic and pharmacokinetic parameters across the innumerable nucleotide sequences possible within any single oligonucleotide chemical class. For example, rates of drug release from a dosage form can be estimated from precedent data as well as distribution and elimination estimates even where the biopharmaceutic phenomena are dependent upon biophysical interactions such as protein binding. The exception to this axiom may be in instances where intra- or intermolecular Watson–Crick base pairing occurs. Such base pairing, if significant, may result in a stable secondary structure (if self-complementary) or in a noncovalent polymeric structure consistent with “nearest neighbor” thermodynamic data [2]. Such altered states need to be anticipated if oligonucleotide sequences permit such associations beyond four or five base pairs per 20-mer oligonucleotide. Phosphorothioate oligodeoxynucleotides are synthesized as complex mixtures of diastereomers. In the solid state they are amorphous, electrostatic, hygroscopic solids with low-bulk densities, possessing very high surface areas, and poorly defined melting points. Their good chemical stability allows them to be stored both as lyophilized powders and as concentrated, sterile, aqueous solutions. Stability data demonstrate only minimal change in impurity profile when (1) lyophilized powders are stored at 25°C for more than three years, or (2) sterile solutions are stored refrigerated for more than three years. Oligonucleotides are extremely water-soluble under neutral and basic conditions, consistent with their polyanionic nature. Consequently, drug-product concentrations are often only limited by an increase in solution viscosity at very high concentrations, e.g. 300 mg/ml. Extremes of pH and ionic strength influence the apparent solubility. In acidic environments such as the stomach, ionized

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portions of the molecule are partially neutralized by protonation resulting in a marked decrease in oligonucleotide solubility, an event that can be easily reversed by subsequently elevating the pH. All the physical-chemical characteristics discussed here are important to designing viable, efficacious formulations with acceptable storage and in vivo stability. Each delivery system presents its own formulation challenges, as will be seen in the remainder of this chapter. Phosphorothioate oligonucleotide degradation has been primarily attributed to two mechanisms: desulfurization and acid-catalyzed hydrolysis. Desulfurization of the backbone, observed at elevated temperatures and under intense UV light, leads to (pharmacologically active) oligonucleotides primarily containing one or more phosphate diester linkages where oxygen has replaced sulfur [3]. Note that while such oligonucleotides are pharmacologically active, the accumulation of phosphodiester linkages—particularly if on adjacent or terminal nucleotides—renders the compound more susceptible to metabolism by nucleases. Thus, the presence of numerous phosphodiester linkages may shorten the compound’s in vivo half-life, particularly if the phosphodiesters are located in the central deoxy-gap of the oligonucleotide (e.g., MOE gap-mer oligonucleotides). This gap is between the nuclease-resistant wings of the molecule where 2
-O-alkoxyalkane ribonucleosides afford protection from enzymatic cleavage. Indeed, there is evidence that chimeric oligonucleotides containing phosphodiester linkages along with 2
-O-alkoxyalkane derivatives improve in vivo activity [4]. Under acidic conditions, a phosphodiester oligonucleotide may be more prone to depurination, resulting in an oligonucleotide containing one or more abasic sites. The loss of bases decreases the base-pair binding affinity to the target mRNA, rendering the oligonucleotide less effective. Also, abasic sites are more susceptible to base-mediated cleavage of the adjacent phosphorothioate linkages. Formulation development must ensure adequate stability and therefore must include analytical techniques for monitoring these attributes. Desulfurization can be monitored by a variety of analytical techniques including, for example, capillary gel electrophoresis, anion-exchange and ion-pair liquid chromatography; however, liquid chromatography coupled to mass spectroscopy is required to monitor other products of which some may be stability limiting. As such, this technique is rapidly becoming the method of choice for assessing oligonucleotide quality and stability. 8.2.2

Intravenous Infusion

Twenty-mer oligonucleotides generally are isotonic (300 mOsm/kg) in solution at concentrations of approximately 150 mg/mL. Simple salt solutions can be used to achieve isotonicity for less concentrated solutions if desired. Additionally, buffering salts can be added as a manufacturing aid and stabilizing agent. These simple solutions have been administered intravenously to mice, monkeys, dogs, pigs, and humans without limitation other than total dose and rate of run-in considerations. 8.2.3

Subcutaneous Injection

Many consider the practical limitation of the subcutaneous route to be a dose volume of approximately one milliliter. Given the high water-solubility of oligonucleotides and that pain of injection studies (using hypertonic salt solutions) that show only slight and transient discomfort up to threefold tonicity, the only limiting factor for subcutaneous dosing is the solution viscosity. Viscosity of oligonucleotide solutions increase exponentially as a function of concentration (Figure 8.1). Not unexpectedly, different sequences have different intermolecular interactions that manifest as significantly different viscosity profiles. This is one of the few sequence-dependent physical-chemical properties noted to date. While it is possible to inject solutions of 100 cPs through a 29-gauge needle, it is very difficult to draw up the dose, or inject with the newer microbore needles (31 gauge) especially if the dosing solution is not equilibrated to room temperature since viscosity is a function of temperature (Figure 8.2). Practical solutions to this limitation include using prefilled syringes, dose withdrawal with a large bore needle, room-temperature storage, and of course limiting the solution concentration.

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400

Viscosity (cP)

300

200

100

0 0

50

100

150

200

250

300

350

25

30

400

Oligo conc. (mg/mL) Figure 8.1

Effect of oligonucleotide concentration on viscosity.

500 ISIS 104838

Viscosity (cP)

400 ISIS 113715

300 200 100 ISIS 107248

0 0

5

10

15

20

Temperature (°C) Figure 8.2

Effect of temperature (C) on viscosity (cP) for various 200 mg/mL oligonucleotides.

Table 8.1 Systemic Bioavailability by the Subcutaneous Route Is Essentially Complete for a Second-Generation Oligonucleotide, ISIS 104838 Administration Route Monkey IV Subcutaneous IV Subcutaneous Human IV Subcutaneous a b

Dose (mg/kg) 3 4 10 20 2 2.5

Cmax (
g/mL)

AUC (
gh/mLa)

Plasma BAV (%)

Tissue BAV (%)

33 5.0 92 15.2

46 36 149 364

100 59 100 122

100 96 100 105a

26 5.5

49 49

100 80

NMb NM

Liver. Not measured.

Bioavailability after a subcutaneous injection has been studied extensively in multiple species. While bioavailabilities measured in plasma show nonlinear dose proportionality, the bioavailabilities measured in tissue are essentially complete at all doses. Table 8.1 presents data from monkey

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[5] and man [6] that illustrates apparently low plasma bioavailability at low subcutaneous doses. Since the majority of putative targets for oligonucleotides reside in tissue, it is appropriate to think of subcutaneous dosing as 100% bioavailable. With the potency and biological half-life achieved with the newer generation of oligonucleotides, the subcutaneous route is being advanced in the clinic for a variety of indications.

8.3 LOCAL ADMINISTRATION 8.3.1

Brain

In general, the blood–brain barrier (BBB) is only permeable to lipophilic molecules having a molecular weight less than about 600 Da, while even smaller and electrostatic water-soluble molecules generally can not be transported [7]. This precludes delivery of oligonucleotides to the brain from the systemic circulation without the assistance of specialized drug-delivery systems. Unmodified phosphodiester oligodeoxynucleotides have been directly injected intracerebrally to down-regulate the expression of neurotransmitters in a sequence-specific manner [8]. As expected, phosphodiester oligodeoxynucleotides are rapidly degraded in the brain and while the phosphorothioate oligodeoxynucleotides are more metabolically stable, they are nevertheless rapidly cleared via the cerebrospinal fluid bulk flow. Whitesell et al. demonstrated extensive brain penetration and marked cellular uptake after continuous intracerebral infusions with a mini-osmotic pump [9]. Direct intracerebral administration has been utilized by Sommer et al. [10], to study phosphorothioate oligodeoxynucleotides directed to c-fos and by Mustafa and Dar [11], to down-regulate the adenosine A1 receptor. One report in the literature details the accumulation of systemically administered phosphorothioate oligodeoxynucleotides in implanted subcutaneous and intracranial glioblastoma tumors in mice [12]. A similar phosphorothioate oligonucleotide was shown not to cross the BBB in normal animals [13], so it was hypothesized that the presence of a glioma may sufficiently disrupt the BBB to allow pharmacological concentrations of oligonucleotide to accumulate within the tumor. Accumulation was sufficient in this model to demonstrate antitumor activity. Boado et al. devised delivery systems based on conjugates of streptavidin and the OX26 monoclonal antibody directed to the transferrin receptor as a carrier for the transport of oligonucleotides [14]. These delivery systems were found to transport peptide nucleic acid antisense molecules, but not phosphorothioate oligonucleotides, across the BBB. The authors attributed this difference to preferential binding of phosphorothioate oligonucleotide to plasma protein instead of the antibody complex, which reduced transport. 8.3.2

Ocular

The first approved antisense oligonucleotide therapeutic, Vitravene (fomivirsen) is indicated for the local treatment of cytomegalovirus (CMV) retinitis in patients with AIDs [1]. Local treatment is accomplished by intravitreal injection of 50
L of a sterile, aqueous solution with a 30gauge needle. Fomivirsen is cleared from the vitreous of rabbits over the course of 7 to 10 days, by a combination of tissue distribution and metabolism. Following direct intravitreal injection, fomivirsen concentrations were greatest in the retina and iris. Fomivirsen was detectable in retina within hours after injection, and concentrations increased over 3 to 5 days. Metabolism is the primary route of elimination from the eye. Metabolites of fomivirsen are detected in the retina and vitreous in animals. Systemic exposure to fomivirsen following single or repeated intravitreal injections in monkeys was below limits of quantization. In monkeys treated every other week for up to 3 months with fomivirsen, there were isolated instances when fomivirsen’s metabolites were observed in liver, kidney, and plasma at a concentration near the level of detection [15].

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An alternative to direct intravitreal injection is to use a noninvasive method of oligonucleotide delivery employing iontophoresis. The advantage of using low-current-density iontophoresis is that the oligonucleotide intraocular distribution will allow for therapeutic concentrations to be achieved across a large number of tissues, including the anterior uvea and the cornea as well as neural retinal areas. Voigt et al. [16] evaluated this intraocular oligonucleotide delivery approach in the rat model of endotoxin-induced uveitis, which is characterized by NOSII (nitrous oxide synthase II) up-regulation. Iontophoresis facilitated the penetration of both anti-NOSII oligonucleotide and a control oligonucleotide with scrambled nucleotide sequence across the intraocular tissues of the eyes. Both active and control oligonucleotide were 21-base oligodeoxynucleotides. This biodistribution was demonstrated by histological examination of the fluorescently labeled oligonucleotides and by the down-regulation of NOSII in the iris/ciliary body of the active treated eyes compared to the saline or scrambled oligonucleotide-treated eyes. The application of iontophoresis demonstrates a unique and noninvasive approach to efficiently deliver oligonucleotide into the eye—increasing the likelihood of successful therapy for debilitating eye diseases manifesting posterior segments of the eye. 8.3.3

Topical Delivery

The barrier properties of human skin have long been an area of multidisciplinary research. Skin is one of the most difficult biological barriers to penetrate and traverse, primarily due to the presence of the stratum corneum. The stratum corneum is composed of corneocytes laid in a brick-and-mortar arrangement with layers of lipid. The corneocytes are partially dehydrated, anuclear, metabolically active cells completely filled with bundles of keratin with a thick and insoluble envelope replacing the cell membrane [17]. The primary lipids in the stratum corneum are ceramides, free sterols, free fatty acids, and triglycerides that form lamellar lipid sheets between the corneocytes [18]. These unique structural features of stratum corneum provide an excellent barrier to the penetration of most molecules, particularly, large hydrophilic molecules such as oligonucleotides. To penetrate the stratum corneum barrier, a number of approaches can be pursued. These approaches either alter the oligonucleotide’s physiochemical properties directly, the biophysical properties of the barrier, or provide some other driving force or influence onto both drug and the stratum corneum barrier. Altering the thermodynamic properties of oligonucleotide molecules is possible by either utilizing a hydrophobic counter cation (e.g., benzalkonium) or by chemically modifying the oligonucleotide chemistry to eliminate the anionic backbone charges (e.g., morpholino or PNA oligonucleotide chemistries). These modifications have resulted in increased penetration across isolated hairless mouse skin. This was explained on the basis of greater partitioning into the lipid phase [19]. Alternatively, physically changing the stratum corneum to disrupt its integrity can result in improved skin penetration. There have been a number of creative approaches to decrease the integrity of the skin without overtly causing long-term damage. Some examples of these breaching methods to induce transient openings in the stratum corneum include both ultrasound-induced sonoporation [20] and electroporation [21]. Iontophoresis, which involves application of an electric field across the skin to induce electrochemical transport of charged molecules, has been studied extensively for transdermal delivery of oligonucleotides [22]. Lastly, the use of topical formulations containing permeation enhancers (PEs) offers several advantages in regard to the tailoring of formulation compositions and on the practical ease of application—particularly being able to avoid the use of application devices. Chemical penetration enhancers have recently been studied for increasing transdermal delivery of oligonucleotides or other polar macromolecules [23]. Chemically induced transdermal penetration results from a transient reduction in the barrier properties of the stratum corneum. The reduction may be attributed to a variety of factors such as opening of intercellular junctions due to hydration [24], solubilization of the stratum corneum [25,26], or increased lipid bilayer fluidization [27]. Combining various surfactants and co-solvents can be used to achieve skin penetration, purportedly resulting in therapeutically relevant concentrations of oligonucleotide

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in the viable epidermis and dermis [28]. In summary, it appears feasible to deliver oligonucleotide to the skin using a number of different delivery techniques and formulations. 8.3.4

Pulmonary Delivery

The ability to efficiently deliver oligonucleotides to the lungs makes it possible to treat a number of important diseases. Preclinical data demonstrate delivery to lung tissue by way of intravenous (IV) administration or by the pulmonary route. However, parenteral delivery of oligonucleotides does not lead to substantial lung tissue accumulation, and is therefore not an ideal route for treatment of disease in lung tissue. After a 5-mg/kg IV bolus injection in mice, for example, the concentration in lung tissue has been shown to be less than 2
g/gm [5]. Direct administration to the pulmonary airways through inhalation targets specific cell types not accessible via the parenteral route, increases efficiency of dosing with higher concentrations at much lower administered dose, and results in profound pharmacological response in murine models for asthma (e.g., [29]). The pulmonary route is an increasingly attractive option for local delivery of a wide variety of macromolecules including oligonucleotides. Delivery options include nebulizers, metered dose inhalers, and dry powder inhalers. Any of these pulmonary devices are far less invasive than parenteral administration and, in addition to potentially increasing efficacy, are likely to increase patient compliance.

8.3.4.1 Formulation Considerations Wu-Pong and Byron have contributed a review of the issues associated with the pulmonary delivery of oligonucleotides [30]. Since oligonucleotides are freely soluble and highly hygroscopic, it would be reasonable to assume first-dosage forms will rely upon aerosolization of simple aqueous solutions. Our data suggest that commercially available nebulization devices will generate suitable aerosolizations of oligonucleotide solutions at concentrations of up to 180 mg/mL (Table 8.2) [31]. Ultrasonic and jet nebulizations were found to have essentially no effect on phosphorothioate stability of the oligonucleotide over 40 min, which is longer than typical treatment times (Figure 8.3). Depending on particle size, delivery may primarily be to the upper airways or to the alveoli, where systemic delivery may also be possible [30,32]. Particles with diameters of approximately 5–10
m will generally deposit in the upper airways, while those in the range of approximately 0.5–5
m will reach the alveoli. Smaller particles are often exhaled; larger particles are generally swallowed. Due to the relatively small doses of drug that may conveniently be delivered by aerosolization, this route may be more practical for treatment of the local tissue rather than systemic dosing for oligonucleotides.

8.3.4.2 Deposition and Uptake The local and systemic bioavailability of simple solutions of a second-generation phosphorothioate oligonucleotide (partially modified 2
-MOE) in monkeys by direct pulmonary administration was compared to parenteral administration (Table 8.3). Not surprisingly, direct delivery to the lung

Table 8.2 Nebulization of ISIS-2503 Solutions by Healthline Nebulizer Model NEB-3A at 10 mL /min [31] Concentration (mg/mL)

MMADa (
m)

100 150 180

1.02 0.77 0.70

a b

Mass mean aerodynamic diameter. Geometric standard deviation.

GSD (
m)

% Particles 5
m

Solution Delivery Rate (mg/min)

1.96 2.27 2.29

98.6 98.0 97.8

258 213 198

b

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Ultrasonic

Jet

% Full length, full PS

100 98 96 94 92 90 0

Figure 8.3

10

20 Time (min)

30

40

Effect of different nebulization methods on phosphorothioate oligonucleotide stability.

Table 8.3 Bioavailability of ISIS 141923 (2

MOE chimera) after pulmonary administration (0.5/mg/kg) Tissue or Biofluid Lung Plasma Peribronchial lymph nodes Liver Kidney cortex a

Concentrationa (
g/g or
g/mL)

% Bioavailability (Relative to IV)

6.6 (2.5) nd 1.0 (0.6) nd 0.4 (0.1)

1260 1% 0.2 0.1 0.07

Tissues analyzed by LC/MS/MS LLOQ = 0.125
g/g; plasma analyzed by hybridization ELISA–LLOQ = 2 ng/mL.

demonstrated the highest bioavailability to the lung and very little was absorbed systemically. This preferential exposure of the target tissue is ideal for optimization of effect and minimization of offtarget adverse effect. At Isis Pharmaceuticals, Inc., antisense compounds were successfully delivered to cells of the rat lung using simple aqueous formulations aerosolized by commercial nebulization equipment [33]. Single or multiple nose-only administrations led to significant oligonucleotide concentrations in lung tissues and bronchiolar lavage fluid with low or mild toxicity, depending on dose. Using immunohistochemistry, oligonucleotides could be visualized in pneumocytes, vascular endothelium, and alveolar macrophages. Single and multiple exposures differed only in the amount of oligonucleotide present and not the distribution pattern. A similar study in mice determined that the cellular uptake of the phosphorothioate 2
-MOE oligonucleotide ISIS 13920 was specific: predominantly into macrophages, and to lesser extent, eosinophils [34]. Cellular uptake was also observed in both tracheal and bronchial epithelium. Furthermore, the use of newer oligonucleotide chemistries, such as the sugar-modified (2
-O-methoxyethyl) derivatives that significantly enhance tissue accumulation allows higher steady-state concentrations or less frequent dosing to be achieved, due to their longer tissue elimination half-lives (Table 8.4).

8.3.4.3 Demonstration of Pharmacology by the Pulmonary Route In addition to these studies that characterize deposition and uptake, there are several reports in the literature that demonstrate antisense pharmacology using the pulmonary route. Administration of an aerosolized phosphorothioate oligodeoxynucleotide (first-generation antisense to adenosine A1

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Table 8.4 Comparison of Lung Tissue Exposure 24 hrs after Nose-Only or Intratracheal Administration of ISIS 141923 Species

0.1 mg/kg

0.5 mg/kg

5 mg/kg

50 mg/kg

Lung Tissue t1/2 (days)

Mouse Monkey

0.35 1.0

– 6.6

5.2 –

73 –

4 7

Note: Dose level expressed as the pulmonary dose, i.e., amount estimated to be deposited in lung. Necropsy conducted 24 h after the end of exposure (EOE) or at selected time intervals after exposure. Oligonucleotide concentration data expressed as mean
g oligonucleotide/g lung tissue. n  3 to 5 animals.

receptor) desensitized rabbits to subsequent challenge with either adenosine or dust mite allergen. Rabbits treated with the sequence-specific oligonucleotide demonstrated a dose-dependent desensitization, while animals treated with a mismatched sequence control showed no desensitization [35]. Specifically, the authors showed that aerosols created with ordinary nebulizers (which create 80% of droplets  5
m in diameter) effectively reach the bronchial smooth muscle in sufficient quantities to specifically attenuate the target up to 75% [36]. Similar results were found using bradykinin B2 receptor-specific oligonucleotide and a mismatched control [35]. Isis Pharmaceuticals has reported preclinical activity of nebulized 2
-MOE ASOs targeting IL-3R
, CD86, p38
MAP kinase, and TNF-
mRNAs in mouse models of asthma at inhaled doses in the microgram-per-kilogram range [29,37] administered as simple solutions. These ASOs have demonstrated a broad profile of pharmacological activity in acute and chronic models of inhaled allergen challenge in mice. Sequence-specific reductions in targeted protein observed on pulmonary epithelial cells, dendritic cells, macrophages, and eosinophils were seen consistent with the distribution of the ASOs observed by immunohistochemical staining. The literature reviewed here shows that both formulated and unformulated (simple aqueous solution) oligonucleotides can be delivered to the lungs where they subsequently suppress local gene expression, opening a wide variety of diseases to antisense therapy.

8.4 ORAL AND GASTROINTESTINAL DELIVERY Oral and rectal dosage forms are ideal for patient convenience and compliance. For traditional small molecules, these routes have been used for both systemic delivery and delivery to local gastrointestinal (GI) tissues. For 6000 Da, polyanionic, hydrophilic oligonucleotide molecules, a variety of issues must be considered when designing such dosage forms, particularly to enable systemic delivery by the oral route. Among these are chemical instability and precipitation at gastric pH, metabolic instability within the intestinal environment, low intestinal permeability, high protein binding, and first-pass hepatic clearance. These challenges have led many to conclude that effective oral systemic administration of oligonucleotides is not feasible [38]. Indeed, the early reports of oral bioavailability approaching 35% from gavage solutions containing radioactive phosphorothioate oligodeoxynucleotides in rats were consequently determined to be the uptake of degraded forms [39]. The above-mentioned barriers to oral bioavailability of oligonucleotides were delineated by Nicklin et al. [40]. Of these, our experience demonstrates that two stand out as critical: instability in the GI tract and low permeability across the intestinal mucosa. However, progress is being made to address and understand each of these barriers by way of changes to oligonucleotide chemistry and use of appropriate formulations. 8.4.1

Presystemic Metabolism

Natural DNA and RNA are rapidly digested by the ubiquitous nucleases found within the gut. As a consequence, oligonucleotides require stabilization to achieve a reasonable GI residence time

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to allow absorption to occur. To impart enzyme resistance, modifications can be made to the phosphate backbone, the nucleotide sugar, or both. It has been demonstrated that such modifications need to be incorporated at only a subset of the sites available on the oligonucleotide molecule. Such hybrid structures have demonstrated significantly improved nuclease stability. This was shown by Zhang for both backbone modifications (methylphosphonates) and for sugar-modified (2
-O-methyl) oligonucleotides [41,42]. However, care must be taken to ensure that mRNA binding affinity is not impaired by such modifications—particularly, modifications involving an increase in the size of the 2
-O-substituent. Therefore, it is not surprising that small 2
-O-alkoxyalkyl substituents (i.e., groups related to ethylene glycol) retain or improve upon the RNA binding affinity and have greatly improved nuclease resistance. The 2
-MOE derivative was proposed as a promising candidate from this class of derivatives [43] and has since been evaluated for presystemic enzyme stability within the digestive system of the rat. This stability is proportional to the degree of sugar substitution (Figure 8.4). Relative to the unmodified phosphorothioate oligodeoxynucleotides, these data demonstrate the increasing benefit of MOE substituents present as either a hemimer, with MOEs at the 3
termini only; or a gapmer, with MOEs present at both the 3
and 5
termini; and lastly, a full MOE substitution at every sugar location [44]. These derivatives possess promising properties but are still awaiting clinical demonstration of their utility as therapeutic agents. Selection of a specific chemistry for development depends upon a number of criteria, of which nuclease stability is but one. An alternative approach to improving enzyme stability of oligonucleotides is by way of formulations that impose a physical barrier, preventing nuclease from reaching the oligonucleotide cleavage site [45–47]. Examples of such formulations include (1) conventional modified release dosage forms that incorporate the oligonucleotide within the solid matrix of the dosage unit, and (2) oligonucleotides complexed with cationic species formulated through a process referred to as complex coacervation. In addition to enzyme stability, such formulations have been purported to impart additional characteristics favorable to oligonucleotide absorption, including modified delivery (e.g., site-specific or increased retention by bioadhesion), a change in oligonucleotide physicochemical properties (e.g., charge neutralization or lipophilicity), and a change or impact on the absorbing membrane itself (e.g., opening of tight junctions or fluidization of mucosal membrane). These concepts are more fully discussed below.

P=S full 2'-MOE

P=S 2′-MOE gapmer − capped

P=S ODN

P=S 3′-2′-MOE hemimer

% Intact presystemically

120 100 80 60 40 20 0 0

Figure 8.4

5

10

15 Time (h)

20

25

30

Stability of various oligonucleotide chemistries in the rat intestine prior to absorption.

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227

Permeability

The physicochemical properties of phosphorothioate oligonucleotides present a significant barrier to their GI absorption into the systemic circulation or the lymphatics. These properties include their size (approximately 7 kDa for 20-mers), hydrophilic nature (log Do/w approximating 3.5), and multiple ionization pKas (e.g., unpublished titration data, using a Sirius GlpKa instrument on a 20-mer sequence, noted over 17 pKas for the unmodified oligodeoxynucleotide and over 32 pKas for the MOE hemimer form). While the baseline permeability values for 20-mer oligodeoxynucleotides approximate 2  106 cm/s in the rat small intestine (mucosal-to-serosal), this can be improved by chemistry modifications (Figure 8.5), these values are still far below those of compounds exhibiting high permeability, such as naproxen [48]. The use of formulations can further improve upon this permeability. When formulating oligonucleotide drugs to improve oral bioavailability, the mechanism of oligonucleotide absorption, either paracellular via the epithelial tight junctions or transcellular by direct passage through the lipid membrane bilayer, must be considered. Modeling permeation data using paracellular and transcellular models appropriate for water-soluble, hydrophilic macromolecules suggests that oligonucleotides predominantly traverse the GI epithelium via the paracellular route [49–52]. Therefore, one must select permeation enhancers (PEs) that facilitate paracellular transport in a transient and reversible manner while meeting other formulation criteria including suitable biopharmaceutics, safety considerations, manufacturability, physical and chemical stability, and practicality of the dosage-form configuration (i.e., regarding production costs, dosing regimen, patient compliance, etc.). Initial attempts at selecting PEs have identified certain surfactants, such as bile salts and fatty acids, which appear to facilitate oligonucleotide absorption. The advantages of these components are many in that they are endogenous to foods and body constituents, and the literature is rich with information about the use and exposure of these two classes of compounds [53]. The precise mechanism of action for these PEs are unknown but are believed to involve a disruption of the mucus layer barrier, an increase in the fluidity of the mucosal membrane, and potentially opening the paracellular, tight-junction opening. The mucolytic effect coupled with the increased membrane fluidity

80 × 10 −6 60 × 10 −6 40 × 10 −6

Figure 8.5

P=O full MOE

P=O full MOE

P=S full MOE

P=S 2′-MOE 3′-hemimer

P=S 2′-MOE gapmer

0

P=S ODN

20 × 10 −6 Naproxen

Permeation coefficient (cm/sec)

100 × 10 −6

Effect of oligonucleotide chemistry on permeability in the rat intestine in situ [49].

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imparted by these excipients appears to allow increased concentrations of oligonucleotide to enter the villi crypt wells. Experimental data suggest that tight junctions at the villi crypts are larger and therefore more permeable than their counterparts at the villi tips [54]. When PE-formulated drug is administered orally to mice, the oligonucleotide can be visualized by immunohistochemistry throughout the brush border areas of the murine ileum [52]. This enhanced intimacy of the oligonucleotide to the absorbing surface should increase its GI residence time and therefore its potential absorption via entry into the paracellular tight junctions. 8.4.3

Systemic Bioavailability

When the previously mentioned formulation (oligonucleotide and penetration enhancer in solution for intrajejunal [IJ] administration) was assembled as a conventional tablet with a one-hour dissolution profile, the plasma concentrations after oral dosing were negligible (data not shown). Upon reformulation to promote immediate dissolution (in less than 15 min), the absolute bioavailability approached 3%, as indicated by the plasma concentration plots in (Figure 8.6). These results, obtained with a rudimentary solid dosage form are ⬃40% that obtained with the IJ dosing. From these results, it may be concluded that the following factors are important for oligonucleotide absorption in the above PE system: coincident presentation of oligonucleotide with the PE components, high concentrations at the absorbing site, and enteric protection to prevent component dilution and oligonucleotide exposure to low pH, which has been associated with rapid degradation. It should be noted that plasma concentrations represent the absorption and distribution of oligonucleotide, but not the true tissue or body elimination. A more relevant metric for pharmacokinetics and bioavailability would be the total tissue levels. This is particularly true for oligonucleotide chemistries that are rapidly cleared from the bloodstream but are accumulated in target tissues or organs. In line with this hypothesis, a study was performed to evaluate plasma pharmacokinetics and tissue distribution of ISIS-2302 and ISIS-15839, a phosphorothioate oligodeoxynucleotide and a MOE hemimer with the same sequence. Pharmacokinetic parameters for each chemical class were assessed in beagle dogs during and following 14-day oral dosing of tablets containing oligonucleotide and PEs in enteric-coated tablets [55]. The in vitro performance of these tablets was such that they remained intact in acid medium, yet rapidly dissolved (⬃15 min) at pH 6.5. In plasma, the 15839 IJ solution (n = 2) 2302 IJ solution (n = 3)

10 Plasma conc. (µg/mL)

2302 oral tablets (n = 6) 15839 oral tablets (n = 4)

8 6 4 2 0 0

30

60

90

120

150

180

Time (min) Figure 8.6

Intact oligonucleotide in dogs after either IJ administration of a solution formulation (open symbols with broken lines, 10 mg/kg) or oral administration of a tablet formulation (filled symbols with solid lines, ⬃15 mg/kg) of two oligonucleotide chemistries. ISIS-2302 (squares) is a phosphorothioate oligodeoxynucleotide; ISIS-15839 (triangles) is a hemimer MOE derivative with the same sequence. Error bars are standard error.

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Table 8.5 Summary of Plasma Oligonucleotide Pharmacokinetics after the First Single 200-mg Oral Dose in a Multiple-Dose Study

AUC (
g min/mL) Cmax (
g/mL) % Plasma BAV

ISIS 15839

ISIS 2302

91 1.2 1–4%

62 1.1 0.1–3%

Table 8.6 Tissue Oligonucleotide Concentrations and Approximate Bioavailabilities Based on Data Obtained at the End of Two-Week Daily Dosing Study in Dogs Oligonucleotide Chemistry (20-Base Phosphorothioates) ISIS 2302—antisense to ICAM-1 (deoxy) ISIS 15839—antisense to ICAM-1 (hemimer MOE of ISIS 2302) a

Liver Concentration (
g/g)

Kidney Cortex Concentration (
g/g)

% BAVa

4.9 33

12 109

1.3 5.5

Average bioavailabilities as ratio of final tissue concentrations (oral to IV), dose adjusted.

ISIS 15839 gave approximately 150% the bioavailability found for ISIS 2302 (Table 8.5). However, oligonucleotide tissue concentrations at the end of the study for both liver and kidney cortex were approximately tenfold higher for ISIS 15839 than for ISIS 2302 (Table 8.6). In this multiple dosing study, the distribution of oligonucleotides out of the plasma circulation was very rapid with a distribution half-life of 30–60 min. Therefore, as mentioned earlier, the bioavailability assessments based upon plasma data may be inaccurate or misleading for this class of compounds and this relative range of absolute bioavailabilities. Alternatively, the bioavailability determined from sentinel tissues, such as the kidney and liver, may be a more credible representation of the true systemic exposure since these tissues reflect true elimination kinetics as well as accumulation to steady-state concentrations during chronic dosing. On this basis, our data indicate that bioavailabilities based upon organ concentrations (after oral and IV dosing) would be 50% higher for the kidney and 100% higher for the liver than those calculated by plasma concentrationtime area under the curves (AUC). In consideration of the practical difficulties involved in experimental determinations of organ bioavailability, we have performed simulations of organ accumulation after oral dosing of first-generation phosphorothioate oligodeoxynucleotide versus a MOE gapmer (Figure 8.7). Each simulation is generated assuming a random input metric of 2–15% oral bioavailability (geometric mean 7%) with all other pharmacokinetic parameters consistent for these two chemistries remaining fixed. These simulations indicate that even with variable absorption, long tissue half-lives provide sufficiently improved accumulation, making it feasible to give the newer chemistry MOE gapmer compounds by the oral route. Two studies of oligonucleotide oral administration in human volunteers have been reported in the literature. In the first, the oral absorption of the 2
-MOE-modified antisense oligonucleotide, ISIS 104838, was demonstrated in healthy volunteers with an average 9.5% plasma bioavailability across four formulations tested (Figure 8.8). The greatest average performance achieved in this study for a single formulation was 12.0% bioavailability within an individual dose and subject range of 1.96 to 27.5%. This study suggests that oral formulations can be devised that maintain target tissue concentrations associated with inhibition of targeted human mRNA [56]. The second study reports the oral bioavailability (BAV) and pharmacology of these formulations upon repeated oral administration of a second-generation oligonucleotide [57]. In this study, 36 healthy volunteers received an IV loading dose of 200 mg of ISIS 301012 on day 1, after which they received either oral ISIS 301012 or placebo once daily for 30 or 90 days. The oral formulations used in this study utilized a controlled release of the penetration enhancer, sodium caprate, to enhance oligonucleotide bioavailability. Subjects were monitored for plasma (trough) drug levels and serum lipids weekly. Additionally, complete (24-h) plasma pharmacokinetic profiles were

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Predicted conc. (µg/g)

100 80 60 40 20 0 0

Figure 8.7

200

400

600 Time (h)

800

1000

1200

Simulation comparison of tissue oligonucleotide concentrations for a daily oral dosing regimen of 1-month duration of a phosphorothioate oligodeoxynucleotide and a MOE gapmer oligonucleotide at 2 mg/kg using a variable bioavailability input metric randomly generated from 2% to 15% (geometric mean 7%).

A  IR minitabs  FASTED B  50/50 fast pulsatile  FASTED C  50/50 slow pulsatile  FASTED D  70/30 fast pulsatile  FASTED E  70/30 fast pulsatile  FED

3.5

ISIS 104838 conc. in plasma (µg/mL)

3.0

2.5

2.0

1.5

1.0

0.5

0.0 -2

Figure 8.8

0

2

4 6 Time (h)

8

10

12

Mean ISIS 104838 concentration–time profiles in plasma following oral administration of various test formulations. Each data point represents the mean of 6–13 individual measurements and the error bars are standard deviation.

conducted on the first and last oral dosing. A mean oral bioavailability of 6% was observed in this study (Figure 8.9). Maximal pharmacological responses were achieved on days 55 and 69 with a statistically significant decrease of 12–15% apoB and commensurate reductions in LDL-C (p0.005) (Figure 8.10). The oral formulation was generally, well tolerated with mild, intermittent GI symptoms noted. While oral bioavailability was modest, averaging 6%, this study was the first to report significant gene down-regulation and pharmacological activity after oral administration of an antisense oligonucleotide and provides key information for future formulation enhancements.

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IV 100 mg (n = 6) ORAL 500 mg, Day 3 (n = 24) ORAL 500 mg, Day 43 (n = 24)

100

ISIS 301012 in plasma (µg/mL)

231

10

1

0.1

0.01

0.001 0

5

10 15 Time (h)

20

25

30

Figure 8.9 Mean ISIS 301012 concentration–time profiles in plasma following IV administration (day 1) and oral administrations (days 3 and 43). Each data point represents the mean of 6 or 24 individual measurements and the error bars are standard deviation.

Placebo Oral active 10 5

%Baseline

0 −5 −10 −15 −20

* p < 0.01 ** p < 0.001

*

**

−25 apoB100

LDL-C

Figure 8.10 Day 69 primary endpoints, plasma apoB100 and LDL-C. Mean changes from baseline for placebo and orally treated groups.

8.4.4

Local GI Uptake

Direct delivery of oligonucleotides to GI tissues, by way of oral or rectal dosage forms, may lead to significantly higher local oligonucleotide concentrations than when dosed by the parenteral route [58]. These dosage forms may therefore be effective in delivering oligonucleotides for the

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treatment of a variety of GI disease indications such as certain malabsorption syndromes, inflammatory conditions, and carcinomas. In support of this local delivery approach are observations in mice (dextran sodium sulfate colitis model) and rats (trinitrobenzene sulfonate [TNBS] colitis model) that locally administered solutions of oligonucleotides accumulate to a greater extent in inflamed GI tissue than in normal tissue [55]. In the rat colitis model, ISIS-2302 in water was administered by IV or intracolonic injection. The oligonucleotide concentration was determined in the colon (Figure 8.11). While there was no significant difference in tissue or plasma concentrations between normal and colitic rats when given oligonucleotide by IV administration, large differences were found between normal and colitic rats for intracolonic administration. Concentrations in the colon, in particular, were considerably higher in the diseased tissue. The data also suggest that diseased tissue may be targeted with simple oral formulations, without the need for PEs. Neurath et al. [58] locally administered a single 150-
g dose of the antisense NF-B phosphorothioate oligodeoxynucleotide targeted to mice with TNBS-induced colitis. The oligonucleotide abrogated the clinical and histological signs of colitis more effectively than daily glucocorticoids, suggesting a treatment for Crohn’s disease. Zhang and Agrawal have introduced oligonucleotides into the colon of rats as solutions and suppositories to test stability and uptake [59]. Their work demonstrated the necessity of stabilizing the oligonucleotides to intestinal degradation. A retention enema formulation to treat inflammation of the colon is currently under investigation at Isis Pharmaceuticals, Inc. Absorption into local tissues was achieved in animal enema models using oligonucleotides in an aqueous buffered, viscous formulation. Two hours after a 1-h retention, levels of intact oligonucleotide in healthy tissue were about hundredfold higher than those achieved with an IV injection of an equivalent dose (Table 8.7) [55]. Furthermore, the presence of oligonucleotide was still evident the following day in this single-dose study. A variety of oral dosage forms may be pursued to more conveniently present oligonucleotides for local GI indications. A pharmacokinetic study in dogs shows the feasibility of creating therapeutic levels of drug in the GI tissue by a simple enterically coated solid dosage form. In this study ISIS 13920, a 3-9-8 MOE gapmer, was administered for 5 consecutive days as a 400-mg oral tablet or 200-mg retention enema. GI tissue from the jejunum to the distal colon was analyzed for oligonucleotide concentration either 6 or 24 h after the last dose. The 6-h results are presented in

16 Colon

% dose/whole organ

14 12 10 8 6 4 2 0 0

10

20

30

40

50

Time (h) Figure 8.11 Rat colon tissue oligonucleotide concentrations as a percent of administered radioactive dose for normal rats (filled symbols) or treated with TNBS (open symbols) as a model for colitis. Solid lines represent the IV route and broken lines designate rectal dosing—both at 100 mg/kg of ISIS 2302 with nonexchangeable 3H label.

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Table 8.7 Dog Colon Tissue Biopsy Concentrations of Intact ISIS 2302 Oligonucleotide after Single-Dose IV (2 and 10 mg/kg) or Retention Enema (10 mg/kg) Administrations CGE (
g/g)

Formulation (Enemas at 5 mg/mL Oligonucleotide)

3h

24 h

1.5% Hydroxypropylmethylcellulose 1.0% Carrageenan Emulsion with Captex, Labrasol, and Crill 0.5% Tween 80, 0.75% HPMC 5% Sorbitol, 0.75% HPMC IV dosing at 2 mg/kg IV dosing at 10 mg/kg

660 558 224 621 417 2 11

7 3 1 6 1 NA 0.6

Tablets

Enema

400

Tissue concentration (µg/g)

350 300 250 200 150 100 50 0

Jejujum

Mid ileum Term. ileum Prox. colon Mid colon Distal colon Tissues

Figure 8.12 Tissue concentrations of ISIS 13920 6 h after last dose for enterically coated tablets or a 1-h retention enema.

Figure 8.12; from these data it can be seen that the enteric tablet formulation delivered the drug along the GI tract with the highest concentrations appearing in the colon. Importantly, the colon concentrations of intact oligonucleotide approximate those produced by the retention enema (albeit at twice the dose). Colon concentrations of intact drug fell to approximately 1
g/gm of tissue by 24 h for both formulations. Stool analysis accounted for 85–95% of the administered dose in 48 h with greater than 90% of the drug intact. These data suggest that with the use of proper formulations and stable chemistry, local delivery of therapeutic amounts of oligonucleotides is possible using the oral and rectal routes. 8.5 CONCLUSIONS Using fundamental biopharmaceutical principles and guidance from relevant animal models, it is possible to consider new routes of administration that have the potential for increased patient convenience and compliance. To date, antisense oligonucleotide clinical investigations have been limited to the use of local or parenteral administration of simple saline solutions. However, animal

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models have shown that formulations and delivery routes can be used to alter the tissue distribution of oligonucleotides. By making use of the knowledge gained through these studies, antisense oligonucleotides may be selectively delivered to a variety of target indications that would otherwise not be possible, such as the lung, and colon. Early studies in man have shown it possible to deliver therapeutic doses of oligonucleotides orally. However, to be cost-effective the efficiency of these dosage forms must increase.

REFERENCES 1. De Smet, M.D., Meenken, C., and Van Den Horn, G.J., Fomivirsen - a phosphorothioate oligonucleotide for the treatment of CMV retinitis. Ocul. Immunol. Inflammation, 7(3–4): 189–198, 1999. 2. SantaLucia, J., Jr., A unified view of polymer, dumbbell, and oligonucleotide DNA nearest-neighbor thermodynamics. Proc. Natl. Acad. Sci. USA, 95: 1460–1465, 1998. 3. Krotz, A.H., Mehta, R.C., and Hardee, G.E., Peroxide-mediated desulfurization of phosphorothioate oligonucleotides and its prevention. J. Pharm. Sci., 94: 341–352, 2005. 4. Altmann, K.-H. et al., Second generation of antisense oligonucleotides: from nuclease resistance to biological efficacy in animals. Chimia, 50: 168–176, 1996. 5. Geary, R.S. et al., Pharmacokinetics of a tumor necrosis factor-
phosphorothioate 2
-O-(2-methoxyethyl) modified antisense oligonucleotide: comparison across species. Drug Metab. Dispos., 31(11): 1419–1428, 2003. 6. Sewell, K.L. et al., Phase I trial of ISIS 104838, a 2
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. J. Pharmacol.Exp. Ther., 303(3): 1334–1343, 2002. 7. Pardridge, W.M., Transport of small molecules through the blood-brain barrier: biology and methodology. Adv. Drug Delivery Rev., 15(1–3): 5–36, 1995. 8. Le Corre, S.M. et al., Critical issues in the antisense inhibition of brain gene expression in vivo: experiences targeting the 5-HT1A receptor. Neurochem. Int., 31(3): 349–362, 1997. 9. Whitesell, L. et al., Stability, clearance, and disposition of intraventricularly administered oligodeoxynucleotides: implications for therapeutic application within the central nervous system. Proc. Nat. Acad. Sci USA, 90(10): 4665–4669, 1993. 10. Sommer, W. et al., The spread and uptake pattern of intracerebrally administered oligonucleotides in nerve and glial cell populations of the rat brain. Antisense Nucleic Acid Drug Dev., 8(2): 75–85, 1998. 11. Mustafa, S.J. and Dar, M.S., Adenosine A1 receptor antisense oligonucleotide treatment of alcohol and marijuana-induced psychomotor impairments. US Patent 5932557, 1999. 12. Yazaki, T. et al., Treatment of glioblastoma U-87 by systemic administration of an antisense protein kinase C-alpha phosphorothioate oligodeoxynucleotide. Mol. Pharmacol., 50(2): 236–242, 1996. 13. Crooke, S.T. et al., Pharmacokinetic properties of several novel oligonucleotide analogs in mice. J. Pharmacol. Exp. Ther., 277(2): 923–937, 1996. 14. Boado, R.J., Tsukamoto, H., and Pardridge, W.M., Drug delivery of antisense molecules to the brain for treatment of Alzheimer’s disease and cerebral AIDS. J. Pharm. Sci., 87(11): 1308–1315, 1998. 15. Vitravene™ product insert. 16. Voigt, M. et al., Down-regulation of NOSII gene expression by iontophoresis of anti-sense oligonucleotide in endotoxin-induced uveitis. Biochem. Biophys. Res. Commun., 295: 336–341, 2002. 17. Holbrook, K.A. and Wolf., K., The structure and development of skin, in Dermatology in General Medicine. Fitzpatrick, T.B., Eigen, A.Z., Wolff, K., Freedberg, I.M., and Austen, K.F., eds., McGraw Hill, New York, 97–145, 1993. 18. Lampe, M.A. et al., Human stratum corneum lipids: characterization and regional variations. J. Lipid Res., 24: 120–130, 1983. 19. Lee, Y.M. et al., Effect of benzalkonium chloride on percutaneous absorption of antisense phosphorothioate oligonucleotides. Arch. Phar. Res., 19: 435–440, 1996. 20. Mitragotri, S., Blankschtein, D., and Langer, R., Ultrasound-mediated transdermal protein delivery. Science, 269: 850–853, 1995. 21. Regnier, V., Le Doan, T., and Preat V., Parameters controlling topical delivery of oligonucleotides by electroporation. J. Drug Target., 5: 275–289, 1998.

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22. Banga, A.K. and Prausnitz, M.R., Assessing the potential of skin electroporation for the delivery of protein- and gene-based drugs. Trends Biotechnol., 16: 408–412, 1998. 23. Roberts, M.S. and Walker, M., Water. The most natural penetration enhancer, in Pharmaceutical Skin Penetration Enhancers. Hadgraft J., ed., Marcel Dekker, New York, 1995, 1–30. 24. Catz, P. and Friend, D.R., Mechanism of skin penetration enhancer: Ethyl acetate. Pharm. Res., 6: 108, 1988. 25. Millns, J.L. and Maibach, H.I., Mechanisms of sebum production and delivery in man. Arch. Dermatol. Res., 272: 351–362, 1982. 26. Hadgraft, J. and Williams, D.G., Azone. Mechanism of action and clinical effects, in Pharmaceutical Skin Penetration Enhancers. J. Hadgraft, ed., Marcel Dekker, New York, 1993, 175–197. 27. Michniak, B., In-vitro evaluation of a series of azone analogs as dermal penetration enhancers. Int. J. Pharm., 91: 85–93, 1993. 28. Geary, R.S. et al., Pharmacokinetics and metabolism in mice of a phosphorothioate oligonucleotide antisense inhibitor of C-RAF-1 kinase expression. Drug Metab. Dispos., 25(11): 1272–1281, 1997. 29. Duan, W. et al., Inhaled p38 Mitogen-activated protein kinase antisense oligonucleotide attenuates asthma in mice. Am. J. Respir. Crit. Care Med., 171(6): 571–578, 2005. 30. Wu-Pong, S. and Byron, P.R., Airway-to-biophase transfer of inhaled oligonucleotides. Adv. Drug Delivery Rev., 19(1): 47–71, 1996. 31. Deshmukh, H., unpublished results, 2002. 32. Yu, J. and Chien, Y.W., Pulmonary drug delivery: physiologic and mechanistic aspects. Crit. Rev. Ther. Drug Carrier Syst., 14(4): 395–453, 1997. 32. Danahay, H. et al., Distribution of a 20-mer phosphorothioate oligonucleotide, CGP69846A (ISIS 5132), into airway leukocytes and epithelial cells following intratracheal delivery to Brown-Norway rats. Pharm. Res., 16(10): 1542–1549, 1999. 33. Templin, M.V. et al., Pharmacokinetic and toxicity profile of a phosphorothioate oligodeoxynucleotide following inhalation delivery to lung in mice. Antisense Nucleic Acid Drug Dev., 10: 359–368, 2000. 34. Hung, E., unpublished observations, 2005. 35. Nyce, J.W. and Metzger, W.J., DNA antisense therapy for asthma in an animal model. Nature (London), 385(6618): 721–725, 1997. 36. Nyce, J.W., Respirable antisense oligonucleotides as novel therapeutic agents for asthma and other pulmonary diseases. Expert Opin. Invest. Drugs, 6(9): 1149–1156, 1997. 37. Karras, J.G., Geary, R.S., and Gregory, S.A., Inhaled antisense oligonucleotide therapies: inspiration and progress. Drug Discov. Today, in press. 38. Humphrey, M.J., The oral bioavailability of peptides and related drugs, in Delivery Systems for Peptide Drugs. Davis, S.S., Illum, L., and Tomlinson, E., eds., Plenum Press, New York, pp. 139–151, 1986. 39. Agrawal, S. and Zhang, R., Pharmacokinetics and bioavailability of antisense oligonucleotides following oral and colorectal administrations in experimental animals. Handb. Exp. Pharmacol., 131 (Antisense Research and Application): 525–543, 1998. 40. Nicklin, P.L., Craig, S.J., and Phillips, J.A., Pharmacokinetic properties of phosphorothioates in animals—absorption, distribution, metabolism and elimination. Handb. Exp. Pharmacol., 131(Antisense Research and Application): 141–168, 1998. 41. Zhang, R. et al., In vivo stability, disposition and metabolism of a “hybrid” oligonucleotide phosphorothioate in rats. Biochem. Pharmacol., 50(4): 545–556, 1995. 42. Zhang, R. et al., Pharmacokinetics and tissue disposition of a chimeric oligodeoxynucleotides phosphorothioate in rats after intravenous administration. J. Pharmacol. Exp. Ther., 278(2): 971–979, 1996. 43. Altmann, K.-H. et al., Second generation of antisense oligonucleotides. From nuclease resistance to biological efficacy in animals. Chimia, 50(4): 168–176, 1996. 44. Bennett, C.F. et al., Antisense oligonucleotide-based therapeutics, in Gene and Cell Therapy: Therapeutic Mechanisms and Strategies. Templeton, N.S., ed., Marcel Dekker, New York, pp. 347–374, 2004. 45. Kolbe, H.V.J. and Boussif, O., Use of a cationic polymer for the preparation of a complex with nucleic acid and related compositions, European Patent Application, EP 987029, 2000. 46. Richardson, S.C.W., Kolbe, H.V.J., and Duncan, R., Potential of low molecular mass chitosan as a DNA delivery system: biocompatibility, body distribution and ability to complex and protect DNA. Int. J. Pharm., 178(2): 231–243, 1999.

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47. Zobel, H.P. et al., Evaluation of aminoalkylmethacrylate nanoparticles as colloidal drug carrier systems. Part 2. Characterization of antisense oligonucleotides loaded copolymer nanoparticles. Eur. J. Pharm. Biopharm., 48(1): 1–12, 1999. 48. Khatsenko, O. et al., Absorption of antisense oligonucleotides in rat intestine: effect of chemistry and length. Antisense Nucleic Acid Drug Dev, 10(1): 35–44, 2000. 49. Anderberg, E.K., Lindmark, T., and Artursson, P., Sodium caprate elicits dilatations in human intestinal tight junctions and enhances drug absorption by the paracellular route. Pharm. Res., 10(6): 857–864, 1993. 50. Lane, M.E., O’Driscoll, C.M., and Corrigan, O.I., The relationship between rat intestinal permeability and hydrophilic probe size. Pharm. Res., 13(10): 1554–1558, 1996. 51. Soderholm, J.D. et al., Reversible increase in tight junction permeability to macromolecules in rat ileal mucosa in vitro by sodium caprate, a constituent of milk fat. Dig. Dis. Sci., 43(7): 1547–1552, 1998. 52. Teng, C.-L., unpublished results, 1998. 53. Lee, V.H., Yamamoto, A., and Kompella, U.B., Mucosal penetration enhancers of facilitation of peptide and protein drug absorption. Crit. Rev. Ther. Drug Carrier Syst., 8(2): 91–192, 1991. 54. Hollander, D., The intestinal permeability barrier. A hypothesis as to its regulation and involvement in Crohn’s disease. Scand. J. Gastro., 27: 721–726, 1992. 55. Geary, R.S., unpublished results, 2004. 56. Tillman, L.G., Geary, R.S., and Hardee, G.E., Oral delivery of antisense oligonucleotides: plasma bioavailability of ISIS 104838 in man. Accepted for publication, J. Pharm. Sci., 2007. 57. Hardee, G.E. et al., A Phase I Study of Orally Administered ISIS 301012 for the Reduction of ApoB-100 and LDL-cholesterol. Manuscript in preparation, 2006. 58. Neurath, M.F. et al., Local administration of antisense phosphorothioate oligonucleotides to the p65 subunit of NF-B abrogates established experimental colitis in mice. Nat. Med. (NY), 2(9): 998–1004, 1996. 59. Zhang, R. and Agrawal, S., Down-regulation of gene expression by colorectal administration of synthetic oligonucleotides, PCT Int. Appl., WO 9840058, 1998.

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CHAPTER

9

Liposomal Formulations for Nucleic Acid Delivery Ian MacLachlan

CONTENTS 9.1 9.2

Liposomes for the Delivery of Nucleic Acid Drugs.............................................................237 Liposome Constituents .........................................................................................................239 9.2.1 Cationic Lipids .........................................................................................................239 9.2.2 The Role of Helper Lipids in Promoting Intracellular Delivery ..............................240 9.2.3 PEG–Lipids...............................................................................................................241 9.2.4 Active Targeting........................................................................................................242 9.3 Methods of Encapsulating Nucleic Acids ............................................................................242 9.3.1 Passive Nucleic Acid Encapsulation.........................................................................243 9.3.2 The Ethanol Drop (SALP) Method of Nucleic Acid Encapsulation........................247 9.3.3 Encapsulation of Nucleic Acid in Ethanol-Destabilized Liposomes .......................247 9.3.4 The Reverse-Phase Evaporation Method of Nucleic Acid Encapsulation ...............248 9.3.5 The Spontaneous Vesicle Formation by Ethanol Dilution (SNALP) Method of Nucleic Acid Encapsulation .....................................................................................249 9.4 Analytical Methods...............................................................................................................251 9.4.1 Measuring Particle Size ............................................................................................251 9.4.2 Zeta Potential............................................................................................................253 9.4.3 Encapsulation............................................................................................................253 9.5 Pharmacology of Liposomal NA..........................................................................................254 9.5.1 Pharmacokinetics and Biodistribution of Liposomal NA Following Systemic Administration ..........................................................................................................254 9.5.2 Toxicity of Liposomal NA Formulations .................................................................256 9.5.3 Immune Stimulation .................................................................................................258 9.5.4 Immunogenicity........................................................................................................259 9.5.5 The Efficacy of Liposomally Formulated NA Drugs...............................................260 References ......................................................................................................................................262 9.1 LIPOSOMES FOR THE DELIVERY OF NUCLEIC ACID DRUGS Liposomes are artificial vesicles made up of one or more bilayers of amphipathic lipid encapsulating an equal number of internal aqueous compartments. They are distinguished on the basis of their size and the number and arrangement of their constituent lipid bilayers (Figure 9.1). 237

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Figure 9.1

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Liposomes. Mulilamellar vesicles (MLVs) are large (hundreds of nm in diameter) complex structures containing a series of concentric bilayers separated by narrow aqueous compartments. Large unilamellar vesicles (LUVs) are between 50 and 500 nm in diameter, while the smallest liposomes namely small unilamellar vesicles (SUVs) are ⬍50 nm. LUVs are the preferred systems for delivery of NA drugs. Lipids are drawn roughly to scale.

Multilamellar vesicles (MLVs) are formed by the aqueous hydration of dried lipid films. Typically hundreds of nanometers in diameter, they are large, complex structures containing a series of concentric bilayers separated by narrow aqueous compartments. Simple unilamellar vesicles between 50 and 500 nm in diameter are referred to as large unilamellar vesicles (LUVs) while the smallest liposomes, vesicles smaller than 50 nm in diameter, are small unilamellar vesicles (SUVs). Liposomes have received attention not only for their utility as model membrane systems, but also for use in drug delivery. Typically, liposomes are used as drug carriers, with the solubilized drug encapsulated in the internal aqueous space formed by the liposomal lamellae. Liposomal drug formulations can be used to overcome a drug’s nonideal properties, such as limited solubility, serum stability, circulation half-life, biodistribution, and target tissue selectivity. Experience with conventional small molecule drugs has shown that the drugs that benefit the most from liposomal delivery, are those that are chemically labile, subject to enzymatic degradation and have an intracellular site of action [1]. For this reason, there is considerable interest in exploiting liposomes as carriers of nucleic acids (NAs), either as plasmid vectors for gene therapy applications or to deliver smaller NA species such as antisense oligonucleotides, ribozymes and, more recently, siRNA for the purposes of downregulating target genes. Because of their ability to achieve favorable drug/lipid ratios and their more predictable drug release kinetics LUV are the preferred liposome delivery system for NA drugs. An advantage of liposomal drug delivery is that the pharmacokinetics, biodistribution, and intracellular delivery of the liposome payload are largely determined by the physicochemical properties of the carrier. For example, the biodistribution of a NA entrapped within a small, long circulating liposome is independent of the type of NA, which can be a relatively stable double-stranded plasmid DNA molecule or single-stranded antisense DNA, or one of the more labile ribonucleotide molecules such as ribozymes or a duplex siRNA. This is only true if the liposome is truly acting as a carrier, rather than a mere excipient. Liposomes function as excipients when used to formulate hydrophobic drugs that would otherwise be difficult to administer in aqueous dosage form. Hydrophobic drugs rapidly exchange into lipoproteins or other lipid-rich environments soon after injection, resulting in comparably uncontrolled pharmacology. In the context of NA drug delivery, liposomes are considered excipients if used to enable vialing and aqueous dosing of hydrophobic lipid–NA conjugates [2–5]. (These applications are not considered in this chapter, nor are those that use preformed, cationic lipid-containing vesicles to form “lipoplex” or “oligoplex” systems.)

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An objective inherent in all pharmaceutical development is to minimize the risks associated with treatment while maximizing the benefit to patient health. The most important risk to patients is the toxicity associated with the administration of poorly tolerated compounds, often exacerbated by attempts to increase efficacy by escalating the administered dose. A well-designed liposomal delivery system will be capable of reducing the toxicity and increasing the potency of NA-based drugs by optimizing NA delivery to target tissues. Liposomal NA delivery will be determined by the physical and biochemical properties of the liposome including stability, size, charge, hydrophobicity, interaction with serum proteins, and interaction with nontarget cell surfaces. Ideally, liposomal carriers for NA delivery will have the following properties: (i) they will be safe and well tolerated; (ii) they will have appropriate pharmacokinetic attributes to ensure delivery to intended disease sites; (iii) they will mediate effective intracellular delivery of intact NA; (iv) they will be nonimmunogenic, enabling the use of multidosing treatment regimes; and (v) they will be stable upon manufacture so that large batches can be prepared with uniform, reproducible specifications. In this chapter we discuss the physical makeup, manufacturing methods, and pharmacological considerations specific to liposomal systems for the delivery of NA-based drugs, with emphasis on those that enable systemic delivery of synthetic polynucleotides such as antisense ODN, ribozymes, and siRNA.

9.2 LIPOSOME CONSTITUENTS NA encapsulation was first described in the late 1970s, prior to the development of cationic lipidcontaining lipoplex, using naturally occurring, neutral lipids to encapsulate high-molecular-weight DNA [6–8]. The first reports of low-molecular-weight oligo- or polynucleotide encapsulation similarly used passive techniques to entrap NA in neutral liposomes [9–11]. The advent of cationic lipid-mediated lipofection [12] saw a shift in emphasis away from encapsulated systems in favor of “lipoplex” or “oligoplex” systems. More recently, improvements in formulation technology have allowed for a return to encapsulated systems that contain cationic lipids as a means of facilitating both encapsulation and intracellular delivery. More advanced systems typically contain multiple lipid components, each of which play a role in determining the physical and pharmacological properties of the system as a whole. 9.2.1

Cationic Lipids

Cationic lipids play two roles in liposomal NA formulations. In the first case, they encourage interaction between the lipid bilayer and the negatively charged NA, allowing for the enrichment of NA concentrations over and above that which would be achieved using passive loading in charge neutral liposomes. Cationic lipids allow for encapsulation efficiencies greater than 40% when using coextrusion methods, and greater than 95% when using more sophisticated techniques [13–15]. Cationic lipids also function by providing the liposome with a net positive charge, which in turn enables binding of the NA complex to anionic cell surface molecules. The most abundant anionic cell surface molecules, sulfated proteoglycans and sialic acids, interact with and are responsible for the uptake of cationic liposomes [16–18]. The role of cationic lipids in liposomal uptake presents a dilemma: highly charged systems are rapidly cleared from the blood, thereby limiting accumulation in target tissues. Particles with a neutral charge however, display good biodistribution profiles, but are poorly internalized by cells. This supports the concept of a modular delivery solution, that is, an engineered nanoparticle with individual components fulfilling different functions in the delivery process, and in particular, a system which responds to the microenvironment in a manner that facilitates transfection. Titratable, ionizable lipids are components that allow for the adjustment of the charge on the system by simply changing the pH after encapsulation [19]. At reduced pH when the system is strongly charged, NAs are efficiently encapsulated. When liposomes containing titratable, ionizable lipids are at a pH closer to the pKa of the cationic lipid, such as

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physiological pH, they become more charge neutral and are able to avoid opsonization by blood components [19]. More recently, the use of novel, pH titratable cationic lipids with distinct physicochemical properties that regulate particle formation, cellular uptake, fusogenicity, and endosomal release of NA drugs have been described [20]. The chemical and biological properties of pH-titratable cationic lipids are influenced by their degree of lipid saturation. In particular, the phase transition properties, as measured using 31P-NMR, are affected. Above the phase transition temperature, Tc, lipids adopt the more highly fusogenic reverse hexagonal HII phase [20–22]. By noting the temperature at which this phase transition occurs, the relative ease with which lipids form the HII phase and become “fusogenic” can be determined. On this basis it has been shown that the fusogenicity of liposomal systems increases as the titratable cationic lipid becomes less saturated. The lipid pKa also correlates with the degree of saturation. pK measurements confirm that saturated lipids carry more residual charge at physiological pH. For this reason, liposomes containing the more highly saturated cationic lipids are taken up more readily by cells in vitro [20]. However, liposomes containing the more fusogenic unsaturated cationic lipids DLinDMA and DLenDMA are more effective at mediating RNA interference in both in vitro cell culture systems and in vivo. The apparently conflicting results between cellular uptake and silencing potency are a reminder that cellular uptake per se is insufficient for effective delivery of NA. Cellular uptake, fusogenicity, and endosomal release are distinct processes, each of which need to be enabled by the delivery vehicle and each of which are profoundly affected by the physicochemical properties of the cationic lipids used. 9.2.2

The Role of Helper Lipids in Promoting Intracellular Delivery

Although we have just shown that cationic lipids may have inherent fusogenic properties of their own, cationic lipids were originally believed to require fusogenic “helper” lipids for efficient NA delivery [23–26]. Fusogenic liposomes facilitate the intracellular delivery of complexed plasmid DNA by fusing with the membranes of the target cell. Fusion may occur at a number of different stages in delivery, either at the plasma membrane, endosome or nuclear envelope. Fusion of first-generation, nonencapsulated lipoplex systems with the plasma membrane is expected to be a particularly inefficient method of introducing NA into the cytosol. Since lipoplex-NA is predominantly attached to the surface of the liposome, lipoplex fusion events resolve with NA, formerly attached to the liposome surface, deposited on the outside surface of the plasma membrane. Encapsulated systems are significantly different from lipoplex in this respect. Upon fusion with either the plasma or endosomal membrane(s), encapsulated carriers deliver their contents directly into the cytosol. Lipids that preferentially form nonbilayer phases, in particular the reverse hexagonal HII phase, such as the unsaturated phosphatidylethanolamine DOPE, promote destabilization of the lipid bilayer and fusion. Similar to fusogenic cationic lipids, decreasing the degree of lipid saturation increases the lipid’s affinity for the fusogenic HII phase [27–32]. However, some cationic lipids can function in the absence of these so-called helper lipids, either alone [24,25] or in the presence of the nonfusogenic lipid cholesterol [33]. This would suggest that either these lipids have properties which promote delivery through a mechanism which does not require membrane fusion, or that their own fusogenic properties are adequate to support delivery. As described above, cationic lipids are readily designed for optimal fusogenicity by controlling lipid saturation. This provides for multiple opportunities for modulating the fusogenicity of a liposomal lipid bilayer [20]. Attempts to address the role of fusogenic lipids in vivo have yielded confounding results. In this regard it is important to distinguish the effect of fusogenic lipids on NA delivery to target tissue from their effect on intracellular delivery. Fusogenic formulations are more likely to interact with the vascular endothelium, blood cells, lipoproteins, and other nontarget systems while in the blood compartment. For this reason there may be an advantage to transiently shield the fusogenic potential of systemic carriers using shielding agents such as polyethylene glycol (PEG).

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241

PEG–Lipids

An ideal delivery system would be one that is transiently shielded upon administration, facilitating delivery to the target site, yet becomes increasingly charged and fusogenic as it reaches the target cell. PEG lipids partially address this challenge. PEG–lipid conjugates are readily incorporated in liposomal NA formulations. They provide a benefit during the formulation process, stabilizing the nascent particle and contribute to formulation stability by preventing aggregation in the vial [13]. PEG conjugates sterically stabilize liposomes by forming a protective hydrophilic layer that shields the hydrophobic lipid layer. By shielding the liposome’s surface charge they prevent the association of serum proteins and resulting uptake by the reticuloendothelial system when liposomes are administered in vivo [34,35]. In this way, cationic liposome NA formulations are stabilized in a manner analogous to PEGylated liposomal drug formulations that exhibit extended circulation lifetimes [36–41]. Although this approach has been investigated with a view towards improving the stability and pharmacokinetics of lipoplex containing either plasmid DNA [42] or antisense oligonucleotides [43], PEG–lipid-containing lipoplex systems suffer from the heterogeneity and suboptimal pharmacology common to most nonencapsulated NA–cationic lipid complexes. Although PEG–lipid-containing systems are promising with respect to their ability to deliver NA to disease sites, improvements are required to increase their potency. Early PEGylated liposomes for the delivery of small molecule chemotherapeutic drugs utilized stably integrated PEG lipids such as PEG-DSPE [39]. These systems are designed to function as carriers that facilitate the accumulation of active drug compound at disseminated disease sites. The drug is released at the cell surface at a “leakage rate” determined by the liposomal bilayer composition. NA-based drugs differ in this respect in that they require effective intracellular delivery, hence the use of the cationic and fusogenic lipids described earlier. PEGylated systems typically exhibit relatively low-transfection efficiencies. This is mainly due to the ability of the PEG coating to inhibit cell association and uptake [23,44,45]. Ideally, PEG–lipid conjugates would have the ability to dissociate from the carrier and transform it from a stable, stealthy particle to a transfection-competent entity at the target site. Various strategies have been applied to this problem. A number of investigators have explored the use of chemically labile PEG–lipid conjugates [46–52], in particular those that are “pH sensitive.” Typically, these systems invoke a chemically labile linkage between the lipid and PEG moieties that reacts via acid-catalyzed hydrolysis to destabilize the liposomes by removal of the sterically stabilizing PEG layer. Although this approach results in improved performance both in vitro and in vivo, it may be regarded as suboptimal for two reasons. First, pH-sensitive PEG lipids are designed to be rapidly hydrolyzed in the reduced pH environment encountered within the endosome, but since PEG lipids are known to inhibit cellular uptake, a prerequisite to endosomal localization and hydrolysis, their use actually limits the amount of material delivered to the endosome [53]. Second, the incorporation of pH-sensitive or otherwise chemically labile lipids results in a truncation of formulation shelf life relative to systems that use more stable PEG–lipids. An alternative to the use of acid-labile PEG–lipids involves the use of chemically stable, yet diffusible PEG lipids. The concept of diffusible PEG lipids arose from the observation that the length of the PEG lipid anchor has an influence on PEG lipid retention and the stability and circulation lifetime of empty lipid vesicles [54]. It has been found that by modulating the alkyl chain length of the PEG lipid anchor [55–59], the pharmacology of encapsulated NA can be controlled or “programmed” in a predictable manner. Upon formulation, the liposome contains a full complement of PEG in steady-state equilibrium with the contents of the vial. In the blood compartment, this equilibrium shifts and the PEG–lipid conjugate is free to dissociate from the particle over time, revealing a positively charged and increasingly fusogenic lipid bilayer that transforms the particle into a transfection-competent entity. Diffusible PEG lipids differing in the length of the their lipid anchors have been incorporated into liposomal systems containing plasmid DNA (SPLP) [13,55], antisense oligonucleotides (PFV, SALP) [19,56,60], and siRNA (SNALP) [14,15,61]. This approach may help to resolve the two conflicting demands imposed upon NA carriers. First, the carrier must be stable and circulate long

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enough to facilitate accumulation at disease sites. Second, the carrier must be capable of interacting with target cells to facilitate intracellular delivery. 9.2.4

Active Targeting

Active targeting refers to processes that aim to increase the accumulation, retention or internalization of a drug through the use of cell-specific ligands. This is to be distinguished from the passive “disease site targeting” or the “enhanced permeability and retention” (EPR) effect, which results in the accumulation of appropriately designed carriers in target sites such as tumor tissue. Active targeting has been successfully applied to liposomal small molecule drug formulations and generally has the effect of improving the therapeutic index of the liposomal drug when measured in preclinical studies. NA delivery systems stand to benefit from targeting in two ways, first through improving the accumulation and binding of formulations to target cells and second by facilitating intracellular delivery through endocytosis. The perceived benefits of active targeting have encouraged numerous investigators in this area and targeting of NA formulations has been achieved through the use of molecules as diverse as antibodies directed against cell surface proteins [62–65], protein ligands of cell surface receptors [66–69], vitamins [70–72], and glycolipids [73,74]. The earliest reports of targeted liposomal formulations of encapsulated NA were attempts to improve the intracellular delivery characteristics of charge neutral liposomes encapsulating either synthetic antisense DNA [63,65] or in vitro transcribed antisense RNA [64]. The results of these studies were encouraging, suggesting a significant benefit associated with the use of targeted systems. Although these in vitro studies effectively demonstrated the potential advantage of targeting at the level of intracellular delivery, they were unable to address important pharmacological considerations such as those that influence accumulation at disease sites. It is unlikely that addition of targeting ligands to delivery systems that are rapidly removed from the circulation will result in delivery exceeding that achieved by systems that display passive disease site targeting. For this reason many investigators have pursued approaches involving the addition of targeting ligands to sterically stabilized and charge shielded systems, such as those containing PEG lipids [71,72,75–77]. This approach has been advanced, in part, by the development of the so-called postinsertion technique [78]. Postinsertion allows for the insertion of ligand–PEG–lipid conjugates into preformed liposomes containing encapsulated NA. This represents a significant improvement on earlier approaches in which ligands were chemically coupled to preformed liposomes, an approach limited by suboptimal coupling efficiencies, or where ligand–lipid conjugates were incorporated in the first stages of the formulation process, an approach limited by the resulting negative impact on NA encapsulation efficiency and subsequent suboptimal presentation of the targeting ligand. A number of reports suggest that it is possible to design encapsulated systems containing targeting ligands that retain extended circulation lifetimes and passive disease site targeting the following systemic administration. It remains to be seen if the benefits of active targeting outweigh the increased cost, manufacturing complexity and immunogenicity that often accompanies the use of such technology.

9.3 METHODS OF ENCAPSULATING NUCLEIC ACIDS To capitalize on the pharmacology of liposomal drug carriers it is necessary to completely entrap NA within the contents of a liposome. In this regard it is important to distinguish first-generation “lipoplex” or “oligoplex” systems from those that truly encapsulate their NA payload. Lipoplex are electrostatic complexes formed by mixing preformed cationic lipid-containing vesicles with NA [12,79,80]. The result is a heterogenous, metastable aggregate that is effective when used to transfect cells in culture but has relatively poor performance in vivo. Upon systemic administration, lipoplex systems are rapidly cleared from the blood, accumulating in the capillary bed of first-pass organs such as the lung.

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243

Lipoplex are effectively taken up by the cells of the innate immune system, contributing to their profound toxicities and off-target effects. These side effects may manifest as “efficacy” in antitumor or antiinfective applications, confounding data interpretation and encouraging the acceptance of false-positive results. For these reasons, an abundance of caution is encouraged when initiating in vivo studies that use liposomes to deliver NA. Of particular importance is the use of appropriate analytical methodology, described in Section 9.4, to properly characterize lipid-based systems prior to and during use. 9.3.1

Passive Nucleic Acid Encapsulation

Liposomal encapsulation of small molecule drugs may be achieved by either “passive” or “active” loading. Unlike small molecule drugs, NAs are not readily packaged in preformed liposomes using pH gradients or other similar active loading techniques. This is predominantly due to the large size and hydrophilic nature of NA, which conspire to prevent them from crossing intact lipid bilayers. For this reason, much of the work on NA encapsulation has utilized passive loading technology. Passive encapsulation typically involves the preparation of a “lipid film,” the lipidic residue that remains after evaporation of the organic phase of a lipid solution (Figure 9.2). Rehydration of the

Lipid solution in solvent

Nucleic acid solution in buffer

Dried lipid film

Lipid hydration with nucleic acid solution

MLV formation by freeze/thaw (5−10×)

MLV extrusion (10×)

LUV collection

Free nucleic acid removal

Sample concentration

Sterile filtration Figure 9.2

Passive method of NA encapsulation. Passive encapsulation utilizes a dried lipid film prepared by evaporating the organic phase of a lipid solution. The resulting lipid film is rehydrated in an aqueous solution of NA in buffer, forming MLV. Multiple freeze-thaw cycles increase the extent of NA encapsulation within the MLV bilayers. The vesicles are then extruded through polycarbonate filters producing LUV.

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lipid film in aqueous media, typically buffer containing NA, followed by vigorous mixing, results in the formation of MLV. This is followed by multiple cycles of freezing and thawing to increase the extent to which the NA solute is entrapped by the MLV bilayers. The MLV preparation is then subjected to multiple rounds of extrusion through polycarbonate filters to produce LUV (Figure 9.2 and Figure 9.3) [81]. The size of the LUV is determined by the size of the filter pores. This process suffers from a number of limitations. When used to encapsulate NA, the efficiency of passive encapsulation is generally quite low, ranging from 3 to 45%, depending on the composition of the lipid bilayer and other factors (Table 9.1). The low encapsulation efficiency, consequently, necessitates the incorporation of a postencapsulation separation step such as dialysis, size exclusion chromatography or ultrafiltration to remove nonencapsulated NA. In an effort to improve the efficiency of encapsulation, excess lipid is often incorporated in the formulation process, resulting in low NA/lipid ratios which ultimately impact toxicity and cost of goods. Finally, the extrusion process is inherently difficult to scale. Preparation of large batches requires the use of custom-built extruders to accommodate large filters. The probability of filter tears, resulting in batch failure, increases as the size and cost of the batch increases. In spite of these process limitations, extrusion-based methods for liposome preparation have been successfully adopted by many laboratories, presumably because the technology is readily accessible to the casual investigator. Furthermore, significant progress has been made adapting or enhancing extrusion-based processes for the liposomal formulation of NA-based drugs. These include the use of cationic and anionic lipids [82,83], ionizable cationic lipids [19,84], PEG lipids [85], and detergent or organic solvents such as ethanol [19,60] to control bilayer assembly.

Figure 9.3

The Lipex™ thermobarrel extruder for the preparation of uniformly sized liposomes by extrusion. An MLV or other vesicle preparation is introduced to the top of the extruder and the extruder is pressurized with nitrogen, forcing the MLV through a polycarbonate filter of defined pore size. The resulting LUVs are collected via the outlet port at the bottom of the device. Extrusion is repeated, typically for a total of 10 passes. The unit permits thermostatic operation by virtue of the thermobarrel, which can be coupled to a circulating water bath. (Photo courtesy Northern Lipids Inc., Vancouver, Canada, http://www.northernlipids.com.)

Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive Passive

Passive Passive Passive Passive Passive Passive Passive Passive Passive

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28

29 30 31 32 33 34 35 36 37

Formulation Method DOPE:Chol:Oleic Acid:Palmitoyl-CD4 DPPC:CH:SPDP-PE EPC:Chol:DMPG;EPC:DMPG DOPE:Chol:Oleic Acid DPPC:Chol:SPDP-PE PC:Chol:PS EPC:Chol⫾Folate-PEG-DSPE PC:Chol DOPE:Chol:Oleic Acid:Palmitoyl-CD4 DDAB:PC:Chol PC:Chol:PS HVJ liposome: PC:Chol: DC-Chol DOGS:DOPE HVJ liposome HVJ liposome:Chol:PC:PS DSPC:Chol EPC:Chol:Folate-PEG-DSPE DPPC:Chol:DPPS or DPPA HVJ liposome: PS:PC:Chol HVJ liposome: PC:DOPE: Sph:PS:Chol DPPE:Cetyltrimethyl ammonium bromide HVJ liposome DOPE:CHEMS or SPC PC40:Chol:PEG-DSPE:DOTAP EPC:Chol HVJ liposome: PS: PC:Chol DPPC:DMPG HVJ liposome: PE-DTP:PS:PC:Chol immunoliposomes CHEMS:DOPE or conventional SPC liposomes DDAB:EPC:Chol HVJ liposome: PS:PC:Chol PE:CHEMS:LLO Folate liposomes: EPC:Chol:DSPE-PEG-Pteroate PE:CHEMS:LLO Thiocationic lipid: oleic acid:Vitamin D HVJ liposome: PS:PC:Chol DSPC:Chol:CPL

Lipid Composition

Table 9.1 Liposomal Formulations of Oligo- and Polynucleotide Drugs

ODN ODN ODN Various ODN ODN ODN ODN

ODN ODN ODN ODN ODN ODN ODN ODN ODN ODN ODN Ribozyme ODN ODN Plasmid/ODN RNA Aptamer ODN ODN pDNA ODN ODN TFD, ODN ODN ODN ODN ODN ODN ODN

⬍10 3 ⬍2 10 2–3 ⬍10 30–40 ⬍10 ⬍10 ⬎90 ⬍10 60 88 ND 10–75 ND 15–20 24–32 ND ND 10 ND ⬃16 80–100 10–15 ND 43.5⫾ 4 ND

LIPOSOMAL FORMULATIONS FOR NUCLEIC ACID DELIVERY (Continued)

[176] [177] [178] [179] [72] [180] [181] [182] [183]

[153] [63] [154] [155] [65] [156] [85] [157] [158] [159] [160] [161] [162] [163] [164] [165] [166] [92] [167] [168] [169] [170] [171] [93] [172] [173] [174] [175]

Reference

22:36

Up to 20 ⬎85 ND 7–15 ND 10–30 ND 2–5 ND

Payload

Encapsulation (%)

5/24/2007

250–300 467.2⫾72.0 ND 240–370 90–110 90–100 ND ND 130

220⫾55 ⬃200 460⫾200 170 100–140 ND 100–140 110⫾40 220⫾55 ⬍2000 ND ND 100–150 ND ND 50–65 ND 50–70 ND ND ND ND 200–300 ⬍200 110⫹30 ND 316–562 400–500

Size (nm)

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(Continued)

Passive Passive Passive Passive Passive Ethanol drop—SALP Ethanol drop—SALP Ethanol drop—SALP Ethanol drop—SALP Ethanol drop—SALP Ethanol drop—SALP Ethanol drop—SALP Ethanol drop—SALP Ethanol drop—SALP Reverse-phase evaporation Reverse-phase evaporation Reverse-phase evaporation Reverse-phase evaporation Reverse-phase evaporation Reverse-phase evaporation Reverse-phase evaporation

Reverse-phase evaporation Reverse-phase evaporation Reverse-phase evaporation Ethanol-destabilized liposomes Ethanol dilution—SNALP Ethanol dilution—SNALP Ethanol dilution—SNALP Ethanol dilution—SNALP Ethanol dilution—SNALP Ethanol dilution—SNALP

59 60 61 62 63 64 65 66 67 68

100 ND 880 ND 50–200 110⫾30 100–120 110⫾30 100–120 ⬃130 ⬃130 100–200 80–90 100–150 188 70–120 ND 150–190 ⬎200 ⬍200 110–130

EPC:DPPC:Chol DOPC: Tween 20 PC:DMPA:Chol HVJ liposome: EPC:ESM:Chol:DC-Chol EPC:Chol:PEG-PE: DOTAP PC:Chol:DODAP:PEG-Cer-C14 or -C20 DOPE:Chol:DODAC:PEG-Ceramides DSPC:Chol:DODAP:PEG-Cer-C14 DOPE:Chol:DODAC:PEG-C er-C14 DODAP:DSPC:Chol: PEG-Cer-C14 DODAP:DSPC:Chol:PEG-Cer-C14 EPC:Chol:DODAP DC-Chol:EPC:PEG-DSPE DC-Chol:EPC:PEG-DSPE, Transferrin-PEG-DSPE “Charge-neutralized liposome” HSPC:DSPE-PEG:DOTAP:DSPE-PEG-MAL:Chol CHEMS:DOPE, CHEMS:DOPE:PEG-PE HSPC:Chol:PEG-DSPE PE:CHEMS:Chol DPPC:DPPG:Chol DODAC:DOPE:PEG-DSPE:PEG:DMPE HSPC:DSPE-PEG:DOTAP:Rho-PE: DSPE-PEG-Maleimide DOTAP:Chol:HSPC: PEG-DSPE or MAL-PEG-DSPE DODAP:Chol:PC:PEG-DSPE DOTAP, POPC,CHOL, MPB-PE, PEG-DSPE DSPC:Chol:PEG-Cer-C14:DOTAP DSPC:Chol:PEG-C-DMA:various cationic lipids DSPC:Chol:PEG-C-DMA:DLinDMA or DODMA DSPC:Chol:PEG-C-DMA:DLinDMA DSPC:Chol:PEG-C-DMA:DLinDMA DSPC:Chol:PEG-C-DMA:DLinDMA DSPC:Chol:PEG-C-DMA: DLinDMA ODN ODN ODN ODN/Plasmid siRNA siRNA siRNA siRNA siRNA siRNA

ODN siRNA ODN, siRNA ODN siRNA ODN ODN ODN ODN ODN ODN ODN ODN ODN ODN ODN ODN ODN ODN ODN ODN

Payload

[200] [201] [202] [60] [20] [127] [14] [143] [15] [61]

[184] [185] [186] [187] [188] [19] [56] [189] [190] [191] [192] [193] [194] [195] [76] [75] [196] [77] [197] [198] [199]

Reference

22:36

90–95 80–100 ⬎90 90 67–85 90–95 93⫾3 90–95 92–97 90–95

ND 65 ND ND ND 50–80 43–57 ND 43–57 ND ND 57–85 70–80 70–80 85–95 80–90 ND 80–100 10–14 ⬎95 90

Encapsulation (%)

5/24/2007

100–140 150–200 ⬍180 70–120 132–182 100–130 140⫾12 100–130 73–83 71–84

Size (nm)

Lipid Composition

246

38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58

Formulation Method

Table 9.1

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247

The Ethanol Drop (SALP) Method of Nucleic Acid Encapsulation

Stabilized antisense-lipid particles (SALPs) were developed as a means of improving both the limited efficiency of passive NA encapsulation and the pharmacology of the resulting particles. SALPs are prepared by dropwise addition or injection of an ethanolic lipid solution to an aqueous solution of NA, followed by extrusion through polycarbonate filters [19] (Figure 9.4). By utilizing an ionizable aminolipid at an acidic pH, where the aminolipid is fully charged, highly efficient (up to 70%) encapsulation may be achieved. Furthermore, the use of an ionizable lipid facilitates adjustment of the total charge of the system by simply changing the pH after the encapsulation step. In this manner, antisense oligonucleotides may be encapsulated in lipidic systems at NA/lipid ratios as high as 0.25 (w/w) [19]. At the higher NA/lipid ratios novel small multilamellar vesicles (SMLVs) are formed, consisting of numerous (typically 6–9) lamellae arranged concentrically around a dense core. At lower drug to lipid ratios more typical LUVs or capped-LUVs are formed. 9.3.3

Encapsulation of Nucleic Acid in Ethanol-Destabilized Liposomes

An alternative to the SALP method uses ethanol-destabilized cationic liposomes [60,86] (Figure 9.5). This method requires empty liposome formation by extrusion prior to addition of NA. Once cationic liposomes of the desired size have been prepared, they are destabilized by the addition of ethanol to 40% v/v. Destabilization of preformed vesicles requires the controlled addition of ethanol to a rapidly mixing aqueous suspension of vesicles, to avoid formation of localized areas of high ethanol concentration (⬎ 50% v/v) that promote the fusion and conversion of liposomes into large lipid structures. The addition of NA to ethanol-destabilized liposomes must also be accomplished carefully, in a dropwise manner, to avoid aggregation of the resulting particle suspension. The required extrusion step and the sensitive nature of both the vesicle destabilization

Lipid solution in ethanol

Nucleic acid solution in buffer

Dropwise addition while mixing NA solution

Vesicle formation

Vesicle sizing by extrusion (10×)

Sample concentration

Free nucleic acid and ethanol removal

Sterile filtration

Figure 9.4

Ethanol drop (SALP) method of NA encapsulation. The ethanol drop or SALP method involves the dropwise addition of an ethanolic solution of lipid to an aqueous solution of NA, resulting in the formation of MLV. Vesicles are then sized by extrusion through polycarbonate filters. This method allows for the encapsulation of antisense oligonucleotides with up to 70% efficiency. Either SMLV or LUV can be prepared using this process, depending on the starting NA/lipid ratio.

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Lipid solution in solvent

Dried lipid film

Lipid hydration in buffer MLV formation by freeze/thaw (5−10×)

MLV extrusion (10×)

LUV collection

Nucleic acid solution in buffer

LUV destabilized in ethanol

Nucleic acid encapsulation

Free nucleic acid and ethanol removal

Sample concentration

Sterile filtration Figure 9.5

Encapsulation of NA in ethanol destablized liposomes. A dried lipid film is rehydrated in buffer, resulting in the formation of MLV. Multiple freeze-thaw cycles follow, and the empty vesicles are then extruded through polycarbonate filters, producing LUV. The LUVs are then destabilized by the controlled addition of ethanol to the rapidly mixing aqueous suspension of vesicles. NA solution is added to the destabilized liposomes in a drop wise manner resulting in encapsulation.

and NA addition represent process challenges that must be overcome prior to adopting this method for the reproducible preparation of encapsulated NA at a scale suitable for clinical evaluation. 9.3.4

The Reverse-Phase Evaporation Method of Nucleic Acid Encapsulation

Reverse-phase evaporation, an effective means of preventing the aggregation of charged liposomes, has previously been used to encapsulate plasmid DNA [87–91] and more recently antisense oligonucleotides [75,92]. The coated cationic liposomes (CCL) developed by Allen et al. utilize a reverse-phase evaporation procedure to accomplish NA encapsulation [75,76,93]. The CCL process is comprised of two stages (Figure 9.6). In the first, hydrophobic cationic lipid–NA seed particles are formed. In the second, the cationic particles are coated with neutral lipids and vesicles are formed by reverse-phase evaporation. The formation of the cationic lipid–NA intermediate is performed by combining two immiscible fluids, an organic solution of cationic lipid in chloroform and an aqueous solution of NA. Addition of methanol results in the generation of a Bligh–Dyer

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Cationic lipid solution in chloroform

249

Nucleic acid solution in buffer

Methanol

Biphasic intermediate

Chloroform & water

Bligh−Dyer monophase

Neutral & PEG lipids

Phase separation & recovery

Sonication of organic phase

Gel formation by rotary evaporation

Rehydration

Figure 9.6

Reverse-phase evaporation method of NA encapsulation. The combination of cationic lipid solution in chloroform, and aqueous NA solution in the first step of the reverse-phase evaporation method results in the formation of hydrophobic cationic lipid–NA seed particles. Methanol is added, producing a Bligh–Dyer monophase. Upon reconstitution with excess chloroform and water, the hydrophilic NA is drawn into the organic phase along with the cationic lipid. Neutral lipids are then added, and the organic phase is sonicated and subsequently evaporated to a gel phase. The rehydration step results in NA encapsulated in lipid vesicles ranging from 300 to 600 nm in size.

monophase [94]. When the two-phase system is reconstituted by the addition of excess chloroform and water, the hydrophilic NA is drawn into the organic phase in association with the cationic lipid. Neutral lipids are added and the organic phase is sonicated prior to evaporation to a gel phase. Rehydration results in formation of 300–600 nm vesicles encapsulating NA. Sizing is accomplished via extrusion and unencapsulated NA is removed by size exclusion chromatography. 9.3.5

The Spontaneous Vesicle Formation by Ethanol Dilution (SNALP) Method of Nucleic Acid Encapsulation

The previously described formulation methods rely on the incorporation of an extrusion step to facilitate preparation of small, monodisperse liposomes. The stable nucleic acid lipid particle (SNALP) method was developed specifically as an alternative to these extrusion-based methods [13]. Originally conceived as an alternative to a detergent dialysis method used to encapsulate plasmid DNA, the method has subsequently been adapted to the encapsulation of smaller NA payloads. The detergent dialysis method of plasmid encapsulation involves the simultaneous solubilization of hydrophobic (cationic and helper lipid) and hydrophilic (PEG lipid and plasmid DNA) components in a single detergent-containing phase [55,57]. Particle formation occurs spontaneously upon removal of the detergent by dialysis. This technique results in the formation of small (⬃100 nm diameter) stabilized plasmid

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lipid particles (SPLPs) containing one plasmid per vesicle in combination with optimized plasmid trapping efficiencies approaching 70%. Although SPLP show considerable potential as systemic gene transfer agents [55,95,96], the detergent dialysis manufacturing method suffers from a number of limitations. Detergent dialysis is exquisitely sensitive to minor changes in the ionic strength of the formulation buffer. Changes as small as 10 mM result in a dramatic decrease in encapsulation efficiency [55,57]. Even when SPLPs are formed under ideal conditions the detergent dialysis method results in the formation of large numbers of empty vesicles that require separation from SPLP by gradient ultracentrifugation. The detergent dialysis process is also difficult to scale to the size required to support preclinical and clinical development of the technology. Finally, detergent dialysis is very inefficient when used to encapsulate smaller NA species such as siRNA duplexes or antisense DNA oligonucleotides. For these reasons, alternative methods of preparing SPLP were explored and a more simple, robust, and fully scalable method for the encapsulation of plasmid DNA has been developed. This method, termed “stepwise ethanol dilution,” produces SPLP with the same desirable properties as those prepared by detergent dialysis [13]. Lipid vesicles encapsulating plasmid DNA are formed instantaneously by mixing lipids dissolved in ethanol with an aqueous solution of DNA in a controlled, stepwise manner (Figure 9.7). Combining DNA and lipid flow streams result in rapid dilution of ethanol below the concentration required to support lipid solubility. Using this method, vesicles are prepared with particle sizes ⬍150 nm and DNA encapsulation efficiencies as high as 95%. When the method is adapted to the encapsulation of smaller NA species, vesicle sizes as low as 45 nm are readily obtained and encapsulation efficiencies of 95% are routine. The term SNALP, is used to differentiate from particles prepared using the SALP and SPLP methods, and to denote the more generally applicable methodology which can be applied to any charged NA species. The ability of the ethanol dilution method to rapidly prepare liposomes of desirable size and encapsulate NA with high efficiency is thought to result from the precise control of the conditions

Nucleic acid solution in buffer

Lipid solution in ethanol

Spontaneous vesicle formation by mixing

Vesicle stabilization by dilution

Sample concentration

Ethanol removal

Sterile filtration Figure 9.7

Ethanol dilution (SNALP) method of NA encapsulation. The ethanol dilution or SNALP method involves in-line mixing of lipids dissolved in ethanol with nucleic acid dissolved in buffer, resulting in the spontaneous formation of lipid vesicles. As the solutions are mixed, ethanol is diluted below the concentration required to maintain lipid solubility, resulting in vesicle stabilization. Controlled particle sizes from 40 to 150 nm, and encapsulation efficiencies of up to 95% are routinely observed. No extrusion steps are required.

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under which the lipids enter the aqueous environment, self-arrange into lipid bilayer fragments, and then form liposomes. By analogy, similar parameters have been shown to be critical for SPLP formation and plasmid encapsulation when using detergent dialysis [95,97]. Ionic strength, cationic lipid, and PEG lipid content must be optimized to maximize plasmid entrapment and minimize aggregation or the formation of empty vesicles [97]. The first stage of dilution is proposed to result in the formation of macromolecular intermediates, possibly lamellar lipid sheets or micelles. NA is recruited to these bilayer fragments by electrostatic attraction. If the cationic lipid content is too low, the plasmid fails to associate with these intermediates, favoring the formation of empty vesicles. If the cationic lipid concentration is too high, the surface charge on the lipid intermediate attracts excess NA, leading to the formation of polydisperse aggregates. At optimal cationic lipid concentrations, NA is proposed to associate with the lipid intermediates in such a way as to reduce the net positive charge on the lipid surface. Association of additional lipid leads to the formation of vesicles containing encapsulated NA. Similar to detergent dialysis, SNALP formation by ethanol dilution is optimized by balancing ionic strength, cationic lipid, and PEG lipid content. However, the ethanol dilution method appears much more robust than detergent dialysis, with good results achieved through a wide range of formulation conditions. In summary, a variety of techniques are available for encapsulating NA into lipid-based systems. Stepwise ethanol dilution, the SNALP approach, generates small (diameter ⬍100 nm), well-defined, stable systems with high encapsulation efficiencies (⬎95%) and a broad range of NA/lipid ratios (⬎0.1 w/w) that exhibit the extended circulation lifetimes required to achieve preferential accumulation at target sites such as solid tumors or liver. Among the various methods for encapsulating NA, stepwise ethanol dilution most adequately satisfies demands related to scalability and reproducibility.

9.4 ANALYTICAL METHODS An important adjunct to any method of preparing liposomes for NA delivery is the characterization of the resulting system using appropriate analytical methodology. The critical measurements are those that determine the size and monodispersity of the particle preparation, the degree of NA encapsulation, and the particles’ surface charge. Since each of these attributes has the potential to affect the pharmacology of a liposomal NA delivery system and each has the potential to change over time, it is critical to develop an understanding of each system’s properties and their stability by monitoring each of these parameters using the appropriate methodology. 9.4.1

Measuring Particle Size

Two methods are commonly used to determine the size of a liposome preparation. The first is direct visualization using scanning or transmission electron microscopy. The second is an indirect method, dynamic light scattering, also referred to as quasi-elastic light scattering (QELS) or photon correlation spectroscopy (PCS). Dynamic light scattering measures the size of liposomes suspended in a liquid. A colloidal liposome preparation is in a state of random movement due to Brownian motion. The speed of any given particle is inversely proportional to its size and smaller liposomes move more quickly than their larger counterparts. When a suspension of liposomes is illuminated with a laser, the movement, and therefore the size of the liposomes, can be measured by analyzing the rate at which the light intensity fluctuates as a result of light scatter. It is important to understand that depending on which method is used to measure the size of a liposome preparation, one can, and will, generate different results. Examination of liposomes under an electron microscope provides a two-dimensional image. Generally, we assume that the ideal liposome is spherical, while in reality, especially on an electron microscope grid, there is infinite number of diameters that can be measured. If the maximum length is used as the diameter, then the

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particle is assumed to be a sphere of this maximum dimension. Using the minimum diameter will obviously produce a different result for the particle size. The situation becomes more complex when we consider the problem of describing a liposome preparation that consists of one or more populations of particles with different sizes. If we imagine a photograph taken with an electron microscope of a liposome preparation consisting of three spheres of diameters 50, 100 and 150 nm, how do we determine and express the average size of the liposomes? If we simply add all the diameters (冱d ⫽ 50 nm ⫹ 100 nm ⫹ 150 nm) and then divide by the number of liposomes (n ⫽ 3), the average diameter is 100 nm. This is the mean, or more specifically the number–length mean diameter [98]. The designation “number–length” mean is used, because the number of particles appears in the equation: D[1,0]

Mean diameter ⫽ (50 nm ⫹ 100 nm ⫹ 150 nm) Ⲑ 3 ⫽ 100 nm ⫽ ∑ dⲐn

This value is referred to as D[1,0] because the diameter terms in the numerator are to the power of one (d 1) and there are no diameter terms (d 0) in the denominator of the equation [98]. Manual analysis of photomicrographs yields D[1,0]. Automated image analysis of the same photomicrograph would typically begin by measuring the surface area of each liposome to determine the average size. This compares liposomes on the basis of their surface area. Since the surface area of a sphere is 4
r2, the diameters are squared, divided by the number of particles, and the square root is taken to derive the mean diameter: D[2,0]

⫽ {(50 nm 2 ⫹ 100 nm 2 ⫹ 150 nm 2 ) Ⲑ 3} ⫽ 108 nm ⫽

∑ d 2Ⲑn

This yields the number–surface mean diameter. Since the diameter terms in the numerator are to the power of two (d 2) and there are no diameter terms (d 0) in the denominator of the equation, this value is described as D[2,0] [98]. Our hypothetical example, when analyzed in this way, gives a number–surface mean diameter of 108 nm. These calculations require explicit knowledge of the absolute number of liposomes analyzed (n), however many instrumental methods determine D[4,3], the volume moment mean, using methods which do not require explicit knowledge of the number of particles analyzed. For example, dynamic light scattering instruments often generate the D[4,3] or the equivalent–volume mean diameter [98]. D[4,3] (50 nm 4 ⫹ 100 nm 4 ⫹ 150 nm 4 ) 冒 (50 nm 3 ⫹ 100 nm 3 ⫹ 150 nm 3 ) ⫽ 136 nm ⫽ ∑ d 4冫 ∑ d 3 In this case, the calculated equivalent–volume mean diameter is 136 nm, a difference of 36% relative to the value of D[1,0], the result of manual analysis of data acquired using an electron microscope. These examples, derived from the work of Rawle [98], illustrate how different methods of determining average particle size may yield different results. Often, investigators give extra weight to data acquired by electron microscopy, perhaps because the data acquisition methods seem more direct or “hands on” or because the lower numbers are thought to reflect a higher quality liposome preparation. However, size measurements made using photomicroscopy typically contain ⫾3 – ⫾5% error. If number–length diameter measurements containing ⫾4% error are then used to calculate volume mean diameter, a cubic function of the diameter, the error will be cubed upon conversion and will increase to ⫾64%. However, dynamic light scattering can be used to calculate the volume mean diameter with reproducibility approaching ⫾0.5% [98]. Converting this figure into a number mean gives an error that is the cube root of 0.5%.

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Furthermore, while electron microscopy allows for the direct examination of liposomes, it is not suitable as an in-process or quality control technique. Sample preparation for electron microscopy is laborious and slow, and a limited number of particles can be examined, increasing the danger of unrepresentative sampling and magnification of error. 9.4.2

Zeta Potential

Zeta potential is a measure of the electric charge acquired by a liposome. This is of interest for two reasons. In the first case, the charge affects particle stability; in the second case the charge affects liposomal pharmacology. Liposomes, as colloidal particles, are subject to the DVLO theory [99,100]. This theory suggests that the stability of a colloidal system is governed by both the repulsive electrical double layer and the attractive van der Waals forces which the particles experience as they approach one another. The energy barrier presented by the repulsive forces must be large enough to prevent particles from contacting one another, adhering and forming aggregates. If this energy barrier is overcome the attractive van der Waals forces will pull the particles into contact and keep them together, an unsatisfactory situation for a liposomal preparation designed to be used as a drug. The goal of liposomal formulation is to prepare a stable, monodisperse particle preparation that retains both monodispersity and particle size in an effort to yield consistent performance. Since charge is a good measure of the magnitude of the interaction between particles, the zeta potential gives an indication of the potential stability of a liposomal system. Liposomes with a large negative or positive zeta potential will repel each other and remain monodisperse and stable. If liposomes have low zeta potential values then the attractive van der Waals forces are able to overcome the repulsive electrical double layer forces, the particles come together, aggregate, and the formulation tends to be unstable. As a rule, liposomes with zeta potentials more positive than ⫹30 mV or more negative than ⫺30 mV are considered stable. Particles with low zeta potentials between ⫺30 and ⫹30 mV are normally unstable. This would suggest that liposomes should be prepared such that they carry substantial surface charge to enhance their stability as a monodisperse particle preparation. This does not take into account the complex electrostatic milieu encountered once the liposome leaves the vial and enters the blood compartment. Once in the blood, liposomes are free to interact with blood components such as proteins, lipoproteins, and cell surface membranes. Many of these entities are charged and as such, exert either attractive or repulsive forces on the liposomes depending on the charge differential. For this reason, liposomes with substantial positive or negative charge (zeta potential), although stable upon formulation, are rapidly cleared upon systemic administration [101,102]. This presents a dilemma in the design of liposomal systems for the delivery of NA. NA formulations generally incorporate cationic lipids to encourage interaction of the anionic NA with the lipid bilayer. The resulting systems are often highly charged, and accordingly have no appreciable circulation lifetime in systemic applications. In an effort to improve upon the pharmacology of liposomes containing cationic lipids a number of strategies have been adopted including steric stabilization using lipid conjugates of hydrophilic polymers such as PEG. PEG lipids have the undesired side effect of confounding zeta potential readings. For this reason other methods may be necessary for determining the apparent surface charge of PEGylated systems, such as those that utilize fluorescent dyes for example the toluene nitrosulfonic acid (TNS) assay [20]. The situation is further complicated when using titratable lipids in which case surface charge measurements are specific to the medium in which they are obtained. 9.4.3

Encapsulation

The pharmacology of a liposomal formulation of NA will be largely determined by the extent to which the NA is encapsulated inside the liposome bilayer(s). Encapsulated NA will be protected from nuclease degradation, while those that are merely associated with the surface of a liposome will be less protected. Encapsulated NA shares the extended circulation lifetime and biodistribution

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of the intact liposome, while those that are surface associated will adopt the pharmacology of naked NA once they disassociate from the liposome surface. For this reason encapsulation must be accurately determined. An acceptable method is the use of a membrane-impermeable fluorescent dye exclusion assay. This method requires a dye that has enhanced fluorescence when associated with NA. Specific dyes are available for the quantitative determination of plasmid DNA, single-stranded deoxyribonucleotides, and single- or double-stranded ribonucleotides. Encapsulation is determined by adding the dye to a liposomal formulation, measuring the resulting fluorescence and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergentmediated disruption of the liposomal bilayer releases the encapsulated NA, allowing it to interact with the membrane-impermeable dye. NA encapsulation is calculated as E ⫽ (Io⫺I)ⲐIo, where I and Io refer to the fluorescence intensities before and after the addition of detergent [55]. Although other methods have been used to determine the liposomal encapsulation of NAs, including nuclease protection assays, chromatographic separation [43], density gradient ultracentrifugation [103], and capillary electrophoresis [104], this method is the most accurate, rapid, and cost-effective. Methods that rely on nuclease protection or chromatographic separation often fail to differentiate encapsulated NA from that which is merely surface associated or trapped in lipid–NA aggregates.

9.5 PHARMACOLOGY OF LIPOSOMAL NA Systemic delivery to disseminated target tissues requires the use of a “stealthy,” relatively charge neutral delivery system, since indiscriminate interaction with blood components, lipoproteins or serum opsonins, can cause aggregation before the carrier reaches the target site. This is especially important in the case of systems containing large polyanionic molecules such as NA, which have a greater potential for inducing toxicity through interaction with complement and coagulation pathways [105]. Other barriers to delivery may include the microcapillary beds of the “first-pass” organs, the lungs and the liver, and the phagocytic cells of the reticuloendothelial system. Accessing target cell population requires the ability to extravasate from the blood compartment to the target site. Charge neutral carriers of appropriate size can pass through the fenestrated epithelium found in sites of clinical interest such as tumors, sites of infection, inflammation, and in the healthy liver and accumulate via the EPR effect [106] (also referred to as “passive” targeting or “disease site” targeting). To take advantage of this EPR effect, which can result in profound enrichment at the target site, carriers must be small (diameter on the order of 100 nm) and long circulating (extended circulation lifetimes following intravenous injection in mice). Clearly, NA stands to benefit from the pharmaceutical enablement conferred by encapsulation in appropriately designed liposomal carriers. 9.5.1

Pharmacokinetics and Biodistribution of Liposomal NA Following Systemic Administration

Following intravenous injection, the clearance of properties of encapsulated NA can be assessed by lipid and/or NA markers. (As methods of determining the pharmacokinetics and biodistribution of NA themselves are described elsewhere in this volume they will not be discussed here.) Previous experience shows that, if NA is fully encapsulated in stable liposomes, the lipid and NA components are cleared from the blood compartment at the same rate and the NA remains intact, protected from nuclease degradation while encapsulated within the liposome [14,15,107]. As long as the liposome remains intact, the biodistribution of a nonexchangeable lipid marker [108] incorporated in the formulation is representative of the biodistribution of the entire particle, including the NA component. This finding may be applied to analysis of liposomal clearance and biodistribution up to 24 h after administration, after which time even the most stable lipid markers will begin to experience some remodeling or exchange [109].

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A comparison of the clearance properties of liposomal formulations of siRNA in three different species is shown in Figure 9.8. Liposomes were formulated containing distearyl phosphotidylcholine (DSPC), cholesterol, DLinDMA and PEG-c-DMA encapsulating siRNA. The specific liposome composition, manufactured using the SNALP process, was selected for efficient delivery to the liver, with a view towards avoiding accumulation in distal tissue or in nontarget tissues of the reticuloendothelial system such as the spleen. The dose remaining in plasma and tissue samples obtained at various times after intravenous administration in either mice or guinea pigs was determined using the radiolabeled lipid marker ([3H]-cholesteryl hexadecyl ether [CHE]) [15,61]. The plasma clearance properties of liposomally encapsulated siRNA in cynomolgus monkeys was determined directly by ion exchange high-performance liquid chromatography (HPLC) [15]. Four hours after tail vein injection in mice, 3.3⫾1.3% of the injected dose remains in the plasma with a half-life of 38 min. The half-life of unprotected, unmodified phospodiester siRNA has been shown to be ⬍ 2 min in mice [14]. When liposomal siRNA is administered intravenously via ear vein injection in guinea pigs, 3.0 ⫾ 1.0% of the injected dose remains in the plasma 4 h after administration, corresponding to a plasma half-life of 39.3 min [61]. When encapsulated siRNA is administered to cynomolgus monkeys as a bolus injection in the saphenous vein, 17% of the injected dose remains in the plasma after 4 h, corresponding to a plasma half-life of 72 min. The agreement between the clearance properties in mice and guinea pigs, especially given the different routes of administration, is remarkable. Also noteworthy is the extent to which the doubling in the plasma half-life as measured in mice and primate species is predicted based on the comparative pharmacologic studies which have given rise to the technique of allometric scaling, whereby the pharmacological parameters of a given drug can be predicted in different species [110]. Using either radiolabeled lipid markers or direct analysis of NA, the biodistribution of liposomal NA following intravenous administration may be determined. Figure 9.9 illustrates the accumulation of liposomal siRNA in various tissues 24 h after the administration in mice and guinea pigs. The liver and spleen typically demonstrate the highest levels of liposome accumulation. In this case the liver has accumulated 70.7 ⫾ 5.4 and 83.4 ⫾ 6.5% of the injected dose per gram, in mice and guinea pigs, respectively and the spleen has accumulated 0.94 ⫾ 0.15 and 2.2 ⫾ 0.2% of the injected dose per gram in mice and guinea pigs, respectively; whereas, the kidney, heart and brain accumulate the least amount of liposomal NA. Of note, the kidney, the prototypical target tissue associated with the toxicity of naked antisense drugs, accumulates ⬍1% of the injected dose per gram, in both mice and guinea pigs.

Percent injected dose

100 Cynomolgus monkey

80

Guinea pig 60

Mouse

40 20 0 0

Figure 9.8

2

4

6 Time (h)

8

10

12

Plasma clearance of liposomal (SNALP) encapsulated siRNA. Plasma clearance of SNALP siRNA determined in mice, guinea pigs, and cynomolgus monkeys. Each animal received a single intravenous injection of SNALP-formulated siRNA. Data represent percent of the total injected dose in blood at the indicated time points after treatment. Mouse and guinea pig data are presented as mean ⫾ s.d., n ⫽ 5. Cynomolgus monkey data represent the mean of two treated animals.

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Percent injected dose per tissue

100 Guinea pig 10

Mouse

1

0.1

0.01 Liver Figure 9.9

Spleen

Lung

Kidney

Heart

Brain

Biodistribution of liposomal (SNALP) encapsulated siRNA. Biodistribution of SNALP siRNA was determined in mice and guinea pigs. Each animal received a single intravenous injection of 3 H-labeled SNALP-formulated siRNA. Data represent percent of the total injected dose in each tissue 24 h after treatment. Data are represented as mean ⫾ s.d., n ⫽ 5.

While these results are typical of freely circulating liposomal systems, the extent to which liposomes accumulate in certain tissues, especially the liver, spleen, and distal disease sites such as tumors, can be modulated by affecting changes in the liposome formulation. Manipulation of the chemistry of the individual lipid components and their relative molar ratios within the system can significantly alter a formulation’s pharmacokinetics, biodistribution. and transfection efficiency. One such example of this plasticity is illustrated in Figure 9.10 and Figure 9.11. The plasma clearance and liver accumulation of three liposomal siRNA formulations (SNALP) that differ only in the alkyl chain length of the incorporated PEG–lipid are shown. Shorter PEG–lipid anchor lengths decrease the blood circulation times of the SNALP and increase the rate and extent of nanoparticle accumulation in the liver of mice. SNALP containing PEG lipids with distearyl (C18), dipalmityl (C16), and dimyristyl (C14) lipid anchors have circulation half-lives of 4 h, 2 h, and 40 min, respectively. In this example, up to 75% of the total injected dose of PEG-cDMA-containing particles accumulates in the liver after intravenous administration, while 35% of the dose accumulates in the liver when the more stably integrated PEG-cDSA is used. Further manipulation of the liposomal bilayer composition can result in ⬍20% of the total injected dose accumulating in the liver, with concomitant increases in the extent of accumulation in non–reticuloendothelial system (non-RES) tissues such as disseminated tumors [58]. The extent of NA distribution in tissues following administration of liposomal systems is markedly greater than what has been observed in other systems. This can be attributed to the extended blood circulation lifetimes of liposomal formulations and their ability to protect encapsulated NA from degradation, greatly extending the available timeframe for delivery to and accumulation within tissues. While liposomal formulations may provide plasma half-lives for intact NA of 0.5–60 h [14,15,61,107,114] “naked” NA and cationic lipoplex typically have half-lives of minutes or less [14,111–114]. 9.5.2

Toxicity of Liposomal NA Formulations

The raison d’être of drug delivery technology is to improve a drug’s effectiveness by increasing availability of the drug at the intended target site. However, an unintended by-product that often accompanies the use of drug delivery technology is a shift in drug-associated toxicity. In many cases these drug-related toxicities may be anticipated by previous experience with the free drug in that the mechanism of toxicity is conserved; however, a shift in the target organ of toxicity is common [115,116]. While many small molecule chemotherapeutic drugs in their free form give rise to nephrotoxicity or

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100 PEG-C-DSA

Percent injected dose

80

PEG-C-DPA 60

PEG-C-DMA

40 20 0 0

4

8

12 16 Time (h)

20

24

Figure 9.10 Plasma clearance of SNALP containing PEG–lipids with increasing alkyl chain lengths. Plasma clearance of 3H-labeled SNALP containing PEG-C-DMA, PEG-C-DPA or PEG-C-DSA in ICR mice. SNALP were administered i.v. at 5 mg/kg siRNA. Data represent percent of the total injected dose in blood at the indicated time points after treatment. Values are mean ⫾ s.d., n ⫽ 4 mice.

Percent injected dose in liver

100 1h 80

4h 24 h

60 40 20 0 PEG-C-DMA

PEG-C-DPA

PEG-C-DSA

Figure 9.11 Liver accumulation of SNALP containing PEG–lipids with increasing alkyl chain lengths. Liver accumulation of 3H-labeled SNALP containing PEG-C-DMA, PEG-C-DPA or PEG-C-DSA in ICR mice. SNALP were administered i.v. at 5 mg/kg siRNA. Data represent percent of the total injected dose in liver at the indicated time points after treatment. Values are mean ⫾ SD, n ⫽ 4 mice.

hepatotoxicity as a result of accumulation in the kidneys or liver, the same drugs, once encapsulated and delivered in liposomal form, may give rise to previously unobserved mucocutaneous toxicities or peripheral neuropathy [115,116]. These changes in toxicity are similar to those observed when using liposomal NA drugs. When naked antisense phosphorothioate oligonucleotides are injected intravenously in mammals, ⬃20% of the injected dose accumulates in the kidney [117]. At high doses in monkeys this level of accumulation manifests as toxicity, first in the form of focal tubular regeneration, and at even higher doses as perturbation in N-acetylglucosamine, total protein, and retinol-binding protein levels [118]. Similarly, high levels of accumulation in the liver lead to hypertrophy of Kupffer cells and ultimately increases in transaminases such as AST and ALT [119]. Since liposomal encapsulation results in a dramatic shift in the biodistribution of NA we would expect a concomitant shift in the target organs of toxicity. Indeed, as intravenous administration of liposomal NA results in accumulation of ⬍1% of the total injected dose in the kidney when measured in either mice or guinea pigs [61], little or no nephrotoxicity results, even at doses greater than those required to elicit hepatic toxicity as measured by elevations of serum transaminases [15].

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A special consideration when working with liposomal systems is their potential to activate the complement system. In particular, liposomes possessing cationic or anionic lipids are capable of binding complement proteins and triggering the activation of the complement cascade [105]. To test liposomal systems for their ability to activate complement, standard in vitro assays may be performed, or complementary experiments may be performed in vivo. Results of numerous studies indicate that complement activation by liposomal NA may be prevented by controlling the amount and presentation of liposomal surface charge, either through the use of PEG–lipids or by adjusting the amount or type of cationic lipid used. PEGylated systems containing modest amounts of titratable cationic lipid appear to be particularly stealthy in this regard [120]. 9.5.3

Immune Stimulation

NA can cause activation of the mammalian innate immune system leading to the release of interferons and proinflammatory cytokines. In the case of DNA, immune stimulation is triggered primarily by the recognition of unmethylated CpG sequence motifs by Toll-like Receptor-9 (TLR9) [121] located within the endosomal compartment of certain antigen presenting cells (APC) [122,123]. Similar immune recognition pathways are also activated by exogenous single [124,125] and double-stranded RNA [126] through TLR7/8 and TLR3, respectively. It has been recently reported that synthetic siRNA can also induce potent immune stimulation [127–129]. The immune responses elicited by NAs are greatly potentiated by the use of delivery vehicles, including either liposomal- or polycation-based vehicles that facilitate intracellular delivery via endosomes, the primary intracellular location of the affected TLRs [127,130]. Although the immunomodulatory effects of CpG DNA have potential therapeutic utility in oncology and allergy applications [131], in many other applications immune activation represents an additional hurdle to drug development. The consequences of an unmanaged activation of the innate immune response can be severe, particularly in more sensitive species including humans [132–134] due to the multitude of local and systemic inflammatory reactions that can be triggered by activation. Many of the toxicities associated with the administration of siRNA in vivo have been attributed to this response [14,127]. On the basis of the finding that immune activation by siRNA is sequence-dependent, it is possible to design active siRNA with negligible immunostimulatory activity by selecting sequences that lack GU-rich or other immunostimulatory motifs [127,135]. Although this strategy has proven successful, it significantly limits the number of novel siRNA sequences that can be designed against a given target. Furthermore, it currently requires some degree of screening due to the relatively ill-defined nature of putative RNA immunostimulatory motifs. Another approach involves the use of stabilization chemistries that were previously developed for ribozymes or antisense oligonucleotide drugs [136] that have more recently been applied to the chemical stabilization of synthetic siRNA. siRNA may be designed containing 2⬘OMe [137–139], 2⬘F [137,139–141], 2-deoxy [140] or “locked nucleic acid” (LNA) [128,142] modifications yet retaining functional RNAi activity, indicating that these chemistries can be compatible with the RNAi machinery. However, modification of siRNA appears to be tolerated only in certain positional or sequence-related contexts, and in most cases, indiscriminate modification has a negative impact on RNAi activity [128,138–140,142]. Until recently, the design of chemically modified siRNA has required laborious screening in an effort to identify duplexes that retain potent gene silencing activity. Recently, a more rational approach to the design of highly active, nonstimulatory siRNA molecules has been described [143]. Surprisingly, minimal 2⬘OMe modifications within one strand of a double-stranded siRNA duplex are sufficient to fully abrogate the immunostimulatory activity of siRNA, irrespective of sequence. Remarkably, incorporation of as few as two 2⬘OMe guanosine or uridine residues in highly immunostimulatory siRNA molecules completely abrogate siRNA-mediated interferon and inflammatory cytokine induction in human peripheral blood mononuclear cells (PBMC) and in mice in vivo. This degree of chemical modification

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represents ⬃5% of the native 2⬘-OH positions in the siRNA duplex. Since complete abrogation of the immune response requires only one of the RNA strands to be selectively modified, 2⬘OMe modifications can be restricted to the sense strand of the duplex, therefore minimizing the potential for attenuating the potency of siRNA, which is predominantly determined by the antisense “guide” strand. Minimally modified siRNA retains potent gene silencing in vivo, without evidence of cytokine induction, immunotoxicities or off-target effects associated with immune activation triggered by unmodified siRNA. This provides a simple method of designing nonimmunostimulatory siRNA based on native sequences with proven RNAi activity. It is presently unclear how the introduction of 2⬘OMe nucleotides into one strand of an siRNA duplex prevents recognition of siRNA by the immune system. The trans-inhibitory effect of 2⬘O-methylation, whereby 2⬘OMe-modified ssRNA annealed to unmodified immunostimulatory ssRNA generates a nonimmunostimulatory duplex, is consistent with a hypothesis that involves recognition of the siRNA by its putative immune receptor, thought to be TLR-7 [128], as a doublestranded molecule. It is conceivable that 2⬘OMe-modified siRNA may avoid recognition by the innate immune system using a mechanism that has evolved to allow for the differentiation of self from pathogen-derived RNA. Other NA modification chemistries have the potential to influence immune stimulation. LNAs containing a 2⬘-O, 4⬘-C methylene bridge in the sugar ring have been shown to partially reduce the immunostimulatory activity of siRNA [128]. However, siRNAs containing inverted deoxy abasic end caps retain immunostimulatory activity [14]. No evidence of a trans-inhibitory effect was observed with LNA-modified duplexes. These observations suggest that, for reasons we do not currently understand, immune stimulation by siRNA may be particularly sensitive to inhibition by 2⬘OMe modifications versus other stabilization chemistries. Minimal 2⬘OMe modification to prevent the induction of interferons and inflammatory cytokines has been shown to both limit the potential for nonspecific effects on gene expression and improve the tolerability of siRNA formulations. Intravenous administration of liposomal 2⬘OMe-modified siRNA is efficacious and well tolerated in mice [143]. This approach, coupled with ongoing improvements in delivery technology and siRNA design, may be an important component in the development of synthetic siRNA therapeutics. 9.5.4

Immunogenicity

The potential for a drug to be immunogenic is a serious concern in drug development since the establishment of an antibody (Ab) response can severely compromise both the safety and efficacy of a drug. This has hampered the development of drugs, including protein-based therapeutics such as monoclonal antibodies that contain immunogenic components. It has long been recognized that liposomes can act as immunological adjuvants as a result of their particulate nature, efficient uptake by APC, and ability to cross-link surface receptors [144]. This property is enhanced when immunostimulatory agents such as CpG DNA are incorporated into the liposomes [145,146]. This has been exploited in the design of liposomal vaccines that generate strong Ab responses against weakly immunogenic antigens attached to the liposome surface. It is therefore unsurprising that immunogenicity has proven to be a major obstacle in developing receptor-targeted liposomes that incorporate antibodies, peptides or receptor ligands on their surface to enhance target cell uptake [147–149]. Remarkably, the addition of a PEG coating to these liposomes typically has a minor effect on reducing their immunogenicity [145,147,149]. Experience with stable plasmid lipid particles (SPLPs), a liposomal system for the delivery and expression of therapeutic pDNA [13,58], provides an example of the challenges faced when designing nonimmunogenic NA carriers. The in vivo safety and efficacy of SPLP that contain stably integrated PEG lipids are severely compromised following repeat administration due to a surprisingly robust Ab response against PEG that arises from a single administration. The immunogenicity of PEGylated liposomes containing pDNA can be greatly reduced by using alternative diffusible PEG–lipids that diffuse more readily from the lipid bilayer upon administration. By eliminating

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the Ab response to PEG, these modified liposomes can be safely readministered to mice while maintaining the effective delivery of the pDNA payload to distal tumor sites. Administration of nonimmunogenic SPLP is still associated with substantial cytokine induction, indicating that the reduced immunogenicity is not due to abrogation of the immunostimulatory activity of the pDNA payload. Instead, this supports the hypothesis that robust Ab responses to PEG require the close physical association of the PEG–lipid with pDNA and are driven by the specific binding and internalization of PEGylated liposomes containing stimulatory pDNA by PEG-reactive B cells [120]. An alternative approach to reducing carrier immunogenicity may be the development of less immunostimulatory NAs. CpG-free pDNA and chemically modified antisense ODN or synthetic siRNA may have significantly reduced capacity to activate cytokine responses. Synthetic siRNAs can induce potent immune stimulation in vivo, driving the production of a strong anti-PEG Ab response when immunostimulatory siRNAs are encapsulated in PEGylated liposomes containing stably integrated C18-PEG–lipids [120]. Minimally modified siRNA duplexes, when encapsulated in PEGylated liposomes containing stably integrated PEG–lipids, are nonimmunogenic. Use of minimally modified, nonimmunostimulatory NA and/or diffusible PEG–lipids allows for flexibility in the design of nonimmunogenic liposomal systems. These findings raise important concerns regarding the potential immunogenicity of any delivery vehicle currently under consideration for use with immunostimulatory NA-based drugs. Given that most RNA and DNA species stimulate innate cytokine responses and B cell activation [124–128,143], vehicle immunogenicity may prove to be problematic for a range of NA-based therapeutics. Antibody responses against surface components, especially targeting ligands, of liposomal systems should be closely monitored. However, the ability to abrogate the immunogenicity of liposomal NA formulations by simple modification of either their lipid composition or NA provides multiple paths forward in the design and clinical development of these systems. 9.5.5

The Efficacy of Liposomally Formulated NA Drugs

Currently, the clinical experience with liposomal formulations of NA drugs is limited, requiring us to rely on preclinical results to gauge their promise. In this regard there are many reports of efficacy associated with liposomal formulations of antisense ODN, ribozymes and more recently siRNA (recently reviewed by Behlke [150]). Liposomal NAs have been evaluated in preclinical models of infectious disease, inflammation, cancer, and various metabolic conditions. However, it is only recently that we have come to appreciate the extent to which nonspecific effects, such as stimulation of the innate immune system, may effect the results obtained in preclinical models that are used to measure efficacy. Particularly troublesome is the impact that induction of the innate immune system has on antitumor efficacy in murine models and on models of infectious disease. For these reasons it is especially crucial to adopt appropriate controls when working with these systems. Specifically, the inclusion of nontargeting control NA, with similar immunostimulatory properties to the active compounds, is required. As described in the previous section, with our improved understanding of the chemical modification strategies that abrogate the immune stimulation associated with siRNA, it is now straightforward to design immunologically silent NA that retain their desired mechanism of action [143]. In spite of the well-documented impact that liposomal NA can have on the innate immune system some investigators have failed to fully characterize the immunostimulatory properties of their test article prior to publishing the efficacy results. Others have unwittingly reported falsenegative immune stimulation data obtained by analyzing the immunostimulatory properties of their compounds either in cell lines that are not competent for an innate immune response, or by harvesting preclinical samples at inappropriate time points, days or even weeks after the immune stimulus has been applied. It is noteworthy that in our laboratory we have undertaken a retrospective analysis of published siRNA used in efficacy studies and with one single exception all were shown to be immunostimulatory. All of the siRNA had either been previously described as nonimmunostimulatory, or their immunostimulatory properties had not been described. Even

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more alarming is the fact that the one exception in our analysis was a negative control siRNA that has been used in more than 12 published studies to support the “efficacy” of an assortment of highly immunostimulatory siRNA. Control siRNA with similar immunostimulatory properties to the active compounds would have been preferred. Although the various pathways that may be affected by NA drugs are complex, assaying for interferon alpha and IL-6, either 2–12 h after intravenous administration in mice or 24 h after exposure of primary PBMCs, is all that is required to adequately gauge the immunostimulatory properties of most molecules. When efficacy studies are approached and interpreted with appropriate caution, the results can be substantially more convincing. Rather than provide a retrospective analysis of previously described efficacy studies, here we will describe one example that hopefully illustrates the potential of liposomal formulations of NA drugs, the example of liposomal siRNA targeting apolipoprotein B (apoB). ApoB is an essential component involved in the assembly and secretion of very low-density lipoprotein (VLDL), a precursor to LDL. ApoB is considered “non-druggable” with conventional small molecule therapies yet it is a highly relevant, genetically and clinically validated disease target. Targeting apoB with second-generation antisense oligonucleotides has shown promising preclinical and clinical results [151] and cholesterol-conjugated siRNAs directed against apoB have successfully resulted in knockdown of apoB message yielding a concomitant reduction in total cholesterol [152]. Unlike many oncology, inflammatory or infectious disease targets, the apoB transcript is regulated mainly at the posttranslational level. This is believed to confer some protection from off-target effects that would otherwise result in unintended perturbations in apoB expression levels, making apoB a “good” target for proof of concept efficacy studies. Intravenous administration of high doses, ⬎50 mg/kg, of apoB-specific siRNA, siApoB-1, as naked siRNA, in the absence of chemical conjugation, has previously shown to have no in vivo silencing activity in mice [152]. However, profound silencing of liver apoB mRNA and apoB-100 protein is achieved with a single, low dose of liposomal (SNALP-formulated) siApoB-1 [15]. No detectable reduction in apoB is observed upon treatment with SNALP-formulated mismatch siRNA (siApoB-MM) or empty SNALP vesicles, indicating that silencing is specific to the siRNA and is not an artifact caused by the liposomal carrier or due to other off-target effects. Figure 9.12 is an illustration of the relative potency of siApoB-1 SNALP-mediated silencing

Encapsulated

Conjugate

Relative apoB mRNA (%)

120 100 80

~1000 Fold Greater potency

60 40 20 0 Saline 1

0.5 0.25 0.1 Saline 100 50

25 12.5

Dose (mg/kg) Figure 9.12 The efficacy of liposomal (SNALP) encapsulated siRNA compares favorably to chemically conjugated siRNA. The dose-dependent silencing of liver apoB mRNA after administration of either SNALP siApoB-1 (left panel) or Chol-siApoB-1 (right panel) is shown. Liver apoB mRNA levels were quantified relative to GAPDH mRNA 3 days after i.v. administration of siRNA. Data are mean values relative to the saline treatment group ⫾ s.d. Chol-siApoB-1 was administered at doses of 100, 50, 25 or 12.5 mg/kg (n ⫽ 6 per group), and SNALP siApoB-1 was administered at siRNA doses of 1, 0.5, 0.25, and 0.1 mg/kg (n ⫽ 4 per group).

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compared to cholesterol-conjugated siApoB-1. While a dose of 100 mg/kg of cholesterol-conjugated siRNA is required to achieve 50% knockdown of apoB message, comparable levels of gene silencing are achieved at a dose of 0.1 mg/kg SNALP siApoB-1, corresponding to a 1000-fold increase in the potency of siRNA, when it is encapsulated relative to the cholesterol conjugate. This represents an increase in potency relative to the naked, unconjugated molecule that is more than four orders of magnitude. This degree of silencing is readily achieved in the absence of immune stimulation or other toxicities. A nonhuman primate study, using a considerably less potent siRNA sequence, confirmed that liposomal siRNA can potently silence apoB [15]. Silencing of 90% of the endogenous apoB message was achieved in cynomolgus monkeys treated with a single intravenous administration of 2.5 mg/kg SNALP-formulated siApoB-1. Again, this result was achieved in the absence of any toxicity as measured by general tolerability, complement activation, coagulation or proinflammatory cytokine production. There were no changes in hematology parameters for SNALP-treated animals. The only measurable change in SNALP siApoB-2-treated primates was a moderate, transient increase in liver enzymes in monkeys that received the highest dose of SNALP siApoB-2. This manifested as transient transaminosis that peaked at 48-h posttreatment and was highly variable among individual animals. This effect was completely reversible, normalizing within 6 days, while the reduction in apoB had yet to reach its nadir. It is important to consider that a 90% reduction in apoB levels is unlikely to be a relevant clinical target, meaning that lower doses would be used in a clinical context. In monkeys, a more moderate treatment with a dose of 1.0 mg/kg resulted in a 68% in apoB message and a 50% reduction in plasma LDL in the absence of any transaminosis. Subsequent examination of this type of toxicity in mice has revealed a number of opportunities for improving the therapeutic window of apoB SNALP, including the use of more potent siRNA sequences, formulation refinements, and changes to the dosing regime. While further optimization of NA payloads, formulations, and treatment regimens may be required, the experience with apoB SNALP suggests that effective systemic delivery of NA using liposomes is readily achievable. Together with efforts to develop chemically modified NA with optimal pharmacologic properties, liposomal NA shows considerable promise in a number of applications. It is highly likely that as NA drugs continue to move from bench to bedside, liposomes will increasingly become a component of their success.

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186. Kunisawa, J. et al., Fusogenic liposome delivers encapsulated nanoparticles for cytosolic controlled gene release, J Control Release 105 (3), 344–353, 2005. 187. Akita, H. et al., Nigral injection of antisense oligonucleotides to synaptotagmin I using HVJ-liposome vectors causes disruption of dopamine release in the striatum and impaired skill learning, Brain Res 1095 (1), 178–189, 2006. 188. Zhang, C. et al., siRNA-containing liposomes modified with polyarginine effectively silence the targeted gene, J Control Release 112 (2), 229–239, 2006. 189. Leonetti, C. et al., Encapsulation of c-myc antisense oligodeoxynucleotides in lipid particles improves antitumoral efficacy in vivo in a human melanoma line, Cancer Gene Ther 8 (6), 459–468, 2001. 190. Hu, Q., Bally, M. B. and Madden, T. D., Subcellular trafficking of antisense oligonucleotides and down-regulation of bcl-2 gene expression in human melanoma cells using a fusogenic liposome delivery system, Nucl Acids Res 30 (16), 3632–3641, 2002. 191. Waterhouse, D. N. et al., Combining doxorubicin and liposomal anti-HER-2/NEU antisense oligodeoxynucleotides to treat HER-2/NEU-expressing MDA-MB-435 breast tumor model, J Exp Ther Oncol 3 (5), 261–271, 2003. 192. Waterhouse, D. N. et al., Pharmacodynamic behavior of liposomal antisense oligonucleotides targeting Her-2/neu and vascular endothelial growth factor in an ascitic MDA435/LCC6 human breast cancer model, Cancer Biol Ther 3 (2), 197–204, 2004. 193. Wilson, A. et al., Targeted delivery of oligodeoxynucleotides to mouse lung endothelial cells in vitro and in vivo, Mol Ther 12 (3), 510–518, 2005. 194. Chiu, S. J., Marcucci, G. and Lee, R. J., Efficient delivery of an antisense oligodeoxyribonucleotide formulated in folate receptor-targeted liposomes, Anticancer Res 26 (2A), 1049–1056, 2006. 195. Chiu, S. J. et al., Efficient delivery of a Bcl-2-specific antisense oligodeoxyribonucleotide (G3139) via transferrin receptor-targeted liposomes, J Control Release 112 (2), 199–207, 2006. 196. Duzgunes, N. et al., Enhanced inhibition of HIV-1 replication in macrophages by antisense oligonucleotides, ribozymes and acyclic nucleoside phosphonate analogs delivered in pH-sensitive liposomes, Nucleosides Nucleotides Nucl Acids 20 (4–7), 515–523, 2001. 197. Ponnappa, B. C. et al., In vivo delivery of antisense oligonucleotides in pH-sensitive liposomes inhibits lipopolysaccharide-induced production of tumor necrosis factor-alpha in rats, J Pharmacol Exp Ther 297 (3), 1129–1136, 2001. 198. Wong, F. M. et al., A lipid-based delivery system for antisense oligonucleotides derived from a hydrophobic complex, J Drug Target 10 (8), 615–623, 2002. 199. Pastorino, F. et al., Targeted liposomal c-myc antisense oligodeoxynucleotides induce apoptosis and inhibit tumor growth and metastases in human melanoma models, Clin Cancer Res 9 (12), 4595–4605, 2003. 200. Brignole, C. et al., Targeted delivery system for antisense oligonucleotides: a novel experimental strategy for neuroblastoma treatment, Cancer Lett 197 (1–2), 231–235, 2003. 201. Stuart, D. D., Semple, S. C. and Allen, T. M., High efficiency entrapment of antisense oligonucleotides in liposomes, Methods Enzymol 387, 171–188, 2004. 202. Bartsch, M. et al., Stabilized lipid coated lipoplexes for the delivery of antisense oligonucleotides to liver endothelial cells in vitro and in vivo, J Drug Target 12 (9–10), 613–621, 2004.

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III A

Hybridization-Based Drugs: Basic Properties 2⬘⬘-O -Methoxyethyl Oligonucleotides

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10

Pharmacological Properties of 2⬘⬘-O -MethoxyethylModified Oligonucleotides C. Frank Bennett

CONTENTS 10.1 Introduction .........................................................................................................................273 10.2 Properties of the 2⬘-O-Methoxyethyl Nucleoside ...............................................................275 10.3 Comparison of 2⬘-MOE-Modified Oligonucleotides to First-Generation PS ODNs..................................................................................................276 10.4 Multiple Antisense Mechanisms Can Utilize 2⬘-MOE Modifications................................277 10.5 In Vitro Pharmacological Properties of 2⬘-MOE Gapmers .................................................279 10.6 In Vivo Pharmacological Properties of 2⬘-MOE Gapmers ..................................................282 10.6.1 Liver ......................................................................................................................285 10.6.2 Kidney ...................................................................................................................286 10.6.3 Adipose Tissue ......................................................................................................287 10.6.4 Bone Tissue ...........................................................................................................287 10.6.5 Lymphoid Tissues and Inflammatory Cells ..........................................................288 10.6.6 Oncology Models ..................................................................................................289 10.6.7 Local Administration of 2⬘-MOE Gapmers ..........................................................289 10.7 Human Pharmacology .........................................................................................................290 10.8 Conclusions .........................................................................................................................291 References ......................................................................................................................................292

10.1 INTRODUCTION The 2⬘-O-methoxyethyl (2⬘-MOE) modification (Figure 10.1a) was identified as part of a broad research collaboration between scientists working at Ciba-Geigy Ltd. (now Novartis) and Isis Pharmaceuticals. Different chemical modifications were incorporated into a series of standardized oligonucleotide sequences and evaluated in screens to test for binding affinity to RNA and DNA, nuclease resistance, and potency in cell culture experiments [1–3]. It was noted that increasing the alkoxy chain length at the 2⬘-position of the ribose increased nuclease

273

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O

O

O Base

O O PS O

O Base

Me O O O O PX X = O, S O O Base O

O 2′-Deoxy-phosphorothioate-DNA (2′-H/P=S) (b)

O Base

O

O

Me

2′-O-(2-Methoxyethyl)-RNA

First-generation oligodeoxynucleotide

Supports RNase H

Uniform 2′-MOE

Does not support RNase H

Standard gapmer

Supports RNase H

Gap widened

Supports RNase H

3′-Hemimer

Supports RNase H

Gap disabled

Does not support RNase H

Figure 10.1 Structure of 2⬘-MOE nucleoside and oligonucleotide. (a) Chemical structures of a deoxynucleotide and 2⬘-MOE dinucleotide units. (b) Schematic representation of different oligonucleotide designs incorporating 2⬘-MOE nucleosides. Solid bar represents oligodeoxynucleotides (DNA). Open bar represents 2⬘-MOE modified nucleotides.

resistance, but decreased hybridization affinity for RNA, with the 2⬘-O-propyl appearing to be an optimal compromise between those two parameters [4,5]. Surprisingly, 2⬘-O-alkoxyalky substitutions [6] of varying chain lengths were found to provide both a large increase in nuclease resistance and an increase in hybridization affinity, breaking the trend observed with 2⬘-alkoxy substitutions. Additional work with 2⬘-MOE-modified oligonucleotides demonstrated that the oligonucleotides exhibited a tissue distribution similar to phosphorothioate oligodeoxynucleotides (PS ODNs) and decreased toxicities compared to PS ODNs [7,8]. At the time of this writing, there are six 2⬘-MOE-modified oligonucleotides in clinical trials, and six drugs in investigational new drug (IND)-enabling toxicology studies (Table 10.1). All the 2⬘-MOE-modified oligonucleotides currently in clinical trials are phosphorothioate-modified, chimeric oligonucleotides containing 2⬘-MOE-modified nucleosides on the 5⬘ and 3⬘ ends and a central oligodeoxynucleotide core that supports the RNase H mechanism. These oligonucleotides are referred to as 2⬘-MOE gapmers (Figure 10.1b). 2⬘-MOE gapmers have proven to be a robust drug discovery platform providing numerous drugs in late-stage research. This chapter reviews the current state of knowledge regarding the general pharmacological properties of 2⬘-MOE-modified oligonucleotides, with emphasis on 2⬘-MOE gapmers. Subsequent chapters review the pharmacokinetic and toxicological properties of 2⬘MOE gapmers, as well as more in depth pharmacology in specific therapeutic areas.

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Table 10.1 2⬘-MOE Modified Oligonucleotides in Development Drug

Target

Indication

Status

ISIS 113715 ISIS 301012 OGX-011

PTP-1B Apo B Clusterin

Type 2 diabetes Hyperlipidemia Cancer, various

Phase 2 Phase 2 Phase 2

ATL 1102 LY2181308 LY2275796 ISIS 369645

CD49D (VLA4) Survivin eIF4E IL-4 receptor-


ISIS 325568

Glucagon receptor Heat shock protein 27 C-raf kinase

Multiple sclerosis Cancer, various Cancer, various Asthma, inhaled Type 2 diabetes Cancer, various

Phase 2 Phase 1 Phase 1 Preclinical toxicology Preclinical toxicology Preclinical toxicology Preclinical toxicology

OGX-427 iCo007

ISIS 353512 ISIS 333611

C-reactive protein Superoxide dismutase 1

Macular degeneration intravitreal Cardiovascular Amyotrophic lateral sclerosis

Preclinical toxicology Preclinical toxicology

Sponsor Isis Pharmaceuticals, Inc. Isis Pharmaceuticals, Inc. OncoGenex/ Isis Pharmaceuticals, Inc. Antisense Therapeutics Ltd. Eli Lilly Eli Lilly Isis Pharmaceuticals, Inc. Isis Pharmaceuticals, Inc. OncoGenex iCo

Isis Pharmaceuticals, Inc. Isis Pharmaceuticals/ ALS Association

10.2 PROPERTIES OF THE 2⬘⬘-O -METHOXYETHYL NUCLEOSIDE What it is responsible for the enhanced properties of the 2⬘-MOE modification? Similar to other 2⬘-alkoxy modifications, the sugar of the 2⬘-MOE nucleoside is in a northern C3⬘-endo conformation resulting in an A-form helical geometry when bound to RNA [9]. The preorganization of the oligonucleotide into an A form geometry contributes to the increased binding affinity for RNA. Examination of the crystal structure of 2⬘-MOE-modified nucleotides duplexed to RNA revealed that the ethylene alkyl chain of the 2⬘-MOE-modified sugar is in a gauche conformation [9–11]. The 2⬘-MOE substitution also forms a water bridge to the phosphate oxygen atoms, further adding rigidity to the sugar conformation. The increased nuclease resistance may be due in part to steric hindrance provided by the methoxyethyl substituent and the bound water molecules. Supporting this conclusion was the finding that the 2⬘-O-methyl[tri-(oxyethyl)] substituent (three ethyleneglycol groups) further increased nuclease resistance of the modified oligonucleotide [10]. Thus, 2⬘-MOE differs from other 2⬘-modified oligonucleotides such as 2⬘-O-methyl by the increased hydration in the minor groove, which may decrease interactions with molecules such as nucleases and other proteins with the phosphate backbone. There is a rich history of modified nucleosides producing pharmacological effects as antivirals, cytotoxics, purinergic receptor agonists and antagonists, and kinase inhibitors. Although 2⬘-MOE nucleosides have not been extensively examined for activity in all these various systems, sufficient work has been completed to minimize anxiety that metabolites of 2⬘-MOE-modified oligonucleotides could produce adverse effects in patients. First, all the 2⬘-MOE nucleosides and nucleotides have been evaluated in a variety of antiviral assays without effects (unpublished data). Second, 2⬘-MOE nucleotide triphosphates do not appear to be substrates for viral or mammalian RNA polymerases. Third, treatment of mice with doses as high as 300 mg/kg daily for 16 days failed to produce any detectable effects on body weights, clinical signs, food consumption, hematology, clinical chemistry, and macroscopic evaluation of major organ systems at necropsy.

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Finally, with the extensive evaluations of 2⬘-MOE-modified oligonucleotides in rodents, primates, dogs, and man, there is no evidence that the oligonucleotides or metabolites produce effects attributable to interaction with purinergic receptors or protein kinases. The 2⬘-MOE nucleosides and nucleotides appear to be well tolerated and it is unlikely that 2⬘-MOE oligonucleotide metabolites will be a source of toxicity. Therefore, in addition to the positive attributes the 2⬘-MOE nucleoside confers to antisense oligonucleotides, the nucleoside or nucleotide metabolites have not proven to be a source of anxiety for potential toxicities. This is not true for other high-affinity nucleosides [12,13]. 10.3 COMPARISON OF 2⬘⬘-MOE-MODIFIED OLIGONUCLEOTIDES TO FIRST-GENERATION PS ODNS PS ODNs helped provide a foundation for the development of antisense oligonucleotide–based therapeutics. At least 20 PS ODNs have entered clinical trials with one drug, fomivirsen, receiving regulatory approval in the United States and Europe. In total, over 5000 patients have been treated with PS ODNs and numerous preclinical toxicology studies completed in multiple species. Because of these activities, there is a wealth of information available for PS ODNs as a chemical class. As such, limitations of PS ODNs have been identified and opportunities for further enhancement of the platform have been defined, such as increasing potency, decreasing toxicities, enhancing tissue residence time (half-life), altering tissue distribution, increasing oral bioavailability, and lowering cost of goods. Many of these goals have been met by 2⬘-MOE-modified oligonucleotides (Table 10.2). In general, 2⬘-MOE gapmers are 10- to 50-fold more potent than PS ODNs in cell culture experiments. Both classes of oligonucleotides exhibit cytotoxic effects when transfected into cells with cationic lipids at similar concentrations [14–16]. However, the concentrations at which PS ODNs produce cytotoxic effects in cells overlap with concentrations required Table 10.2 Comparison of First-Generation PS ODNs to Second-Generation 2⬘⬘-MOE Gapmers

a

Property

PS ODNs

IC50 cell culturea LD50 cell cultureb Tissue half-lifec ED50 mouse liver d Human weekly dose cancer e Human weekly dose liver target f

50–300 nM ⬃400 nM 24–72 h ⬃250 mg 1470–2450 mg ⬃840 mg

2⬘-MOE Gapmers 5–50 nM

⬃500 nM

10–30 days 12.5–25 mg/kg 320–480 mg ⬃100 mg

The IC50 in cell culture is based upon publications using optimized first- and secondgeneration antisense oligonucleotides (see the following for examples: [52,92–196]). b The LD50 represents the concentration of oligonucleotide that produces a 50% loss of treated cells in the presence of a cationic lipid for delivery. c The tissue half-life is a range reported for various PS ODNs and 2⬘-MOE gapmers [23,24,130,150,197,198]. d The ED50 for reduction of target mRNA in mouse liver is represented as the total cumulative dose required to produce a 50% reduction in target RNA. For PS ODNs, the PKC-
4189 oligonucleotide was used as a representative PS ODN [196]. The Fas antisense oligonucleotide 22023 is used as an example of a second-generation 2⬘-MOE gapmer [107]. e The doses used for PS ODN are based upon the weekly doses of oblimersen used for the CLL and melanoma (http://www.fda.gov/ohrms/dockets/ac/06/briefing/2006-4235B1index.htm) [199]. The dose for 2⬘-MOE gapmers is based on the dose of OGX-011 producing a maximal reduction of clusterin in tumor samples [22]. f The human weekly dose for a liver expressed target is based on the maximal doses of Isis 14803 used to treat hepatitis C virus [186]. The dose for the 2⬘-MOE gapmer is based on the dose of ISIS 301012 required to produce a statistical significant reduction of ApoB in plasma [21].

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to inhibit target gene expression, confounding interpretation of pharmacology studies performed in cell culture. In contrast, 2⬘-MOE gapmers generally exhibit at least a tenfold difference in concentrations required to produce maximal inhibition of target gene expression and concentrations at which toxicities are detected. The 2⬘-MOE modification confers significant nuclease resistance to the oligonucleotide, allowing the use of phosphodiester modifications in the internucleosidic linkages connecting the 2⬘-MOE nucleosides. Such oligonucleotide, referred to as “mixed backbone” further decrease toxicity in cell culture experiments. The toxicities of PS ODNs in multiple species have been well characterized [17,18]. In rodents, one of the major toxicities observed at higher doses of PS ODNs is immune stimulation, manifest as splenomegaly and mononuclear cell infiltrate in a number of peripheral tissues. Oligonucleotides incorporating the 2⬘-MOE modification exhibit at least a fivefold reduction in immune stimulation in mice, compared to PS ODNs [7]. 2⬘-MOE-modified oligonucleotides also exhibit decreased anticoagulant effects and potential for complement activation in nonhuman primates [19]. Thus, 2⬘-MOE-modified oligonucleotides exhibit an improved therapeutic index both in vitro and in vivo, including in man [20–22]. The tissue half-life of 2⬘-MOE gapmers is significantly longer than PS ODNs (Table 10.2) [23,24]. Typically, in mouse studies PS ODNs are dosed daily, whereas 2⬘-MOE gapmers are routinely dosed weekly. Although the oral bioavailability of 2⬘-MOE-modified oligonucleotides is relatively low, it is improved compared to PS ODNs [25,26]. Similar to results in cell culture, 2⬘-MOE gapmers are more potent than PS ODNs in animals and man (Table 10.2). In mice, 10- to 20-fold less 2⬘-MOE gapmer is required to produce a similar reduction in target RNA as a PS ODN. The potency enhancement for man is more difficult to determine as there has been no direct comparison of a PS ODN to a 2⬘-MOE gapmer in the clinic. In addition, there are very few studies with PS ODNs in which reduction in targeted RNA or protein has been reported. The best we can do is look at oligonucleotides targeting similar tissues, but different molecular targets. Given these caveats, one could conclude that 2⬘-MOE gapmers are 5- to 10-fold more potent in man than PS ODNs (Table 10.2). If one considers the increase in potency and longer tissue half-life, a 20- to 50-fold decrease in the total amount of 2⬘-MOE gapmer administered for a course of therapy compared to a PS ODN would be a reasonable assumption. At the time of this writing, the 2⬘-MOE nucleosides building blocks have similar cost as deoxynucleosides; therefore, on a weight basis, the cost to manufacture a 2⬘-MOE gapmer is similar to PS ODNs. At commercial scale, 2⬘-MOE nucleosides will be cheaper to manufacture than deoxynucleosides. Given that the cost of drug product will be less than PS ODNs, and less drug will be used for a course of therapy, the cost of therapy for 2⬘-MOE gapmers should be commercially quite attractive. In fact, the cost of goods is projected to be less than most protein-based therapeutics.

10.4 MULTIPLE ANTISENSE MECHANISMS CAN UTILIZE 2⬘⬘-MOE MODIFICATIONS As discussed in the previous chapters, there are multiple antisense mechanisms through which oligonucleotides can be utilized to regulate gene expression. Oligonucleotides incorporating 2⬘-MOE-modified nucleotides, if properly designed (Figure 10.1), can support most, if not all, antisense mechanisms. The design of the oligonucleotide needs to be tailored to the mechanism. The most broadly exploited application of the 2⬘-MOE nucleosides is in oligonucleotides that work through the RNase H–based mechanism. RNase H hydrolyzes RNA in an RNA/DNA duplex, which typically forms an H-form helix. 2⬘-alkoxy- and alkoxyalkoxy-modified nucleosides, including 2⬘-MOE, exhibit a northern C3⬘-endo sugar conformation resulting in an A-form helical geometry when bound to RNA [9,10,27]. Therefore, to exploit the RNase H mechanism of action, chimeric oligonucleotides consisting of 2⬘-MOE and DNA nucleosides are used (Figure 10.1b). Previous studies have shown that a minimum of five contiguous oligodeoxynucleotides are required to

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support RNase H–mediated cleavage of RNA in mammalian cells with optimal length being 10 or more contiguous oligodeoxynucleotides [28,29]. Optimal length and placement of the oligodeoxynucleotides segment within the oligonucleotide can vary from sequence to sequence and should be investigated with each oligonucleotide sequence [29]. The oligodeoxynucleotide segment is typically placed in the center of the oligonucleotide, with the 2⬘-MOE nucleosides providing nuclease resistance to the 3⬘ and 5⬘ ends (2⬘-MOE gapmers in Figure 10.1b). However, for some sequences, placing the oligodeoxynucleotide segment on the 5⬘ or 3⬘ ends (hemimer in Figure 10.1b) may be optimal for activity. A second antisense mechanism resulting in degradation of target RNA is RNA interference (RNAi). As discussed in previous chapters, RNA interference is a natural antisense pathway that is being broadly exploited to inhibit the expression of genes in experimental systems. The enzyme that cleaves the target RNA, Argonaute-2 (Ago-2, also called eIF2C2), is structurally similar to RNase H and utilizes a similar enzymatic mechanism [30,31]. One of the first publications of data utilizing 2⬘-MOE-modified nucleosides for the siRNA mechanism was a report by Dorn et al., in which they incorporated two 2⬘-MOE phosphorothioate-modified nucleosides on the 3⬘ ends of the sense and antisense strands as an overhang to help protect the oligonucleotide from 3⬘ exonuclease activity [32,33]. These modified siRNA duplexes were found to exhibit equal efficacy in cell culture experiments as unmodified siRNA and also to inhibit expression of the target protein when infused locally into CSF fluid [32,33]. Prakash et al. extended this work by incorporating additional 2⬘-modified nucleosides into various positions of the sense and antisense strand [34]. The 2⬘-MOE nucleoside decreased activity when incorporated into the antisense strand. Incorporation of three consecutive 2⬘-MOE modifications on the 3⬘ end or 5⬘ end resulted in almost complete loss of activity and incorporation in the center of the molecule resulted in marked loss of activity. In contrast to the antisense strand, the sense strand was more tolerant of 2⬘-MOE modifications, with modifications throughout the sense strand having minimal or no effect on potency of the siRNA molecule [34]. The finding that the 2⬘-MOE modification significantly reduces potency of the siRNA molecule when incorporated into the antisense strand but not sense strand can be exploited to ensure that the appropriate strand is loaded into the RNA-induced silencing complex (RISC), decreasing potential for off-targets effects of the sense strand binding to and promoting degradation of an RNA as has been previously described [35–38]. Design and optimization of siRNA oligonucleotides for in vitro and in vivo use is still early; however, based upon these preliminary results the 2⬘-MOE modification has utility. The 2⬘-MOE modification has also been shown to support antisense mechanisms that do not result in degradation of target RNA. Such mechanisms can take better advantage of the increased affinity provided for by the modification, in that uniformly modified or “gap-disabled” oligonucleotides (Figure 10.1b) can be used. As an example, Baker et al. demonstrated that a uniform 2⬘-MOE-modified oligonucleotide targeting the extreme 5⬘ terminus of the ICAM-1 (CD54) mRNA was very effective at reducing ICAM-1 protein expression [39]. In fact, uniform 2⬘-MOE-modified oligonucleotides were found to be as potent and effective in inhibiting ICAM-1 expression as optimized 2⬘-MOE gapmers that support RNase H activity. These results demonstrate that catalytic activity is not required for potent antisense effects. Given the low abundance of most mRNAs and the vast excess of oligonucleotides typically delivered to cells, turnover of the oligonucleotide is not required for efficient antisense effects [40,41]. A broadly utilized non-RNase antisense mechanism is modulation of RNA maturation. Most messenger RNAs have different splice variants or alternate polyadenylation signals that can be regulated by oligonucleotide binding to pre-mRNA. Uniform modified 2⬘-MOE oligonucleotides have been successful used to redirect splicing in cultured cells and animals [42–50]. In addition, uniform 2⬘-MOE oligonucleotides have been shown to modulate polyadenylation site selection [51]. Uniform 2⬘-MOE antisense oligonucleotides were designed to regions on the E-selectin mRNA near the polyadenylation signal and found to alter polyadenylation site selection [51]. Oligonucleotides targeting the third polyadenylation site increased expression of transcripts utilizing the first and second site, increasing expression of E-selectin protein [51]. These results

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demonstrate that 2⬘-MOE-modified oligonucleotides can be used to modify maturation of pre-mRNA transcripts resulting in increased expression of specific RNA and protein variants. Although inhibition of protein coding RNAs has by far been the major focus for experimental and therapeutic uses of antisense oligonucleotides, it is becoming increasingly clear that noncoding RNAs play an important role in normal and diseased cells. Antisense oligonucleotides are one of the few approaches for modulating function of noncoding RNAs. RNase H–based oligonucleotides are capable of binding to and promoting cleavage of pre-mRNAs [52,53], thus 2⬘-MOE gapmers are ideal candidates for interfering with nuclear retained RNAs, as was recently demonstrated by Prasanth et al. [54] and Lanz et al [55]. The telomerase RNA is one of the most broadly studied noncoding RNAs as a target for antisense-based therapeutics [56–63]. A variety of different antisense molecules have been designed and tested for inhibition of telomerase RNA, with one drug currently in clinical trials [56]. Corey and colleagues have utilized several different oligomer modifications, including uniform 2⬘-MOE oligonucleotides targeting telomerase RNA to inhibit telomerase activity in biochemical assays, cultured cells, and animals [59,61,62,64]. A 13-nucleotide uniform 2⬘-MOE oligonucleotide complementary to telomerase RNA was found to be particularly effective, inhibiting telomere length and cell growth in vitro and in vivo, and as such represents an interesting candidate for further development [62,64]. MicroRNA is another noncoding RNA that regulates expression of messenger RNA (mRNA) by binding to the mRNA in an antisense orientation [65–68]. The roles of microRNAs in normal physiological and pathophysiological states are still poorly understood; however, there is sufficient data available to suggest that modulation of microRNA function could be of therapeutic value [69–78]. Activity of microRNAs can either be antagonized using antisense oligonucleotides or increased using siRNA-like oligonucleotides as agonists. Uniform 2⬘-MOE oligonucleotides have been effectively used to inhibit function of microRNAs in cell culture and animal tissues [75,76,79]. Davis et al. recently compared several different oligonucleotide modifications for efficiency in antagonizing a microRNA and found that uniformly 2⬘-MOE-modified oligonucleotides were the most effective, followed by 2⬘-fluoro and LNA [79]. Thus, 2⬘-MOE-modified oligonucleotides can be used to help identify functions of microRNAs in cells and animals and potentially as microRNA targeting drugs. In summary 2⬘-MOE-modified oligonucleotides can be broadly used for all antisense mechanisms. The design of the oligonucleotide needs to be optimized for each mechanism. The basic pharmacokinetic, class-specific toxicities and manufacturing of the oligonucleotide will be the same regardless of the mechanism. Thus, one can take advantage of the investments that have been made in characterizing the properties of this class of oligonucleotides optimizing manufacturing and generating a clinical safety database, by exploiting 2⬘-MOE-modified oligonucleotides broadly for multiple antisense mechanisms. 10.5 IN VITRO PHARMACOLOGICAL PROPERTIES OF 2⬘⬘-MOE GAPMERS Antisense oligonucleotides utilizing the 2⬘-MOE modification have been used to inhibit the expression of over 4000 mammalian genes in cell culture–based assays. The vast majority of oligonucleotides were designed to work through the RNase H–based mechanism, i.e., 2⬘-MOE gapmers. We have successfully identified 2⬘-MOE gapmers to thousands of different types of genes including transcription factors, receptors, adapter proteins, protein kinases, phosphatases, other enzymes, apoptosis-related proteins, structural proteins, and proteins of unknown or poorly characterized function [80–103]. In fact, all nuclear-encoded genes appear to be amenable to RNase H–based oligonucleotides. As expected, based upon the diversity of genes inhibited, the pharmacological effects produced by these oligonucleotides in cell culture is equally diverse. For most cultured mammalian cells, it is necessary to utilize physical or chemical means to facilitate introduction of oligonucleotides into the cytoplasmic compartment of the cell, which contrasts with the in vivo situation [40,104]. There are a few notable exceptions, such as some primary cell cultures. These results suggest that natural cellular uptake pathways that facilitate delivery of modified oligonucleotides to the target RNA in animal tissues are lost as cells adapt to

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growth in cell culture. In fact, studies using primary hepatocytes demonstrate efficient delivery of antisense oligonucleotides for the first 24 hours after cells are isolated, and then a decline in efficiency afterward (C. Thompson and C.F. Bennett, unpublished data). Efficiency of oligonucleotide delivery depends on the delivery methodology, cell line, growth state of the cell, etc.; therefore, making comparisons of potencies of oligonucleotides between different laboratories and investigators is without taking into account the specific experimental details is not appropriate. For a fair comparison, the oligonucleotides should be tested in the same experiment. Cationic lipids are most broadly used for delivery of 2⬘-MOE-modified oligonucleotides. We have not found a particular cationic lipid formulation to be vastly superior to other formulations. We have found that with certain cell lines, one lipid formulation may work better while for another cell line a second formulations works better. For each new cell line tested, it is recommended to test several cationic lipid formulations and oligonucleotide to lipid ratios, to optimize delivery and minimize toxicity. Once optimized, we typically find that 2⬘-MOE gapmers (Figure 10.1) reduce targeted mRNA in a concentration-dependent manner with IC50 values typically between 1 and 20 nM and maximal effects achieved between 50 and 100 nm (as an example, see Figure 10.2). Most adherent cell lines are amenable to cationic lipid-mediated delivery of 2⬘-MOE gapmers, while electroporation is a better delivery option for nonadherent cell lines. Significantly higher concentrations of oligonucleotides are required when electroporation is used as the delivery technology; typically 1 to 10 M [14,105]. At these concentrations, it is still possible to demonstrate specific inhibition of targeted gene expression. 2⬘-MOE gapmers exhibit exquisite sequence specificity. Introduction of a single mismatch in the RNase H cleavage region results in a two- to five-fold loss of activity, and three or more mismatches generally result in complete loss of activity [2,52,106,107]. 2⬘-MOE gapmers have proven very useful to help functionalize genes in cell culture–based experiments. As an example, 2⬘-MOE gapmers have been used extensively to characterize functions of a variety of phosphatases and kinases [80,83,84,90,96,98,108–127]. Antisense oligonucleotide based approaches are increasingly being used to create libraries of validated antisense inhibitors that have been used in high-throughput cell culture–based screens to identify novel functions of targeted genes. As an example, Koller et al. used a library of validated 2⬘-MOE gapmers targeting 1500 different human genes to identify those genes regulating cell cycle progression in T47D and MCF7 breast cancer cells [80]. The 2⬘-MOE gapmers were prescreened as described in Chapter 5 to identify potent and selective inhibitors of the gene of interest before incorporating into the library. 2⬘-MOE gapmers targeting approximately 3.5% of the genes tested either increased accumulation of cells in a particular phase of the cell cycle or promoted apoptosis (sub G1). In that this was a biased library focused on genes of interest as potential drug candidates, the percent of positives identified is very reasonable. Antisense oligonucleotides targeting several well-known cell-cycle genes were positive in the assay, as well as some less well-characterized genes. Of particular note were 2⬘MOE gapmers targeting Eg5 (a kinesin-5 family member), which produced a marked increase in cells with 4N DNA content. Cells undergoing mitosis, pretreated with the Eg52⬘-MOE gapmer, developed monopolar spindles and rosette-like microtubule arrays with chromosomes at the periphery, implicating Eg5 in centrosome separation and bipolar spindle formation, confirming results obtained with small-molecule inhibitors [128]. On the basis of cell culture experiments, the oligonucleotides were tested in a human tumor xenograft model and demonstrated inhibition of tumor growth in vivo. One advantage of 2⬘-MOE gapmers is that results obtained in cell culture models can be easily confirmed in more complex animal models. 2⬘-MOE gapmers, similar to any other pharmacological agent, will produce undesired “off-target” effects (or toxicities) at higher concentrations. These undesired effects may be hybridization-dependent or hybridization-independent. Off-target hybridization-dependent effects are due to binding of the oligonucleotide to identical or closely related sequences in another target RNA and apply to all antisense-based approaches. These effects can be controlled with appropriate selection of the antisense sequence, using two different 2⬘-MOE gapmers targeting the same gene to confirm pharmacological activity, appropriately designed mismatch control oligonucleotides, and using bioinformatics to identify potential cross-reactive target RNAs, followed by experiments to determine if the “cross-reactive” gene product is modulated by the antisense oligonucleotide. Oligonucleotides

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(a)

125

Inhibition of G6Pt in mouse hepatocellular carcinoma cells

(b)

Oligo 2 Oligo 1 Control oligo

150

Oligo 2 Oligo 1 Control oligo

125 100

100

% Control expression

% Control expression

281

75 50

75 50 25

25 0

0 1

10

100

1000

0.1

1

10 [nM]

[nM] Inhibition of SRB-1 in mouse liver

(c)

1000

Inhibition of G6Pt in mouse liver

(d) 140

140 120

120

100

100

% Control expression

% Control expression

100

80 60

80 60

40

40

20

20 0

0 0 Saline Control

8

20

50

8

20

Dose (mg/kg) Oligo 2 Oligo 1

50

0

8

20

50

0

8

20

50

Dose (mg/kg) Oligo 2 Oligo 1

Figure 10.2 2⬘-MOE Gapmers inhibit target gene expression in cell culture and rodent liver. (a) Primary hepatocytes were treated with increasing concentrations of two different 2⬘-MOE gapmers (Oligo 1 and Oligo 2) designed to bind to scavenger receptor B1 (SRB1) mRNA in the presence of a cationic lipid. SRB1 RNA in the cells determined by a quantitative RT-PCR assay. (c) Mice were treated with the same oligonucleotides at increasing doses administered intraperitoneally twice a week for 3 weeks. (b) Mouse hepatocellular carcinoma cells were treated with increasing concentrations of 2⬘-MOE gapmer oligonucleotides designed to bind to glucose 6-phospahtase translocase (G6Pt) or a control 2⬘-MOE gapmer in the presence of cationic lipid. (d) Mice were dosed intraperitoneally with increasing doses of the G6Pt oligonucleotide twice per week for 3 weeks. Total RNA was isolated and the amount of G6Pt RNA determined by a quantitative RT-PCR assay.

also produce off-target effects through non-hybridization-dependent interactions. These interactions are well described for PS ODNs and often result in decreased cell proliferation, apoptosis, or induction of inflammatory mediators, depending on cell type and delivery methods. As already discussed, second-generation 2⬘-MOE gapmers tend to produce less of these off-target effects. 2⬘-MOE oligonucleotides have an increased specificity in cell culture–based experiments based upon their increased potency and decreased off-target effects. For cell culture–based experiments, it is possible to utilize 2⬘-MOE-modified oligonucleotides with reduced or no phosphorothioate linkages, further reducing some of the unwanted effects due to the phosphorothioate modification [39,79]. We have found that some 2⬘-MOE gapmers do produce antiproliferative effects and induce cell stress and pro-inflammatory genes in a sequence-specific manner at high concentrations [14,129]. These effects do not appear to be hybridization-dependent and occur at concentrations three- to tenfold higher (⬃200 to 1000 nM) than are normally used to inhibit the expression of a target gene. In a study published by Drygin et al., 6 out of 43 (14%) 2⬘-MOE gapmer oligonucleotides designed such that

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they are not predicted to hybridize to any known human gene (i.e., controls), produced greater than a 20% decrease in cell number at a concentration of 100 nM, which typically produces 90% reduction in target gene expression [14]. In contrast, at a concentration of 200 to 500 nM typically required to inhibit expression for PS ODNs, the majority of PS ODNs would produce more profound cytotoxic effects. Because cell proliferation and apoptosis assays are routinely used to screen for pharmacological activity, it is important to keep in mind the potential for these non-antisense-mediated effects when interpreting data from cell culture screens. The same types of controls described above are very useful in ruling out a non-antisense effect as being responsible for pharmacological activity. In particular, a second oligonucleotide targeting the same target is a valuable control, confirming that a specific gene product is responsible for producing the observed phenotype. One could also use an oligonucleotide that works by a different antisense mechanism such as siRNA for confirmation of an observation. In that siRNA oligonucleotides also produce a number of off-targets effects through different mechanisms [35,37,38], the use of both an siRNA and 2⬘-MOE gapmers to confirm phenotypic effects in cell culture is an attractive additional set of controls to consider including in critical experiments. In summary, 2⬘-MOE gapmers have proven to be very useful to help ascribe function to genes in cell culture–based experiments. If properly optimized as described in Chapter 5, they can be as potent as siRNA oligonucleotides [52]. It is important to use the appropriate controls to facilitate interpretation of phenotypic results, as they, like all oligonucleotides, are capable of producing nonantisense effects at high concentrations. 10.6 IN VIVO PHARMACOLOGICAL PROPERTIES OF 2⬘⬘-MOE GAPMERS 2⬘-MOE gapmers have been found to be very effective inhibitors of gene expression in multiple mammalian species, including mice, rats, hamsters, rabbits, dogs, and nonhuman primates. We have shown that optimized oligonucleotides reach target RNAs in cells within tissues, including liver, kidney, adipose tissue, and spleen, producing specific reductions of the targeted RNA (Figure 10.3). The mechanism(s) responsible for oligonucleotide distribution across the plasma membrane to sites where the target RNA is present is not well understood. A detailed discussion of oligonucleotide pharmacokinetics can be found in Chapters 7 and 11. However, to understand the general pharmacological properties of 2⬘-MOE gapmers it is important to have some knowledge of the tissue and SCD-1

FBP-1

FKHR

Liver ASO

Saline

Saline

ASO

p85α (PI-3K)

IKK-β

ASO

Saline

PTP-1B

Adipose tissue Saline

ASO

Saline

ASO

IKK-β

ASO

SGLT-2

Spleen

Kidney Saline

Figure 10.3

Saline

ASO

Saline

ASO

Pharmacodynamic effects of 2⬘-MOE gapmers in different mouse tissues. Mice were treated with optimized 2⬘-MOE gapmers directed to stearoyl-coenzyme A desaturase-1 (SCD-1), fructose1,6-bisphosphatase 1 (FBP-1), forkhead box O1A (FKHR), inhibitor kappa B kinase  (IKK-), phospatidylinositol 3-kinase, p85
(PI-3K), protein tyrosine phosphatase-1B (PTP-1B), and sodium-dependent glucose cotransporter 2 (SGLT2) for 3 weeks. Animals were sacrificed and total RNA isolated from the indicated tissues. Target mRNA levels were determined based upon northern blotting or protein determined by western blotting (IKK- in spleen only).

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cellular distribution. We have shown that phosphorothioate oligonucleotides and 2⬘-MOE oligonucleotides with phosphorothioate linkages broadly distribute to most peripheral tissues, with the highest concentration occurring in kidney, liver, and lymphoid tissues [23,130,131]. Within these tissues, the oligonucleotide distribution is heterogenous with regard to cell types [132,133]. As an example, Figure 10.4 demonstrates cellular localization of a 2⬘-MOE gapmer, ISIS 13920, in several representative tissues. Some cells such as macrophage-derived cells accumulate high concentrations of oligonucleotides, while epithelial-derived cells accumulate intermediate concentrations (proximal convoluted epithelial cells of kidney being a notable exception, as is apparent in Figure 10.4). Muscle cells (skeletal, smooth and cardiac) and lymphocytes tend to accumulate low levels of oligonucleotides [133]. As expected on the basis of the distribution of 2⬘-MOE gapmers, they do not homogenously inhibit expression of targeted genes in all tissues of the body, nor all cell types within tissues. Pharmacodynamic effects tend to correlate with cell and tissue concentrations [132]. A panel of northern blots prepared from mouse tissues treated with various optimized 2⬘-MOE gapmers as

Figure 10.4

Liver

Kidney

Adipose

Bone

Spleen

Human tumor xenograft

(See color insert following page 270.) Immunolocalization of 2⬘-MOE gapmer in different mouse tissues following systemic treatment. Mice were dosed with ISIS 13920, a 2⬘-MOE gapmer recognized by the 2E1 monoclonal antibody. Tissues were collected and stained with the 2E1 antibody for presence of oligonucleotide as described by Butler et al. [133]. Antibody bound to the oligonucleotide appears as the brown stain in the histological sections. The blue stained structures are the result of staining with hematoxylin, used as a counterstain. G—glomerulus, PT—proximal tubules, O—osteoclast, E—endosteum, T—tumor xenograft, S—stromal cells.

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shown in Figure 10.3. Treatment of mice with optimized 2⬘-MOE gapmers reduced gene expression of the three target genes in liver by 85–90% as determined by northern blot analysis (Figure 10.3). Several hundred genes have been inhibited in mouse liver with 2⬘-MOE gapmers. 2⬘-MOE gapmer antisense oligonucleotides have also been shown to produce specific inhibition of gene expression in adipose tissue, spleen, and kidney after systemic administration (Figure 10.3), demonstrating that effects are not restricted to one primary target tissue. For systemic applications, 2⬘-MOE gapmers can be formulated in saline or buffered saline and administered by intravenous, subcutaneous, and intraperitoneal routes. Bioavailability of subcutaneousand intraperitoneal-injected 2⬘-MOE gapmers is greater than 85%. Pharmacological affects can be produced in the lung, colon, eye, and central nervous system (CNS) by local delivery routes such as pulmonary, colonic, intravitreal, intrathecal, and intraventricular. The distribution in these tissues following local administration is shown in Figure 10.5 and is discussed in more detail in the following text.

Granular cells Purkinje cell

Motor neuron Molecular layer

Lumbar cord, ICV

Cerebellum, IT

R/C

Lung, inhalation

IN

Eye, intravitreal

Figure 10.5 (See color insert following page 270.) Immunolocalization of 2⬘-MOE gapmer in different tissues following local administration. Animals were dosed with the 2⬘-MOE gapmer ISIS 13920 and oligonucleotide localized using immunocytochemistry as previously described [133]. Lumbar cord, ICV: 13920 was infused into the lateral ventricle for 14 days and ISIS 13920 localized (brown stain) in the lumbar cord. Cerebellum, IT: Rhesus monkeys were infused with ISIS 13920 into the intrathecal space for 14 days and ISIS 13920 localized in the cerebellum. Lung, inhalation: Mice were treated with ISIS 13920 by aerosol administration and localization of ISIS 13920 determined in lung tissue. Eye, intravitreal: ISIS 13920 was injected intravitreally into a mouse eye and localized in different cell population by immunohistochemical staining (brown stain). R/C—cell bodies of the rods and cones in the retina, IN— inner nuclear layer of the retina.

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2⬘-MOE-modified oligonucleotide that have reduced phosphorothioate linkages tend to accumulate in the kidney and are more rapidly excreted in the urine [23,130]. The phosphorothioate linkage increases serum protein binding [23,134], and is responsible for prevention of filtration of the oligonucleotide through the glomerulus into the urine where they are either reabsorbed in proximal tubules or excreted in the urine. For local applications, it may be possible to reduce the number of phosphorothioate linkages due to the enhanced nuclease resistance provided by the 2⬘-MOE modification. However, extensive analyses of phosphorothioate requirements for local applications have not been performed. The role of the phosphorothioate modification in distribution of the oligonucleotide inside cells and binding to target RNA is currently unknown. The best characterized class of 2⬘-MOE-modified oligonucleotides for in vivo applications are 5-10-5 2⬘-MOE gapmers (Figure 10.1b). Uniformly modified 2⬘-MOE oligonucleotides have also been shown to be active in vivo [46,48,75]. The increased potency and specificity provided by the 2⬘-MOE modification have dramatically enhanced the utility of 2⬘-MOE-modified oligonucleotides for in vivo studies. Even though non-antisense effects observed with 2⬘-MOE-modified oligonucleotides are dramatically reduced, it is important to utilize appropriate controls to help interpret the pharmacology of the oligonucleotide. Similar controls as described for cell culture experiments can be used, i.e., two different oligonucleotides targeting the same gene, sequence mismatched controls, etc. With appropriate controls, 2⬘-MOE-modified oligonucleotides, in particular, 2⬘-MOE gapmers have proven to be very valuable for in vivo applications. 10.6.1 Liver The most widely studied target organ for antisense effects is liver. Hundreds of different target genes have been inhibited in mouse and rat liver with 2⬘-MOE gapmers (e.g., [97,98,100,101,107,135–148]). There is a good correlation between activity of oligonucleotides tested in cell culture assays and in vivo. As an example, Figure 10.2 shows a dose-response analysis of oligonucleotides against two molecular targets in cultured liver cells and liver tissue after systemic administration. Two oligonucleotides targeting mouse scavenger receptor B1 (SRB-1, CD36) were tested in primary hepatocytes, with one oligonucleotide (Oligo 1) demonstrating potent inhibition of SRB-1 expression while the second oligonucleotide failed to inhibit SRB-1 expression. Similar results were obtained when the two different 2⬘-MOE gapmers were tested in mice (Figure 10.2). A second example is provided by 2⬘-MOE gapmers targeting glucose 6-phosphatase translocase (G6Pt). In this case, both oligonucleotides inhibited G6Pt expression. However, there was a marked difference in potency, with Oligo 1 being approximately tenfold more potent than Oligo 2. Similar rankings were observed when livers from mice treated with both 2⬘-MOE gapmers were analyzed for G6Pt expression (Figure 10.2). In general, optimized 2⬘-MOE gapmers inhibit expression of the target mRNA in liver after a single dose between 50 and 100 mg/kg, with maximal effects occurring 24– 48 h after the dose [107,132]. As discussed, elsewhere in this volume, most 2⬘-MOE gapmers have a tissue half-life between 10 and 30 days. With repeat administration, it may take 50 to 150 days to reach steady state tissue concentrations (five half-lives), which is often longer than most experimental protocols in rodents. To increase the onset for maximal effects, a loading dose regimen is often used. A typical dosing schedule is 10–25 mg/kg dosed two or three times a week for the first week and then weekly administration thereafter. This dosing regimen works for multiple species including mice, rats, and primates (Table 10.3). In fact, the potency of 2⬘-MOE gapmers is similar in these species, with rats tending to be a little less sensitive than mice and primates (Table 10.3). The potency for optimized 2⬘-MOE gapmers targeting different genes in liver may vary three- to fivefold. The reason for the differences in potencies are not understood; however, the most likely explanation is that the more potent oligonucleotides may have been “better optimized” in cell culture experiments before advancing into animal experiments. As discussed in Chapter 5, the cell type in which the target gene is expressed can also affect apparent potency of the oligonucleotide based upon differential distribution to the

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Table 10.3 Comparison of Doses in Mouse, Rat, and Monkey Target

Mouse Liver (mg/kg/week)

Rat Liver (mg/kg/week)

Rhesus Monkey Liver (mg/kg/week)

PTP-1B Glucagon Receptor

10–20 5–10

25–50 10–20

10–20 5–10

Note: Comparison of dose required to produce a 50% reduction in target mRNA in liver following treatment with different antisense oligonucleotides. Animals were generally dosed twice per week by subcutaneous or intraperitoneal injection for 3–6 weeks. Reported dose is total weekly dose.

different cell population. The possibility that some molecular targets may be inherently more sensitive to 2⬘-MOE gapmers than others also cannot be ruled out. To date, the main uses of the 2⬘-MOE gapmers targeting liver genes have been to investigate roles of various genes in metabolic control, lipid and lipoprotein biosynthesis, and prevention of hepatitis. The first two topics will be dealt with in more detail in other chapters in this volume. One of the first studies demonstrating that 2⬘-MOE oligonucleotides can be used to protect the liver from injury was a study published by Zhang et al. [107]. In this study, mice were pretreated with an antisense oligonucleotide targeting Fas (CD95) and then challenged with various agents known to produce hepatic damage in mice. Reduction of Fas expression by 90% in liver with the Fas antisense oligonucleotide completely blocked liver damage due to Fas antibody (Jo-2) and reduced liver damage due to acetaminophen. The Fas antisense oligonucleotide failed to protect against liver damage induced by the T-cell mitogen, concanavalin A [107]. These results have been extended to include over 20 additional anti-apoptotic and pre-apoptotic genes and additional liver toxins such as endotoxin and bile acids [136,137]. Of particular note is a study in which antisense oligonucleotides directed towards two different targets were co-administered in mice. Administration of 2⬘-MOE gapmers targeting BCl-2 family members Bcl-xL and Bid, individually, inhibited expression of the respective genes by at least 80% [137]. Treatment with the Bid 2⬘-MOE gapmer completely attenuated Fas antibody–induced apoptosis, while the Bcl-xL 2⬘-MOE gapmer sensitized the liver to Fas antibody–mediated apoptosis. Co-administration of both oligonucleotides resulted in a similar level of mRNA suppression as either oligonucleotide dosed individually. The Bid 2⬘-MOE gapmer protected against Fas antibody–induced liver damage, even when the levels of BCL-xL protein were reduced by the BCL-xL antisense. These results demonstrate that administration of two different 2⬘-MOE gapmers is well tolerated. Furthermore, the oligonucleotides do not antagonize each other, suggesting that whatever mechanisms are responsible for accumulation of oligonucleotides in cells and activation of RNase H and cleavage of RNA are not saturated at these dose levels. These results have been extended to as many as four different 2⬘-MOE gapmers [149]. In summary, numerous published and unpublished studies demonstrate that 2⬘-MOE gapmers are valuable tools to help determine the function of genes in rodent liver and are complementary to other approaches such as genetic knockouts. 10.6.2 Kidney The highest concentration of 2⬘-MOE-modified oligonucleotides following systemic administration is found in the kidney cortex [23,130,150]. Concentrations in kidney cortex are typically two- to fivefold greater than liver concentrations. Examination of the cellular localization of 2⬘-MOE gapmer in kidney reveals that the highest oligonucleotide concentrations are found in proximal tubule epithelial cells (Figure 10.4). Less oligonucleotide is detectable in glomerulus and distal tubular epithelial cells (Figure 10.4). Given the heterogeneity of oligonucleotide concentration in different cell populations in kidney, it is not surprising that demonstrating pharmacological effects in kidney has been variable depending on the molecular target and where it is expressed. Targets enriched in proximal tubules are more sensitive to 2⬘-MOE gapmers than targets expressed elsewhere in the kidney [151]. Lower doses of 2⬘-MOE gapmers may be required to inhibit the

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expression of a gene expressed in proximal tubular cells compared to liver. The kidney is the one tissue where it has been possible to demonstrate potent antisense effects with systemically administered mixed backbone 2⬘-MOE gapmers (i.e., no phosphorothioate modifications in the internucleosidic 2⬘-MOE linkages). Replacement of phosphorothioate linkages with phosphodiester linkages decreases plasma protein binding and increases oligonucleotide filtration through the glomerulus [23]. Effects of 2⬘-MOE gapmers in other cell types of the kidney have not been well characterized and warrant additional investigation. 10.6.3 Adipose Tissue 2⬘-MOE gapmers and uniformly modified 2⬘-MOE oligonucleotides typically have 17 to 19 negative charges on the phosphorothioate linkages, and as such are hydrophilic molecules. It is counterintuitive that a hydrophilic molecule produce pharmacological effects in adipose tissue; however, 2⬘-MOE gapmers, gap-disabled, and uniform 2⬘-MOE oligonucleotides have all been shown to modulate gene expression in adipose tissue [97,98,100,146,147,152]. Examination of the distribution of a 2⬘-MOE gapmer in adipose tissue revealed that the drug readily accumulates in adipocytes, with the drug localizing to the cell body rather than in the fat droplets (Figure 10.4). 2⬘-MOE gapmers also accumulate in macrophages often found in adipose tissue. Because lipid droplets contribute the bulk of the mass in adipose tissues, the total tissue concentration of 2⬘-MOE gapmers in adipose tissue is modest. In fact, it is more accurate to model dosing based upon lean body mass rather than total body mass [153]. The doses of 2⬘-MOE gapmers required to inhibit the expression of target gene in adipose tissue are similar to liver. With the increased awareness of the importance of adipose tissue in contributing to a variety of disease states including obesity, inflammatory diseases, malignancy, and cardiovascular disease [154–156], 2⬘-MOE-modified antisense oligonucleotides are ideally suited to help determine gene function in adipose tissue and as potential therapeutics for targets expressed in adipose tissue. 10.6.4 Bone Tissue It has been known for some time that PS ODNs distribute to bone marrow [131,133,157], and so do 2⬘-MOE gapmers (Figure 10.4). Unfortunately, little information is currently available on the pharmacological effects of PS ODNs or 2⬘-MOE gapmers in this tissue. The 2⬘-MOE gapmer is found in cells forming the endosteum lining the bone marrow cavity, multinucleated osteoclasts, and additional cell present in the marrow (Figure 10.4). The nature of these other cells has not been well characterized. The finding that 2⬘-MOE gapmers accumulate in osteoclasts was further confirmed by using 2⬘-MOE gapmers directed toward tumor necrosis factor receptor superfamily member 11A (RANK, OPG), which is highly expressed by osteoclasts. Two different 2⬘-MOE gapmers targeting mouse RANK mRNA reduced the RANK expression by 40–50% in a dose-dependent manner in total bone tissue [158]. Further characterization of RANK expression on bone marrow cells by flow cytometry demonstrated an 80% decrease in RANK protein expression on monomyeloid and monocytic subpopulations. RANK antisense oligonucleotides were found to decrease PTH-hypercalcemia and ovariectomy-induced bone loss in mice [158,159]. Additionally, the RANK antisense oligonucleotides exhibit anti-inflammatory activity in a rat model of adjuvantinduced arthritis [160]. These results support that osteoclasts are a sensitive cell type to 2⬘-MOE gapmer–mediated antisense effects. Antisense oligonucleotides were also identified to tumor necrosis factor (ligand) superfamily member 11 (RANKL, OPGL), the binding partner and signaling molecule for RANK, which is predominantly expressed in osteoblasts and bone marrow stromal cells. Treatment of mice with RANKL 2⬘-MOE gapmer (30 mg/kg/day for 18 days) resulted in a 40% reduction in RANKL expression in bone tissue and also decreased PTH-induced hypercalcemia and bone loss, although the effects were not as pronounced as those produced by RANK 2⬘-MOE gapmers [159,160]. These results suggest that 2⬘-MOE gapmers also produce pharmacological

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effects in osteoblasts, although it cannot be ruled out that the effects were due to inhibition of RANKL expression in stromal cells. Additional studies are warranted to characterize the pharmacological effects of 2⬘-MOE gapmers in other cell population present in bone marrow. 10.6.5 Lymphoid Tissues and Inflammatory Cells After kidney and liver, lymphoid tissues such as lymph nodes and spleen accumulate the highest concentration of 2⬘-MOE gapmers [23,130,150,161]. The concentrations of 2⬘-MOE gapmers in these tissues are typically two- to fivefold less than in liver. Histological examination of localization in spleen and normal lymph nodes reveals that highest concentrations are found in macrophages and dendritic cells and lowest in lymphocytes, which correlates with sensitivity to antisense effects (Figure 10.4). Therefore, macrophages and dendritic cells are much better cellular targets for antisense pharmacology than T or B lymphocytes. Typically, mice must be dosed with very high doses of 2⬘-MOE gapmers (80 mg/kg every day for 14 days) to obtain a modest 30–50% reduction of target gene in lymphocytes. Much lower doses of oligonucleotide (10–25 mg/kg ) will produce a more robust inhibition of targeted gene expression in macrophages and dendritic cells. The sensitivity of other immune cells to 2⬘-MOE gapmers has not been well characterized, although preliminary data suggest eosinophils may be sensitive to 2⬘-MOE gapmers at reasonable doses (15–20 mg/kg) [162]. 2⬘-MOE gapmers readily accumulate at sites of active inflammation [161,163]. As an example, Figure 10.6 shows the localization of a 2⬘-MOE gapmer (ISIS 13920) in normal and inflamed mouse

Figure 10.6

(See color insert following page 270.) Immunolocalization of 2⬘-MOE gapmer in normal and inflamed tissue. Inflammation was induced in mouse ears by either treating topically with DNFB [200] or in intestinal tissue by treating with dextran sulfate [163]. Once the inflammation was established, mice were treated with ISIS 13920 and tissue collected approximately 24 h after dosing. The 2⬘-MOE gapmer was localized in normal and inflamed tissue by staining with 2E1 antibody [133]. Tissues were counterstained with hematoxylin (blue stain). Brown staining represents localization of ISIS 13920.

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ear and colon tissues. As described in Chapter 29, 2⬘-MOE gapmers have been evaluated in a variety of inflammatory models. The oligonucleotides produced anti-inflammatory activity comparable to that seen with a monoclonal antibody directed to the same molecular target [161–166]. When evaluating 2⬘-MOE gapmers in models of inflammation, it is important to keep in mind that oligonucleotides can produce sequence-dependent modulation of the immune system through non-antisense mechanisms [167,168]. Although the 2⬘-MOE modification markedly decreases immune stimulation through interactions with Toll-Like Receptor 9 (TLR9), 2⬘-MOE gapmers can still produce sequence dependent activation of immune cells at high doses. With proper controls and appropriate selection of molecular targets, it is possible to discovery 2⬘-MOE gapmers drugs for the treatment of immune related disorders (Table 10.1). 10.6.6 Oncology Models In contrast to results in the liver, demonstrating antisense effects in mouse tumor models has been more variable. The most common tumor models used to study anticancer drugs are human tumor xenografts. It is well recognized that these models probably do not recapitulate many feature of human cancer other than cell growth, yet are broadly used. A major source of variability of antisense oligonucleotides in tumor models is the inherent variability of human tumor xenografts from laboratory to laboratory. Another source of variability is the proliferation rates of the tumor cells as well as the tumor–stromal cell interactions that affect biodistribution of the oligonucleotide [169]. PS ODNs distribute more readily to tumors early after transplantation and poorly to wellestablished or necrotic tumor xenografts. Similar observations have been confirmed for 2⬘-MOE gapmers. An example of the distribution of a 2⬘-MOE gapmer in a human tumor xenograft is provided in Figure 10.4. Highest concentrations of 2⬘-MOE gapmer can be found in stromal cells surrounding the xenograft and inflammatory cells infiltrating the tumor tissue, although some drug is present in xenograft cells as well (Figure 10.4). It should also be kept in mind that every time the cells divide, the cellular concentration of oligonucleotide is diluted, thus the half-life of 2⬘-MOE gapmers in tumor cells is shorter than in nonproliferating cells. We have found that initiating treatment within 3–5 days of implanting the cells produces more reproducible effects than initiating treatment later. In addition, because of the loss of drug in cells due to expansion, we have found that more frequent administration of the 2⬘-MOE gapmers (i.e., daily or every other day) produces more reproducible effects. Using tumor models in which the pharmacokinetic/pharmacodynamic effects of 2⬘-MOE gapmers have been characterized, it has been possible to demonstrate sequence-specific antisense effects [80,170–175]. These results have lead to several 2⬘-MOE gapmers advancing into clinical trials for the treatment of various malignancies. Thus, even though tumor models are more variable than targeting normal tissues, it is possible to demonstrate specific antisense effects in the models. 10.6.7 Local Administration of 2⬘⬘-MOE Gapmers 2⬘-MOE gapmers have been administered locally to the lung, eye, and CNS; with good distribution to multiple cell types in these tissues (Figure 10.6) [176–185]. As would be expected with local therapy, lower doses of oligonucleotides are used. Doses as low as 1 ng/kg were found to be effective in reducing airway hyper-reactivity and lung eosinophilia for an inhaled IL-4 receptor-
2⬘-MOE gapmer [177]. Inhaled 2⬘-MOE oligonucleotides are well tolerated with no adverse effects in mice observed at doses up to 1 mg/kg; a dose 10,000- to 100,000-fold higher than required to produce pharmacology. A 2⬘-MOE-modified oligonucleotide targeting IL-4 receptor-
is currently in development for the treatment of asthma [177]. Oligonucleotides, including 2⬘-MOE-modified oligonucleotides do not cross the blood–brain barrier. Therefore, to gain access to neural tissues, the oligonucleotide must be administered directly into the CNS space. Delivery of 2⬘-MOE gapmers into the cerebral spinal fluid (CSF) results in

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broad distribution throughout the CNS [182,183]. As an example, a 2⬘-MOE gapmer infused into the lateral ventricle of rats distributes to motor neurons present in the lumbar portion of the spinal cord, and a 2⬘-MOE gapmer infused into the intrathecal space of rhesus monkeys distributes to neurons present in the cerebellum (Figure 10.6). Although this is an invasive procedure, it is routinely performed in preclinical models and devices for delivery of drugs into CSF of patients have been approved by various regulatory agencies. For CNS applications in rats, doses are generally between 50 and 200 g/day with drug being delivered by constant infusion using Alzet osmotic pumps. In general, the antisense drugs are well tolerated at these doses; however, adverse effects have been noted at higher dosages, including somnolence and paralysis. To mitigate potential for adverse effects, it is important to use highly purified oligonucleotides that have been desalted to reduce salt load into CSF and also characterized for endotoxin levels. 2⬘-MOE gapmers have proven to be very effective antisense agents when administered locally to target tissues. Local administration increases the therapeutic opportunities for 2⬘-MOE gapmers by delivering effective concentrations of drug to tissues normally not accessible from systemic routes. Additionally, local administration of 2⬘-MOE gapmers reduces systemic exposure, diminishing potential for systemic side effects. Several locally administered 2⬘-MOE gapmers are advancing into clinical trials (Table 10.1). More detailed discussions of the pharmacological effects after local administration is discussed elsewhere in this volume.

10.7 HUMAN PHARMACOLOGY Several 2⬘-MOE gapmers have entered clinical trials (Table 10.1). Although still early in development, data generated to date suggest that 2⬘-MOE gapmers are performing significantly better than first-generation PS ODNs. Although it is difficult to demonstrate unequivocally, as the oligonucleotides were designed to different molecular targets and different therapeutic indications, data suggest the 2⬘-MOE gapmers are more potent in man than PS ODNs (Table 10.2). Perhaps the best example to make this point is a 2⬘-MOE gapmer targeting Apo B (ISIS 301012), a liver-expressed target gene. ISIS 301012 produced dose-dependent reductions in serum Apo B concentrations in normal volunteers [21]. Doses as low as 100 mg dosed three times per week for one week followed by three additional weekly doses produced statistical significant reductions in serum Apo B and a dose of 200 mg produced a 50% decrease in Apo B. Dosing schedules have not been optimized for this drug in patients, but it would be reasonable, considering the drug has a 30-day tissue half-life, to expect that doses lower than 100 mg per week will suffice to maintain a therapeutic effect following a loading dose regimen. A first-generation PS ODN targeting the hepatitis C virus in liver was found to reduce viral titer after treating for 12 weeks at a dose of 840 mg per week [186]. Another second-generation antisense drug thought to work predominantly in the liver is ISIS 113715 [98,187,188]. ISIS 113715 targets protein tyrosine phosphatase 1B (PTP-1B), a protein phosphatase that has been shown to attenuate insulin signaling in tissues. Inhibiting expression of PTP-1B with ISIS 113715 increases insulin sensitivity in rodents and normalizes glucose levels in diabetic animals [98,188,189]. A phase 1 trial of ISIS 113715 in normal volunteers confirmed the insulin-sensitizing effects of ISIS 113715 in man [187]. The primary purpose of the phase 1 study was to establish safety of the drug; however, at the two highest doses of 5.0 mg/kg and 7.5 mg/kg for three doses, a 27–32% reduction in insulin AUC following an intravenous glucose tolerance test (IVGTT) was observed [187]. The drug is currently being evaluated in type 2 diabetic patients, with lower doses showing evidence of efficacy. Several 2⬘-MOE gapmer drugs targeting tissues other than liver are being investigated in clinical trials (Table 10.1). ISIS 104838, an antisense oligonucleotide targeting TNF-
produced a dosedependent reduction in TNF-
expression from ex vivo activated mononuclear cells [20]. A dose of 6 mg/kg produced a 40–50% reduction in TNF-
production, while a dose of 2 mg/kg failed to produce a significant reduction in TNF-
. In a phase 1 study, Gleave and coworkers examined the

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effects of OGX-011, an antisense drug targeting clusterin, in high-risk prostate cancer patients scheduled for prostatectomy [22]. In the study, patients were treated with OGX-011 on days 1, 3, and 5 and then weekly from days 8–29. Prostatectomy was performed between days 30 and 36. Tumor tissue was analyzed for drug levels and reductions in clusterin expression. Increasing doses of the drug produced increased drug levels in tumor tissue and reductions in clusterin expression in tumor tissue and draining lymph node, with maximal effects observed between 320 and 640 mg. A dose of 640 mg produced a 93% decrease in clusterin mRNA and similar reductions in protein based upon immunohistochemistry. Two additional 2⬘-MOE gapmers are being investigated for the treatment of cancer. LY2181308 is an 18-mer 2⬘-MOE gapmer targeting survivin, a gene that regulates cell division and apoptosis. LY218308 is in phase 1 clinical trials. LY2275796 targets the eukaryotic translation initiation factor 4E (eIF4E) and is also in phase 1 trials. Although it is too early to draw any conclusions regarding efficacy of these drugs, they have been well tolerated, at doses greater than 10 mg/kg. In comparison, first-generation PS ODNs produced significant side effects at doses above 4–7 mg/kg. 2⬘-MOE gapmers have been found to produce less pro-inflammatory effects in patients compared to first-generation PS ODNs. Most PS ODNs are administered as intravenous infusions, with fatigue, fever, rigors, and other flu-like symptoms being commonly reported side effects. One 2⬘-MOE gapmer, OGX-011, has also produced mild flu-like syndrome when infused intravenously, but at higher doses than typically observed for PS ODNs [22]. This drug differs from other secondgeneration oligonucleotides in that it does not contain the 5-methyl deoxycytosine modification that further reduces immune cell activation [7,167]. 2⬘-MOE gapmers also produce less local inflammation when injected subcutaneously. One PS ODN drug, alicaforsen (ISIS 2302), has been administered as a subcutaneous injection with a dose of 0.5 mg/kg being the maximally tolerated dose due to injection site reactions and lymphadenopathy [190]. Several 2⬘-MOE gapmers have been dosed subcutaneously at doses as high as 6 mg/kg with minimal to mild injection site reactions reported [20,21]. Thus, 2⬘-MOE gapmers appear to be more potent than PS ODNs and better tolerated.

10.8 CONCLUSIONS The 2⬘-MOE modification represents a true advancement for antisense-based therapeutics. 2⬘-MOE gapmers are broadly used in cell culture and rodents to identify gene function and validate genes as potential drug targets. They distribute to a number of tissues, producing pharmacological effects in those tissues when administered systemically. The pharmacological effects of 2⬘-MOE gapmers can be further expanded to additional tissues by local administration, with effects restricted to the dosed tissue. Doses of drug required to produce pharmacological effects in cell culture and in preclinical models are well tolerated. Furthermore, these attributes are translating to the clinic, with significant increases in potency and tolerability reported. With advances in understanding the basic principles of antisense technology, more informed decisions regarding selection of molecular targets, therapeutic indications, and clinical trial designs are being implemented, building off the successes and failures of first-generation oligonucleotides. Hopefully, these drugs will be proven to benefit patients and become approved therapeutics. Is there potential to further improve upon the performance of 2⬘-MOE gapmers? The answer is clearly yes. We are already finding that potency of 2⬘-MOE gapmers can be further improved twoto fivefold in rodents by optimizing the design of the oligonucleotide to serve as a better substrate for RNase H and to improve the pharmacokinetics. The gap-widened design (or version 2.2) is an example of such a design (Chapter 17). Further optimization of design of 2⬘-MOE gapmers and dosing schedules can enhance accumulation in target tissues and potentially decrease accumulation in nontarget tissues, resulting in improved specificity. Additional investment into the medicinal chemistry of oligonucleotides is warranted and will likely yield dividends by producing chemical

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modifications that exhibit further improvements over 2⬘-MOE. Preliminary data suggest that it is possible to further enhance potency of oligonucleotides over 2⬘-MOE gapmers, but unfortunately the chemical modifications used also produce severe hepatoxicity [191]. These data demonstrate potential increases in potency can be achieved, and hopefully the toxicities will be separated from potency. Even though there has been tremendous progress in developing antisense-based therapeutics, there are still gaps in our fundamental knowledge about the technology. As examples we know very little about: ●

●

●

●

●

The mechanisms responsible for the distribution of 2⬘-MOE gapmers or any oligonucleotide out of plasma and accumulation in cells. The molecular pathways that the 2⬘-MOE gapmers or other oligonucleotide types exploit to selectively bind to their receptors (target RNA) inside cells. The enzymes are responsible for the degradation of 2⬘-MOE gapmers and other types of oligonucleotides in plasma and in tissues. The factors that limit 2⬘-MOE gapmers from crossing mucosal membranes in the intestine and other mucosal membranes. The molecular mechanisms for untoward effects produced by 2⬘-MOE gapmers and other oligonucleotides.

Continued progress toward answering these and other basic questions is important to facilitate improvements in the technology, leading to the rational-hypothesis driven practice of medicinal chemistry and formulation efforts. These investments in the platform will lead to continued success with the technology and better drugs for our patients.

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11. Lind, K.E., Mohan, V., Manoharan, M., and Ferguson, D.M., Structural characteristics of 2⬘-O-(2-methoxyethyl)-modified nucleic acids from molecular dynamics simulations, Nucleic Acids Res., 26, 3694, 1998. 12. Shen, L., Siwkowski, A., Wancewicz, E.V., Lesnik, E.A., Butler, M., Witchell, D., Vasquez, G., Ross, B., Acevedo, O., Inamati, G., Sasmor, H., Manoharan, M., and Monia, B.P., Evaluation of C-5 propynyl pyrmidine-containing oligonucleotides in vitro and in vivo, Antisense Nucleic Acid Drug Dev., 13, 129, 2003. 13. Richardson, F.C., Zhang, C., Lehrman, S.R., Koc, H., Swenberg, J.A., Richardson, K.A., and Bendele, R.A., Quantification of 2⬘-fluoro-2⬘-deoxyuridine and 2⬘-fluoro-2⬘-deoxycytidine in DNA and RNA isolated from rats and woodchucks using LC/MS/MS., Chem. Res. Toxicol., 15, 922, 2002. 14. Drygin, D., Barone, S. and Bennett, C.F., Sequence-dependent cytotoxicity of second-generation oligonucleotides, Nucleic Acids Res., 32, 6585, 2004. 15. Lai, J.C., Tan, W., Benimetskaya, L., Miller, P., Colombini, M., and Stein, C.A., A pharmacologic target of G3139 in melanoma cells may be the mitochondrial VDAC., Proc. Natl. Acad. Sci. USA, 103, 7494, 2006. 16. Lai, J., Benimetskaya, L., Santella, R., Wang, Q., Miller, P., and Stein, C.A., G3139 (Oblimersen) may inhibit prostate cancer cell growth in a partially bis-CpG-dependent non-antisense manner, Mol. Cancer Ther., 2, 1031, 2003. 17. Levin, A.A., Monteith, D.K., Leeds, J.M., Nicklin, P.L., Geary, R.S., Butler, M., Templin, M.V., and Henry, S.P., Toxicity of oligodeoxynucleotide therapeutic agents, in Antisense Research and Application, Vol. 131, Crooke, S.T., ed., Springer-Verlag, Berlin Heidelberg, 169, 1998. 18. Levin, A.A., Henry, S.P., Monteith, D., and Templin, M.V., Toxicity of antisense oligonucleotides, in Antisense Drug Technology: Principles, Strategies, and Applications, Crooke, S.T., ed., Marcel Dekker, New York, 201, 2001. 19. Nicklin, P.L., Ambler, J., Mitchelson, A., Bayley, D., Phillips, J.A., Craig, S.J., and Monia, B.P., Preclinical profiling of modified oligonucleotides: anticoagulation and pharmacokinetic properties, Nucleos. Nucleot., 16, 1145, 1997. 20. Sewell, L.K., Geary, R.S., Baker, B.F., Glover, J.M., Mant, T.G.K., Yu, R.Z., Tami, J.A., and Dorr, F.A., Phase 1 trial of Isis 104838, a 2⬘-methoxyethyl modified antisense oligonucleotide targeting tumor necrosis factor -alpha, J. Pharm. Exp. Ther., 303, 1334, 2002. 21. Kastelein, J.J.P., Wedel, M.K., Baker, B.F., Su, J., Bradley, J.A., Yu, R.Z., Chuang, E., Graham, M.J., and Crooke, R.M., Potent reduction of Apolipoprotein B and LDL cholesterol by an antisense inhibitor of Apolipoprotein B, Circulation, In press, 2006. 22. Chi, K.M., Eisenhauer, E., Fazli, L., Jones, E.C., Goldenberg, S.L., Powers, J., and Gleave, M.E., A phase I pharmacokinetic and pharmacodynamic study of OGX-011, a 2⬘-methoxyethyl antisense to clusterin, in patients with localized prostate cancer prior to radical prostatectomy, J. Natl. Cancer Inst., 97, 1287, 2005. 23. Geary, R.S., Watanabe, T.A., Truong, L., Freier, S., Lesnik, E.A., Sioufi, N.B., Sasmor, H., Manoharan, M., and Levin, A.A., Pharmacokinetic properties of 2⬘-O-(2-methoxyethyl)-modified oligonucleotide analogs in rats, J. Pharm. Exp. Ther., 296, 890, 2001. 24. Geary, R.S., Leeds, J.M., Fitchett, J., Burckin, T., Truong, L., Spainhour, C., Creek, M., and Levin, A.A., Pharmacokinetics and metabolism in mice of a phosphorothioate oligonucleotide antisense inhibitor of C-RAF-1 kinase expression, Drug Metab. Dispos., 25, 1272, 1997. 25. Geary, R.S., Khatsenko, O., Bunker, K., Crooke, R., Moore, M., Burckin, T., Truong, L., Sasmor, H., and Levin, A.A., Absolute bioavailability of 2⬘-O-(2-methoxyethyl)-modified antisense oligonucleotides following intraduodenal instillation in rats., J. Pharm. Exp. Ther., 296, 898, 2001. 26. Khatsenko, O., Morgan, R.A., Truong, L., York-Defalco, C., Sasmor, H., Conklin, B., and Geary, R.S., Absorption of antisense oligonucleotides in rat intestine: effect of chemistry and length, Antisense Nucleic Acid Drug Dev., 10, 35, 2000. 27. Egli, M., Minasov, G., Tereshko, V., Pallan, P.S., Teplova, M., Inamati, G.B., Lesnik, E.A., Owens, S.R., Ross, B.S., Prakash, T.P., and Manoharan, M., Probing the influence of stereoelectronic effects on the biophysical properties of oligonucleotides: comprehensive analysis of the RNA affinity, nuclease resistance, and crystal structure of ten 2⬘-O-ribonucleic acid modifications, Biochemistry, 44, 9045, 2005. 28. Monia, B.P., Lesnik, E.A., Gonzalez, C., Lima, W.F., McGee, D., Guinosso, C.J., Kawasaki, A.M., Cook, P.D., and Freier, S.M., Evaluation of 2⬘-modified oligonucleotides containing 2⬘-deoxy gaps as antisense inhibitors of gene expression, J. Biol. Chem., 268, 14,514, 1993.

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161. Myers, K.J., Witchell, D.R., Graham, M.J., Koo, S., Butler, M., and Condon, T.P., Antisense oligonucleotide blockade of alpha 4 integrin prevents and reverses clinical symptoms in murine experimental autoimmune encephalomyelitis, J. Neuroimmunol., 160, 12, 2003. 162. Lach-Trifilieff, E., McKay, R.A., Monia, B.P., Karras, J.G. and Walker, C., In vitro and in vivo inhibition of interleukin (IL)-5-mediated eosinopoiesis by murine IL-5alpha antisense oligonucleotide, Am. J. Respir. Cell Mol. Biol., 24, 116, 2001. 163. Myers, K.J., Murthy, S., Flanigan, A., Witchell, D.R., Butler, M., Murray, S., Siwkowski, A., Goodfellow, D., Madsen, K., and Baker, B., Antisense oligonucleotide blockade of tumor necrosis factor-alpha in two murine models of colitis., J. Pharmacol. Exp. Ther., 304, 411, 2003. 164. Chen, W., Langer, R.M., Janczewska, S., Furian, L., Geary, R., Qu, X., Wang, M., Verani, R., Condon, T., Stecker, K., Bennett, C.F., and Stepkowski, S., Methoxyethyl-modified ICAM-1 antisense phosphorothioate oligonucleotides inhibit allograft rejection, ischemic/reperfusion injury, and cyclosporine-induced nephrotoxicity, Transplantation, 79, 401, 2005. 165. Johnston, B., Chee, A., Issekutz, T.B., Ugarova, T., Fox-Robichaud, A., Kickey, M.J., and Kubes, P., Alpha 4 integrin-dependent leukocyte recruitment does not require VCAM-1 in a chronic model of inflammation., J. Immunol., 164, 3337, 2000. 166. Qu, X., Kirken, R.A., Tian, L., Wang, M.-E., Bennett, C.F., and Stepkowski, S., Selective inhibition of IL-2 gene expression by IL-2 antisense oligonucleotides blocks heart allograft rejection, Transplantation, 72, 915, 2001. 167. Krieg, A.M., Yi, A.-K., Matson, S., Waldschmidt, T.J., Bishop, G.A., Teasdale, R., Koretzky, G.A., and Klinman, D.M., CpG motifs in bacterial DNA trigger direct B-cell activation, Nature, 374, 546, 1995. 168. Senn, J.J., Burel, S., and Henry, S.P., Non-CpG-containing antisense 2⬘-methoxyethyl oligonucleotides activate a proinflammatory response independent of toll-like receptor 9 or myeloid differentiation factor 88, J. Pharm. Exp. Ther., 314, 972, 2005. 169. Choon, Q., Achenbach, T.V., Borchers, O., Muller, R., and Slater, E.P., In vivo pro-apoptotic and antitumor efficacy of a c-Raf antisense phosphorothioate oligonucleotide: relationship to tumor size, Antisense Nucleic Acid Drug Dev., 12, 11, 2002. 170. Zellweger, T., Miyake, H., Cooper, S., Chi, K., Conklin, B.S., Monia, B.P., and Gleave, M.E., Antitumor activity of antisense oligonucleotides is improved in vitro and in vivo by incorporation of 2⬘-O-(2-methoxy)ethyl chemistry., J. Pharm. Exp. Ther., 298, 934, 2001. 171. Bertram, J., Peacock, J.W., Fazil, L., Mui, A.L., Chung, S.W., Cox, M.E., Monia, B., Gleave, M.E., and Ong, C.J., Loss of PTEN is associated with progression to androgen independence, Prostate, 66, 895, 2006. 172. Miyake, H., Monia, B.P., and Gleave, M.E., Inhibiton of progression to androgen-independence by combined adjuvant treatment with antisense BCL-XL and antisense Bcl-2 oligonucleotides plus taxol after castration in the Shionogi tumor model, Int. J. Cancer, 86, 855, 2000. 173. Fennell, D.A., Corbo, M.V., Dean, N.M., Monia, B.P., and Cotter, F.E., In vivo suppression of Bcl-XL expression facilitates chemotherapy-induced leukaemia cell death in SCID/NOD-Hu model, Brit. J. Haemat., 112, 706, 2001. 174. Chiarle, R., Simmons, W.J., Cai, H., Dhall, G., Zamo, A., Raz, R., Karras, J.G., Levy, D.E., and Inghirami, G., Stat3 is required for ALK-mediated lymphomagenesis and provides a possible therapeutic target, Nat. Med., 11, 623, 2005. 175. Li, W.-C., Ye, S.-L., Sun, R.-X., Liu, Y.-K., Tang, Z.-Y., Kim, Y., Karras, J.G., and Zhang, H., Inhibition of growth and metastasis of human hepatocellular carcinoma by antisense oligonucleotide targeting Stat3, Clin. Cancer Res., In press, 2006. 176. Duan, W., Chan, J.H.P., Kelly, M., Crosby, J.R., Choo, H.H., Leung, B.P., Karras, J.C., and Wong, W.S.F., Inhaled p38alpha mitogen-activated protein kinase antisense oligonucleotide attenuates asthma in mice, Am. J. Respir. Crit. Care Med., 171, 571, 2005. 177. Karras, J.G., Crosby, J.R., Guha, M., Tung, D., Miller, D.A., Gaarde, W.A., Geary, R.S., Monia, B.P., and Gregory, S.A., Anti-inflammatory activity of inhaled interleukin-4 receptor-alpha antisense oligonucleotide in mice, Am. J. Resp. Cell Mol. Biol., In press, 2006. 178. Corderio, M.F., Mead, A., Ali, R.R., Alexander, R.A., Murray, S., Chen, C., York-Defalco, C., Dean, N.M., Schultz, G.S., and Khaw, P.T., Novel antisense oligonucleotides targeting TGF-beta inhibit in vivo scarring and improve surgical outcome, Gene Ther., 10, 59, 2003.

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179. Danis, R.P., Criswell, M.H., Orge, F., Wancewicz, E.V., Stecker, K., Henry, S.P., and Monia, B.P., Intravitreous anti-raf-1 kinase antisense oligonucleotide as an angioinhibitory agent in porcine preretinal neovascularization., Curr. Eye Res., 26, 45, 2003. 180. Hua, X.-Y., Moore, A., Malkmus, S., Murray, S.F., Dean, N., Yaksh, T.L., and Butler, M., Inhibition of spinal protein kinase Calpha expression by an antisense oligonucleotide attenuates morphine infusion-induced tolerance, Neuroscience, 113, 99, 2002. 181. Barclay, J., Patel, S., Doern, G., Wotherspoon, G., Moffatt, S., Eunson, L., Abdel’al, S., Natt, F., Hall, J., Winter, J., Bevan, S., Wishart, W., Fox, A., and Ganju, P., Functional downregulation of P2X3 receptor subunit in rat sensory neurons reveals a significant role in chronic neuropathic and inflammatory pain, J. Neurosci., 22, 8139, 2002. 182. Butler, M., Hayes, C.S., Chappell, A., Murray, S.F., Yaksh, T.L., and Hua, X.-Y., Spinal distribution and metabolism of 2⬘-O-(2-methoxyethyl)-modified oligonucleotides after intrathecal administration in rats., Neuroscience, 131, 705, 2005. 183. Smith, R.A., Miller, T.M., Yamanaka, K., Monia, B.P., Condon, T.P., Hung, G., Lobsiger, C.S., Ward, C.M., McAlonis-Downes, M., Wei, H., Wancewicz, E.V., Bennett, C.F., and Cleveland, D.W., Antisense oligonucleotide therapy for neurodegenerative disease, J. Clin. Invest., 116, 2290, 2006. 184. Honore, P., Kage, K., Mikusa, J., Watt, A.T., Johnston, J.F., Wyatt, J.R., Faltynek, C.R., Jarvis, M.F., and Lynch, K., Analgesic profile of intrathecal P2X3 antisense oligonucleotide treatment in chronic inflammatory and neuropathic pain states in rats, Pain, 99, 11, 2002. 185. Adams, M.R., Nikkel, A.L., Donnelly-Roberts, D.L., Watt, A.T., Johnston, J.F., Cowsert, L.M., Butler, M., Kroeger, P.E., Frost, L., Curzon, P., Decker, M.W., and Bitner, R.S., In vitro and in vivo effects of an alpha3 neuronal nicotinic acetylcholine receptor antisense oligonucleotide, Brain Res. Mol. Brain Res., 129, 67, 2004. 186. Gordon, S.C., Bacon, B.R., Jacobson, I.M., Shiffman, M.L., Afdhal, N.H., Yu, R.Z., McHutchison, J.G., and Kwoh, J.J., Treatment of chronic hepatitis C with ISIS 14803, an antisense inhibitor of HCV, given for 12 weeks, Hepatology, 38, 312, 2003. 187. Kjems, L.L., Bhanot, S., Bradley, J.D., Monia, B., Kwoh, J., and Wedel, M.K., Increased insulin sensitivity in humans by protein tyrosine phosphatase 1B (PTP-1B) inhibition – evaluation of ISIS 113715, an antisense inhibitor of PTP-1B., Diabetes, 54, A530, 2005. 188. Waring, J.F., Ciurlionis, R., Clampit, J.E., Morgan, S., Gum, R.J., Jolly, R.A., Kroeger, P.E., Frost, L., Trevillyan, J.M., Zinker, B.A., Jirousek, M., Ullrich, R.G., and Rondinone, C.M., PTP1B antisensetreated mice show regulation of genes involved in lipogenesis in liver and fat, Mol. Cell. Endocrin., 203, 155, 2003. 189. Gum, R.J., Gaede, L.L., Koterski, S.L., Heindel, M., Clampit, J.E., Zinker, B.A., Trevillyan, J.M., Ullrich, R.G., Jirousek, M.R., and Rondinone, C.M., Reduction of protein tyrosine phosphatase-1B increases insulin-dependent signaling in ob/ob mice., Diabetes, 52, 21, 2003. 190. Schreiber, S., Nikolaus, S., Malchow, H., Kruis, W., Lochs, H., Raedler, A., Hahn, E.G., Krummenerl, T., Steinmann, G., and Group, G.I.-S., Absence of efficacy of subcutaneous antisense ICAM-1 treatment of chronic active Crohn’s disease, Gastroenterology, 120, 1339, 2001. 191. Swayze, E.E., Siwkowski, A.M., Wancewicz, E.V., Migawa, M.T., Wyrzykiewicz, T.K., Hung, G., Monia, B.P., and Bennett, C.F., Antisense oligonucleotides containing locked nucleic acid (LNA) improve potency but cause significant hepatotoxicity in animals, Nucleic Acid. Res., in press, 2006. 192. Dean, N.M., McKay, R., Condon, T.P., and Bennett, C.F., Inhibtion of protein kinase C-a expression in human A549 cells by antisense oligonucleotides inhibits induction of intercellular adhesion molecule 1 (ICAM-1) mRNA by phorbol esters, J. Biol. Chem., 269, 16,416, 1994. 193. Miraglia, L., Geiger, T., Bennett, C.F., and Dean, N.M., Inhibition of interleukin-1 type I receptor expression in human cell-lines by an antisense phosphorothioate oligodeoxynucleotide, Int. J. Immuno pharmacol., 18, 227, 1996. 194. Taylor, J., Zhang, Q., Monia, B., Marcusson, E., and Dean, N., Inhibition of Bcl-xL expression sensitizes normal human keratinocytes and epithelial cells to apoptotic stimuli, Oncogene, 18, 4495, 1999. 195. Monia, B.P., Johnston, J.F., Geiger, T., Muller, M., and Fabbro, D., Antitumor activity of a phosphorothioate oligodeoxynucleotide targeted against C-raf kinase, Nat. Med., 2, 668, 1995. 196. Dean, N.M., and McKay, R.A., Inhibition of protein kinase C-alpha expression in mice after systemic administration of phosphorothioate antisense oligodeoxynucleotides, Proc. Natl. Acad. Sci. USA, 91, 11,762, 1994.

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197. Yu, R.Z., Geary, R.S., Leeds, J.M., Watanabe, T., Moore, M., Fitchett, J., Matson, J., Burckin, T., Templin, M.V., and Levin, A.A., Comparison of pharmacokinetics and tissue disposition of an antisense phosphorothioate oligonucleotide targeting human Ha-ras mRNA in mouse and monkey, J. Pharm. Sci., 90, 182, 2000. 198. Geary, R.S., Yu, R.Z., Leeds, J.M., Watanabe, T.A., Henry, S.P., Levin, A.A., and Templin, M.V. Pharmacokinetic properties in animals, in Antisense Drug Technology: Principles, Strategies, and Applications, Crooke, S.T., ed., Marcel Dekker, Inc., New York, 2001. 199. Bedikian, A.Y., Millward, M., Pehamberger, H., Conry, R., Gore, M., Trefzer, U., Pavlick, A.C., Deconti, R., Hersh, E.M., Hersey, P., Kirkwood, J.M., and Haluska, F.G., Bcl-2 antisense (oblimersen sodium) plus dacarbazine in patients with advanced melanoma: the oblimersen melanoma study group, J. Clin. Oncol., in Press, 2006. 200. Klimuk, S.K., Semple, S.C., Nahirney, P.N., Mullen, M.C., Bennett, C.F., Scherrer, P., and Hope, M.J., Enhanced anti-inflammatory activity of a liposomal intercellular adhesion molecule-1 antisense oligodeoxynucleotide in an acute model of contact hypersensitivity, J. Pharm. Exp. Ther., 292, 480, 2000.

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11

Pharmacokinetic/Pharmacodynamic Properties of Phosphorothioate 2⬘⬘-O-(2-Methoxyethyl)-Modified Antisense Oligonucleotides in Animals and Man Richard S. Geary, Rosie Z. Yu, Andrew Siwkowski, and Arthur A. Levin

CONTENTS 11.1 Introduction .........................................................................................................................306 11.1.1 Pharmacokinetics and Metabolism .......................................................................307 11.1.2 Pharmacokinetics/Pharmacodynamics ..................................................................307 11.2 Pharmacokinetics/Pharmacodynamics in Animals .............................................................308 11.2.1 Target Tissues ........................................................................................................308 11.2.1.1 Systemic Administration ......................................................................308 11.2.1.2 Local or Topical Administration...........................................................310 11.2.1.3 Unique Challenges of Human Tumor Xenografts................................310 11.2.2 Onset of Action .....................................................................................................311 11.2.3 Duration of Action.................................................................................................312 11.2.4 Multiple-Dose Regimens.......................................................................................314 11.2.5 Linking Plasma to Tissue across Species: Accumulation and Clearance ................................................................................314 11.3 Pharmacokinetics/Pharmacodynamics in Man ...................................................................316 11.3.1 ISIS 301012, ApoB-100 Antisense .......................................................................316 11.3.1.1 Onset of Action.....................................................................................316 11.3.1.2 Duration of Action................................................................................317 11.3.1.3 Plasma Trough EC50 Determination from Phase 1 Experience ..........317 11.3.1.4 Subchronic Dosing ...............................................................................318 11.3.2 ISIS 104838, TNF␣ Antisense ..............................................................................320 11.3.3 OGX-011, Clusterin Antisense..............................................................................320 11.4 Conclusions .........................................................................................................................321 References ......................................................................................................................................321

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11.1 INTRODUCTION Antisense compounds are short synthetic chemically modified oligonucleotides, usually between 15 and 25 nucleotides in length, designed to hybridize to RNA through Watson–Crick base pairing. Upon binding the target RNA, the oligonucleotide prevents translation of the encoded protein product in a sequence-specific manner. Since the rules for Watson–Crick base pairing are well characterized [1], antisense oligonucleotides (ASOs) represent, in principle, a simple method for rationally designing drugs. In practice, exploitation of ASO technology for therapies has presented a unique set of challenges, some of which relate to their pharmacokinetic behavior. There have been significant resources applied towards identification of chemical modifications that further improve upon the pharmacokinetic and pharmacodynamic properties of ASOs. The vast majority of these modifications includes fully phosphorothioated backbones and have followed a model of mixing 2⬘-O-methoxyethyl (2⬘-MOE) modifications placed at the 3⬘ and 5⬘ termini of the oligonucleotides with -deoxynucleotides in the intervening gap (Figure 11.1). This phosphorothioate (PS) 2⬘-MOE/DNA chimeric construct has provided even greater biological stability to the molecule, higher binding affinity to its target mRNA while maintaining susceptibility to RNase H, increased in vitro and in vivo potency, and decreased general nonhybridization toxicities [2–4]. Within this second chemical class there exist a growing number of compounds that are in development (Table 11.1). As expected, there is significantly more information regarding the pharmacokinetic properties of first-generation phosphorothioate oligodeoxynucleotides (PS ODNs) available in the literature [5–20] (to site just a few). Proprietary PS 2⬘-MOE partially modified ASOs are relatively early in their development and published data are less plentiful, but represent a growing body of literature [4,21–29].

HO O

HO O

O S P O O

B

B O S P O O O

O S P O O

HO

O OCH3 O

5

B

B O S P O O

18 O

O

B

B O S P O O

8 O

O S P O O

B

O

O

B O OCH3

5

Figure 11.1 Represents chemical structure of first- and second-generation phosphorothioate oligonucleotides (B ⫽ G, C, T, or A).

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Table 11.1 List of Systemically Administered 2⬘⬘-MOE-Modified Antisense Oligonucleotides Currently in Development Product ISIS 113715 ISIS 301012 ATL 1102 OGX-011 LY2181308 LY2275796 OGX 427 ISIS 325568

Lead Indication

Target

Company

Diabetes High cholesterol Multiple sclerosis Cancer Cancer Cancer Cancer Diabetes

PTP-1b apoB-100 VLA4 Clusterin Survivin EIF4e Hsp27 GCGR

Isis Isis Antisense Therapeutics Oncogenix Eli Lilly Eli Lilly Oncogenix Isis

Source: Isis Pharmaceuticals, Inc. WebPage (www.isispharm.com).

11.1.1 Pharmacokinetics and Metabolism An extensive review of the principles that guide an understanding of the pharmacokinetics of PS oligonucleotides in general and proprietary PS partial 2⬘-MOE-modified ASOs as a second chemical class has been presented in Chapter 7 (Levin et al.) of this book. In general, the pharmacokinetics of the PS 2⬘-MOE chimeras are represented by rapid distribution from the blood compartment to tissues following parenteral administration (30 min to 2 h distribution half-life) with little to no measurable metabolism in plasma. These PS 2⬘-MOE chimeras circulate in blood primarily bound to plasma proteins and do not distribute into red cells. The plasma concentration–time profile is characterized by a polyexponential decline that is dominated by the early rapid distribution phase such that ⬍1% of the administered 2⬘-MOE chimera dose remains in circulation by 24 h after administration. The terminal elimination half-life, however, is quite long and closely tied to slow metabolic clearance of the 2⬘-MOE chimeras from distributed tissues and cells in the body (half-lives ranging from 8 to 30 days, depending on sequence and tissues). Less than 10% of the dose in this chemical class is excreted in urine over the first 24 h following administration. Biodistribution is extensive with organs displaying highest concentrations being kidney, liver, spleen, lymph nodes, and bone/bone marrow. These PS 2⬘-MOE chimeras are generally poorly distributed in skeletal muscle, lung, and heart, and do not cross the blood–brain barrier (BBB) following parenteral administration. In fact, the biodistribution observed for PS 2⬘-MOE ASOs is remarkably similar to the first-generation PS ODNs. The similarity in the distribution of first-generation PS ODNs and the modified second-generation PS 2⬘-MOE ASOs is a function of the shared identical chemistry of the phosphate linkage, a replacement of one nonbridging oxygen with sulfur (PS). While altering the number and positioning of the 2⬘-MOE modifications in the sequence does little to alter biodistribution, reducing the number of PSs in the backbone results in marked reduction in biodistribution to liver and other systemic tissues and increases renal distribution and urinary excretion [22] (see Chapter 7, Levin et al., this volume for details). These unique distribution properties of PS oligonucleotides, independent of sequence, provide a rationale for the selection of targets that may have therapeutic benefit. While distribution is predominantly driven by the PS backbone, improved potency and duration of action observed for this chemical class of antisense compounds are driven by the 2⬘-MOE modifications placed at the 3⬘ and 5⬘ termini. 11.1.2 Pharmacokinetics/Pharmacodynamics Applying and integrating pharmacokinetic and pharmacodynamic analyses to guide and expedite drug development have shown increasing interest in recent years [30]. While these principles are well accepted and widely used for low-molecular-weight drugs, there have been limited reports of the pharmacokinetic/pharmacodynamic relationship with antisense therapeutic agents [31–34]. The vast majority of studies with PS ODNs or 2⬘-MOE chimeras in laboratory animals and in the clinic have characterized the pharmacology with respect to structure–activity relationships (i.e., sequence and mismatched sequence data) and dose–response relationships [35–38].

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Although the mechanism of action for first- and second-generation PS antisense compounds is the same (RNAse H-mediated degradation of the target), the partial 2⬘-O-(2-MOE) modifications provide increased affinity to the target mRNA while maintaining favorable RNase H activity and, therefore, provide enhanced potency and specificity over the first-generation PS ODNs [36,39–41]. This chapter will review exposure–activity relationships for fully phosphorothioated oligonucleotides that are partially modified with 2⬘MOE at the 5⬘ and 3⬘ terminus but contain an internal “gap” of -deoxynucleotides to maintain susceptibility to RNase H degradation of the target mRNA [42,43].

11.2 PHARMACOKINETICS/PHARMACODYNAMICS IN ANIMALS Investigations of the pharmacological effects of ASO in vivo have focused primarily on (1) target mRNA reduction and subsequent reduction in protein translation; (2) downstream effects that result from target protein reduction, which are dependent on the target studied; or (3) clinical outcomes dependent on target and specific to the disease indication. This chapter will include a review of pharmacokinetic/pharmacodynamic relationships to include the investigations between effective concentrations at the target sites of ASOs in preclinical animal models with each of the pharmacological effects discussed above. We will also present relatively new understanding of the relationship between postdistribution phase 2⬘-MOE chimeric concentrations in plasma with underlying tissue accumulation and clearance. Establishment of the relationship between plasma 2⬘-MOE chimeric concentrations and the concentrations of parent 2⬘-MOE ASOs at the target site is required to allow the use of plasma concentration of drug as a surrogate in clinical studies where collection of target site tissues may be prohibitive. 11.2.1 Target Tissues

11.2.1.1 Systemic Administration The natural biodistribution of PS 2⬘-MOE ASOs in animals following parenteral administration of simple solutions provides primary direction regarding organs that may be successfully treated, as well as those unlikely to be affected. It is obvious from the relatively large body of literature available that liver and kidney are important organs for PS ASO distribution and activity [12,23,26,33,44,45]. Since tissue distribution is largely independent of sequence and similar across species [29,46,47], pharmacokinetic and biodistribution information from one 2⬘-MOE chimera provides information for the platform within the chemical class. Much of the pharmacokinetic/pharmacodynamic data available to date have been generated based on the known property of phospohorothioate oligonucleotide distribution to the liver. The hepatocyte is a rich target site for drug-discovery activities given the importance of the liver in metabolic and lipid homeostasis. Therefore, both in regard to metabolic and cardiovascular disease, examples of antisense-specific inhibition of gene expression in liver are numerous [31,33,35,45,48]. A summary of measured PS 2⬘-MOE-modified ASO exposure–response relationships (Table 11.2) has begun to provide insights for which tissue types and what exposure levels are likely to produce antisense-mediated reduction in target mRNA expression. First, similar effective inhibitory concentrations of 2⬘-MOE-modified chimeras are observed for numerous gene targets in a given tissue. For example, the 2⬘-MOE chimera concentrations that produce
50% reduction in target mRNA in liver appear to be consistently achieved at ⭐15 ␮M (
100 ␮g/g of liver tissue) in mice. Second, exposure relationships vary with different tissues. For example, effective adipose tissue drug levels appear to be somewhat lower than those required for inhibition in liver. However, comparison of wet mass concentration data may be somewhat misleading for at least two reasons: (1) a large fraction of the adipose cell is reflected in the lipid portion of the cell to which oligonucleotides do not distribute; (2) liver is a heterogeneous tissue in which nonparenchymal cells (Kupffer and endothelial cells) effectively take up substantially

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more PS oligonucleotides per cell than do parenchymal (hepatocytes) cells [32,49–51]. When corrections are made for these two disparate known effects, the concentration of 2⬘-MOE chimera within the cell does not appear to be substantially different between hepatocytes and adipocytes. Beyond liver, adipose tissue, and kidney, other likely organs in which PS 2⬘-MOE ASOs may be active based on biodistribution data include bone, bone marrow [52], and intestine [53,54] as well as macrophages [55], which tend to actively ingest PS oligonucleotides independent of tissue localization. A study conducted with a PS 2⬘-MOE-modified chimera (ISIS 116847), a sequence targeting a ubiquitously expressed gene (PTEN), has shown various levels of activity to no activity in numerous tissues that roughly reflect the pharmacokinetic distribution properties of PS oligonucleotides (Table 11.3). In other words, as expected, the native distribution of these 2⬘-MOE-modified PS oligonucleotides correlates strongly with the observed activity of this representative 2⬘-MOE chimera. As expected liver exhibited high concentrations of the 2⬘-MOE chimera and marked reduction in mRNA and the target protein. Also, marked mRNA and PTEN protein reduction was localized in the renal proximal tubular epithelium of the kidney, a cell-type known to take up Table 11.2 Listing of Tissue/Cell and Gene Target Inhibition Tissue/cell Whole liver Whole liver Hepatocyte Whole liver Whole liver Whole liver Kidney cortex Adipose tissue Tumor xenograft

Gene Target

apoB-100 Fas ligand Fas ligand GCGR PTP-1b PTEN PTEN PTP-1b Clusterin

Exposurea
g/g) (
85 49 16 24 52 94 320 14 4.2

Strain

Reference

C57BL/6 Balb/c Balb/c Balb/c ob/ob Balb/c Balb/c ob/ob Nude

[33] [32] [32] Unpublished Unpublished [51] Unpublished Unpublished [36]

Note: Both 20-mer 2⬘-MOE-modified phosphorothioate gapmer antisense oligonucleotide exposure and activity (either mRNA or protein target) exist in mice following parenteral administration (i.v., i.p. or s.c.). a 2⬘-MOE chimera concentrations required to induce a 50% reduction of target mRNA or inhibition of protein expression in specified tissues; full-length (parent) 2⬘-MOE chimera was measured using quantitative capillary gel electrophoresis with UV detection; mRNA was measured using qRT-PCR, unless otherwise specified; protein was quantified by Western blot densitometry. Table 11.3 Tissue Profiling for Measurable Inhibition of PTEN Expression in Mice following Treatment with ISIS 116847 Tissue/Cell

Exposure a

Inhibition b

Liver Kidney cortex Kidney medulla Fat Heart Lung Spleen Skeletal muscle Brain Adrenal glands Small intestine

High Highest Medium Low Low Very low Medium Very low None detected Low Medium

Good (hepatocytes) Good (proximal tubular epithelium) Low Low Low (myocytes) None None None None None Low (epithelial cells)

Note: ISIS 116847 is a representative 2⬘-MOE-modified gapmer antisense oligonucleotide targeting PTEN mRNA. a Relative levels based on quantitative CGE-UV analysis (40 mg/kg dose administered i.p. in Balb/C mice), where High ⫽ 100–200 µg/g, Medium ⫽ 5–10-fold lower than High, and Low ⫽ 50–100-fold lower than High, and Very Low is ⬎ 100-fold lower than High. b IHC PTEN semiquantitative PTEN protein immunostaining.

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highest concentrations of PS oligonucleotides. Less effect was observed in the kidney medulla where lower overall 2⬘-MOE chimera concentrations were measured. Perhaps, one interesting exception to the rule was the spleen where relatively high levels of 2⬘-MOE-modified oligonucleotide are consistently measured without any measurable antisense effect to date. While the reason for this discrepancy is not known, one explanation for this difference may be the nature of this organ as a scavenger of xenobiotics and a predominant phagocytotic mechanism of cellular uptake in this tissue that may prevent free access of the ingested 2⬘-MOE ASOs to the nucleus of these cells. This is also a dynamic tissue, in that cells traffic both to and from this organ from other locations. Many of these circulating cells do not take up the PS 2⬘-MOE ASOs. Immunohistochemical staining of oligonucleotide in the spleen also suggests that much of the drug may be trapped in extracellular compartments (E. Hung, unpublished observation). Other organs or tissues, where antisense activity is unlikely following parenteral dosing due to poor distribution of PS 2⬘-MOE ASOs, include skeletal muscle, heart, lung, testis, ovaries, pancreas, and brain (as PS oligonucleotides do not cross the BBB).

11.2.1.2 Local or Topical Administration Finally, for those organs or tissues that are poorly served by the natural distribution of PS 2⬘-MOE-modified ASOs following parenteral administration, local administration has been shown to consistently result in positive antisense effects. For example, local administration to eye following intravitreal injection for retinal targets [56–60], to lung by aerosol or intratracheal inhalation [61–65], and to brain by direct infusion into the central nervous system (CNS) via intrathecal or intracerebroventricular (ICV) administration [66–71] have all provided local gene target inhibition by direct application of the compounds to the site. Thus, alternative routes of administration may be indicated for significant unmet medical needs for which therapeutic targeting requires exposure of these tissues. Nevertheless, the focus of this chapter will be on the pharmacokinetics of parenteral or systemic 2⬘-MOE chimera administration and its relationship to antisense effects (pharmacodynamics) in organs, cells, or tissues that are well served by their natural biodistribution.

11.2.1.3 Unique Challenges of Human Tumor Xenografts Effective tumor 2⬘-MOE chimera levels are achieved at overall lower concentrations than those seen in liver as well (see Table 11.1). One possible explanation for this observed difference may be partial or selective uptake of the oligonucleotide in specific cells within the tumor where concentrations viewed in whole tumor tissue may be diluted by cells that take up little to no 2⬘-MOE ASOs. Alternatively, it is possible that tumor uptake may be facilitated by rapid turnover of cells as has been observed with gene therapy [72] thus requiring less 2⬘-MOE chimera to elicit an antisense effect. Indeed, much interest has been generated for over a decade regarding antisense treatment of neoplastic cells and more specifically solid tumors. Recent preclinical human xenograft data with 2⬘-MOE-modified chimeras have been encouraging [36,73]. It is important to note that the animal models for tumor PK/PD are compromised somewhat in their predictive power by the nature of the solid tumor (subcutaneous placement and rapid protein encapsulation due to the xenobiotic nature of the implant). The unnatural placement and protein encapsulation of xenograft human tumors produce a difficult distribution hurdle for PS 2⬘-MOE chimeras in general and may in fact underestimate the distribution of the 2⬘-MOE chimeras to solid tumors in the clinical setting. There is a significant additional issue with interpretation of xenograft studies due to the fact that antisense sequences targeting tumors are designed to cause tumors to stop growing. While tumor growth can be directly measured, the proper quantitation of the target mRNA or protein is challenging and requires proper selection of methods and controls. In addition, appropriate methods are required to select for the specific human mRNA or protein selective from the murine mRNA or protein. Real time polymerase

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chain reaction (RT-PCR) approaches that utilize selective sequences allow differentiation in mRNA, but protein antibodies often cross-react with the murine protein target complicating interpretation. Even with these limitations, preclinical data indicate that the biodistribution of 2⬘-MOE chimeras into human xenograft tumors is measurable and pharmacodynamic effects have been recently reported for 2⬘-MOE-modified ASOs in numerous properly controlled models [36,74]. 11.2.2 Onset of Action The kinetics of drug distribution from the site of administration to the binding of the targeted mRNA is foundational to understanding the kinetics of onset of action. As previously described, PS 2⬘-MOE ASOs are rapidly distributed from blood to tissues with plasma half-lives ranging from 30 min to 2 h dependent on dose and species [26] with nearly complete plasma clearance by 24 h after intravenous administration. However, distribution out of the plasma compartment does not translate to immediate antisense inhibition of targeted mRNA. The kinetics of the onset of action can be separated into pharmacokinetic and pharmacodynamic events. The pharmacokinetics of distribution and transport predict relatively short lag times associated with direct antisense inhibition of the target mRNA (by 24 h following administration). Immunohistochemistry of suborgan and subcellular distribution indicates that the PS class of ASOs first associates with cellular membranes and extracellular matrix proteins [52,75]. The immunohistochemical data closely mimic early experiments that involved “cold” quantitative analysis of PS ODNs in various cell types overtime in liver [49,50]. Within the liver, for example, endothelial and Kupffer (nonparenchymal fraction) cells accumulate higher concentrations of either PS ODNs or the second-generation 2⬘-MOE-modified ASOs with distribution to parenchymal fraction delayed and at an overall lower concentration. The initial distribution kinetics from plasma is therefore dominated by apparent binding to cell surface proteins (including the endothelial surface of the circulatory system itself) and extracellular matrix. While intracellular distribution can be seen as early as 1 h after dosing for some cell types (macrophages, Kupffer cells in the liver, and renal proximal tubular epithelial cells), PS oligonucleotides in these cell types and at this early time point appear to be predominately associated with cytoplasmic vesicles (endosomes or lysosomes) representative of a dominant endo- or pinocytotic uptake mechanism. By 24 h, however, nuclear distribution can be seen in some cell types, including hepatocytes. Cytoplasm to nuclear distribution is hypothesized to involve a protein shuttling mechanism [76] based on high capacity, low-affinity “hand-off” to higher affinity targets ultimately leading to the highest affinity association of the complementary mRNA or pre-mRNA sequence and the 2⬘-MOE ASOs. Endosomal capture of PS oligonucleotides may also provide a subsequent longer term “sink” from which additional 2⬘-MOE chimera may be available overtime as it is released from endosomes and eventually trafficked to ultimate interaction with its cognate mRNA, although this cannot be directly confirmed by existing data. In general, the pharmacodynamics of 2⬘-MOE ASOs follow closely in time with these observations with some inhibition observed as early as 24 h and maximal inhibition observed by 48–72 h after a single-dose administration [31,32] for several different PS 2⬘-MOE-modified oligonucleotides targeting different genes in mouse liver. As an example, Figure 11.2 illustrates the time profile for pharmacodynamic decrease in Fas mRNA in liver following a single subcutaneous injection in mice at a dose of
1 mg per animal (50 mg/kg) of ISIS 22023, a PS 2⬘-MOE-modified gapmer oligonucleotide with a sequence complementary to murine Fas mRNA. Additional experience targeting murine hepatic targets indicate that mRNA reduction is tightly correlated with hepatic tissue concentration of the 2⬘-MOE-modified oligonucleotide and that the relationship generally is similar during accumulation and clearance from the organ, suggesting very little hysteresis under the conditions of these experiments (Figure 11.3). Both of the experiments illustrated in Figure 11.3 were dosed twice weekly for 4 weeks, during which time organ

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FAS mRNA (% control)

150

100

50

0 0

10

20

30

40

Time (day) Figure 11.2 Kinetics of Fas mRNA reduction following a single dose (50 mg/kg, s.c.) of 2⬘MOE chimera (ISIS 22023) with maximum reduction at 48 h and return to baseline levels by 14 days after administration. Each symbol represents the average of three mice and error bars are standard errors of the mean.

concentrations continued to increase, and then allowed to recover without further dosing for an additional 4–8 weeks to monitor clearance of the antisense 2⬘-MOE oligonucleotides and its effect. Further delay in the onset of downstream pharmacological effect can be explained by slow degradation of protein translated prior to mRNA destruction by antisense-mediated RNase H cleavage. Long half-life target proteins will maintain their activity for some time after reduction of mRNA and protein translation in direct proportion to their half-life. The turnover kinetics for many proteins is unknown. Nevertheless, with prolonged suppression of translation, the proteins decay overtime and effects on protein activity are observed. Knowledge of this property emphasizes the importance of proper maintenance of 2⬘-MOE oligonucleotide exposure by appropriate dose and frequency of dosing which will be ultimately guided by tissue half-life and accumulation. Therefore, for rapidly cleared proteins, the antisense effect at the protein target level is observed in parallel with the mRNA reduction. However, downstream pharmacology can incur additional lag from the initial reduction in mRNA of target. One well-known example would be the reduction of hemoglobin A1c following inhibition of a target that results in lower fasting serum glucose. Maximal hemoglobin A1c reduction takes several months (following maximal reduction of target) to manifest due to the known slow kinetics of the glycosylated hemoglobin turnover. A thorough understanding of the target biomarker dynamics provides guidance for design and ultimate success in clearly defining the activity of potentially therapeutic antisense approaches, not unlike any drug treatment paradigm. 11.2.3 Duration of Action While onset of action relies on the kinetics of trafficking from the circulation to the ultimate intracellular cognate mRNA and downstream effects, duration of antisense action relies directly on the clearance kinetics of the ASO from the tissue or cell. Clearance of PS 2⬘-MOE ASOs from tissues is mediated by nuclease hydrolysis. Because 2⬘-MOE-modified chimeras modified at the 3⬘ and 5⬘ termini are resistant to exonuclease hydrolysis, their retention time in tissues is greatly prolonged. Tissue half-lives for 2⬘-MOE-modified oligonucleotides range from 2 to 4 weeks dependent on sequence and tissue type in preclinical animal models including mice [26]. The rate-limiting step in the nuclease-catalyzed hydrolysis of 2⬘-MOE chimeras is the initial endonuclease cleavage of the unmodified -deoxynucleotide gap of the molecule as described in Chapter 7 (Levin et al.).

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(a) GCGR

mRNA (% control)

100 80 60 40 20 0 0

50

100

150

200

250

ISIS 18075 concentration in liver (µg/g)

(b) apoB-100 100

mRNA (% control)

90 80 70 60 50 40 0

20

40

60

80

100

120

140

ISIS 147764 concentration in liver (µg/g) Figure 11.3

Murine mRNA reduction hysteresis analysis for two antisense PS 2⬘-MOE chimeras, targeting (a) glucagon receptor, and (b) apoB-100 mRNA in the liver of mice treated twice weekly for 4 weeks at a dose of 50 mg/kg/week.

The duration of action of the 2⬘-MOE ASOs’ effect on target mRNA has been shown to be tightly correlated with measured pharmacokinetic tissue half-lives. For example, Yu et al. [32] have reported that the reduction in Fas mRNA following a single anti-Fas 2⬘-MOE chimeric dose in mouse returned to baseline after
10–14 days (Figure 11.2). While the whole liver tissue half-life for ISIS 22023 was
30 days, the half-life for clearing this oligonucleotide from hepatocytes (the cell type in the liver that expresses Fas mRNA) was
14 days. Yu and colleagues describe a two-compartment liver tissue model that accounted for the differential degradation and clearance rates of this 2⬘-MOE chimeric in the hepatocytes and nonparenchymal cells in the liver of mice. In another study involving an anti-apoB-100 PS partially 2⬘-MOE-modified ASO, ISIS 147764, dosed repeatedly (twice weekly for over a month) in mice, the liver expression of apoB mRNA remained more than 50% reduced for
4 weeks following cessation of dosing, consistent with the long residence time for the parent oligonucleotide in the liver (Figure 11.4). In addition, downstream pharmacological markers in serum (secreted apoB-100, LDL-C, and total cholesterol) had not returned to baseline levels in these high fat-fed C57BL/6 mice even by 8 weeks after cessation of dosing [33], the last time point monitored in this study.

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Predicted concentration Observed concentration apoB mRNA Predicted mRNA

120

250

100

200

80

150

60

100

40

50

20

Liver apoB mRNA (% control)

ISIS 147764 concentration in liver (µg/g)

300

0

0 0

20

40

60

80

100

120

End of treatment Time (day) Figure 11.4 Kinetics of a murine sequence 2⬘-MOE-modified apoB-100 chimera (ISIS 147764) in liver and effect on apoB-100 mRNA in liver tissue following 6 weeks of treatment. The half-life for ISIS 301012 in liver is
1 month and the antisense-mediated reduction in mRNA levels returns to
50% of baseline
1 month after the treatment ends. Each symbol is the average of four mice and the error bars are standard deviations.

11.2.4 Multiple-Dose Regimens Since the effects of the 2⬘-MOE oligonucleotides are tightly correlated with tissue pharmacokinetics, multiple-dose regimens that maintain the effect are facilitated by the knowledge of the compounds’ kinetics in the target tissue. Conventional dosing frequency rationale (pharmacokinetic modeling) can be applied to ensure maintenance of active tissue levels. Using this approach, Yu and colleagues reported that three doses of 50 mg/kg administered once every 2 weeks (approximately once every half-life as determined from the hepatocyte kinetics) with the anti-Fas ligand 2⬘-MOE ASO, ISIS 22023, maintained suppression of the Fas mRNA for up to 60 days [32]. Additional pharmacokinetic modeling indicated that a 24-mg/kg dose administered once weekly would maintain liver concentrations of ISIS 22023 consistent with previous suppression of the targeted mRNA. In a follow-up study [77] performed at this dose and schedule, the 2⬘-MOE ASO treatment successfully suppressed translation of the Fas-ligand for up to 5 months (duration of repeat dose study) without loss of effect and ultimately provided 100% protection from a lethal dose of agonistic Fas antibody challenge. These data taken together with additional experience with longer-term studies conducted to date in mice, rats, and monkeys indicate that pharmacokinetics can be used to design schedules that produce optimal suppression of target expression overtime. 11.2.5 Linking Plasma to Tissue across Species: Accumulation and Clearance The plasma disposition curves for PS 2⬘-MOE chimeras overtime are polyexponential with at least one additional kinetic phase following the initial rapid distribution phase. We have come to understand that the terminal elimination phase of the plasma concentration–time profile for PS 2⬘-MOE ASOs represents an equilibrium with tissue [78] (Figure 11.5). The concentrations of the PS 2⬘-MOE ASOs in plasma following distribution are very low relative to observed maximum plasma concentrations, generally 3–4 orders of magnitude less. This

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ISIS 22023 concentration in plasma or liver (µg/mL or g)

known pharmacokinetic property has placed sensitivity demands on the bioanalytical assays heretofore unrealized with standard high-performance liquid chromatography (HPLC) or capillary gel electrophoresis (CGE) technologies. Sequence-specific hybridization enzyme-linked immunosorbent assay (ELISA) methods have been developed to assist in sensitive and selective quantitation of full-length parent 2⬘-MOE ASOs in plasma [79,80]. The relationship of the postdistribution plasma concentrations to concentrations of full-length PS 2⬘-MOE oligonucleotides in liver, for example, has now been monitored in multiple species. Remarkably, the relationship between whole liver concentrations and the plasma concentrations is similar across species and appears to be independent of sequence at least for this chemistry (2⬘-MOE-modified chimeras). In general, the ratio between liver 2⬘-MOE chimera concentrations and the terminal phase plasma 2⬘-MOE chimera concentrations range from 4000 to 6000 (Table 11.4). The consistency of this partitioning between liver tissue and plasma concentrations from mouse to nonhuman primates provides confidence that this relationship can be used to estimate tissue levels in man. Because of the observed equilibrium property for 2⬘-MOE ASOs between tissue and plasma, tissue half-lives can be estimated based on the terminal phase plasma oligonucleotide concentration half-life [26]. The terminal plasma half-life of the 2⬘-MOE ASOs is likely a hybrid half-life for whole body tissue elimination, but appears to be in good agreement with estimated whole liver clearance, likely because such a large fraction of the dose resides in this tissue. In fact, as a surrogate 1000 Plasma 100

Liver

10 1 0.1 0.01 0.001 0

5

10

15

20

25

30

35

Time (day) Figure 11.5 Terminal plasma concentrations of ISIS 22023, a 2⬘-MOE-modified chimera (ISIS 22023) targeting Fas mRNA, are in equilibrium with tissue concentrations of this compound (liver in this example) following single-dose administration in mice. Each symbol represents three mice and the error bars are standard deviations. Table 11.4 Comparison of Average Liver to Plasma Concentration Ratios ([Liver] / [Plasma]) at Equilibrium in Mice and Monkeys Species

Compound

Ratio

Mouse

muFas ASO HuApoB-100 ASO HuVLA4 ASO HuTNF-␣ ASO PTP-1b ASO HuApoB-100 ASO

4500 3900 5000 6500 5300 5500

Monkey

Note: All compounds are 20 nucleotides in length, phosphorothioate 2⬘-MOE partially modified oligonucleotides.

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for tissue concentrations, the plasma PS 2⬘-MOE oligonucleotide concentration trough (Cmin) can be used to monitor tissue accumulation as well as clearance. It is this property of PS 2⬘-MOE oligonucleotide pharmacokinetics that now begins to allow modeling of both plasma and tissue pharmacokinetics and ultimate pharmacodynamic response in man. 11.3 PHARMACOKINETICS/PHARMACODYNAMICS IN MAN Clinical proof of concept for PS 2⬘-MOE oligonucleotide-specific pharmacodynamics that are well predicted by its pharmacokinetics is required to bridge the considerable experience in preclinical animal models to the human patient. Recent experience with multiple PS 2⬘-MOE-modified ASOs in early clinical trials has begun to provide a compelling critical mass of evidence for in vivospecific and selective antisense pharmacology that is well correlated with their respective pharmacokinetics. A human antisense inhibitor of apoB-100 (ISIS 301012) is ideally suited for PK/PD assessment in the clinic as its direct secreted protein target (apoB) is readily measured in plasma allowing dose-, concentration-, and time-dependent analysis of the target and its downstream pharmacological effects: LDL-C and total cholesterol lowering. Additional Phase 1 and early Phase 2a studies that have provided PK/PD information for multiple PS 2⬘-MOE oligonucleotides including ISIS 104838 (antisense to TNF-␣) indicated for inflammation, and OGX-011 (antisense to clusterin) being evaluated in cancer will be discussed. In general three approaches have been used in estimating PK/PD relationships in the clinic: ●

● ●

Correlation between clinical endpoint with total plasma drug concentration exposure (plasma AUC or cumulative AUC) [34,81,82] Estimation of plasma EC50 by inhibitory effect Emax model at equilibrium (example to follow) Estimation of plasma EC50 by linking plasma kinetics to target dynamics and building plasma PK/PD link model (internal development in progress, not published)

An example of the second approach is described below for a specific PS 2⬘-MOE chimerics antisense inhibitor to apoB-100 [33,83,84]. ApoB-100 is a structural component of low-density lipoprotein carrier of cholesterol (LDL-C). Thus measurement of apoB (target) in plasma and its correlated lipoprotein particles, VLDL and subsequently LDL-C, provides a direct measure of antisense effect and makes this 2⬘-MOE chimera antisense compound an ideal candidate for more elaborate PK/PD model development. 11.3.1 ISIS 301012, ApoB-100 Antisense ISIS 301012, a 2⬘-MOE partially modified ASO that specifically inhibits human apoB-100 translation, is being tested for its ability to modulate atherogenic lipids in view of treating dyslipidemias associated with cardiovascular risk. In addition to being an ideal target for treating hypercholesteremia and its putative role in atherogenesis, this antisense target is ideal for study and development of PK/PD models due to its target’s expression site, the hepatocyte, and the fact that the protein target is secreted into the blood and can be directly measured. A Phase 1 study was designed as a single- and multipledose escalation study to assess the safety, pharmacokinetics, and pharmacodynamics of ISIS 301012 in healthy volunteers with modestly elevated LDL-Cholesterol (LDL-C). Doses evaluated in this Phase 1 study ranged from 50 to 400 mg ISIS 301012 administered once weekly.

11.3.1.1 Onset of Action Aggressive 1-week induction dosing (three i.v. doses given over a 1-week period) resulted in significant reductions in serum apoB, with concomitant and well-correlated reductions in LDL-C

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LDL (% baseline) 100

40 35 Trough plasma [ISIS 301012] (ng/mL)

25

80

20 70

15 10

Mean LDL (% baseline)

90 30

60

5 50

0 0

7 14 21 28 35 42 49 56 63 70 77 84 91 98 105 112 119 Days

i.v.

s.c.

Figure 11.6 Onset and duration of action of LDL-cholesterol lowering (closed squares) were well correlated with the trough plasma concentrations of ISIS 301012 (closed diamonds) overtime. The arrows detail the dosing days and route of administration. The duration was in good agreement with the estimated terminal half-life of 31.2 days for ISIS 301012. Each symbol represents the average of eight subjects and the error bars are standard deviations.

in healthy volunteers by the end of the second week (Figure 11.6). Once weekly subcutaneous administration during a 3-week maintenance period resulted in additional decrease in apoB and LDL-C levels with maximum effects observed at or just following the last maintenance dose consistent with the highest plasma trough (Cmin) concentration.

11.3.1.2 Duration of Action While maximum reductions were observed at or soon after the final weekly dose administered, recovery of apoB and LDL-C back to baseline levels was a slow process exceeding 3 months after the treatment ended (Figure 11.6). This prolonged duration of action was well correlated with the terminal half-life of ISIS 301012 (31.2 days at a dose of 200 mg) measured in man. Overall the pharmacodynamics of ISIS 301012 were in excellent agreement with the pharmacokinetics observed both in preclinical models and in the healthy human volunteers. Pharmacodynamics were best correlated with the trough ISIS 301012 concentrations (measured just prior to the subsequent dose—7 days or greater after dosing). These observations in man are in good agreement with the preclinical data that suggested plasma trough concentrations observed in the terminal elimination phase of the polyexponential plasma concentration–time curve were in equilibrium with target tissue levels of 2⬘-MOE-modified oligonucleotides.

11.3.1.3 Plasma Trough EC50 Determination from Phase 1 Experience Plasma trough concentration–effect relationships have been analyzed using inhibitory effect models utilizing a modified Hill equation. Utilizing Phase 1 human volunteer pharmacokinetic/pharmacodynamic data, the plasma EC50 was estimated to be 14 ng/mL (Figure 11.7).

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Trough ISIS 301012 concentration (ng/mL) Figure 11.7 Relationship of plasma trough concentrations to serum apoB levels as determined using a sigmoidal inhibitory Emax model. The estimated Cmin EC50 was 14 ng/mL. All individual data were included in the plot with the Cmin concentrations taken just prior to the last dose treated (168 h after last dose).

That is, a plasma trough concentration of 14 ng/mL corresponded to a 50% decrease in circulating apoB protein in normal volunteers with slightly elevated LDL-cholesterol. This equilibrium plasma concentration corresponds to
80 ␮g/g liver ISIS 301012 concentration based on the ratio between plasma and liver ISIS 301012 concentrations in monkeys. The reader will recognize that this liver concentration level is remarkably similar to the liver tissue EC50s established in murine models for multiple PS 2⬘-MOE-modified ASOs (Table 11.1) including the apoB-100 murine analog.

11.3.1.4 Subchronic Dosing In a separate clinical Phase 2a study in polygenic hypercholesteremic patients, various subchronic dosing regimens were compared. On the basis of the long half-life of ISIS 301012 determined in Phase 1, every other week maintenance dosing (s.c. injection) was evaluated following a 2-week induction regimen of twice weekly dosing. This regimen was compared to a separate cohort of patients that were treated by s.c. injection once weekly for 13 weeks with no induction dosing. A pharmacokinetic/pharmacodynamic model developed at Isis based on preclinical and Phase 1 data shown in the previous sections, predicted both drug concentration and effect reasonably well for all dosing regimens studied to date (Figure 11.8). These data suggest that the models developed in normal volunteers would extrapolate well to the patient population. Additional optimization of the models to include variability in response and prediction of individual response will require additional work with population PK/PD models that incorporate experience and additional covariate analysis in many more individuals. Consistent with the predictability of the previous PK/PD model, a second inhibitory Emax evaluation for this study combined with the Phase 1 experience was essentially superimposable (Figure 11.9). The Cmin EC50 was less variable (lower standard error) due to the higher number of subjects included

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Pharmacokinetic/pharmacodynamic model simulations provided reasonable predictions of actual onset and degree of response to the predicted antisense oligonucleotide concentrations in plasma in polygenic hypercholesteremics not previously treated. Observed data (filled symbols) are medians of the sample (n ⫽ 8).

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Trough ISIS 301012 concentration (ng/mL) Figure 11.9 Overlay of both Phase 1 and 2a clinical data from early proof of concept studies indicate similar concentration–response profiles. The Cmin EC50 with the combined data is estimated to be 11 ng/mL with a standard error of 6 ng/mL. The data are regressed using a sigmoidal inhibitory Emax model.

in the estimate and the estimate was slightly lower at 11 ng/mL. The reproducibility and predictability of the exposure–response model suggests that these models may provide a powerful tool for developing optimal dose and dose regimen for later stage testing. ␣ Antisense 11.3.2 ISIS 104838, TNF␣ ISIS 104838 is a 2⬘-MOE partially modified ASO targeting human TNF␣. Initial reports described dose-dependent reduction in circulating TNF-␣ response to LPS stimulation of peripheral blood mononuclear cells (PBMCs) isolated from subjects following treatment with this antisense inhibitor [24] in healthy volunteers. In a Phase 2a study in patients with rheumatoid arthritis, investigators demonstrated that treatment with ISIS 104838 reduced TNF␣ mRNA and protein in synovial tissue biopsies that were positively correlated with concentrations of intact ISIS 104838 in synovial tissue [82]. The group average tissue levels that represented measurable concentration-dependent reduction in synovium-associated TNF␣ were
2–14 µg/g. These levels were achieved with weekly doses of 100–300 mg administered either by intravenous or subcutaneous routes. Synovial biopsy concentrations were dose-dependent and route (i.v. versus s.c.)-independent. These results are consistent with our observation that s.c. bioavailability is essentially equal to intravenous dosing for this class of PS 2⬘-MOE-modified oligonucleotide, and that tissue biodistribution is not altered by s.c. injection. 11.3.3 OGX-011, Clusterin Antisense Another example of PK/PD relationships can be found in a reporting of Phase 1 dose-ranging study (weekly dose of 120–640 mg) in patients with prostate cancer with OGX-011, a 2⬘-MOE chimera targeting clusterin mRNA [38,85]. The clusterin gene encodes a cytoprotective chaperone protein that promotes cell survival and confers broad-spectrum resistance to chemotherapy treatment.

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Inhibition of clusterin expression in prostate cancer cells that were removed surgically following antisense treatment, as assessed by several methods, including quantitative RT-PCR (qRTPCR) and immunohistochemistry, occurred in a dose and prostate drug concentration-dependent manner [38]. It was concluded from this study that the recommended Phase 2 dose of OGX-011 was 640 mg administered once weekly based on
90% inhibition of the clusterin expression in surgically removed prostate tumor tissue together with acceptable tolerability in humans at this dose. OGX-011 concentrations measured over the dose range were
1–5 ␮g/g. The average prostate tumor tissue concentration of 5 ␮g/g corresponded to
90% inhibition. This exposure–response relationship is in surprisingly good agreement with preclinical model (xenograft mouse) data [36], where tumor tissue concentrations of
600–700 nM (
4–5 ␮g/g) produced marked reductions of
80% in clusterin expression in xenograft tumor.

11.4 CONCLUSIONS Antisense-mediated reduction of target mRNA and subsequent expression of target protein using PS 2⬘-MOE-modified chimeras is dose- and concentration-dependent. As a chemical class, 2⬘-MOE partially modified PS oligonucleotides have a native biodistribution pattern that appears to be largely sequence-independent and thus instructive to the class. The relative concentrations of the 2⬘-MOE chimeras as determined by wet tissue weight (␮g/g) that result in measurable reduction in target expression are dependent on the organ, cell, or tissue type. As a general rule, those tissues that are well served by the native biodistribution of the PS oligonucleotide chemical class following systemic administration are the tissues that exhibit measurable 2⬘-MOE-modified antisense effects. These properties of 2⬘-MOE ASO pharmacology direct the use of pharmacokinetics to design appropriate dose and schedule regimens for efficient and successful development in the clinic. The added biological stability and increased mRNA-binding affinity of the 2⬘-MOE partially modified PS ASO as compared to other oligonucleotide analogs, confers a much longer biological half-life and greater potency impacting both dose and schedule of dosing. High-sensitivity bioanalytical methods provide a tool that ensures characterization of terminal elimination kinetics as well as measurement of trough plasma concentrations that correspond to tissue accumulation and clearance. Appropriate characterization of the relationship between plasma and tissue pharmacokinetics in nonclinical models will allow for better clinical trial designs. Finally, the remarkably similar pharmacokinetics independent of sequence and therefore similar across the platform of oligonucleotides in this chemical class, provide guidance for the application of lessons learned from one compound to inform the development of those that follow in the same chemical class.

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58. Henry, S. P., Miner, R. C., Drew, W. L., Fitchett, J., York-Defalco, C., Rapp, L. M., and Levin, A. A., Antiviral activity and ocular kinetics of antisense oligonucleotides designed to inhibit CMV replication, Invest. Ophthalmol. Vis. Sci. 42 (11), 2646, 2001. 59. Roy, S., Zhang, K., Roth, T., Vinogradov, S., Kao, R. S., and Kabanov, A., Reduction of fibronectin expression by intravitreal administration of antisense oligonucleotides, Nat. Biotechnol. 17, 476, 1999. 60. Leeds, J. M., Henry, S. P., Truong, L., Zutsi, A., Levin, A. A., and Kornbrust, D. J., Pharmacokinetics of a potential human cytomegalovirus therapeutic, a phosphorothioate oligonucleotide, after intravitreal injections in the rabbit, Drug Metab. Dispos. 25 (8), 921, 1997. 61. Duan, W., Chan, J. H., McKay, K., Crosby, J. R., Choo, H. H., Leung, B. P., Karras, J. G., and Wong, W. S., Inhaled p38 alpha mitogen-activated protein kinase antisense oligonucleotide attenuates asthma in mice, Am. J. Respir. Crit Care Med. 171 (6), 571, 2005. 62. Templin, M. V., Levin, A. A., Graham, M. J., Aberg, P. M., Axelsson, B. I., Butler, M., Geary, R. S., and Bennett, C. F., Pharmacokinetic and toxicity profile of a phosphorothioate oligonucleotide following inhalation delivery to lung in mice, Antisense Nucl. Acid Drug Dev. 10 (5), 359, 2000. 63. Rojanasakul, Y., Weissman, D. N., Shi, X. L., Castranova, V., Ma, J. K. H., and Liang, W. W., Antisense inhibition of silica-induced tumor necrosis factor in alveolar macrophages, J. Biol. Chem. 272 (7), 3910, 1997. 64. Nyce, J. W. and Metzger, W. J., DNA antisense therapy for asthma in an animal model, Nature 385 (6618), 721, 1997. 65. Ali, S., Leonard, S. A., Kukoly, C. A., Metzger, W. J., Wooles, W. R., McGinty, J. F., Tanaka, M., Sandrasagra, A., and Nyce, J. W., Absorption, distribution, metabolism, and excretion of a respirable antisense oligonucleotide for asthma, Am. J. Respir. Crit. Care Med. 163 (4), 989, 2001. 66. Whitesell, L., Geselowitz, D., Chavany, C., Fahmy, B., Walbridge, S., Alger, J. R., and Neckers, L. M., Stability, clearance, and disposition of intraventricularly administered oligodeoxynucleotides: implications for therapeutic application within the central nervous system, Proc. Natl. Acad. Sci. USA 90, 4665, 1993. 67. Wojcik, W. J., Swoveland, P., Zhang, X. F., and Vanguri, P., Chronic intrathecal infusion of phosphorothioate or phosphodiester antisense oligonucleotides against cytokine responsive gene-2/IP-10 in experimental allergic encephalomyelitis of Lewis rat, J. Pharmacol. Exp. Ther. 278 (1), 404, 1996. 68. Szklarczyk, A. and Kaczmarek, L., Antisense oligodeoxyribonucleotides: stability and distribution after intracerebral injection into rat brain, J. Neurosci. Methods 60 (1–2), 181, 1995. 69. Schlingensiepen, R., Goldbrunner, M., Szyrach, M. N., Stauder, G., Jachimczak, P., Bogdahn, U., Schulmeyer, F., Hau, P., and Schlingensiepen, K.-H., Intracerebral and intrathecal infusion of the TGFbeta2-specific antisense phosphorothioate oligonucleotide AP 12009 in rabbits and primates: toxicology and safety, Oligonucleotides 15 (2), 94, 2005. 70. Godfray, J. and Estibeiro, P., The potential of antisense as a CNS therapeutic, Expert Opin. Ther. Targets 7 (3), 363, 2003. 71. Butler, M., Hayes, C. S., Chappell, A., Murray, S. F., Yaksh, T. L., and Hua, X. Y., Spinal distribution and metabolism of 2⬘-O-(2-methoxyethyl)-modified oligonucleotides after intrathecal administration in rats, Neuroscience 131 (3), 705, 2005. 72. van der Aa, M. A., Mastrobattista, E., Oosting, R. S., Hennink, W. E., Koning, G. A., and Crommelin, D. J., The nuclear pore complex: the gateway to successful nonviral gene delivery, Pharm. Res. 23 (3), 447, 2006. 73. So, A., Sinnemann, S., Huntsman, D., Fazli, L., and Gleave, M., Knockdown of the cytoprotective chaperone, clusterin, chemosensitizes human breast cancer cells both in vitro and in vivo, Mol. Cancer Ther. 4 (12), 1837, 2005. 74. Koller, E., Propp, S., Zhang, H., Zhao, C., Xiao, X., Chang, M. -Y., Hirsch, S. A., Shepard, P. J., Koo, S., Murphy, C., Glazer, R. I., and Dean, N. M., Use of a chemically modified antisense oligonucleotide library to identify and validate Eg5 (kinesin-like 1) as a target for antineoplastic drug development, Cancer Res. 66 (4), 2059, 2006. 75. Butler, M., Stecker, K., and Bennett, C. F., Histological localization of phosphorothioate oligodeoxynucleotides in normal rodent tissue, Nucleosides Nucleotides 16 (7–9), 1761, 1997. 76. Lorenz, P., Misteli, T., Baker, B. F., Bennett, C. F., and Spector, D. L., Nucleocytoplasmic shuttling: a novel in vivo property of antisense phosphorothioate oligodeoxynucleotides, Nucl. Acids Res. 28 (2), 582, 2000.

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77. Yu, R. Z., Geary, R. S., and Levin, A. A., Pharmacokinetics and pharmacodynamics of antisense oligonucleotides, in Encyclopedia of Molecular Cell biology and Molecular Medicine, Meyers, R. A., ed., Wiley-VCH, Weinheim, Germany, 2007. 78. Yu, R. Z., Matson, J., and Geary, R. S., Terminal elimination rates for antisense oligonucleotides in plasma correlate with tissue clearance rates in mice and monkeys, in Annual Meeting of American Association of Pharmaceutical Scientists, Denver, CO, 2001. 79. Yu, R. Z., Baer, B., Chappel, A., Geary, R. S., Chueng, E., and Levin, A. A., Development of an ultrasensitive noncompetitive hybridization–ligation enzyme-linked immunosorbent assay for the determination of phosphorothioate oligodeoxynucleotide in plasma, Anal. Biochem. 304 (1), 19, 2002. 80. Yu, R. Z., Geary, R. S., and Levin, A. A., Application of novel quantitative bioanalytical methods for pharmacokinetic and pharmacokinetic/pharmacodynamic assessments of antisense oligonucleoutides, Curr. Opin. Drug Discov. Dev. 7 (2), 195, 2004. 81. Yacyshyn, B. R., Chey, W. Y., Goff, J., Salzberg, B., Baerg, R., Buchman, A. L., Tami, J., Yu, R., Gibiansky, E., and Shanahan, W. R., Double blind, placebo controlled trial of the remission inducing and steroid sparing properties of an ICAM-1 antisense oligodeoxynucleotide, alicaforsen (ISIS 2302), in active steroid dependent Crohn’s disease, Gut 51 (1), 30, 2002. 82. Wei, N., Fiechtner, J., Boyle, D., Kavanaugh, A., Delauter, S., Rosengren, S., Firestein, G. S., Tami, J., Yu, R., and Sewell , L., Synovial Biomarker Study of ISIS 104838, An antisense oligodeoxynucleotide targeting TNF-alpha, in rheumatoid arthritis, in The 67th Annual Meeting of the American College of Rheumatology (ACR), Orlando, FL, 2003. 83. Crooke, R., Second-generation antisense drug for cardiovascular disease demonstrates significant and durable reductions in cholesterol, in The 9th Drug Discovery Technology World Congress, Boston, MA, 2004. 84. Bradley, J. D., Crooke, R., Kjems, L. L., Graham, M., Leong, R., Yu, R., Paul, D., and Wedel, M., Hypolipidemic effects of a novel inhibitor of human APO-B 100 in humans, in The American Diabetes Association’s 65th Annual Meeting, San Diego, CA, 2005. 85. Gleave, M. and Chi, K. N., Knock-down of the cytoprotective gene, clusterin, to enhance hormone and chemosensitivity in prostate and other cancers, Ann. NY Acad. Sci. 1058, 1, 2005.

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12

Toxicologic Properties of 2⬘-O-Methoxyethyl Chimeric Antisense Inhibitors in Animals and Man Scott P. Henry, Tae-Won Kim, Kimberly Kramer-Stickland, Thomas A. Zanardi, Robert A. Fey, and Arthur A. Levin

CONTENTS 12.1

Background .........................................................................................................................328 12.1.1 Scope of the Review: Single-Strand ASO, siRNA, and Oligonucleotide Aptamers..............................................................................328 12.1.2 Phosphorothioate Deoxy versus 2⬘-O-Methoxyethyl Chimeric ASO...................328 12.1.3 Sequence Motifs....................................................................................................329 12.1.4 Safety Assessment Strategy for ASOs ..................................................................330 12.1.4.1 Exaggerated Pharmacology ..................................................................330 12.1.4.2 Off-Target Antisense Effects ................................................................331 12.1.4.3 Class-Related Toxicologic Properties...................................................331 12.1.4.4 Sequence-Specific Hybridization-Independent Effects (Aptameric Effects) ..............................................................................331 12.2 Properties of 2⬘-O-Methoxyethyl ASO ...............................................................................332 12.2.1 Toxicokinetic Properties........................................................................................332 12.2.2 Systemic versus Local Administration..................................................................333 12.2.2.1 Local Pulmonary, CNS, or Ocular Administration ..............................333 12.2.3 Oral Administration...............................................................................................334 12.2.4 General Toxicology ...............................................................................................336 12.2.4.1 Acute and Transient Changes Related to Binding of Plasma Proteins ....337 12.2.4.2 Target Organ Accumulation and Effect ................................................340 12.2.4.3 Proinflammatory Effects.......................................................................346 12.2.4.4 Chronic Administration ........................................................................351 12.2.5 Reproductive Toxicology ......................................................................................352 12.2.6 Genetic Toxicology ...............................................................................................353 12.2.7 Safety Pharmacology ............................................................................................354 12.3 Species-Specific Effects ......................................................................................................356 12.4 Conclusions .........................................................................................................................357 Acknowledgments ..........................................................................................................................357 References ......................................................................................................................................357

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12.1 BACKGROUND Antisense inhibitors continue to mature as integral research tools for understanding biological mechanisms, and have made great progress as therapeutic agents since the last comprehensive reviews in this area were published [1]. Of course, therapeutic applications are the ultimate goal for the technology, requiring a solid understanding of the molecular mechanisms, pharmacologic activity (both in vitro and in vivo), pharmacokinetic properties, toxicology, and clinical efficacy in specific disease areas. There are now numerous examples of pharmacologic activity in animal models, illustrating the ability of these inhibitors to regulate the expression of the targeted genes in vivo [2–4]. Evidence of antisense activity in patients has also been demonstrated for several targets in preliminary clinical trials [5–9], and more examples will likely follow. One pivotal area of therapeutic development where questions remain is toxicology. This is particularly the case in the context of clinical indications that have expanded beyond cancer therapy, and now include treatments for more chronic and non-life-threatening diseases, such as diabetes and cardiovascular disease. These latter indications obviously require a very thorough examination of the safety and tolerability profiles. This review will update the current state of knowledge surrounding the toxicologic assessment of these inhibitors. Much of the basics about this technology, both with regard to the toxicology, and specific mechanisms of action, have previously been reviewed and will not be repeated here [10]. 12.1.1 Scope of the Review: Single-Strand ASO, siRNA, and Oligonucleotide Aptamers This review will focus on the latest information for single-strand antisense oligonucleotides. The therapeutic properties of antisense inhibitors have been studied extensively since the early 1990s, examining many clinical indications, routes of administration, and durations of treatment. Currently, at Isis Pharmaceuticals, we have performed investigational new drug (IND)-enabling toxicology studies on eight different phosphorothioate oligodeoxynucleotides (PS ODNs; socalled first-generation oligonucleotides) (Table 12.1). Mice and monkeys are the typical species used for nonclinical safety assessment, but studies have also been performed in rats and dogs. The doses examined in repeat-dose subchronic studies range up to 200 mg/kg/week in mice and 80 mg/kg/week in monkeys. Chronic duration studies have been performed for a number of different inhibitors as well. In clinical studies resulting from these development programs, more than 4000 subjects and patients have been treated with this class of compound (for review, see Chapter 13). There have been a number of other PS ODNs advanced into toxicology evaluation and clinical trials, including an inhibitor of Bcl-2 that is currently in Phase 3 trials for cancer, that are not listed [7,8]. Thus, this experience provides a broader base of experience from which to review the toxicologic properties. 12.1.2 Phosphorothioate Deoxy versus 2⬘⬘-O-Methoxyethyl Chimeric ASO The more contemporary single-stranded antisense oligonucleotides studied since 2001 have been modified in several ways to improve hybridization affinity, increase metabolic stability, and decrease certain toxicities [10–12]. These so-called second-generation antisense inhibitors are specifically designed to have greater potency through increased hybridization affinity, and more favorable pharmacokinetic properties through decreased metabolism [11,13]. The specific modification conferring the greatest increase in potency was the 2⬘-O-methoxyethyl (2⬘-MOE) substituent on the ribose sugar of nucleobases [14–16]. The oligonucleotides still possess phosphorothioate linkages throughout, with the 2⬘-MOE substituents placed on several residues (most typically 5) each on the 3⬘ and 5⬘ ends of the oligonucleotide (Table 12.2). The center residues remain deoxy phosphorothioate nucleotides (usually at least 8–10 residues) to maintain the necessary substrate conditions for the RNase H enzyme. Thus, the strategy is for the terminal residues to provide

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Table 12.1 First- and Second-Generation Antisense Oligonucleotides That Have Progressed into Clinical Development Compound Name (Isis/Company #) PS ODN 2105

mRNA Target

2922/Vitravene GEM 91 Oblimersen 2302/Alicaforsen

Human Papilloma virus Human CMV HIV TAT Bcl-2 ICAM-1

3521/Affinitak 5132 2503 5320 14803

PKC-␣ c-Raf Kinase H-ras G-quartet aptamer Hepatitis C virus

2⬘⬘-MOE ASO 13312 13650/iCo-007

Human CMV c-Raf kinase

112989/OGX-11 107248/ATL 1102 113715 301012 23722/LY2181308 183750/LY2275797 OGX 427 ISIS 325568

Clusterin VLA-4 PTP-1b apoB-100 Survivin EIF4e HSP 27 GCGR

Indication Genital warts CMV retinitis HIV Cancer Crohn’s/ulcerative colitis Cancer Cancer Cancer HIV HCV CMV retinitis Diabetic macular edema Cancer Multiple sclerosis Diabetes Hypercholesterolemia Cancer Cancer Cancer Diabetes

Phase of Development 2 Registered 2 3 3 3 2 2 2 2 1 IND pending 2 2 2 2 2 1 1 Preclinical

Table 12.2 Structure of Typical PS ODNs and 2⬘⬘-MOE ASOs Compound

Molecular Target

Sequence (5⬘⬘ → 3⬘⬘)

ISIS 5132 ISIS 13650

Human C-raf kinase Human C-raf kinase

TsCsCsCsGsCsCsTsGsTsGsAsCsAsTsGsCsAsTsT TsC*sC*sC*sGsCsCsTsGsTsGsAsCsAsTsGsC*sAsTsT

Note: C represents deoxynucleotide; C*, 5 methyl cytosine; T, 2⬘-methoxyethyl; S, phosphorothioate linkage; and O, phosphodiester linkage.

increased hybridization affinity, resulting in an increase in potency, without compromising the mechanism of action [16]. At Isis Pharmaceuticals, attention has been focused on these 2⬘-MOE oligonucleotides since 1999, and IND-enabling toxicology evaluation has been performed on more than six unique sequences (Table 12.1). 12.1.3 Sequence Motifs There are two other important changes to most second-generation ASOs. First, with the knowledge of potential aptameric effects of the oligonucleotides, certain sequence motifs are avoided in the selection process for therapeutic compounds. One motif that is avoided is runs of 4 guanosine nucleotides in a row. These have the potential to form “G-quartets” or aggregates between multiple oligonucleotides, and thus affect the biologic activity. Another sequence motif that is avoided is CpG dinucleotide motifs [17]. This dinucleotide pair, especially when flanked by two purines on the 5⬘ end of the oligonucleotide and two pyrimidines on the 3⬘ end, results in a particular high level of proinflammatory activity for the oligonucleotide. Avoiding these motifs does not eliminate the proinflammatory effects all together, but does greatly reduce the level of this activity. Also related to minimizing the proinflammatory effect is the incorporation of 5-methyl cytosine (5-MeC) as

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opposed to cytosine. As discussed in more detail below, the proinflammatory effects result from the recognition of oligonucleotides as foreign DNA by receptors on macrophages and monocytes of the innate immune system that recognize certain patterns [18]. Bacterial DNA has a much lower content of methylated cytosine as compared to mammalian DNA. Thus, the incorporation of 5-MeC makes the oligonucleotide appear more like mammalian DNA [19]. The lower degree of proinflammatory effects for second-generation ASOs relative to first-generation ASOs results in significant improvement in tolerability. The other property that is significantly affected by incorporation of 2⬘-MOE residues is the metabolic stability. The 2⬘-modified residues are much more resistant to nuclease-mediated degradation than deoxy residues. Since clearance of phosphorothioate oligodeoxynucleotides from tissues was primarily mediated by metabolic degradation, this modification has resulted in a much longer tissue residence time [11] (Chapter 11). Since the activity of antisense oligonucleotides is determined by the tissue concentration, the longer half-life of 2⬘-MOE ASOs is managed by less frequent administration [20]. As described below, the tissue kinetics generally follow first-order kinetics and as such the tissue concentration for second-generation ASOs reach steady-state levels. These 2⬘-MOE ASOs will be the main focus of this toxicology overview. 12.1.4 Safety Assessment Strategy for ASOs The toxicology evaluation for 2⬘-MOE ASOs follows the traditional path of evaluating a range of doses in both a rodent and nonrodent species treated by the same route and duration as intended to use in clinical trials. Previous reviews have summarized that similar to any pharmacologic agent, toxicities can arise from several different sources [10]. For antisense inhibitors, consideration must be given to the potential for hybridization-dependent toxicities (i.e., those arising from hybridization to mRNA) and hybridization-independent toxicities (i.e., those arising from the interaction of the oligonucleotide with a specific protein/receptor or resulting from accumulation). The hybridization-independent effects can be either sequence-independent, which is the case with most toxicities, or occasionally sequence-dependent. The majority of observations in toxicology studies are related to hybridizationindependent effects. Since the antisense molecules tend to have common charge-to-mass ratios and pharmacokinetic behavior, the toxicologic properties also tend to be similar. Thus, while there is more quantitative variation in the toxicology properties between sequences than observed in the pharmacokinetic properties, there are a number of changes that are considered common to the chemical class. These concepts are important for the safety assessments of antisense inhibitors and are discussed in further detail below.

12.1.4.1 Exaggerated Pharmacology For gene targets where the sequence is well conserved across species, oligonucleotides that cross-hybridize from human to monkey, and even to rodent species, are selected to further facilitate the toxicologic evaluation. Oligonucleotides with sequences and activities that are homologous among species allow the examination of both exaggerated pharmacology and sequence-dependent hybridization-independent effects in the same molecule in multiple species. However, it is not always possible to get active antisense agents that are homologous across species. The strategy for these cases has generally been through the examination of both the human-specific inhibitor and another oligonucleotide with a sequence homologous to at least one of the species examined (mice or monkey) in parallel [21–23]. In this way, the toxicity profiles of pharmacologically active and inactive compounds can be compared to determine if toxicities are due to nonspecific interactions or attributable to the inhibition of intended target mRNA (so-called exaggerated pharmacology). An example of an area for specific concern would be antisense inhibitors for oncology where the intended pharmacological effects might be to increase apoptosis. Studies should be designed to insure this effect is specific to tumor cells and does not occur in parenchymal cells. Although

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exaggerated pharmacology has not been a major issue for drug development to date, species-specific antisense inhibitors must continue to be used to test for this phenomenon.

12.1.4.2 Off-Target Antisense Effects Off-target antisense effects, while theoretically possible, are statistically improbable and to date have not been observed. For these to occur and be biologically important, the oligonucleotide with homologous sequence must be able to access and hybridize with the mRNA, the mRNA must be expressed at the time of exposure and in the tissues exposed to oligonucleotide, and finally the nontarget mRNA must be critical for cell function. Concerns over the potential for off-target antisense oligonucleotides have increased with the 2⬘-MOE ASO in that the modifications increase hybridization affinity. One of the consequences of this increased affinity is that hybridization can occur with shorter regions of homology and also that hybridization events can occur with 1 or 2 sequence mismatches. Essentially, the 2⬘-MOE modifications are able to overcome the destabilization that occurs from a mismatched sequences or that exists from small regions of homology. Still, the best way to address this potential is through the careful design and interpretation of rodent and primate toxicology studies. Also, the sequence of potential therapeutic ASOs are blasted against the human genome to evaluate the potential for unintended targets. Gene arrays have been employed to a limited degree to address the possible off-target antisense effect with greater sensitivity and specificity than standard biological assessment. However, the consistency and reproducibility of these changes need to be better established and these techniques are not currently used for this purpose.

12.1.4.3 Class-Related Toxicologic Properties It still remains the case, as it was for both PS ODNs and 2⬘-MOE ASOs, that the majority of the toxicities observed are generally independent of sequence, and highly correlated with their pharmacokinetic properties. Furthermore, the class-specific effects for the 2⬘-MOE ASOs are very similar to those for PS ODNs [12,24]. The similarity is based on the fact that these molecules are all polyanions that are highly water-soluble and approximately 7200 daltons. There are subtle differences between these 2 classes of ASO. These stem from a slightly lower degree of protein binding for 2⬘-MOE ASOs that is presumably due to the steric hindrance resulting from the 2⬘-alkyl substituents, as well as greater metabolic stability than seen for PS ODNs [25]. Just as important as the consistency in the toxicity profile for 2⬘-MOE ASOs, there are a number of organ systems that are consistently unaffected by ASO treatment. This includes consistently no effect on skeletal or cardiac muscle, gastrointestinal tract, pulmonary, central nervous system (CNS), bone marrow, or mitochondrial effects. In part, this is due to the fact that the oligonucleotides are not equally distributed to all tissues. For example, oligonucleotides are not taken up into muscle cells and do not cross from systemic circulation to the CNS [11,26]. These properties help eliminate certain target organs from concern for both hybridization-dependent and hybridization-independent toxicities.

12.1.4.4 Sequence-Specific Hybridization-Independent Effects (Aptameric Effects) Despite the consistency of the toxicity profile, there remain certain effects that are sequencedependent. The most well studied are the sequence-dependent proinflammatory effects. While 2⬘-MOE ASOs tend to avoid CpG motifs altogether, the proinflammatory effects have not been completely eliminated by this procedure. While certainly of lower magnitude than for PS ODNs, proinflammatory effects are still observed in mice for 2⬘-MOE ASOs and the degree of the effect is still sequence-dependent. Since no specific motif has been identified for this, the best way to evaluate the potential is through direct experimentation. There may be other sequence motifs that control toxicities, but as of now there is no understanding of the receptor or mechanism of action. These sequence-specific effects are discussed in greater detail in the context of specific toxicities.

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12.2 PROPERTIES OF 2⬘⬘-O -METHOXYETHYL ASO 12.2.1 Toxicokinetic Properties The toxicology properties of oligonucleotides are well correlated with exposure, and thus, the PK/ADME properties of oligonucleotides are very relevant to the overall toxicology profile. In plasma, the behavior of a 2⬘-MOE ASO is very similar to that of a PS ODN with regard to Cmax, AUC, clearance, etc. [11,26]. Therefore, the monitoring of the acute and transient changes that can occur between oligonucleotides at high concentration and plasma proteins has not changed. The one parameter that has changed slightly is related to protein binding. The PS ODNs are typically about 98% bound to plasma proteins at clinically relevant concentration, whereas the 2⬘-MOE ASO are typically 92–98% bound to plasma proteins [25,27]. This lower degree of protein binding is correlated with a slightly lower propensity for complement activation as detailed below. This change in the protein binding properties may also contribute to the lower degree of proinflammatory effects observed for 2⬘-MOE ASOs. The other consideration for toxicologic evaluation is the uptake and clearance in tissues. While the profile of tissue distribution has not changed for 2⬘-MOE ASOs, the metabolic stability has increased [11,26]. The 2⬘-MOE modifications on the termini effectively protect the oligonucleotide against exonuclease degradation. The metabolism of 2⬘-MOE ASO is initiated by an endonuclease cleavage event, followed by exonuclease degradation of deoxyphosphorothioate residues [11]. The effect of this difference in metabolism is a longer half-life that has increased from 1 to 2 days for PS ODNs, to 14 to 30 days for 2⬘-MOE ASOs [11,26,28]. This obviously has the potential to result in higher accumulation of oligonucleotide in tissue and accumulation in kidney is one of the primary target organ effects in monkey. However, the longer half-life of 2⬘-MOE ASO has been balanced by a less frequent dosing regimen. Dose regimens used in 2⬘-MOE ASO toxicology studies are typically either once or twice a week, compared to the every-other-day treatment used for most PS ODNs. In recent clinical trials, dose regimens of once weekly administration have shown activity. The most important principle in this change is that the tissue concentration kinetics of oligonucleotides follow first-order kinetics [11,26]. Thus, less frequent administration of a compound with a long half-life will accumulate to a similar degree in tissues. Another critical point is that consistent with first-order kinetics, tissue concentrations reach a steady state concentration, where the rate of uptake and clearance are balanced [11,26]. As shown in the kidney concentrations for a representative 2⬘-MOE ASO, the tissue concentrations tend to increase with dosing for the first few weeks, but reach a steady-state concentration that is predictable based on the tissue elimination half-life (Figure 12.1). This is a very important consideration in the 600

Predicted steady-state max

113715 concentration kidney cortex (µg/g)

500 400 300 200 100 0 0

Figure 12.1

20

40

60 Time (days)

80

100

120

Concentrations of oligonucleotide in kidney follow first-order kinetics and achieve steady-state levels with repeated administration. The concentrations (⫾ standard deviation) of ISIS 113715 are shown (filled circles) after the first and fourth doses (after loading) as well as the washout from kidney over time. First-order models of the data were then used to simulate once-weekly dosing (loading dose model).

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context of chronic administration in that accumulation does not continue for the full duration of treatment. Instead, as illustrated below, steady-state concentrations are achieved in approximately 6–8 weeks, depending on the dose regimen used. From the toxicology perspective, the primary changes in PK/ADME properties for 2⬘-MOE ASOs relative to PS ODNs are a slight decrease in protein binding and increase in metabolic stability. The plasma PK properties and tissue distribution profiles are not significantly changed. The resulting longer tissue half-life for 2⬘-MOE ASO is compensated for by less frequent administration, resulting in more convenient treatment regimens for a parenterally administration therapeutic. 12.2.2 Systemic versus Local Administration One requirement on the effective use of antisense inhibitors is that the oligonucleotides need to get into tissues and target cells of interest in order to interact with the receptor of interest, mRNA [29]. If oligonucleotide is taken up into cells, the potential for antisense activity is good [20,30,31]. However, simple parenteral administration does not result in uniform or universal distribution to all tissues and cell types. Thus, it is difficult to get activity in some organs, such as skeletal muscle, using parenteral administration. One obvious way around this in many cases is to administer antisense inhibitors directly to the desired site of action. There are certain advantages to this approach that include more efficient delivery to the target cell type, less exposure to systemic organs that are not the intended target (e.g., kidney), and thus less concern for systemic toxicities. Of course, this approach presents certain challenges also, most notably from a toxicology perspective is the need to evaluate the local tolerability in tissues that are not typically exposed following parenteral administration. The sections that follow summarize some of the recent experience with local administration and tolerability of oligonucleotides.

12.2.2.1 Local Pulmonary, CNS, or Ocular Administration Ocular delivery of antisense inhibitors has proven to be an effective and well tolerated means of treating ocular disease with the approval of Vitravene for the treatment of CMV retinitis, and is still a route being studied for other indications using both 2⬘-MOE ASO and siRNA. The ocular application of antisense inhibitors will be reviewed in a separate chapter in this volume [32]. Delivery of nebulized solutions of oligonucleotides by aerosol exposure systems for the treatment of asthma has been investigated for several antisense inhibitors, and two separate programs using PS ODN antisense inhibitors that have progressed to clinical trials [33,34]. There are CpG optimal oligonucleotides being examined for treatment of asthma as well (for review, see Chapter 28). Oligonucleotide solutions have proved to be reasonably easily to nebulize and deliver by this method. Distribution of oligonucleotide to the lung is much more extensive and complete to both the large and small airways relative to parenteral administration, and thus provides a better opportunity for antisense activity [33,35]. At the same time, the relative exposure to systemic tissues such as kidney and liver are reduced [35]. Studies performed to look at the immunolocalization of PS ODNs have shown the oligonucleotide to be taken up in bronchiolar epithelium, as alveolar epithelium and endothelium, and well as alveolar macrophages [35]. The concentrations of oligonucleotide achieved in lung are dose-dependent, and while oligonucleotides can be measured in liver and kidney at higher doses (ⱖ3 mg/kg), the concentrations are lower than would be expected if the dose were administered systemically. Thus, the overall systemic therapeutic window is wider both because there is a lower proportion of oligonucleotide distributed to tissues by local administration and also because the total dose needed is lower on a bodyweight basis for local therapy. Aerosol delivery of antisense inhibitors has been not been associated with significant local irritation or toxicity. The primary effects associated with pulmonary delivery of PS ODN have been the increased number of cell infiltrates in the lungs of mice and the hypertrophy of alveolar macrophages [35]. The observation of increases in cell infiltrates in mice is not a surprising finding given the known

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sensitivity of rodents to PS ODN–mediated proinflammatory effects. This change is likely exaggerated in mice and similar cell infiltrates have not been observed in lungs of monkeys treated with up to 0.5 mg/kg with a representative 2⬘-MOE ASO. However, this is likely to be the most common finding associated with pulmonary delivery of oligonucleotides and a close analysis of the inflammatory effects in monkeys will reflect the actual pulmonary tolerability in people. It is important to note that even in mice, the infiltrates are composed primarily of lymphohistiocytic cells and not associated with fibrosis or other morphologic changes. Obviously, the increased presence of inflammatory cells in the lungs of asthma patients is not desirable, and is the primary focus of toxicology studies by this route for these compounds. However, given the careful selection of oligonucleotides, the comparison of relative dose responses for pharmacology and toxicity, and the appropriate interpretation of relative species sensitivity, there is a clear path for clinical development. As has been well documented, the proinflammatory effect of oligonucleotides is highly sequence- and chemistry-dependent. Thus, the selection and testing of more recent 2⬘-MOE ASO and siRNA inhibitors being studied for pulmonary indications have emphasized the need for greater potency and lower proinflammatory effects. Of the antisense inhibitors to make it to clinical trials, the adenosine A1 receptor from Epigenesis was the first [36]. More recently, Topigen has entered clinical trials with a combination of antisense inhibitors, one targeting CCR3 and the other targeting the common ␤c subunit of IL-3, IL-5, and GMCSF [34]. Both report successful completion of phase 1 and phase 2 studies with no issues of pulmonary tolerability in either normal subjects or patients with asthma. These clinical data, along with the monkey toxicology data that show little or no increase in pulmonary cell infiltrates, likely suggest that the mouse is overly sensitive to the possibility of cell infiltrates. 2⬘-MOE ASO inhibitors for asthma are also progressing to clinical trials. For a P38-␣ inhibitor in a mouse model of ovalbumin-sensitized asthma model, efficacy was reported at doses of 0.003–0.3 mg/kg every other day for five doses [37]. These doses are well below the 1–3 mg/kg dose range associated with increased cell infiltrates even in mice. If this potency observed for the P38-␣ ASO is translated to other species, pharmacologically active doses should be well below doses require to increase cell infiltrates, thus supporting the use of antisense inhibitors to treat asthma or other pulmonary disorders. Another target tissue requiring local administration is the CNS. Oligonucleotides administered by intravenous (IV) or subcutaneous (SC) injection do not cross the blood-brain barrier and therefore are restricted from the CNS [38,39]. For many uses, this is an attribute in that concern for CNS toxicity is eliminated, but if the brain or spinal cord is the desired target for pharmacology it requires that the antisense inhibitor be administered directly to the CNS. Despite this technical hurdle, the brain has been a popular target for antisense inhibitors as there are many examples of using antisense inhibitors to regulate expression of genes in the brain [40,41]. However, very little has been done to thoroughly investigate the tolerability. In fact, the data of CNS tolerability of PS ODNs or 2⬘-MOE ASOs is not well defined. Still, therapeutic application of ASOs in the brain is progressing. Another chapter in this volume describes the various indications being considered and pursued (for review, see Chapter 27). Of the more recent preliminary safety studies performed in rats and mice, there is evidence that these compounds can be tolerated in the brain. However, these studies also underscore the great difficulty in performing intracerebro ventricular (ICV) or intrathecal (IT) infusion. 12.2.3 Oral Administration Oligonucleotides for systemic diseases are administered by intravenous infusion or subcutaneous injection. As the technology moves toward more chronic applications, a more convenient route of administration would be a considered benefit to patients. Therefore, the feasibility of oral administration of oligonucleotides has been investigated for several antisense inhibitors including two separate programs using 2⬘-MOE ASOs that have progressed to clinical trials. While antisense inhibitors have attractive therapeutic potential, their oral delivery is challenging in light of their physicochemical properties such as hydrophilic-polyanionic chemistry, molecular weight, and gastrointestinal

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instability. These factors have limited antisense inhibitor administration to parenteral routes for systemic indications. Phosphorothioate oligonucleotides are negatively charged molecules with a high molecular weight (⬃7000 g/mol) and, therefore, exhibit poor bioavailability following oral administration (⬍1% dose absorbed). However, so-called absorption enhancers, or permeation enhancers, such as sodium caprate (designated C10), are known to facilitate the passage of medium-sized molecules across the mucosa of the gastrointestinal tract. It is thought that sodium caprate exerts its effect by eliciting dilations in intestinal mucosal-cell tight junctions, thereby allowing drug absorption by the paracellular route [42]. Chemically modified 2⬘-MOE ASOs have an increased resistance to nuclease metabolism that enhances both gut stability and tissue accumulation. These properties together with the longer half-life provided by 2⬘-MOE allows orally administered ASOs to achieve and maintain therapeutic levels for selected systemic indications. Oral tolerability and intestinal absorption of ISIS 301012 (human-specific 2⬘-MOE apoB ASO) with permeation enhancer have been characterized in dogs after 4 and 13 weeks of treatment. In dogs, ISIS 301012 was administered for 4 or 13 weeks by daily administration of enteric-coated tablets with C10. Daily oral administration at doses up to 100 mg/kg/day ISIS 301012 was well tolerated in stomach and intestine and produced expected changes in systemic target organs. The most consistent microscopic changes in dogs were considered indicative of cellular uptake of ISIS 301012 and included the presence of basophilic granules in the renal proximal tubular epithelial cells and Kupffer cells in the liver (Table 12.3). Basophilic granules were most evident in the renal proximal tubular epithelium, which was consistent with the highest measured concentrations in the renal cortex (up to 1641 ␮g/g total oligonucleotide). Concentrations of oligonucleotide in kidney were high enough to be associated with minimal to moderate tubular epithelial cell vacuolation in all dogs treated with ISIS 301012 at 13 weeks of treatment (Table 12.3), and the vacuolation is described in more detail in Section 12.2.4.1 (Inhibition of Clotting Time). However, no functional changes were associated with the tubular vacuolation, indicating normal renal function. There were no other treatment-related toxicities in dogs by oral administration of ISIS 301012. The dog exhibited measurable systemic absorption of oligonucleotides as determined by plasma and tissue concentrations of ISIS 301012 and its oligonucleotide metabolites (Table 12.4). The estimated bioavailability as compared to parenteral (SC) administration of ISIS 301012 in dogs ranged from approximately 2% at the highest dose of 100 mg/kg to 10% at a dose of 20 mg/kg. These data suggest saturation of oral absorption of ISIS 301012. Saturation of absorption is the most likely explanation for the relative absence of dose-dependent exposure in the dogs. Liver and kidney drug concentrations measured at the end of the study confirmed this observation (Table 12.4). The distribution profile in tissues was similar to that achieved following parenteral administration, with the highest concentrations seen in kidney cortex, followed by liver. The oral formulation of antisense inhibitor with C10 has been shown to significantly improve the absorption of orally administered oligonucleotides in animal models and because distribution is similar is not expected to produce toxicity uniquely related to oral route. Table 12.3 Comparison of Histologic Changes in the Kidneys of Dogs Treated with Oral ISIS 301012 for 13 Weeks Week 13 Parameter (Kidney) Tissue concentration (␮g/g) Basophilic granules Tubular vacuolation a

Oral dose route.

20 mg/kg/day (p.o.a)

40 mg/kg/day (p.o.)

100 mg/kg/day (p.o.)

1399 ⫾ 800 6 of 6 (minimal) 6 of 6 (minimal to mild)

1641 ⫾ 649 6 of 6 (minimal) 6 of 6 (mild to moderate)

1579 ⫾ 825 6 of 6 (minimal) 6 of 6 (minimal to moderate)

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Table 12.4 ISIS 301012 Plasma AUC and Tissue Concentrations after Three Months of Repeated Oral Dose Administration in Dogs Concentration (␮g/g) Dosea, Route

AUCb

Intact ISIS 301012 Liver

Dogs 20 mg/kg/day, p.o. 40 mg/kg/day, p.o. 100 mg/kg/day, p.o.

Day 1/29 2.05/3.58 5.04/2.08 5.43/1.11

Kidneyc

Total Oligonucleotide Liver

Kidneyc

Day 92 (24 h following last p.o. dose) 220 ⫾ 100 300 ⫾ 120 253 ⫾ 106

1340 ⫾ 684 1573 ⫾ 607 1386 ⫾ 731

237 ⫾ 110 333 ⫾ 123 282 ⫾ 108

1399 ⫾ 800 1641 ⫾ 649 1579 ⫾ 825

Note: Values are mean ⫾ standard deviation. a ISIS 301012 was administered once daily throughout the study. b Plasma AUC (␮g h/mL). c Kidney cortex for dog.

12.2.4 General Toxicology As our nonclinical development experience expands, the majority of toxicologic properties can still be characterized as “class-related” effects, that is, toxicities that are largely independent of sequence. The basis for the consistency is the remarkably similar pharmacokinetic properties from one sequence to the next. It does appear that there is a little more variability in tissue half-lives for 2⬘-MOE ASOs than was observed for PS ODNs, but the behavior is still relatively consistent for the class (Chapter 11). The basis for this consistency is that the interactions of oligonucleotides with plasma proteins, cell-surface receptor, or uptake/accumulation within cells are the source or various toxicities and these are dependent on the physical chemical characteristics of the ASOs. This consistency greatly improves the efficiency of development of this class of compound in that what is learned from one particular compound can be inferred in the design and characterization of another compound for a completely unrelated disease. Thus, the design and conduct of a development program is much more efficient and less prone to failure as development proceeds. The consistency in behavior that comes with a platform technology also contributes to the overall safety evaluation. The primary reason is that through repetition, one learns from experience how consistent or variable the changes are. It also allows one to thoroughly study a mechanism underlying specific changes and extrapolate those findings to clinical studies to define the correlation. Detailed in Table 12.5 are examples of five of the most common class-related toxicities observed in mouse and monkey, the current understanding of the mechanism, and a listing of the clinical correlates, if any. While 2⬘-MOE ASO toxicology properties tend to be similar, they are not quantitatively the same, and there is certainly more sequence-dependent variability than in the PK/ADME properties. The best examples of this variability are the proinflammatory effects that are recognized as a hybridization-independent class effect, but the potency of the effect can vary dramatically with sequence [10]. Even for 2⬘-MOE ASOs that avoid the CpG dinucleotide motifs, there is a spectrum of potency for these effects. And thus, it is still important to address the tolerability of each sequence thoroughly [12,43]. Although the cellular receptors for non-CpG oligonucleotides are not well characterized, it is likely that this variable potency is attributed at least in part to binding affinity. The proinflammatory properties of 2⬘-MOE ASOs are discussed further below. Other toxicities that are potentially sequence-dependent are those dependent upon pharmacokinetic properties and the degree of accumulation, such as renal accumulation. While the toxicologic properties are largely independent of sequence, there are notable differences in the behavior between species. The most notable and well characterized is the difference in sensitivity to proinflammatory effects between rodents and primates. This is manifested as a relative

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Table 12.5 Tabulation of the Five Most Common Toxicities with Corresponding Mechanism and Clinical Correlates Toxicity

Mechanism

Clinical Correlates

Increased aPTT

Inhibition of tenase complex

Peak plasma concentration related/transient prolongation

Proinflammatory effects

Release of cytokines via activation of monocytes/dendritic cells

Fever chills for ⬎4mg/kg with PS ODNs, less effect with 2⬘-MOE ASO, injection site erythema

Complement activation (in monkeys)

Inhibition of complement inhibitor, Factor H

Not clinically significant in man

Renal tubular changes: granules, vacuoles, degen/regen

Oligo concentration in kidneys

No renal effects observed in man to date

Thrombocytopenia

Transient sequestration: mechanisms not known

Occasionally observed. Affected by disease, concurrent medication, and sequence

absence of the lymphoproliferation and multiorgan lymphohistiocytic cell infiltrates in monkey compared to mouse. Another example is the toxicities related to the accumulation of oligonucleotide in proximal tubular epithelium. Because of the more rapid clearance of oligonucleotide from tissues in mice compared to monkeys, it is uncommon to achieve the kidney concentration in mice comparable to those in monkeys. Therefore, mouse is not a good model for studying the renal effects of oligonucleotide drugs in the kidney. Thus, in characterizing the nonclinical toxicology profile it is important to understand the species used and how best to interpret the data. For these examples, although the mice tend to be hypersensitive to the proinflammatory effects, they provide some idea of the relative, but not absolute, potential for proinflammatory effects of a given sequence. However, the monkey more directly reflects the potential for accumulation and possible effects of target organs for oligonucleotide distribution. In fact, the pharmacokinetics in humans have been shown to be very closely related to those observed in monkeys [44]. On the basis of these observations, monkey is considered more directly reflective of the potential behavior of an oligonucleotide in human. More specific details on the nonclinical development of antisense inhibitors can be found in the “points to consider” documents published by FDA [21,22,45], and previous reviews. The development still follows a traditional path, examining dose response in a rodent and nonrodent species using both male and female animals for the duration of treatment that equals the intended clinical duration of treatment.

12.2.4.1 Acute and Transient Changes Related to Binding of Plasma Proteins A tendency for alterations in clotting time and activation of the alternative complement pathway by PS ODNs has been well characterized with regard to dose-response, time-course, plasma oligonucleotide concentration relationships, and mechanism [10]. In general, inhibition of activated partial thromboplastin time (APTT) has not been a significant safety issue in clinical trials, but concern has affected the administration in clinical trials by favoring either 1- to 2-h IV infusion or SC injection as a way to manage and minimize peak plasma concentrations [46–48]. Monkey toxicology studies for 2⬘-MOE ASOs are still designed to address the immediate effects of dose administration on clotting time and complement activation. However, as summarized below, 2⬘-MOE ASOs tend to produce less potent effects on complement activation relative to PS ODNs, presumably due to lower degree of protein interaction.

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Inhibition of Clotting Time The effects of 2⬘-MOE ASOs on clotting time are very similar to those described previously for PS ODNs [49–51]. For example, the effects are specific for the intrinsic clotting pathways, affecting APTT but not prothrombin time (PT), are largely independent of sequence and species, well correlated with plasma oligonucleotide concentration, and are readily reversible as oligonucleotide is cleared from plasma. In monkeys, the typical observation of this change occurs at doses of ⱖ10 mg/kg administered by 1-h IV infusion where the peak plasma concentration of 90–100 ␮g/ml is associated with a 20–30% increase in APTT (Figure 12.2). The return to normal values over the next couple of hours is attributed to clearance of oligonucleotide from plasma. Specific mechanistic studies have not been performed for 2⬘-MOE ASOs, but it is presumed that the increase in APTT is due to the reversible interaction between oligonucleotide and components of the intrinsic tenase complex as described for PS ODNs [50,51]. Consistent with the stable pharmacokinetic properties, the APTT values are highest at the plasma Cmax and return to baseline within hours after each dose administration. There is no progression of the effect over repeated administration, even in chronic studies. In subjects, similar dose-dependent increases in APTT that peak at the end of infusion and return to baseline have been reported for 2⬘-MOE ASO [48]. Despite these effects on APTT, there are typically no signs of altered coagulation status, with no evidence of internal organ hemorrhage. Fibrinogen and d-dimer levels are also routinely measured in monkey studies, but are unaffected. Superficial bruising associated with the site of venipuncture or restraint is the one observation associated with prolongation of clotting times at doses ⱖ50 mg/kg [52], but the observation has been less frequent with 2⬘-MOE ASOs even with treatment up to 80 mg/kg by SC injection. With the advent of other classes of antisense inhibitors, this effect on clotting time is likely to be mostly associated with the oligonucleotides containing phosphorothioate linkages. siRNA inhibitors with diester linkages, or even those that are only partially stabilized with phosphorothioate linkages are less likely to affect clotting times due to much lower degree of plasma protein binding and more rapid clearance from plasma. Morpholino oligonucleotides also will have no effect of clotting time because of their neutral charge and lack of protein interaction.

40

Control 1 mg/kg 3 mg/kg 10 mg/kg

35

APTT (s)

20 mg/kg

30

25

20

0

20 40 60 80 100 Time from start of infusion/dose (min)

120

Figure 12.2 Effects of ISIS 113715 on APTT following 1-h IV infusion of 1–10 mg/kg or by SC injection at 20 mg/kg. Values represent the mean and standard deviation from 6–8 monkeys in each group.

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One unique application of oligonucleotides in this context worth mentioning in this section is the thrombin-binding oligonucleotide aptamers [53,54]. In this case, oligonucleotides have been designed and selected to specifically interact with thrombin and inhibit its function. These various aptamers have specific consensus sequences that are composed largely of guanosine and thymidine residue and have been shown to interact specifically with either anion Exosite I or Exosite II. While these specific aptamers function through a distinct mechanism and are not representative of the antisense oligonucleotides, it is evidence of some sequence-specific interactions, and thus illustrates the importance of evaluating this parameter as part of the toxicology evaluation.

Complement Activation The mechanism and secondary changes associated with complement activation by PS ODNs in monkeys has been reviewed previously and will not be repeated here [10]. Complement activation has been shown to be the source of hemodynamic changes reported in monkeys treated with rapid infusion of PS ODNs [55]. The activation of the alternative pathway of complement by oligonucleotides has been more of an issue in the monkey toxicology studies due to the consequence of the anaphylactic-like response to C5a production, but has not been observed in clinical trials because of the well-defined threshold plasma concentration in monkey required for activation that is avoided as part of the clinical dose regimens [48,56]. Also, there are some data which suggest that humans are less sensitive to alternative pathway activation by oligonucleotides [10]. Similar to the effects of PS ODNs, the activation of complement is attributed to interaction between the oligonucleotide and regulatory proteins of the complement cascade, specifically Factor H [56]. This oligonucleotide–protein interaction is highly concentration-dependent and readily reversible as oligonucleotide is cleared from plasma, so the effect is transient. Complement activation by 2⬘-MOE ASOs is possible and has been observed, but the overall potency appears lower than observed for PS ODNs [24]. Again, this is attributed to the lower degree of protein binding by 2⬘-MOE ASOs. The lower degree of potency is illustrated by comparing the relationship between plasma oligonucleotide concentration, and the concentration of the alternative pathway split product Bb, used to monitor for complement activation (Figure 12.3). Compared to a representative PS ODN where the threshold oligonucleotide concentration is approximately 70 ␮g/ml, the threshold Control

6

1 mg/kg 3 mg/kg

Complement Bb (µg/ml)

10 mg/kg 20 mg/kg

4

2

0 0

4

8

12

16

20

24

Time from start of infusion/dose (min) Figure 12.3 Complement activation following 1-h IV infusion of 1–10 mg/kg ISIS 113715 or by SC injection of 20 mg/kg. Values represent the mean and standard deviation from 6–8 monkeys in each group.

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concentration for 2⬘-MOE ASOs is in the range 90–100 ␮g/ml or higher [24]. There are several 2⬘-MOE ASO where complement activation was not observed. Because of the doses used in clinical trials and the ability to minimize the peak plasma concentrations by prolonging the duration of IV infusion, no complement activation has been observed in clinical trials [48].

12.2.4.2 Target Organ Accumulation and Effect Similar to many pharmacologic agents, there is a reasonably close correlation between exposure to drug and target organ effect for antisense oligonucleotides. This relationship for oligonucleotides is particularly well defined since it is the tissue concentrations that are required for pharmacologic action as well. This section primarily focuses on the class-specific target organ exposure and toxicity properties. The one notable exception to the strict exposure/effect relationship for oligonucleotides is the potential for proinflammatory effects where the primary target organs of spleen and lymph nodes are not among the tissues containing the highest concentrations. However, these effects are still dose- and concentration-dependent and are described separately (Section 12.2.4.3). This section will also focus primarily on 2⬘-MOE ASOs, pointing out similarities and a few differences with respect to PS ODNs that have been reviewed previously [10]. One aspect of the exposure/effect relationship for oligonucleotides worth emphasizing is that the basis of the class-specific toxicities are the very well conserved pharmacokinetic properties for oligonucleotides within a given class (i.e., PS ODN, 2⬘-MOE ASO, or siRNA). There is clearly more variability between sequences in the toxicity profiles than pharmacokinetic properties, but the exposure basis for potential changes is remarkably consistent. For the same reasons, there are a number of potential toxicities or potential target organs that are consistently not affected by treatment. For example, the absorption of oligonucleotide into brain, skeletal muscle, or heart muscle is essentially absent following systemic administration, and no toxicities in these particular organs have ever been reported [10,39]. Similarly, very low levels of oligonucleotide are delivered to gastrointestinal, ocular, testes, or lung tissue following IV or SC administration, and these are also not considered target organs for toxicity. The only changes reported in intestine and lung have been the increased presence of lymphohistiocytic cell infiltrates in mice or rats, which are more attributable to the increased presence of proinflammatory cytokines and chemokines produced by the oligonucleotide systemically rather than a direct effect of the oligonucleotide on these organs specifically. No intestinal, ocular, or pulmonary effects have been reported in monkeys. As a result of the typical target and nontarget organs, the design, conduct, and interpretation of toxicology evaluations is very efficient for this class of compound.

Kidney Kidney is the organ that contains the highest oligonucleotide concentration following oligonucleotide administration, and is arguably the primary target organ for toxicity in monkeys and rats. The high concentration of oligonucleotide in kidney is the result of the physiological handling of this material by the kidney in which oligonucleotide that is filtered at the glomerulus is readily reabsorbed by the proximal tubular epithelium [57]. The oligonucleotide filtered by the glomerulus has been shown to interact with brush boarder epithelium and is taken up into these cells by endocytosis, much in the same way as these cells reabsorb low-molecular-weight proteins from the filtrate [58]. Thus, the uptake and processing of oligonucleotide by these cells is consistent with the functional role that these cells play in the kidney. Immunohistochemistry and other techniques have been used to determine that concentrations of oligonucleotide within kidney are particularly high in the proximal tubular epithelium [57,59]. Following uptake, the majority of oligonucleotide in these cells resides in membrane-bound subcellular compartments, such as endosomes and lysosomes. At the higher doses examined in toxicology studies, these cells can achieve very high concentrations

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of oligonucleotide [12,60]. It is these high concentrations of oligonucleotide that lead to the histologic changes in kidney, which are typically limited to proximal tubular epithelium [52,61]. A thorough description of the histologic changes for a 2⬘-MOE ASO along with the interpretation of the findings and assessment of functional consequences is provided below. The basic toxicological properties of a PS ODN and a 2⬘-MOE ASO in kidney are the same [12]. The difference is that the stability, and thus longer clearance half-life for the 2⬘-MOE ASO, can result in higher kidney concentrations. This difference in half-life is compensated for in dose regimens that have increased from every-other-day administration of PS ODNs, to once-weekly dose regimens for 2⬘-MOE ASOs. The summary below focuses mostly on the tissue–concentration relationship that is relevant for any particular class of antisense oligonucleotide or dose regimen used. The renal tolerability of siRNAs is not discuss here, but kidney is not likely to be a target organ for toxicity in their current unstabilized iterations because they will not achieve high enough concentrations to produce effects. The preferential uptake of oligonucleotide by proximal tubular epithelium and processing into endosomes and lysosomes is similar in mice, rats, and monkeys. Our experience with numerous PS ODN and 2⬘-MOE ASO oligonucleotides indicates that that the uptake and absorption are also very consistent within the class. However, since effects in the tissue are dictated by the concentration, subtle differences in pharmacokinetics that alter renal accumulation either due to distribution or clearance will impact the overall profile (for review, see Chapter 8). Also the relative rate of clearance for a given species will dictate the changes observed, and thus changes are more commonly observed in monkeys relative to mice, where the tissue half-lives are much shorter. The summary that follows focuses primarily on our experience in monkeys.

Summary of Tubular Morphologic Changes For many animals treated with either PS ODNs or 2⬘-MOE ASOs, the histologic hallmark of oligonucleotide exposure in kidney is the presence of basophilic granules in the proximal tubular epithelium. Similar to endogenous nucleic acids, these oligonucleotides stain with hematoxylin. Therefore, cells that accumulate 2⬘-MOE ASOs or their metabolites will have increased basophilic staining in the cytoplasm. This is particularly noteworthy in cells that take up oligonucleotide in endosomes or phagolysosomes. Endosomal accumulation in cells of the proximal tubular epithelium appears microscopically as basophilic granules [52,60,61]. Basophilic granules, therefore, are considered evidence of oligonucleotide uptake and compartmentalization in these cells. The pattern and distribution of these basophilic granules (i.e., oligonucleotide) in the tubular epithelium is the same as for normal low-molecular-weight protein substrates, such as ␤2-microglobulin. Thus, in the absence of changes in cell morphology, basophilic granules are not considered toxicologically significant. In addition to basophilic granulation, minimal to moderate cytoplasmic vacuolation of tubular epithelium was seen in most monkeys dosed with ⬎10 mg/kg/week [52]. Vacuolation of the tubular epithelium is occasionally observed as a background observation because of the very extensive endosomal/lysosomal activity in these cells. However, the size and incidence of these vacuolar changes can be significantly increased by oligonucleotide treatment. The vacuolation is typically characterized by single-to-multiple large clear cytoplasmic vacuoles. Occasionally, this vacuolation was associated with apparent swelling and rupture of epithelial cells. Electron microscopic analysis showed that vacuoles were distended phagosomes or phagolysosomes containing electron-dense material (data not shown). The electron-dense material has characteristics of nucleic acid material that is contained within subcellular membrane. Vacuolation appeared greater in S1-S2 segments compared to S3 segments, consistent with the greater complexity and efficiency of the lysosomal/vacuolar apparatus in the S1-S2, particularly S2 segment. Changes were generally not observed in the basolateral membrane. Vacuolation, and the mild degenerative changes, were all reversible and not associated with evidence of tubular dysfunction in serum chemistry parameters, specific measures of renal physiology, or urinalysis parameters [62]. On the basis of the data available, we reason that the vacuoles result from the extraction of oligonucleotide during processing and/or osmotic imbalances induced by the localized high concentrations

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of hydroscopic material in phagolysosomes. Oligonucleotides are highly water-soluble molecules that could easily be extracted if not completely cross-linked by fixative in tissues. Because of these properties, it is also possible that high concentrations of oligonucleotide contained in membrane-bound subcellular compartments would be osmotically active upon fixation, particularly in slower fixation processes, such as immersion in formalin. The type of cytoplasmic vacuolation observed in 2⬘-MOE ASO–treated monkeys is most closely correlated with osmotic nephrosis that has been described for a number of hydrophilic compounds including sugars, proteins, and pegylated proteins, and hypokalemia [63–65]. In each of these examples, a relatively innocuous compound (e.g., mannitol or sucrose) is concentrated in membrane-bound vesicles in the proximal tubule and it is at these sites that influxes of water and osmotic imbalances result in swelling of these membrane-bound aggregations of solute, giving the appearance of vacuoles. Ultrastructural examination of the tissues differentiates the vacuolation at high concentrations of 2⬘-MOE ASO from vacuolar degeneration produced by tubular cell toxicants such as cisplatin, cyclosporine, mercuric chloride, and methotrexate [66–69]. In contrast to the swelling of phagolysosomes observed at high concentrations of ISIS 113715, cytotoxic compounds produce generalized swelling of organelles and vacuolar degeneration as the osmotic balance in the entire cell is disrupted in the process of cell death. Generalized organelle swelling is not manifested in the tubules in which 2⬘-MOE ASO has accumulated in granules or vacuoles. Thus, it appears that the vacuolation induced by ISIS 113715 is markedly different than vacuolar degeneration. Data suggest that membrane-bound accumulations of oligonucleotide develop into vacuoles during the handling and fixing of tissues. Because oligonucleotide is concentrated in membrane-bound vesicles in vivo, at the time of tissue harvesting and fixation there are osmotic changes that result in the appearance of the artifact. The best evidence for a role of tissue processing was that little to no vacuolation was associated with more rapid and optimal fixation of tissues with paraformaldehyde and gluteraldehyde even at the higher dose levels tested (40 mg/kg/week) [62]. Thus, significant care needs to be taken in the handling and processing of kidney for histologic evaluation. We recommend fixing separate portions of kidney cortex by both emersion in 10% formalin and 3% paraformaldehyde or 3% gluteraldehyde. There are other dose- and concentration-dependent changes in renal morphology associated with oligonucleotide treatment. The changes range from minimal reductions in the height of the brush border and height of the proximal tubular cells (i.e., tubular cell atrophy), to tubular cell regeneration, focal tubular epithelial cell degeneration, and finally frank epithelial cell degeneration at high doses and concentrations of oligonucleotide (⬎5000 ␮g/g in renal cortex). Minimal proximal tubular cell degeneration is a common dose-related finding in studies with this and other related oligonucleotides, and is often characterized by damage or sloughing of individual tubular epithelial cells. These types of minimal changes would be expected in a cell that was sequestering high concentrations of oligonucleotide in phagolysomes. At doses of 10 and 20 mg/kg/week 2⬘-MOE ASO in monkey, the degree of cellular degeneration is minimal and not considered toxicologically important, based on the scattered incidence of single degenerative epithelial cells and the absence of associated functional alterations. At doses of ⱖ40 mg/kg/week, the tubular cell degenerative changes can begin to change tubular function as indicated by a slight increase in urine protein [61,62]. The accumulation in proximal tubules and associated changes are not unique to 2⬘-MOE ASOs. At doses of PS ODNs ⱖ40 mg/kg/week, significant alterations in proximal tubular structure are observed at tissue concentrations ⱖ3000 ␮g/g [61]. Our experience with 2⬘-MOE ASOs suggests that marked tubular degeneration is achieved only at doses of ⱖ80 mg/kg/week and tissue concentrations ⱖ5000 ␮g/g. The mechanism of tubular epithelial cell degeneration at these very high dose effects is unknown, but appears to be related to excessive accumulation of oligonucleotides in these cells. Renal tubular changes such as the presence of granules, inclusions, cellular atrophic, and degenerative changes are highly concentration-dependent and the relationships between dose, concentration, and morphologic changes have been characterized. Figure 12.4 represents a qualitative representation of these changes and the relationship to the estimated tissue concentrations at

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Marked

Moderate

Basophilic Kidney concentration at granulation 50 to 200 mg/week in patients

Atroph/regen Tubular vacuolation

Mild

Tubular cell degen

Minimal 500

1000

2000

3000

4000

Kidney cortex concentration (µg/g) Figure 12.4 Qualitative representation of the correlation between tissue concentration– and histopathology-related changes in monkey kidney after 2⬘-MOE ASO treatment for 13 weeks. The indicated range of kidney concentration in patients treated with doses of 50–200 mg (approximately 0.7–3 mg/kg for a 70-kg individual) is estimated from the pharmacokinetic parameters in monkeys, which are similar to humans.

clinically relevant doses [2]. This illustrates that kidney concentrations were three- to fivefold lower than the concentrations associated with minimal epithelial cell degeneration. This characterization of the dose–concentration effect relationship is the basis for our assessment of the safety of these compounds. The extrapolation from our understanding of the concentration–response relationships in monkeys to predicting these relationships in humans is rooted in the similarities of the pharmacokinetics and disposition of oligonucleotides in monkey and man. Monkey and human pharmacokinetic data with 2⬘-MOE ASOs are superimposable and generally predictable from one oligonucleotide to another [11].

Tubular Morphologic Changes and Treatment Duration in the Monkey While a safety margin exists between clinical exposure and effect in the kidney, the obvious unanswered question is for the long-term progression and tolerability. It is important first to appreciate that while there is a long half-life for oligonucleotide in tissue, the accumulation of oligonucleotide reaches a steady-state level. The experience from numerous 2⬘-MOE ASOs in development demonstrates that these compounds follow first-order elimination kinetics and, therefore, tissue concentrations attain a steady state with time (Figure 12.1). It is possible to predict steady-state tissue concentrations of oligonucleotide using dose, dose frequency, and tissue half-life [11]. Data from 4- and 13-week studies confirm the predictions that concentrations reach steady state (Isis Pharmaceuticals, Inc., unpublished data). Furthermore, the degree of accumulation can be managed by adjusting dose and frequency of administration. Often, 13-week studies employ a loading regimen in which three to four doses are administered in the first week to approach steady state (70–80%). After the first week, the dose regimen for 2⬘-MOE ASOs are typically either once or twice weekly. Because of first-order kinetics, tissue concentrations of oligonucleotide reach steady state and do not continue to increase over time. These kinetics drive reversibility as well. Thus, while it may take 13 weeks or longer to get complete clearance, approximately 80% clearance is obtained by 2 half-lives. Since the effects on kidney appear to be determined by the tissue concentration, the clearance of drug and reversibility will occur in a reasonable time frame. If renal tubular epithelial cell changes are strictly concentration-dependent, then the duration of exposure should be less important than dose in producing changes. This in fact appears to be the

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Table 12.6 Concentrations and Morphologic Changes in Monkeys Treated with 20mg/kg/week ISIS 113715 at 6 and 13 Weeks Parameter

6 Weeks a

Tissue concentration (␮g/g) Tubular vacuolation Tubular degeneration

2233 ⫾ 1085 2 of 4 (mild to moderate) 2 of 4 (minimal)

13 Weeks 2644 ⫾ 887 3 of 4 (minimal to moderate) 2 of 4 (minimal)

a Presented as mean with standard deviation. Dose regimen used included every fourth day dosing regimen following by-weekly dosing at 20 mg/kg.

case, as kidney cortex concentration is a more critical factor in determining the response of the renal tubular epithelium than exposure time. For example, as duration of treatment with 2⬘-MOE ASOs increases from 1 to 3 months, kidney cortex concentrations were similar and there was no progression of the morphologic changes in the proximal tubules of monkeys (Table 12.6). This absence of progression was observed for other sequences as well. These observations suggest that once a steady-state kidney cortex concentration is achieved in monkeys, there is no further progression of tubular effects up to 13 weeks. Since the changes in tubular epithelial cell morphology are so closely associated with concentrations in this cell, and this cell has evolved to reabsorbing/processing solutes from lumen, it is logical that the effects will not be progressive. At more clinically relevant doses, the tubular changes were less frequent and less pronounced than that observed at the high dose. Furthermore, toxicity studies have used once-weekly dosing regimens. The clinical regimen may ultimately be a less frequent regimen or lower doses that would produce lower renal cortex concentrations and thus less exposure.

Correlation of Tubular Morphology with Renal Functional Changes in the Monkey The effect of oligonucleotide exposure and accumulation in cortex on the renal function was studied in monkeys treated with a 2⬘-MOE ASO, ISIS 113715, using a loading regimen followed by weekly dosing for a month. The high dose in this study, 40 mg/kg/week (administered on days 1, 3, 5, 7, 14, 21, and 28), produced a typical pattern of vacuoles and minimal proximal tubular degeneration after a month of treatment [62]. There was mild vacuolation and tubular epithelial cell degeneration at the 40 mg/kg/week dose level with no evidence of other tubular or glomerular changes. In this study, there was a thorough evaluation of the standard markers of renal function as well as detailed assessment of tubular epithelial cell function. The only change associated with these histologic changes at the higher doses is a low incidence of mild increase in urine protein. The proteinuria was measured as an increase in the protein/creatinine ratio from approximately 0.2 at baseline to 1.2. Electrophoretic characterization of protein excreted in monkeys treated with oligonucleotides has been shown to be composed of low-molecular-weight proteins, and are best correlated with the degree of tubular degeneration [61, unpublished Isis Pharmaceuticals, Inc.]. With the exception of these changes in protein excretion, there were no other functional changes observed. There were no changes in mean serum creatinine or blood urea nitrogen (BUN) and no changes in electrolytes. Effective renal plasma flow rate and glomerular filtration rate were unchanged in treated monkeys and there were no changes in the excretion of substrates for tubular reabsorption, including glucose, amino acids, or ␤2-microglobulin [62]. Nor were there treatmentrelated changes in typical markers of tubular damage such as N-acetyl glucosaminidase (NAG). Stresses such as water deprivation and glucose challenge were also assessed and there were no changes in concentrating ability and threshold for glucose spillage in the kidneys of treated monkeys even at the highest dose of 40 mg/kg/week. These data represent the best assessment possible of renal and tubular epithelial cell function and clearly demonstrate that there are no functional changes associated with the high concentration of oligonucleotide in tubular epithelium or cytoplasmic vacuolation. The slight increase in low-molecular-weight protein was correlated with minimal degenerative changes in epithelial cells.

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Table 12.7 Reversibility of Histologic Changes in Monkeys Treated with 10 mg/kg/week ISIS 113715 for 13 Weeks Parameter Tissue concentration (␮g/g) Basophilic granules Cytoplasmic vacuolation Tubular epithelial cell degeneration

End of Treatment (13 Weeks) 1800 ⫾ 880 6 of 6 (mild to moderate) 5 of 6 (mild to mild) 3 of 6 (minimal)

End of Recovery (13 Week Tx Free) 153 ⫾ 115 2 of 4 (minimal) 1 of 4 (minimal) None

Importantly, there has been no suggestion of altered glomerular morphology or function in the studies performed in monkeys with ISIS 113715. The supporting data includes routine histologic, ultrastructural, clinical pathology, and urinalysis evaluation at doses up to 20 mg/kg/week administered for 13 weeks or 40 mg/kg/week administered for 4 weeks. Additionally, the glomerular filtration rate was assessed as part of the renal physiology study at 40 mg/kg/week. The absence of apparent changes in glomerular morphology or function is consistent with the relative concentration of oligonucleotide in the cortex, which is much higher in tubular epithelium than glomerulus [57,59].

Reversibility of Renal Effects in the Monkey The morphologic changes in tubular epithelium observed in monkeys are reversible. In monkeys treated with ISIS 113715, the basophilic granules, cytoplasmic vacuolation, and tubular epithelial cell degenerative changes reverse with the cessation of treatment and the clearance of drug from the kidney (Table 12.7). The clearance rate is closely correlated with the rate of oligonucleotide clearance from kidney. Proximal tubular epithelium normally undergoes a normal turnover process, and so is easily capable of recovering once drug is cleared. For most 2⬘-MOE ASOs, the half-life in kidney is approximately 2 weeks. Thus, to get close to complete clearance takes about 13 weeks. However, it is important to remember that these compounds follow first-order kinetics. As such, 75% of clearance will occur after two half-lives, which is approximately 1 month. In cases where multiple time points have been taken during recovery, there is evidence for reversibility of histologic changes in kidney once concentrations are below 1200–1500 ␮g/g, as would be predicted by Figure 12.4. Liver Liver is the organ that contains the second highest oligonucleotide concentration in all species studied [11,70]. Similar to kidney, the pharmacokinetic and toxicologic properties of both PS ODN and 2⬘-MOE ASO are quite similar, differing primarily the rate of clearance and therefore the concentration at any given dose and regimen [26]. But, the concentration–effect relationship was similar between the two classes of oligonucleotides. Liver is different from kidney, however, in that oligonucleotide is present in all the cell types within liver [59,71]. Of course, it is the presence of oligonucleotide in hepatocytes that makes those cells good targets for antisense activity. Antisense activity has also been demonstrated in Kupffer cells [72,73]. Of the 2⬘-MOE ASOs that progress to development, the toxicologic properties in liver are thus very much like those reviewed previously for PS ODNs [10]. The most commonly observed histopathologic change in liver from treated mice and monkeys is the presence of basophilic granules in the cytoplasm of Kupffer cells and Kupffer cell hypertrophy. This reflects the normal function of Kupffer cells to take up foreign material, and the basophilic granules are oligonucleotide staining with hematoxylin. In monkeys, these are the only histologic observations and they are typically not associated with any increase in the enzyme markers of hepatocyte damage or liver dysfunction. This includes dose regimens for a couple of different 2⬘-MOE ASOs that range up to 140 mg/kg/week for 5 weeks, or 80 mg/kg/week for 13 weeks (Isis Pharmaceuticals, Inc., unpublished observations).

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Rodents treated with 2⬘-MOE ASOs, as was the case with PS ODNs, have the additional finding of increased lymphohistiocytic cell infiltrates as a consequence of the proinflammatory effects of oligonucleotides in mice and rats [10,12]. These infiltrates are dose-dependent and can be associated with single hepatocyte necrosis and increases in serum ALT and AST. The connection between the cell infiltrates and hepatoxicity observed in mice has been well established using chemical modifications of oligonucleotide to affect the degree of proinflammatory effects [74,75]. Consistent with the monkey data, PS ODNs and 2⬘-MOE ASOs have been largely deficient of hepatotoxicity in clinical trials (Chapter 13). The only notable exception was the report of mild and transient increases in ALT of patients treated with the anti-HIV PS ODN, GEM 91 [76]. This was also likely attributable to the proinflammatory effects that were present for this early generation of ASO and included 27 nucleotide residues along with CpG motifs. The most notable development in the area of hepatotoxicity for 2⬘-MOE ASOs, and some other chemical modifications, including linked nucleic acids (LNA)-modified oligonucleotides is the relatively recent observation of a small subset of ASOs with certain sequences that have a propensity for producing particularly potent hepatotoxicity in mice and rats (E. Swayze et al., unpublished observation). The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. The relevance of these findings to monkeys is also not well defined, but there is one example where an oligonucleotide that was hepatotoxic in mice was not hepatotoxic in monkeys (Isis Pharmaceuticals, Inc., unpublished data). Intense effort has been made to understand the sequence specificity of these effects and progress toward defining particular motifs is being made. Still, the best method for addressing these potential effects is through screening of multiple potential lead compounds in mice. Our typical screen is to dose mice at 50–100 mg/kg/week for 4 weeks and measure ALT and AST. Some increases in transaminases with this regimen are expected, but oligonucleotides that produce more than a tenfold increase in transaminases at this dose level are not selected for therapeutic development. The species specificity of these more hepatotoxic ASOs is not known. There is some evidence that the mechanism may be related to proinflammatory effects, and therefore is species-specific. We have experience with one compound that was hepatotoxic in mice, but was not hepatotoxic in monkeys, further pointing to some species specificity in the mechanism. Still, these compounds are avoided, to the degree possible, for development purposes.

Hematopoietic An effect of oligonucleotide administration on platelets is something that has been observed for PS ODNs in mice and correlated with splenomegaly associated with the proinflammatory effects [10]. Some transient effects on platelet count in patients in clinical trials with PS ODNs also have been reported [77–80]. The observation of thrombocytopenia for 2⬘-MOE ASOs in rodents is far less than observed with PS ODNs, consistent with the lower degree of splenomegaly [12]. Still, there is at least one 2⬘-MOE ASO that has produced decrease in platelet count both in monkey and man [48]. Thrombocytopenia in both species was dose-dependent and transient. The cause of the decrease in platelet count in the clinical cases for both PS ODNs and 2⬘-MOE ASOs are not considered to be related to splenomegaly, but could nonetheless be related to some increase in proinflammatory cytokines. It is possible that these cytokines or chemokines may have secondary effects on platelet production or platelet sequestration. For example, IL-10 has been associated with mild and transient underproduction of platelets in people [81]. This effect on platelets has not been a common observation with 2⬘-MOE ASOs, but is a parameter that should be closely monitored for novel oligonucleotides.

12.2.4.3 Proinflammatory Effects The most common and well-studied of the hybridization-independent effects associated with oligonucleotide administration is the tendency to stimulate a proinflammatory reaction [10,82].

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However, while this is appropriately considered a class effect, the degree of proinflammatory effect depends on several variables, and so the context of the type of oligonucleotide is very important to consider. There are a number of factors that influence the potency of the proinflammatory effects, including oligonucleotide sequence, base modifications, and backbone chemistry, which are discussed in more detail below. The most extreme example of the proinflammatory effects is the class of oligonucleotide aptamers containing specific sequence motifs designed to produce a shift toward Th1-type immunity for therapeutic purposes [18]. These so-called immunomodulatory oligonucleotides have been studied in great detail and are reviewed specifically in Chapter 27. This review will focus on the effect and consequence of proinflammatory effects associated with antisense oligonucleotides, which differ in meaningful way from the immunomodulatory oligonucleotides. It is known that the basis for the sequence specificity of this effect are specific motifs in the oligonucleotides that closely mimic bacterial DNA, and are therefore recognized by pathogenassociated molecular pattern (PAMP) receptors of the innate immune system to activate B cells, monocytes, macrophages, and dendritic cells [83,84]. As it turns out, a sequence motif as simple as a Cytosine-Guanosine (CpG) dimer flanked by two 5⬘-purine and two 3⬘-pyrimidines is sufficient to be recognized by these PAMPs as bacterial-like in rodents [85]. The basis for this recognition is the under representation of the CpG dimers in mammalian DNA [85]. As a result, the design of antisense oligonucleotides has been greatly influenced by the emergence of the immunomodulatory oligonucleotides. Without this knowledge, early antisense inhibitors, such as GEM 91, fomivirsen, and others contained CpG dimers, and although they did not contain the optimal flanking sequences, they were relatively effective proinflammatory agents [60,86–88]. These effects were the basis of some speculating that the pharmacologic effects of these compounds were not mediated by antisense activity [89–91]. Obviously, these proinflammatory effects are undesirable for antisense oligonucleotides designed to have other specific pharmacological effects, and so more contemporary ASOs avoid CpG dimers all together. Another observation based on differences between bacterial and mammalian DNA was that a CpG dimer that contained 5-methyl Cytosine (5-mehtyl C), as opposed to Cytosine was far less proinflammatory [85]. This was consistent with the relative higher presence of 5-methyl C in mammalian DNA. So now, many ASOs contain 5-methyl C to make them more like mammalian nucleic acids. These two simple modifications have themselves greatly reduced the level of proinflammatory effects in ASO. Addition of 2⬘-alkoxy substituents on the ribose has further decreased the degree of proinflammatory effects [74,75]. The modified oligonucleotides we have the most experience with are the 2⬘-MOE ASOs, which produce far less splenomegally than PS ODNs (Figure 12.5). Consistent with less effect on spleen weight, 2⬘-MOE ASOs selected for therapeutic development produce less increase in chemokines and essentially no increase in IL-6 or IL-12 (Figure 12.5). Other modifications of the backbone can be made to further decrease the proinflammatory effects. For example, decreasing the phosphorothioate content in the context of other changes decreases the proinflammatory effects [92]. Morpholino or PNA oligonucleotides have virtually no proinflammatory effects due to their neutral charge and distinct chemical nature. From the perspective of toxicology evaluation and safety assessment, the single most important aspect of this proinflammatory effect is that the changes are mediated by direct activation of cells of the innate immune system. ASOs are not recognized by the adaptive immune system as a foreign antigen [18]. This means no production of oligonucleotide-specific antibody response, and thus, no drug neutralizing antibodies and little risk of serious antigenic or hypersensitivity response. The more proinflammatory PS ODNs are associated with B-cell proliferation resulting in increased total immunoglobulin production, but this response has been shown to be polyclonal in nature [93–95]. As a result, there is little concern for hypersensitivity reaction and no evidence thereof observed or reported (for review, see Chapter 13). This is not to say that the proinflammatory effects of oligonucleotides are not of concern or at least considerable nuisance in many examples. However, it does avoid the most potentially critical sensitization/challenge-type reactions that could potential trigger

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Spleen weight 4 2 mg/kg 10 mg/kg

Spleen/BW ratio (fold increase)

3

50 mg/kg

2

1

0 ISIS 5132 First generation

ISIS 13650 Second generation

Figure 12.5 2⬘-MOE ASOs result in less splenomegaly in mice treated for 2 weeks than PS ODNs. ISIS 13650 is representative of the 2⬘-MOE ASO, while ISIS 5132 is a PS ODN with the same sequence. Values are plotted as the fold increase in spleen to body weight ratio.

a hypersensitivity reaction at very low doses. Instead, the proinflammatory effects associated with PS ODNs or 2⬘-MOE ASOs follow much more typical dose-response patterns. The types of changes associated with the proinflammatory effects of oligonucleotides are largely a consequence of cellular activation and subsequent cytokine and chemokine release. This is manifested as the well-described dose-dependent increase in lymphoid organ weight, lymphoid hyperplasia, and multiorgan-lymphohistiocytic cell infiltrate observed primarily in mice [10]. For PS ODNs, these effects are well correlated with increases in Th1-type cytokines, which include IL-1, IL-6, IL-12, IFN-
, and chemokines MCP-1 and MIP-2 among others [96–98]. The magnitude of these effects varies with any particular sequence, but the qualitative pattern of change is consistent. Importantly, the efforts to mitigate the proinflammatory effects by avoiding specific sequence motifs and adding 2⬘-alkoxy substituents have generally resulted in less proinflammatory ASOs [75]. One relatively subtle difference in the proinflammatory effects for 2⬘-MOE ASOs is the relative absence of IL-6 and IL-12 production and correspondingly less lymphoproliferative changes in mice [43] (Figure 12.6). The chemokine production and presence of lymphohistiocytic cell infiltrates are still present with 2⬘-MOE ASOs, but it appears that the signal for B-cell proliferation and polyclonal immunoglobulin production is reduced [12]. Characterization of the mechanism for proinflammatory effects of 2⬘-MOE ASO treatment continues, but it appears that chemokine production is the source of lymphohistiocytic cell infiltrates. A correlation between MCP-1 and increased tissue infiltrates has been established [98,99]. The reason for the preferential production of chemokines by 2⬘-MOE ASOs is not entirely well defined, but may have to do with the specific PAMP receptors that are bound and activated. In monkeys, the evidence of proinflammatory effects is very limited for both PS ODNs and 2⬘-MOE ASOs [12,52,60]. There is typically little or no increase in spleen weight, and multi-organ lymphohistiocytic cell infiltrates are rare. Mild lymphoproliferative effects in monkeys have been reported, but are typically only seen with SC administration and primarily occur in the draining lymph node. The only consistent site of inflammatory cell infiltrate is in the SC injection–site skin.

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Concentration (pg/mL)

IL-6

mMCP-1

25

500

20

400

15

300

10

200

5

100

0

Ctrl

5132

13650

Placebo Figure 12.6

0 100 mg/kg

Ctrl

5132

13650

300 mg/kg

2⬘-MOE ASOs produce lower amounts of cytokines and chemokines in plasma relative to PS ODNs. ISIS 13650 is representative of the 2⬘-MOE ASO, while ISIS 5132 is a PS ODN with the same sequence. Values are plotted as the mean and standard deviation relative to control values following a single dose in mice. MCP-1 values are increased twofold relative to control at the 300 mg/kg dose level

The relative absence of proinflammatory effects observed in monkeys is well correlated with a relative absence in cytokine or chemokine production. Our experience with SC injection of 2⬘-MOE ASOs in clinical trials is the development of mild and transient erythema and induration, much like those associated with other subcutaneously administered therapeutics (for review, see Chapter 13). These effects are dose-dependent, but have not been dose-limiting. Earlier versions of PS ODNs were associated with symptoms in patients consistent with cytokine activation including fever, nausea, aches, and chills [78,100]. However, relatively little cytokine syndrome has been reported in our trials with 2⬘-MOE ASOs [48].

Mechanism of Proinflammatory Activity The mechanism by which CpG optimal oligonucleotides activate the innate immune response is well documented. CpG oligonucleotides bind to TLR9 receptors that recognize bacterial DNA [101–103]. This TLR9 receptor is a member of a large family of innate immune receptors that each recognizes various conserved motifs on infectious agents such as bacteria and viruses [104]. In addition to bacterial DNA and CpG oligonucleotides, other TLRs include lipopolysaccharide, peptidoglycan, and techoic acid [104]. The siRNA also induce proinflammatory effects, but may interact and signal through distinct TLR receptors such as TLR3 or TLR7 (Nature REF). All the various TLR receptors signal through a common pathway including MyD88, IRAK, and TRAF6 leading to NF-␬B and production of cytokines including IL-1, IL-6, and IL-12 [101,103]. The production of lymphoproliferative cytokines such as IL-6 and IL-12 likely accounts for the increase in B-cell number and serum immunoglobulin [97]. These TLRs are most highly expressed on professional immune cells such as monocytes, macrophages, dendritic cells, and T cells, but some members of the family are also expressed at low levels on other cell types such as dermal endothelial cells, intestinal epithelial cells, and hepatocytes [105]. Thus, the interaction of CpG oligonucleotide with TLR9 on these cells leads to production of the inflammatory cytokine cascades that lead to the lymphoproliferative changes observed in mice. The relative distribution of these receptors among different tissues and in different species may account for some of the species sensitivity and pattern of tissue cell infiltrates [106]. For example, macrophages and other myeloid-derived mononuclear

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cells were sensitive to activation by CpG oligonucleotides in mice, but were insensitive to treatment in cells of human origin. One important point on sequence motifs for those interested in ASO therapeutics is that while avoiding CpG dinucleotides will help mitigate the proinflammatory effects, it is not the only motif associated with cytokine production. Other oligonucleotide structures, including G-rich and T-rich sequences, have also been implicated in these effects [107–109]. Elias and colleagues defined a specific T-rich motif that contained PyNTTTTGT (Py—pyrimidine; N—any nucleotide) [109]. Interestingly, sequence motifs have also been found that tend to be anti-inflammatory or at least block the proinflammatory effects of CpG oligonucleotides through a competitive type interaction [110]. In short, we understand some of the rules for avoiding proinflammatory oligonucleotides, but not all of them, and novel sequences must still be tested in either mice or cell-based systems to know for sure if any given sequence is proinflammatory. For 2⬘-MOE ASOs that do not contain CpG motifs, the precise mechanism leading to the residual proinflammatory effects that exist is less well defined. It is possible, especially for oligonucleotides containing CpG dinucleotides without the optimal flanking sequences, that some of the effect may still be mediated through TLR9 interaction [111]. However, it has recently been reported that the proinflammatory effects of non-CpG-containing oligonucleotides is TLR9independent [43]. Still, the proinflammatory effects of these non-CpG oligonucleotides are the result of direct stimulation of macrophages and monocytes. Thus, it is hypothesized that oligonucleotides can bind other receptors of the innate immune system (Senn et al., unpublished obervations). Experiments to directly compare the relative potency of CpG and non-CpG oligonucleotides indicate there is roughly a ⬎100-fold difference in potency [112]. However, because of the phosphorothioate linkages these oligonucleotide are highly protein-bound and there may be some interaction with other innate immune receptors. Possible candidates are TLR3 and TLR7, which are the receptors for single-strand and double-strand DNA [113,114]. There are also other non-TLR receptors that are under investigation. Still, these interactions appear to be relatively low affinity for non-CpG 2⬘-MOE ASOs, which is consistent with the relative decrease in proinflammatory effects observed in treated mice.

Species Specificity of Proinflammatory Effects Understanding the mechanistic basis of the proinflammatory effects is useful for toxicology evaluation. With this knowledge, we can approach the safety assessment in a very traditional manner, evaluating the dose-dependent proinflammatory effects of each sequence, against the proposed clinical doses and indication. The safety margins are based on the dose-response curves in toxicology studies with an understanding of the clinical relevance of these effects based on a wealth of data compiled with numerous sequences tested in animals and man. For the examples we have experience with, the proinflammatory effects of a 2⬘-MOE ASO in mice occur at doses that are greater than studied in clinical trials for arthritis with good tolerability [12,48]. From our experience with the class, the relative species specificity is understood. Rodents seem to overpredict for the proinflammatory effects and primates are much more predictive. Very little cytokine/chemokines release would be expected in patients, particularly at the doses used in current trials. There are clear differences in the biology between rodents and primates, and sensitivity of primates appear to be much more restricted to a sequence motif [111,115]. One example is the optimal motif characterized for primates and marked alteration of potency with single nucleotide substitutions [116,117]. Other potential explanations for differences in species sensitivity have to do with the relative expression pattern of potential receptors. For example, Hornung and colleagues report that macrophages and myeloid cells in mice can be directly activated by CpG oligos, whereas human cells of a similar type were insensitive [106].

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The low level of proinflammatory effects associated with 2⬘-MOE ASO currently in clinical development does not limit current clinical activity. On the basis of the mechanistic work in animal models, we are trying to validate biomarkers that can be used to monitor for potential proinflammatory effects in clinical trials. Although the proinflammatory effects of 2⬘-MOE ASOs do not limit dosing in clinical trials currently, some concern for chronic low-grade proinflammatory effects still exists. Once validated, these markers will be used to ensure the absence of these effects in patients at clinically relevant doses.

12.2.4.4 Chronic Administration The tolerability of chronic administration of oligonucleotides is probably the largest remaining unanswered question concerning the toxicologic properties of antisense oligonucleotides. The two specific areas that need to be addressed for chronic administration are the consequence of long-term proinflammatory effects and the renal tolerability. Chronic studies of 6 months duration or longer have been performed for at least three different PS ODN projects at Isis Pharmaceuticals, Inc. (e.g., ICAM-1, PKC-␣, and Hepatitis C). In these studies, the findings after chronic administration were consistent with that observed at comparable dose levels following subchronic administration (Isis Pharmaceuticals, Inc., unpublished data). Most notably, the proinflammatory effects observed in mice do tend to progress with chronic administration, but there was no evidence of progression of renal effects in either mice or monkeys. The most relevant question that remains is the chronic renal tolerability of 2⬘-MOE ASOs. As these second-generation antisense oligonucleotides are only now reaching the later stages of development, the data to answer this question are not mature enough to make broad conclusions. We have completed 6 months of treatment for a representative oligonucleotide in this class, ISIS 141923. In this study, monkeys were treated with ISIS 141923 by once-weekly administration of 20 mg/kg/week for 26 weeks. As expected, the target organ effect at this dose level was in kidney and correlated with the highest tissue concentration. As discussed above, the kidney cortex concentration achieved steady state concentration by 4–6 weeks and was maintained through the treatment duration for this dose level. Importantly, the histologic changes observed after chronic treatment were similar to those observed after subchronic duration of treatment, suggesting no progression of toxicity in monkeys at least with up to 6 months of treatment (Table 12.8). There were also no changes in markers of renal function in either serum or urine. No other significant changes were noted in body weight, clinical pathology, organ weight, or histopathology. For sure, more data are needed to address this issue and more data will be forthcoming. However, present data begin to address the chronic tolerability of 2⬘-MOE ASOs.

Table 12.8 Oligonucleotide Concentration and Histologic Finding in Kidney Following 6 Months of 20 mg/kg/week ISIS 141923 Treatment in Monkeys Parameter Renal cortex concentration Basophilic granules Cytoplasmic vacuolesa: proximal tubular epithelium Degeneration: proximal tubular epithelium a

Finding 1900 ␮g/ml 4 of 4 (mild to moderate) 2 of 4 (minimal) 0 of 4

Cytoplasmic vacuoles were not observed in tissues fixed with paraformaldehyde/glutaraldehyde.

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12.2.5 Reproductive Toxicology Reproductive toxicity of PS ODNs and now several 2⬘-MOE ASOs have been evaluated in male and female mice and in rabbits, examining the effects of fertility, fetal development, and development and reproductive function of offspring. Oligonucleotide effects on reproductive organs are limited, in part because of the very low degree of oligonucleotide exposure, particularly to male reproductive organs or developing fetus. For example, the oligonucleotide concentrations in testes is typically among the lowest of any of the organs examined, and immunohistochemical examination indicates that what oligonucleotide is present does not cross the blood-testes barrier, but instead is contained in interstitial tissue macrophages. This is important, as it reduces concern for exposure of the germ line cells to oligonucleotides. No effect on testes morphology or male reproductive function has been observed for any oligonucleotide studied to date [118,119]. In pregnant females, very little oligonucleotide is associated with placenta and the concentrations of oligonucleotide in fetal kidney or liver are typically below the level of quantitation [118,119]. This indicates that very little oligonucleotide is transferred across the placenta. Detailed analysis of distribution to placenta has indicated that oligonucleotide concentrations are higher on the maternal side of the placenta in rats, documenting the distribution to fetus, and quantifying that fetal kidney concentrations are 1% of those measured in maternal kidney [120]. This limited fetal exposure to oligonucleotide greatly decreases the concerns for fetal development. Reproductive organs that have been a target for toxicity in rodents include ovaries, uterus, and vagina. Oligonucleotide is distributed to ovaries and uterus, and in mice there has been observation of dose-dependent increase in lymphohistiocytic cell infiltrates in these tissues associated with the proinflammatory effects so commonly observed in this species. In our experience, however, there has only been one case where this exposure to oligonucleotide and increase in cell infiltrate was correlated with a functional change in mice. The 2⬘-MOE ASO inhibitor of TNF-␣ produced a particular marked level of cell infiltrates in mice at doses ⱖ35 mg/kg/week. For this particular compound, the infiltrates were significant enough to contribute to a decrease in ovarian weight and produce ovarian atrophy. This effect on ovaries was associated with a dose-dependent decrease in the fertility index in mice at doses ⱖ20 mg/kg/week. This change was attributed to proinflammatory effects that include an increase in proinflammatory cytokines and chemokines, thus causing the cell infiltrates. Proinflammatory cytokines have been directly associated with ovarian atrophy and the change is therefore considered secondary to the proinflammatory effects rather than an intrinsic toxicity of the oligonucleotide to female reproductive organs [121,122]. Because of the species specificity of the proinflammatory effects discussed earlier, and the doses required to produce these effects (i.e., ⱖ20 mg/kg/week), this effect is of little concern to human safety assessment. The other more common observation in fetal development studies is a dose-dependent decrease in maternal body weight and food consumption in female rabbits that is associated with spontaneous abortion and decrease in fetal body weight at the higher dose levels [10,119]. These effects are also associated with the proinflammatory effects of oligonucleotides, and cytokine release has been associated with increases in the rate of abortions [121,122]. Certain of the more potent proinflammatory PS ODNs have been described to have a relatively high rate of abortion. While spontaneous abortions have been observed in rabbits, the incidence is lower for 2⬘-MOE ASOs compared to first-generation ASO, despite higher target organ concentrations (Table 12.9). This relative decrease in the reproductive effects of 2⬘-MOE ASOs in rabbit compared to PS ODNs is consistent with the generally lower degree of proinflammatory effects and further suggests that these effects are not attributable to an intrinsic reproductive toxicity of this class. There are several examples where investigators have used antisense inhibitors to study the effects on reproduction function. The most notable was the use of a 2⬘-O-methyl-modified oligonucleotide targeted to vascular endothelial growth factor (VEGF). In this study, antisense treatment produced a 50% inhibition of VEGF mRNA and was associated with developmental abnormalities similar to that observed in the VEGF-deficient mice [123]. These effects were attributed to the inhibition of VEGF both because of the similarity of the phenotype as well as

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Table 12.9 Comparison of the Relative Dose, Tissue Concentration, Maternal Toxicity, and Fetal Toxicity Correlation between Several Representative PS ODNsand 2⬘⬘-MOE ASOs Maternal Tissue Concentration (␮g/g)

ASO

a

Highest Dose Tested Chemistry (mg/kg/week) Kidney

Maternal Toxicity

Fetal Toxicity

Maternal Increased BWa Gain Abortion Reduction in Fetal Liver (% of control) Frequency Litter Size Alterations

2302 3521 2922

PS ODN PS ODN PS ODN

60 35 60

273 343 426

15 19 44

62 ⫺38 ⫺82

⫺ ⫹ ⫹⫹⫹⫹

⫺ ⫺ ⫺

⫺ ⫺ ⫹

113715 104838 301012

2⬘-MOE 2⬘-MOE 2⬘-MOE

60 52.5 52.5

1400 1321 2336

285 231 194

82 74 100

⫺ ⫹ ⫺

⫺ ⫺ ⫺

⫺ ⫺ ⫺

BW⫽body weight.

the lack of effect on VEGF expression or fetal development of a 5-base mismatched control. While these effects are clearly related to the targeted pharmacology, it does raise important points about the accessibility of the fetus to antisense inhibitors during development. It is possible in this case that the exposure of the vascular endothelium to the VEGF inhibitor in the placenta may be sensitive to antisense inhibition at least at certain points in development, or it is possible these effects were related to inhibition of maternal VEGF systemically during an important phase of development. 12.2.6 Genetic Toxicology To date, oligonucleotide therapeutics are routinely examined in the standard battery of genetic toxicology assays that include the Ames bacterial mutagenicity assay, the CHO cell chromosomal aberrations assay, the L5178Y/TK(⫹/–) mouse lymphoma mammalian gene mutation assay, and the mouse micronucleus assay. Each of these assays has been performed with 10–12 unique compounds, of which 5–7 have been 2⬘-MOE ASOs. Less common is the in vitro rat hepatocyte unscheduled DNA synthesis assay. To our knowledge, antisense oligonucleotides have been uniformly negative in these assays with no evidence of mutagenicity or clastogenicity. Representative examples of these assays have been thoroughly reviewed and published for PS ODNs [124]. Importantly, experiments have been performed in parallel for representative compounds to document the exposure of cells to oligonucleotide and the production of metabolites. In vitro assays are performed in the presence and absence of metabolic activation and employ concentrations up to the limit of the assays (5000 ␮g/plate or 5000 ␮g/ml) in the presence and absence of metabolic activation. The metabolism of oligonucleotide and liberation of the mononucleotide metabolites were among the most significant concerns initially as imbalances in the nucleotide pools have been associated with increased rate of mutations. However, these studies showed that significant accumulation of nucleotides was required to affect the fidelity of DNA repair. For PS ODNs, it was shown that the liberation of mononucleotides is not associated with accumulation in nucleotide pools, and many of the mononucleotides are further catabolized. A new concern for 2⬘-MOE ASOs was the presence of modified nucleosides. However, in the testing of several 2⬘-MOE ASOs, no genetic toxicity has been observed and it turns out that the 2⬘-O-methoxyethyl-modified residues are highly resistant to exonuclease degradation. Thus, as a consequence, metabolism of these oligonucleotides is initiated by an endonuclease cleavage event, following sequential degradation of the exposure deoxyphosphorothioate nucleotides [11]. Mass spectral characterization of the metabolites largely finds the 2⬘-O-methoxyethyl-modified portions of the oligonucleotide intact. The EMEA has published a paper on the genetic toxicity potential for antisense oligonucleotides [126]. It sites the two primary concerns as being the potential for base mispairing due to

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liberation of metabolites resulting in point mutations in newly synthesized DNA, and site-specific mutations resulting from triplex formation with DNA [125]. The traditional gene mutation and clastogenicity assays are considered to be of suitable specificity and sensitivity to address the first potential issue. However, based on the negative response in these assay, in light of the documentation of exposure and metabolism, the EMEA concluded that PS ODNs were not likely to pose a genotoxic hazard by this mechanism. As a result, the report suggested that in vitro testing of novel antisense inhibitors in these assays was not necessary. The EMEA report did go on to indicate that the traditional assays were not sufficient to detect site-specific mutations possible; results inform triplex formation. Those assays would require PCR or restriction length polymorphism endpoints to be detected. Such standardized assays are not available. Still, the likelihood of triplex formation between oligonucleotides and 2⬘-MOE ASOs is very remote. Triplex formation has been shown to require specific polypurine sequences and it is likely that the 2⬘-MOE substituents would sterically interfere with binding into the major grove of DNA. 12.2.7 Safety Pharmacology In examining the immediate effects on major organ system function, there are obviously a number of ways to design the program. Our approach at Isis to safety pharmacology evaluation for antisense inhibitors has been strategically based on the known pharmacokinetic and toxicity parameters for these compounds. The strategy for safety pharmacology is also consistent with the guidance provided in the FDA Points to Consider documents that have been published [21,22]. The organ system that has received the most attention for this class of compound has been cardiovascular function because of the potential for complement activation and the potential secondary effects on vascular tone. A definitive link has been made between complement activation and the reported alterations in blood pressure and heart rate in monkeys treated with PS ODNs [55]. Since the complement activation is only observed in monkeys, we do the cardiovascular safety pharmacology in monkeys. Our approach is to implant telemetry units in selected monkeys (2/sex in at least two different treatment groups including the high dose) to measure ECG, mean arterial pressure, heart rate, and body temperature. These parameters are then recorded at frequent intervals for a 24-h period prior to treatment to establish circadian fluctuations and normal response to various stimuli encountered during the day. The physiology is then measured prior to and immediately after treatment for up to 24 h on the first and last dose administered in the study. This approach assures examination of both the potential for acute alterations related to complement activation after single and repeated administration. In addition, external ECG (qualitative and quantitative) is performed on all animals prior to treatment and the week prior to schedule sacrifice. Thus, a thorough assessment of cardiovascular function is performed in monkeys. As discussed previously, alterations secondary to complement activation have been occasionally observed, more so for PS ODNs, but there has been no primary alteration of cardiovascular function attributed to an oligonucleotide. Thus, there is no concern for an intrinsic effect of an antisense oligonucleotide on cardiovascular function at doses in patients that do not produce complement activation. Since the availability of the in vitro hERG assay, several PS ODNs and 2⬘-MOE ASOs have been examined in this assay. There was no strategic or scientific reason to examine these compounds in this assay and there is no reason to speculate that there would be any specific interaction with ion channels. The experiments were performed, however, up to the limit concentration of 1050 ␮g/ml in the assay and there has been no associated alteration in ion channel function (Table 12.10 and Figure 12.7). The concentrations of 2⬘-MOE ASOs examined in these assays are 150-fold greater than the typical peak plasma concentration in clinical trials associated with a 200 mg dose (approximately 3 mg/kg: Cmax⫽6 ␮g/ml), and 100-fold greater than the oligonucleotide concentration in heart associated with a 35 mg/kg/week dose in monkeys. Thus, based on the very large margins of safety there is no concern for QT wave prolongation in patients. This is not surprising considering the chemical nature of the oligonucleotides, which are polyanionic molecules

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Table 12.10 Summary of Effect of a 2⬘⬘-MOE ASO (ISIS 301012) on hERG Current (Mean % Inhibition) ISIS 301012

Treatment (N a ⫽ 3)

Vehicle

10 ␮M

50 ␮M

100 ␮M

150 ␮M

1 2 3 Meanb ⫾ SDc SEMd

0.5 ⫺0.5 0.4 0.1 ⫾ 0.6 0.3

0.5 1.4 ⫺1.0 0.3 ⫾ 1.2 0.7

0.0 1.6 ⫺0.7 0.3 ⫾ 1.2 0.7

0.4 0.5 ⫺0.2 0.2 ⫾ 0.4 0.2

1.4 ⫺0.5 0.0 0.3 ⫾ 1.0 0.6

a

Number of cells. Mean percent inhibition at each ISIS 301012 concentration. c Standard deviation. d Standard error of the mean. b

800

Control (black)

Current (pA)

600 400 200

150 µM ISIS 301012 (gray)

Potential (mV)

0 100 0 −100 0

Figure 12.7

2000 Time (ms)

4000 Sw 86/87

2⬘-MOE ASOs have no effect on the in vitro hERG assay. ISIS 301012 is representative of the 2⬘-MOE ASO. Upper panel, hERG currents (current in pA; time in ms) were evoked by voltage protocol shown in the lower panel (voltage in mV).

of approximately 7200 Mw. Most of the typical compounds that have been associated with alteration of ion channel function have been small compounds, capable of interacting with the ion channel. Assessment of renal function is also important for PS ODNs and 2⬘-MOE ASOs because this is the organ with the highest concentrations of oligonucleotide. As described previously, it is the renal proximal tubular epithelial cells that contain the vast majority of the oligonucleotides. Since the concerns are largely attributed to the concentration of oligonucleotide in tissue, it does not make sense to examine renal function in the context of the typical single-dose experiments. Thus, our typical approach is to examine renal function as part of the repeat-dose toxicology studies. Particular attention is focused on monkeys where the tissue concentrations tend to be the highest. Evaluation of renal function relies primarily on routine serum chemistry, urinalysis, and histopathology. In addition, we try to also measure quantitative urine total protein and creatinine in monkeys, so that we can report urine protein/creatinine ratio. Other measures of tubular function have occasionally been examined, such as urine glucose, urine amino acids, measurement of specific low-molecular-weight proteins in urine and enzyme markers of epithelial cell damage in urine, but no changes in these markers have been observed. The most reliable measure has been total urine protein or protein creatinine ratios. Other measures of CNS, pulmonary, and gastrointestinal function have been performed for several oligonucleotides from both the PS ODN and 2⬘-MOE ASO classes, and found to be

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uniformly negative. There is no scientific rationale for conducting these studies with antisense oligonucleotides because there is little to no distribution to lung or brain, respectively, and no morphologic changes observed in these organs. The results from the oligonucleotides examined in these specific studies confirm that there is no intrinsic toxicity of the chemical class on these organ systems when administered systemically. This strategy is based on the assumption of parenteral administration. As more projects use local administration to specific target organs, the strategy will need to be adapted to those circumstances. For example, CNS and pulmonary function assessment will be performed in projects that utilize intrathecal or intracerebroventricular administration. Pulmonary safety pharmacology will also be added to projects where oligonucleotides are administered by aerosol delivery of nebulized solutions.

12.3 SPECIES-SPECIFIC EFFECTS Another important aspect of toxicity evaluation in animal models, especially rodents, is relative species sensitivity of various effects. The greater sensitivity of mice to the proinflammatory effects relative to primates has been well described and is obviously important to understand in the process of safety assessment. Rats have been used less often than mice in the toxicology evaluation of these inhibitors, but are also an important component of the testing regimen. Recently, however, unique species-specific effects have been observed in rats that are not even observed in mice. The ratspecific effect of ASO treatment is change in kidney function that is manifested as an increase in low-molecular-weight proteins excreted in urine. Proteinuria in rats is dose-dependent (usually observed at doses ⱖ10 mg/kg) and independent of sequence, but this effect is not seen in mice or monkeys. Although the mechanism is not well established yet, proteinuria is thought to be attributable to the decrease in tubular reabsorption of these low-molecular-weight proteins. Since oligonucleotides themselves are taken up in these particular cells, it is not clear at this point if the decrease in tubular protein reabsorption is attributable to a simple competitive interaction for a particular receptor or if expression levels for a receptor(s) responsible protein reabsorption are decreased (Figure 12.8). Regardless, these changes have been shown to be reversible and are not associated with other changes in kidney function. The reasons for the greater sensitivity of rats are not known,

Urinary protein/creatinine

5

4

3 Rat 10 mg/kg 2 Rat control 1

0 0

2

4

6

8

10

12

Time (weeks) Figure 12.8 Comparison of quantitative urine protein/creatinine ratio in rats and monkeys. Values are plotted as the mean and standard deviation of 4–8 animals. Baseline and control values of protein excretion in rats are generally 10 times higher in rats than in monkeys. Rats are subsequently more sensitive to an increase in urine protein excretion that is observed at doses as low as 10 mg/kg. No proteinuria is observed in monkeys at doses up to 80 mg/kg.

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but may be related, in part, to certain species-specific effects, such as proinflammatory effects, or may be a reflection of the greater susceptibility of rats to the development of renal abnormalities (i.e., proteinuria). Male rats, in particular, are notoriously sensitive to kidney insult as evidenced by the aging rat nephropathy that develops in this species, and also the sensitivity to certain agents, such as the nephropathy associated with puromycin treatment. Rats also normally excrete far more protein in their urine than monkeys or humans. The sensitivity of rats is particularly evident in male rats and is consistent with known species sensitivities. This observation is thoroughly characterized by studying dose-response, time course, progression, and reversibility in the same manner as other ASO effects, but an understanding of the relative species specificity of this effect and the potential mechanistic basis are instrumental for interpretation of the significance and the impact on relative safety margins.

12.4 CONCLUSIONS The safety and toxicology evaluation of oligonucleotide therapeutics continues to progress. With the experienced gained with 2⬘-MOE ASOs, this class of inhibitors appears to have a tolerability profile sufficient to support the safe clinical application for many different types of indications. A thorough understanding of the dose, time course, and mechanistic basis for most target organ effects has now been established. This knowledge and the consistency of properties from one sequence to another contribute to the safe and effective application of these inhibitors in clinical trials. With other types of oligonucleotide therapies including siRNA and other chemical modifications that increase potency, such as LNA modified oligonucleotides, the field of therapeutic oligonucleotides will continue to evolve, fine-tuning the balance between efficacy and tolerability.

ACKNOWLEDGMENTS We would like to thank Robert Saunders and Tracy Reigle for their assistance in preparation of this manuscript. In addition, we would like to acknowledge the contributions of other members of our lab including Lijiang Shen, Hong Zhang, and Russel Watt.

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION and steroid sparing properties of an ICAM-1 antisense oligodeoxynucleotide, alicaforsen (ISIS 2302), in active steroid dependent Crohn’s disease, Gut 51(1), 30, 2002. Yacyshyn, B., Bowen-Yacyshyn, M. B., and Shanahan, W., The clinical experience of antisense therapy to ICAM-1 in Crohn’s disease, Curr. Opin. Mol. Ther. 1(3), 332, 1999. Sewell, L. K., Geary, R. S., Baker, B. F., Glover, J. M., Mant, T. G. K., Yu, R. Z., Tami, J. A., and Dorr, A. F., Phase I trial of ISIS 104838, a 2⬘-methoxyethyl modified antisense oligonucleotide targeting tumor necrosis factor-alpha, J. Pharmacol. Exp. Ther. 303(3), 1334, 2002. Henry, S. P., Novotny, W., Leeds, J., Auletta, C., and Kornbrust, D. J., Inhibition of coagulation by a phosphorothioate oligonucleotide, Antisense Nucleic Acid Drug Dev. 7, 503, 1997. Sheehan, J. P. and Lan, H.-C., Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex, Blood 92(5), 1617, 1998. Sheehan, J. P. and Thao, P. M., Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex by an allosteric mechanism, Biochemistry 40(16), 4980, 2001. Henry, S. P., Bolte, H., Auletta, C., and Kornbrust, D. J., Evaluation of the toxicity of ISIS 2302, a phosphorothioate oligonucleotide, in a 4-week study in cynomolgus monkeys, Toxicology 120, 145, 1997. Jeter, M. L., Ly, L. V., Fortenberry, Y. M., Whinna, H. C., White, R. R., Rusconi, C. P., Sullenger, B. A., and Church, F. C., RNA aptamer to thrombin binds anion-binding exosite-2 and alters protease inhibition by heparin-binding serpins, FEBS Lett. 568(1–3), 10, 2004. Paborsky, L. R., McCurdy, S. N., Griffin, L. C., Toole, J. J., and Leung, L. L., The single-stranded DNA aptamer-binding site of human thrombin, J. Biol. Chem. 268(28), 20,808, 1993. Henry, S. P., Beattie, G., Yeh, G., Chappel, A., Giclas, P. C., Mortari, A., Jagels, M. A., Kornbrust, D. J., and Levin, A. A., Complement activation is responsible for acute toxicities in rhesus monkeys treated with a phosphorothioate oligodeoxynucleotide, Int. J. Immunopharmacol. 2(12), 1657, 2002. Henry, S. P., Giclas, P. C., Leeds, J., Pangburn, M., Auletta, C., Levin, A. A., and Kornbrust, D. J., Activation of the alternative pathway of complement by a phosphorothioate oligonucleotide: potential mechanism of action, J. Pharmacol. Exp. Ther. 281(2), 810, 1997. Oberbauer, R., Schreiner, G. F., and Meyer, T. W., Renal uptake of an 18-mer phosphorothioate oligonucleotide, Kidney Int. 48, 1226, 1995. Rappaport, J., Hanss, B., Kopp, J. B., Copeland, T. D., Bruggeman, L. A., Coffman, T. M., and Klotman, P. E., Transport of phosphorothioate oligonucleotides in kidney: implications for molecular therapy, Kidney Int. 47, 1462, 1995. Butler, M., Stecker, K., and Bennett, C. F., Histological localization of phosphorothioate oligodeoxynucleotides in normal rodent tissue, Nucleos. Nucleot. 16(7–9), 1761, 1997. Monteith, D. K., Geary, R. S., Leeds, J. M., Johnston, J., Monia, B. P., and Levin, A. A., Preclinical evaluation of the effects of a novel antisense compound targeting C-raf kinase in mice and monkeys, Toxicol. Sci. 46(2), 365, 1998. Monteith, D. K., Horner, M. J., Gillett, N. A., Butler, M., Geary, R. S., Burckin, T., Ushiro-Watanabe, T., and Levin, A. A., Evaluation of the renal effects of an antisense phosphorothioate oligodeoxynucleotide in monkeys, Toxicol. Pathol. 27(3), 307, 1999. Henry, S. P., Johnson, M., Zanardi, T. A., Fey, R., Auyeung, D., Lappin, P. B., and Levin, A. A., Renal uptake and tolerability of a 2⬘-O-methoxyethyl modified antisense oligonucleotide (ISIS 113715) in monkey, Toxicol. Sci., submitted. Bendele, A., Seely, J., Richey, C., Sennello, G., and Shopp, G., Short communication: renal tubular vacuolation in animals treated with polyethylene-glycol-conjugated proteins, Toxicol. Sci. 42(2), 152, 1998. Conover, C. D., Linberg, R., Gilbert, C. W., Shum, K. L., and Shorr, R. G., Effect of polyethylene glycol conjugated bovine hemoglobin in both top-load and exchange transfusion rat models, Artif. Organs 21(10), 1066, 1997. Merski, J. A. and Meyers, M. C., Light- and electron-microscopic evaluation of renal tubular cell vacuolation induced by administration of nitrilotriacetate or sucrose, Food Chem. Toxicol. 23(10), 923, 1985. Kone, B. C., Racusen, L. C., Whelton, A., and Solez, K., Acute renal failure produced by combining cyclosporine and brief renal ischemia in the Munich Wistar rat, Clin. Nephrol. 25, Suppl. 1, S171, 1986. Kintzel, P. E., Anticancer drug-induced kidney disorders, Drug Safety 24(1), 19, 2001. El-Badawi, M. G., Abdalla, M. A., Bahakim, H. M., and Fadel, R. A., Nephrotoxicity of low-dose methotrexate in guinea pigs: an ultrastructural study, Nephron 73(3), 462, 1996.

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91. Stein, C. A., Phosphorothioate antisense oligodeoxynucleotides: questions of specificity, Tibtech 14, 147, 1996. 92. Vollmer, J., Jepsen, J. S., Uhlmann, E., Schetter, C., Jurk, M., Wader, T., Wullner, M., and Krieg, A. M., Modulation of CpG oligodeoxynucleotide-mediated immune stimulation by locked nucleic acid (LNA), Oligonucleotides 14(1), 23, 2004. 93. Branda, R. F., Moore, A. L., Lafayette, A. R., Mathews, L., Hong, R., Zon, G., Brown, T., and McCormack, J. J., Amplification of antibody production by phosphorothioate oligodeoxynucleotides, J. Lab Clin. Med. 128(3), 329, 1996. 94. McIntyre, K. W., Lombard-Gilloly, K., Perez, J. R., Kunsch, C., Sarmiento, U. M., Larigan, J. D., Landreth, K. T., and Narayanan, R., A sense phosphorothioate oligonucleotide directed to the initiation codon of transcription factor NF-κB p65 causes sequence-specific immune stimulation, Antisense Res. Dev. 3, 309, 1993. 95. Liang, H., Nishioka, Y., Reich, C. F., Pisetsky, D. S., and Lipsky, P. E., Activation of human B cells by phosphorothioate oligodeoxynucleotides, J. Clin. Invest. 98(5), 1119, 1996. 96. Klinman, D. M., AE-Kyung, Y., Beaucage, S. L., Conover, J., and Krieg, A. M., CpG motifs present in bacterial DNA rapidly induce lymphocytes to secrete interleukin 6, interleukin 12, and interferon γ, Proc. Natl. Acad. Sci. USA 93, 2879, 1996. 97. Zhang, Y., Shoda, L. K., Brayton, K. A., Estes, D. M., Palmer, G. H., and Brown, W. C., Induction of interleukin-6 and interleukin-12 in bovine B lymphocytes, monocytes, and macrophages by a CpG oligodeoxynucleotide(ODN 2059) containing the GTCGTT motif, J. Interferon Cytokine Res. 21(10), 871, 2001. 98. Takeshita, S., Takeshita, F., Haddad, D. E., Ishii, K. J., and Klinman, D. M., CpG oligodeoxynucleotides induce murine macrophages to up-regulate chemokine mRNA expression, Cell Immunol. 206(2), 101, 2000. 99. Kanda, H., Tateya, S., Tamori, Y., Kotani, K., Hiasa, K.-i., Kitazawa, R., Kitazawa, S., Miyachi, H., Maeda, S., Egashira, K., and Kasuga, M., MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity, J. Clin. Invest. 116(6), 1494, 2006. 100. Cotter, F. E., Antisense therapy for cancer, Eur. J. Cancer 1(2), 19, 2003. 101. Takeshita, F., Gursel, I., Ishii, K. J., Suzuki, K., Gursel, M., and Klinman, D. M., Signal transduction pathways mediated by the interaction of CpG DNA with Toll-like receptor 9, Semin. Immunol. 16(1), 17, 2004. 102. Takeshita, F., Leifer, C. A., Gursel, I., Ishii, K. J., Takeshita, S., Gursel, M., and Klinman, D. M., Cutting edge: Role of toll-like receptor 9 in CpG DNA-induced activation of human cells, J. Immunol. 167, 3555, 2001. 103. Hacker, H., Vabulas, R. M., Takeuchi, O., Hoshino, K., Akira, S., and Wagner, H., Immune cell activation by bacterial CpG-DNA through myeloid differentiation marker 88 and tumor necrosis factor receptor-associated factor (TRAF)6, J. Exp. Med. 192(4), 595, 2000. 104. Aderem, A. and Ulevitch, R. J., Toll-like receptors in the induction of the innate immune response, Nature 406(6797), 782, 2000. 105. Tsuboi, N., Yoshikai, Y., Matsuo, S., Kikuchi, T., Iwami, K.-I., Nagai, Y., Takeuchi, O., Akira, S., and Matsuguchi, T., Roles of toll-like receptors in C-C chemokine production by renal tubular epithelial cells, J. Immunol. 169(4), 2026, 2002. 106. Hornung, V., Rothenfusser, S., Britsch, S., Krug, A., Jahrsdorfer, B., Giese, T., Endres, S., and Hartmann, G., Quantitative expression of toll-like receptor 1-10 mRNA in cellular subsets of human peripheral blood mononuclear cells and sensitivity to CpG oligodeoxynucleotides, J. Immunol. 168(9), 4531, 2002. 107. Ho, P. P., Fontoura, P., Ruiz, P. J., Steinman, L., and Garren, H., An immunomodulatory GpG oligonucleotide for the treatment of autoimmunity via the innate and adaptive immune systems, J. Immunol. 171(9), 4920, 2003. 108. Vollmer, J., Janosch, A., Laucht, M., Ballas, Z. K., Schetter, C., and Krieg, A. M., Highly immunostimulatory CpG-free oligodeoxynucleotides for activation of human leukocytes, Antisense Nucleic Acid Drug Dev. 12(3), 165, 2002. 109. Elias, F., Flo, J., Lopez, R. A., Zorzopulos, J., Montaner, A., and Rodriguez, J. M., Strong cytosineguanosine-independent immunostimulation in humans and other primates by synthetic oligodeoxynucleotides with PyNTTTTGT motifs, J. Immunol. 171(7), 3697, 2003.

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An Overview of the Clinical Safety Experience of First- and Second-Generation Antisense Oligonucleotides T. Jesse Kwoh

CONTENTS 13.1 13.2

Introduction .........................................................................................................................365 The Clinical Experience for Gen-1 and Gen-2 ASO ..........................................................367 13.2.1 Subjects Treated and Indications Studied .............................................................367 13.2.2 Systemic Administration Schedules and Doses Studied.......................................369 13.2.3 Systemic Treatment Durations..............................................................................370 13.2.4 Local Treatment with ASO ...................................................................................371 13.3 Clinical Safety Results for Systemic Administration .........................................................371 13.3.1 Prolongation of aPTT............................................................................................374 13.3.2 Complement Activation ........................................................................................375 13.3.3 Proinflammatory Effects .......................................................................................377 13.3.3.1 Infusion-Associated Constitutional Symptoms ...................................378 13.3.3.2 Subcutaneous Injection Site Responses...............................................383 13.3.3.3 Hypersensitivity Reactions ..................................................................385 13.3.4 Kidney Effects.......................................................................................................386 13.3.5 Thrombocytopenia ................................................................................................389 13.3.6 Liver Effects..........................................................................................................391 13.4 Clinical Safety Results for Local Administration...............................................................393 13.5 Conclusions .........................................................................................................................394 References ......................................................................................................................................395

13.1 INTRODUCTION Helping patients by finding new therapeutic agents that specifically reduce the key protein causing the patient’s condition/disease is a major theme in current pharmaceutical discovery and development efforts. The stimulus for this focus is the expectation that as selectivity increases, a greater proportion of the drug in each dose will be bound to the therapeutic target and reduced amounts of drug will be tied up by other proteins. The net effect of increased selectivity, therefore, 365

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should be lower drug doses that are needed to achieve target inhibition and disease improvement, and fewer side effects resulting from suppression of nontarget proteins. With the high selectivity inherent in Watson–Crick hybridization, the use of antisense oligonucleotides (ASOs) has been repeatedly shown to be an effective means for specifically reducing the expression of target mRNA and proteins in numerous in vitro and in vivo model systems. These demonstrations are reviewed extensively in other chapter of this volume and in the previous edition of this book [1]. Reductions of target mRNA and/or protein expression have also been shown in the clinical studies of several ASOs. For example, dose-related reductions in the serum levels of both apoB and LDL-cholesterol were observed in healthy volunteers with baseline, fasting, total cholesterol levels between 200 and 300 mg/dL, who were repeatedly dosed with an ASO inhibitor of apoB100, ISIS 301012, for 4 weeks [2]. At the end of the treatment period, the subjects treated weekly with 200 mg ASO (n ⫽ 8) had a mean 50% reduction from baseline in their apoB levels and mean 35% reduction in their LDL-cholesterol levels. Both changes were significantly different ( p ⫽ 0.002 and 0.001, respectively) from those of subjects treated with placebo (n ⫽ 7). In another example, patients with localized prostate cancer were treated with ASO and androgen blockade in an openlabel Phase I study of OGX-011, an ASO inhibitor of clusterin [3]. At the end of the 4-week treatment period, radical prostatectomies were performed on the patients. A dose-related reduction ( ptrend ⫽ 0.008) of clusterin mRNA levels was measured in tumor tissue isolated from the excised organs. At the 640 mg/week dose, the mean clusterin mRNA level was 7.1% of the levels in tumor tissues collected from untreated historical controls and from patients treated with lower (40 and 80 mg) OGX-011 doses. Additional examples of target reductions have been observed in the ASO clinical studies of oblimersen (bcl-2 inhibitor) [4,5], ISIS 5131 (c-raf kinase inhibitor) [6,7], and alicaforsen (ICAM-1 inhibitor) [8]. Besides potent inhibition of the target protein, and the target having a critical role in disease, the successful development of an ASO into a marketed therapeutic will also depend upon the clinical safety profile of the drug candidate. In this regard, the clinical development programs for ASOs have an advantage over analogous programs for small-molecule drug candidates. While each smallmolecule compound has a unique nonclinical and clinical safety profile that must be discovered and characterized de novo, nonclinical and clinical studies for new ASOs can rely on previously characterized chemical class members for guidance. This is because ASOs of the same chemical class have very similar pharmacokinetic and toxicological profiles. The commonalities between members of the phosphorothioate oligodeoxynucleotide class were reviewed in the first edition of this book [9,10]. Chapters 11 and 12 of this book review the pharmacokinetic and toxicological behavior of 2⬘-O-(2-methoxyethyl) (MOE)-modified phosphorothioate oligonucleotides. This chapter will review the clinical safety results for these two related ASO classes. Substituting sulfur for one of the two nonbridging oxygens in each internucleotide linkage was one of several initial approaches used to stabilize synthetic oligodeoxynucleotides against nuclease attack and thereby permit the development of ASOs into therapeutic agents [11]. With at least 15 class members having been studied clinically [12–69], phosphorothioate oligodeoxynucleotides are the most extensively studied ASO class and will be referred to as Gen-1 or first-generation ASOs for the remainder of this chapter. MOE-modified ASOs are the product of medicinal chemistry efforts to increase the potency of Gen-1 ASOs [70]. These oligonucleotides have 2⬘-O-(2-MOE) substitutions of the sugars for the 4–5 nucleotides at each terminus of the ASO. The MOE modification increases the binding affinity of the ASOs to mRNA and increases the resistance of the ASOs to exonucleolytic attack. Using a chimeric structure (oligodeoxynucleotide core surrounded by MOE-modified nucleotides) for MOE-containing ASOs preserves their capacity to serve as RNase H substrates [70]. With eight of these second-generation (Gen-2) ASOs having entered the clinical study [2,3,71], the clinical experience for this chemical class is expanding rapidly. In addition to being limited to Gen-1 and Gen-2 ASOs, this review of ASO clinical safety will also be focused predominantly on the clinical results from (1) studies containing control groups because they allow distinguishing between ASO and non-ASO effects; (2) larger studies because

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the impact of subject to subject variations upon study results are minimized; (3) studies evaluating systemic exposure to ASOs because they generally provide the most information on drug-related adverse effects; and (4) studies that were directly managed by Isis Pharmaceuticals because of access to the primary study data. Where applicable, results from small and/or uncontrolled studies will be included. This chapter contains both published and unpublished results from Isis studies. Published results will be indicated where appropriate. In addition, any relevant results that are available from Gen-1 ASO clinical studies performed by others and from Gen-2 ASO studies performed by Isis’ development partners will be included in this review. The structure of this review will be to consider the significant adverse events (AEs) observed in the clinical studies of Gen-1 and Gen-2 ASOs and to consider the laboratory and AE results related to the nonclinical toxicological findings for these compounds. The five common effects identified for Gen-1 and Gen-2 ASOs in animal studies are prolongation of activated partial thromboplastin time (aPTT) in monkeys, activation of the alternative pathway of complement in monkeys, proinflammatory effects in mice, histological changes in the proximal tubules epithelial cells of both mice and monkeys, and thrombocytopenia in both species [72]. In addition, liver was identified as a possible target organ for toxicity, despite the absence of observed adverse effect in monkeys, because the largest amount, and the second highest concentration after kidney, of ASOs are found in this organ [73].

13.2 THE CLINICAL EXPERIENCE FOR GEN-1 AND GEN-2 ASO 13.2.1 Subjects Treated and Indications Studied The greatest amount of clinical experience developed for any ASO class is with Gen-1 compounds. Isis Pharmaceuticals has clinically evaluated seven Gen-1 ASOs (Table 13.1). Systemic (i.e., intravenous or subcutaneous) administration was studied for five of these compounds (alicaforsen, ISIS 2503, aprinocarsen, ISIS 5132, and ISIS 14803) and local administration was evaluated for three (afovirsen, alicaforsen, and fomivirsen). In the studies of these drugs, ⬃3700 study subjects were given an ISIS Gen-1 ASO, a considerable growth in experience from the ⬃1800 subjects treated at the time of the last review [74]. Currently, ⬃2500 subjects have been dosed using intravenous or subcutaneous administration and 1200 received Gen-1 ASOs as a local therapy. The studies of ISIS Gen-1 ASOs span 11 treatment populations. The largest study population (1660) is patients with cancer and the tumor types that have been studied include nonsmall cell lung (NSCLC), small cell lung, prostate, breast, colorectal, and ovarian cancers and non-Hodgkin’s lymphoma, melanoma, and glioblastoma, in addition to some others [6,7,25–49]. Seventy-six of the 85 studies on ISIS Gen-1 ASOs were small (⬍100 subjects). The typical study size was 10–40 study subjects. Three large randomized, double-blind, placebo-controlled studies of alicaforsen given intravenously have been conducted. These three studies enrolled 150, 180, and 300 patients with Crohn’s disease and randomized two-thirds of the patients to treatment with alicaforsen and one-third to placebo treatment [15]. Two large (590 and 640 patients) randomized, controlled clinical trials have been conducted for aprinocarsen [31,32]. In these open-label trials, patients with previously untreated NSCLC were randomized 1:1 between treatment with a standard chemotherapy regimen and treatment with the standard regimen plus aprinocarsen given intravenously. Two chemotherapy regimens (carboplatin plus paclitaxel and gemcitabine plus cisplatin) were studied. For local Gen-1 ASO administration, large studies have been conducted for intradermal afovirsen (275 patients), intravitreal fomivirsen (172 patients) [24], and alicaforsen enema (112 and 189 patients) [20,21]. In addition to the ISIS Gen-1 ASOs listed in Table 13.1, clinical studies have been conducted for at least eight other Gen-1 ASOs [51–69]. Roughly 1000 subjects were treated with ASO in these trials. For the most part, the ASOs were administered by intravenous infusion. The majority of the study subjects were patients with cancer although patients with human immunodeficiency virus (HIV) [62]

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Table 13.1 Clinical Experience with First- and Second-Generation ISIS Antisense Oligonucleotides Subjects Treateda ASO Name

Target

Route

First-Generation ASO Afovirsen (ISIS 2105)

HPV-11 E2

Intradermal

Alicaforsen (ISIS 2302)

ICAM-1

i.v. or s.c.

Rectal ISIS 2503 Fomivirsen (ISIS 2922) Aprinocarsen (ISIS 3521) ISIS 5132 ISIS 14803

H-ras HCMV IE2 PKC-
c-raf kinase HCV IRES

Topical i.v. Intravitreal i.v. i.v. i.v. or s.c.

Second-Generation ASO ISIS 13312 LY2181308c (ISIS 23722) ISIS 104838

HCMV IE2 Survivin TNF-


Intravitreal i.v. i.v. and/or s.c.

ATL 1102e (ISIS 107248) OGX-011d (ISIS 112989) ISIS 113715

VLA-4 Clusterin PTP-1B

Topical i.v. or s.c. i.v. i.v. or s.c.

LY2275796c (ISIS 183750) ISIS 301012

eIF-4E apoB100

i.v. i.v. and/or s.c. Oral

Study Populations

ISIS

Healthy volunteers Genital warts Healthy volunteers Crohn’s disease Rheumatoid arthritis Psoriasis Renal transplantation Ulcerative colitis Pouchitis Psoriasis Cancer CMV retinitis Cancer Cancer Chronic hepatitis C

76 335 91 593 38 17 42 263 12 94 288 412 1152 221 104

CMV retinitis Cancer Healthy volunteers Rheumatoid arthritis Psoriasis Healthy volunteers Cancer Healthy volunteers Type 2 diabetes Cancer Healthy volunteers Hypercholesterolemia Healthy volunteers

2 14f 50 162 60 36f 73f 87 80 2f 139 85 24

Control 37 63 28 252 11 14 93 — b

— 8 622 — — — — 16 53 b

18f — 5 24 — 7 19 —

a

Enrollment as of August, 2006 except for studies conducted by Eli Lilly and Company, OncoGeneX, and Antisense Therapeutics. b Separate psoriatic plaques of each subject were treated with ASO- and placebo-containing creams. c In development by Eli Lilly and Company. d In development by OncoGeneX Technologies, Inc. e In development by Antisense Therapeutics, Ltd. f

Incomplete enrollment information available.

represent one-quarter (⬃250) of the population. Evaluations of oblimersen, a Gen-1 inhibitor or Bcl-2, comprise the largest segment of the cancer patient population. Numerous small studies have been conducted for oblimersen [53–59]. At least one large randomized controlled trial (770 patients with advanced melanoma randomized between treatment with dacarbazine versus dacarbazine plus oblimersen) has been conducted for this ASO [51,52]. The clinical experience with Gen-2 ASOs is smaller but growing rapidly (Table 13.1). Eight of these compounds have entered clinical evaluation with active development programs ongoing for six (ATL 1102, ISIS 113715, ISIS 301012, LY2181308, LY2275796, and OGX-011). Approximately 800 subjects have been treated with a Gen-2 ASO with all but 74 subjects receiving the ASOs systemically. With the development efforts for four of the Gen-2 ASOs focused on chronic disease conditions (type 2 diabetes for ISIS 113715, hypercholesterolemia for ISIS 301012, rheumatoid arthritis for ISIS 104838, and multiple sclerosis for ATL-1102), the proportion of cancer patients in the Gen-2 ASO clinical experience is less (17%) than in the Gen-1 experience (66% of the subjects treated intravenously or subcutaneously). In line with greater emphasis on development for

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noncancer indications, more studies of healthy volunteers have been part of the Gen-2 ASO clinical experience (11 of 25 versus 5 of 85 for Gen-1 ASOs). Approximately 40% of the subjects that have received Gen-2 ASOs by systemic administration were healthy volunteers. 13.2.2 Systemic Administration Schedules and Doses Studied Whereas intravenous infusion was mainly used for systemic delivery of Gen-1 ASOs (96% of subjects), subcutaneous administration is being more widely studied as the delivery route for Gen-2 ASOs (63% of subjects). Table 13.2 summarizes the Gen-1 and Gen-2 dosing experience for ISIS ASOs. Three intravenous infusion schedules have been studied for Gen-1 ASOs. For noncancer indications, the standard infusion for Gen-1 ASOs was 2-h long. Owing to the tissue half-lives of these Table 13.2 Number of Subjects Treated with Gen-1 and Gen-2 ASOs by Duration for Selected Doses Number of Subjects Treated By Duration (weeks)

ASO Generation

Total

1–4

5–12

13–26

⬎26

325 132 6 6 55 3 59 34 12 27

150 130 1 3 5 3 59 1 — n/a

162 — 5 3 50 — — 33 12 n/a

— — — — — — — — — n/a

13 2 — — — — — — — n/a

6 4 9 3 6 7

— — 3 2 1 4

6 4 5 1 2 3

— — 1 — 1 —

— — — — — —

Continuous Intravenous Infusion (14 or 21 days per treatment cycle) ISIS 3521 1 2 mg/kg/day 1098 130 3 mg/kg/day 11 5 ISIS 5132 1 2 mg/kg/day 95 21 4 mg/kg/day 41 6 5 mg/kg/day 4 3 ISIS 2503 1 6 mg/kg/day 243 55 10 mg/kg/day 4 2

489 5 49 13 1 113 2

386 — 11 4 — 21 —

38 1 2 — — 7 —

— — 2 116 — — — 24 2 —

— — — — — — — — — —

— — — — — — — — — —

Drug

2-h Intravenous Infusions ISIS 2302 1 ISIS ISIS ISIS ISIS ISIS ISIS

3521 5132 14803 104838 301012 113715

OGX-011

1 1 1 2 2 2 2

24-h Intravenous Infusions ISIS 3521 1 ISIS 5132

1

ISIS 2503

1

Subcutaneous Injection ISIS 2302 1 ISIS 14803 ISIS 104838

1 2

ISIS 117315

2

ISIS 301012

2

ATL 1102

2

Dosea

b

2 mg/kg 300 mg 6 mg/kg 6 mg/kg 6 mg/kg 6 mg/kg 200 mg 200 mg 600 mg 640 mg 18 24 24 30 18 24

mg/kg mg/kg mg/kg mg/kg mg/kg mg/kg

0.5 mg/kg 2 mg/kg 2 mg/kg 200 mg 6 mg/kg 200 mg 400 mg 200 mg 400 mg 6 mg/kg

64 3 2 148 3 30 23 60 24 8

64 3 — 32 3 30 23 36 22 8

Note: n/a, not available. a Doses shown are those at which substantial number of subjects were treated and/or the highest doses tested. b 200 mg ISIS 113715 and 6 mg/kg ISIS 104838 were administered by 1-h infusion.

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drugs (3–4 days) [9], infusions were typically given on a thrice-weekly schedule. The inconvenience of this dosing schedule was an impediment to the clinical evaluation of Gen-1 ASOs. Some Gen-2 ASOs have also been studied using 1- or 2-h intravenous infusions. However, these compounds only require once weekly dosing because of their improved stability (tissue half-lives of 11–30 days) [73]. Doses up to 6 mg/kg given by 2-h infusion have been studied for three Gen-1 ASOs (aprinocarsen, ISIS 5132, and ISIS 14803) [6,46]. For Gen-2 ASOs, doses up to ⬃600 mg have been studied for ISIS 113715 and OGX-011 [3]. Alternative intravenous infusion schedules have been evaluated for Gen-1 ASOs, in cancer patients to reduce the number of clinic visits required of these patients. Once-weekly 24-h intravenous infusions were studied for three drugs (aprinocarsen, ISIS 5132, and ISIS 2503) [27,45]. All three could be given at doses up to 24 mg/kg. However, this infusion schedule required patients to make clinic visits on two consecutive days each week. Most of the studies for aprinocarsen, ISIS 5132, and ISIS 2503 (40 of 45 studies) and oblimersen [51–59] have used 14- or 21-day continuous intravenous infusions as part of repeated 21- and 28-day treatment cycles, respectively. With this schedule, patients carry an ambulatory infusion pump that delivers a constant flow of ASO during the treatment period. This infusion schedule allowed patients to make only one clinic visit per week to either start the infusion, change the seven-day infusate reservoir, or disconnect the pump. More than 2000 cancer patients have received ISIS Gen-1 ASOs in this manner. Unlike the 1-, 2-, and 24-h infusion schedules where there were no differences between ASOs as to the doses that could be given [6,16,27,45,46,50], the maximum tolerated dose (MTD) varied between ASOs with the continuous infusion schedule. For aprinocarsen, the MTD was 2 mg/kg/day. At 3 mg/kg/day, the principal dose-limiting toxicity was Grade 3 fatigue [44]. Although ISIS 5132 at 5 mg/kg/day did not technically meet dose-limiting criteria, the AE profile at this dose was sufficient to discontinue the dose escalation [28]. ISIS 2503, in contrast, had a good safety profile and good tolerability at 10 mg/kg/day given by 14-day continuous intravenous infusion [47]. For this drug, further dose escalation was abandoned because the concentration of the drug product available at that time would have required patients to carry a 7-day drug reservoir in excess of 1 L. Oblimersen was studied at 3 mg/kg/day given by 14-day infusion in the Phase III study of patients with advanced melanoma [51,52]. The reason for the differences between Gen-1 ASOs using the continuous infusion format is unknown. For subcutaneous injection of Gen-1 ASOs, tolerability issues (local skin reactions and lymphadenopathy) limited the doses that could be given (see Section 13.3.3.2). Although single doses of alicaforsen up to 4 mg/kg were injected into healthy volunteers, the dose explored in a Phase II study of patients with Crohn’s disease was 0.5 mg/kg because of the side effects [17]. For oblimersen, the MTD for 14-day subcutaneous infusion in patients with non-Hodgkin’s lymphoma was found to be 147.2 mg/m2/day [4]. As a result, the development programs for both alicaforsen and oblimersen have relied on intravenous infusion for the bulk of their systemic studies [13–16,51–59]. As noted earlier, 63% of subjects treated with Gen-2 ASOs received drug by subcutaneous injection and this is the main route of administration in the development programs for ISIS 104838, ISIS 113715, ISIS 301012, and ATL 1102. Doses of, or equivalent to, 200 and 400 mg have been given in all of these programs without tolerability being dose limiting. ATL 1102 has been given subcutaneously at 6 mg/kg to eight healthy subjects in a Phase I study without dose-limiting effects. 13.2.3 Systemic Treatment Durations Studies in which ASOs were given intravenously or subcutaneously for 12 weeks or longer have been conducted for five Gen-1 ASOs (alicaforsen, aprinocarsen, ISIS 5132, ISIS 2503, and ISIS 14803) and four Gen-2 ASOs (ISIS 104838, ISIS 113715, ISIS 301012, and OGX-011). Table 13.2 shows the number of patients treated by duration for selected doses. The bulk of the subjects (72%) in the table with treatment durations of 5–12 weeks or longer were cancer patients dosed by continuous intravenous infusion. Those cancer studies also had a substantial number (422) of

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patients treated for 13–26 weeks. The reason for early treatment discontinuation was cancer progression in the majority of cases. Among the 487 NSCLC patients that discontinued early from treatment in a 670-patient Phase III study, cancer progression (tumor progression, lack of efficacy, and death due to disease) was the reason 58% of the patients that received aprinocarsen, cisplatin, and gemcitabine and 64% of the patients that received only chemotherapy [32]. AE or death due to study drug toxicity was the reason for 25 and 19% of the patients in the two study arms, respectively [32]. The differences between the two study arms were not significantly different. Similarly, in the 586-patient study of aprinocarsen in combination with carboplatin and paclitaxel, 63 and 11% of the NSCLC patients receiving aprinocarsen discontinued treatment early because of disease progression and toxicity, respectively. These frequencies were similar to those of patients receiving only chemotherapy (53 and 10%, respectively). Across ASOs and dose levels, more than 500 subjects have been treated for more than 3 months and more than 60 subjects for more than 6 months. Among the longer treatment durations for alicaforsen are 13 patients with Crohn’s disease that received 2–4 treatment courses (2 mg/kg or 300 mg given thrice weekly for 4 weeks) over a span of 1–4 years. The subject that received aprinocarsen for the longest duration was a patient with non-Hodgkin’s lymphoma that received 17 treatment cycles composed of thrice weekly 2-h intravenous infusions of doses ranging from 0.5 to 1.5 mg/kg given for three weeks per 28-day cycle [46]. This patient’s dosing was discontinued after the patient achieved a complete tumor response. The subject that received the greatest number of treatment cycles was a patient with pancreatic cancer who was dosed at 6 mg/kg/day with ISIS 2503 by 14-day continuous intravenous infusion for 24 cycles (21 days/cycle) in combination with gemcitabine [49]. 13.2.4 Local Treatment with ASO Gen-1 ASOs have been studied as intravitreal therapy (fomivirsen) for acquired immune deficiency syndrome (AIDS)-associated cytomegalovirus (CMV) retinitis, intradermal therapy (afovirsen) for genital warts, topical therapy (alicaforsen) for psoriasis, and rectal enema therapy (alicaforsen) for ulcerative colitis and pouchitis. The number of subjects studied for each of these indications is provided in Table 13.1. Fomivirsen was granted marketing approval by the US Food and Drug Administration in 1998 and by other regulatory agencies in 1999 based on those studies. The Gen-2 clinical experience with local therapy formulations is more limited. The development of the Gen-2 version of fomivirsen (ISIS 13312) was terminated because of the shrinking size of the disease population due to the advent of highly active antiretroviral therapy (HAART). A topical cream containing ISIS 104838 was examined for the treatment of psoriasis in a single exploratory study.

13.3 CLINICAL SAFETY RESULTS FOR SYSTEMIC ADMINISTRATION Overall, the AE profile for patients treated intravenously or subcutaneously with Gen-1 and Gen-2 ASOs is similar to that of control subjects. Table 13.3 shows the number and frequency of patients with AEs and the number of events for ASO-treated and control patients that were observed in large randomized, controlled trials of two Gen-1 ASOs and one Gen-2 ASO. The results for alicaforsen, a Gen-1 ASO inhibitor of ICAM-1, come from two nearly identical studies of the 300 mg dose given by 2-h intravenous infusion to patients with Crohn’s disease. Patients were dosed thrice weekly for 4 weeks. The median weight-adjusted alicaforsen dose in these studies was 4.2 mg/kg. The per-patient and the per-patient-year rates of any AE, severe AEs, serious adverse events (SAEs), and deaths for patients treated with alicaforsen were similar to those for patients that received placebo. A higher number of events (918 versus 276) were recorded for alicaforsen-treated subjects than expected based on the 2:1 randomization ratio. The difference was due to patients

c

b

a

Number of Patients % of Patients

Per Patient-Years

10.72 1.70 0.98 0 918 55 17 0 72 6 8 0

66.1 5.5 7.3 0 9.35 0.78 1.04 0

91.6 16.8 5.3 0.8 5.00 0.92 0.29 0.04 820 33 10 1 38 3 3 0

84.4 6.7 6.7 0

4.87 0.38 0.38 0

ISIS 104838 (100, 200, or 400 mg/week) or Placebo for 12 Weeks in Patients with Rheumatoid Arthritis c ASO (131 patients, 24.0 Pt-Yrs) Placebo (45 patients, 7.8 Pt-Yrs)

73.9 11.7 6.8 0

100.0 86.1 58.5 10.9 3.93 3.38 2.30 0.43 6295 1162 289 32

291 205 89 31

99.7 70.2 30.5 10.6

4.59 3.23 1.40 0.49

99.4 89.4 50.3 6.5 4.79 4.31 2.43 0.31

5456 1355 402 29

319 256 114 21

99.4 79.8 35.5 6.5

4.38 3.51 1.56 0.29

5225 912 244 21

5074 690 135 32

Event count includes multiple recordings for some events due to changes in severity or seriousness. Alicaforsen dose was 100 mg for first infusion; for next 11 infusions dose was 300 mg for patients weighing ⬎50 kg and was 200 mg for those weighing 36–50 kg. Two-hour infusions were performed thrice weekly for 4 weeks. All ISIS 104838 patients received two 200 mg injections in the first week; 47 patients received one 200 mg injection every other week beginning in week 2; 44 patients received on 200 mg injection once weekly during weeks 2–12; and 40 patients received two 200 mg injections each week during weeks 2–12. Injections of placebo were given when injections of ISIS 104838 were not so that all subjects received two injections per week.

320 288 162 21

Aprinocarsen (2 mg/kg/day) ⫹ Cisplatin ⫹ Gemcitabine or Chemotherapy Alone in Patients with Non-Small Cell Lung Cancer ASO ⫹ chemotherapy (322 patients, 66.8 Pt-Yrs) Chemotherapy alone (321 patients, 72.9 Pt-Yrs)

294 253 172 32

155 3 3 0

276 13 9 0

Number of Eventsa

372

All Severe Serious Deaths

Number of Eventsa

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All Severe Serious Deaths

120 22 7 1

All Severe Serious Deaths

Per Patient-Years

Alicaforsen (300 mg) or Placebo Thrice Weekly for Four Weeks in Patients with Crohn’s Disease b ASO (222 patients, 15.3 Pt-Yrs) Placebo (109 patients, 7.7 Pt-Yrs)

% of Patients

5/5/2007

Aprinocarsen (2 mg/kg/day) ⫹ Carboplatin ⫹ Paclitaxel or Chemotherapy Alone in Patients with Non-Small Cell Lung Cancer ASO ⫹ chemotherapy (294 patients, 74.8 Pt-Yrs) Chemotherapy alone (292 patients, 63.4 Pt-Yrs)

164 26 15 0

Number of Patients

All Severe Serious Deaths

Adverse Event Criteria

Table 13.3 Overview of Adverse Events in Randomized Controlled Trials of Gen-1 and Gen-2 ASOs

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experiencing a variety of transient constitutional symptoms (e.g., fever, rigors, headache, nausea, and other flu-like symptoms) following ASO infusions. These are discussed in greater depth in Section 13.3.3.1. Of the 17 SAEs experienced by alicaforsen-treated patients and 9 SAEs by placebo-treated patients, 12 and 8 of the events, respectively, were either Crohn’s disease or Crohn’s disease aggravated (preferred terms of the Medical Dictionary for Regulatory Activities, MedDRA). AEs of the gastrointestinal disorder MedDRA system organ class (SOC) were similarly the most common AEs (excluding the infusion-associated constitutional symptoms) observed for both ASO- and placebo-treated patients. In the study of ISIS 104838, a Gen-2 ASO inhibitor of TNF-
, 176 patients with rheumatoid arthritis gave themselves two subcutaneous injections each week for 12 weeks. Since animals given ASO by subcutaneous injection have the same tissue concentrations of ASO as animals dosed intravenously, the two routes provide identical systemic exposure to ASO [73]. Patients were randomized to receive either only injections of placebo, one 200 mg ASO injection every other week, one 200 mg injection every week, or two 200 mg injections every week. While patient rates for AEs were not significantly different between ASO- and placebo-treated patients, higher rates and higher number of events than expected based on the 3:1 population ratio were observed for ISIS 104838treated patients for both all AEs and severe AEs (Table 13.3). The difference resided almost entirely in the incidence of dermatologic reactions at subcutaneous injection sites. For ASO-treated patient, 307 injection site events were observed for 70 patients. In comparison, two injection site events involving two patients occurred in the placebo-treatment group. Further information about these injection site reactions is presented in Section 13.3.3.2. The one fatality in the study was due to bacteroides sepsis at 8 weeks after the patient’s last subcutaneous injection. The patient had received one injection of ISIS 104838 every other week. The sepsis was thought to be the result of a perforated diverticulum and the event was considered not related to study drug by the Investigator. Two large controlled studies of aprinocarsen, a Gen-1 ASO inhibitor of PKC-
, have been performed [31,32]. In both, patients with NSCLC were treated with standard chemotherapy regimens with one-half of the patients randomized to also receive ASO. Aprinocarsen was given at 2 mg/kg/day by 14-day continuous infusion per 3-week treatment cycle. Treatment cycles were repeated until either the patient completed six cycles (more cycles were allowed if tumor reduction was continuing), had progression of their disease, or experienced unacceptable toxicity. As expected for cancer studies involving chemotherapeutics, the frequency of events for all categories were substantially higher than observed in either the alicaforsen or ISIS 104838 studies. For the combination with carboplatin and paclitaxel, patients receiving aprinocarsen had higher frequencies (e.g., 58.5 versus 30.5% for SAEs) and incidences (e.g., 289 versus 135 SAEs) of AEs. Catheter-related infections and thrombocytopenia were the two main reasons for this. Patients randomized to receive aprinocarsen were required to have a central venous access line surgically implanted. Events in the MedDRA Infection and Infestation SOC constituted 83 SAEs involving 67 of the patients who received ASO versus 17 SAEs involving 14 control subjects. Thrombocytopenia and platelet reduction events represented 364 AEs involving 151 ASO-treated patients compared to 122 AEs in 115 control patients. The higher frequency and number of AEs for aprinocarsen added to chemotherapy did not extend to deaths. The number of on-study fatalities was equal between the two treatment groups. With the cisplatin and gemcitabine combination, the frequency and number of severe AEs and SAEs were also higher for aprinocarsen-treated patients than control patients (Table 13.3). As with the carboplatin and paclitaxel combination, increased thrombocytopenia and catheter-related infections rates were the principal reasons. For SAEs, 378 thrombocytopenia or plateletdecreased events occurred in 212 ASO-treated patients. In comparison, only 157 events occurred in 99 control subjects. SAEs in the infections of SOC were 99 in 72 ASO-treated patients versus 25 in 21 control patients. Deaths on study occurred at equal frequency in the two study arms for this chemotherapy regimen.

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The above results indicate that treatment with ASO generally has a similar AE profile to control patients. The areas where ASO treatment contributes to greater AEs numbers are infusion-associated constitutional symptoms, subcutaneous injection site reactions, thrombocytopenia at least when ASO is given in combination with chemotherapy, and infections due to implanted catheters. The constitutional symptoms and subcutaneous injection site reactions are likely to be alternative manifestations of the proinflammatory effects seen with Gen-1 and Gen-2 ASOs in nonclinical studies performed on mice. Thrombocytopenia has also been observed in those studies. In addition, nonclinical studies of ASOs have also identified prolongation of aPTT, complement activation, and renal effects as toxicities common to the Gen-1 and Gen-2 ASO chemical classes and liver as a potential target organ for toxicity. The clinical findings related to these are reviewed below. 13.3.1 Prolongation of aPTT As predicted by nonclinical studies in monkeys, both first- and second-generation ASOs administered by intravenous infusion produced reversible, dose-related prolongations of aPTT in the clinical trials of these ASOs [6,12,46,50,71]. No differences between the two ASO generations were distinguishable. Figure 13.1A shows the mean intact ASO concentration and aPTT levels at various times before, during, and after infusion for six cancer patients given aprinocarsen, a Gen-1 inhibitor of PKC-
, at 6 mg/kg by 2-h intravenous infusion. The change in

(D)

aPTT (% change)

aPTT (% change)

2h

Predose

50 45 40 35

140 120 100 80 60 40 20 0 −20 −40

Day 3 Day 5 ISIS 2302 dose (mg/kg)

0 0.2 0.5 1.0 2.0

0 0.2 0.5 1.0 2.0

0 0.2 0.5 1.0 2.0

30 25

Day1

(C)

70 y = 0.1218x − 2.0467 60 R 2 = 0.801 50 40 30 20 10 0 y = 0.006x + 1.1008 −10 R 2 = 0.0658 −20 0 100 200 300 400 500 ISIS 301012 (mg)

55

0 0.2 0.5 1.0 2.0

(B) aPTT (s), mean ± SEM

32 aPTT 28 ASO 24 100 20 80 16 60 12 40 8 20 4 0 0 0 100 200 300 400 Time from start of 2-h infusion (min) 140 120

ISIS 3521 (µg/ml)

aPTT (% change)

(A)

Day 7

y = 1.8752x + 1.0038 R 2 = 0.8618 0

10

20 30 40 50 ISIS 104838 (µg/mL)

60

70

Figure 13.1 Effect of Gen-1 and Gen-2 ASO administration on aPTT. (A) Mean percent change in aPTT and ISIS 3521 plasma concentrations at various times after the start of infusion for six cancer patients given 6 mg/kg ASO by 2-h intravenous infusion. Error bars indicate standard error. (B) Mean aPTT before and at the end of infusion for three healthy volunteers per alicaforsen dose level, and four healthy volunteers treated with placebo. Placebo and ASO were given by 2-h infusions performed every other day. (C) Percent change in aPTT at time of Cmax (i.e., Tmax) for healthy volunteers given 50, 100, 200 or 400 mg ISIS 301012, or placebo, by subcutaneous injection (closed diamonds) and 1-h intravenous infusion (open boxes). (D) Percent change in aPTT versus plasma ASO concentration at the end of infusion for healthy volunteers given 0.1, 0.5, 1, 2, 4, or 6 mg/kg ISIS 104838, or placebo, by 1-h infusion. Results were pooled from infusions on days 1, 8, 10, and 12.

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aPTT approximately parallels the plasma concentration of ASO with maximum prolongation occurring when Cmax is reached at the end of the infusion. All Gen-1 and Gen-2 ASOs produce similar effects [6,12,46,50,71]. Following the end of infusion, plasma concentrations of both Gen-1 and Gen-2 rapidly decline with plasma half-lives of ⬃1 and 2 h, respectively, as the ASOs are distributed to tissues [73,75]. APTT similarly declines during this period returning to predose levels by 2–6 h after the end of infusion [6,12,46,50,71]. These results are consistent with the results of in vitro studies on alicaforsen added to human plasma which show that the prolongation is due to nonspecific ASO binding to the intrinsic tenase complex (factors IXa and VIIIa, phospholipids, and calcium) [76]. The in vitro studies also showed that ASO effects on the extrinsic pathway were substantially less than the effect on aPTT [76]. In agreement, prolongation of prothrombin time (PT) and thrombin time (TT) during clinical dosing was found to be minimal, ⬃10% increased when aPTT was increased 40% [12]. Since the in vitro results predict that ASO levels in plasma determine the extent of aPTT prolongation and the plasma pharmacokinetics for ASOs have been found to be unchanged by repeated administration, aPTT prolongation should be the same infusion to infusion. Figure 13.1B shows the dose–response for aPTT at the end of infusion for healthy subjects given four infusions of alicaforsen, a Gen-1 inhibitor of ICAM-1. The extent of aPTT prolongation was similar between all four infusions at 0.2, 0.5, 1.0, and 2.0 mg/kg ASO. Prolongation of aPTT is not observed following administration of ASO by subcutaneous injection. Figure 13.1C shows the percent change in aPTT coinciding with Cmax following administration of ISIS 301012 in healthy volunteers by intravenous infusion and subcutaneous injection. APTT increased ⬃12% for each 100 mg of ISIS 301012 given intravenously. However, aPTT was not increased at the same doses given subcutaneously. This is the result of the Cmax following subcutaneous delivery being only a fraction of that following intravenous administration (3 versus 22 g/mL, respectively, at the 200 mg dose). Similarly, aPTT prolongation has not been observed for Gen-1 ASOs administration by continuous intravenous infusion [28,44]. Like subcutaneous administration, this infusion schedule results in ASO plasma concentrations (Css of 0.5–0.9 g/mL for 2 mg/kg/day) lower than those associated with 1- and 2-h intravenous infusions [28,37,38]. The above results confirm the prediction from nonclinical studies that the extent of aPTT prolongation is dependent upon plasma concentration [10]. Figure 13.1D shows the percent change in aPTT at the end of 1-h intravenous infusions for ISIS 104838 given to healthy volunteers is directly proportional to the plasma concentration of the ASO at the end of infusion, that is, the plasma Cmax. The linear relationship shown in Figure 13.1D has been observed for Gen-1 (alicaforsen, aprinocarsen, and ISIS 5132) and Gen-2 ASOs (ISIS 104838, ISIS 113715, and ISIS 301012) and has been observed in healthy volunteers, and patients with cancer, type 2 diabetics, and subjects with elevated total cholesterol levels. This class effect of Gen-1 and Gen-2 ASOs on aPTT is not associated with any adverse clinical events probably due to the short duration of the effect. Across ASOs, no evidence has been found for an increased incidence of bleeding, bruising, hemorrhaging or any other clinical sign or symptom indicative of a coagulation disorder following the administration of Gen-1 and Gen-2 ASOs. As a result, the prolongation of aPTT is without clinical significance. 13.3.2 Complement Activation Ten-min intravenous infusion of Gen-1 ASOs in monkeys has been shown to activate the alternative pathway of the complement cascade and produce lethargy, vomiting, hypotension, and transient neutropenia followed by neutrophilia in the affected animals [10]. In the severest cases, cardiovascular system collapses and death ensues [10,77]. Like aPTT prolongation, the effect on the complement pathway is dependent on ASO levels in plasma. However, unlike the coagulation parameter, complement activation depends on ASO in plasma increasing above at

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threshold concentration [10]. Increasing the length of the intravenous infusion from 10 min to 2 h, thereby lowering the ASO plasma Cmax, was found to be a readily applicable approach for preventing the increased appearance of complement split products in the plasma [10]. The threshold concentration in monkeys for intact Gen-1 ASOs is ⬃40–50 g/mL and ⬃70 g/mL for total oligonucleotide. For Gen-2 ASOs, the threshold concentration is 90–100 g/mL, or higher, for intact ASO [72]. The mechanism by which ASO-mediated complement activation occurs is ASO binding to Factor H, a negative regulator that prevents constitutive activation of the alternative pathway [10]. Although monkeys are much more susceptible to this class effect of Gen-1 and Gen-2 ASOs than other species [10], the dose levels and administration schedules that have been used in the clinical trials of ASOs have been chosen to avoid attaining plasma concentrations of ASO above the thresholds identified in monkeys. In keeping with these measures, no clinical signs, symptoms, or sequelae indicative of complement cascade activation have occurred in the clinical studies of ISIS ASOs [6,12,16,27,45,46]. Further, studies that included regular and/or continuous hemodynamic measurements and intensive sequential neutrophil counts during and following intravenous infusion did not observe any remarkable changes [8]. Measurements of complement split products (i.e., C3a, C5a, and/or Bb) have been regularly included in the clinical studies of both Gen-1 and Gen-2 ASOs to monitor for possible complement cascade activation. Except for the three studies evaluating high doses (18–30 mg/kg) of Gen-1 ASO administration by 24-h intravenous infusion, the results for C5a and Bb have consistently indicated that ASO administration does not result in increased levels of these two complement split products. However, increases in the C3a split product have been reported in a number of small studies [6,12,13,44,46]. Since corresponding increases in C5a and Bb were not observed and the development of clinical signs or symptoms was absent, those C3a observations were viewed by Investigators as unlikely to be indicative of any clinically meaningful activation of the complement system. That C3a levels is not increased by intravenous Gen-1 ASO administration was shown in a Phase III study of alicaforsen given to patients with Crohn’s disease. Table 13.4 summarizes the results from C3a measurements performed on preinfusion and end-of-infusion specimens collected from 100 patients given with alicaforsen, and 50 patients given placebo, by 2-h intravenous infusion. Mean C3a levels were similar between preinfusion and end-of-infusion regardless of whether the infusions contained 100 or 300 mg alicaforsen or placebo. The standard deviations and the range of results at each time point, however, indicate that the assay had a high variability. Further, higher C3a level before a dose than after was observed for 42% of the infusions. Preinfusion C3a levels ⬎500 ng/mL (501–6032 ng/mL) were observed for 13% of the infusions for both alicaforsen- and placebo-treated patients. These findings suggest that the high C3a results (⬎500 ng/mL) observed might be due to assay variability arising from either the assay methodologies or more likely from variances in specimen collection, processing, and handling. As a consequence, caution should be exercised in interpreting the C3a results from small populations. Given the absence of clinical signs or symptoms of complement activation, the Phase III results for alicaforsen also suggest the C3a levels seen in the study, 39–7182 ng/mL, were not clinically meaningful. Increases in complement split products as a result of ASO administration likely did occur in the Gen-1 ASO studies of weekly 24-h intravenous infusion. Figure 13.2 shows the mean end-of-infusion levels for each of the Gen-1 ASOs (aprinocarsen, ISIS 5132, and ISIS 2503). Dose-related increases in Bb as well as C3a were observed. Although the studies were small (15–25 patients per trial) and uncontrolled, the similarity of the results from the three studies, each performed by a different clinical site and Investigator and each evaluating a different ASO, suggests that the findings are reliable. However, the Bb and C3a levels observed in these trials are unlikely to be clinically meaningful since no clinical signs or symptoms of complement cascade activation were observed in any of the patients. Further, the C3a levels in these studies were not in excess of those shown in Table 13.4. The finding of increased complement split products following 24-h intravenous infusions is also contrary to the model of complement activation occurring when ASO concentrations rise above a threshold where Factor H is sufficiently prevented from suppressing the alternative pathway.

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Table 13.4 Complement Split Product C3a Levels before and after Intravenous Infusions of Alicaforsen in Patients with Crohn’s Disease Complement C3a (ng/mL) Alicaforsen

Placebo Preinfusion

End-of-Infusiona

First Infusion (100 mg Alicaforsen or Placebo) N 91 88 Mean ⫾ SD 305.7 ⫾ 309.5 361.8 ⫾ 795.5 Range 60–1743 55–7182

45 289.4 ⫾ 229.2 39–1251

46 251.2 ⫾ 203.1 45–1179

Seventh Infusion (300 mg Alicaforsen or Placebo) b,c N 94 95 Mean ⫾ SD 494.0 ⫾ 780.5 571.4 ⫾ 529.1 Range 110–6032 146–3622

47 309 ⫾ 440.4 65–2804

47 245.7 ⫾ 226.9 72–1163

Statistics

Preinfusion

End-of-Infusiona

a

Alicaforsen and placebo administered by 2-h intravenous infusion. See Table 3 for dosing schedule.

b

Includes unscheduled specimen collections from other 300/200 mg infusion days. Dose was 300 mg for patients weighing ⬎50 kg and 200 mg for patients weighing 36–50 kg.

c

(B)

(A) 3000

4

Bb levels (mcg/mL)

C3a levels (ng/mL)

2500 2000 1500 1000

3

2

1

500 0

0 3

6 12 18 24 Dose levels (mg/kg/24 h)

ISIS 5132

ISIS 3521

30

ISIS 2503

3

6 12 18 24 Dose level (mg/kg/24 h)

ISIS 5132

ISIS 3521

30

ISIS 2503

Figure 13.2 Mean complement split product C3a (A) and Bb (B) levels at the end of 24-h intravenous infusion. Aprinocarsen, ISIS 5132, and ISIS 2503 were administered to cancer patients by 24-h intravenous infusion once weekly. Error bars indicate standard error.

The mean steady-state plasma concentration (mean Css) of ISIS 3521 in patients given 24 mg/kg was 12.9 g/mL [45]. For patients given 24 and 30 mg/kg ISIS 5132, the mean Css were 13.4 and 19.1 g/mL [27]. These plasma concentrations are much lower than the threshold concentration (40–50 g/mL), identified in studies of monkeys dosed by 2-h intravenous infusion. Further, both aprinocarsen and ISIS 5132 have been given to cancer patients by 2-h intravenous infusions, which resulted in higher plasma concentrations of ASO (29.4 and 22.7 mg/mL, respectively, for 6 mg/kg dose) without evidence for substantial increases in the levels of complement split products [6,46]. While the increased levels of complement split products following 24-h intravenous infusion probably is due to the length of time that ASO plasma levels are at the Css, ⬃20 h, further nonclinical studies will be required to understand the mechanism. 13.3.3 Proinflammatory Effects Immune stimulation in mice and other rodents is a class effect of both Gen-1 and Gen-2 ASOs with the Gen-2 ASOs being less potent for this effect than Gen-1 ASOs [72]. Following repeated

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administration of ASO, the typical findings indicative of proinflammatory effects in mice are splenomegaly, lymphoid hyperplasia, and diffuse mixed mononuclear cell infiltrates in liver, kidney, lung, heart, thymus, pancreas, and other organs [10]. ALT/AST increases and platelet count reductions have been observed in association with these proinflammatory effects and generally are secondary consequences of the immune stimulation [10]. It should be noted that some individual nonclinical ASOs have been shown to have ALT/AST and platelet effects that are separate from the proinflammatory effects. For Gen-1 ASOs, the proinflammatory effects are well correlated to increases in serum cytokine (e.g., IL-1, IL-6, IL-12, and IFN-) and chemokine (e.g., MCP-1 and MIP-2) levels [72]. Gen-2 ASOs are less proinflammatory and they appear to preferentially stimulate chemokines such as MCP-1, however this remains an area of active research [72]. The dosing of monkeys with Gen-1 and Gen-2 ASOs does not produce findings similar to the ones in mice. Generally, the only findings indicative of proinflammatory effects that are observed in monkeys given ASOs are mild lymphoproliferative effects at draining lymph nodes following subcutaneous administration [72]. There are phosphorothioate oligodeoxynucleotides, especially ones with CpG dinucleotides contained within palindromic motifs, which can stimulate B-cell maturation and proliferation in monkeys [78]. However, the current discovery process for clinical ASOs selects for development candidates with low immunostimulatory activity. While signs, symptoms, or laboratory results indicative of the proinflammatory effects typical of rodents have not been observed in the clinical studies of Gen-1 and Gen-2 ASOs, the administration of these compounds does produce two effects, which are likely to be related to the proinflammatory properties of phosphorothioate oligodeoxynucleotides. These effects are the dose-related occurrences of constitutional symptoms (e.g., fever and chills) in association with intravenous infusions and dermatologic reactions (e.g., painless erythema) following subcutaneous injection of ASOs. These effects are discussed below. The development of constitutional symptoms appears to be mainly a class effect of Gen-1 ASOs whereas the subcutaneous injection site reactions are a class effect for both Gen-1 and Gen-2 ASOs. In addition, rare hypersensitivity-like (allergic-like or anaphylactoid) reactions have been observed in the clinical studies of some firstgeneration ASOs. Since phosphorothioate oligodeoxynucleotides are poorly or not antigenic [72], these hypersensitivity-like reactions may also be related to the proinflammatory properties of these compounds if they are actually due to ASO administration.

13.3.3.1 Infusion-Associated Constitutional Symptoms The development of several flu-like symptoms is temporally associated with the intravenous infusion of Gen-1 ASOs. The incidence and severity of these symptoms are dose-related but the symptom profile varies with the length of infusion.

Constitutional Symptom Profile Two-Hour Intravenous Infusions Fever and chills/rigors were the most common adverse effects experienced by patients given high doses (⬃4–6 mg/kg) of the Gen-1 ASOs such as alicaforsen (ISIS 2302) [16], aprinocarsen (ISIS 3521) [46], ISIS 5132 [6], and ISIS 14803. Typically, these symptoms were mild to moderate in intensity, arose within 4 h of the end of infusion, and resolved overnight, either spontaneously or with acetaminophen, ibuprofen and/or corticosteroid treatment [6,16]. Additional symptoms experienced by the patients included headache, nausea, vomiting, fatigue, myalgia, and arthralgia. A good characterization of the symptom profile associated with 2-h intravenous infusions was obtained from two Phase III studies of alicaforsen treatment for Crohn’s disease. In these two nearly identical double-blind, placebo-controlled studies that essentially only differed in geography, patients with Crohn’s disease were randomized 2:1 between treatment with 300 mg alicaforsen (200 mg if weight ⬍51 kg) and placebo given thrice weekly for 4 weeks by 2-h infusion. The study

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design included identification of any AEs that had an onset within 8 h of the start of an infusion (i.e., until 6 h after the end of infusion). Table 13.5 presents the most commonly observed infusionassociated AEs in the two studies. As with smaller studies, fever and rigors were the most common events (16 and 11% of alicaforsen-treated patients) and both were observed more frequently in patients given ASO than in patients treated with placebo. The table also shows that the infusionassociated events constituted ⬎50% of the incidence for these events throughout the study (e.g., 64% of the fever episodes for alicaforsen-treated patients occurred in association with infusions). Headache, nausea, arthralgia, vomiting, myalgia, and dizziness were also observed in association with alicaforsen infusions. However, headache and nausea occurred with similar frequencies in patients given placebo. While dizziness occurred less frequently following alicaforsen infusions than placebo infusions (3.7 versus 5.5% of patients, respectively), the ratio of the number of events between the two treatment groups (23:4) was greater than the randomization ratio (2:1) indicating that dizziness is likely to be an effect of alicaforsen infusion in some patients. As a result the effects due to alicaforsen administration were pyrexia, rigors, arthralgia, vomiting, myalgia, and dizziness. While these results are qualitatively similar to the results from smaller studies, the frequency of the AEs in the Phase III studies was lower. There are two reasons for this. First, the alicaforsen Phase III studies treated patients with a fixed dose of 300 mg for all patients weighing more than 50 kg. Patients weighing 36–50 kg were dosed at 200 mg. On the basis of the weights of the patients, the median weight-adjusted dose was 4.2 mg/kg and the range of doses that were given was 1.6–5.9 mg/kg. As a result the average patient in these two alicaforsen studies was treated at lower doses than some of those studied (6 mg/kg) in the smaller studies of other Gen-1 ASOs. Second, the design of the alicaforsen Phase III studies included a dose schedule and a premedication regimen both of which were adopted as means for reducing the incidence of the infusionassociated reactions. The dosing schedule for these studies required the dose for the first infusion to be 100 mg. Since symptoms are usually worst after the first infusion, administering a lower dose at the first infusion should aid acclimation to ASO treatment. The study protocols also called for patients to receive preinfusion corticosteroid treatment equivalent to a low dose of prednisone (10 mg) and end-of-infusion treatment with 1000 mg acetaminophen with each of their first six infusions. These medications were included in the study to ameliorate the development of symptoms. While no controlled studies have been performed to demonstrate that the dose schedule and the premedication regimen are effective, it is likely one or both reduced the frequency of

Table 13.5 Common Adverse Events Associated with 2-h Intravenous Infusions of Alicaforsen Given to Patients with Crohn’s Disease Number of Patients (%) Number of Eventsa Alicaforsen (N ⫽ 221) b Adverse Events (MedDRA Preferred Terms) Pyrexia Rigors Headache NOS Nausea Arthralgia Vomiting NOS Myalgia Dizziness a b c

Placebo (N ⫽ 109) b

Infusion Associatedc

All Occurrences

Infusion Associatedc

All Occurrences

34 (15.6%) 67 25 (11.3%) 58 24 (10.9%) 53 13 (5.9%) 20 13 (5.9%) 18 10 (4.5%) 13 8 (3.6%) 12 6 (3.7%) 23

50 (27.5%) 104 31 (14.0%) 70 39 (17.6%) 90 26 (11.7%) 36 16 (7.3%) 23 19 (8.6%) 25 11 (5.0%) 16 12 (5.4%) 36

7 (6.4%) 11 0 10 (9.2%) 32 7 (6.4%) 15 0 0 1 (0.9%) 1 3 (5.5%) 4

11 (10.1%) 16 1 (0.9%) 1 16 (14.7) 44 10 (9.2%) 24 2 (1.8%) 2 4 (3.5%) 5 1 (0.9%) 1 5 (4.6%) 6

Event count includes multiple recordings for some events due to changes in severity or seriousness. See Table 13.3 for doses and infusion schedule. Infusion-associated adverse events are those that began in the 8 h following the start of a 2-h intravenous infusion.

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infusion-associated AEs in these alicaforsen studies because the observed frequencies for fever and rigors were substantially lower than the expected frequency of 50–100%. The alicaforsen Phase III studies also provided a clear demonstration of the reduction in infusionassociated symptom incidence that occurs with repeated infusions. Figure 13.3 shows the frequency of patients with any infusion-associated symptoms and the rate for all infusion-associated AEs for each of the 12 infusions administered. For both parameters, alicaforsen- and placebo-treated patients had similar responses at the first infusion. The frequency of patients with events and the rate of events increased for the alicaforsen-treated patients with the second infusion due to the dose escalation from 100 to 300 mg alicaforsen between the two infusions. Thereafter, the frequency and the rate both declined with repeated infusion. By the sixth infusion, the patient frequency and the event rate for patients receiving alicaforsen was similar to those of patients receiving placebo. While the dose-related occurrence of infusion-associated constitutional symptoms is a Gen-1 ASO class effect, the effect has not been generally observed for Gen-2 ASOs. Fever and chills have been reported for OGX-011, a Gen-2 ASO inhibitor of clusterin [3]. Prostate cancer patients given OGX-011 by 2-h intravenous infusion experienced fever and chills with increasing frequency and severity as the OGX-011 dose was escalated from 40 to 640 mg during the Phase I study of the drug. At both 480 and 640 mg OGX-011, five of six patients at each dose experienced fever and six of six patients experienced rigors. In contrast, fever and chills were not experienced by healthy volunteers given up to 6 mg/kg ISIS 104838, a Gen-2 ASO inhibitor of TNF-
, by 1-h intravenous infusion (zero of three subjects at 6 mg/kg) [71]. Nor were they experienced by healthy volunteers and type 2 diabetics given ISIS 113715, at Gen-2 ASO inhibitor of PTP-1B, doses of 7.5 mg/kg (zero of three subjects) and 600 mg (0 of 12 patients), respectively. These results suggest that the development of infusion-associated symptoms is not a Gen-2 class effect although testing of additional Gen-2 ASOs is needed. Why OGX-011 produces infusion effects is not known. There are two structural differences between OGX-011 and other ISIS Gen-2 ASOs. First, OGX-011 has a 4-13-4 MOE-deoxy-MOE structure in contrast to the 5-10-5 structures of ISIS 104838 and ISIS 113715. As a result, OGX-011 is more Gen-1-like than the other Gen-2 ASOs because of its higher proportion of phosphorothioate oligodeoxynucleotides. Second, OGX-011 does not contain 5-methyl-cytosines in its deoxynucleotide region. Again, this makes OGX-011 more Gen-1-like. However, the presence of (A)

(B) 70

Percent of patients

25

ISIS 2302 Placebo

20 15 10 5

Events per 100 patients

30

60

40 30 20 10 0

0 1

2

3

4

5

6 7 8 Infusion

9 10 11 12

ISIS 2302 Placebo

50

1

2

3

4

5

6 7 8 Infusion

9 10 11 12

Figure 13.3 Frequency (A) and incidence (B) of infusion-associated adverse events. Patients with Crohn’s disease were given alicaforsen (N ⫽ 221) or placebo (N ⫽ 109) by 2-h intravenous infusions thrice weekly for 4 weeks. For alicaforsen-treated patients, the first dose was 100 mg. The dose for subsequent infusions was 300 mg for patients weighing ⬎50 mg and was 200 mg for patients weighing 36–50 kg. Before infusions 1–6, patients were pretreated with 10 mg prednisone or the equivalent dose of another corticosteroid. Patients also received 1000 mg acetaminophen at the end of these infusions. Infusionassociated adverse events were those events that began within 8 h following the start of an infusion.

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5-methyl-cytosines in ISIS 104838 and ISIS 113715 is not likely to be the reason they do not elicit infusion-related symptoms. ISIS 14803, a Gen-1 ASO that has 5-methyl-cytosine replacements for all five of its cytosine positions, elicits infusion-associated symptoms (fever in 70% and chills in 55% of patients treated with 6 mg/kg twice a week for 12 weeks).

Twenty-Four Hour Intravenous Infusions Three first-generation ASOs (aprinocarsen, ISIS 5132, and ISIS 2503) have been studied in Phase I dose-escalation studies of patients with advanced cancer using administration by once weekly 24-h intravenous infusion [27,45]. Like 2-h intravenous infusions, 24-h infusions of Gen-1 ASOs result in the dose-related appearance of fever, chills, and other flu-like symptoms. Aprinocarsen and ISIS 2503 doses up to 24 mg/kg were studied. ISIS 5132 was studied up to 30 mg/kg. Mild and moderate fever (National Cancer Institute Common Toxicity Criteria for Adverse Events, NCI CTCAE, grades 1 and 2) and severe chills (NCI CTCAE grade 3) were common toxicities with all patients experiencing them at the 24 and 30 mg/kg doses. Fevers could be managed with 650 mg acetaminophen given before and every 4 h during the infusion [27]. Fatigue, nausea/vomiting, myalgia were also common although the dose relationships for these symptoms were not as clear as the relationship to fever [27,45]. There does not appear to be a difference in the nature, frequency or severity of infusion-associated constitutional symptoms that result from Gen-1 ASO administration of 6 mg/kg by 2-h infusion and 24 mg/kg by 24-h infusion. The main difference between the two schedules is that the onset of symptoms is during the 24-h infusion but is after the end-of-infusion for the 2-h schedule. This difference probably is due to Cmax occurring at the end-of-infusion for 2-h administration [6,46] and Css being achieved by 4 h of the start of the longer infusions [27,45]. Continuous Infusion (14- and 21-day infusions) Aprinocarsen, ISIS 5132, and ISIS 2503 have been studied as single agents and in combination with a variety of chemotherapy regimens in patients with different types of cancers using 14- and 21-day continuous intravenous infusions for ASO administration. [25,27–44,47–48]. The main study dose for aprinocarsen and ISIS 5132 was 2 mg/kg/day although these drugs were studied at up to 3 and 5 mg/kg/day, respectively. ISIS 2503 was routinely dosed at 6 mg/kg/day and doses up to 10 mg/kg/day of the drug were studied in small numbers of subjects. In contrast to the shorter infusion schedules, fever and rigors were less frequently associated with ASO infusion. For aprinocarsen given at 2 mg/kg/day by 14-day infusion in combination with carboplatin and paclitaxel to 292 patients with NSCLC, 25.2% experienced fever and 12.9% experienced rigors. Fatigue was the most common infusionassociated side effect (69% of patients) although the extent of alicaforsen’s role is difficult to dissect from this controlled study because 62% of patients receiving chemotherapy alone also had fatigue. Other commonly reported adverse effects were malaise, headache, nausea, and vomiting. For aprinocarsen monotherapy, severe (NCI CTCAE grade 3) fatigue was dose limiting at 3mg/kg/day. Patients described the fatigue as general lack of energy that led them to spend most of day in bed. Fatigue, and the other infusion-related symptoms, typically appeared midway through the 21-day infusion. Symptoms improved markedly during 7-day rest period between repeated infusions [44]. Origin of Constitutional Symptoms Except for the 24-h infusion schedule, post- or peri-infusion increases in complement split products have not been reproducibly detected in subjects treated with either Gen-1 or Gen-2 ASOs. Therefore, activation of the complement system is unlikely to be the source of fever and chills and other constitutional symptoms experienced by subjects receiving high doses of Gen-1 ASOs by short infusion. The infusion-associated symptoms are more likely related to the increased serum levels of proinflammatory cytokines observed after the end of infusion for several Gen-1 ASOs.

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In a Phase II study of ISIS 14803 in patients with chronic HCV, serum levels of interleukin (IL)-1
, IL-1, IL-1 receptor antagonist (IL-1Ra), tumor necrosis factor (TNF)-
, IL-6, and interferon (INF)- were measured at 2 and 4 h after the end of 2-h infusions administering 2.5 and 6 mg/kg doses of the Gen-1 ASO. In this study, fever was experienced by 70% of the patients treated at 6 mg/kg twice weekly and chills were experience by 55%. Three of 37 patients initially receiving 6 mg/kg required dose reduction to 4 mg/kg because of the intensity of their symptoms. Postinfusion increases in serum levels of TNF-
, IL-6, and IL-1Ra were observed at both doses but the increases were greater at the 6 mg/kg dose. Figure 13.4 shows the percent change for these cytokines. The greatest changes were observed for IL-6. For each of these three cytokines, the percent change following the 6 mg/kg dose was significantly greater than the percent change at 2.5 mg/kg dose. No significant changes were observed for IL-1
, IL-1, or INF-. In the studies of 24-h intravenous infusion, measurements of serum cytokine levels were performed for a subset of the patients. Table 13.6 shows the mean and range of results obtained for TNF-
, IL-6, and IL-1Ra. While quantitative differences between the drugs were observed for the pre- and postinfusion cytokine levels at the 24 mg/kg dose, postinfusion serum levels were clearly higher than preinfusion levels for all three cytokines at doses examined. IL-1 was also measured for these patients and no changes in the levels of that cytokine were observed. While a role for complement activation in the development of the infusion-associated symptoms in patients dosed by 24-h intravenous infusion cannot be ruled out, the common observation of increased proinflammatory cytokine levels following both 2- and 24-h infusions points to release of cytokines as the probable root of the symptoms. The extent of cytokine release is likely to be associated with ASO plasma Cmax, although this requires further investigation.

Treatment of Constitutional Symptoms Treatment with antipyretics, antihistamines, and corticosteroids have been reported to ameliorate the infusion-associated constitutional symptoms. Yacyshyn et al. [16] reported that the symptoms from high dose (300–350 mg) alicaforsen given by 2-h infusion were effectively managed with 700 600

2.5 mg/kg

Mean % change ± SEM

6 mg/kg 500 400 300 200 100 0 TNF-α

IL-6

IL-1Ra

Figure 13.4 Percent change in serum cytokine levels following infusions of ISIS 14803. Patients with chronic hepatitis C were treated with ISIS 14803 given by 2-h intravenous infusions. During the first 2 weeks of dosing, patients received 2.5 mg/kg ASO thrice weekly. Subsequently, the patients were dosed at 6 mg/kg either once or twice weekly for another 10 weeks. Measurements of serum cytokine levels were performed before the start of the first 2.5 and 6 mg/kg infusions and at 2 and 4 h after the end of these infusions. Percent change was calculated for each patient based on the higher result from the two postinfusion cytokine measurements.

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Table 13.6 Serum Cytokine Levels at the Beginning and End of 24-h Intravenous Infusions of Gen-1 ASO Mean Cytokine Level (Range) IL-6 (pg/mL)


(pg/mL) TNF-


IL-1Ra (pg/mL)

ASO Dose

N

0h

24 h

0h

24 h

0h

24 h

ISIS 2503 18 mg/kg

3

9.67 (0–29)

1026 (324–2063)

3.33 (0–10)

119 (100–130)

427 (0–731)

5009 (3086–5971)

ISIS 2503 24 mg/kg

5

13 (0–42)

1232 (315–1864)

18.5 (0–70)

152 (76–199)

541 (382–727)

5745 (5403–5971)

ISIS 3521 24 mg/kg

3

42 (0–127)

892 (205–1854)

45 (0.6–121)

210 (169–239)

335 (300–394)

5719 (5391–5971)

ISIS 5132 24 mg/kg

3

44 (16–63)

120 (97–162)

24 (6–34)

160 (129–214)

428 (165–769)

1613 (805–2425)

ISIS 5132 30 mg/kg

4

68 (7–110)

985 (186–2025)

49 (8–147)

186 (115–369)

1533 (679–2274)

5784 (5552–5971)

acetaminophen and/or corticosteroids. They report antihistamines were not effective [16]. However, Advani et al. [45] noted that symptoms from 24-h infusions of aprinocarsen could be ameliorated or prevented with premedication with acetaminophen and diphenylhydramine. For 24-h infusions of ISIS 5132, Rudin et al. [27] noted that fever could be managed through treatment with 650 mg acetaminophen before and every 4 h during the infusions. Stevenson et al. [6] reported that ibuprofen was effective in patients given ISIS 5132 by 2-h infusion. Although no controlled studies of the effectiveness of antipyretic, antihistamine, or corticosteroid treatment for preventing or mitigating infusion-related constitutional symptoms were reported, the results of the previously mentioned alicaforsen Phase III studies (Section 13.3.3.1.1.1) are suggestive that pretreatment with low doses of prednisone and end-of-infusion treatment with acetaminophen does lower the incidence of these symptoms.

13.3.3.2 Subcutaneous Injection Site Responses The Gen-1 ASO subcutaneous administration experience is principally based on two drugs: alicaforsen and oblimersen. Two studies were performed with alicaforsen. In a Phase I placebo-controlled study, different alicaforsen doses and administration schedules were explored in healthy volunteers. Five single doses ranging from 50 to 400 mg and five multiple-dose regimens were tested. Each of these was tested in a four-subject cohort where one of the subjects was randomized to dosing with placebo. All 30 subjects given alicaforsen in the study experienced erythema, induration and/or pain at the injection site. At the higher doses (single doses of 200 and 400 mg, and four doses of 1 and 2 mg/kg given every other day), subjects often experienced all three effects. Lymphadenopathy was reported for 12 subjects treated with alicaforsen, 10 of whom were in multiple-dose groups. In most cases, lymph- adenopathy was tender and inguinal (located at those nodes draining the injection site). In the cohorts that were given 1 or 2 mg/kg alicaforsen every other day, and in the cohort that was given 0.5 mg/kg/day by continuous subcutaneous infusion for 6 days, all of the subjects that received alicaforsen developed lymphadenopathy. Daily injection of 0.5 mg/kg for 7 days was better tolerated than continuous subcutaneous infusion. A Phase II study of alicaforsen in patients with Crohn’s disease was conducted using daily subcutaneous injection of 0.5 mg/kg [17]. In this 75-patient, double-blind, placebo-controlled study, patients were equally randomized to receive 0.5 mg/kg alicaforsen injections for either 0, 1, 5, 10, or 20 days over a 4-week treatment period. Injection site reactions occurred in 23% of the alicaforsen-treated patients but in none of the placebo-control group. Aside from the early study discontinuation of one alicaforsen patient in the 20-day group due to an injection site reaction, subcutaneous injections of 0.5 mg/kg were well tolerated in this study [17].

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However, further study of subcutaneous administration was not pursued for alicaforsen because clinical evaluation of higher doses (4–6 mg/kg) was desired. Subcutaneous administration of oblimersen, a Gen-1 ASO inhibitor of Bcl-2, was evaluated as a 14-day subcutaneous infusion in 21 patients with non-Hodgkin’s lymphoma [4]. Oblimersen doses ranging from 4.6 to 195.8 mg/m2/day were evaluated. All subjects experienced inflammation at the infusion site, which could be managed for most patients by changing the site of infusion. Skin biopsies of two patients with severe infusion site reaction showed mild perivascular lymphocytic infiltrates in the superficial dermis. Five patients (1 of 3 at 110.4 mg/m2/day, 3 of 5 at 147.2 mg/m2/day, and 1 of 3 at 195.8 mg/m2/day) developed tender enlarged lymph nodes during oblimersen infusion in regions draining the injection site. Because none of the three patients given 195.8 mg/m2/day were able to complete the 14-day treatment course due to systemic AEs (e.g., fatigue, fever, thrombocytopenia, hypercalcemia, generalized maculopapular rash, and acute renal failure), the maximum tolerated oblimersen dose given by subcutaneous infusion was set at 147.2 mg/m2/day. Intravenous administration was subsequently used for the clinical studies of oblimersen [51–59]. With Gen-2 ASOs, subcutaneous injection reactions have also been observed following subcutaneous injection; however, these reactions have not been found to be dose limiting as was the case for alicaforsen. The typical reaction to subcutaneous injections of 200 mg Gen-2 ASO is painless erythema at the injection site. Table 13.7 shows the injection site reactions that reported a study of the TNF-
inhibitor, ISIS 104838, in which 171 patients with rheumatoid arthritis selfinjected 200 mg ISIS 104838 or placebo twice weekly for 12 weeks. The patients were equally randomized between receiving no ISIS 104838 doses, one dose every other week, one dose each week, or two doses each week with placebo given when ISIS 104838 was not. Overall, 53% of the patients receiving ASO had at least one injection site reaction. Erythema was the most frequent of these events (37%) of patients. Assuming the patients with the MedDRA preferred terms of Injection Site Reaction NOS and Injection Site Inflammation had erythema as a component of their reaction, the frequency could be as high as 45%. Other reactions (bruising, pain, induration, etc.) each occurred in ⬍20% of the patients receiving ISIS 104838. For the most part, the injection site reactions did not interfere with continued dosing and only 3 of 131 patients withdrew early from dosing due to the injection site reactions. The basis for the subcutaneous injection site reactions is suspected to be proinflammatory responses to transient high ASO concentrations at the injection site. Although injection site reactions

Table 13.7 Frequency and Incidence of Injection Site Reactions in Patients with Rheumatoid Arthritis Treated Given ISIS 104838 by Subcutaneous Injection Number of Patients (Frequency) Number of Events MedDRA Preferred Term Overall Injection Injection Injection Injection Injection Injection Injection Injection Injection Injection Injection Injection Injection Injection Injection a

site site site site site site site site site site site site site site site

erythema bruising pain induration pruritus reaction NOS burning swelling vesicles inflammation pigmentation changes discomfort edema rash urticaria

See Table 13.3 for dosing schedule.

ISIS 104838 (N ⫽ 131)a 70 49 21 16 15 11 8 6 6 5 3 3 2 2 2 2

(53.4%) 307 (37.4%) 115 (16.0%) 40 (12.2%) 29 (11.5%) 46 (8.4%) 25 (6.1%) 10 (4.6%) 7 (4.6%) 6 (3.8%) 7 (2.3%) 3 (2.3%) 3 (1.5%) 2 (1.5%) 2 (1.5%) 2 (1.5%) 10

Placebo (N ⫽ 45) 2 (4.4) 2 2 (4.4) 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0

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similar to those observed in man have not been seen in animals treated with ASOs, perivascular lymphoid infiltration of subcutaneous injection sites suggestive of local inflammatory responses have been observed in nonclinical studies [72]. The lower proinflammatory activity of Gen-2 ASOs observed in nonclinical studies is consistent with the better clinical tolerability of Gen-2 ASO subcutaneous injections.

13.3.3.3 Hypersensitivity Reactions In the Gen-1 ASO clinical experience, there have been rare reports of anaphylactoid reactions in patients treated with ASOs. These reports have been concerning due to the complement activation and proinflammatory potentials of Gen-1 ASO. At present, it is not possible to distinguish whether these events are due to ASO administration or are natural occurrences in the study populations. Table 13.8 provides the frequencies and number of cases for anaphylactoid reactions in the pooled clinical experience of three ASOs that also have results available from a sufficiently large control population. The two larger populations are for the Gen-1 ASOs alicaforsen and aprinocarsen. The control population for alicaforsen is Crohn’s disease patients treated with placebo. For aprinocarsen, the control population is NSCLC patients treated with chemotherapy regimens (gemcitabine with cisplatin or carboplatin with paclitaxel) lacking aprinocarsen. The one Gen-2 ASO in the table is the TNF-
inhibitor ISIS 104838 and the control population is patients with rheumatoid arthritis treated with placebo. Besides the data for anaphylactoid reactions, Table 13.8 also provides data for hypersensitive and drug hypersensitivity AEs. All three of these events have a low incidence, ⬍2%, in ASOtreated subjects with the frequency of AEs coded as drug hypersensitivity being the highest of the three categories and anaphylactoid reaction being the lowest. For all three types of AEs, the incidence in ASO-treated patient is approximately the same as in control-treated subject. However, it is not currently possible to rule out a role for Gen-1 ASOs in these hypersensitivity reactions because of the small number of cases and because some of the events were temporally associated with ASO infusions.

Table 13.8 Frequency of Hypersensitivity Reaction among Patients Treated with ASO Alicaforsen Gen-1 Crohn’s Diseasea Single Agent

ISIS 104838 Gen-2 Rheumatoid Arthritisc Single Agent

Frequency

n /N d

Frequency

n /N d

0.3% 0.4%

2/593 1/252

0.6% 0.5%

4/616 3/613

0% 0%

0/131 0/45

Hypersensitivity NOS ASO Control

0.8% 0%

5/593 0/252

1.3% 1.1%

8/616 7/613

0% 0%

0/131 0/45

Drug Hypersensitivity ASO Control

0% 0%

0/593 0/252

1.9% 2.8%

12/616 17/613

1.5% 0%

2/131 0/45

Study Treatment

Anaphylactoid Reactions ASO Control

a

Aprinocarsen (ISIS 3521) Gen-1 NSCLCb Combination Therapy

Frequency

n /N d

e

Patients pooled from six placebo control trials and one open-label study of alicaforsen. Patients pooled from 586 patient trial of carboplatin ⫹ paclitaxel combination and 643 patient study of cisplatin ⫹ gemcitabine combination. c Patients from single Phase 2 trial; see Table 13.3 for dosing schedule. d Number of patients with event, n, and number of patients in the study population, N. e Includes events coded to the MedDRA preferred terms anaphylactic shock, anaphylactic reaction, and angioneurotic edema. b

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If these hypersensitivity-like reactions are due to ASO administration, the mechanism giving rise to the observed AEs is unclear. Phosphorothioate oligonucleotides are poorly antigenic and nonclinical attempts to raise antibodies against these compounds have largely been unsuccessful [10,72]. For example, no IgG or IgM antibodies were detected in mice and monkeys treated with alicaforsen for up to 6 months [10]. Attempts to sensitize monkeys to alicaforsen by coadministering it with Freund’s complete adjuvant were uniformly negative [10]. To test for IgE and/or anaphylactic antibodies in the serum of chronically treated monkeys, a passive anaphylaxis model was used in which the serum from alicaforsen-treated monkeys was injected intradermally into a second set of monkeys. Alicaforsen challenge of the second set of monkeys did not reveal any evidence of immediate hypersensitivity. Further, immunotoxicologic evaluation of alicaforsen and ISIS 3082 (the murine-specific analog of alicaforsen) in mice showed only the expected reductions in delayedtype hypersensitivity that would be expected with pharmacologically active ISIS 3082 [79]. In humans, anti-alicaforsen IgG/IgM antibody testing was performed for 91 of 198 patients with Crohn’s disease that were treated with 2 mg/kg alicaforsen. A positive result was found for only one patient [15]. That patient had a low titer (1:10) of an antibody that reacted with alicaforsen on study day 82. Baseline values were not analyzed, so it is not clear if the antibody had developed during treatment or not. These nonclinical and clinical results suggest that the observed allergicand anaphylaxis-like reactions were not due to antibody recognition of Gen-1 ASOs. 13.3.4 Kidney Effects In animals treated with Gen-1 and Gen-2 ASOs, the highest tissue concentrations of ASOs are found in kidney, particularly in the epithelial cells of the proximal tubules. Although no effects on kidney function, except for a slight increase in urine protein/creatinine ratio (0.2 to 1.2), have been found in monkeys given doses far greater than those routinely used clinically (40 compared to 2–5 mg/kg/week for a Gen-2 ASO), histological changes have been observed [10,72]. The most common observation is the dose-related appearance of basophilic granules in proximal tubule epithelial cells. The prevalence of granules is minimal and mild at 1 and 3 mg/kg/week doses, respectively, for Gen-2 ASOs. These granules represent endosomal accumulation of ASO and are not considered toxicologically significant [10,72]. At doses of 10–20 mg/kg/week, scattered instances of single-cell degeneration can be detected [10,72]. Although, the observation of marked tubular cell degeneration required doses at ⭓80 mg/kg/week and tissue concentrations ⭓5000 g/g [72], the potential for effects on kidney is an area for concern in the clinical development of Gen-1 and Gen-2 ASOs. Clinically, no evidence has been observed for a Gen-1 or Gen-2 ASO class effect on renal performance. Figure 13.5 shows the serum creatinine results from controlled clinical studies of the two Gen-1 and Gen-2 ASOs. Patients with Crohn’s disease given two 4-week courses of 2 mg/kg alicaforsen and one 4-week course of 300 mg alicaforsen are shown in Figure 13.5A and Figure 13.5B, respectively. NSCLC patients treated with 2 mg/kg/day aprinocarsen and either carboplatin and paclitaxel or cisplatin and gemcitabine are shown in Figure 13.5C and Figure 13.5D, respectively. Patients with rheumatoid arthritis that were given 200 mg ISIS 104838 twice weekly for 12 weeks are shown in Figure 13.5E and patients with type 2 diabetes given 200 mg ISIS 113715 once weekly for 12 weeks are shown in Figure 13.5F. In each of these trials, the serum creatinine results for patients treated with ASO were indistinguishable from those of control patients. Figure 13.6 provides the BUN results for the same studies, except BUN measurements were not collected in the aprinocarsen plus cisplatin and gemcitabine study. As with serum creatinine, the BUN results for patients given ASO in these studies were indistinguishable from those of control patients. Table 13.9 shows the incidence for increases in urine protein for patients treated with alicaforsen and ISIS 104838 in comparison to control groups. With the two ASOs, the frequencies for any increase in proteinuria and for increases greater than or equal to two grades were similar to ASO- and placebo-treated subjects. These findings extend to other tests diagnostic for renal function changes such as serum albumin and bicarbonate and urine blood.

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1.2 1.0 0.8 0.6 0.4 0.2

ISIS 2302 (n = 99) Placebo (n = 101)

1.4 1.2 1.0 0.8 0.6 0.4 Placebo (n = 109)

0.2

S

B

ISIS 2302 (n = 221)

2 3 Event / Week

4

5

(D) 1.8 1.6 1.4 1.2 1.0 0.8 0.6 ISIS 3521 (n = 294) Control (n = 292)

0.4 0.2

(E)

S B 2 3 4 5 6 7 8 9 10 11 12 13 14 Event / Week

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 0.2 0.0

Placebo (n = 45) ISIS 104838 (n = 40)

Scr

Bsln

5

13

Event / Week

25

Creatinine (mg/dL), mean ± S.D.

Creatinine (mg/dL), mean ± S.D.

1.6

S B 1 2 3 4 5 8 9 10 11 12 13 Event / Week

(C)

Creatinine (mg/dL), mean ± S.D.

Creatinine (mg/dL), mean ± S.D.

(B) 1.4

1.8 1.6 1.4 1.2 1.0 0.8 0.6 0.4 ISIS 3521 (n = 322) Control (n = 321)

0.2 0.0

S

B

1

2 3 4 Event / Month

(F) Creatinine (mg/dL), mean ± S.D.

Creatinine (mg/dL), mean ± S.D.

(A)

387

5

6

1.4 1.2 1.0 0.8 0.6 0.4

ISIS 113715 (n = 22) Placebo (n = 8)

0.2 S B 2 4

6

8 10 12 13 17 21 24 Event / Week

Figure 13.5 Mean serum creatinine levels during treatment with Gen-1 and Gen-2 ASOs. (A) Patients with Crohn’s disease were given two 4-week courses of alicaforsen or placebo. During each course, ASO or placebo was administered by 2-h intravenous infusions performed thrice weekly.There was a 4-week treatmentfree period between the two courses. The alicaforsen dose was 2 mg/kg. (B) Patients with Crohn’s disease were given one 4-week course of alicaforsen or placebo. See Figure 13.3 for alicaforsen dose and administration schedule. (C) NSCLC patients were given repeated 21-day treatment cycles of aprinocarsen, carboplatin, and paclitaxel or chemotherapeutics without ASO. Aprinocarsen was administered by 14-day continuous infusion of ASO at 2 mg/kg/day during the first 2 weeks of each treatment cycle. (D) Same as (C) except the chemotherapeutics were cisplatin and gemcitabine. (E) Patients with rheumatoid arthritis were treated with placebo or 200 mg ISIS 104838 given twice weekly (400 mg ASO/week) by subcutaneous injection for 12 weeks. (F) Patients with type 2 diabetes were given placebo or 200 mg ISIS 113715 once weekly by 1-h intravenous infusion for 12 weeks.

With one exception, there has been no evidence that Gen-1 or Gen-2 ASO administration has any negative consequence on kidney function at the doses studied among the 105 studies of ISIS ASOs. This includes a study in which 29 patients undergoing renal allograft were treated with alicaforsen for 2 weeks beginning with ASO administration during the transplant operation [13]. The only exception

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BUN (mg/dL), mean ± S.D.

28 24 ISIS 2302 (n = 99) Placebo (n = 101)

20 16 12 8 4 0

BUN (mg/dL), mean ± S.D.

(B)

(A)

20 16 12 8 4 S

B

2 3 4 Events / Weeks

5

(D) 25 20 15 10 5 0

ISIS 3521 (n = 294)

Control (n = 292)

S B 3 4 5 6 7 8 9 10 11 12 13 14 Events / Weeks

BUN (mg/dL), mean ± S.D.

30 BUN (mg/dL), mean ± S.D.

Placebo (n =109) ISIS 2302 (n = 221)

24

0

S B 1 2 3 4 5 8 9 10 11 12 13 Events / Weeks

(C)

28

30 25 20 15 10 5 Placebo (n = 45)

0

Scr

Bsln

ISIS 104838 (n = 40)

5 13 Event / Week

25

BUN (mg/dL), mean ± S.D.

(E) 30 25 20 15 10 5 0

ISIS 113715 (n = 22) Placebo (n = 8)

S B 2 4

6

8 10 12 13 17 21 24 Event / Week

Figure 13.6 Mean BUN levels during treatment with Gen-1 and Gen-2 ASOs. (A)–(C) Same clinical studies as Figures 13.5A–C. (D) and (E) Same clinical studies as Figure 13.5E and Figure 13.5F, respectively. BUN results were not collected for the study of aprinocarsen in combination with cisplatin and gemcitabine.

occurred in the treatment of chronic HCV patients with ISIS 14803, a Gen-1 ASO. Two of 104 patients developed cryoglobulinemic glomerulonephritis. While cryoglobulinemia is a common condition in patients infected with HCV, with estimated prevalences up to 50% [80], the occurrence of two glomerulonephritis cases resulting from the condition in a 100-patient sample size is highly unusual. However, the two cases are probably the result of the proinflammatory activity of ISIS 14803 rather than a direct effect on kidney function. The proinflammatory effects likely led to exacerbation of the preexisting cryoglobulinemia in these two patients with deposition of the immunoglobulins in kidneys.

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Table 13.9 Frequency of Protein in Urine for Patients Treated with Gen-1 and Gen-2 ASOs Number of Patients (Frequency)a Alicaforsen (Gen-1 ASO) Crohn’s Disease Number of Patients Total patients Any increase in urine protein ⭓2 grade increase in urine protein a

ISIS 104838 (Gen-2 ASO) Rheumatoid Arthritis

ASO-Treated

Placebo-Treated

ASO-Treated

Placebo-Treated

220 21 (9.5%) 2 (0.9%)

111 9 (8.1%) 1 (0.9%)

131 9 (6.9%) 3 (2.3%)

42 2 (4.8%) 1 (2.4%)

See Table 13.3 for doses and administration schedule.

13.3.5 Thrombocytopenia Platelet reductions have been regularly observed in mouse studies of ASOs, especially those of Gen-1 ASOs [10,72]. These reductions are likely due to sequestration of the platelets secondary to the splenomegaly induced by the proinflammatory activity of Gen-1 and Gen-2 ASOs. Clinically, thrombocytopenia has been reported in cancer studies of Gen-1 ASOs (aprinocarsen, ISIS 5132, ISIS 2503, oblimersen) [4,25–49,51–59] but not in studies of other Gen-1 ASOs (alicaforsen, ISIS 14803) [12–17,50]. Figure 13.7A and Figure 13.7B show the platelet counts for Crohn’s disease patients treated with two 4-week courses of thrice weekly 2 mg/kg alicaforsen (Gen-1 ASO inhibitor of ICAM-1) and patients given one 4-week course of thrice weekly 300 mg alicaforsen, respectively. The platelet counts for patients given alicaforsen were indistinguishable from those of patients given placebo. However, NSCLC patients given aprinocarsen (Gen-1 ASO inhibitor of PKC-
) in combination with carboplatin and paclitaxel had lower platelet levels than those of patients that received only carboplatin and paclitaxel (Figure 13.7C). The reason for the platelet count differences between the alicaforsen and aprinocarsen studies appears to be the infusion schedules used. Figure 13.7D shows the platelet results taken from separate uncontrolled trials of aprinocarsen for patients that received 4–6 mg/kg by 2-h intravenous infusion compared to the results for patients that received 2 mg/kg/day by 21-day continuous intravenous infusion. The platelet levels for patients treated by continuous infusion declined during the first 4-week treatment cycle. In contrast, the platelet levels during treatment for patients receiving 2-h infusions were not significantly reduced from pretreatment levels, despite these patients receiving weekly aprinocarsen doses of 12–18 mg/kg compared to the 14 mg/kg given by continuous infusion. Administration of ISIS 5132 by 2-h infusion similarly did not result in platelet reduction whereas reductions were observed for continuous infusion [6,25–30]. The mechanism for this infusion-schedule effect is not clear. Limited clinical investigations into platelet aggregation were not instructive [45]. The results from 24-h infusions suggest that sequestration may possibly have a role. Figure 13.7E shows the platelet count results for patient treated with aprinocarsen given once weekly by 24-h infusion. Substantial platelet count reductions were observed between preinfusion and end-of-infusion measurements with recovery between infusions. These transient reductions are consistent with temporary sequestration during infusion. However, further investigations are needed to establish the mechanism. For Gen-2 ASO, thrombocytopenia was observed in the clinical study of one compound, ISIS 104838. Figure 13.7F shows the platelet count results for patients with rheumatoid arthritis treated with the ASO at 200 mg/week for 12 weeks. By the end of treatment, platelet levels were significantly reduced compared to baseline- and to placebo-treated patients. However, treatment of type 2 diabetics with 200 mg/week ISIS 113715 for 12 weeks did not result in similar platelet reductions (Figure 13.7G). Treatment of healthy volunteers with 200 mg/week ISIS 301012 for 4 weeks also did not produce noticeable reduction in platelet count (Figure 13.7H), although the duration of dosing could be argued to be too short to detect an effect similar to that seen with ISIS 104838.

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION (A) Platelet count (/nL), mean ± SEM

400 350 300 250 200 150 100 ISIS 2302 (n = 99) Placebo (n = 101)

50 0

S

B

1

3

4

5

8

9

10

11

Platelet count (/nL), mean ± SEM

(B)

450

12 13

450 400 350 300 250 200 150 100 Placebo (n = 109) ISIS 2302 (n = 221)

50 0

S

B

2

3

Event/ Week 450

(D) 500

400

450

Platelet count (/nL), mean ± SEM

Platelet count (/nL), mean ± SEM

(C)

350 300 250 200 150 100 ISIS 3521 (n = 294) Control (n = 292)

50 0

S

B

2

3

4

5

6

7

8

8

6

7

8

300 250 200 150 100

2-h infusion (n = 12) 21-day infusion (n = 36)

50 0

2

3

4

5

Event / Week

(F) 400

Platelet count (/nL), mean ± SEM

450 400 350 300 250 200 150 100 50

24-h infusion ISIS 3521 (n = 11)

0 S

0-1

350 300 250 200 150 100 Placebo (n = 45) 50 0

7-8 14-15 21-22 28-29 35-36 42-4349-50 56-57

ISIS 104838 (n = 44) Scr

Bsln

5

Event / Day

(G)

(H)

450 400 350 300 250 200 150 100 ISIS 113715 (n = 22) Placebo (n = 8)

50 0 S

B

2

4

6

8

10

13

25

Event / Week

12 13

Event / Week

17 21 24

Platelet count (/nL), mean ± SEM

Platelet count (/nL), mean ± SEM

6

350

S

500

Platelet count (/nL), mean ± SEM

5

400

9 10 11 12 13 14

Event / Week

(E)

4

Event/ Week

450 400 350 300 250 200 150 100

ISIS 301012 (n = 8) Placebo (n = 7)

50 0 S

B

2

8

15

22

55

Event / Day

Figure 13.7 Platelet levels during treatment with Gen-1 and Gen-2 ASOs. (A)–(C) Same clinical studies as Figures 13.5A–C. (D) Cancer patients were given repeated 28-day treatment cycles of aprinocarsen administered during the initial 3 weeks/cycle either by thrice weekly 2-h intravenous infusion or by continuous intravenous infusion. The 2-h infusion results were pooled from three patients treated at 4 mg/kg, three patients treated at 5 mg/kg, and six patients treated at 6 mg/kg. The dose given by continuous infusion was 2 mg/kg/day. (E) Cancer patients were given aprinocarsen by once weekly 24-h infusion. The results were pooled from two patients given 12 mg/kg, five patients given 18 mg/kg, and four patients given 24 mg/kg. (F) Same clinical study as Figure 13.5E except patients were treated with 200 mg ISIS 104838 once weekly. (G) Same study as Figure 13.5F. (H) Healthy volunteers were treated with 200 mg ISIS 301012 thrice during first treatment week and then once weekly for the next 3 weeks.

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The above results indicate that thrombocytopenia is a clinical class effect of Gen-1 ASOs but only in the context of 14- or 21-day continuous intravenous infusions and 24-h infusions. Platelet count reductions are not a class effect of Gen-1 ASOs when 2-h infusions are used to administer the ASOs. For Gen-2 ASOs, clinical results for more class members are needed but preliminarily, thrombocytopenia appears to be an effect limited to one ASO, ISIS 104838. Interestingly, platelet reductions in monkey studies of clinical Gen-2 ASOs have only been observed for ISIS 104838 and not for ISIS 113715 or ISIS 301012. These findings indicate that nonclinical studies may be predictive of which Gen-2 ASO will have clinical effects on platelet levels. 13.3.6 Liver Effects Elevations in ALT and/or AST, and other signs of hepatotoxicity, have been absent in the monkey studies of Gen-1 and Gen-2 ASOs [10,72]. In mouse studies of clinical ASOs, mild ALT and AST elevations have been observed with these compounds [10,72]. However, these elevations are thought to be secondary to the mononuclear cell infiltrations into the liver arising from the proinflammatory effects of Gen-1 and Gen-2 ASOs [10,72]. The absence of hepatic effect in monkeys, despite these animals having higher liver concentrations of ASO than mice, supports this view. Nevertheless, the high distribution of Gen-1 and Gen-2 ASO to the liver, ⬃40–50% of a dose [9,73], suggests that adverse effects in liver could potentially occur during the clinical evaluation of these compounds. In the clinical studies of Gen-1 ASOs, elevations in serum transaminases or other signs of hepatotoxicity have largely been absent. Table 13.9 shows the incidence of ALT increases for two Gen-1 ASOs, which have been subjected to randomized controlled clinical studies. For alicaforsen, the incidence of on-study ALT results ⬎50 U/L in 544 Crohn’s patients with baseline ALT levels ⬍41 U/L was 7.0%. This frequency was similar to that seen in 232 Crohn’s disease patients treated with placebo. Alicaforsen treatment also did not result in a greater incidence of moderate or severe ALT elevations. The frequencies for patients with ALT levels ⬎150 U/L were similar between those treated with alicaforsen and placebo. For patients with baseline ALT levels ⬎40 U/L, there were also no differences in the frequencies of ALT increases between alicaforsen- and placebo-treated patients. Similarly, there were no differences between NSCLC patients given standard chemotherapy regimens and aprinocarsen compared to those given only standard chemotherapeutics. Further, the frequencies for on-study ALT levels ⬎50 or ⬎150 U/L were not remarkably different between the two Gen-1 ASOs. Significant increases in serum transaminases, especially ALT, have been observed with one Gen-1 ASO [50]. Of 71 patients with chronic HCV given ISIS 14803 as a single agent, 22 (31%) had transient ALT increases to ⬎5 ⫻ the upper limit of the reference range (ULN). The entry criteria for the studies allowed inclusion of patients with ALT levels up to 5 ⫻ ULN. Three of the patients had peak ALT levels of ⬃1200 U/L, or about 30 ⫻ ULN. These studies did not have control patients so the background frequency for ALT elevation in the studies is not known. However, the spontaneous occurrence of ALT elevations as high as 30 ⫻ ULN is highly unusual even for the chronic HCV patients. Reductions in HCV levels ⬎0.9 log10 were observed for 13 of 71 patients with three of the patients having reductions of 3.0–4.0 log10. All 13 also had ALT elevations. While the ALT elevations in chronic HCV patients treated with ISIS 14803 could possibly be due to the deposition of ISIS 14803 in the liver, it is probable that the ALT elevations were secondary to changes in virus replication. Patients on treatment with peginterferon and ribavirin and given ISIS 14803 at the same doses as in the single-agent studies had far fewer (3%) ALT elevations. Since the patients on antiviral therapy had substantially lower viral titers, these results point to changes in viral replication in the single-agent studies as the primary reason for the ALT elevations observed with ISIS 14803. It is also possible that the ALT elevations were secondary to a proinflammatory effect from administration of ISIS 14803, since treatment with interferon causes changes in the immune system. However, it is unclear that interferon treatment would negate the proinflammatory activity of Gen-1 ASOs. For Gen-2 ASOs, the hepatic effects of ASO administration are less clear. Table 13.10 shows the frequency of ALT increases observed in patients with rheumatoid arthritis who were treated with

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Table 13.10 Frequency of ALT Increases during Treatment with ASOs Number of Patients (Frequency)a

ALT Subgroup Baseline ALT ⭐ 40 U/L On-study ALT ⬎ 50 U/L On-study ALT ⬎ 150 U/L a

Alicaforsen (ISIS 2302)

Aprinocarsen (ISIS 3521)

ISIS 104838

Gen-1 Crohn’s Disease

Gen-1 NSCLC

Gen-2 Rheumatoid Arthritis

ASO 544 38 (7.0%) 4 (0.7%)

Control

ASO

232 12 (5.2%) 1 (0.4%)

479 73 (15.2 %) 3 (0.6%)

Control 507 48 (9.5%) 1 (0.2%)

ASO

Control

120 4 (3.3%) 0

44 3 (6.8%) 0

See Table 13.8 for patient populations.

ISIS 104838 for 12 weeks in a 176-patient, randomized, placebo-controlled, double-blind study. The frequency for any elevations of ALT was similar between those receiving ASO (3.3%) and those receiving placebo (6.8%). No moderate or severe ALT elevations were observed in either treatment group. However, ALT increases have been observed in the early clinical studies of three other Gen-2 ASOs. In a Phase I study in healthy volunteers with fasting total cholesterol levels between 200 and 300 mg/dL, 5 of 29 (17%) subjects treated with ISIS 301012, a Gen-2 ASO inhibitor of apoB-100 for 4 weeks, and 1 of 7 (14%) subjects treated with placebo had ALT increases ⬎1.1 ⫻ ULN (ULN ⫽ 55 U/L) [2]. The peak ALT level for four of the ASO-treated subjects were 2–3 ⫻ ULN and was 5.3 ⫻ ULN for the fifth subject whereas the peak for the placebo-treated subject was 1.6 ⫻ ULN. With ISIS 113715, a Gen-2 ASO inhibitor of PTP-1B, in a Phase II study, 19 of 72 (26%) type 2 diabetics treated with the ASO for 6 or 12 weeks and 3 of 24 (13%) patients treated with placebo had ALT increases ⬎36 U/L (the span of the reference range). Peak ALT levels for 11 ASO-treated patients and all three placebo-treated subjects were ⭐3 ⫻ ULN. For the remaining ASO-treated patients, six had peak ALT of 3–5 ⫻ ULN and two had peak levels of 8–15 ⫻ ULN. In a Phase I study in patients with localized prostate cancer receiving androgen blockade therapy and OGX-011 for 4 weeks, 14 of 25 (56%) patients had AST and/or ALT increases to ⬎1 ⫻ ULN [3]. Six of the patients had peak ALT levels of 2–3.1 ⫻ ULN. While the role of the Gen-2 ASOs in these serum transaminase elevations cannot be ruled out, there are alternative possibilities that need additional study. For ISIS 301012, treatment with the ASO resulted in reduced serum levels of apoB and LDL cholesterol, which is the desired pharmacological action of the drug. While the dataset is small, there was an inverse association between the magnitude of LDL/apoB reductions and the serum transaminase elevations. Similarly, a correlation between percent triglyceride level reduction and ALT increases was also observed. Although based on small datasets, these linkages between signs of efficacy (LDL, apoB, and triglyceride reductions) and transaminase elevations suggest that the ALT increases may be secondary to perturbations in hepatic lipid metabolism resulting from the pharmacological action of ISIS 301012. Similar transient ALT elevations are observed after initiation of therapy with other lipid-lowering drugs [81,82]. For ISIS 113715, ALT elevations are normal occurrences in the type 2 diabetic population with incidence rate estimates ranging up to 20% [83]. Further, some of the ALT elevations in ASO-treated patients that were observed in the study have known non-ASO etiologies (e.g., hepatitis B infection and alcohol intoxication). For OGX-011, it was noted that changing the antiandrogen used from flutamide to bicalutamide mitigated the transaminase elevations [3]. Since serum transaminase elevations is a known side effect of flutamide, with frequencies as high as 60% [84], it is possible that the antiandrogen had a primary role in the transaminase elevations observed in the prostate cancer study. Three additional factors impact the assessment of the transaminase elevations observed in the above studies for ISIS 301012, ISIS 113715, and OGX-011. First, the observation of significant transaminase elevations unrelated to study drug is not infrequent in clinical studies. Several reports have demonstrated the occurrence of such elevations even in healthy volunteers receiving placebo [85,86].

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Factors such as sucrose intake and changes in the physical activity levels of the subjects have been linked to transaminase increases. Second, the studies for the three Gen-2 ASO were small and either had control groups that were one-third the size of the ASO-treated group (ISIS 113715 and ISIS 301012) or had no control group (OGX-011). As a result, it is not clear to what extent the transaminase elevations observed for the three ASO are deviations from those that are normally observed in the study populations. Third, the studies for ISIS 113715, ISIS 301012, and OGX-011 performed weekly serum chemistry measurements, which could lead to overestimation of the frequency of laboratory changes. While intensive laboratory assessments are the norm for Phase I and early Phase II studies, laboratory assessments are usually performed less frequently, often monthly, during Phase III trial. Therefore, clarification of whether administration of these three ASOs produced an increased incidence in serum transaminase elevations and whether Gen-2 ASOs have class effect on liver must await, in part, the performance of studies with larger control groups.

13.4 CLINICAL SAFETY RESULTS FOR LOCAL ADMINISTRATION Four local treatment applications have been investigated clinically for Gen-1 ASOs. These are intravitreal treatment of AIDS-related CMV retinitis with fomivirsen, intradermal therapy for genital warts with afovirsen, topical treatment of psoriasis with alicaforsen, and rectal enema therapy for ulcerative colitis and pouchitis with alicaforsen. Limited exploration into local administration has been conducted for two Gen-2 ASOs: intravitreal treatment of CMV retinitis with ISIS 13312 and topical treatment of psoriasis with ISIS 104838. With the above four routes of administration, the amount of ASO entering systemic circulation is very low because of the amount of ASO applied to the local site (e.g., 1 mg afovirsen per wart [23]), the low amount of ASO able to cross from the local site to systemic circulation (e.g.,⬍1% for the 240 mg enema formulation of alicaforsen [22]), or both (e.g., no detectable plasma levels of fomivirsen following intravitreal injection of 165 and 330 g doses [24]). As a result, most or all of the clinical effects for ASOs that have been identified for systemic administration (Section 13.3) should not occur in subjects receiving localized ASO therapy. Intravitreal administration of fomivirsen, a Gen-1 ASO inhibitor of CMV, was granted marketing approval by the US Food and Drug Administration in 1998 and by European regulatory agencies in 1999. The most frequently reported ocular side effects of fomivirsen treatment were increased ocular pressure (pressure ⬎24 mm Hg), anterior chamber inflammation, uveitis, vitritis, and ocular pain [24]. For eyes treated with 330 g given once weekly for 3 weeks and then every other week thereafter, the pooled event rates from two studies were 1.39, 0.85, 0.53, 0.50, and 0.46 per patient-year, respectively. Lower rates were observed when the 330 g dose was given less frequently (i.e., Days 1 and 15 and then every 4 weeks thereafter) or when the 165 g dose was given. The frequencies for these and other ocular AEs in fomivirsen-treated patients are reviewed in Ref. [24]. All of the nonocular AEs and laboratory changes observed in the patients treated with fomivirsen were attributed by the clinical investigators to be consequences of HIV disease or to other systemic treatments [24]. Intradermal injection of afovirsen, a Gen-1 ASO inhibitor of human papillomavirus type 11 (HPV-11), was evaluated as a treatment for genital warts. No systemic AEs related to the ASO were observed for afovirsen doses of 0.1–3.75 mg/injection with some subjects receiving injections into multiple sites and a total dose of 20 mg/day. The most commonly observed dermatological effects were erythema and induration. While the occurrence and severity of these effects were highly variable between subjects, both appeared to increase with repeated afovirsen injections. The most severe dermatological effects (desquamation, erosion, and ulceration) were only observed when the cumulative dose per site reached ⬃5 mg. These effects at the site of ASO injection are probably due to the same proinflammatory effects that give rise to the subcutaneous injection site reactions discussed earlier in Section 13.3.2. Histological examination of biopsies from injection sites of two subjects that were dosed with 1.02 mg/injection twice weekly for 3 weeks revealed a dense inflammatory

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reaction, with both T and B lymphocyte involvement. For the most part, the dermatological events did not cause discomfort to the subjects. Cream formulations of alicaforsen (0.03–4% concentrations) and of ISIS 104838 (0.5–4% strengths) have been studied for the topical treatment of mild to moderate plaque-type psoriasis vulgaris. Ninety-four patients, in two studies, were treated with alicaforsen-containing creams and 31 patients (one study) were treated with ISIS 104838 formulations. In these studies, each patient applied placebo and ASO creams to different plaques. All of the SAEs were considered unrelated to ASO by the clinical investigators and the AEs that were observed in these studies were typical of the patient population. The application of ASO cream was found to improve induration at the psoriatic plaques. Erythema at the plaques was unchanged. No adverse dermatological changes were reported in the cream formulations. The absence of dermatological effects similar to those seen with intradermal and subcutaneous injections is likely due to the limited penetration of topically applied ASOs through the skin. Rectal enema formulations of alicaforsen have been studied for the treatment of ulcerative colitis and pouchitis [18–22]. Recently, the results from a 189-patient study comparing two alicaforsen doses (120 and 240 mg) to mesalamine enema and from a 112-patient study comparing four dosing regimens of alicaforsen enema to placebo enema were published [20,21]. The AE profile of patients treated with the alcaforsen enema formulations was indistinguishable from the profiles observed for patients treated with placebo enema or treated with mesalamine enema. In particular, there was no increased incidence of AEs involving the colon or the rectum in patients treated with alicaforsen compared to control patients. The results from the studies of intravitreal, intradermal, topical, and rectal delivery of ASOs confirm the expectation that localized treatment would not result in AEs at sites other than where the ASO was applied. In studies of intradermal injection and rectal enema administration where control groups were included, the AE profile and laboratory results for patients receiving ASO were indistinguishable from the safety results observed for the control patients. Where separate control patients were not included in the studies (intravitreal injection and topical adminstration), no AEs or laboratory results unusual for the disease population outside of the site of ASO application were not found. As for the site of ASO application, there were local side effects with intradermal and intravitreal injection of ASO. However, no local effects have been found for the topical cream and enema formulations of ASO.

13.5 CONCLUSIONS Our understanding of the Gen-1 ASO clinical safety profile has grown substantially since the last edition of this chapter in 2001. The ensuing years have seen the completion of at least five Phase IIIrandomized, controlled studies of these compounds (two for alicaforsen in Crohn’s disease, two for aprinocarsen in NSCLC [31,32], and one for oblimersen in malignant melanoma [51,52]), which have allowed greater definition of the clinical safety profile for Gen-1 ASOs. In addition, a growing clinical experience is developing for Gen-2 ASOs. The clinical effects of the Gen-1 ASO drug class are prolongation of aPTT, infusion-associated constitutional symptoms, and subcutaneous injection site reactions. Additionally, thrombocytopenia is a class effect when these ASOs are given by 14- and 21-day continuous intravenous infusions. In nonclinical studies, prolongation of aPTT in monkeys and thrombocytopenia in mice are class effects of Gen-1 ASOs. The clinically observed infusion-associated constitutional symptoms and subcutaneous injection site reactions are likely to be alternative manifestations of the proinflammatory effects of Gen-1 ASOs that give rise to the immune stimulation effects observed in mice and other rodents. Whether the rare hypersensitivity reactions that have been seen in some clinical trials of Gen-1 ASOs are due to the proinflammatory effects of these compounds remain to be determined. Clinical evidence for the Gen-1 ASO class effects on complement activation in monkeys and

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histological changes in the kidneys of mice and monkeys has not been observed. Gen-1 ASOs also do not have a clinical class effect on the liver. Aside from the above clinical class effects, the clinical studies of Gen-1 ASOs have not uncovered any safety issues that were not previously identified by the nonclinical studies of these drugs. None of the clinical safety class effects are sufficiently concerning to prevent development of drug class members with demonstrated clinical benefit. For Gen-2 ASOs, the clinical safety picture is still developing. Prolongation of aPTT is certainly a class effect for Gen-2 ASOs and there does not appear to be a difference between Gen-1 and Gen-2 ASOs for this effect. Subcutaneous injection site reactions also are a class effect, however it appears that higher Gen-2 ASO doses can be tolerated compared to Gen-1 ASO doses. As for the other two Gen-1 ASOs clinical class effects, studies of additional class members are needed. Intravenous administration of two Gen-2 ASOs (ISIS 104838 and ISIS 113714) at doses ⭓600 mg were not associated with fevers and chills that are routinely observed with the infusions of Gen-1 ASOs. However, these infusion-associated symptoms were seen with OGX-011. Thrombocytopenia was observed with ISIS 104838 given subcutaneously for 12 weeks. However, it was not observed with ISIS 113715 given intravenously at equivalent doses for 12 weeks. As with Gen-1 ASOs, the clinical studies of Gen-2 ASOs have not found any indications for complement activation or adverse effects on kidney function. The studies have also not detected any hypersensitivity events to date. Gen-2 ASOs currently do not appear to have a class effect on liver. ALT/AST increases associated with ASO administration were not observed in a 176-patient, randomized, placebo-controlled, double-blind trial of ISIS 104838 but were seen in smaller trials of three other Gen-2 ASOs. Until larger controlled trials for these three ASOs are conducted, and other Gen-2 ASOs are tested, the extent to which these ASO contributed to the observed ALT/AST elevations cannot be fully assessed for a number of reasons (see Section 13.3.6). Like Gen-1 ASOs, the clinical studies of Gen-2 ASOs have not uncovered any additional safety issues to date that were not already identified by the nonclinical toxicology studies. The clinical safety effects observed for Gen-2 ASO to date clearly are not barriers to the continued clinical development of these compounds. REFERENCES 1. Crooke, S.T., Ed., Antisense Drug Technology Principles, Strategies, and Applications, Marcel Dekker, New York, 2001, 929pp. 2. Kastelein, J.J.P. et al., Potent reduction of apolipoprotein B and LDL cholesterol by short-term administration of an antisense inhibitor of apolipoprotein B, Circulation, 114, 1729, 2006. 3. Chi, K.N. et al., A phase I pharmacokinetic and pharmacodynamic study of OGX-011, a 2⬘-methoxyethyl antisense oligonucleotide to clusterin, in patients with localized prostate cancer, J. Natl. Cancer Inst., 97, 1287, 2005. 4. Waters, J.S. et al., Phase I clinical and pharmacokinetic study of Bcl-2 antisense oligonucleotides therapy in patients with non-Hodgkin’s lymphoma, J. Clin. Oncol., 18, 1812, 2000. 5. Webb, A. et al., Bcl-2 antisense therapy in patients with non-Hodgkin lymphoma, Lancet, 349, 1137, 1997. 6. Stevenson, J.P. et al., Phase I clinical/pharmacokinetic and pharmacodynamic trial of the c-raf-1 antisense oligonucleotide ISIS 5132 (CGP 69846A), J. Clin. Oncol., 17, 2227, 1999. 7. O’Dwyer, P.J. et al., c-raf-1 depletion and tumor responses in patients treated with the c-raf-1 antisense oligodeoxynucleotides ISIS 5132 (CGP 69846A), Clin. Cancer Res., 5, 3977, 1999. 8. Yacyshyn, B.R. et al., A placebo-controlled trial of ICAM-1 antisense oligonucleotide in the treatment of Crohn’s disease. Gastroenterology, 114, 1133, 1998. 9. Geary, R.S. et al., Pharmacokinetic properties in animals, in Antisense Drug Technology Principles, Strategies, and Applications, Crooke, S.T., Ed., Marcel Dekker, New York, 2001, chapter 6. 10. Levin, A.A. et al., Toxicity of antisense oligonucleotides, in Antisense Drug Technology Principles, Strategies, and Applications, Crooke, S.T., Ed., Marcel Dekker, New York, 2001, chapter 9.

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administered as a 24-hour weekly infusion schedule in patients with advanced cancer, Invest. New Drugs, 23, 467, 2005. 46. Nemunaitis, J. et al., Phase I evaluation of ISIS 3521, an antisense oligodeoxynucleotide to protein kinase C-alpha, in patients with advanced cancer, J. Clin. Oncol., 17, 3586, 1999. 47. Cunningham, C.C. et al., A phase I trial of a H-ras antisense oligonucleotide ISIS 2503 administered as a continuous intravenous infusion in patients with advanced carcinoma, Cancer, 92, 1265, 2001. 48. Adjei, A.A. et al., A phase I trial of ISIS 2503, an antisense inhibitor of H-ras, in combination with gemcitabine in patients with advanced cancer, Clin. Cancer Res., 9, 115, 2003. 49. Alberts, S.R. et al., Gemcitabine and ISIS-2503 for patients with locally advanced or metastatic pancreatic adenocarcinoma: a North Central Cancer Treatment Group Phase II trial, J. Clin. Oncol., 22, 4944, 2004. 50. McHutchison, J.G. et al., A phase I trial of an antisense inhibitor of hepatitis C virus (ISIS 14803), administered to chronic hepatitis C patients, J. Hepatol., 44, 88, 2006. 51. FDA Briefing Material for Oncologic Drugs Advisory Committee meeting on May 3, 2004 to consider oblimersen NDA, www.FDA.gov. 52. Genta Incorporated, FDA Advisory Committee Briefing Document, NDA 21-649, www.FDA.gov. 53. Chi, K.N. et al., A phase I dose-finding study of combined treatment with an antisense Bcl-2 oligonucleotide (Genasense) and mitoxantrone in patients with metastatic hormone-refractory prostate cancer, Clin. Cancer Res., 7, 3920, 2001. 54. Morris, M.J. et al., Phase I trial of BCL-2 antisense oligonucleotide (G3139) administered by continuous intravenous infusion in patients with advanced cancer, Clin. Cancer Res., 8, 679, 2002. 55. Rudin, C.M. et al., A pilot trial of G3139, a bcl-2 antisense oligonucleotide, and paclitaxel in patients with chemorefractory small-cell lung cancer, Annals Oncol., 13, 539, 2002.

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56. Marcucci, G. et al., Phase 1 and pharmacodynamic studies of G3139, a Bcl-2 antisense oligonucleotide, in combination with chemotherapy in refractory or relapsed acute leukemia, Blood, 101, 425, 2003. 57. Rudin, C.M. et al., Phase I study of G3139, a bcl-2 antisense oligonucleotide, combined with carboplatin and etoposide in patients with small-cell lung cancer, J. Clin. Oncol., 22, 1110, 2004. 58. Tolcher, A.W. et al., A phase I pharmacokinetic and biological correlative study of oblimersen sodium (Genasense, G3139), an antisense oligonucleotide to the Bcl-2 mRNA, and of docetaxel in patients with hormone-refractory prostate cancer, Clin. Cancer Res., 10, 5048, 2004. 59. Tolcher, A.W. et al., A phase II, pharmacokinetic, and biological correlative study of oblimersen sodium and docetaxel in patients with hormone-refractory prostate cancer, Clin. Cancer Res., 11, 3854, 2005. 60. Bishop, M.R. et al., Phase I trial of an antisense oligonucleotide OL(1)p53 in hematologic malignancies, J. Clin. Oncol., 14, 1320, 1996. 61. Séréni, D. et al., Pharmacokinetics and tolerability of intravenous trecovirsen (GEM®91), an antisense phosphorothioate oligonucleotide, in HIV-positive subjects, J. Clin. Pharmacol., 39, 47, 1999. 62. Hybridon halts development of GEM91; developing new antisense HIV drug, Antiviral Agents Bull., 10, 226, 1997. 63. Andrews, D.W. et al., Results of a pilot study involving the use of an antisense oligonucleotide directed against the insulin-like growth factor type I receptor in malignant astrocytomas, J. Clin. Oncol., 19, 2189, 2001. 64. Kutryk, M.J.B. et al., Local intracoronary administration of antisense oligonucleotide against c-myc for the prevention of in-stent restenosis, J. Am. Coll. Cardiol., 39, 281, 2002. 65. Rudin, C.M. et al. Delivery of a liposomal c-raf-1 antisense oligonucleotide by weekly bolus dosing in patients with advanced solid tumors: a phase I study, Clin. Cancer Res., 10, 7244, 2004. 66. Dritschilo, A. et al., Phase I study of liposome-encapsulated c-raf antisense oligodeoxyribonucleotide infusion in combination with radiation therapy in patients with advanced malignancies, Clin. Cancer Res., 12, 1251, 2006. 67. Gewirtz, A.M., Developing oligonucleotide therapeutics for human leukemia, Anti-Cancer Drug Design, 12, 341, 1997. 68. Kronenwett, R. and Haas, R., Antisense strategies for the treatment of hematological malignancies and solid tumors, Ann. Hematol., 77, 1, 1998. 69. Marcucci, G. et al., A phase I study of GTI-2040 (G), an antisense to ribonucleotide reductase (RNR), in combination with high-dose AraC (HiDAC) in acute myeloid leukemia (AML), J. Clin. Oncol., 24, 352s, 2006. 70. Dean, N.M. et al., Pharmacology of 2⬘-O-(2-methoxy)ethyl-modified antisense oligonucleotides, in Antisense Drug Technology: Principles, Strategies, and Applications, Crooke, S.T., Ed., Marcel Dekker, New York, 2001, chapter 12. 71. Sewell, K.L. et al., Phase I trial of ISIS 104838, a 2⬘-methoxyethl modified antisense oligonucleotide targeting tumor necrosis factor-
, J. Pharmacol. Exptl. Therap., 303, 1334, 2002. 72. Henry, S.P. et al., Toxicologic properties of 2⬘-methoxyethyl chimeric antisense inhibitors in animals and man, Antisense Drug Technology: Principles, Strategies, and Applications, Crooke, S.T., Ed., Taylor & Francis, Boca Raton, 2007, chapter 12. 73. Geary, R.S. et al., Pharmacokinetic/pharmacodynamic properties of phosphorothioate 2⬘-O-(2methoxyethyl) modified antisense oligonucleotides in animals and man, Antisense Drug Technology: Principles, Strategies, and Applications, Crooke, S.T., Ed., Taylor & Francis, Boca Raton, 2007, chapter 11. 74. Dorr, F.A., Glover, J.G. and Kwoh, T.J., Clinical safety of phosphorothioate oligodeoxynucleotides, in Antisense Drug Technology: Principles, Strategies, and Applications, Crooke, S.T., Ed., Marcel Dekker, New York, 2001, chapter 10. 75. Yu, R.Z. et al., Pharmacokinetic properties in humans, in Antisense Drug Technology: Principles, Strategies, and Applications, Crooke, S.T., Ed., Marcel Dekker, New York, 2001, chapter 8. 76. Sheehan, J.P. and Lan, H.-C., Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex, Blood, 92, 1617, 1998. 77. Galbraith, W.M. et al., Complement activation and hemodynamic changes following intravenous administration of phosphorothioate oligonucleotides in the monkey, Antisense Res. Dev., 4, 201, 1994. 78. Hartman, G. et al., Delineation of a CpG phosphorothioate oligodeoxynucleotides for activating primate immune responses in vitro and in vivo, J. Immunol., 164, 1617, 2000.

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79. Henry, S.P. et al., Assessment of the effects of ISIS 2302, an antisense inhibitor of human ICAM-1 on cellular and humoral immunity in mice, J. Immunotoxicol., 3, 199, 2006. 80. Agnello, V., Mixed cyroglobulinemia and other extrahepatic manifestations of hepatitis C virus infection, in Hepatitis C, Liang, T.J. and Hoofnagle, J.H., Eds., Academic Press, San Diego, CA, 2000, chapter 15. 81. Chalasani, N., Statins and hepatotoxicity: focus on patients with fatty liver, Hepatol., 41, 690, 2005. 82. Kaplowitz, N., Idiosyncratic drug hepatotoxicity, Nat. Rev. Drug Disc., 4, 489, 2005. 83. Everhart, J.E., Digestive diseases and diabetes, in Diabetes in America, 2nd Edition, Harris M.I. et al., Eds., National Institutes of Health, Bethesda, Washington, DC, chapter 21. 84. Rosenthal, S.A. et al., Flutamide-associated liver toxicity during treatment with total androgen suppression and radiation therapy for prostate cancer, Radiology, 199, 451, 1996. 85. Rosenweig, P., Miget, N. and Brohier, S., Transaminase elevation on placebo during Phase I trials: prevalence and significance, Br. J. Clin. Pharmacol., 48, 19, 1999. 86. Purkins, L. et al., The influence of diet upon liver function tests and serum lipids in heathy male volunteers resident in a phase I unit, Br. J. Clin. Pharmacol., 57, 199, 2003.

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CHAPTER

14

Manufacturing and Analytical Processes for 2⬘⬘-O- (2-Methoxyethyl)-Modified Oligonucleotides Daniel C. Capaldi and Anthony N. Scozzari

CONTENTS 14.1 14.2

14.3

Introduction .........................................................................................................................402 Manufacturing Process........................................................................................................403 14.2.1 Starting Materials..................................................................................................404 14.2.1.1 Nucleoside Phosphoramidites ..............................................................404 14.2.1.2 Precursors of Nucleoside Phosphoramidites........................................404 14.2.1.3 Impurities in Nucleoside Phosphoramidites ........................................405 14.2.1.4 Nucleoside-Loaded Solid Support .......................................................406 14.2.1.5 Sulfur Transfer Reagent .......................................................................407 14.2.2 Reagents ................................................................................................................408 14.2.2.1 Detritylation Reagent ...........................................................................408 14.2.2.2 Activator Solution ................................................................................409 14.2.2.3 Capping Reagents ................................................................................409 14.2.3 Solid Support ........................................................................................................410 14.2.4 Synthesizers ..........................................................................................................411 14.2.4.1 Millipore 8800 .....................................................................................411 14.2.4.2 GE OligoProcess Synthesizer ..............................................................412 14.2.5 Solid-Phase Synthesis, Purification and Isolation ................................................413 14.2.5.1 Solution Preparation.............................................................................413 14.2.5.2 Oligonucleotide Synthesis ...................................................................413 14.2.5.3 Deprotection.........................................................................................415 14.2.5.4 Purification...........................................................................................415 14.2.5.5 Final Detritylation ................................................................................417 14.2.5.6 Precipitation .........................................................................................418 14.2.5.7 Freeze-Drying ......................................................................................419 14.2.5.8 Yield and Purity ...................................................................................419 Analytical Processses..........................................................................................................419 14.3.1 Starting Materials..................................................................................................419 14.3.1.1 Phosphoramidites .................................................................................419 14.3.1.2 Phenylacetyl Disulfide .........................................................................420 14.3.2 Reagents and Solvents ..........................................................................................421 401

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14.3.3

Drug Substance Intermediates ..............................................................................421 14.3.3.1 Analysis of Crude Samples..................................................................421 14.3.3.2 Analysis of Purified Samples...............................................................421 14.3.3.3 Analysis of Detritylated Samples ........................................................422 14.3.4 Drug Substance .....................................................................................................422 14.3.4.1 Identity Testing.....................................................................................422 14.3.4.2 Impurity Tests and Assay .....................................................................422 14.4 Conclusions .........................................................................................................................430 Acknowledgments ..........................................................................................................................430 References ......................................................................................................................................430

14.1 INTRODUCTION The approval of Vitravene in 1998 for the treatment of CMV retinitis in AIDS sufferers stands as an early testament to the promise of antisense technology. More recently, a second oligonucleotide-based therapeutic, Macugen,* was approved for the treatment of wet, age-related macular degeneration (AMD). These successes and encouraging clinical trial results obtained using other oligonucleotides† suggest that substantial quantities of high-quality modified DNA and RNA will be required in the near future. Most current large-scale manufacturing efforts utilize the four-step solid-phase phosphoramidite approach first described 25 years ago by Beaucage and Caruthers [1] (Scheme 14.1). That the core chemistry should remain essentially unchanged for a quarter century is evidence of the inherent simplicity, efficiency, and ruggedness of the approach. While no sea change has occurred, advances in reagent, solid support, synthesizer, purification, and isolation technologies have transformed the art so that the synthesis of multi-hundred-kilogram quantities of modified DNA and RNA at reasonable cost is now a reality. In the first part of this chapter, we discuss some of the manufacturing process improvements that have been developed in our laboratory and in others over the past 15 years. Using 2⬘-O-methoxyethyl-2⬘-deoxyphosphorothioate gapmers (MOE gapmers) as an example, we illustrate the importance of understanding the starting material and reagent supply chains. Advances in synthesizer design, and the development and implementation of optimized reagents, solid supports, and synthesis parameters that result in improved yield and purity are described. Finally, in this section, we discuss the purification, deprotection, and isolation strategies employed at Isis Pharmaceuticals, Inc. for the synthesis of MOE gapmers. Clearly, the synthesis of large quantities of a complex drug substance in compliance with current good manufacturing practices (cGMP) can only be contemplated in an environment that encompasses appropriate quality controls on starting materials, reagents, intermediates, and the drug substance itself. The establishment of a useful control strategy is reliant on the development of analytical methodologies capable of measuring those critical product characteristics, be they of starting materials, reagents, intermediates, or drug substance, which affect the safety and

* In December of 2004, Eyetech Pharmaceuticals, Inc. and Pfizer, Inc. received FDA approval for Macugen (pegaptanib sodium injection) for the treatment of neovascular (wet) age-related macular degeneration (AMD). † Antisense oligonucleotide examples include ISIS 301012 (Hyperlipedemia, Isis Pharmaceuticals, Inc.); ISIS 113715 (type II diabetes, Isis Pharmaceuticals, Inc.); Genasense (cancer, Genta, Inc.); LY2181308 (cancer, Eli Lilly and Co.); AEG35156 (Aegera Therapeutics, Inc.); AP12009 (cancer, Antisense Pharma, Gmbh); OGX-011 (cancer, Oncogenex Technologies, Inc. and Isis Pharmaceuticals, Inc.); and ATL-1102 (multiple sclerosis, Antisense Therapeutics Limited).

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pg

HO

pg

B1

O

403

B1

O

O

O

a O

O

N H

O

N H

O

pg

RO O

pg Bn

RO O

O

c

O R′O P X

O

n-1

R′O P

B1

n -1

O O

N H

O

b n=n+ 1

d, a

e

pg

HO

Bn

O

OLIGONUCLEOTIDE

OR′

B1

O

N H

O

P

pg

O

O O

O (R″)2N

O

pg

O

b n=2

pg Bn

RO

Bn

O R′O P X

pg

O O n-1

B1

O O O

N H

R = acid-labile protecting group; R′ = base-labile protecting group; R″ = alkyl group; X = O or S; n = number of nucleobases; H Bnpg = protected nucleobase at position n; H2N = solid support

Key a—Deprotection; b—Coupling; c—Oxidation or sulfurization; d—Capping; e—Ammonolysis

Scheme 14.1

Solid-phase synthesis of oligonucleotides via the phosphoramidite approach.

efficacy of formulated drug product. One such integrated analytical approach for MOE gapmers is described in the second part of this chapter.

14.2 MANUFACTURING PROCESS The manufacture of MOE gapmers is a multi step process that can be divided into two distinct stages: solid-phase synthesis and downstream processing. In the first operation, the desired sequence is assembled by a computer-controlled solid-phase synthesizer. Subsequent downstream processing includes deprotection steps, reversed-phase (RP) chromatographic purification, isolation, and drying to yield the drug substance. During the chemical synthesis, phosphoramidite monomers are sequentially coupled to an elongating oligonucleotide that is covalently bound to a solid support. After the final elongation cycle, the solid support–bound oligonucleotide is deprotected and liberated from the solid support. The crude product is then purified by RP high-performance liquid chromatography (HPLC) and the purified material treated with acetic acid to remove the final protecting group. Following precipitation, the product is redissolved in purified water, filtered, and freeze-dried to yield the drug substance. Our description of the process used to manufacture MOE gapmers begins upstream of solidphase synthesis with a discussion of starting materials, i.e., those components that contribute atoms to the drug substance.

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14.2.1 Starting Materials

14.2.1.1 Nucleoside Phosphoramidites The majority of the atoms that make up the drug substance is contributed by the nucleoside phosphoramidites shown in Figure 14.1. The residues of the 2⬘-deoxy gap, whose presence is required to preserve Rnase H–mediated degradation of target mRNA strands, are provided by 2⬘-deoxy phosphoramidites 1–4. The nucleotides of the MOE wings, which are responsible for the improved pharmacological properties of MOE gapmers relative to their all-2⬘-deoxy phosphorothioate counterparts, are assembled by coupling 2⬘-MOE phosphoramidites 5–8. Depending on the sequence, up to eight different nucleoside phosphoramidites are required.

14.2.1.2 Precursors of Nucleoside Phosphoramidites The precursors of nucleoside phosphoramidites 1–8 are the corresponding 2⬘-deoxy- and ribonucleosides. The latter have long been available inexpensively as products of fermentation. Likewise, until recently, 2⬘-deoxynucleosides were derived from natural sources, being most often isolated following enzymatic digestion of DNA obtained from salmon milt. The limited nature of this resource means that naturally obtained 2⬘-deoxynucleosides are considerably more expensive than ribonucleosides. In the past few years, this natural source has been supplanted, and 2⬘-deoxynucleosides are now

O

OCH3

OCH3

HN N H3CO

O

O N

P

N

N

N

O

O

N

H3CO

O

O CN

O

N

O

R N

1: R = H 5: R = OCH2CH2OCH3

P

R CN

O

O

OCH3 O

H3CO

N

O

N

O P

HN H3C

NH

O

O

H3CO

CN

3: R = H 7: R = OCH2CH2OCH3

N

O

N

O

R O

N

2: R = H 6: R = OCH2CH2OCH3

OCH3 H3C

NH

N

O P

O

R O

CN

4: R = H 8: R = OCH2CH2OCH3

Figure 14.1 Nucleoside phosphoramidites used to manufacture MOE gapmers.

N H

O

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produced synthetically [2]. Predictably, the development of efficient routes to 2⬘-deoxynucleosides has resulted in considerable cost reductions [3]. In addition to lower cost, synthetic 2⬘-deoxynucleosides have several other advantages over materials obtained by digestion of DNA, not the least of which is that they can be made available in essentially unlimited quantities. A second advantage is that quantities of each 2⬘-deoxynucleosides may be specified independently. This is important because MOE gapmers are unlikely to contain the roughly equal proportions of 2⬘-deoxyadenosine, 2⬘-deoxyguanosine, and thymidine that are obtained by digestion of salmon milt DNA. Relying on DNA for 5-methyl-2⬘-deoxycytidine is even less appealing, as this nucleoside accounts for only about 0.6% of the nucleosides present [4]. A third advantage of synthetic over naturally sourced 2⬘-deoxynucleosides is that the chemical process avoids the hydrolytic enzymes that are used to digest DNA. These enzymes are often derived from bovine sources and their use raises concerns about the possibility of contamination with transmissible spongiform encephalopathy (TSE)-inducing agents.

14.2.1.3 Impurities in Nucleoside Phosphoramidites A detailed understanding of the types of impurities associated with starting materials is critical to the continued successful synthesis of any drug substance. These data are often gathered during the early stages of drug development and are used to help establish specification limits for the starting materials. With appropriate limits in place, the drug substance manufacturer can predict with some degree of confidence that when passing lots of starting materials are fed into the process, drug substance of acceptable quality will be produced. Acceptable levels for starting material impurities vary depending upon the nature of the particular impurity and the manufacturing process. For example, some impurities may form components that can be easily separated from the drug substance; these types of impurities might be tolerated at relatively high levels. Others may be unreactive so that although they may be controlled to mitigate an effect on yield, their presence has no effect on drug substance quality. In most processes, however, there is some subset of impurities that must be carefully controlled to ensure successful drug substance manufacture. There are several aspects of solidphase oligonucleotide manufacturing that make controlling starting material impurities more critical and more challenging than for other processes. First, oligonucleotide synthesis consists of an extended series of reactions in which the products from one reaction are fed into the next without any attempt at purification. In a typical synthesis of a 20-mer oligonucleotide, a series of 19, or in some cases 20 coupling reactions involving the addition of phosphoramidite starting materials is performed before any purification takes place. This means that even low levels of reactive impurities in phosphoramidites can have a detectable effect on drug substance quality. For example, a reactive impurity present at 0.5% in each phosphoramidite might result in a 10% impurity in crude 20-mer drug substance. Second, it is often the case that an oligonucleotide drug substance impurity caused by an impurity in a phosphoramidite has very similar chromatographic properties to the parent drug. This means that once formed, an impurity may often be difficult to remove. That significant amounts of difficult-to-remove drug substance impurities can be caused by low-level phosphoramidite impurities requires critical components in the starting materials be controlled at low levels. Many of the impurities present in nucleoside phosphoramidites 1–8 have been identified. The impact of these impurities on the drug substance manufacturing process was assessed, and impurities classified as nonreactive and noncritical, reactive and noncritical, or critical [5]. A 2⬘-MOE A example of each type of impurity is shown in Figure 14.2. Impurities belonging to the first group, e.g., 9, which is a hydrolysis product of MOE A phosphoramidite, do not react with the nascent oligonucleotide under the conditions required for phosphoramidite coupling. The presence of these components has no effect on drug substance quality. Compounds belonging to the second group are reactive under the coupling conditions, but again have no impact on drug substance quality because they result in the production of the parent oligonucleotide. Compound 10, which contains a modified amino group attached to phosphorus, is a good example of this type of impurity. Reactive and critical compounds, e.g., 11, react with the growing oligonucleotide

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION O

O

HN N O O O HO P O O

9

N

HN N

DMTO

O

N O

N

OCH3 CN

N

DMTO N O

O N

P

O

10

HN N

N

DMTO

OCH3 CN

N

O

N

N

O P

N

OCH3 O

CN

11

Figure 14.2 Examples of nonreactive noncritical (9), reactive noncritical (10), and critical (11) phosphoramidite impurities of MOE A phosphoramidite.

chain to produce drug substance impurities. The presence of 11 in MOE A phosphoramidite leads to the incorporation of a 2⬘-O-methyl residue in place of the desired 2⬘-MOE residue. By determining which impurities are critical to drug substance quality, we have established a rational, scientifically based approach to specification setting for 2⬘-deoxy and 2⬘-MOE phosphoramidites. In this scenario, those impurities that are critical to quality are tightly controlled, while those that do not contribute drug substance impurities are more loosely controlled. This helps ensure a reproducible manufacturing process while avoiding forcing phosphoramidite suppliers to purify materials to the point beyond which there is no impact on drug substance quality.

14.2.1.4 Nucleoside-Loaded Solid Support In the solid-phase approach, the 3⬘-terminal nucleoside of an oligonucleotide is normally contributed by the solid support, which by definition is regarded as a starting material. There are several disadvantages associated with using nucleoside-loaded solid supports. First, because it is attached to an insoluble polymer, complete characterization of the nucleoside after it is loaded is difficult. This can have a negative impact on the ability of a manufacturer to defend a choice of starting material, with the consequence that starting material status may have to be transferred to a precursor and the support-bound nucleoside itself manufactured under GMP conditions. Second, in addition to these characterization studies that are normally performed on a single lot of material some time during the drug development stage, it is important to determine, on a lot-to-lot basis, the starting material attributes that impact drug substance quality. It is difficult to accomplish this for nucleosides that are attached to insoluble polymers, and most manufacturers resort to performing impurity profile analyses on material that has been hydrolyzed from the support. Unfortunately, this tactic is only partially successful for N 4-benzoyl-5-methylcytosine and N 6-benzoyladenine nucleoside loaded supports, where important impurities are the corresponding N-unprotected nucleosides [6]. These components obviously cannot be readily quantitated following hydrolysis. A final disadvantage of nucleoside-loaded supports, and one that adds complexity in the areas of material procurement, method and specification development, testing, and inventory management, is that each oligonucleotide that possesses a unique 3⬘ residue requires its own support. We recently introduced a novel universal linker, termed Unylinker, which overcomes many of the problems inherent to nucleoside-loaded solid supports [7]. Unylinker-loaded solid support 13 is prepared by reaction of succinate 12 with amino or hydroxyl derivatized support in the presence of HBTU [8] (Scheme 14.2). Oligonucleotide synthesis is initiated by removal of the 5⬘-O-4,4⬘-dimethoxytrityl (DMT) group from the Unylinker molecule, and the 3⬘-terminal residue of the target sequence added by coupling the appropriate phosphoramidite. At the end of synthesis, the products are incubated in ammonium

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O

B

O

O

DMTO

O O

O

O

N

O

O

b

O

X

O

O

DMTO

a

N

O

O

O O

O

13

O N

O O

O

B

O

O

= Solid support; X = NH or O

O

O

X

12

O

P O

c

O

P O O

O O

HO N O

Key a—Amino or hydroxyl derivatized solid support, O-benzotriazol-1-yl-N,N,N ′,N ′-tetramethyluronium hexafluorophosphate, diisopropylethylamine, MeCN, then acetic anhydride, N-methylimidazole, pyridine, MeCN; b—Solid phase oligonucleotide synthesis then TEA-MeCN; c, NH4OH

Scheme 14.2

Synthesis and mechanism of cleavage of Unylinker support.

hydroxide. Under these conditions, the ester bond linking the oligonucleotide via the Unylinker molecule to the support cleaves, freeing the hydroxyl group of the oxabicycloheptane ring system to attack the cis-oriented, vicinal phosphorothioate group liberating a 3⬘-hydroxyl oligonucleotide. The advantages of this approach can be summarized as follows: ●

●

●

●

Because the identity of the 3⬘-residue is determined by the first phosphoramidite coupled, Unylinkerloaded support can be used to make any sequence. Because it does not contribute atoms to the drug substance, the support need no longer be regarded as a starting material. This reduces characterization expectations for the support. Impurities in the Unylinker molecule that impact drug substance quality are not affected by hydrolysis. This means that the critical product characteristics of the Unylinker may be assessed following removal of the molecule from the solid support. Branched impurities of the types described by Kurata et al. [6] are avoided when Unylinker-loaded support is used.

MOE gapmer oligonucleotides have been manufactured at large scale at Isis using both nucleosideand Unylinker-loaded solid supports. Because there appear to be clear advantages associated with the latter approach, we intend using Unylinker-loaded materials in all future campaigns.

14.2.1.5 Sulfur Transfer Reagent The sulfur atoms of the internucleotide linkages of MOE gapmers are introduced following addition of each nucleoside phosphoramidite (Scheme 14.1). Over the years, many compounds have been evaluated for their ability to transfer sulfur [9–25]. To our knowledge, however, only phenylacetyldisulfide (PADS, 14) [11,20], 3H-1,2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent, 15) [10], and 3-amino-1,2,4-dithiazole-5-one (ADTT, 16) [22] have been used at scales of 100 mmol or greater. Currently, the reagent of choice, and the one used to manufacture MOE gapmers at Isis, is 14, which is readily synthesized from phenyacetyl chloride and sodium sulfide. As the source of the sulfur atoms that are incorporated into the drug substance, PADS meets the definition of a starting material (Figure 14.3).

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION O

O S

S

S S

O O

14

15

O

NH2 S S

N S

16

Figure 14.3 Sulfur transfer reagents that have been used in large-scale phosphorothioate oligonucleotide manufacture.

Several features underlie PADS’s popularity as a sulfur transfer reagent. First, because the reagent is simple to produce from readily accessible starting materials, it is relatively inexpensive. For example, we estimate that PADS is approximately 20 times cheaper than the Beaucage reagent on the per gram basis [26]. Second, PADS produces crude oligonucleotides of high phosphorothioate diester content. In our hands, under optimized conditions at scales ranging from 200 to 750 mmol, per linkage phosphorothioate–phosphate diester ratios of greater than 99.9 : 0.1 are achievable [27]. For a 20-mer oligonucleotide, this results in crude product containing about 2% of molecules that contain a single phosphate diester linkage. Furthermore, both the rate and extent of sulfurization using PADS are high, with the reaction proceeding to completion within a couple of minutes. The extent of reaction, which can be judged by analyzing crude oligonucleotides for the presence of DMT-C-phosphonates [28], is often overlooked in discussions of sulfur transfer reagents. The rapid rate of sulfur transfer helps maintain short synthesis times. Third, 14 has several ease-of-use characteristics that enhance its practicality. For example, PADS is a stable, crystalline compound that is freely soluble in MeCN. Once in solution, the reagent remains so, and PADS solutions are not plagued by the precipitation problems that sometimes accompany solutions of other reagents. Finally, the sulfurization reaction with PADS does not appear to be affected by the presence of small amounts of water [29]. This is in contrast to other reagents whose preferred use under strictly anhydrous conditions limits their practicality at large scale. 14.2.2 Reagents

14.2.2.1 Detritylation Reagent The first step in each synthesis cycle is removal of the DMT group from the support-bound oligonucleotide. Although different reagents have been evaluated for their ability to remove DMT groups from nucleosides [30–32], many have limited utility in solid-phase oligonucleotide synthesis. The most commonly used reagents are solutions of trichloroacetic (TCA) or dichloroacetic acid (DCA) in methylene chloride. Early GMP syntheses performed at Isis utilized a 3% v/v solution of dichloroacetic acid (DCA) in methylene chloride, a combination that remained in place through 1999, and that forms part of the commercial manufacturing process for Vitravene. Concerns regarding the toxicity and cost of disposal of methylene chloride prompted Krotz et al. to evaluate a number of different solvents for detritylation, efforts that resulted in the identification of toluene as a “green” alternative [33]. At about the same time, we became aware of a report by Paul and Royappa [34] that showed that increasing the concentration of DCA could reduce the total volume of detritylation solution required and cut the detritylation time, thereby potentially shortening overall synthesis times and increasing the maximum synthesis scale attainable with our existing tank farm. The two ideas were combined, and MOE gapmers are currently manufactured at Isis using a 10% v/v solution of DCA in toluene for detritylation. As discussed, the potential for low-level impurities in nucleoside phosphoramidites to lead to detectable drug substance process impurities is due to the linear nature of oligonucleotide synthesis. The situation is similar for other aspects of oligonucleotide chemistry, where the progression of a side reaction even to a small extent in each cycle can have a dramatic impact on the purity of the final

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O

B

O O +−

Na

S P

Cl3C

409

R O

O O

B

O O

R

R = H or OCH2CH2OCH3

I Figure 14.4 Chloral-modified oligonucleotide.

product. Chloral-modified oligonucleotides I, which are formed due to the presence of low concentrations of trichloroacetaldehyde in DCA, provide a good example of this phenomenon [35] (Figure 14.4).

14.2.2.2 Activator Solution To promote reaction with the 5⬘-hydroxyl group of the support-bound oligonucleotide, phosphoramidites must first be activated by a suitable reagent. The ideal activator would be a cheap, stable, nonhygroscopic compound, which promotes rapid activation and coupling without the formation of side-products. Although many compounds have been evaluated on a small scale [30,36–41], only 1H-tetrazole (17) [1] and 4,5-dicyanoimidazole (DCI, 18) [42,43] have been reported for use in the large-scale production of phosphorothioate oligonucleotides (Figure 14.5). These compounds have been used at Isis to manufacture MOE gapmer oligonucleotides with similar results. 1H-tetrazole, used at a concentration of 0.45 M in MeCN, is a crystalline, nonhygroscopic solid, which is relatively inexpensive when purchased in bulk. 17 is an effective activator of 2⬘-deoxy and relatively unhindered analogs such as 2⬘-MOE phosphoramidites, with maximum coupling efficiencies being attained with coupling times of ca. 2 min. 1H-tetrazole does possess some disadvantages, however, not the least of which is its reported explosive nature [44]. In addition, the solubility of 17 at room temperature in MeCN is only around 0.5 M, making it prone to crystallize from 0.45 M activator solutions in the event of falling ambient temperature. These difficulties led Vargeese et al. to introduce DCI as an alternative activator for oligonucleotide synthesis. 18, which is employed as a 0.5-M solution in MeCN, is an inexpensive, crystalline material that activates 2⬘-deoxy and 2⬘-MOE phosphoramidites with at least the same efficiency as 1H-tetrazole. It is not a classified explosive and is considerably more soluble in MeCN (up to 1.1 M at room temperature). At Isis, DCI recently displaced 1H-tetrazole as the activator of choice for the synthesis of all new MOE gapmer oligonucleotides.

14.2.2.3 Capping Reagents The final step in each synthetic cycle consists of treating the support-bound products with an acylating mixture. This step, commonly referred to as capping, is believed to acylate any 5⬘-hydroxy groups that failed to react with phosphoramidite during the preceding coupling reaction and prevent them from reacting in any subsequent cycle. The most widely used reagent system, and the one

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION NC N

N N N H

17

N NC

N H 18

Figure 14.5 Activators that have been used in large-scale phosphorothioate oligonucleotide manufacture.

used at Isis to manufacture 2⬘-MOE gapmers, consists of two separate solutions, one containing N-methylimidazole, pyridine, and MeCN (Cap A), the other acetic anhydride and MeCN (Cap B), which are added simultaneously to the reactor. Data from our laboratory suggest that capping provides no improvement in quality in those instances where coupling yields are uniformly high and synthesis proceeds as expected [45]. However, upon observation of a lower than expected coupling efficiency, perhaps due to a reagent delivery problem, capping appeared to prevent subsequent elongation, allowing isolation of high-quality product, albeit in reduced yield [46]. Although such instances are rare, the drug substance is sufficiently precious and capping reagents sufficiently inexpensive to make capping worthwhile. The other claimed benefit of capping, provided it is performed prior to oxidation (or presumably sulfurization), is that it is effective at reversing phosphitylation of the O-6 position of N-isobutyrylguanine residues [47]. In fact, this is often cited as the reason why capping should precede oxidation in DNA synthesis. In phosphorothioate synthesis, however, the order of the steps is normally reversed, possibly over concerns that oxidation, i.e. phosphate triester formation, might occur during the capping step. Although we have not explored these issues extensively, our experience to date is that neither the alleged benefit nor the assumed danger of capping prior to sulfurization is significant. Currently, the reactions are performed in order: sulfurization, then capping. 14.2.3 Solid Support At Isis, the earliest GMP syntheses were performed using controlled pore glass (CPG) supports contained in argon-sparged reaction vessels—runs that were essentially scaled-up versions of the familiar micromolar-scale syntheses performed today on conventional single- and multicolumn DNA synthesizers. A major drawback of the sparged reactor approach to synthesis is that reaction volumes must be sufficient to suspend the solid support. This is readily achieved at small scales where one can use large excesses of reagents. At large scales, however, where for economic reasons using the minimum amounts of reagents is paramount, the required volumes cannot be achieved except by dilution. Dilution is deleterious in two ways. First, decreased reactant concentrations result in decreased reaction kinetics, which in turn lead to unacceptably long cycle times. Second, because solvents are never completely anhydrous, the increased dilution means that a greater portion of phosphoramidite delivered in each cycle undergoes hydrolysis before it has a chance to react. These specific issues led Pharmacia Biotech (now GE Healthcare) to introduce the packed-bed or flow-through approach to DNA synthesis, which remains probably the single most important advance in large-scale oligonucleotide synthesis of the past 15 years. In GE’s new approach, a suitable solid support is packed into a steel column and reagents flow through the support in a manner similar to column chromatography. Reaction volumes are reduced dramatically because there is no need to suspend the support. This allows for increased reactant concentrations, which in turn result in faster reaction kinetics and greatly reduced reagent excesses. To exploit the potential of the packed-bed approach, a solid support with adequate compressibility resistance that could be packed evenly into a synthesis column was required. Given the obvious parallel to HPLC, it is not surprising that GE’s original solid support, Primer Support 30 HL, was based on a chromatography resin. 30 HL is a rigid, nonswelling, cross-linked polystyrene

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matrix, fashioned into 30-
m spheres that are coated with dextran. The dextran surface is functionalized by treating with epichlorohydrin, then ammonium hydroxide, to provide material containing 140
mol/g of NH2 groups [48]. The amino-derivatized support is then loaded with nucleoside to the extent of ca. 90
mol/g. At Isis, the use of 30 HL in the packed-bed approach enabled an approximately 100-fold increase in synthetic scale over what was achievable using CPG in a sparged reactor. Although 30 HL functioned remarkably well in the packed-bed approach, GE felt several performance characteristics might be improved by engineering a support specifically designed for oligonucleotide synthesis. First, it was clearly understood that 30 HL was most effective when used to synthesize oligonucleotides containing fewer than about 30 residues, and that attempted synthesis of longer sequences resulted in lower yields. Second, despite the jump in scale provided by 30 HL, there remained a desire to synthesize still larger amounts of oligonucleotide using existing hardware. It was realized that the second goal would be accomplished if the support were modified to allow efficient synthesis at higher support loadings (when 30 HL was loaded at ⬎100
mol/g reduced yields were observed). In fact, it turns out that the two goals are really the same, and that what limits both length and loading is the amount of oligonucleotide that can be forced into the support. In seeking to improve the situation, one could imagine that the ideal support would occupy no space at all. While such a support cannot be made, it is apparent that allowing the material to expand as synthesis proceeds could increase the maximum amount of oligonucleotide that could be packed into the support. On the surface, however, there appears to be a problem with such an approach; that is, if one allows space for the support to expand as synthesis proceeds, how does one ensure the proper flow of reagent through the support in earlier cycles? Conceivably, this issue might be addressed by allowing the synthesis column itself to expand. Alternatively, one could design a support that swells when wetted with solvent so that the support volume remains relatively constant throughout synthesis, with oligonucleotide gradually displacing an increasing proportion of solvent molecules as synthesis proceeds. It was with this second scenario in mind that GE developed Primer Support 200. Similar to 30 HL, Primer Support 200 consists of uniform (monodisperse) 30-
m beads of amino-derivatized, divinylbenzene cross-linked polystyrene. The degree of cross-linking, however, is lower than that of 30 HL, and it is this difference that allows Primer Support 200 to expand when wetted with solvents such as MeCN and toluene. Scientists at GE demonstrated that 20-mer oligonucleotide synthesis using Primer Support 200 loaded with nucleoside at 200
mol/g proceeded with the same efficiency as synthesis conducted at 90
mol/g on 30 HL [49]. At Isis, Primer Support 200 has been used to synthesize 20-mer MOE gapmers at scales of up to 750 mmol. Recently, an alternative solid support for use in packed-bed reactors was reported [50]. This material, developed jointly by Isis and the Japanese company NittoDenko, is marketed under the trade name NittoPhase. NittoPhase is composed of nonuniform (polydisperse) spheres of divinylbenzene cross-linked polystyrene with an average diameter of 90
m. Due to its polydisperse nature, it is anticipated that NittoPhase will be simpler and less costly to manufacture than Primer Support 200. NittoPhase, similar to Primer Support 200, swells when wetted with solvent, and in our hands when nucleoside or Unylinker loaded at 200
mol/g delivers essentially equivalent yields and purities of 2⬘-MOE gapmers. At Isis, NittoPhase has been used to manufacture MOE gapmers at scales in excess of 500 mmol. 14.2.4 Synthesizers

14.2.4.1 Millipore 8800 Prior to 1995, Isis used Millipore 8800 DNA synthesizers for GMP oligonucleotide synthesis. In essence, these instruments are scaled-up versions of the familiar ABI-type bench-top synthesizers. Typically, between 1 and 15 g of nucleoside-loaded solid support, most commonly CPG, is placed in a cylindrical glass reactor. The reactor has a glass frit at the base and two openings at the top for introducing reagents and venting the reactor. The reactor is plumbed through the glass frit to a waste

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port and a gas inlet. Reagents and solvents are housed in a reagent delivery module in pressurized reservoirs and are delivered to the reactor by positive pressure gas displacement. To ensure proper mixing, the support is gently agitated by flowing inert gas through the glass frit. After the reaction has proceeded for the prescribed time, excess reagents and solvent are flushed from the reactor to waste through the glass frit under positive pressure. The most serious limitation of this approach is that relatively large excesses of reagents are needed to provide the minimum reaction volume required for proper fluidization of the support. For example, for 5 g of CPG, it is recommended that approximately 5 equivalents of phosphoramidite be used in each coupling reaction [51]. Although the number of equivalents required diminishes with increasing scale, results from our laboratories indicate that at least 2.5 equivalents of phosphoramidite are required at the 50-g scale [52]. In addition to this issue, in our hands the use of positive pressure gas displacement for reagent delivery often results in inconsistent reagent delivery volumes due to fluctuations in back pressure. Despite these limitations, Millipore 8800 synthesizers generally provide good-quality oligonucleotides at reasonable cost and many such units remain in operation today. At Isis and elsewhere, however, it was ultimately the issue of maximum synthesis scale (ca. 2 mmol) that relegated these instruments from the GMP manufacturing suite to the research laboratory.

14.2.4.2 GE OligoProcess Synthesizer At Isis, large-scale GMP manufacture of MOE gapmers is conducted using a GE OligoProcess synthesizer (Figure 14.6). Two of the eight synthesizers currently in operation are located at Isis. Each of these instruments is based on prototypes that were installed at Isis and Hybridon in 1996. At its most basic level, the OligoProcess synthesizer is a device for delivering precise volumes of reagents and solvents to a steel reactor packed with a suitable solid support. Unlike sparged reactor systems, which rely on proper fluidization of the solid support to ensure adequate mixing, packed-bed synthesizers function by ensuring a uniform flow of reagents through the solid support in a manner analogous to column

Figure 14.6 Large-scale GE OligoProcess DNA synthesizer.

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chromatography. Because there is no requirement to suspend the support, the need for a minimum reaction volume is avoided, allowing one to work at higher reagent concentrations and lower excesses than are possible in a sparged reactor system. The reactor consists of a stainless steel tube fitted with filters at either end. Solvents and reagents are introduced from the top of the reactor through a distribution plate that helps ensure uniform flow through the support. Synthetic scale is adjusted by varying the amount of solid support in the reactor; at a support loading of 200
mol/g, a 600-mmol synthesis requires 3 kg of support. At Isis, phosphoramidite solutions are contained in dedicated, stainless steel tanks, housed inside the manufacturing facility. The phosphoramidite tanks are connected to the synthesizer through braided stainless steel hoses lined with convoluted Teflon. Reagent solutions are contained in stainless steel tanks located in purpose-built sheds standing adjacent to the manufacturing suite. The 900-L tank used to prepare and store detritylation reagent, i.e., 10% dichloroacetic acid in toluene, is glass-lined to prevent corrosion. The reagent tanks are hard piped in stainless steel or, in the case of the detritylation reagent in glass-lined stainless steel to outlets located inside the suite; braided stainless steel hoses lined with convoluted Teflon connect the outlets to the synthesizer. Each phosphoramidite and reagent delivery line is connected via an inlet port to a dedicated mechanical pump that controls delivery. The pumps are capable of delivering precise volumes over a wide range of well-defined flow rates, permitting strict control of reagent excesses and reaction times. Specialized software automatically controls and monitors the pumps and associated valves and flow-meters; once programmed and initiated oligonucleotide synthesis proceeds without the need for operator intervention. 14.2.5 Solid-Phase Synthesis, Purification and Isolation

14.2.5.1 Solution Preparation Typically, solution preparation for a large-scale GMP synthesis of a MOE gapmer begins a day or two in advance of the start of synthesis. Phosphoramidites are prepared directly in dedicated tanks by adding the required amount of material to anhydrous MeCN under a blanket of Ar gas. 1H-tetrazole (0.45 M) or DCI (0.5 M) solution is prepared similarly, by adding the solid directly to the appropriate tank filled with anhydrous MeCN. PADS is prepared by adding solid reagent directly to equal volumes of MeCN and 3-picoline. Krotz et al. showed that PADS is relatively stable in MeCN, but, upon addition of 3-picoline is converted with a half-life of ca. 12 h to a mixture of as yet unidentified compounds [27]. Interestingly, at least one of these compounds appears to be a more potent sulfur transfer reagent than PADS. A practical upshot of these findings is that the phosphate diester content of phosphorothioate oligonucleotides is reduced by allowing the PADS reagent system to age for at least 24 h prior to use. For example, at the laboratory scale, when PADS solutions were allowed to sit at room temperature for between 24 and 30 h before use, the average proportion of molecules formed that contained a single phosphate diester linkage [(P⫽O)1] was 2.7%. In contrast, when the same solution was used within 6 h of preparation, the average (P⫽O)1 content was 8.6%. At large scale, we routinely use PADS solutions that have been aged for ⬎24 h to manufacture crude drug substance containing ⱕ2% (P⫽O)1.

14.2.5.2 Oligonucleotide Synthesis The synthetic steps of MOE gapmer manufacture are performed without operator intervention using a large-scale OligoProcess DNA synthesizer. As a prelude to synthesis, the calculated amount of solid support, loaded with either the 3⬘-terminal nucleoside, or more commonly now, Unylinker [7], is placed into the synthesis column. A technician selects the desired sequence from a list of preprogrammed options, makes the required modifications based on scale, and initiates the synthesis. Following a series of preliminary steps during which the reagent lines are primed and the support

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washed with MeCN to remove residual moisture, synthesis begins according to Scheme 14.1 with removal of the 4,4⬘-dimethoxytrityl group (DMT) from the support-bound nucleoside or Unylinker molecule. This is accomplished using a 10% v/v solution of DCA in toluene. It has long been recognized that the length of the detritylation reaction is important in determining crude oligonucleotide purity and yield. Detritylation times that are too short result in incomplete deprotection, which may ultimately lead to an increase in the amount of oligonucleotides that lack mainly a single nucleotide (the so-called n-1 impurity) [53–55]. Overexposure to acid results in depurination. It should be noted that the 2⬘-oxygen atom renders the MOE purine residues essentially stable toward depurination; hence, depurination in MOE gapmers is restricted to 2⬘-deoxyadenosine and 2⬘-deoxyguanosine residues in the gap. The extent of depurination depends on the purine, 2⬘-deoxyadenosine versus 2⬘-deoxyguanosine, and its location within the gap. We are not aware of any studies that directly compare the susceptibility of N 6-benzoyl-2⬘-deoxyadenosine (dABz) and N 2-isobutyryl-2⬘-deoxyguanosine (dG ib) residues toward DCA-catalyzed depurination in toluene, but in methylene chloride dABz depurinates five- to sixfold faster than dG ib [56]. Regarding position dependence, it might be imagined that a residue’s propensity toward depurination would be related to its proximity to the 3⬘ end of the gap. This is because residues located at the 3⬘ end of the gap are exposed to a greater number of acid treatments than those located closer to the 5⬘ side. However, because 3⬘ and 5⬘ phosphate triester groups destabilize the transition state leading to the oxonium ion formed by cleavage of the glycosidic bond, the rate of depurination of any residue is faster during the cycle immediately following its addition than in all subsequent cycles [56]. This effect distributes more evenly the abasic sites within the sequence. It is apparent then that there exists a balance between deprotection and depurination; one would like to maximize the former and minimize the latter. Optimization of the process of detritylation is complicated by several factors. First, the rates of detritylation of the four nucleotides are different and observe the order dA ⬎ dG ⬎ dC ⬎ T. Second, the nascent oligonucleotide adsorbs approximately 2 mmol of DCA per mmol of nucleotide [33]. This means that the amount of acid required to effect detritylation increases as the synthesis proceeds. Unpublished observations from our laboratory suggest that the conditions currently used at Isis represent those minimally required to achieve complete deprotection of the slowest detritylating residue of a 20-mer oligonucleotide [57]. This indicates that more than the minimum amount of acid is used for the fastest detritylating nucleotide. Efforts to establish the minimum acid treatment times for each nucleotide of a given sequence, which should result in the minimum amount of depurination, are underway in our laboratories and in others [58]. Following detritylation, the support is washed with dry MeCN in preparation for coupling. This solvent wash, similar to those that separate the other steps in a cycle, may appear rather trivial at small scale, but it is certainly not so at large scale, where overall synthesis time and solvent consumption are important cost drivers. As noted, packed-bed synthesis, where solvents flow through the support in a chromatographic-like manner, is inherently less solvent consumptive than sparged reactor synthesis. In addition, over the course of the past 10 years we have been able to reduce gradually the total amount of solvent consumed so that in 2006 a large-scale synthesis consumes only 50% of the MeCN it did in 1996. The oligonucleotide is extended through coupling of activated 2⬘-deoxy and 2⬘-MOE phosphoramidites. Both 1H-tetrazole and DCI have been used at Isis for the manufacture of MOE gapmers with similar results, although for a number of reasons (Section 14.2.2.2) we prefer DCI. Since phosphoramidites comprise the majority of the raw materials cost for MOE gapmer synthesis, it is not surprising that much effort has been expended in trying to establish the required minimum excess. Current practice at Isis is to use 1.75 equivalents of phosphoramidite per coupling; at large scale we observe no increase in crude purity or yield by working at higher phosphoramidite excess, and we experience yield reductions if the excess is reduced [59]. Sulfurization follows coupling. In this step, the newly formed phosphite triester is converted to the corresponding phosphorothioate triester (Scheme 14.1, reaction c). The benefits of the PADS-3-picoline reagent system for effecting sulfur transfer were discussed in Section 14.2.1.5.

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Using properly aged PADS solutions [27], we obtain crude 20-mer MOE gapmers that contain less than 2% (P⫽O)1 and undetectable levels of the DMT-phosphonate esters that mark incomplete sulfurization [28]. The final step in each synthetic cycle is capping (Scheme 14.1, reaction d). In this step, any 5⬘-hydroxyl groups that failed to react in the preceding coupling step are apparently converted to acetate esters by simultaneous treatment with N-methylimidazole, pyridine and MeCN (Cap A), and acetic anhydride and MeCN (Cap B). This four-step cycle—detritylate, couple, sulfurize, and cap—is repeated until the desired sequence is assembled. Analysis of crude products (Section 14.3.3.1) indicates that under the conditions described above, average cycle-wise coupling efficiencies approach 97%. For 20-mer oligonucleotides, this translates to an overall synthesis yield of approximately 56% based on the support loading, or 32% based on the amount of phosphoramidite consumed. By using optimized contact times and wash volumes, the time required to complete one cycle has been reduced to between 18 and 22 min, allowing complete assembly of a 20-residue MOE gapmer within 6–7 h. The RP purification strategy employed at Isis relies on separation of the DMT-on full-length product from the capped failure sequences; for this reason, the oligonucleotide is synthesized with its 5⬘-DMT-group intact.

14.2.5.3 Deprotection Upon completion of the synthetic steps of oligonucleotide synthesis, the support-bound products are normally next treated with ammonium hydroxide to effect cleavage from the support and remove base and phosphate triester protecting groups. Treatment of cyanoethyl-protected oligonucleotides in this manner, however, liberates acrylonitrile, which in the presence of ammonium hydroxide can add to the N-3 position of thymine. This results in a process impurity that contains primarily one (2-cyanoethyl)-N 3-thymine (CNET) residue. The extent of CNET formation depends on a variety of factors, e.g., the amount of ammonium hydroxide used, but as a general rule approximately 1% of CNET is formed for every thymine residue present in the parent compound. To overcome this problem, we developed a two-stage deprotection process that first involves treatment of the support-bound oligonucleotide with a 1:1 v/v solution of triethylamine (TEA) in MeCN [60]. Under these conditions, the acrylonitrile liberated is not caused to react with thymine residues and instead is simply washed from the support. Once the cyanoethyl groups have been removed, the oligonucleotide can safely be treated with ammonium hydroxide to cleave the products from the support and deprotect the bases. Following ammonolysis, the crude products are pumped through a filter to remove the solid support into a jacketed stripping tank. The stripping tank is connected to a vacuum pump, which is used to remove ammonium hydroxide from the crude product solution. The ion-pair (IP) HPLC chromatogram of a typical crude MOE gapmer is shown in Figure 14.7. Approximately 75% of the UV absorbance of the crude product is due to two peaks that are attributed mainly to DMT-on parent oligonucleotide. The DMT-on oligonucleotide resolves into two peaks on the basis of the stereochemistry at the final internucleotide linkage. The remaining 25% of the UV absorbance of a crude MOE gapmer is accounted for by shorter oligonucleotides, most of which are formed due to incomplete reaction during each coupling step.

14.2.5.4 Purification The topic of oligonucleotide purification was recently reviewed by Deshmukh [61]. Anion exchange (AX) HPLC, which takes advantage of the negatively charged internucleotide linkages, has been used widely for oligonucleotide purification. Typical purifications involve the use of strong anion exchange resins such as POROS HQ (PE BioSystems), which are eluted with an increasing gradient of sodium chloride at high pH. This technique has been investigated for the analysis of phosphorothioate oligonucleotides by Bergot [62], Agrawal [63], and Srivatsa [64]. Deshmukh et al. demonstrated the large-scale purification (75 g) of a crude 20-mer phosphorothioate using AX-HPLC

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UV absorbance at 260 nm (mAU)

DMT- on parent 200 150

Protecting group debris

100 50

Failure sequences

10

20 Time (min)

Figure 14.7 IP-HPLC UV chromatogram of a crude 20-mer MOE gapmer.

on polymeric chromatographic media (SOURCE 30Q) [65]. AX displacement chromatography using high-molecular-mass dextran sulfate as the displacer was evaluated for the purification of a 24-mer phosphorothioate [66]. More recently, Tugcu et al. reported the purification of a 20-mer phosphorothioate by AX displacement chromatography using low-molecular-weight amaranth as displacer [67]. Deshmukh et al. reviewed the application of self-displacement techniques in AX chromatography to phosphorothioate oligonucleotide purification [68]. AX-HPLC offers several advantages as a method for the purification of oligonucleotides. First, because the feed for purification is often a DMT-off, crude oligonucleotide, the need to perform a final detritylation reaction (vide infra) postpurification is avoided [69].* Second, AX-HPLC is performed at relatively low pressure without the use of organic solvents—features that help reduce capital outlay and the cost of waste disposal. Third, AX-HPLC is able to resolve at least partially, oligonucleotides that contain one phosphate diester linkage [(P⫽O)1] from fully-thioated material [62–64], thus providing a means of increasing the phosphorothioate diester content of the sample. One significant disadvantage associated with AX-HPLC is the requirement to desalt and concentrate the purified product, a task normally accomplished using RP-HPLC or tangential flow filtration. Another serious drawback is that at least in our hands, the recovery of full-length product is substantially less than 100%. It is for these last two reasons that we prefer to purify MOE gapmers by RP HPLC. RP-HPLC purification of DMT-on oligonucleotides utilizes the hydrophobic nature of the DMT group as a handle for purification. Resolution between DMT-on and off sequences is easily achieved, permitting quantitative removal of capped failure sequences from the crude product. Silicate or organic polymer C18 derivatized stationary phases and weakly buffered sodium or ammonium acetate mobile phases containing methanol or MeCN are normally used. The use of sodium acetate permits isolation of the desired sodium salt of the drug substance without a subsequent salt-exchange step, while methanol is preferred at large scale for economic reasons. Typically, an aqueous solution of crude DMT-on product is loaded onto the column at low mobile-phase organic content. The organic content of the mobile phase is then increased to elute the DMT-off failure sequences and protect group debris, e.g., benzamide, before being stepped up a second time to elute the DMT-on material. The recovery of full-length product in this process approaches 100%. The IP liquid chromatogram of an RP-HPLC-purified MOE gapmer is shown in Figure 14.8. Comparison of the chromatograms shown in Figures 14.7 and 14.8 illustrates how effectively the purification process removes sequences that do not possess a DMT group. A typical MOE gapmer has a UV purity of approximately 95% under these conditions. *

AX-HPLC, as well as RP-HPLC, has also been used to purify, then detritylate on-column, DMT-on oligonucleotides.

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200

100

20

10 Time (min) Figure 14.8 IP-HPLC UV chromatogram of RP-HPLC-purified 20-mer MOE gapmer.

14.2.5.5 Final Detritylation The final chemical transformation in MOE gapmer synthesis is removal of the DMT group from the 5⬘ terminus of the purified product. This commonly performed reaction is seldom given much consideration at small scale, where the objective is often simply quick and complete deprotection. As a consequence, most published procedures [70] for detritylation are rather general in nature, and often do not consider, for example, the effects of pH, oligonucleotide concentration, solvent composition, and sequence on detritylation kinetics. At large scale, however, it is important to establish a rugged, practical detritylation process that results in complete deprotection with minimal concurrent depurination. Initial efforts aimed at developing such a process focused on direct acidification with acetic acid of the HPLC fraction obtained from purification [71]. Although operationally simple, the presence of NaOAc in the HPLC fraction meant that large volumes of acetic acid, ca. 25–30% of the final reaction volume, were required to lower the pH to 3.5. In addition, both NaOAc and methanol slow the reaction, making extended reaction times at pH 3.5 necessary. Partial precipitation during acetic acid addition further limited the usefulness of this procedure. To eliminate these issues, a precipitation step was inserted between the purification and detritylation steps. Precipitation at large scale is accomplished at room temperature by addition of the HPLC fraction to ethanol (see Section 14.2.5.6). The resulting precipitate is reconstituted in water. Because this solution is essentially free from NaOAc, much less acetic acid is required to lower the pH. Additionally, because salt and methanol have been removed, the rate of detritylation at any given pH is increased. With a process for removing salt and methanol in place, it remained necessary to develop an understanding of the influence of pH, concentration, and sequence on reaction kinetics. These factors and others were considered by Krotz et al. [71] who established pH versus rate profiles for a variety of phosphorothioate oligonucleotides. These workers also determined the relationship between pH and oligonucleotide concentration at fixed acetic acid concentrations and demonstrated that detritylation rates depend on the identity of the 5⬘ nucleoside, with the order of detritylation rates being dG ⬎ dA ⬎ dC ⬎ T. These results were used to establish a rugged detritylation process for MOE gapmers, which is described as follows. The first task is to establish at small scale a half-life time (t1/2) at fixed pH, temperature, and oligonucleotide concentration. The extent of deprotection is conveniently followed by HPLC. The t1/2 for most oligonucleotides is 5–10 min at pH 3.5. Once the t1/2 has been determined at small scale, the remaining solution of oligonucleotide is adjusted to the same pH with acetic acid and the reaction allowed to proceed at the same temperature for a total of 15 half-life times, which is sufficient to reduce DMT-on levels to much less than 0.1%. For oligonucleotides that contain 40% 2⬘-deoxypurine residues, this results in the formation of approximately 1% of molecules that contain an abasic site. Selecting a pH that results in a t1/2 of

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5–10 min ensures the reaction proceeds quickly enough so that it is complete within a reasonable period, but slowly enough to protect against overreaction and increased depurination. Once the reaction is complete, the products are neutralized by addition of NaOH.

14.2.5.6 Precipitation Following neutralization, the fully deprotected MOE gapmer is precipitated by addition of NaOAc and ethanol. The precipitation step as it is performed at Isis has evolved considerably over the course of the past 15 years. For the earliest syntheses, the process was little more than a direct scale-up of the classical small-scale precipitations that are used to isolate cellular DNA. In the original process, drug substance was precipitated by addition of NaOAc and ethanol at –20°C and the product collected in polypropylene bottles by centrifugation at 15,000g using a fixed-rotor, high-speed centrifuge. This process was capable of yielding approximately 30 g of material per run. The process was scaled from here by replacing the bottles and fixed rotor centrifuge with a continuous-flow high-speed centrifuge. In this setup, the cold suspension was pumped into a titanium and stainless steel Carr Powerfuge continuous-flow centrifuge operating at 15,000g. The product was removed from the supernatant and deposited on baffles inside the centrifuge. Using this equipment, the amount of oligonucleotide that could be isolated from a single run was increased to 250 g. Although possible technically, scaling up this precipitation and isolation process would have been expensive. For example, we estimated it would have cost ca. $1.5 M (1999 currency rates) to fabricate a continuous-flow high-speed centrifuge capable of isolating 1 kg of oligonucleotide per run, which represented only a fourfold increase in scale over the then current process. In addition to the isolation issue, we had by this time scaled our synthesis and purification processes to the extent that the task of cooling sufficient ethanol for precipitation to near –20°C was both time consuming and expensive. Fortunately, the solution to both problems presented itself with the observation that provided a minimum oligonucleotide concentration was met, the product could be precipitated quantitatively by addition of an aqueous solution of detritylated oligonucleotide to an agitated mixture of ethanol and NaOAc at room temperature. By carefully controlling the oligonucleotide concentration, its rate of addition, the amount of NaOAc, and the mixing speed, the product could be persuaded to form relatively dense agglomerates, which could be isolated by low-speed (⬍ 2000g) centrifugation. Implementation of room-temperature precipitation and the installation of a low-speed centrifuge allowed us to scale the isolation process to 2 kg/run. Despite the fact that this third-generation, room-temperature precipitation–low-speed centrifugation process was used successfully to isolate a variety of phosphorothioate oligonucleotides, there remained a desire to move away from centrifugation altogether. From our room-temperature precipitation studies, we had observed that the density of the precipitate was determined by temperature and the volume of ethanol used. We reasoned that if the precipitate could be made sufficiently dense, it might prove possible to isolate it simply by allowing it to sink to the bottom of the tank. In fact, this proves to be the case; upon addition to less than 3.5 volumes of ethanol at room temperature, the product precipitates essentially as one mass that collects at the bottom of the tank. The supernatant is then conveniently removed via a dip-tube. This sedimentation process has replaced low-speed centrifugation as our preferred method for precipitating MOE gapmers both from RP-HPLC fractions and following detritylation. In addition to concentrating the product in preparation for freeze-drying (Section 14.2.5.7), the post detritylation precipitation step performs the important function of removing 4,4⬘-dimethoxytritanol and sodium acetate, the latter formed by neutralization of acetic acid with NaOH, from the product. 4,4⬘-Dimethoxytritanol and NaOAc remain in the ethanolic supernatant and are removed by decantation as described above. To ensure complete removal of the former and reduction of the latter to acceptable levels, the sediment is redissolved in water and the precipitation and decantation processes repeated. Following this second “polishing” precipitation, a slight vacuum is applied to the tank to remove most of the ethanol that is displaced from the sediment as it settles under its own weight. The sediment is then dissolved in water in preparation for freeze-drying.

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14.2.5.7 Freeze-Drying The aqueous solution of MOE gapmer, which contains sufficient ethanol to render it bactericidal and fungicidal, is filtered into disposable trays, which are then transferred to the freeze drier. The trays and their contents are cooled to –50°C and a vacuum applied. Following the primary drying step, the tray temperature is increased and a secondary drying step performed. This is sufficient to reduce the water content of the drug substance to between 3% and 5%. Following completion of the lyophilization process, the drug substance is transferred from the trays into high-density polyethylene bottles and stored at –20°C to await release testing and conversion to drug product.

14.2.5.8 Yield and Purity Typically, between 3.4 and 3.7 g of “as-is” 20-mer MOE gapmer oligonucleotide per millimole of support is isolated following lyophilization. For a 600-mmol synthesis, this translates into an as-is weight of between 2.0 and 2.2 kg. The as-is weight includes between 3% and 5% residual moisture, between 0.5% and 1% ethanol, and less than 0.3% NaOAc. The purity of the isolated drug substance is determined by a selective and sensitive IP HPLC method that utilizes both UV and mass spectrometry (MS) detectors (Section 14.3.4.2). A typical 20-mer MOE gapmer has a purity of 90–92% when analyzed by this methodology. These values may be used to convert the as-is weight into a weight of pure, anhydrous, solvent- and salt-free drug substance; typically this results in yields of between 2.9 and 3.3 g/mmol of solid support, or between 1.7 and 2.0 kg from a 600-mmol synthesis.

14.3 ANALYTICAL PROCESSES Analytical testing provides data that are used to help assess the quality of drug substances. Analytical chemistry also provides important information that helps guide manufacturing process improvement and formulation development. Starting materials, reagents, drug substance intermediates, and the drug substance itself are all often tested for their suitability for use. Careful selection of methods and testing points leads to an understanding of those attributes that are critical to product quality. Once appropriate tests and limits have been established, one can state with a certain degree of confidence that any materials that pass specification at each of these points will result in product of the desired quality. In this way, analytical testing actually helps build quality into the manufacturing process. In addition to these so-called release tests, it is important to understand the stability of drug substances and products so that appropriate storage conditions and retest or expiry periods are chosen. Release and stability indicating analytical methods useful for the control of MOE gapmers are discussed in the following sections. Because analytical data are so critical to the drug development process, it is vital that one understands the characteristics of the methods used to acquire them. This is the process of analytical method validation, where experiments designed to assess the accuracy, precision, selectivity, sensitivity, and robustness of analytical methods are performed. These characteristics have been established for all the methods used to analyze MOE gapmer starting materials, intermediates, and drug substances. Some of the results obtained in these studies are discussed below. 14.3.1 Starting Materials

14.3.1.1 Phosphoramidites The phosphoramidites used to manufacture MOE gapmers are shown in Figure 14.1. In Section 14.2.1.3, we introduced the concept of critical and noncritical phosphoramidite impurities, defining them, respectively, as impurities that do and do not result in the formation of process impurities in the drug substance. We discussed the necessity of being able to detect and control low

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UV absorbance at 260 nm (mAU)

420

O HN N O

N

O

O

30 O N

P

O O

CN

8

10

10

20

30

Time (min) Figure 14.9 RP-HPLC UV chromatogram of MOE C phosphoramidite 8.

levels of critical impurities, arguing that the linear nature of solid-phase DNA synthesis acts to magnify the effect of these components. We also acknowledged that noncritical impurities can be tolerated at much higher levels. From this discussion, it is apparent that a useful analytical method for phosphoramidites would be able to identify and differentiate critical impurities, which might be present at low levels, from noncritical ones. To accomplish this, we developed a novel RP-HPLC method with ultraviolet (UV) and MS detection. Other groups have reported the use of MS for the identification of phosphoramidites [72,73] and their impurities [74], but we are unaware of any reports concerning the use of HPLCMS for this purpose. The UV chromatogram of a typical batch of MOE C phosphoramidite (8) that was obtained using this method is shown in Figure 14.9. The UV chromatogram shows the presence of two main peaks (Rt ⫽ 24.5 and 26.0 min) that are attributed to the two diastereoisomers of 8. Also present are a number of impurities that may be detected and quantitated by their UV absorbance. After passing through the UV detector, the analyte is directed into an electrospray ionization mass spectrometer, which enables one to determine the mass of any of the UV peaks. Satisfactory mass spectra are usually obtained for species present at approximately 0.01% and above the concentration of the parent phosphoramidite. The mass spectral dimension of the analysis enables the analyst to determine an impurity’s identity and from it, its critical or noncritical nature. In our opinion, the mass spectral dimension is indispensable in this regard as the number of impurities and their diastereoisomeric nature (some critical impurities contain two P atoms and appear as four resolvable peaks) makes it impossible to rely solely on relative retention times for identification. In addition to confirming the identities of previously observed impurities, the mass spectrometer permits one to begin identifying new impurities and facilitates comparisons between different starting material suppliers and routes of synthesis. This is especially important during the development phase when quality agreements with suppliers have not yet been established and when synthetic routes and lot sizes are not fixed. The accuracy of the method was evaluated by determining its ability to recover various amounts of unambiguously synthesized critical and noncritical impurities in the presence of the parent phosphoramidite. In all cases, straight lines (R2 ⬎ 0.995) with negligible y-intercepts and slopes close to unity were obtained. Limits of quantitation (LOQ) for all impurities were ⬍ 0.05% of the parent phosphoramidite.

14.3.1.2 Phenylacetyl Disulfide Given its structural simplicity and its straightforward route of preparation, it is not surprising that PADS is somewhat easier to analyze and control than a nucleoside phosphoramidite. We have

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developed an isocratic RP-HPLC method for this task. The method utilizes a C8 column eluted with a mixture of MeCN and water and is accurate, precise, and sufficiently selective to be able to resolve all major impurities, e.g., both phenylacetyl monosulfide and trisulfide are baseline resolved from PADS. 14.3.2 Reagents and Solvents Most of the reagents and solvents used in the manufacture of MOE gapmers are widely available articles of commerce that can be obtained from large established suppliers. Often, these materials are accepted on certificates of analysis and only identity testing is performed on receipt. USP identity tests are available for many of these chemicals; the identity of others can be established by simple techniques such as infrared spectroscopy. When assay tests are required, simple titration or gas chromatography (GC) techniques are employed. Where appropriate, water content is determined by Karl Fischer titrimetry. 14.3.3 Drug Substance Intermediates

14.3.3.1 Analysis of Crude Samples At Isis, analytical testing is performed at three intermediate stages en route to the final drug substance. The first of these tests is performed on the crude MOE gapmer obtained following ammonolysis. Analysis is conducted by IP HPLC with UV and MS detection (IP-HPLC-UV-MS); a typical UV chromatogram was shown in Figure 14.7. The IP-HPLC system resolves, baseline to baseline, DMT-off phosphorothioate oligonucleotides that differ in length by a single nucleotide up to the nonadecamer level. As mentioned, the crude material consists of two DMT-on peaks and some earlier eluting DMT-off impurities, which are present mainly due to incomplete coupling. The relative amount of DMT-on material and the levels of the individual failure sequences reflect the coupling efficiency in each cycle. Coupling efficiency has a direct impact on the final yield of drug substance, but because the subsequent RP-HPLC purification method is adept at removing DMT-off failures, it has little influence on drug substance purity. In contrast, DMT-on process impurities that cannot be removed by RP-HPLC purification will almost certainly be carried through to the drug substance. Many of these species cannot be resolved chromatographically from the DMT parent oligonucleotide using IP or other HPLC techniques. The MS dimension of the crude analysis allows one to detect and quantitate coeluting components based on differences in mass rather than retention time. Because the details of this part of the method are very similar to those of the IP-HPLC-UV-MS method used for analysis of the drug substance, we will put off a detailed discussion until Section 14.3.3.4. For the moment, suffice it to say that by inspecting the mass spectrum of the DMT-on peaks of the crude MOE gapmer, the analyst is able to predict with some certainty levels of various process impurities in the drug substance.

14.3.3.2 Analysis of Purified Samples A second intermediate check is performed following RP-HPLC purification of the crude MOE gapmer. This is accomplished using the same IP-HPLC method used to analyze the crude product. For crude product, it was important to determine levels of DMT-on impurities that could not be removed by purification because these species impact directly the purity of the drug substance. As noted, this is accomplished using MS detection. Because these impurities are not removed by purification, however, it is not required that their levels be determined a second time in purified samples. For this reason, only the UV portion of the IP-HPLC analysis is performed on the purified samples. The results from this analysis are used to confirm that the purification process functioned to remove all DMT-off failures from the crude material. The IP-HPLC-UV trace of a typical purified DMT-on

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MOE gapmer was shown in Figure 14.8. It will be noted that not all of the early eluting impurities present in crude material are removed by purification and that consequently the UV purity of RP-HPLC-purified MOE gapmers is somewhat less than 100%. The earlier eluting impurities remaining after purification consist of DMT-on sequences that lack nucleotides from the 3⬘ end of the molecule. These components form due to ammonium hydroxide–mediated cleavage of depurinated full-length material that arises during solid-phase synthesis.

14.3.3.3 Analysis of Detritylated Samples The final chemical transformation in MOE-gapmer synthesis is removal of the 5⬘-DMT group that proved so useful in the preceding purification step. The completeness of this reaction is determined by a simple RP-HPLC method. 14.3.4 Drug Substance The results from analytical testing of any drug substance, at least under currently accepted paradigms, constitute an important part of the information that is used to determine whether or not the material is fit for use. The types of tests performed vary to some degree based on the source, nature, and intended use of the drug substance, but normally include at the minimum, tests for identification and assay of the parent compound and for quantitation of related impurities. Other commonly performed tests might include analyses for residual water and solvents and determination of inorganic salts and heavy metals. The methods that have been developed at Isis for identity testing and for assay and impurity profiling of MOE gapmer drug substances are discussed in detail in the following sections.

14.3.4.1 Identity Testing Identity testing seeks to establish the identity of a sample. To be useful, an identity test must be able to discriminate between closely related structures, i.e., it must be specific for the analyte in question. In some cases, it may not be possible to establish identity through a single test; in these instances appropriate additional analyses are performed so that the combination of tests is specific. Determining the identity of a 20-mer MOE gapmer is not trivial. Traditional approaches that work for small molecules, such as comparing the HPLC retention time of a sample to that of a standard of known identity, are not specific. Mass spectrometry cannot distinguish between oligonucleotides of the same base composition but different sequence. In fact, for many sequences, even extremely accurate mass measurements provide no guarantee of base composition, or, for that matter, oligonucleotide length [75]. Oligonucleotide sequencing establishes both the base composition and the order in which the bases are arranged. Phosphorothioate oligonucleotides can be sequenced in a number of ways that include desulfurization followed by controlled enzymatic digestion and matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS analysis [76], failure sequence analysis [77], and MS fragmentation [78]. We currently favor the latter technique. In our method, infrared multiphoton decomposition (IRMPD) at various laser powers is used to fragment the MOE gapmer and the resulting fragment ions detected by Fourier transform–ion cyclotron resonance mass spectrometry (FTICR-MS) [79]. Fragment ions for the sequence under consideration are calculated based on known fragmentation patterns of oligonucleotides [80] and the mass spectra checked for the presence of these species. Sufficient fragments from the 5⬘ and 3⬘ termini are identified to permit unambiguous reconstruction of the full nucleotide sequence.

14.3.4.2 Impurity Tests and Assay Impurity tests are intended to establish levels of impurities in the sample. Testing can be quantitative or limiting in nature. Assay tests seek to provide a quantitative measure of the main

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component of the drug substance, or for drug products, a determination of the percent label claim. The methods used for these purposes should be accurate, precise, specific, sensitive, and robust. Phosphorothioate oligonucleotide drug substance and product purities may be determined by a variety of electrophoretic, chromatographic, and spectroscopic techniques. At Isis, early lengthbased separations of clinical trials material were accomplished using denaturing polyacrylamide gel electrophoresis (PAGE) with quantitation by UV densitometry. The 1980s saw the introduction of capillary gel electrophoresis (CGE) methods capable of resolving nucleic acids of differing lengths. The technique was adapted for the analysis of phosphorothioate oligonucleotides by increasing the theoretical plate count of the gel-filled capillary [81,82]. By including an internal standard, Srivatsa et al. showed that CGE could be used to determine assay in addition to impurity profile [83]. The CGE method developed at Isis provides single nucleotide resolution for 20-mer 2⬘-deoxy phosphorothioate oligonucleotides. Phosphorothioate diester oligonucleotides normally contain low levels of full-length molecules that contain a phosphate diester linkage in place of a phosphorothioate diester linkage. Often termed (P⫽O)1, this component is not resolved from the fully thioated oligonucleotide by CGE. However, resolution can be achieved by strong anion exchange (SAX) chromatography, with (P⫽O)1 eluting earlier than the uniform phosphorothioate diester [62–64]. The ratio of phosphorothioate diester to phosphate diester linkages may also be estimated using 31P NMR spectroscopy [84], by integrating the signals at δ ⫽ 56 and 0 ppm, respectively. Notice that 31P NMR measures only the ratio of the two types of linkage and on its own provides no information about sample purity. Additionally, because 31P NMR measures the ratio of linkage types, it is much less sensitive to changes in (P⫽O)1 content than SAX HPLC. For example, using SAX HPLC it is relatively straightforward to determine the 1% difference in UV peaks that exists between 20-mer phosphorothioate oligonucleotides that contain 2% and 3% (P⫽O)1. It is more difficult to determine the approximately 0.05% difference between these two samples that is detected by 31P NMR. Prior to 2000, Isis determined assays and impurity profiles of phosphorothioate oligonucleotides intended for clinical use by a combination of CGE for separation of length-based impurities, and SAX HPLC for control of (P⫽O)1. This combination approach suffered from several drawbacks. First, it required every sample be analyzed by two separate methods. Running each sample twice adds cost, especially in the QC environment where each method has associated with it standards that must be prepared and system suitability checks that must be performed, and each instrument requires calibration and maintenance. Additionally, there are several characteristics of CGE that make it less than ideal for its stated purpose. First, limits of detection (LOD) values are relatively high, being on the order of 0.5%. Second, it is less robust than one might like. Third, it provides little, if any structural information. Although SAX HPLC is certainly more sensitive and robust than CGE, it too provides very little structural information. Finally and perhaps most seriously, even together CGE and SAX HPLC cannot be used to resolve and quantitate many of the process impurities and degradation products that are present in MOE gapmers. HPLC-MS is a powerful tool for the analysis of singly and doubly stranded oligonucleotides. Of the two commonly used ionization techniques suitable for oligonucleotide analysis, i.e., MALDI and electrospray ionization (ES), the ability of the latter to produce ions directly from a flowing solution makes it most suited to chromatographic hyphenation. Both RP and IP mobile phases are compatible with ES-MS, although the increased resolving power of IP chromatography makes it the preferred separation mode for HPLC-MS of oligonucleotides. For the analysis of oligonucleotides by IP-HPLC, a hydrophobic stationary phase is used in combination with an aqueous-organic mobile phase containing an IP reagent, which consists of an amphiphilic cation and a small hydrophilic counterion. It is believed that amphiphilic cations are adsorbed at the interface between the liquid and stationary phases, forming a positively charged layer. Interaction of the negatively charged phosphodiester backbone with this layer provides a means of resolving oligonucleotides on the basis of length. Under optimal conditions, single nucleotide resolution of oligonucleotides that contain up to at least 80 nucleotides is achievable.

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It is accepted, however, that IP mobile phases optimized for chromatographic separation are not ideal for ES-MS. For example, although 100 mM triethylammonium acetate (TEAA) permits excellent length-based separations, its use in ES-MS results in a dramatic reduction in signal intensity [85,86]. Reasoning that preferential evaporation of triethylamine from the electrospray droplet, which would result in a drop in pH, was at least partly responsible for signal reduction, Apffel developed a 400-mM hexafluoroisopropanol-triethylamine (HFIP-TEA) IP system [86], which displayed resolving power comparable to 100 mM TEAA. When sprayed, however, the acidic component, HFIP (boiling point of 57°C), evaporates preferentially from the droplet, leading to a higher surface pH and increased signal intensity. The improvement in signal intensity was sufficient to permit Gilar to detect minor process impurities, e.g., depurinated products, in synthetic oligonucleotides [87]. In another approach aimed at improving signal intensity while maintaining chromatographic separation, Huber [88] demonstrated that postchromatographic addition of MeCN to the column eluent increased signal intensity; this effect was attributed to a decrease in the surface tension of the electrospray droplet caused by addition of an organic solvent. These and other advances, which are summarized in an excellent review of the topic [89], have helped establish IP-HPLC-ES-MS as the method of choice for in-depth analysis of oligonucleotides. In recent years, this methodology has been used for, among other things, analysis of phosphorothioate oligonucleotides [90,91] and their metabolites [92–95], and for analysis of the ocular metabolites of siRNA duplexes [96]. The lack of specificity inherent in the combination of CGE and SAX HPLC, coupled with a growing frustration with, at least in our hands, the lack of robustness of CGE, prompted us in 2000 to examine IP-HPLC-ES-MS as a method for the routine analysis of phosphorothioate oligonucleotides. Having used IP-HPLC-ES-MS in various guises as a method for identifying phosphorothioate metabolites [92–94], we felt confident that the technique could be adapted for use in the QC laboratory. To meet this goal, however, several important issues would need to be addressed, the most pressing of which was how to use the methodology not only to detect but also to quantitate impurities. We imagined one solution would involve extracting ions due to the parent oligonucleotide and any process impurities present from the total ion current detected by the mass spectrometer. The resulting ion chromatograms, one for each ion extracted, could then be integrated and the relative amounts of each component determined. Although conceptually simple, there are several technical challenges associated with this approach. First, under ES ionization conditions, the MS signal due to an oligonucleotide is distributed across several different charge states. As a consequence, quantitation of the parent compound and any impurities might require the analyst to identify and extract several different ions due to the same component; as the number of components increased such an approach would quickly become unwieldy. We realized that for the method to be practical in a QC environment, quantitation would need to be restricted to only a single charge state. To do this without a substantial reduction in sensitivity required development of conditions that favored one charge state over all others. Charge-state distribution in ES is influenced by factors such as solution pH and the concentration and identity of the IP reagent [89]. With this is mind, several trialkyl ammonium buffers were evaluated for their influence on the charge state distribution of a 20-mer phosphorothioate oligonucleotide. The results from these studies indicated that by using 5 mM tributylammonium acetate (TBuAA) in water–acetontrile mixtures, we could force between 70% and 80% of the parent compound into the –4 charge state [97]. TBuAA is also a more effective IP reagent than, for example TEAA, providing better separation and retention of the oligonucleotide. The benefits of this are twofold: first, good separations can be achieved using low concentrations of IP reagent and second, high concentrations of MeCN are required to elute the sample from the column. The low IP reagent concentration and high organic content of the electrospray feed combine to increase ionization efficiency. A second issue associated with using ion currents for quantitation is that oligonucleotides of different lengths ionize with different efficiencies, i.e., short oligonucleotides ionize more readily than long oligonucleotides. Unless the relative ionization efficiencies are known a priori, one risks overestimating the amount of truncated products and underestimating the amount of longer

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components present in the sample. To avoid having to establish response factors for components of different lengths, we decided to restrict the use of MS quantitation to those components that are not resolved chromatographically from the parent compound, and to use UV detection to quantitate the remainder. Under our IP-HPLC conditions, the main UV peak of 20 and 21-mer 2⬘-deoxy phosphorothioate and MOE gapmers contains oligonucleotides that are between one nucleotide shorter and one nucleotide longer than the parent compound. We have demonstrated through recovery experiments (vide infra) that the ionization efficiencies of these, the smallest and largest components that are quantitated by MS, are essentially the same as the parent oligonucleotide. A third challenge inherent to using MS for quantitation is the phenomenon of ion suppression [98], an effect manifested by a plateauing of the MS signal with increasing analyte load. Ion suppression results from the fact that at high analyte concentrations, there is insufficient space on a single electrospray droplet to accommodate in a linear fashion more and more molecules for ionization. To illustrate this effect, the response due to the full-length, fully thioated parent component of a MOE gapmer is plotted against column load in Figure 14.10. Why is ion suppression important? First, impurities are present at much lower levels than the parent oligonucleotide. Second, with the exception of the (P⫽O)1 component, impurities are partially resolved chromatographically from the parent component. These factors mean that the amount of analyte entering the mass spectrometer is lower for the impurities than for the parent and (P⫽O)1 components, which in turn means the impurities experience less ion suppression. Unless this effect is corrected for, any determinations of low-level components based on a comparison of ion current areas will tend to be overestimates. Of course, the nonlinearity of a response does not de facto preclude its use for determining analyte levels. Instead, all that is required is that the relationship between analyte concentration and response be understood. In the present case, we quickly realized that the change in MS response with increasing oligonucleotide concentration could be described by a quadratic equation, which could then be used to interpret the MS responses due to each component of the sample accurately. The procedure is as follows. Various volumes of a standard solution of oligonucleotide are injected and the MS responses due to the parent and (P⫽O)1 components in each injections integrated. The summed responses, parent plus (P⫽O)1, for each injection are plotted against injection volume, and the resulting points fit by a second-order polynomial equation. The resulting equation is used to interpret the MS responses due to components in the sample. These values, which are in effect linearized MS responses, are compared to each other to determine the relative amounts of each component present. As a result of these method development efforts, we have been able to obsolete our CGE-SAX combination in favor of IP-HPLC-ES-MS for QC analysis of all 2⬘-deoxyphosphorothioate oligonucleotides and MOE gapmers intended for toxicology studies and human clinical trials use [99]. The method is used for release and stability testing of drug substances and products. A related method is used for in-process testing of crude drug substance (Section 14.3.3.1). Assay and purity analysis of a batch of MOE gapmer drug substance follows the route described below.

EIC Area (x10−7)

4.0 3.0 2.0 1.0 0.0 0.0

0.5

1.0 1.5 2.0 Amount on column (µg)

2.5

Figure 14.10 Relationship between MS response and column load for a MOE gapmer.

3.0

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UV absorbance at 260 nm (mAU)

426

250

8

4

150 10

18

14

50

10

20 Time (min)

Figure 14.11 Full-scale and expanded view of IP-HPLC-UV chromatogram of a 20-mer MOE gapmer.

An aqueous solution of drug substance is chromatographed on a C18 column held at 50°C and eluted with a gradient of MeCN in aqueous 5 mM TBuAA. A typical UV chromatogram is shown in Figure 14.11. Inspection of the UV chromatogram (expanded view) reveals the presence of some earlier eluting components, which are quantitated using UV detection. These components lack nucleotides from the 3⬘ or 5⬘ends of the parent oligonucleotide and terminate either in hydroxyl groups at both ends and or in a hydroxyl group at one end and a phosphorothioate monoester at the other. In addition, 3⬘ truncated sequences that end in a 2-deoxyribose moiety are also present. Also evident from Figure 14.11 are a group of later-eluting impurities. These components, which are also quantitated by UV detection, include high-molecular-weight impurities of the types described in reference [6]. Integration of the UV chromatogram in the manner shown establishes a UV purity for the sample; the sample shown in Figure 14.11 has a UV purity of 95.2%. After passing through the UV detector, the eluent is fed directly into a single-quadrupole, ES ionization mass spectrometer. The mass window is set to extend from 150 amu below to 150 amu above the calculated most abundant mass of the –4 charge state of the parent oligonucleotide, a range that is sufficient to permit detection of all components that elute within the main UV peak. The expanded average mass spectrum of the main UV peak of the material shown in Figure 14.11 is shown in Figure 14.12. The most striking feature of the average mass spectrum is its exceptionally high quality. The spectrum displays little background noise and only low levels of the noncovalent sodium and potassium adducts that often plague MS analysis of oligonucleotides (the sodium adduct in this spectrum has a relative abundance of 2.5%). The excellent signal-to-noise ratio and the low adduct levels, evidence of the ability of IP-HPLC to remove ubiquitous metal cations, facilitates detection of a number of process impurities that co-elute with the main UV peak. By virtue of our choice of IP conditions, these components are detected as their quadruply charged anions. The major ion at m/z ⫽ 1795.0 (relative abundance ⫽ 100%) corresponds to the –4 charge state of the parent MOE gapmer (calculated most abundant mass ⫽ 7184.1 amu). Also present is a signal at m/z ⫽ 1791.1, consistent with the (P⫽O)1 component of the parent and a series of signals at m/z ⫽ 1690.4, 1694.2, 1696.6, 1708.4, and 1715.0, which can be attributed to oligonucleotides that lack the nucleoside thiophosphates of MOE G, MOE A, MOE MeC (and MOE MeU), dG, dMeC (and T), respectively (the so-called n – 1 components [53–55]). The signal at m/z ⫽ 1718.1 is an oligonucleotide that lacks the 3⬘ terminal nucleoside, in this case MOE A, but retains a 3⬘-phosphorothioate monoester [100]. As expected, the sample also contains some oligonucleotides that have suffered depurination; the signal at m/z ⫽ 1765.8 corresponds to molecules that have lost adenine and added water, that at m/z ⫽ 1761.9 to those that have lost guanine and added water. The signal at m/z ⫽ 1784.0 is due to an oligonucleotide

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1.2

1795.0 Na +-adduct

1784.0 1.0

A

1791.1

Relative abundance

1696.6 0.8 A

1761.9 1765.8

A

A

0.6

0.4

1694.2

1881.6

A

1708.4 1715.0

A

A A

1690.4 1718.1

0.2

1650

1700

1750

1800

1850

1900

m/z Figure 14.12 Expanded average mass spectrum of main UV peak of Figure 14.11.

with a most abundant mass that is 44 amu less than the parent sequence, a mass difference that corresponds to replacement of a 2⬘-MOE residue with the corresponding 2⬘-OMe residue. This process impurity arises due to the presence of 2⬘-OMe phosphoramidite, e.g., 11, in some lots of MOE phosphoramidite. Finally, the signal at m/z ⫽ 1881.6 is attributed to molecules that contain an additional dG thionucleotide. The presence of this particular n ⫹ 1 component is consistent with the relative detritylation rates of the nucleotides under the coupling conditions [101]. The average mass spectrum also contains some buffer adducts (marked A in Figure 14.12) that must be excluded from the subsequent quantitation. In the next step of the analysis, the ion currents due to each of the components present under the main UV peak are extracted; ion currents due to metal and buffer adducts of the parent component are not extracted. The extraction process results in a series of ion chromatograms, one for each ion extracted, which are then integrated. Representative ion chromatograms are shown in Figure 14.13. As noted, because the MS response in not linear, direct comparison of ion current areas leads one to overestimate impurity levels. To avoid this, the responses are linearized using a quadratic curve produced by plotting ion current against injection volumes for a reference standard. Once linearized, the ion current areas of the sample are compared one to another to determine the relative amounts of each component contained within the main UV peak. In the present example, the parent oligonucleotide accounts for 93.5% of the main UV peak, whereas the (P⫽O)1 and n ⫹ pdG components (m/z ⫽ 1791.1 and 1881.6, respectively) are present at 2.6% and 0.25%, respectively. Having determined how much each component contributes to the main UV peak, the analyst multiplies by the UV purity to calculate the contribution of each component to the sample. For example, in the present case we know that the UV purity is 95.2% and that 93.5% of the main UV peak is due to the parent oligonucleotide. The overall sample purity is, therefore, 93.5% ⫻ 0.952, or 89.0%. Similarly, the (P⫽O)1 and n ⫹ pdG species account, respectively, for 2.6% ⫻ 0.952, or 2.5%, and 0.25% ⫻ 0.952, or 0.24% of the sample. The accuracy, precision, robustness, and the limits of detection and quantitation (LOD, LOQ) of this approach have been determined. Accuracy was evaluated by analyzing samples of drug substance to which were added various amounts of authentic impurities. Example recovery plots for (P⫽O)1 and N 3-(2-cyanoethyl)thymine-modified oligonucleotides are shown in Figure 14.14.

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION 800 m/z = 1795.0

Intensity (x10−3)

30

m/z = 1791.1

6

m/z = 1696.6

4

m/z = 1881.6

10

20

10

20

10

20

10

20

Time (min) Figure 14.13 Extracted ion chromatograms for m/z ⫽ 1795.0, 1791.1, 1696.6, and 1881.6.

14

(P=O)1

% Measured

12 10 8 6

y = 0.9139x + 2.3624

4

R 2 = 0.998

2 0 0

2

4

6

8

10

12

% Added

% Measured

5

CNET

4 3

y = 1.2112x + 0.2499 2 R = 0.9987

2 1 0 0

1

2

3

4

% Added Figure 14.14 Recovery plots for (P⫽O)1 and CNET impurities.

Responses for each component were linear (R2 ⱖ 0.998), the slopes of the recovery lines close to unity, and the y-intercepts good approximations of native levels. Similar recovery results were obtained for all other impurities tested, which suggests that the method is capable of determining sample purity accurately.

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Method precision was evaluated by repeated analysis of homogeneous samples of a number of different 2⬘-deoxy phosphorothioate oligonucleotides and MOE gapmers. The combined effect of different analysts and instruments has been determined. Relative standard deviations (RSD) between independent analyses of the same sample were on the order of 0.5% for the parent oligonucleotide and are generally less than 15% for impurities. Robustness was evaluated by making deliberate, small changes to MS parameters, e.g., needle voltage, nebulizer pressure, and drying gas temperature, or chromatography parameters, e.g., column age and temperature; none of these modifications elicited a significant impact on method performance. Finally, LOD and LOQ values were determined for a number of different process impurities using the slopes and standard deviations of the y-intercepts of the recovery lines; typical LOD and LOQ values are 0.1% and 0.2%, respectively. As indicated, in addition to determining impurity profiles we use IP-HPLC-ES-MS to establish drug substance assays and drug product label claims. This is done by comparing the absorbance of the main UV peak of the sample to that of a standard of known concentration. The mass spectrometer is then used to correct for the presence of co-eluting impurities. It is generally accepted that of currently available analytical techniques, IP-HPLC-ES-MS provides the most accurate and complete picture of oligonucleotide purity. Over the past 5 years, we have used this methodology to detect and quantitate a variety of known process impurities of phosphorothioate oligonucleotides, e.g., (P⫽O)1, n ⫺ 1, and n ⫹ 1. The real power of the method, however, lies in its ability to detect, quantitate, and provide structural information on previously unidentified impurities. In some instances, this new knowledge spurred process changes that improved drug substance purity. For example, IP-HPLC-ES-MS analysis of older lots of drug substance revealed they contained substantial amounts, up to 5%, of an impurity with a molecular weight of 53 amu more than the parent compound, which we later discovered contained an N 3-(2-cyanoethyl)thymine residue in place of a thymine residue. We demonstrated that this component, which forms when 2-cyanoethyl-protected oligonucleotides are treated with ammonium hydroxide, could be completely avoided by pretreatment with triethylamine [60]. IP-HPLC-ES-MS not only enabled the discovery of this impurity, but guided the process development effort aimed at its avoidance. In another example, IP-HPLC-ES-MS of some lots of drug substance showed the presence of an impurity with a most abundant mass 147 amu greater than the parent, which we subsequently demonstrated contained the atoms of trichloroacetaldehyde inserted into an internucleotide linkage [35]. This impurity forms due to the presence of low levels of chloral in some lots of dichloroacetic acid (DCA). Here again, IP-HPLC-ES-MS was not only crucial to the discovery of the impurity, but also guided development of an appropriate limit on chloral in DCA. IP-HPLC-ES-MS can also provide useful data to drug product formulators. For example, Krotz et al. showed that impurities in some excipients of a novel topical formulation led to the formation of oligonucleotides containing modified guanine residues [102]. The method has also provided new insights into oligonucleotide degradation profiles. For example, it was discovered that on standing, oligonucleotides react to produce an impurity with a most abundant mass 80 amu larger than the parent oligonucleotide. This unexpected result led to the discovery of a new class of cytosine-modified degradation products [103]. Although the power of IP-HPLC-ES-MS as a method for oligonucleotide analysis is undisputed, the notion that the technique can be used successfully in a QC environment is far less widely accepted. Experience gained at Isis, however, indicates that it is in fact possible to implement such a method, and over the past 5 years we have successfully used IP-HPLC-ES-MS for routine impurity profile and assay analysis of twelve 2⬘-deoxyphosphorothioate and MOE gapmer oligonucleotides at various stages of clinical development. During this time, over 30 analysts have been trained in the method; only a handful of these scientists possessed any prior experience in MS. The methodology has been transferred to Isis collaborators and contract testing laboratories. Finally, in addition to confirming that it is possible to run IP-HPLC-ES-MS in the QC environment, experience gained in the past 5 years has convinced us that at least during the development phase, when the material supply chain and manufacturing processes are evolving, proper control of oligonucleotides, and hence a high level of assurance of patient safety relies heavily on the use of

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highly specific release methods. Of course, as a compound moves through development toward commercialization, the raw material supply chain matures to a point where it is possible to establish quality agreements with suppliers. In addition, the manufacturing process itself is locked and validated. These agreements and activities build quality into the drug substance and relieve some of the burden of release testing. Although we subscribe fully to this approach of quality by design, we believe that given the complexity of oligonucleotide drug substances and the propensity of solid-phase synthesis to magnify the effect of even small changes in the quality of raw materials, IP-HPLC-ES-MS, or other similarly specific analytical methods will continue to be required for release testing of commercial oligonucleotide drug substances and products. 14.4 CONCLUSIONS The large-scale manufacture of oligonucleotides suitable for human clinical use has evolved considerably over the course of the past 15 years. From the earliest syntheses, which were conducted using CPG supports contained in sparged bed reactors and yielded roughly 5 g of material, the process has been scaled up to the extent that 750 mmol syntheses, which provide ca. 2.5 kg of MOE gapmer drug substance, are now routine. The increase in scale resulted chiefly from the development of solid supports and synthesizers that function in a packed-bed approach. At the same time, the cost of goods has decreased nearly as dramatically, so that a gram of MOE gapmer today costs only about 3% of what it did in 1997. Much of this reduction is due to decreases in the cost of phosphoramidites, made possible by the development of optimized routes for the production of protected nucleosides from wholly synthetic precursors. Additional cost savings have been realized through optimization of the processes of solid-phase synthesis, and by reducing phosphoramidite excesses and solvent consumption. Improvements in purification and isolation strategies, for example, the development of a room-temperature precipitation-sedimentation process to replace a more costly, high-speed centrifugation one, have resulted in larger batch sizes and reduced cost. We now understand more completely the structures and routes of formation of many oligonucleotide process impurities. This knowledge has permitted establishment of rational, performancebased specifications for phosphoramidites and other reagents and has led to the development of new processes that avoid or help minimize process impurities, e.g., TEA treatment to avoid CNET formation [60]. As a consequence of these improvements, MOE gapmer drug substances are considerably purer today than they were 10 years ago. Advances in analytical techniques have been realized. The development of sensitive HPLC methods with MS capability has revolutionized the process of phosphoramidite analysis. The combination of CGE and SAX HPLC for assay and impurity-profile determination has been superceded by a vastly more specific IP-HPLC-ESI-MS method that provides, in our opinion, the most accurate assessment of oligonucleotide purity, the most useful development information to the manufacturing and formulation scientists, and the greatest assurance of patient safety. ACKNOWLEDGMENTS The authors acknowledge Dr Lars Holmberg of GE Healthcare, and Max Moore, Chris Coffin, Mark Andrade, Dr Vasulinga Ravikumar, Dr Claus Rentel and Dr Hans Gaus of Isis Pharmaceuticals, Inc. for useful discussions. The authors thank Myrna Lettow for editorial assistance. REFERENCES 1. Beaucage, S.L. and Caruthers, M.H., Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis, Tetrahedron Lett. 22, 1859, 1981. 2. Ravikumar, V.T., personal communication, 2006.

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3. Ravikumar, V.T., 2⬘-deoxynucleosides prices are 8 to 10 times lower in 2006 than they were in 1990, personal communication, 2006 4. Ravikumar, V.T., personal communication, 2006. 5. Gaus, H., New insights into phosphoramidites using HPLC-MS, TIDES, Las Vegas, April 28–May 1, 2003. 6. Kurata, C. et al., Characterization of high molecular weight impurities in synthetic phosphorothioate oligonucleotides, Bioorg. Med. Chem. Lett. 16, 607, 2006. 7. Kumar, R.K. et al., Efficient synthesis of antisense phosphorothioate oligonucleotides using a universal solid support, Tetrahedron 62, 4528, 2006. 8. Pon, R.T. et al., Reusable solid-phase supports for oligonucleotides and antisense therapeutics, J. Chem. Soc. Perkin Trans. 1 2638, 2001. 9. Kamer, P.C.J. et al., An efficient approach toward the synthesis of phosphorothioate diesters via the Schonberg reaction, Tetrahedron Lett. 30, 6757, 1989. 10. Iyer, R.P. et al., 3H-1, 2-Benzodithiole-3-one 1, 1-dioxide as an improved sulfurizing reagent in the solid-phase synthesis of oligodeoxyribonucleoside phosphorothioates, J. Am. Chem. Soc. 112, 1253, 1990. 11. Roelen, H.C.P.F. et al., A study on the use of phenylacetyl disulfide in the solid-phase synthesis of oligodeoxynucleoside phosphorothioates, Recl. Trav. Chim. Pays-Bas 110, 325, 1991. 12. Vu, H. and Hirshbein, B.L., Internucleotide phosphite sulfurization with tetraethylthiuram disulfide. Phosphorothioate oligonucleotide synthesis via phosphoramidite chemistry, Tetrahedron Lett. 32, 3005, 1991. 13. Rao, M.V., Reese, C.B., and Zhengyun, X., Dibenzoyl tetrasulfide – a rapid sulfur transfer agent in the synthesis of phosphorothioate analogues of oligonucleotides, Tetrahedron Lett. 33, 4839, 1992. 14. Arterburn, J.B. and Perry, M.C., Rhenium catalyzed sulfurization of phosphorus (III) compounds with thiiranes: new reagents for phosphorothioate ester synthesis, 38, Tetrahedron Lett. 38, 7701, 1992. 15. Stec, W.J. et al., Bis(O, O-diisopropoxyphosphinothioyl)disulfide – a highly efficient sulfurizing reagent for cost-effective synthesis oligo(nucleoside phosphorothioate)s, Tetrahedron Lett. 34, 5317, 1993. 16. Rao, M.V. and Mcfarlane, K., Solid phase synthesis of phosphorothioate oligonucleotides. Benzyltriethylammonium tetrathiomolybdate as a rapid sulfur transfer reagent, Tetrahedron Lett. 35, 6741, 1994. 17. Efimov, V.A. et al., New efficient sulfurizing reagents for the preparation of oligodeoxyribonucleoside phosphorothioate analogues, Nucleic. Acids. Res. 23, 4029, 1995. 18. Xu, Q. et al., Use of 1, 2, 4-dithiazolidine-3, 5-dione (DtsNH) and 3-ethoxy-1, 2, 4-dithiazolidine-5-one (EDITH) for synthesis of phosphorothioate-containing oligodeoxyribonucleotides, Nucleic Acids Res. 24, 3643, 1996. 19. Zhang, Z. et al., Solid phase synthesis of oligonucleotide phosphorothioate analogues using bis(ethyoxythiocarbonyl)tetrasulfide as a new sulfur transfer reagent, Tetrahedron Lett. 2467, 39, 1998. 20. Cheruvallath, Z.S. et al., Use of phenylacetyl disulfide (PADS) in the synthesis of oligodeoxyribonucleotide phosphorothioates, Nucleos. Nucleot. 18, 485, 1999. 21. Zhang, X. et al., Solid phase synthesis of oligonucleotide phosphorothioate analogues using 3-methyl-1, 2, 4-dithiazolidine-5-one (MEDITH) as a new sulfur transfer reagent, Tetrahedron Lett. 40, 2095, 1999. 22. Tang, J.Y. et al., Large-scale synthesis of oligonucleotide phosphorothioates using 3-amino-1, 2, 4-dithiazole-5-thione as an efficient sulfur transfer reagent, Org. Process Res. Dev. 4, 194, 2000. 23. Ju, J. and McKenna, C.E., Synthesis of oligodeoxyribonucleoside phosphorothioates using Lawesson’s reagent for the sulfur transfer step, Bioorg. Med. Chem. Lett. 12, 1643, 2002. 24. Cheruvallath, Z.S. et al., Solid phase synthesis of phosphorothioate oligonucleotides utilizing diethyldithiocarbonate disulfide (DDD) as an efficient sulfur transfer reagent, Nucleos. Nucleot. Nucleic Acids 22, 629, 2003. 25. Krotz, A.H. et al., Polysulfide reagent in solid-phase synthesis of phosphorothioate oligonucleotides: greater than 99.8% sulfurization efficiency, Nucleos. Nucleot. Nucleic Acids 24, 1293, 2005. 26. Fullerman, D., At the 10-20 kg-scale, Beaucage and PADS can be purchased for approximately $6/g and $0.30/g, respectively, personal communication, 2006. 27. Krotz, A.H. et al., Phosphorothioate oligonucleotides of low phosphate diester content: Greater than 99.9% sulfurization efficiency with “aged” solutions of phenylacetyl disulfide (PADS), Org. Process. Res. Dev. 8, 852, 2004.

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28. Capaldi, D.C. et al., Formation of 4, 4⬘-dimethoxytrityl-C-phosphonate oligonucleotides, Bioorg. Med. Chem. Lett. 14, 4683, 2004. 29. Cheruvallath, Z.S. et al., Synthesis of antisense oligonucleotides: Replacement of 3H-1, 2-benzodithiole3-one 1, 1-dioxide (Beaucage reagent) with phenylacetyl disulfide (PADS) as efficient sulfurization reagent: From bench to bulk manufacture of active pharmaceutical ingredient, Org. Proc. Res. Dev. 4, 199, 2000. 30. Beaucage, S.L. and Iyer, R.P., Advances in the synthesis of oligonucleotides by the phosphoramidite approach, Tetrahedron 48, 2223, 1992 (and references therein). 31. Sekine, M., A new method for removal of modified trityl and pixyl groups by use of an acid species generated by reaction of diethyl oxomalonate and methanol, Nucleos. Nucleot. 13, 1397, 1994. 32. Leonard, N.J. and Neelima, 1,1,1,3,3,3-Hexafluoro-2-propanol for the removal of the 4,4⬘-dimethoxytrityI protecting group from the 5⬘-hydroxyl of acid-sensitive nucleosides and nucleotides, Tetrahedron Lett. 36, 7833, 1995. 33. Krotz, A.H., Cole, D.L., and Ravikumar, V.T., Synthesis of an antisense oligonucleotide targeted against C-raf kinase: Efficient oligonucleotide synthesis without chlorinated solvents, Bioorg. Med. Chem. 7, 435, 1999. 34. Paul, C.H. and Royappa, T., Acid binding and detritylation during oligonucleotide synthesis, Nucleic Acids Res. 24, 3048, 1996. 35. Gaus, H. et al., Trichloroacetaldehyde modified oligonucleotides, Bioorg. Med. Chem. Lett. 15, 4118, 2005. 36. Gryaznov, S.M. and Letsinger, R.L., Synthesis of oligonucleotides via monomers with unprotected bases, J. Am. Chem. Soc. 113, 5876, 1991. 37. Brill, W.K.-D., Nielsen, J., and Caruthers, M.H., Synthesis of deoxydinucleoside phosphorodithioates, J. Am. Chem. Soc. 113, 3972, 1991. 38. Vinayak, R. et al., Advances in the chemical synthesis and purification of RNA, Nucleic Acids Symp. Ser. 33, 123, 1995. 39. Hayakawa, Y., Kataoka, M., and Noyori, R., Benzimidazolium triflate as an efficient promoter for nucleotide synthesis via the phosphoramidite approach, J. Org. Chem. 61, 7996, 1996. 40. Karl, R.M. et al., The 1,1-dianisyl-2,2,2-trichloroethyl moiety as a new protective group for the synthesis of dinucleoside trifluoromethylphosphonates, Nucleosides Nucleotides 15, 379, 1996. 41. Dabkowski, W. et al., Trimethylchlorosilane: a novel activating reagent in nucleotide synthesis via the phosphoramidite route, J. Chem Soc., Chem. Commun. 877, 1997. 42. Vargeese, C. et al., Efficient activation of nucleoside phosphoramidites with 4, 5-dicyanoimidazole during oligonucleotide synthesis, Nucleic Acids Res. 26, 1046, 1998. 43. Wang, Z. et al., Antisense oligonucleotides: Efficient synthesis of 2⬘-O-methoxyethyl phosphorothioate oligonucleotides using 4,5-dicyanoimidazole. Are these oligonucleotides comparable to those synthesized using 1H-tetrazole as coupling activator? Bioorg. Med. Chem. 14, 5049, 2006. 44. Stull, D.R., Fundamentals of Fire and Explosion; AIChE Monograph Series, No. 10, New York, 1977, 73, 22. 45. Capaldi, D.C., unpublished observations, 1997. 46. Moore, M. and Capaldi, D.C., unpublished observations, 2004. 47. Pon, R.T. et al., Prevention of guanine modification and chain cleavage during the solid phase synthesis oligonucleotides using phosphoramidite derivatives, Nucleic Acids Res. 14, 6453, 1986. 48. Holmberg, L., personal communication, 2006. 49. Holmberg, L., Primer support 200: A new and cost efficient support for pilot and process scale synthesis of oligonucleotides, TIDES, Hamburg, November 20, 2002. 50. Isis Pharmaceuticals, Inc. press release, October 2006. 51. Millipore 8800 User’s Guide, Documentation number 8800 OM 2.0, Millipore Corporation, Bedford, 1993, pp. 3–19. 52. Andrade, M., personal communication, 1995. 53. Temsamani, J., Kubert, M., and Agrawal, S., Sequence identity of the n-1 product of a synthetic oligonucleotide, Nucleic Acids Res. 23, 1841, 1995. 54. Fearon, K.L. et al., Investigation of the ‘n–1’ impurity in phosphorothioate oligodeoxynucleotides synthesized by the solid-phase -cyanoethyl phosphoramidite method using stepwise sulfurization, Nucleic Acids Res. 23, 2754, 1995.

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55. Chen, D. et al., Analysis of internal (n-1)mer deletion sequences in synthetic oligodeoxyribonucleotides by hybridization to an immobilized probe array, Nucleic Acids Res. 27, 389, 1999. 56. Septak, M., Kinetic studies on depurination and detritylation of CPG-bound intermediates during oligonucleotide synthesis, Nucleic Acids Res. 24, 3053, 1996. 57. Moore, M., personal communication, 2000. 58. McCormac, P., The difference between scaling up and making bigger – more than just contact time and equivalents, TIDES, La Costa, CA, May 1–3, 2006. 59. Andrade, M., personal communication, 1996. 60. Capaldi, D.C. et al., Synthesis of high-quality antisense drugs. Addition of acrylonitrile to phosphorothioate oligonucleotides: Adduct characterization and avoidance, Org. Process Res. Dev. 7, 832, 2003. 61. Deshmukh, R.R. et al., Large-scale chromatographic purification of oligonucleotides, in Handbook of Bioseparations, vol. 2 (Separation Science and Technology), Ahuja, S., ed., Academic Press, San Diego, 2000, p. 511. 62. Bergot, B.J. and Egan, W., Separation of synthetic phosphorothioate oligodeoxynucleotides from their oxygenated (phosphodiester) defect species by strong-anion-exchange-high-performance liquid chromatography, J. Chromatogr. 599, 35, 1992. 63. Metelev, V. and Agrawal, S., Ion-exchange high-performance liquid chromatography analysis of oligodeoxyribonucleotide phosphorothioates, Anal. Biochem. 200, 342, 1992. 64. Srivatsa, G.S. et al., Selectivity of anion exchange chromatography and capillary gel electrophoresis for the analysis of phosphorothioate oligonucleotides, J. Pharm. Biomed. Anal. 16, 619, 1997. 65. Deshmukh, R.R. et al., A case study: Oligonucleotide purification from gram scale to hundred gram scale, Nucleos. Nucleot. Nucleic Acids 20, 567, 2001. 66. Gerstner, J.A. et al., Gram-scale purification of phosphorothioate oligonucleotides using ion-exchange displacement chromatography, Nucleic Acids Res. 23, 2292, 1995. 67. Tugcu, N. et al., Purification of an oligonucleotide at high column loading by high affinity, lowmolecular-mass displacers, J. Chromatogr. A 923, 65, 2001. 68. Deshmukh, R.R, Leitch II, W.E., and Cole, D.L., Application of sample displacement techniques to the purification of synthetic oligonucleotides and nucleic acids: a mini-review with experimental results, J. Chromatogr. A 806, 77, 1998. 69. Deshmukh, R.R., Cole, D.L., and Sanghvi, Y.S., Purification of antisense oligonucleotides, in Methods in Enzymology, Phillips, M.I., ed., Academic Press, San Diego, 1999, vol. 313, p. 203. 70. Zon, G. and Stec, W.J., Phosphorothioate oligonucleotides, In Oligonucleotides and Analogues, A Practical Approach, Eckstein, F., ed., Oxford University Press, Oxford, 1991, chap. 4. 71. Krotz, A. H. et al., Controlled detritylation of antisense oligonucleotides, Org. Process. Res. Dev. 7, 47, 2003. 72. Kele, Z. et al., Electrospray mass spectrometry of phosphoramidites, a group of acid-labile compounds, J. Mass Spectrom. 34, 1317, 1999. 73. Fujitake, M. et al., Accurate molecular weight measurements of nucleoside phosphoramidites: a suitable matrix of mass spectrometry, Tetrahedron, 61, 4689, 2005. 74. Toren, P.C. et al., Determination of impurities in nucleoside 3⬘-phosphoramidites by fast atom bombardment mass spectrometry, Anal. Biochem. 152, 291, 1986. 75. Keough, T. et al., Detailed characterization of antisense oligonucleotides, Anal. Chem. 68, 3405, 1996. 76. Schuette, J.M. et al., Sequence analysis of phosphorothioate oligonucleotides via matrix-assisted laser desorption ionization time-of-flight mass spectrometry, J. Pharm. Biom. Anal. 13, 1195, 1995. 77. Alazard, D. et al., Sequencing of production-scale synthetic oligonucleotides by enriching for coupling failures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, Anal. Biochem. 301, 57, 2002. 78. Banoub, J.H. et al., Recent developments in mass spectrometry for the characterization of nucleosides, nucleotides, oligonucleotides and nucleic acids, Chem. Rev. 105, 1869, 2005. 79. Sannes-Lowery, K.A. and Hofstadler, S.A., Sequence confirmation of modified oligonucleotides using IRMPD in the external ion reservoir of an electrospray ionization Fourier transform ion cyclotron mass spectrometer, J. Am. Soc. Mass Spectrom. 14, 825, 2003. 80. McLuckey, S.A. et al., Tandem mass spectrometry of small, multiply charged oligonucleotides, J. Am. Soc. Mass Spectrom. 3, 60, 1992.

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81. Cohen, A.S. et al., High-performance liquid chromatography and capillary gel electrophoresis as applied to antisense DNA, J. Chromatogr. A 638, 293, 1993. 82. DeDionisio, L., Capillary gel electrophoresis and the analysis of DNA phosphorothioates, J. Chromatogr. A 652, 101, 1993. 83. Srivatsa, G.S. et al., Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations, J. Chromatogr. A 680, 469, 1994. 84. Hirschbein, B.L. and Fearon, K.L., 31P NMR spectroscopy in oligonucleotide research and development, Antisense Nucleic Acid Drug Dev. 7, 55, 1997. 85. Bleicher, K. and Bayer, E., Analysis of oligonucleotides using coupled high performance liquid chromatography-electrospray mass spectrometry, Chromatographia 39, 405, 1994. 86. Apffel, A. et al., Analysis of oligonucleotides by HPLC-electrospray ionization mass spectrometry, Anal. Chem. 69, 1320, 1997. 87. Gilar, M., Analysis and purification of synthetic oligonucleotides by reversed-phase high-performance liquid chromatography with photodiode array and mass spectrometry detection, Anal. Biochem. 298, 196, 2001. 88. Huber, C.G. and Krajete, A., Analysis of nucleic acids by capillary ion-pair reversed-phase HPLC coupled to negative-ion electrospray ionization mass spectrometry, Anal. Chem. 71, 3730, 1999. 89. Huber, C. G. and Oberacher, H., Analysis of nucleic acids by on-line liquid chromatography-mass spectrometry, Mass Spectrom. Rev. 20, 310, 2001. 90. Bothner, B. et al., Liquid chromatography mass spectrometry of antisense oligonucleotides, Bioorg. Med. Chem. Lett. 5, 2863, 1995. 91. Tengvall, U. et al., Characterization of antisense oligonucleotide-peptide conjugates with negative ionization electrospray mass spectrometry and liquid chromatography-mass spectrometry, J. Pharm. Biomed. Anal. 32, 581, 2003. 92. Gaus, H.J. et al., On-line HPLC electrospray mass spectrometry of phosphorothioate oligonucleotide metabolites, Anal. Chem. 69, 313, 1997. 93. Griffey, R.H. et al., Characterization of oligonucleotide metabolism in vivo via liquid chromatography/ electrospray tandem mass spectrometry with a quadrupole ion trap mass spectrometer, J. Mass Spectrom. 32, 305, 1997. 94. Graham, M.J. et al., Hepatic distribution of a phosphorothioate oligodeoxynucleotide within rodents following intravenous administration, Biochem. Pharmacol. 62, 297, 2001. 95. Dai, G. et al., Characterization and quantification of Bcl-2 antisense G3139 and metabolites in plasma and urine by ion-pair reversed phase HPLC coupled with electrospray ion-trap mass spectrometry, J. Chromatogr. B 825, 201, 2005. 96. Beverly, M. et al., Liquid chromatography electrospray ionization mass spectrometry analysis of the ocular metabolites from a short interfering RNA duplex, J. Chromatogr. B 835, 62, 2006. 97. Gaus, H., personal communication, 1999. 98. Tang, L. and Kebarle, P., Dependence of ion intensity in electrospray mass spectrometry on the concentration of the analytes in the electrosprayed solution, Anal. Chem. 65, 3654, 1993. 99. Rentel, C., Development of an LC-MS Method for Quality Control of Oligonucleotides, TIDES, La Costa, CA, May 1–3, 2006. 100. Ravikumar, V.T. et al., Antisense phosphorothioate oligodeoxyribonucleotide targeted against ICAM-1: synthetic and biological characterization of a process-related impurity formed during oligonucleotide synthesis, Bioorg. Med. Chem. 11, 4673, 2003. 101. Krotz, A.H. et al., On the formation of longmers in phosphorothioate oligodeoxyribonucleotide synthesis, Tetrahedron Lett. 38, 3875, 1997. 102. Krotz, A.H., Gaus, H., and Hardee, G.E., Formation of oligonucleotide adducts in pharmaceutical formulations, Pharm. Dev. Technol. 10, 283, 2005. 103. Rentel, C. et al., Formation of modified cytosine residues in the presence of depurinated DNA, J. Org. Chem. 70, 7841, 2005.

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Hybridization-Based Drugs: Basic Properties Duplex RNA Drugs

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15

Utilizing Chemistry to Harness RNA Interference Pathways for Therapeutics: Chemically Modified siRNAs and Antagomirs Muthiah Manoharan and Kallanthottathil G. Rajeev

CONTENTS 15.1 15.2 15.3

Introduction .......................................................................................................................437 Molecular Mechanism of RNAi ........................................................................................438 Role of Chemical Modifications .......................................................................................439 15.3.1 Backbone Modifications ......................................................................................440 15.3.2 Sugar Modifications.............................................................................................440 15.3.3 Base Modifications ..............................................................................................442 15.4 Design of siRNAs..............................................................................................................443 15.5 Chemical Methods for Improving Specificity...................................................................445 15.6 Chemical Methods of Reducing Immune Response .........................................................446 15.7 Chemical Synthesis of RNA and siRNAs .........................................................................446 15.8 Systemic Delivery of Synthetic siRNAs: Conjugation Approaches .................................448 15.9 Systemic Delivery of Synthetic siRNAs: Complexation Approaches ..............................451 15.10 MicroRNAs and microRNA Silencing with Antagomirs..................................................453 15.10.1 miRNA Pathway ................................................................................................453 15.10.2 Role of miRNAs in Vivo ....................................................................................456 15.10.3 Antagomirs.........................................................................................................456 15.10.4 Viral miRNAs ....................................................................................................457 15.10.5 Viral Suppression of Silencing ..........................................................................458 15.11 Conclusions and Perspectives............................................................................................458 Acknowledgments ..........................................................................................................................459 References ......................................................................................................................................459

15.1 INTRODUCTION Small molecule pharmaceutical drugs meet the “Lipinski Rules,” which state that drugs with favorable absorption, distribution, metabolism, and elimination (ADME) properties have certain physical characteristics, including a high lipophilicity, presence of not more than five hydrogen bond 437

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donors and ten hydrogen bond acceptors, and molecular weight not more than 500. In sharp contrast, short interfering RNAs (siRNAs) lack these drug-like properties due to their large size (two turns of a nucleic acid double helix), nearly 40 anionic charges (due to the phosphodiester backbone), and high-molecular-weight (over 13 kDa). In aqueous solution, with their sugar-phosphate backbone exposed to water, siRNAs are extremely hydrophilic and heavily hydrated. Furthermore, siRNAs are very unstable in serum due to susceptibility to degradation by serum nucleases, contributing to their short half-life in vivo. Despite their lack of “drug-like” properties, siRNAs offer great therapeutic promise as these agents specifically inhibit production of certain gene targets through RNA (RNAi) interference mechanisms. The antisense strand of the siRNA duplex serves as a guide to identify a complementary sequence in an mRNA; the targeted mRNA is degraded or, under certain conditions, translation of protein is repressed, with the result that the targeted gene is silenced.

15.2 MOLECULAR MECHANISM OF RNAi The ribonuclease Dicer initiates RNAi by cleaving double-stranded RNA (dsRNA) substrates into small fragments of 21–25 nucleotides in length. The RNAi pathway is shown in Figure 15.1. The human Dicer consists of distinct domains: an amino terminal helicase domain, a PAZ domain, tandem RNase III domains responsible for cleavage, and a carboxy terminal dsRNA-binding domain. In the crystal structure of an intact Dicer enzyme derived from Giardia intestinalis, the PAZ domain—the module that binds the end of dsRNA—is found to be separated from the two catalytic ribonuclease III (RNase III) domains by a flat, positively charged surface of about 65 Å in length, which matches the distance spanned by two turns of a helix or ⬃21–25 base pairs of RNA. Thus, Dicer is a “molecular ruler” that recognizes dsRNA and cleaves at a specified distance from the helical end of a dsRNA to produce siRNAs [1]. The short interfering duplexes are incorporated into a protein complex called the RNA-induced silencing complex or RISC (Figure 15.1). The two strands of the siRNA duplex are dissociated and the

Synthetic siRNA

Dicer RNase III

dsRNA trigger

ATP ADP + Pi

siRNAs

transgene, transposon, or virus RISC = RNA-induced silencing complex

ATP ADP + Pi

Selective gene

RISC RISC

Argonaute RNase H-like cleavage RISC

mRNA Cleaved mRNA

RISC

Figure 15.1 The RNAi pathway. Synthetic siRNA or natural siRNAs are processed and incorporated into the RNA-induced silencing complex or RISC. This complex recognizes and selectively silences (prevents translation of) mRNA complementary to the guide strand of the siRNA.

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“sense” (also called “passenger”) strand is discarded. siRNA-RISC functions as an enzymatic, multipleturnover complex that recognizes and cleaves mRNA strands complementary to the “antisense” or “guide” strand of the siRNA. The mRNA is cleaved between the nucleotides paired to bases 10 and 11 of the siRNA guide strand [2]. The RISC catalytic site appears, therefore, to be fixed relative to the 5⬘ end of the siRNA. Cleavage is magnesium-ion-dependent and yields 3⬘ hydroxyl and 5⬘ phosphate groups on the resulting mRNA degradation products [3,4]. Argonaute proteins are the catalytic cores of the RISC. Biochemical analysis identified Argonaute 2 (Ago2) as the protein responsible for mRNA cleavage [5]. Argonaute proteins are characterized by an N-terminal PAZ domain and a C-terminal PIWI domain. The crystal structure of the PAZ domain from human Ago eIF2c1 bound to a 9-mer siRNA duplex has been solved [6]. In a sequence-independent manner, PAZ anchors the two nucleotide, 3⬘ overhang of the siRNA-like duplex within a highly conserved binding pocket. It further secures the duplex by binding to the phosphodiester backbone of seven nucleotides of the overhang-containing strand and to the 5⬘-terminal residue of the complementary strand. It has been proposed that PAZ serves as a 3⬘-end-binding module for siRNA transfer in the RNA silencing pathway and as an anchoring site for the 3⬘end of the guide strand within silencing effector complexes [6]. The C-terminal PIWI domain adopts an RNase H fold critical for the endoribonuclease cleavage activity of the RISC [7]. The crystal structure of Archaeoglobus fulgidus Piwi protein (AfPiwi) bound to siRNA identified the binding pocket for guide-strand 5⬘-end recognition and provided insight into guide-strand-mediated mRNA target cleavage specificity [8]. This structure shows the phosphorylated 5⬘-end of the guide RNA anchored within a basic pocket, highly conserved across known PIWI proteins, that contains the C-terminal carboxylate and a bound divalent cation. The first nucleotide from the 5⬘ end of the guide strand is unpaired and stacks over a tyrosine residue whereas successive nucleotides form a short RNA duplex with the target mRNA. Prior to RISC activation, the sense strand of the siRNA duplex is cleaved by Ago2. Ago2 directly binds to the siRNA duplex and cleaves the sense strand in the same manner as it cleaves mRNA substrates [9,10]. The passenger strand cleavage is important for efficient RISC activation in vitro, since RISC slicer activity is decreased when the cleavage of passenger strand is blocked. RNAi is conserved from fungi to humans. The effect of endogenous siRNA can be replicated by addition of short, synthetic oligonucleotide duplexes or by expression of shRNAs, expressed in the cell as fold-back stem-loop structures that are processed into siRNAs. This review will focus mostly on synthetic siRNAs. A 5⬘-phosphate group on the guide strand is mechanistically required for cleavage of the target mRNA. Synthetic siRNA bearing a 5⬘-hydroxyl group can also mediate RNAi function, as the RNA is phosphorylated by an endogenous kinase. When the 5⬘-hydroxyl terminus of the guide strand was blocked with 5⬘-OMe, silencing was abolished [11].

15.3 ROLE OF CHEMICAL MODIFICATIONS Although the molecular weight of siRNAs cannot be reduced, these molecules can be made more “drug-like” through judicious use of chemical modification to the sugars, backbone, or bases of the nucleotide components or by conjugating ligands to oligoribonucleotides. First, the thermodynamic stability of the duplex and/or nuclease resistance can be enhanced by chemical modification. Second, modifications can increase the half-life of the siRNA duplexes in circulation in vivo. Unmodified siRNAs are very unstable in serum due to degradation by serum nucleases [12]. Third, chemical modification can be used to improve biodistribution and to target the siRNA to certain cell types. Fourth, one can envision improving the potency of siRNA with appropriate chemical modifications in terms of target-binding affinity, favored conformational features of the modification such as sugar puckering of the nucleoside (C3⬘-endo) and A-helix geometry, altering on- and off-rates of hybridization, and enhancing product release. Fifth, chemical modifications can reduce the off-target effects of siRNAs. Finally, chemical modifications can abrogate potential immunostimulatory effects, as discussed below.

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15.3.1 Backbone Modifications Modifications to the natural phosphodiester backbone (P⫽O, Figure 15.2, 1) significantly improve nuclease stability and pharmacokinetic properties of siRNAs. The phosphorothioate (P⫽S) modification (Figure 15.2, 2) is generally well tolerated on both strands and provides improved nuclease resistance [13,14]. For each P⫽S modification the Tm is reduced by 0.5–0.8°C, but this does not seem to affect the potency significantly [15]. In the boranophosphate (P⫽BH ⫺ 3 , Figure 15.2, 3) linkage, a nonbridging phosphodiester oxygen is replaced with an isoelectronic borane moiety. Boranophosphate siRNAs have been shown to be more active than native siRNAs if the center of the guide strand is not modified and they may be at least 10 times more nuclease resistant than unmodified siRNAs [16,17]. 2⬘,5⬘-Phosphodiester linkages (Figure 15.2, 4 and 5) appear to be tolerated on the nonguide but not on guide strand of the siRNA [18]. 5⬘- and 3⬘-inverted deoxy abasic end caps (Figure 15.2, 6 and 7) provide exonuclease stability as terminal backbone modifications [19]. Another end-cap backbone modification tested was 2-hydroxyethylphosphate (Figure 15.2, 8); it is tolerated only when placed at the 3⬘ end of the sense strand [20]. Introduction of 5⬘-3⬘ and 3⬘-5⬘ amide backbones (Figure 15.2, 9 and 10) provides neutral hydrophobic backbone residues within RNA oligonucleotides that will have potential applications for RNA interference and microRNA [21]. 15.3.2 Sugar Modifications For historical and ease of synthesis reasons, siRNA duplexes often have deoxy residues (Figure 15.2, 11) at the 3⬘ termini of each strand. An siRNA motif consisting entirely of 2⬘-O-methyl (2⬘-OMe, Figure 15.2, 12) and 2⬘-deoxy-2⬘-fluoro (2⬘-F, Figure 15.2, 13) nucleotides has enhanced plasma stability and increased in vitro potency. At one site, this motif showed ⬎500-fold improvement in potency over the unmodified siRNA [22]. In addition to offering resistance to endonuclease cleavage, sugar modifications such as 2⬘-OMe, 2⬘-F, and locked nucleic acid (LNA, Figure 15.2, 14) with a methylene bridge connecting 2⬘ and 4⬘ carbons appear to be able to reduce the immunostimulatory effects of siRNAs [23]. Incorporation of the LNA-like 2⬘-O,4⬘-C-ethyelene sugar modification (ENA, Figure 15.2, 15) at the 3⬘ ends of both sense and antisense strands completely abolishes RNAi activity in vitro [20]. Among these sugar modifications, 2⬘-F, LNA, and ENA provide significant increases in target-binding affinities (with RNA hybridization melting temperature differences,
Tm, of 2–4°C per modification), 2⬘-H causes a decrease in Tm (
Tm ⫽ ⫺0.5°C per modification), and the others did not have much effect [15]. Using phosphatase and tensin homolog (PTEN) as a target, the effect of 2⬘-sugar modifications such as 2⬘-F, 2⬘-OMe, and 2⬘-O-(2-methoxyethyl) (2⬘-O-MOE, Figure 15.2, 16) in the guide and nonguide strands was evaluated in HeLa cells [24]. The activity depended on the position of the modification in the guide strand. The siRNAs with modified residues at the 5⬘ end of the guide strand were less active relative to those modified at the 3⬘ end. The 2⬘-F sugar was generally well tolerated on the guide strand, whereas the 2⬘-O-MOE modification resulted in loss of activity regardless of placement position in the construct. The incorporation of 2⬘-OMe and 2⬘-O-MOE in the nonguide strand of siRNA did not have significant effect on activity [24]. Duplexes containing the 4⬘-thioribose (4⬘-S, Figure 15.2, 17) modification had 600 times greater stability than that of natural RNA [25]. Crystal structure studies revealed that 4⬘-S-containing residues adopt conformations very similar to the C3⬘-endo pucker observed for unmodified sugars in the native duplex [26]. Stretches of 4⬘-S-RNA were well tolerated in both the guide and nonguide strands. However, optimization of both the number and placement of 4⬘-thioribonucleosides was necessary for maximal potency. These optimized siRNAs were generally equipotent or superior to native siRNAs and exhibited increased thermal and plasma stability. Furthermore, significant improvements in siRNA activity and plasma stability were achieved by judicious combination of 4⬘-thioribose with 2⬘-OMe and 2⬘-O-MOE modifications [27].

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O

B

O

OH O P O O O O

B

O

O

OH O P O S O O

B

OH

O

OH

B

O

OH

O

B

H

O

4

5

O Y P O O O

B

O

O

S

O O

O Y P O O O

O

16

B

O

O

U

O

O OH

B

B O

O

O O

B

F

B

O

O

B

B

O

O O

B

OH

10

O O P O Y O O

O

14

15

B

F

O Y P O O O

NH2 O Y P O O O

B

B

F

OH O

17

O

O

O O Y P O O O

B

O

O

9

13

OH O P O Y O S

B

O

O

F O Y P O O O

12

B

OH HN

U

O

O

O

O

11

HN OH

B

O

O

O O P O Y O O

B

OH

O

8

7

O

U

O

O

OH

O

B

U

O

O

O O P O O

B

OH

6

O

O P O O O

B

OH

T

O

O O O P O O O

O

O

O P O O O

3

O

O O P O O

O

O

H

OH

OH

O

O

B

B

O

O

HO

O

2

B

O

O

OH O P O H3B O O

B

O

1 O

B

O

O

441

18

O

NH2

19

Y = O or S

Figure 15.2 Chemical modifications of siRNAs: sugar and backbone.

siRNAs modified with 2⬘-deoxy-2⬘-fluoro-arabinonucleotide (2⬘-araF, Figure 15.2, 18) showed increased RNAi activity and substantially enhanced serum stability relative to unmodified siRNA. siRNA duplexes with fully 2⬘-araF-modified sense strands (FANA) and unmodified guide strands had enhanced RNAi potency relative to unmodified siRNA of up to four fold and considerably increased serum half-life. The 2⬘-araF modification is also tolerated at the termini of the guide strand [28]. The 2⬘-amino modification (Figure 15.2, 19) has not been widely studied. One study

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reported some loss of activity when the modification was placed in the sense strand and caused severe loss of activity when the modification was in the antisense strand [29]. As the study involved long dsRNA in Caenorhabditis elegans, the loss of activity could have arisen from lack of efficient cleavage of the transcript by Dicer or at the amplification step. Additional studies with this modification are warranted. 15.3.3 Base Modifications The nucleobase modifications evaluated in siRNA duplexes are shown in Figure15.3. 4-Thiouridine (Figure 15.3, 20) and 5-bromouridine (Figure 15.3, 21) residues are tolerated in both sense and antisense strands, whereas bulky 5-iodo (Figure 15.3, 22) or cationic 5-(3-aminoallyl) (Figure 15.3, 23) O

S NH N

I

NH N

O

N

N

21

23

22

O N CH3

N

O

N

N

N O

O

N N

O

O OH

O

25

F

O

26

HN

NH

N

N

N

NH

O

O

OH

OH

O

O

O

28

29

O

O NH

31

30 H3C

N

N

N

NH

O

S

N

O

N

O

OH

OH

OH

O

O

O

32

33

34

Figure 15.3 Nucleobase modifications of siRNAs.

H N

O

O

O

O

OH

O

N

O O

O OH

O

NH2

NH

O

O

27

O

O N

O

OH

O

24

F

O O

OH

O

F

NH2

O

OH

O

F

NH2

NH

O

N

O

O

20

O

OH

OH

O

O

O

O

O

N

N O

OH

OH

NH

O

O

O

O

H2N

NH

O

O

O

O

O

Br

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modifications are not. Replacement of guanosine by inosine (Figure 15.3, 24) reduces activity relative to unmodified duplex [29]. As this analysis was carried out with in vitro transcribed long dsRNA, the lack of gene inhibition could be due to inhibition of Dicer processing rather than a step involving RISC. In a different study, N-Me-uridine (Figure 15.3, 25) in place of uridine and 2,6-diaminopurine (Figure 15.3, 26) substituted for adenine-abolished siRNA activity [30]. siRNA duplexes with 2,4-difluorotoluyl ribonucleoside (rF, Figure 15.3, 27) substituted for uridine retain silencing activity, are recognized by the RISC enzymes and kinases, and have improved nuclease resistance in serum relative to unmodified siRNA. The rF:adenosine base pair was destabilizing relative to a uridine:adenosine pair, although it was slightly less destabilizing than other mismatches. The crystal structure of a duplex containing rF:A pairs showed local structural variations relative to a canonical RNA helix. As the fluorine atoms cannot act as hydrogen bond acceptors and are more hydrophobic than uridine, there was an absence of a well-ordered water structure around the rF residues in both grooves [31,32]. Recently it has been shown that use of 1-deoxy-1-(2,4-difluorophenyl)-
-D-ribofuranose (Figure 15.3, 28), a demethylated rF, in place of rU at position 7 relative to the 5⬘ end appears to enhance sequence selectivity beyond that of the natural base [32]. Site-specific insertion of N 2-benzylguanosine (Figure 15.3, 29) into siRNA-blocked interaction of RNA-dependent protein kinase (PKR) with dsRNA-binding motifs (dsRBMs) and inhibited activation of PKR by siRNA. Interestingly, siRNA modified by site-specific incorporation of N 2-benzylguanosine into the sense strand greatly reduced PKR activation but retained the ability to specifically silence a targeted message by RNA interference in HeLa cells [33,34]. Pseudouridine, 5-methylcytosine, 5-methyluridine, 2-thiouridine, and N6-methyladenosine (Figure 15.3, 30, 31, 32, 33, 34, respectively) modifications significantly reduce the immunostimulatory effects of single-stranded RNAs. RNA elicits immunostimulatory effects through interaction with human toll-like receptors (TLRs), TLR3, TLR7, and TLR8, but incorporation of these modified nucleobases reduces immunostimulatory activity [35]. Dendritic cells exposed to such nucleobase-modified RNA express significantly less cytokines and activation markers than those treated with unmodified RNA. Dendritic cells and TLR-expressing cells are potently activated by bacterial and mitochondrial RNA, but not by mammalian total RNA, which is abundant in modified nucleosides.

15.4 DESIGN OF siRNAs Three major designs of siRNA are shown in Figure 15.4, 35–37. The “double overhang” design 35 has TT overhangs at 3⬘ ends of both guide and passenger strands and the duplex has 19 complementary nucleotides (Figure 15.4, Table 15.1) [36,37]. A modification to the classical double overhang design is the “single overhang” design 36; this design eliminates the TT overhang from 3⬘ end of sense strand (Table 15.1, entry 2). It has been shown that the single overhang design is equally potent and efficacious in silencing targeted gene in vivo as design 35 and the blunt-ended duplex provides exonuclease protection relative to a duplex with a 3⬘ overhang [12]. The third design is a blunt-ended perfect duplex 37 as shown in Figure 15.4 (Table 15.1, entry 3). Blunt-ended siRNA targeting PTEN, p110
, and Akt1, members of phosphatidylinositol (P1) 3-kinase pathway, have been reported [22,38]. Duplex designs 38–42 represent siRNAs with chemical modifications. Blunt-ended designs 38-A to 38-F and 39-A to 39-F shown in Table 15.1 represent “staggered” and “matched” designs with alternating 2⬘-OH/2⬘-F, 2⬘-OH/2⬘-OMe, 2⬘-F/2⬘-OMe, 2⬘-F/2⬘-OH, 2⬘-OMe/2⬘-OH and 2⬘-OMe/2⬘-F chemical modifications [22,39]. In the matched design, modified residues are base-pairing partners; in the staggered design, the modified residues are paired with unmodified residues. siRNA designs 40-A and 40-B (Figure 15.4, Table 15.1) consist, respectively, of fully modified 2⬘-F and 2⬘-OMe sense/passenger strand and unmodified guide strand [40]. siRNA designs with all pyrimidines either on the passenger (Figure 15.4, 41, Table 15.1) or on

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35

5′ 5′

36 5′ 5′

37

5′ 5′

38

5′

39 5′

40 5′

41 5′

42

O

,

O

= O

B

O

; OH

B

O

= O

O

and R

R = H, OH or OMe

B

O

= O

R

R = F, OMe

Figure 15.4 Schematic representation of different siRNA designs.

Table 15.1 siRNA Design and Chemical Modifications Description of Modifications Design 35 36 37 38-A 38-B 38-C 38-D 38-E 38-F 39-A 39-B 39-C 39-D 39-E 39-F 40-A 40-B 41 42

Sense Strand (S); 5ⴕ-3ⴕ Ribose with 3⬘ TT overhang Ribose with 3⬘ blunt end Ribose with 3⬘ blunt end Alternating 2⬘-OH/2⬘-F, staggered Alternating 2⬘-OH/2⬘-OMe, staggered Alternating 2⬘-F/2⬘-OMe, staggered Alternating 2⬘-F/2⬘-OH, staggered Alternating 2⬘-OMe/2⬘-OH, staggered Alternating 2⬘-OMe/2⬘-F, staggered Alternating 2⬘-OH/2⬘-F, matched Alternating 2⬘-OH/2⬘-OMe, matched Alternating 2⬘-F/2⬘-OMe, matched Alternating 2⬘-F/2⬘-OH, matched Alternating 2⬘-OMe/2⬘-OH, matched Alternating 2⬘-OMe/2⬘-F, matched All 2⬘-F pyrimidines All 2⬘-OMe pyrimidines All pyrimidines with 2⬘-F or 2⬘-OMe All pyrimidines with 2⬘-F or 2⬘-OMe

Antisense Strand (AS); 5ⴕ-3ⴕ Ribose with 3⬘ TT overhang Ribose with 3⬘ TT overhang Ribose with 3⬘ blunt end Alternating 2⬘-OH/2⬘-F, staggered Alternating 2⬘-OH/2⬘-OMe, staggered Alternating 2⬘-F/2⬘-OMe, staggered Alternating 2⬘-F/2⬘-OH, staggered Alternating 2⬘-OMe/2⬘-OH, staggered Alternating 2⬘-OMe/2⬘-F, staggered Alternating 2⬘-OH/2⬘-F, matched Alternating 2⬘-OH/2⬘-OMe, matched Alternating 2⬘-F/2⬘-OMe, matched Alternating 2⬘-F/2⬘-OH, matched Alternating 2⬘-OMe/2⬘-OH, matched Alternating 2⬘-OMe/2⬘-F, matched Ribose Ribose Ribose with TT overhang All pyrimidine with 2⬘-F or 2⬘-OMe

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both strands (Figure 15.4, 42, Table 15.1) are active in vitro and have increased serum stability relative to duplexes with mixed purines and pyrimidines [40]. Another design has inverted bases at the passenger strand termini, providing improved exonuclease stability in vivo [19].

15.5 CHEMICAL METHODS FOR IMPROVING SPECIFICITY To achieve specificity necessary for therapeutic use of siRNAs, “off-targeting” due to silencing of genes that share partial homology to the siRNA has to be avoided. A combination of bioinformatics methods, chemical modification strategies, and empirical testing is required to address these issues [41]. Recent biochemical studies on the molecular mechanism of RNA interference have highlighted some key features of potent siRNA duplexes (Figure 15.5). Upon examination of the sequences of a large number of miRNA precursor sequences, it was observed that the predicted thermodynamic stability of the two ends of the duplex was unequal [42,43]. Specifically, calculating the
G for the several base pairs at each end of the duplex revealed that the 5⬘ end of the mature miRNA paired less tightly with the carrier strand than did the 3⬘ end. In short, there is a thermodynamic asymmetry of siRNA precursors. It was hypothesized that components of the RISC machinery select the guide strand based on this asymmetry. Experimental evidence supporting this “asymmetry rule” has been derived from studies using chemically synthesized siRNAs in transfection experiments. Using an elegant assay in which each strand of the siRNA targets a different reporter gene, the relative efficiency of RISC incorporation for each of the two strands was quantified [42]. The RISC machinery preferentially incorporated the strand with the 5⬘ end that bound thermodynamically less stably with the other strand. In fact, strand selection could be switched by making a single nucleotide substitution at the end of the duplex to alter relative binding of the ends. A similar conclusion was reached independently by another group based on in vitro screening of a large collection of siRNAs with varying potency. [43,44]. Thus, designing siRNAs with relatively weaker base pairing at the 5⬘ end of the desired guide strand may increase the likelihood of obtaining a potent duplex. However, there is no in vivo demonstration of such asymmetric selection criteria available yet. Another recent work suggests that off-targeting effects can be reduced by chemical modification of the nucleotide at position 2 of the guide strand (Figure 15.5) [45]. The introduction of a 2⬘-OMe modification at position 2 of the guide strand was shown to inactivate the off-target activity of the siRNA without compromising silencing of the intended mRNA. It has been claimed that introduction of the modification at a single nucleotide position was sufficient to suppress the majority of off

Target cleavage site

3′

5′ 5′

3′

Highly discriminatory to chemical modification

Guide (antisense) strand

Weak base pairing increases potency Chemical modification reduces off targeting

Figure 15.5 Critical nucleotide positions in siRNAs. Nucleotides that are important for potency, mRNA recognition, mRNA cleavage, and cleavage specificity, including minimizing off targeting, are shown.

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targeting. The mechanism, as explained by recently published crystal structure data, was based on the perturbation of RISC interaction with the modified 2⬘-OMe nucleotide.

15.6 CHEMICAL METHODS OF REDUCING IMMUNE RESPONSE siRNAs may elicit an immune response. Certain siRNA sequences stimulate the innate immunity via endosomal TLRs, particularly TLR3, TLR7, and TLR8. The mechanism of stimulation is unknown; it remains unclear whether siRNA duplexes act as specific ligands for these receptors. In human peripheral blood mononuclear cells (PBMCs), immunostimulatory RNA motifs in single-stranded RNAs are more effectively recognized by the innate immune system than motifs sequestered in siRNA duplexes. Replacement of the 2⬘-hydroxyl uridines with 2⬘-F, 2⬘-deoxy, or 2⬘-OMe uridines abrogated immune activation. Thus, immune recognition of RNA by TLRs can be evaded by 2⬘-ribose modifications of uridines [46]. Interestingly, CpG motif-containing RNAs may have immunostimulatory effects due to the release of IL-12, IFN-, and IL-6. It has been shown that chemical modifications such as 2⬘-OMe and 5-methylcytosine reduce production of these cytokines [35]. Similarly replacement of uridine with 2⬘-modified uridines also reduces immunostimulatory effects. In the cytosol, factors such as PKR and helicases RIG-1 and melanoma differentiation antigen-5 (MDA-5) are involved in immune stimulation, but these factors may be activated by long dsRNAs and may not be relevant to siRNA-based therapeutics. Another mechanism whereby siRNA duplexes can induce unintended effects is via stimulation of the innate immune system in certain specialized immune cell types. It has been demonstrated that siRNA duplexes harboring distinct sequence motifs can engage TLRs in plasmacytoid dendritic cells leading to increased production of interferon [23]. Such immune stimulation could pose a significant problem in a therapeutic setting. In the case of siRNAs, toll like recptor-7 (TLR-7) appears to mediate this immune stimulation [23]. To prevent immune stimulation by siRNA duplexes, certain sequence motifs can be avoided during siRNA design or chemical modification can be used to inactivate the motifs. The former approach is not viable at present since the full spectrum of stimulatory motifs has not been identified for duplex RNA. Evidence supporting the latter approach comes from studies in which chemical modifications at the 2⬘ position of nucleotides within putative TLR-7-interacting sequences eliminated immune stimulation without compromising silencing activity [23,47]. Delivery strategies that target siRNA to certain organs or cell types and avoid the cell types responsible for immune stimulation may circumvent the immune response.

15.7 CHEMICAL SYNTHESIS OF RNA AND siRNAs The most common methods for RNA synthesis use phosphoramidite chemistry with one of the following chemistries, shown in Figure 15.6, for 2⬘-OH protection: 2⬘-O-t-butyldimethylsilyl (2⬘-O-TBDMS or silyl chemistry), 2⬘-O-bis(2-acetoxyethoxy)methyl (2⬘-ACE chemistry), 2⬘-Otriisopropylsilyloxymethyl (2⬘-O-TOM), or 2⬘-O-(1-(2-fluoro- phenyl)-4-methoxypiperidin-4-yl) (2⬘-O-Fpmp). The most widely used of these is 2⬘-O-TBDMS chemistry (Figure 15.6, 43). The TBDMS-protected phosphoramidite building blocks were originally introduced by Ogilvie in combination with methyl protection of phosphate/phosphite moiety and monomethoxytrityl protection of the 5⬘-hydroxy group [48–51]. The 5⬘-dimethoxytrityl-protected, 2-cyanoethyl-N,N-diisopropylaminophosphoramidites with standard base-protecting groups (Abz, Cbz, Gibu) remain the reagents of choice for the preparation of RNA oligonucleotides and these phosphoramidites are also commercially available with a variety of “fast” base-protecting groups that are analogs of phenoxyacetyl groups [52–54,55].

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t t

Figure 15.6

2⬘-Hydroxyl protecting groups used in solid-phase synthesis of RNA oligonucleotides. Protecting groups are 43: 2⬘-O-t-butyldimethylsilyl (TBDMS); 44: 2⬘-O-triisopropylsilyloxymethyl (TOM); 45: 2⬘-O-t-butyldithiomethyl (DTM); 46: 2⬘-O-(2-cyanoethoxymethyl) (CEM); 47: 2⬘-O-(1-(2-fluorophenyl)-4-methoxypiperidin-4-yl) (FPMP); 48: 2⬘-O-(1-(4-chloro- phenyl)-4-ethoxypiperidin-4-yl) (CPEP); 49: 2⬘-O-bis(2-acetoxyethoxy)methyl (orthoester or ACE); 50: 2⬘-O-acyloxymethyl (AOM) and 51: 2⬘-O-acylthiomethyl (ATM).

Removal of the TBDMS group is achieved by treatment with tetrabutylammonium fluoride (TBAF) [56,57], or by treatment with neat triethylamine trihydrofluoride (Et3N(HF)3) [58,59]. The excess fluoride reagent can be scavenged, for instance by using isopropyl trimethylsilyl ether [60]. Recently, a pyridine/HF-based TBDMS deprotection method has been developed and used for manufacturing siRNAs (Figure 15.7) [61,62]. Another silyl-based 2⬘-hydroxyl protection was achieved by introduction of a methylene bridge between the oxygen of the 2⬘-hydroxyl and the triisopropylsilanol group to obtain TOM (Figure 15.6, 44) protection of the ribonucleosides [63]. According to the manufacturer, protection with TOM gives improved coupling efficiencies; the monomers are available from QIAGEN. Recently, the 2⬘-O-t-butyldithiomethyl (2⬘-O-DTM) (Figure 15.6, 45) group has been reported for 2⬘-hydroxyl protection [64]. Removal of the 2⬘-O-DTM was performed under reductive conditions using aqueous 1,4-dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP) at 55°C in buffered pH of 7.6 for 90 min. The 2-cyanoethoxymethyl (CEM, Figure 15.6, 46) group has been developed for 2⬘-hydroxyl protection for RNA synthesis. It is claimed that the new protecting group allows the synthesis of oligoribonucleotides with an efficiency and final purity comparable to that obtained in DNA synthesis [65]. Deprotection of group is readily achieved by treatment with TBAF for few hours,

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O

O

B

O

O Si O Y P O B O O

O

O Si

Py.HF

O

B

OH O Y P O O O

Y = O or S

O

B

OH

Figure 15.7 Py.HF deprotection of 2⬘-O-TBDMS group from the sugar/RNA.

but oligonucleotides fail to undergo deprotection with Et3N⭈3HF. An RNA oligomer of 82 nucleotides has been successfully synthesized. In the Fpmp technology, blocking of 2⬘-hydroxyl with 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp, Figure 15.6, 47) group provides efficient coupling of the phosphoramidite with the use of 5-(3-nitrophenyl)-1H-tetrazoles as activator [66]. This group is removed by incubation in a medium of pH 3 over an extended period of time. Another analog of Fpmp group, Cpep (Figure 15.6, 48), may be of value as it requires mild acidic conditions for deprotection [67]. Monomers will soon be commercially available for this chemistry and more synthetic results will be forthcoming. In the ACE technology an orthoester, 2⬘-O-bis(2-acetoxyethoxy)methyl (Figure 15.6, 49), protects the 2⬘-OH [68,69]. Rather than a 5⬘-DMT for protection of the 5⬘ terminus and cyanoethyl groups for protection of phosphates, silyl ether and methyl are used, respectively. Following synthesis, the methyl protecting groups on the phosphates are removed by 1 M disodium-2-carbamoyl-2cyanoethylene-1,1-dithiolate trihydrate in DMF. Although more elaborate cycles and reagent changes are required when using ACE chemistry rather than 2⬘-O-TBDMS chemistry, the advantage of ACE is in the stability of the 2⬘-O-protected RNA, which can be purified and stored. The 2⬘ protection is efficiently removed by incubation in aqueous buffers. It is possible to protect the 2⬘ position with groups that are not removed until the siRNA enters cells. The 2⬘ positions have been protected with 2⬘-O-acyloxymethyl (AOM, Figure 15.6, 50) or acylthiomethyl (ATM, Figure 15.6, 51) groups [70]. Oligouridylates modified with these groups are deprotected by cellular esterases to release RNA. Protected oligouridylates exhibit exceptional resistance to nuclease degradation and the 2⬘-AOM groups did not prevent duplex formation. For generating siRNAs from individual RNA strands, guide and passenger strands are usually individually synthesized on either controlled pore glass (CPG) or polystyrene-based support as shown in Figure 15.8 using “silyl” chemistry. After completion of the synthesis simultaneous cleavage from support and base deprotection followed by 2⬘-O-sugar deprotection yields crude RNA oligonucleotide. Ion exchange or reverse-phase high-performance liquid chromatography (RP-HPLC) purification and subsequent desalting affords pure oligonucleotide ready for annealing (Figure 15.9). Annealing of equimolar mixture of guide and passenger strands in appropriate buffer of choice yields the siRNA of interest. 15.8 SYSTEMIC DELIVERY OF SYNTHETIC siRNAs: CONJUGATION APPROACHES siRNA duplexes synthesized without any chemical modification are rapidly cleared from plasma and excreted; unmodified duplexes do not bind measurably to serum proteins like albumin, low-density lipoprotein (LDL), and high-density lipoprotein HDL [12]. Certain terminal conjugates are able to improve bioavailability; for example, siRNAs conjugated at the 5⬘ end of the sense strand

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B1

O

DMTO HO

O N H

O

449

+ Activator

R O

O

B2

O

B1

O

O

N P

R

R

O

CN

O

Deblocking DMTO

Coupling

B2

O

DMTO

CN

B2

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NC O O P Y O

R

O O

O P

B1

O

R B1

O

R

O

Y = O or S

R

O

Oxidation

Capping DMTO

Deprotection, purification

B2

O

XO

CN O P

O

B1

O

O

R

O

B21

O

B1

O

R

O

HO

O O

O

R

O

O

OH P OH Y O

O

5′

3′

Y

P OH O

O

B1

21-mer RNA after 20 cycles OH H/OH

Figure 15.8 Solid-phase synthesis of RNA oligonucleotides. O

O HO

5′

Q

O

HO

5′

O

O Q = O or NH 1. Deprotection 2. Purification 3. Desalting

HO

5′

Q

O

1. Deprotection 2. Purification 3. Desalting

OH

HO

Sense

5′

OH Antisense

Annealing

HO HO

5′ siRNA

5'

OH OH

Figure 15.9 Manufacturing of siRNA. Step 1: syntheses of sense (or passenger) and antisense (or guide) strands on solid support (CPG or PS support); step 2: deprotection, purification and desalting of single-stranded oligonucleotides, and step 3: annealing of equimolar mixture of guide and passenger strands in appropriate buffer to obtain siRNA.

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with cholesterol, lithocholic acid, lauric acid, or long alkyl branched chains (C32) improved in vitro cell permeation in liver cells [71]. Conjugates that have been evaluated in vivo are discussed below. siRNAs conjugated with cholesterol (chol-siRNA, Figure 15.10) have an improved pharmacokinetic profile relative to unmodified siRNA. When radioactively labeled chol-siRNAs or unconjugated siRNA were administered to rats by intravenous injection at 50 mg/kg, the chol-siRNAs had an elimination half-life (t½) of 95 min, whereas unconjugated siRNAs had a t½ of 6 min [12]. Chol-siRNAs showed broad tissue biodistribution 24 h after injection into mice. Although no detectable amounts of unconjugated siRNAs were observed in tissue samples, significant levels of chol-siRNAs were detected in liver, heart, kidney, adipose, and lung tissue samples. The enhanced pharmacokinetics of the chol-siRNA translates into therapeutic activity. Cholesterol-conjugated siRNA silenced an endogenous gene encoding apolipoprotein B (apoB) after intravenous injection in mice. Administration of chol-siRNA resulted in silencing of the apoB messenger RNA in liver and jejunum, decreased plasma levels of apoB protein, and reduced total cholesterol. In this in vivo study, the mechanism of action for the siRNAs was proven to occur through RNAi-mediated mRNA degradation [12]. Cholesterol conjugates have been studied in the classical antisense therapeutics and the liver distribution achieved by cholesterol conjugation has been well documented [72]. A high-dose safety study of cholesterol conjugates has also been reported [73]. To target siRNA to cancer cells, siRNA was conjugated to an aptamer that mediates binding to a cell-surface receptor to siRNA (Figure 15.11) [74]. The aptamer binds to prostate specific membrane antigen (PSMA), a cell-surface receptor overexpressed in prostate cancer cells and tumor 5′

O 3′

Y

P

O O OH N

H N

Lig

O

O

an

O

d

Linker

Y = O or S

Figure 15.10 Conjugates improve bioavailability of siRNA. Design of cholesterol-siRNA conjugate.

G U

C

A U

U

G A

Figure 15.11 Design of aptamer-siRNA conjugate.

siRNA

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vascular endothelium. Aptamers linked to siRNA complementary to a survival gene are internalized by cells expressing PSMA, the siRNA target protein levels are reduced, and cell death results. Cells that do not express PSMA were not killed by incubation with the aptamer-siRNA. The aptamer-siRNA specifically inhibits tumor growth and mediates tumor regression in a xenograft model of prostate cancer. In another development, successful cell-culture delivery of siRNA to PSMA-positive LNCaP cells was achieved by direct administration of streptavidin that was bridged to biotinylated siRNAs and biotinylated PSMA-specific aptamer [75]. The siRNA-mediated inhibition of gene expression was as efficient as that observed with conventional lipid-based transfection agent and was dependent upon conjugation to the aptamer.

15.9 SYSTEMIC DELIVERY OF SYNTHETIC siRNAs: COMPLEXATION APPROACHES A number of liposome formulations of siRNAs have proven effective for systemic delivery in mouse models. Systemically delivered cationic cardiolipin liposomes-containing siRNA specific for Raf-1 inhibited tumor growth in a xenograft model of human prostate cancer [76]. Simple polyethylenimine (PEI) formulations have also shown efficacy in xenograft tumor models [77]. The stable nucleic acid lipid particles (SNALP) liposomal formulation (Figure 15.12) has shown promise for siRNA liposomal delivery. siRNA targeted to a hepatitis B virus (HBV) mRNA was incorporated into a SNALP and administered by intravenous injection into mice carrying replicating HBV [78]. A reduction in HBV DNA was observed with SNALP-formulated siRNA, but not with unformulated siRNA. Reductions were seen in serum HBV DNA for up to 6 weeks with weekly dosing of the SNALP-siRNA. In a major step forward in the development of systemic RNAi for therapeutic applications, dosedependent silencing of apo-B messenger RNA with a SNALP-formulated siRNA was observed in a nonhuman primate model [79]. ApoB is a lipid-associated protein essential for assembly and secretion of LDL. A single dose of 2.5 mg/kg siRNA encapsulated in the SNALP formulation

O

(a) O

N

O O

(b)

O O O

(c) O

O O P O O

H N

O O

N

O

O n

(d) HO Figure 15.12 Lipid components of SNALP formulation: (a) cationic lipid (DLinDMA), (b) distearoylphosphatidylcholine (DSPC), (c) PEG-lipid (mPEG2000-C-DMA), and (d) cholesterol (chol).

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reduced apoB mRNA in the livers of cynomolgus monkeys by ⬎90% compared to saline-treated controls. As in the murine experiments, apoB silencing was accompanied by a significant reduction in serum cholesterol (⬎65%) and LDL levels (⬎85%). Furthermore, silencing was shown to last for at least 11 days following a single dose of encapsulated siRNA. The treatment appeared to be well tolerated, with transient increases in liver enzymes as the only reported evidence of toxicity; a finding that appeared to be related to apoB silencing itself. In this study, a 5⬘ rapid amplification of cDNA ends (5⬘RACE) analysis was used to definitively demonstrate that silencing was due to the synthetic siRNA. This analysis showed that cleavage of apoB mRNA occurred at the predicted cleavage site, exactly 10 nucleotides from the 5⬘ end of the site of hybridization of the antisense strand of siApoB-2. Early in the development of lipid-based complexation for delivery of siRNA, siRNA targeting A549 liver metastasis complexed with cationic liposomes (LIC-101) derived from 2-O-(2-diethylaminoethyl)-carbamoyl-1,3-O-dioleoylglyecerol and egg phosphatidylcholine showed strong antitumor activity in a mouse model after bolus intravenous injection (Figure 13) [80]. The siRNA specific for bcl-2 gene complexed with LIC-101 suppressed expression of bcl-2 protein in a dosedependent manner. Subcutaneous administration of same siRNA/cationic lipid complex (LIC-101) close to the vicinity of xenografted tumor inhibited growth of prostate cancer. Self-assembling nanoparticles have also been used for systemic delivery of siRNA. Nanoparticles—consisting of polyethylenimine derivitized with polyethylene glycol in turn attached to an Arg-Gly-Asp (RGD) peptide—complexed with an siRNA targeting vascular endothelial growth factor receptor-2 (VEGF R2) were tested in vitro and in vivo [81]. Intravenous administration into tumor-bearing mice inhibited both tumor angiogenesis and growth rate; inhibition of VEGF-R2 expression was also demonstrated. Single-walled carbon nanotubes (SWNT) functionalized with -CONH-(CH2)6-NH3Cl were complexed with an siRNA targeting the telomerase reverse transcriptase. These complexes suppressed the growth of human HeLa cells both in vitro and when injected into tumors in nude mice [82]. In another example of a novel delivery system, a protamine-antibody fusion protein has been used to deliver siRNA to HIV-infected or envelope-transfected cells [83]. The fusion protein (F105-P) was designed with protamine linked to the C terminus of the heavy chain Fab fragment of an HIV-1 envelope antibody. siRNAs bound to F105-P induced silencing only in cells expressing HIV-1 envelope (Figure 15.14). Intratumoral or intravenous injection of F105-P-complexed siRNAs into mice targeted HIV envelope-expressing B16 melanoma cells, but not normal tissue or envelopenegative B16 cells. Injection of F105-P with siRNAs targeting c-myc, MDM2, and VEGF inhibited envelope-expressing subcutaneous B16 tumors. Furthermore, an ErbB2 single-chain antibody fused with protamine delivered siRNAs specifically into ErbB2-expressing cancer cells. O N

N H

O O O

(a)

O

O

O

(b)

R R

O O

O

O O P O O

N

Figure 15.13 Lipid components of cationic liposome LIC-101: (a) 2-O-(2-diethylaminoethyl)-carbamoyl-1,3-Odioleoylglyecerol and (b) egg yolk phosphatidylcholine.

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siRNA Protamine

HIV-1 envelop antibody

Figure 15.14 Systemic antibody-mediated cell-type specific delivery of siRNA. Complexation of siRNA with protamine antibody fusion protein facilitates cellular delivery of siRNA.

Another promising approach is nonviral siRNA delivery for cancer targeting. Systemic administration of siRNA complementary to mRNA from the EWS-FLII gene by a targeted nonviral delivery system inhibited tumor growth in a murine model of metastatic Ewing’s sarcoma [84]. The nonviral delivery system consists of cyclodextrin-containing polycation to bind and protect siRNA and transferrin for targeted delivery to transferrin receptor-expressing tumor cells (Figure 15.15). Long-term, low-pressure, and low-volume tail-vein administration of cyclodextrin/transferrin/siRNA into a mouse model did not elicit abnormalities in IL-12, IFN-, liver, kidney functions, total blood counts, or pathology of major organs. In another example of nonviral systemic delivery, endothelial cell-specific uptake of siRNA was demonstrated after single intravenous injection [85,86]. The siRNA–lipoplex complex comprises positively charged liposomes—a mixture of cationic and fusogenic lipids—complexed with negatively charged siRNA (Figure 15.16). Reduction of endogenous CD31 and Tie2 genes expressed in endothelial cells was observed in lung, liver, and heart of animals tested. A few organs, such as kidney and spleen, exhibited a strong uptake of fluorescently labeled lipoplexed siRNA, but significant inhibition of gene expression was not observed. Microscopic analysis suggested strong uptake of siRNA–lipoplexes by respective tissues, not specific delivery to endothelial cells.

15.10 MICRORNAs AND MICRORNA SILENCING WITH ANTAGOMIRS 15.10.1

miRNA Pathway

MicroRNAs (miRNAs) are a class of small (⬃22 nucleotide long) noncoding RNA molecules that negatively regulate gene expression; many miRNAs target genes associated with disease pathways. They were first discovered in C. elegans, but have now been found in plants, invertebrates, and vertebrates, including humans. miRNAs regulate protein expression posttranscriptionally through a process that is biochemically indistinguishable from RNAi (Figure 15.17a). The miRNAs are transcribed as long precursors, called pri-miRNAs, by pol II. The pri-miRNA is processed in the nucleus

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O OH

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O OH

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OH

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O OH

OH O

O

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HO O

OH HO HO

S

OH HO

O OHO

O

HO O

H N

H N NH

OH

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OH O

OH

HO

O HO O

OH S

HO

n NH

HO

O O

OHO OH

H N

S

OH HO

HO N

OH

OH

O

HO

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HN

HO

OH

O

O OH

N O

HO O

NH

O

HO O

NH

OH

X CDP H N

O O

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O PEG

O S

O

m

N H

Transferrin

Tf

PEG-AD

siRNA

Tf-PEG-AD

Tf

(b)

(c)

Tf

Tf Tf

Tf

Untargeted particle

Tf-targeted particle

Figure 15.15 Targeted delivery of siRNA. (a) Cyclodextrin-containing polycation (CDP) condenses siRNA. The ademantyl-PEG (PEG-AD) stabilizes the particles in physiologic fluids via inclusion compound formation. AD-PEG-transferrin (Tf-PEG-AD) conjugate confers a targeting ligand to the particle, promoting their uptake by cells overexpressing cell-surface transferring receptor. Assembly of (b) nontargeted particle and (c) targeted particle.

to pre-miRNA, hairpin intermediates of 60–70 nucleotides by the RNase III endonuclease Drosha. This enzyme activity was discovered and described as early as 2000 [87]. Following export into the cytoplasm, Dicer cleaves the pre-miRNA to produce an imperfect duplex [88,89]. This duplex enters the same gene-silencing pathway described earlier for siRNAs (Figure 15.1). The choice of which strand to degrade appears to be made within the RISC complex, perhaps based on the thermodynamic properties of the ends of the duplex.

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O

O

N

(a)

455

NH3

N H

NH2 N H

NH3

NH2

O O

(b)

O O P O O

O O

NH3

O O

(c) O O

O O P O O

H N

O

O

45

O

Figure 15.16 Lipid components of siRNA–lipoplex complex: (a) cationic lipid (AtuFECT01), (b) helper lipid, and (c) PEG-lipid.

(a)

(b) MicroRNA gene

Primary transcript

Primary transcript Drosha

Drosha

MicroRNA precursor

MicroRNA precursor Dicer

Dicer

miRNA

miRNA Antagomir

miRNA mRNA function modulated

mRNA function unaffected

Figure 15.17 (a) MicroRNA pathway and (b) inhibition of microRNA pathway by an antagomir.

Although the initial observations of miRNA regulation in C. elegans indicated that gene expression was reduced without alteration of mRNA levels [90], cleavage of HOXB8 was detected mRNA in mice, indicating that miRNA-regulated gene expression in animals can occur through a cleavage mechanism [91]. Thus, in mammals miRNAs can downregulate gene expression by one of two posttranscriptional mechanisms: mRNA cleavage or translational repression. Currently, it is assumed that the choice of mechanism is driven by the extent of complementarity between the miRNA and the messenger RNA target. RISC appears to function as an RNA cleavage enzyme when miRNA is fully complementary to RNA target sites [92]. If the duplex formed between the

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target site and the miRNA contains mismatches, cleavage may be precluded, but RISC remains bound to the mRNA target, resulting in translational repression [93]. The cooperative binding of multiple RISCs provides more efficient translational repression than binding of a single complex. This may explain the presence of multiple miRNA complementary sites in the UTRs of messages regulated by miRNA. 15.10.2

Role of miRNAs in Vivo

Conservative predications suggest that up to 30% of human genes are regulated by miRNA [94]. The relevance of these small RNAs to human health should not be underestimated. In model organisms, numerous miRNAs are involved in developmental regulation and this is presumably the case in humans. Fragile X syndrome was the first human disease linked to a dysfunction in an miRNA pathway [95]. Spinal muscular atrophy, early onset parkinsonism, and X-linked mental retardation also appear to involve loss or mutation in miRNA or components of the pathway [96]. Evidence is mounting that miRNA dysregulation plays a role in cancer pathogenesis. Approximately half of known miRNA genes are located in cancer-associated genomic regions. For example, several studies suggest that the oncogene RAS is regulated by the let-7 miRNA family [88]. To delineate the roles of miRNAs in disease processes, two approaches can be conceived in theory. The studies demonstrating the involvement of miRNAs in metabolic disease are illustrative of the two approaches for understanding the precise molecular function of mammalian miRNAs in vivo: one can treat with an agonist (to increase expression of a particular miRNA) or an antagonist (to decrease expression of an miRNA). Both of these approaches could also be used therapeutically to modulate miRNAs and hence to control gene products involved in disease processes. In a demonstration of the first approach, the islet-specific miRNA, miR-375, was overexpressed to study the role of this miRNA in pancreatic endocrine cells [97]. Overexpression of miR-375 suppressed glucose-induced insulin secretion. miR-375 modulates glucose-stimulated insulin secretion and exocytosis by blocking the expression of myotrophin, a protein associated with neuronal secretion. 15.10.3

Antagomirs

The second approach to interfere with miRNAs is based on synthetic anti-miRNA oligonucleotides that can be introduced into cells or animals. Different classes of anti-miRNA oligonucleotides have been tested in cell culture and have been reviewed [96]. The first in vivo demonstration was achieved by a cholesterol-conjugated anti-miRNA named an antagomir [98]. The antagomir, complementary in sequence to the murine miR-122, was modified with three chemistries: uniform 2⬘-OMe nucleotides (for sufficient nuclease stability and binding affinity), terminal phosphorothioate linkages (for nuclease stability), and a cholesterol (for liver targeting) conjugated via a hydroxyprolinol-aminocaproic acid tether (Figure 15.18). The silencing of endogenous miRNAs using this antagomir (Figure 15.17[b]) was observed within 24 h after administration and the silencing was specific, efficient, and long lasting. The biological significance of silencing miR-122, an abundant liver-specific miRNA, was evaluated [98]. Northern blot analysis revealed that miR-122 was completely abolished and the effects were long lasting, at least for 23 days. The effects were sequence-specific (mismatched antagomirs were not effective) and miR-122 specific (other miRs such as let-7 and miR-22 were not affected). Gene expression and bioinformatic analysis of messenger RNA from antagomir-treated animals revealed that the untranslated regions of many upregulated genes are strongly enriched in miR-122 recognition motifs, whereas downregulated genes are depleted in these motifs. For example, the aldolase-A gene was upregulated nearly 600% by antagomir treatment; this was used as one of the positive readouts for this antagomir treatment. Several mRNAs in the cholesterol biosynthesis pathway, including the cholesterol biosynthesis target HMGCR (hydroxymethylglutaryl coenzyme-A reductase, the target for many statins), MVK (mevalonate kinase), and FDPS (farnesyl diphosphate

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(miR-122)

(Antagomir-122)

O

O

= O

O

B

B

O

=

= O

OH

OCH3

O O P O O

=

O O P S O

OH O

=

N O

H N O O

Figure 15.18 Antagomir design.

synthetase), were positively regulated by miR-122. Offering further support for the relevance of the miRNA in cholesterol biosynthesis, plasma cholesterol levels were reduced in antagomir-122treated mice by nearly 40%. In the same study, intravenous administration of antagomir against miR-16, which is expressed in almost all tissues, resulted in a marked reduction of miR-16 levels except brain: levels were reduced in liver, lung, kidney, heart, intestine, fat, skin, bone marrow, muscle, ovaries, and adrenals. This experiment demonstrated the biodistribution properties of the cholesterol conjugate and showed that cholesterol-conjugated, modified oligonucleotides function as effective antagomirs. In a related study, mice were dosed with uniform 2⬘-O-MOE phosphorothioate oligonucleotide complementary to miR-122 [99]. Complete inhibition of miR-122 was observed after a 4-week treatment. Inhibition resulted in reduced plasma cholesterol levels, increased hepatic fatty acid oxidation, and a decrease in hepatic fatty acid and cholesterol synthesis rates. In a diet-induced obese mouse model, miR-122 inhibition resulted in decreased plasma cholesterol levels and a significant improvement in liver steatosis; in addition, expression of several lipogenic genes was reduced. The results from both studies suggest that miR-122 is one of the regulators of cholesterol and fatty acid metabolism in the adult liver and show that antagomirs of microRNAs are powerful tools for silencing of specific miRNAs in vivo. These two reports [99,100] strongly suggest that antagomirs will provide a therapeutic strategy for silencing miRNAs and will allow control the miRNAs involved in the diseases described above. 15.10.4

Viral miRNAs

In small-sized viral genomes, miRNAs offer an efficient strategy for specific inactivation of host cell-defense factors. miRNAs have been cloned from herpes viruses, Epstein–Barr virus, human cytomegalovirus, Kaposi’s sarcoma-associated virus, and are predicted in the genomes of doublestranded DNA (dsDNA) viruses such as herpes simplex virus 1 and 2, variola and vaccinia virus, molluscum contagiosum virus, and human adenoviruses and in the genomes of the single-stranded RNA viruses, measles virus, and yellow fever virus [96]. Clear evidence of the importance of miRNA in the viral life cycle has been shown for the simian virus 40 (SV40). The miRNAs from the circular dsDNA SV40 are perfectly complementary to the early viral mRNAs coding for T antigen. The miRNAs accumulate late in infection and reduce the expression of viral T antigens. The cells

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with miRNAs are less sensitive than cells without miRNA to lysis by cytotoxic T cells and trigger less cytokine production by such cells [100]. 15.10.5

Viral Suppression of Silencing

Given the huge number of host miRNAs and the potential for complementarity with viral genomes, viruses have an incentive to interfere with the silencing pathway. There is evidence that some viruses inhibit the RNAi pathway. Primate foamy virus type 1 (PFV-1) expresses a protein that sequesters siRNAs [101]. Adenovirus-infected cells accumulate polymerase III transcripts known as virus-associated RNAs (VA RNAs). The VA RNAs appear to inhibit the RNAi pathway through binding of Dicer as well as through competition for the nuclear export factor [89]. In contrast, hepatitis C virus (HCV) exploits a cellular miRNA to maintain viral abundance. The liver-specific miRNA, miR-122, discussed above in the section on antagomirs is required for high levels of HCV replication [102]. In Huh7 cells containing replicating HCV genomes, the sequestration of miR-122 with antagomirs resulted in a reduction in the amount of HCV RNA. There are two potential binding sites for miR-122 in the HCV RNA: one in the 3⬘ UTR and the other in the 5⬘ UTR. Surprisingly, experiments showed that a direct interaction occurs between miR-122 and the 5⬘ UTR site of HCV RNA. The regulation is likely to occur during replication, rather than during translation or by interference with RNA stability. The binding of miR-122 might allow a conformational rearrangement in the 5⬘ UTR of the HCV RNA that allows replication to proceed or components of the miRISC that are recruited by miR-122 might be required for viral replication [102]. Current treatments for HCV are often ineffective and a compound directed against conserved sequences of a cellular target such as miR-122 could be attractive. The work described above that dissected the role of miR-122 in cellular metabolism is a first step toward development of an miR-directed therapeutic. A recent study with HSV-1 showed that the latency-associated transcript (LAT) gene is responsible for survival of HSV-1 in infected neurons [103]. The microRNA generated from the exon 1 region of the HSV-1 LAT gene (miR-LAT) downregulates two important genes: transforminggrowth factor-
(TGF-
) and SMAD3. Both genes are involved in the TGF-
pathway and can either inhibit cell proliferation or induce cell death. Antagomir approaches to inhibition of miR-LAT could be a viable therapeutic approach for abolishing HSV-1 in neurons.

15.11 CONCLUSIONS AND PERSPECTIVES RNA interference, the mRNA downregulation process based on double-stranded RNases, has given new excitement to the field of oligonucleotide therapeutics [4]. Clearly the award of the Nobel Prize in 2006 to Fire and Mello for their work attests the importance of RNA interference [104]. The two RNAi approaches presented in this chapter are both extremely attractive. The degradation of target mRNA using siRNAs is straightforward conceptually and in a fairly advanced stage clinically whereas targeting miRNAs using antagomirs remains to be fully validated. The clear proof of the precise position of mRNA cleavage as demonstrated by 5⬘-RACE assays is an impressive proof of the mechanism of RNA interference in vivo. This observation also validates the fact that chemically modified siRNAs are accepted by the RNA interference machinery. However, since there is as yet no structural data available for RISC enzymes bound to siRNA/mRNA/antagomir substrates it is too early to predict what kind of chemical modifications will accelerate the RNAi process so that highly potent siRNA drugs can be developed. The chemical modifications used so far in the RNA interference experiments are all derived from antisense oligonucleotide therapeutics and there have been no chemical modifications specifically designed and developed for enhancing interactions with mammalian Argonaute proteins or other components of the RNAi machinery. The importance of improving cellular delivery of siRNA and antagomir molecules cannot be overemphasized.

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In contrast, the delivery-enhancing chemical modifications and systems should not compromise the safety of the drug molecules and must provide pharmacokinetic properties that offer a good therapeutic window and a reasonable dosing regimen.

ACKNOWLEDGMENTS We are grateful to John Maraganore and all members of the scientific team at Alnylam Pharmaceuticals both at Cambridge, MA and Kulmbach, Germany for their invaluable contributions to the significant progress made in the field of RNAi therapeutics. We thank Laura Ramos for excellent administrative assistance.

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78. Morrissey, D. V.; Lockridge, J. A.; Shaw, L.; Blanchard, K.; Jensen, K.; Breen, W.; Hartsough, K.; Machemer, L.; Radka, S.; Jadhav, V.; Vaish, N.; Zinnen, S.; Vargeese, C.; Bowman, K.; Shaffer, C. S.; Jeffs, L. B.; Judge, A.; MacLachlan, I. and Polisky, B. Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs. Nat. Biotechnol., 2005, 23, 1002–1007. 79. Zimmermann, T. S.; Lee, A. C. H.; Akinc, A.; Bramlage, B.; Bumcrot, D.; Fedoruk, M. N.; Harborth, J.; Heyes, J. A.; Jeffs, L. B.; John, M.; Judge, A. D.; Lam, K.; McClintock, K.; Nechev, L. V.; Palmer, L. R.; Racie, T.; Roehl, I.; Seiffert, S.; Shanmugam, S.; Sood, V.; Soutschek, J.; Toudjarska, I.; Wheat, A. J.; Yaworski, E.; Zedalis, W.; Koteliansky, V.; Manoharan, M.; Vornlocher, H.-P. and MacLachlan, I. RNAimediated gene silencing in non-human primates. Nature, 2006, 441, 111–114. 80. Yano, J.; Hirabayashi, K.; Nakagawa, S.-i.; Yamaguchi, T.; Nogawa, M.; Kashimori, I.; Naito, H.; Kitagawa, H.; Ishiyama, K.; Ohgi, T. and Irimura, T. Antitumor activity of small interfering RNA/cationic liposome complex in mouse models of cancer. Clin. Cancer Res., 2004, 10, 7721–7726. 81. Schiffelers, R. M.; Ansari, A.; Xu, J.; Zhou, Q.; Tang, Q.; Storm, G.; Molema, G.; Lu, P. Y.; Scaria, P. V. and Woodle, M. C. Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle. Nucl. Acids Res., 2004, 32, e149/1–e149/10. 82. Zhang Z.; Yang X.; Zhang Y.; Zeng B.; Wang S.; Zhu T.; Roden R. B S; Chen Y. and Yang R. Delivery of telomerase reverse transcriptase small interfering RNA in complex with positively charged single-walled carbon nanotubes suppresses tumor growth. Clin. Cancer Res., 2006, 12, 4933–4939. 83. Song, E.; Zhu, P.; Lee, S.-K.; Chowdhury, D.; Kussman, S.; Dykxhoorn, D. M.; Feng, Y.; Palliser, D.; Weiner, D. B.; Shankar, P.; Marasco, W. A. and Lieberman, J. Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors. Nat. Biotechnol., 2005, 23, 709–717. 84. Hu-Lieskovan, S.; Heidel, J. D.; Bartlett, D. W.; Davis, M. E. and Triche, T. J. Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma. Cancer Res., 2005, 65, 8984–8992. 85. Santel, A.; Aleku, M.; Keil, O.; Endruschat, J.; Esche, V.; Fisch, G.; Dames, S.; Loeffler, K.; Fechtner, M.; Arnold, W.; Giese, K.; Klippel, A. and Kaufmann, J. A novel siRNA-lipoplex technology for RNA interference in the mouse vascular endothelium. Gene Ther., 2006, 13, 1222–1234. 86. Santel, A.; Aleku, M.; Keil, O.; Endruschat, J.; Esche, V.; Durieux, B.; Loeffler, K.; Fechtner, M.; Roehl, T.; Fisch, G.; Dames, S.; Arnold, W.; Giese, K.; Klippel, A. and Kaufmann, J. RNA interference in the mouse vascular endothelium by systemic administration of siRNA-lipoplexes for cancer therapy. Gene Ther., 2006, 13, 1360–1370. 87. Wu, H.; Xu, H.; Miraglia, L. J. and Crooke, S. T. Human RNase III is a 160-kDa protein involved in preribosomal RNA processing. J. Biol. Chem., 2000, 275, 36957–36965. 88. Ouellet D. L; Perron M. P; Gobeil L.-A.; Plante P. and Provost P. MicroRNAs in gene regulation: when the smallest governs it all. J. Biomed. Biotechnol., 2006, 2006, 69616. 89. Sarnow, P.; Jopling, C. L.; Norman, K. L.; Schuetz, S. and Wehner, K. A. MicroRNAs: expression, avoidance and subversion by vertebrate viruses. Nat. Rev. Microbiol., 2006, 4, 651–659. 90. Wightman, B.; Ha, I. and Ruvkun, G. Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4 mediates temporal pattern formation in C. elegans. Cell, 1993, 75, 855–862. 91. Yekta, S.; Shih, I-h. and Bartel, D. P. MicroRNA-directed cleavage of HOXB8 mRNA. Science, 2004, 304, 594–596. 92. Hutvagner, G. and Zamore, P. D. A microRNA in a multiple-turnover RNAi enzyme complex. Science, 2002, 297, 2056–2060. 93. Zeng, Y. Yi, R. and Cullen, B. R. MicroRNAs and small interfering RNAs can inhibit mRNA expression by similar mechanisms. Proc. Natl. Acad. Sci. USA, 2003, 100, 9779–9784. 94. Lewis, B. P.; Burge, C. B. and Bartel, D. P. Conserved seed pairing, often flanked by adenosines, indicates that thousands of human genes are microRNA targets. Cell, 2005, 120, 15–20. 95. Jin, P.; Zarnescu, D. C.; Ceman, S.; Nakamoto, M.; Mowrey, J.; Jongens, T. A.; Nelson, D. L.; Moses, K. and Warren, S. T. Biochemical and genetic interaction between the fragile X mental retardation protein and the microRNA pathway. Nat. Neurosci., 2004, 7, 113–117. 96. Weiler, J.; Hunziker, J. and Hall, J. Anti-miRNA oligonucleotides (AMOs): ammunition to target miRNAs implicated in human disease? Gene Ther., 2006, 13, 496–502. 97. Poy, M. N.; Eliasson, L.; Krutzfeldt, J.; Kuwajima, S.; Ma, X.; MacDonald, P. E.; Pfeffer, S.; Tuschl, T.; Rajewsky, N.; Rorsman, P. and Stoffel, M. A pancreatic islet-specific microRNA regulates insulin secretion. Nature, 2004, 432, 226–230.

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16

Discovery and Development of RNAi Therapeutics Antonin R. de Fougerolles and John M. Maraganore

CONTENTS 16.1 16.2

Introduction .........................................................................................................................466 In Vitro Selection of Lead Candidates ................................................................................466 16.2.1 Potency..................................................................................................................467 16.2.2 Specificity .............................................................................................................468 16.2.3 Stability .................................................................................................................468 16.2.4 Therapeutic Considerations...................................................................................469 16.3 In Vivo Delivery ..................................................................................................................471 16.3.1 Naked siRNA ........................................................................................................471 16.3.1.1 Ocular...................................................................................................471 16.3.1.2 Respiratory ...........................................................................................472 16.3.1.3 Nervous System ...................................................................................474 16.3.2 Conjugation ...........................................................................................................474 16.3.2.1 Cholesterol ...........................................................................................474 16.3.2.2 Other Natural Ligands..........................................................................475 16.3.2.3 Aptamers ..............................................................................................475 16.3.2.4 Small Molecules...................................................................................475 16.3.3 Liposomes and Lipoplexes....................................................................................475 16.3.4 Peptides and Polymers ..........................................................................................477 16.3.5 Antibodies .............................................................................................................478 16.4 Clinical Trials......................................................................................................................478 16.4.1 Ocular....................................................................................................................478 16.4.1.1 VEGF ...................................................................................................480 16.4.1.2 VEGF Receptor....................................................................................480 16.4.2 Respiratory ............................................................................................................480 16.4.2.1 RSV ......................................................................................................480 16.5 Summary .............................................................................................................................480 References ......................................................................................................................................481

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16.1 INTRODUCTION In less than a decade since its discovery, RNA interference (RNAi) as a novel mechanism to selectively silence messenger RNA (mRNA) expression has revolutionized the biological sciences in the postgenomic era. With RNAi, the target mRNA is enzymatically cleaved, leading to decreased levels of the corresponding protein. The specificity of this mRNA silencing is controlled very precisely at the nucleotide level. Given the identification and sequencing of the entire human genome, RNAi is a fundamental cellular mechanism that can also be harnessed to rapidly develop novel drugs against any disease target. The reduction in expression of pathological proteins through RNAi is applicable to all classes of molecular targets, including those that have been traditionally difficult to target with either small molecules or proteins, including monoclonal antibodies. Numerous proof-of-concept studies in animal models of human disease have demonstrated the broad potential applicability of RNAi-based therapeutics. Further, RNAi therapeutics are now under clinical investigation for age-related macular degeneration (AMD) and respiratory syncytial virus (RSV) infection, with numerous other drug candidates poised to advance into clinical development in the years to come. In this review, we will outline and discuss the various considerations that go into developing RNAi-based therapeutics starting from in vitro lead design and identification, to in vivo preclinical drug delivery and testing, and lastly, to a review of clinical experiences to date with RNAi therapeutics. While both nonviral delivery of small interfering RNAs (siRNA) and viral delivery of short hairpin RNA (shRNA) are being advanced as potential therapeutic approaches based on RNAi, this review will focus solely on development of synthetic siRNA as drugs. Synthetic siRNAs can harness the cellular RNAi pathway in a consistent and predictable manner with regard to the extent and duration of action, thus making them particularly attractive as drugs. As a consequence, siRNAs are the class of RNAi therapeutics that is most advanced in preclinical and clinical studies. 16.2 IN VITRO SELECTION OF LEAD CANDIDATES This section highlights the various steps required to identify potent lead siRNA candidates starting from bioinformatics design through to in vitro characterization. The overall scheme for turning siRNA into drugs is summarized in Figure 16.1. The three most important attributes to take into account when designing and selecting siRNA are potency, specificity, and nuclease stability. 5’

3’

3’

5’

2’ Sugar modification

P=S

Sense -------Anti-Sense

• Select siRNA » In silico design » In vitro assays

P=S

• Stabilize siRNA » Chemistry • Select Delivery » Naked » Conjugation » Liposomes » Peptides/Polymers » Antibodies

Figure 16.1 Turning siRNA into drugs. Outline of steps involved in development of an RNAi therapeutic. This three-step process begins with in silico design and in vitro screening of target siRNA, followed by incorporation of stabilizing chemical modifications on lead siRNA as required, and ending with selection and in vivo evaluation of delivery technologies appropriate for the target cell type/organ and the disease setting.

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With regard to specificity of siRNA, the two issues of “off-targeting” due to the silencing of genes sharing partial homology to the siRNA and “immune stimulation” due to recognition of certain siRNAs by the innate immune system have been of special concern. With an increased understanding of the molecular and structural mechanism of RNAi, all issues around lead siRNA selection are better understood and also are now generally resolvable through the use of bioinformatics, chemical modifications, and empirical testing. Thus, it is now possible to very rapidly, in the span of several months, identify potent, specific, and stable in vitro active lead siRNA candidates to any target of interest. 16.2.1 Potency Work by Tuschl and colleagues [1] represented the first published study to demonstrate that RNAi could be mediated in mammalian cells through the introduction of small fragments of doublestranded RNA (dsRNA), termed small interfering RNA, and that siRNAs had a specific architecture comprised of 21 nucleotides in a staggered 19-nucleotide duplex with a 2-nucleotide 3⬘ overhang on each strand (Figures 16.1 and 16.2). Further elaboration and dissection of the RNAi pathway, including insights from X-ray crystallographic structures, have revealed that long dsRNAs are naturally processed into siRNAs via a cytoplasmic RNaseIII-like enzyme called Dicer. A multienzyme complex known as the RNA-induced silencing complex (RISC) then unwinds the siRNA duplex. The siRNA–RISC complex functions enzymatically to recognize and cleave mRNA strands complementary to the “antisense” or “guide” strand of the siRNA. The target mRNA is then cleaved between nucleotides 10 and 11 (relative to the 5⬘ end of the siRNA guide strand). Loading of RISC with respect to the sense and antisense siRNA strands is not symmetrical. The efficiency with which the antisense or guide strand is incorporated into the RISC machinery (versus incorporation of the sense strand) is the most important determinant of siRNA potency. Through analysis of strand-specific

Synthetic siRNAs

dsRNA Dicer

Dicer Cleavage siRNAs Strand separation Natural process of RNAi

RISC Therapeutic gene silencing

Complementary pairing (A)n mRNA Cleavage

mRNA degradation

Cleaved mRNA

(A)n

Figure 16.2 Harnessing the natural RNAi process with synthetic siRNA. Long double-stranded RNA (dsRNA) is cleaved into short stretches of dsRNA called siRNA. The siRNA interact with the RISC to selectively silence target mRNA. siRNA against any mRNA target can be chemically synthesized and introduced into cells, resulting in specific therapeutic gene silencing.

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reporter constructs [2] and large sets of siRNA of varying potency [3,4], it was found that RISC preferentially associates with the siRNA duplex strand whose 5⬘ end is bound less tightly with the other strand. A detailed description of RNAi-mediated silencing as it relates to siRNA and other small RNAs by Sigova and Zamore can be found in Chapter 3 of the book. 16.2.2 Specificity RNAi-mediated silencing of gene expression has been shown to be exquisitely specific as evidenced by silencing fusion mRNA without affecting an unfused allele [5,6] and by studies showing ability to silence point-mutated genes over wild-type sequence [7]. Nevertheless, along with on-target mRNA silencing, siRNA might have the potential to recognize nontarget mRNA, otherwise known as “off-target” silencing. On the basis of in vitro transcriptional profiling studies, siRNA duplexes have been reported to silence multiple genes in addition to the intended target gene under certain conditions. Not surprisingly, many of these observed off-target genes contain regions that are complementary to one of the two strands in the siRNA duplex [8–10]. More detailed bioinformatic analysis revealed that complementarity between the 5⬘ end of the guide strand and the mRNA was the key to off-target silencing, with the critical nucleotides being in positions 2–8 (from the 5⬘ end of the guide strand) [11,12]. Accordingly, careful bioinformatics design of siRNA can reduce potential off-target effects. Further, published work has shown that the incorporation of 2⬘-O-Me ribose modifications into nucleotides can suppress most off-target effects while maintaining target mRNA silencing [13,14]. In fact, incorporation of a single 2⬘-O-Me modification at nucleotide 2 was sufficient to suppress most off-target silencing of partially complementary mRNA transcripts by all siRNAs tested. Thus, in summary, bioinformatics design and position-specific, sequence-independent chemical modifications can be incorporated into siRNA that reduce off-target effects while maintaining target silencing. A second mechanism whereby siRNA can induce potentially unwanted effects is through stimulation of the innate immune system in certain specialized immune cell types. It has been demonstrated that siRNA duplexes contain distinct sequence motifs that can engage Toll-like receptors (TLRs) in plasmacytoid dendritic cells and lead to increased interferon-alpha production [15]. In much the same way that certain CpG motifs in antisense oligonucleotides are responsible for TLR9-mediated immunostimulation, the interferon induction seen with discrete siRNA nucleotide motifs was found to occur largely via TLR-7. Much additional work remains to be done in identifying the full spectrum of immunostimulatory motifs and whether other receptors might also be involved (reviewed in [16]). Several approaches exist to circumvent the immunostimulatory properties of certain siRNA duplexes. First, in vitro assays exist to rank-order duplexes for their ability to induce interferon when transfected into plasmacytoid dendritic cells [15]. Second, several groups have shown that introduction of chemical modifications, such as 2⬘-O-Me modifications, are capable of abolishing immunostimulatory activity [15,17,18]. Third, siRNA delivery strategies can be employed that would avoid the cell types responsible for immune stimulation. 16.2.3 Stability Not surprisingly, numerous studies have shown that the chemical modification of siRNA duplexes, including chemistries already in use with antisense oligonucleotide and aptamer therapeutics, can protect against nuclease degradation with no effect or intermediate effects on activity [19–21]. For instance, introduction of a phosphorothioate (P⫽S) backbone linkage at the 3⬘ end is used to protect against exonuclease degradation and 2⬘ sugar modification (2⬘-O-Me, 2⬘-F, others) is used for endonuclease resistance. With respect to maintenance of RNAi silencing activity, exonuclease-stabilizing modifications are all very well tolerated. Introduction of internal sugar modifications to protect against endonucleases is also generally tolerated but can be more dependent on the location of the modification within the duplex, with the sense strand being more

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amenable to modification than the antisense strand. Nevertheless, using simple, well-described modifications such as P ⫽ S, 2⬘-O-Me, and 2⬘-F, it is possible in most instances to fully nuclease-stabilize an siRNA duplex and maintain mRNA silencing activity. Importantly, the degree of modifications required to fully stabilize the siRNA duplex can generally be limited in extent, thereby avoiding the toxicities associated with certain oligonucleotide chemistries. Improved nuclease stability is especially important in vivo for siRNA duplexes that are exposed to nuclease-rich environments (such as blood) and are formulated using excipients that do not themselves confer additional nuclease protection on the duplex. As might be expected in these situations, nuclease-stabilized siRNA show improved pharmacokinetic properties in vivo (Alnylam, unpublished results). In other situations, when delivering siRNA directly to more nuclease-amenable sites such as the lung or when delivering in conjunction with delivery agents such as liposomes, the degree of nuclease stabilization that is required can be reduced significantly. While the ability of an siRNA duplex to reach its target cell type intact is vitally important, whether nuclease protection confers a measurable benefit once an siRNA is inside the cell remains to be determined. While in vitro comparisons of naked siRNA versus fully stabilized siRNA do not reveal significant differences in longevity of mRNA silencing [22], these studies have typically been performed using rapidly dividing cells, where dilution due to cell division, and not intracellular siRNA half-life governs the duration of gene silencing [23]. With the recent advent of fluorescence resonance energy transfer studies using siRNA [24], it should be possible in the near future to understand more completely the intracellular benefit of nuclease stabilization on the longevity of RNAi-mediated silencing. 16.2.4 Therapeutic Considerations With the identification of active siRNA, a set of rules were initially proposed for selecting potent siRNA duplex sequences [1]. Subsequently, a number of groups have developed more sophisticated algorithms based on empiric testing to identify multiple criteria that can contribute to defining an active siRNA [25–27]. Using current algorithms, sub-nM IC50 in vitro active siRNA can be routinely identified in a quarter to a half of the designed siRNA with a subset of siRNA often demonstrating low pM activity. In designing siRNAs for therapeutic purposes, other considerations beyond an active target sequence exist. Where possible, it is desirable to identify target sequences that have identity across all the relevant species used in safety and efficacy studies, thus enabling development of a single drug candidate from research stage all the way through clinical trials. Other considerations in selecting a target sequence involve the presence of single-nucleotide polymorphisms and general ease of chemical synthesis. Predicting the nucleotide sequence and chemical modifications required to yield an ideal RNAi therapeutic still remains a work in progress. While much progress has been made in understanding what attributes are required to identify an in vitro active and stable siRNA, much less is known about how well those attributes translate into identifying in vivo active siRNAs. For example, many of the issues around specificity are based on in vitro data and their in vivo relevance remains to be determined. For example, the range of off-target genes identified in tissue culture can differ dramatically depending upon the transfection method used to introduce siRNAs into cells [28]. Likewise, induction of innate immune responses by certain siRNAs has been shown to be cell-type specific [29]. At present, in order to identify robust in vitro active lead candidate siRNAs suitable for subsequent in vivo study, the practical and prudent approach is to synthesize and screen a library of siRNA duplexes for potency, specificity, nuclease stability, and immunostimulatory activity. A good example of such an empiric approach was a screen that we conducted for siRNA targeting all vascular endothelial growth factor (VEGF)-A spliced isoforms to treat AMD. Over 200 siRNA with different sequences and chemistries were evaluated from which an optimized clinical candidate, ALN-VEG01, was selected. This optimization procedure resulted in an siRNA with picomolar in vitro activity and sustained silencing in the relevant ocular cell type than was superior to other published VEGF siRNA compounds (Figure 16.3).

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HeLa Cells 0

24h

Transfect siRNA

48h

Change supernatant

Quantitate VEGF by ELISA

%, VEGF protein (rel. to L2000)

100 L2000 80 ALN-VEG01 MM 60 Luciferase siRNA 40 Cand5 VEGF siRNA 20 ALN-VEG01 0 0.01

0.11

1

10

siRNA (nM) (b)

Human ARPE-19 Cells 5 days

1 day

0 Transfect RPE with VEGF siRNA

10 days

Change supernatant & quantitate VEGF by ELISA

%, VEGF protein (rel. to L2000)

120 100 80

30 nM

60

3.33 nM

40 0.37 nM

20 0

0.04 nM 5 days

10 days

ALN-VEG01

5 days

10 days

ALN-VEG01 MM

5 days

10 days

Cand5

Figure 16.3 Identification of highly potent VEGF siRNA, ALN-VEG01. HeLa cells or ARPE-19 human retinal pigment epithelial cell line were plated in 96 well plates and transfected 24 h later with the indicated concentration of siRNA in Lipofectamine 2000. A Lipofectamine-alone control (L2000) is also indicated. At 24 h post-transfection, culture medium was completely removed and 100
l of fresh 10% FBS in DMEM added. Following this medium change, cultured supernatants from HeLa and ARPE-19 cells were collected 24 h (48 h post-transfection) and 96 h (5-day post-transfection) later, respectively. Fresh culture supernatant was added on day 5 to the confluent ARPE-19 cells and supernatants collected again 5 days later (10-day post-transfection). Thus, the effect of siRNA inhibition on VEGF protein production was measured over different time periods post-transfection (HeLa, 24–48 h; ARPE-19, days 1–5, days 6–10). Quantitation of human VEGF protein in the cultured supernatants was by ELISA. Positive control is Cand5 hVEGF siRNA [32] and negative controls include an irrelevant siRNA (luciferase) and an ALN-VEG01 siRNA containing four inverted nucleotide mismatches (ALN-VEG01 MM).

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16.3 IN VIVO DELIVERY Effective delivery is the most challenging remaining consideration in the development of RNAi as a broad therapeutic platform. To date, animal studies using siRNA either have not employed additional formulation (i.e., “naked siRNA”) or have delivered siRNA formulated as conjugates, as liposome/lipoplexes, or as complexes (peptides, polymers, or antibodies). The route of administration of siRNA has also ranged from local, direct delivery to systemic administration. Local delivery or “direct RNAi” has particular advantages for a developing technology in that as with any pharmacologic approach, doses of siRNA required for efficacy are substantially lower when siRNA are injected into or administered at or near the target tissue. Direct delivery also allows for a more focused delivery of siRNA that might circumvent any theoretical undesired side effects resulting from systemic delivery. Systemic delivery of siRNA especially with cholesterol conjugates and liposome formulations have also been widely explored with considerable success. While this section will provide a review of the different delivery approaches utilized with siRNA, it is not an exhaustive description of all in vivo experimentation. Several recent publications offer such a review [20,30,31]. 16.3.1 Naked siRNA Many reports describing success with RNAi in vivo involve direct delivery of “naked” siRNA to tissues such as eye, lung, and central nervous system. As used here, the term “naked” siRNA refers to the delivery of siRNA (unmodified or modified) in saline or other simple excipients such as 5% dextrose (D5W). The ease of formulation and administration using direct delivery of “naked” siRNA to tissues make this an attractive therapeutic approach. Not surprisingly, the initial development of RNAi therapeutics has focused on disease targets and clinical indications (AMD and RSV infection) that allow for direct administration of siRNA to the diseased organ.

16.3.1.1 Ocular Multiple examples of efficacious local delivery of siRNA in the eye exist, where proof of concept has been attained in animal models of ocular neovascularization and scarring using both saline and lipid-based formulations [32–36]. Much evidence suggests that direct administration of “naked” siRNA is able to target cell types in the back of the eye and have profound diseasemodifying effects. Using the optimized VEGF targeting siRNA ALN-VEG01 described above, we have demonstrated robust specific inhibition of pathologic retinal neovascularization in a rat oxygen-induced model of retinopathy (Figure 16.4). Following a single intravitreal injection of saline-formulated ALN-VEG01, we achieved over 75% inhibition of pathological neovascularization with no effect on the normal retinal vasculature. The inhibition seen with ALN-VEG01 was both dose-dependent and specific as a mismatched siRNA showed no inhibition. Interestingly, the degree of inhibition seen with ALN-VEG01 was dramatically more profound than that seen with either a VEGF165-specific aptamer (pegaptanib, approved for intravitreal use in AMD patients) or a VEGF-receptor immunoglobulin fusion protein (Figure 16.4). Separate earlier studies using lipid-formulated VEGF siRNA had shown a reduction of laser-induced choroidal neovascularization (CNV) in a mouse model of AMD [32]; this initial study was followed by nonhuman primate laser-induced CNV study where it was reported that intravitreal injection of a saline-formulated VEGF siRNA was well tolerated and efficacious [33]. Lastly, intravitreal injection of saline-formulated siRNA targeting VEGF receptor-1 was effective in reducing the area of ocular neovascularization by 1/3 to 2/3 in two mouse models [34]. These encouraging proof-of-concept studies in animal models have lead to clinical trials of siRNA targeting the VEGF pathway in AMD.

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(c)

2

1.5

1

75% MM inhibition p = 0.001 vsMM

* Scheffer’s post hoc analysis

49% inhibition vs MM p = 0.18 vsMM

Neovascularization (mm2)

2.5

Irrelevant siRNA

0.5

0 No Inj

PBS

siMM 60 µg

siVEGF 3 µg

siVEGF Pegaptanib VEGF Rc Ig 60 µg 16 µg 1 µg

(d)

(b) 100

Normal vascular area (%)

90 80 70 60

ALN-VEG01

50 40 30 20 10 0 No Inj

Figure 16.4

PBS

siMM 60 µg

siVEGF 3 µg

siVEGF Pegaptanib VEGF Rc Ig 60 µg 16 µg 1 µg

(See color insert following page 270.) ALN-VEG01 specifically inhibits retinal neovascularization in a rat oxygen-induced retinopathy model. Newborn rats were exposed to alternating high oxygen concentrations from days 0–14 as outlined previously [101]. On day 14, therapeutic agents were given once intravitreally (5
l volume) at the amounts indicated and rats placed in room air for the following 6 days (days 14–20). On day 20, rats were sacrificed and flat mount retinal preparations stained with ADPase was used to quantitate (a) pathologic neovascularization and (b) normal vascular development; representative ADPase flat mount preparations are shown following administration of (c) irrelevant control siRNA or (d) ALN-VEG01. Experimental groups: no injection (No Inj), saline (PBS), high- and low-dose ALN-VEG01 siRNA (siVEGF), high-dose ALN-VEG01 mismatch siRNA (siMM), clinical-grade VEGF aptamer (Pegaptanib), and researchgrade VEGF receptor immunoglobulin fusion protein from R&D Systems (VEGF Rc Ig). All groups were scored blinded; N ⫽ 10 per group. Neovascularization data are expressed as mean neovascular area (mm2) ⫾ SE (A) and normal retinal vasculature data are expressed as percentage vascular area (⫾SE). Scheffe’s post-hoc analysis was employed to identify significant differences in both neovascular area and normal vascular area. One of three representative experiments.

16.3.1.2 Respiratory A number of studies have demonstrated that intranasal and orotracheal administration of formulated siRNA can result in a significant target gene silencing in the lung leading to distinct disease-modifying phenotypes (Table 16.1). Typically, siRNAs were administered in concentrations of ⬃100 µg per mouse and were directed against viral or endogenous disease-related targets. Most of the examples of successful direct delivery of siRNA to lung have involved delivery of “naked” siRNA either in saline or with excipients such as D5W or lung surfactants.

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Table 16.1 Successful in Vivo Delivery of siRNA in Pulmonary Disease Target RSV-P, PIV–P SARS HO-1 KC, MIP-2, Fas Angiopoetin 2 DDR1 FLU ⫺ NP, PA, NP ⫹ PA

Formulation

Route

Disease

Reference

Saline or TKO D5W or Infasurf Saline Saline Saline Saline PEI

Intranasal Intranasal Intranasal Intranasal Intranasal Intranasal Intravenous

Viral RSV and PIV Viral SARS Hyperoxic acute lung injury Septic acute lung injury Hyperoxic acute lung injury Bleomycin-induced fibrosis Viral flu

[37] [38] [39] [40,41] [42] [43] [76,79]

In instances when delivery formulations such as Transit-TKO have been used, they are sometimes shown to be dispensable. For instance, a groundbreaking study reported that intranasal instillation of siRNAs directed against viral targets either unformulated or complexed with Transit-TKO reduced the viral load of RSV and parainfluenza virus (PIV), two relevant pathogens in pediatric and immune-compromised patients [37]. In these studies, viral titers in the lung were reduced by more than 3 orders of magnitude in a mouse model of viral infection using siRNA doses as low as 70
g/animal with no observed adverse events. Intranasal instillation of the siRNAs did not induce a type I or type II interferon response measured 2 days after administration. In addition, pathological features of RSV infection such as elevated leukotriene levels, pulmonary inflammation, as well as increased respiratory rate were reduced to baseline levels after siRNA administration. The RSV-specific siRNA-mediated reduction of viral load could be achieved in both prophylaxis and treatment paradigms. In a similar approach, siRNA formulated in D5W was administered intranasally in a nonhuman primate model of SARS corona virus (SCV) infection [38]. Macaques treated with siRNA prior to, simultaneously with, or with repeated doses after viral infection showed a milder response to viral infection as judged by a reduced elevation of body temperature, a key indicator of the severity for SARS-like symptoms. In addition, siRNA treatment resulted in a significant reduction of interstitial infiltrates and pathological changes to the lung as well as in an inhibition of viral replication in the respiratory tract. Both studies demonstrate the potential of RNAi therapeutics to treat viral infection in the respiratory system. A number of studies showed that siRNAs can achieve delivery in pulmonary tissue in certain disease settings. Lee and colleagues demonstrated that siRNA-mediated silencing of hemeoxygenase-1 (HO-1) after intranasal siRNA administration resulted in enhanced apoptosis in an ischemia-reperfusion (I-R) mouse model [39]. While I-R induced HO-1 in a number of organs, siRNA-mediated HO-1 silencing following intranasal administration was restricted to the lung and resulted in a local elevation of FAS expression and caspase3 activity. In addition, biotinylated siRNA was detected histologically in lung parenchyma up to 16 h after instillation. To further understand the pathogenesis of acute lung injury (ALI), siRNA targeting the two chemokines keratinocyte-derived chemokine (KC) and macrophage-inflammatory protein 2(MIP-2) were administered by intratracheal instillation in a hemorrhage-induced and septic challenge model of ALI [40]. KC and MIP-2 lung mRNA levels were decreased by about 50%, resulting in a significant reduction of local IL-6 concentrations, and in the case of MIP-2 siRNA treatment also resulted in reduced neutrophil influx, interstitial edema, and disruption of lung architecture. Silencing with Fas-siRNAs in the same animal model resulted in the reduction of Fas to a level similar to control animals and ameliorated pulmonary apoptosis and inflammation [41]. Recently, in a hyperoxia-based mouse model of ALI, intranasal administration of an angiopoietin 2 (Ang2) siRNA in saline specifically and dramatically ameliorated hyperoxiainduced oxidant injury, cell death, inflammation, vascular permeability, and mortality [42]. Expression of Ang2 in this model is dramatically induced in lung epithelial cells and this was specifically inhibited by Ang2 siRNA but not control siRNA. Lastly, it was demonstrated that intranasal instillation of siRNA targeting Discoidin Domain Receptor 1 (DDR-1) mRNA

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resulted in 60–70% reduction of DDR1 protein in the lung of animals treated with bleomycin to induce pulmonary fibrosis [43]. DDR1 silencing was accompanied by an attenuation of infiltration of inflammatory cells and a reduction of cytokines. As a consequence, bleomycininduced TGF-beta up-regulation was suppressed and collagen deposition was significantly reduced. In sum, these studies exhibit the potential for direct instillation of “naked” siRNA to effectively silence endogenous lung genes and have disease-modifying effects.

16.3.1.3 Nervous System The nervous system is a third area where direct instillation of siRNA in saline has proven successful in validating disease targets in vivo. Direct administration of saline-formulated siRNA by intracerebroventricular, intrathecal, or intraparenchymal infusion resulted in silencing of specific neuronal mRNA targets in multiple regions of the peripheral and central nervous system [44–47]. While the “naked” unformulated siRNA dose typically required for target silencing in these rodent studies is on the order of ⬃0.5 mg/day, use of polymer or lipid-based delivery systems seems to facilitate cellular uptake as effective in vivo doses are ⬃50
g with these delivery systems [48–50]. 16.3.2 Conjugation Direct conjugation with molecular entities designed to help target or deliver drug into the appropriate target cell type is naturally appealing. Through conjugation, the therapeutic can be fixed as a one-component system, thereby significantly reducing the complexity from a chemistry, manufacturing, and controls perspective. For siRNA, conjugation is especially attractive, given the fact that only one of two strands in the duplex is active. Thus, conjugates can be attached to the sense strand without disrupting the activity of the antisense strand. Typically, conjugates have been placed on either the 5⬘ or 3⬘ end of the sense strand, though they can in some instances also be tolerated on the antisense strand. To date, siRNA conjugates have been made using lipophilic molecules, proteins, peptides, and aptamers.

16.3.2.1 Cholesterol While the use of cholesterol conjugates to aid in vivo delivery to liver had been established using antisense oligonucleotides [51–53], the report in 2004 by Soutschek and coworkers [54] using a cholesterol-siRNA conjugate provided the first mechanistic in vivo proof of concept for RNAi. Effective silencing of the apolipoprotein B (ApoB) in mice was demonstrated following intravenous administration of cholesterol-conjugated siRNA duplexes. In these experiments, injections of cholesterol-conjugated ApoB siRNA at a relatively high dose of 50 mg/kg resulted in silencing of the ApoB mRNA by ⬃55% in the liver and ⬃70% in the jejunum, the 2 principal sites for ApoB expression; control cholesterol-conjugated siRNA demonstrated no silencing activity. Critically, the authors went on to demonstrate that the reduction in ApoB mRNA was a result of RNAi, through 5⬘RACE detection of specific mRNA cleavage product. Paralleling the mRNA decrease in ApoB mRNA, ApoB protein levels in the plasma dropped by ⬃70%, and in addition, consistent with the biological function of ApoB, a 35–40% decrease in serum cholesterol levels was also seen. Cholesterol conjugation imparted critical pharmacokinetic and cellular uptake properties to the duplex as evidenced by the fact that the unconjugated ApoB siRNA was rapidly cleared and unable to effect mRNA silencing. The mechanism by which cholesterol conjugates promote improved distribution and cellular uptake is an area of active research, though speculation has centered on incorporation of the cholesterol-siRNA conjugate into circulating lipoprotein particles and then delivery through receptor-mediated processes into hepatocytes. Whether other tissues or cell types can be efficiently targeted by cholesterol conjugates is also another area of interest.

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16.3.2.2 Other Natural Ligands Beyond cholesterol, other natural ligands have been successfully directly conjugated to siRNA duplexes. Among some siRNA conjugates described are membrane-permeant peptides such as penetratin and transportan [55], and shielding groups such as poly ethylene glycol (PEG) [56]. While these conjugates were successfully synthesized with RNAi-silencing activity preserved, robust in vitro or in vivo evidence indicative of an impact on cellular delivery is still lacking. Nevertheless, work done using natural ligands as targeting agents for siRNA formulated in conjunction with delivery vehicles has been positive. Molecules such as transferrin [57], folate [58], and RGD peptide [59] have been introduced into particle-based delivery vehicles to help provide specific targeting of siRNA to cells bearing the natural receptor.

16.3.2.3 Aptamers The use of RNA aptamers as a conjugate for delivery and targeting of siRNA is a promising and elegant approach. In contrast to most of the other described delivery methods, this approach allows for specific targeting of particular cell types and yet, unlike a similar antibody-based approach, has an additional advantage that the therapeutic can be composed entirely of RNA (i.e., RNA aptamer linked to an siRNA). Recently, in vitro and in vivo proof of concept has been generated using aptamers to PSMA, a cell-surface receptor overexpressed in prostate cancer cells and tumor vascular endothelium [60,61]. PSMA aptamers, when either directly linked to siRNA [60] or conjugated through a modular streptavidin bridge [61], were capable of promoting specific cellular uptake and RNAi-mediated silencing of target mRNA in vitro. Using siRNA directed against survival genes (plk1 and bcl-2) directly linked to PSMA aptamer, it was found these RNA chimeras were internalized by cells and resulted in RNAi-mediated target mRNA silencing and cell death [60]. One concern about aptamer-siRNA chimeras is the potential difficulties in the synthesis of long RNA molecules. Short aptamers (25–35 bases) that bind to a wide variety of targets with high affinity have been described [62]. Thus, in theory it is possible to design siRNA-aptamer chimeras that would have a long strand of 45–55 bases, a synthesis length that is within the range of technical and commercial feasibility.

16.3.2.4 Small Molecules Beyond natural ligands and aptamers as targeting vehicles for siRNA, there also exists the possibility of using small-molecule drugs in this fashion. Conceptually, targeting siRNA to specific cell types through conjugation to small molecules (or as discussed below through complexation to antibodies) is appealing. Developing small-molecule conjugates capable of promoting entry of siRNA into cells requires selection of an appropriate small molecule that is capable of efficient cellular internalization and endosomal escape. 16.3.3 Liposomes and Lipoplexes Drugs have traditionally been formulated in liposomes to provide increased pharmacokinetic properties and decreased toxicity profiles. With the advent of RNAi, research in the use of liposomes to deliver drugs into cells has surged. Liposomes are vesicles that consist of an aqueous compartment enclosed in a phospholipid bilayer with drug typically entrapped in the center aqueous layer. When lipids simply complex with nucleic acids to form particles, they are known as lipoplexes and are typical of most commercial transfection agents, such as Lipofectamine 2000 and TKO. Liposomes are multicomponent lipid-based nanoparticle delivery systems typically comprising a lipid component (often containing a cationic or a fusogenic lipid), cholesterol, and a PEG-lipid. Each of these components has a critical function to play in the fusogenicity

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and pharmacokinetic properties of the liposome (for reviews, see [63,64] and Chapter 9 by MacLachlan in this book). Both liposomes and lipolexes have been extensively utilized to deliver siRNA in vitro and in vivo. In vitro transfection of siRNA using lipid-based delivery agents is now a routine laboratory procedure. More recently, significant success has been demonstrated with both local and systemic administration of siRNA. One of the most important advances in the development of RNAi as a therapeutic came with the demonstration by Zimmermann et al. that systemically delivered siRNA in stable nucleic acid lipid particles (SNALPs [17]) was able to dramatically silence apoB in mice and nonhuman primates [65]. Most importantly, a single intravenous dose of 2.5 mg/kg of SNALP-formulated siRNA in cynomolgus monkeys was able to reduce ApoB mRNA levels in the liver by ⬎90%. As expected, accompanying ApoB mRNA silencing, levels of serum cholesterol and low-density lipoproteins were reduced by greater than 65% and 85%, respectively. Remarkably, the durability of silencing following a single intravenous dose of 2.5 mg/kg SNALPformulated siRNA was shown to last for at least 11 days. Treatment with liposomally formulated siRNA was well tolerated in these experiments, with transient increases in liver enzymes as the only reported evidence of toxicity, and these effects may be related to the targeting of ApoB itself. The general applicability of SNALP formulations for hepatic delivery of siRNA was also demonstrated in animal models of HBV and Ebola virus infection [66,67]. Other “non-SNALP” liposomes have also been demonstrated to be effective in delivering siRNA to different organs. Systemic delivery of a cationic liposome-containing TNF
siRNA specifically inhibited TNF
production in knee joints and alleviated disease in a mouse model of rheumatoid arthritis [68], while a liposomally formulated caveolin-1 siRNA reduced target expression by 90% in mouse lung endothelia with concomitant expected physiological effects [69]. In summary, use of lipid-based formulations for systemic delivery of siRNA, especially to liver, represents one of the most promising near-term opportunities for development of RNAi therapeutics. Local administration of liposomes and lipoplexes has also been successful in delivering siRNA to target cell types in the eye, nervous system, and tumors. Within the ocular context, subretinal injection of VEGF siRNA formulated in Transit-TKO was found to reduce choroidal neovascularization in a laser-induced mouse model [32], and subconjunctival injection of TGB- receptor II siRNA in Transit-TKO to suppress inflammation and fibrosis in a mouse subconjunctival scarring model [35]. With regard to the nervous system, intracranial delivery of lipid-complexed siRNA targeting the viral envelope genes protected mice against Japanese Encephalitis Virus- and West Nile Virusinduced encephalitis [50]. Similarly, intrathecal administration of cationic lipid–formulated delta opioid receptor (DOR) siRNA facilitated delivery and specific target gene silencing in the spinal cord and dorsal root, resulting in blocked antinociception by a DOR-selective agonist [48]. Numerous publications have reported successful antitumoral effects following either direct or systemic tumoral injection of lipid-formulated siRNA (for reviews, see [20,30,31,70]). One recent example demonstrated that either intratumor injection or intraperitoneal injection of TransMessenger-formulated siRNA directed against the human papillomavirus E6 oncogene inhibited tumor growth, suppressed E6 expression, and induced tumor apoptosis in a subcutaneous cervical cancer xenograft model [71]. It is important to note that the necessity for lipid-based delivery systems (and this holds true for other delivery systems as well) in many of these direct RNAi applications must be assessed in a target cell– and disease-specific manner. For eye, lung, and nervous system, there are numerous examples of siRNA being successfully delivered in the absence of such agents, and other examples where the converse is true. Lastly, an area of increasing interest is direct application of lipoplexed siRNA to mucosal surfaces such as the vagina and intestine. Intravaginal delivery of lipid-complexed siRNA directed against herpes simplex 2 virus was found to protect mice when delivered before or after lethal herpes virus challenge [72]. Corroborating these results is evidence for specific lamin A/C and CCR5 silencing using the relevant targeting siRNA complexed with Lipofectamine 2000, while similarly complexed irrelevant siRNA had no effect on either lamin A/C or CCR5 expression [73]. Direct

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delivery of a TNF
siRNA in a Lipofectamine formulation has also recently been demonstrated to not only reduce TNF
levels but also attenuate colonic inflammation after enema administration [73]. Intravaginal and intracolonic administration of siRNA using these lipid-based delivery systems was well tolerated in mice with no reported evidence of toxicity or activation of an interferon response [72,73]. Given the relatively easy physical access, large number of potential unmet medical disorders, general tolerability of the siRNA-lipid formulations, and, importantly, demonstrated robust in vivo delivery success, mucosal administration of lipid-formulated siRNA will be a fertile area for development of future RNAi therapeutics. 16.3.4 Peptides and Polymers Complexation of siRNA with positively charged peptides or polymers has been a field of increasing interest [74]. In general, cationic peptides and polymers are used to form complexes with the negatively charged phosphate backbone of the siRNA duplex. This noncovalent association is very stable and results in nanometer-sized particles. One concern with complexation approaches is the ability to prevent aggregation and thus control particle formation in a reproducible manner. This is most often done by either maintaining a net positive charge on the surface or incorporating molecules such as PEG that help stabilize the particle and prevent aggregation. PEG groups are also used with polymers to improve the pharmacokinetic profile, while agents such as folate, transferrin, antibodies, or sugars such as galactose and mannose can be incorporated for tissue targeting [75]. One of the most widely studied polymers for delivery of nucleic acids is polyethylenimine (PEI). PEI polymers are synthetic linear or branched structures with protonable amino groups and high cationic charge densities. Following complexation with siRNA, the cationic polyplexes are thought to interact with the cell surface through electrostatic interaction and are taken up by cells through endocytosis, where intracellularly they act to buffer the low endosomal pH. Endosomal escape is hypothesized to occur due to a “proton sponge” effect, whereby PEI enhances the influx of protons and water leading to endosomal destabilization and osmotic release of the polyplexes into the cytoplasm [30]. Multiple reports of use of siRNA-PEI complexes in vivo now exist. PEI complexed with influenza siRNA demonstrated profound antiviral effects in infected mice [76], while similar targeting of the Ebola L gene resulted in protection against lethal Ebola infection in guinea pigs [67]. Profound antitumor activity was seen upon administration of PEI-siRNA complexes targeting pleiotrophin [77], VEGF [59], and HER2 [78]. Lastly, local injection of PEIsiRNA polyplexes targeting the NMDA receptor subunit NR2B significantly attenuated formalininduced nociception in the rat [49]. One of the main concerns with the use of PEI as a therapeutic delivery vehicle is the extreme toxicity seen at higher doses. In an attempt to expand the safety margin, several groups are optimizing the physical structure of PEI to enable improved in vivo delivery of siRNA [79–81]. In addition to PEI, other synthetic polycations consisting of histidine and polylysine residues have also been evaluated for delivery of siRNA and appear to be have improved in vitro efficacy as compared to standard PEI [82]. Three other polymer approaches that have yielded in vivo siRNA data are chitosan nanoparticles, cyclodextrin-based nanoparticles, and atelocollagen nanoparticles. Chitosan is a welltolerated natural biodegradable polymer that forms cationic complexes with nucleic acids. Effective in vivo RNAi interference was achieved both in bronchiole epithelial cells of transgenic EGFP mice after intranasal administration of chitosan/siRNA formulations [83] and in subcutaneously implanted breast cancer cells of nude mice after intravenous administration of chitosan/RhoA siRNA complexes [84]. An elegant study using transferrin-targeted cyclodextrin-containing polycation nanoparticles demonstrated targeted silencing of the EWS-FLI1 gene product in transferrin receptor expressing Ewing’s sarcoma tumor cells [57]. Systemic administration of atelocollagen-siRNA complexes had marked effects on subcutaneous tumor xenografts as well as on bone metastases [85,86]. Lastly, other related polymer approaches such as PAMAM dendrimers and Poly (DL-lactic-coglycolic acid) (PLGA) nanoparticles are

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also being investigated as vehicles for siRNA delivery [87,88], though in vivo evidence for RNAi-mediated silencing remains to be demonstrated using these approaches. Peptide-based approaches, including use of cell-penetrating peptides, to deliver oligonucleotides constitute an area of active research [64,89–91]. Several peptide-based gene delivery systems such as MPG [92], Penetratin [93], and cholesteryl oligo-D-arginine (Chol-R9) [94] have been shown to promote siRNA uptake in vitro. In the case of Chol-R9, local in vivo administration of complexed VEGF-targeting siRNA, but not complexed scrambled siRNA, led to tumor regression in a mouse model [94]. Lastly, peptide-based approaches are often coupled with other delivery systems, such as liposomes, to enable more targeted delivery of oligonucleotides [59,64,95]. 16.3.5 Antibodies Antibody-based approaches for specific targeted delivery of siRNA hold much promise. Recently, elegant studies have demonstrated that a protamine-antibody fusion protein was able in vitro to selectively deliver siRNA to HIV-envelope expressing B16 melanoma cells or HIVinfected CD4 T cells [96]. Protamine is used for its nucleic acid–binding properties while the Fab fragment was used to mediate receptor-specific binding to only those cells expressing the gp160 HIV envelope protein. Importantly, using gp160-B16 cells as a tumor model, it was shown that siRNA-antibody-protamine complexes when delivered either intratumorally or intravenously are able to specifically deliver siRNA in vivo and retard tumor growth. This study demonstrates the potential for antibodies as a selective cell-type-specific means to deliver siRNA in vivo.

16.4 CLINICAL TRIALS RNAi has advanced from research discovery to clinical trials in a very short span of time. To date, three different RNAi therapeutics are under investigation in clinical trials, with several more poised to enter trials in the coming years. The initial trials have focused on well-validated therapeutic targets such as the VEGF pathway for the wet form of AMD and the RSV genome for the treatment of RSV infection. Furthermore, in these initial trials, direct administration of siRNA (“Direct RNAi”) to the eye and lung for AMD and RSV, respectively, will maximize the chances of delivering sufficient and therapeutically relevant concentration of drug to the tissue of interest. RNAi therapeutics in earlier phases of development are being developed to combat a series of diseases, of which the most advanced include those targeting viral genomes for pandemic influenza (http://www.alnylam.com/therapeuticprograms/programs.asp) and hepatitis C (Sirna-034; http://www.sirna.com/wt/page/anti_viral), and endogenous disease targets such as RTP801 and p53 for wet AMD and acute renal failure, respectively (http://www.quarkbiotech.com/modules/main). Also in late-stage development are siRNA directed against TNF-
for arthritis (http://www.nastech.com/nastech/pipeline), VEGF pathway for oncology (ICS-283; http://www.intradigm.com/products.html), ApoB for dyslipidemias (Pro-B;http://www.protivabio.com/rd/pipeline/), and a protein kinase target for pancreatic cancer (Atu027; http://www.atugen.com/therapeuticpp). The status of the clinical-stage programs is summarized below. 16.4.1 Ocular Ophthalmic indications have historically been very attractive for oligonucleotide-based therapeutics with the first approved oligonucleotide-based therapeutic being Vitravene® for the treatment of CMV retinitis [97] and the most recently (and only second ever) approved oligonucleotide-based therapeutic being Macugen® for the treatment of AMD [98]. Direct injection of drug into the vitreal eye cavity is an efficient and rapid way to get drug targeted to the back of the eye. Following intravitreal injection of 33P-radiolabeled siRNA, we have been able to demonstrate rapid uptake of radioactivity from the vitreous cavity to the back of the eye, and using autoradiography we

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confirmed localization throughout the retinal layers including the retinal pigment epithelial (RPE) cells (Figure 16.5). The RPE is an important producer of VEGF and a key cell type in the pathogenesis of wet AMD [99]. Another advantage of the ocular compartment as an initial clinical target organ is the relatively favorable nuclease environment. As compared to serum, the nucleases found in eye tissue are less aggressive than those found in serum (Alnylam, unpublished results). While this does allow for unmodified siRNA to be injected directly into the vitreal cavity, it is clear from our study that exonuclease-stabilizing chemistries can dramatically improve the amount of intact siRNA found following intravitreal injection (Figure 16.5). The ease of intraocular drug delivery,

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Figure 16.5 (See color insert following page 270.) Beneficial effect of nuclease stabilization on intact ocular drug levels and anatomical distribution of siRNA following intravitreal injection. A 7-day rabbit study was performed to determine the pharmacokinetics of a chemically modified siRNA (P ⫽ S) and its unmodified counterpart. (a) Rapid uptake and distribution of siRNA within ocular tissues. 33P-radiolabeled unmodified and modified compounds (0.4 mg) were injected intravitreally and ocular tissues analyzed after 0.5, 6, 24, 72, and 168 h. Counts per minute (CPM) were measured for different eye compartments (aqueous humor, vitreous, retina, iris, sclera/choroid). Negligible counts were detected in aqueous humor and iris. Analysis of total CPM in retina for the modified siRNA is shown; unmodified siRNA exhibited a similar profile. Concentration of siRNA/gram of tissue was calculated based on CPM assuming 100% intact duplex. Microautoradiography (as visualized under light and dark field microscopy) shows distribution of radiolabeled modified siRNA throughout retina and sclera two days following intravitreal injection. Radiolabel is detected as dark spots under light field and bright spots under dark field. (b) In vivo benefit of exonuclease protection. Ocular tissues (vitreous and choroid/sclera) were subjected to polyacrylamide gel electrophoresis and percentage of intact 33 P-labeled duplex was determined by autoradioluminogram. Exonuclease protection results in greater stability within the vitreous and ocular tissue than the same duplex in its unmodified form. Estimated ocular levels of intact nucleases protected siRNA (based on CPM ⫻ % intact duplex) are approximately 50-fold above the in vitro IC50 7 days after a single intravitreal injection.

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combined with the clinical validation of VEGF as an attractive therapeutic target, led to the rapid development of several RNAi therapeutics in this space.

16.4.1.1 VEGF Cand5, an siRNA targeting all VEGF-A spliced isoforms, has completed a phase-II trial in patients with serious progressive wet AMD and has been reported to provide dose-related benefits against several endpoints including near vision and lesion size (http://www.acuitypharma.com/press/release10.pdf). Cand5 is also being tested for efficacy against diabetic macular edema patients in a phase-II trial that began in early 2006 (http://www.acuitypharma.com/press/release13.pdf).

16.4.1.2 VEGF Receptor Sirna-027, an siRNA targeting one of the VEGF receptors (VEGF-R1), has recently completed phase I trials in patients with wet AMD and was reported to be well tolerated. Additionally, it was also reported to stabilize or improve visual acuity in a subset of patients (http://www.sirna.com/wt/page/ocular). Whether targeting VEGFR1 alone, one of at least two signaling receptors for VEGF action, will prove sufficient to block choroidal neovascularization in AMD remains to be established. 16.4.2 Respiratory There are many advantages in delivering siRNA therapeutics directly to the lungs for targeting lung epithelial cells. These include a noninvasive method of delivery (locally targeted delivery of drugs acting in the lungs can improve efficacy and decrease potential unwanted systemic side effects); direct access to lung epithelial cells with absence of first-pass metabolism; a local lung environment that is relatively nuclease-friendly; and, lastly, evidence that lung epithelial cells are capable of siRNA uptake and RNAi-mediated silencing [37,39]. There now exist numerous reports for in vivo efficacy with siRNA delivered locally to the lung (Table 16.1). While in many instances delivery of naked siRNA in saline has been shown to be effective [37,39,42], multiple approaches to optimize lung delivery are also being investigated. For instance, local delivery of naked siRNA in lung surfactants has been successful in inhibiting replication of SARS in nonhuman primates [38], and siRNA encapsulated in chitosan nanoparticles has been successful in silencing a reporter GFP gene [83]. Whether utilization of naked siRNA, additional formulations, or delivery vehicles is required will depend very much on the molecular target and disease indication being pursued.

16.4.2.1 RSV For RSV infection, two phase-I intranasal trials with ALN-RSV01, an siRNA targeting the viral nucleocapsid (N) gene, have been completed with over 100 healthy adult volunteers, representing one of the largest human studies with an RNAi therapeutic. ALN-RSV01 was found to be safe and well tolerated (http://phx.corporate-ir.net/phoenix.zhtml?c⫽148005&p⫽ irol-newsArticle2&ID⫽849576&highlight⫽). Clinical development of ALN-RSV01 is progressing with human experimental infection studies on-going.

16.5 SUMMARY RNAi has advanced from research discovery to clinical trials in a very short span of time, starting with the now Nobel-prize winning discovery in 1998 by Fire and Mello that long dsRNA could mediate RNAi in Caernorhabditis elegans [100], to evidence in 2001 by Tuschl and colleagues that short synthetic siRNA could induce RNAi in mammalian cells [1], to the present day when three

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different RNAi therapeutics are in human clinical trials and numerous others poised to enter. Since discovery of its utility in mammalian cells 5 years ago, RNAi has revolutionized biomedical research and has spawned a veritable explosion of scientific knowledge covering all aspects of in vitro and in vivo RNAi biology. The principal challenge that remains in achieving the broadest application of RNAi therapeutics is the hurdle of delivery. Tremendous of progress has been made on this front through the use of different approaches, such as conjugation, complexation, and lipidbased approaches, though it is clear that delivery has yet to be solved for all cell types and tissues. Delivery solutions as they arrive will likely need to be tailored for target cell type and the disease indication. As the challenge of siRNA delivery is met, it will be possible to rapidly advance RNAi therapeutics against potentially any disease target into clinical studies. The ongoing clinical trials with siRNA for the treatment of AMD and RSV infection will be the first indicator of the potential for RNAi therapeutics to be a revolutionary new class of drug molecules.

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22. Layzer JM et al.; In vivo activity of nuclease-resistant siRNAs; RNA; 10; 766; 2004. 23. Bartlett DW and Davis ME; Insights into the kinetics of siRNA-mediated gene silencing from live-cell and live-animal bioluminescent imaging; Nucleic Acids Res.; 34; 322; 2006. 24. Raemdonck K et al.; In situ analysis of single-stranded and duplex siRNA integrity in living cells; Biochemistry; 45; 10,614; 2006. 25. Boese Q et al.; Mechanistic insights aid computational short interfering RNA design; Method. Enzymol.; 392; 73; 2005. 26. Yuan B et al.; siRNA Selection Server: an automated siRNA oligonucleotide prediction server; Nucleic Acids Res.; 32; W130; 2004. 27. Pei Y and Tuschl T; On the art of identifying effective and specific siRNAs; Nat. Meth.; 3; 670; 2006. 28. Fedorov Y et al.; Different delivery methods-different expression profiles; Nat. Meth.; 2; 241; 2005. 29. Reynolds A et al.; Induction of the interferon response by siRNA is cell type- and duplex lengthdependent; RNA; 12; 988; 2006. 30. Aigner A; Gene silencing through RNA interference (RNAi) in vivo: strategies based on the direct application of siRNAs; J. Biotechnol.; 124; 12; 2006. 31. Bumcrot D et al.; RNAi therapeutics: a potential new class of pharmaceutical drugs; Nat. Chem. Biol.; 2; 711; 2006. 32. Reich SJ et al.; Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model; Mol. Vision; 9; 210; 2003. 33. Tolentino MJ et al.; Intravitreal injection of vascular endothelial growth factor small interfering RNA inhibits growth and leakage in a nonhuman primate, laser-induced model of choroidal neovascularization; Retina; 24; 132, 660; 2004 34. Shen J et al.; Suppression of ocular neovascularization with siRNA targeting VEGF receptor 1; Gene Ther.; 13; 225; 2006. 35. Nakamura H et al.; RNA interference targeting transforming growth factor-beta type II receptor suppresses ocular inflammation and fibrosis; Mol. Vision; 10; 703; 2004. 36. Campochiaro PA et al.; Potential applications for RNAi to probe pathogenesis and develop new treatments for ocular disorders; Gene Ther.; 13; 559; 2006. 37. Bitko V et al.; Inhibition of respiratory viruses by nasally administered siRNA; Nat. Med.; 11; 50; 2005. 38. Li BJ et al.; Using siRNA in prophylactic and therapeutic regimens against SARS coronavirus in Rhesus macaque; Nat. Med.; 11; 944; 2005. 39. Zhang X et al.; Small interfering RNA targeting heme oxygenase-1 enhances ischemia-reperfusioninduced lung apoptosis; J. Biol. Chem.; 279; 10,677; 2004. 40. Lomas-Neira JL et al.; In vivo gene silencing (with siRNA) of pulmonary expression of MIP-2 versus KC results in divergent effects on hemorrhage-induced, neutrophil-mediated septic acute lung injury; J. Leukoc. Biol.; 77; 846; 2005. 41. Perl M et al.; Silencing of Fas, but not caspase-8, in lung epithelial cells ameliorates pulmonary apoptosis, inflammation, and neutrophil influx after hemorrhagic shock and sepsis; Am. J. Pathol.; 167; 1545; 2005. 42. Bhandari V et al.; Hypoxia causes angiopoietin-2-mediated acute lung injury and necrotic cell death; Nat. Med.; 12; 1286; 2006. 43. Matsuyama W et al.; Suppression of discoidin domain receptor 1 by RNA interference attenuates lung inflammation; J. Immunol.; 176; 1928; 2006. 44. Makimura H et al.; Reducing hypothalamic AGRP by RNA interference increases metabolic rate and decreases body weight without influencing food intake; BMC Neurosci.; 3; 18; 2002. 45. Thakker DR et al.; Neurochemical and behavioral consequences of widespread gene knockdown in the adult mouse brain by using nonviral RNA interference; Proc. Natl. Acad. Sci. USA; 101; 17,270; 2004. 46. Thakker DR et al.; siRNA-mediated knockdown of the serotonin transporter in the adult mouse brain; Mol. Psychiatry; 10; 782; 2005. 47. Dorn G et al.; SiRNA relieves chronic neuropathic pain; Nucleic Acids Res.; 32; e49; 2004. 48. Luo MC et al.; An efficient intrathecal delivery of small interfering RNA to the spinal cord and peripheral neurons; Mol. Pain; 1; 29; 2005. 49. Tan PH et al.; Gene knockdown with intrathecal siRNA of NMDA receptor NR2B subunit reduces formalin-induced nociception in the rat; Gene Ther.; 12; 59; 2005. 50. Kumar P et al.; A single siRNA suppresses fatal encephalitis induced by two different flaviviruses; PLoS Med.; 3; 505; 2006.

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51. Biessen EA et al.; Targeted delivery of oligodeoxynucleotides to parenchymal liver cells in vivo; Biochem. J.; 340; 783; 1999. 52. Bijsterbosch MK et al.; Delivery of cholesteryl-conjugated phosphorothioate oligodeoxynucleotides to Kupffer cells by lactosylated low-density lipoprotein; Biochem. Pharmacol.; 62; 627; 2001. 53. Bijsterbosch MK et al.; Bis-cholesteryl-conjugated phosphorothioate oligodeoxynucleotides are highly selectively taken up by the liver; J. Pharm. Exp. Ther.; 302; 619; 2002. 54. Soutschek J et al.; Therapeutic silencing of an endogenous gene by systemic administration of modified siRNAs; Nature; 432; 173; 2004. 55. Muratovska A and Eccles MR; Conjugate for efficient delivery of short interfering RNA (siRNA) into mammalian cells; FEBS Lett.; 558; 63; 2004. 56. Kim SH et al.; PEG conjugated VEGF siRNA for anti-angiogenic gene therapy; J. Control. Release; 116; 123; 2006. 57. Hu-Lieskovan S et al.; Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing’s sarcoma; Cancer Res.; 65; 8984; 2005. 58. Kim SH et al.; Target-specific gene silencing by siRNA plasmid DNA complexed with folate-modified poly(ethylenimine); J. Control. Release; 104; 223; 2005. 59. Schiffelers RM et al.; Cancer siRNA therapy by tumor selective delivery with ligand-targeted sterically stabilized nanoparticle; Nucleic Acids Res.; 32; e149; 2004. 60. McNamara JO et al.; Cell type-specific delivery of siRNAs with aptamer-siRNA chimeras; Nat. Biotechnol.; 24; 1005; 2006. 61. Chu TC et al.; Aptamer mediated siRNA delivery; Nucleic Acids Res.; 34; e73; 2006. 62. Nimjee SM, Rusconi CP, and Sullenger BA; Aptamers: an emerging class of therapeutics; Annu. Rev. Med.; 56; 555; 2005. 63. Patil SD et al.; DNA-based therapeutics and DNA delivery systems: a comprehensive review; AAPS J.; 7; E61; 2005. 64. Torchilin VP; Recent approaches to intracellular delivery of drugs and DNA and organelle targeting; Annu. Rev. Biomed. Eng.; 8; 343; 2006. 65. Zimmermann TS et al.; RNAi-mediated gene silencing in non-human primates; Nature; 441; 111; 2006. 66. Morrissey DV et al.; Potent and persistent in vivo anti-HBV activity of chemically modified siRNAs; Nat. Biotechnol.; 23; 1002; 2005. 67. Geisbert TW et al.; Postexposure protection of guinea pigs against lethal ebola virus challenge is conferred by RNA interference; J. Infect. Dis.; 193; 1650; 2006. 68. Khoury M et al.; Efficient new cationic liposome formulation for systemic delivery of small interfering RNA silencing tumor necrosis factor-alpha in experimental arthritis; Arthritis Rheum.; 54; 1867; 2006. 69. Miyawaki-Shimizu K et al.; SiRNA-induced caveolin-1 knockdown in mice increases lung vascular permeability via the junctional pathway; Am. J. Physiol. Lung Cell Mol. Physiol.; 290; L405; 2006. 70. Pai SI et al.; Prospects of RNA interference therapy for cancer; Gene Ther.; 13; 464; 2006. 71. Niu XY et al.; Inhibition of HPV 16 E6 oncogene expression by RNA interference in vitro and in vivo; Int. J. Gynecol. Cancer; 16; 743; 2006. 72. Palliser D et al.; An siRNA-based microbicide protects mice from lethal herpes simplex virus 2 infection; Nature; 439; 89; 2006. 73. Zhang Y et al.; Engineering mucosal RNA interference in vivo; Mol. Ther.; 4; 336; 2006. 74. Juliano RL; Peptide-oligonucleotide conjugates for the delivery of antisense and siRNA; Curr. Opin. Mol. Ther.; 7;132; 2005. 75. Merdan T et al.; Prospects for cationic polymers in gene and oligonucleotide therapy against cancer; Adv. Drug Deliv. Rev.; 54; 715; 2002. 76. Ge Q et al.; Inhibition of influenza virus production in virus-infected mice by RNA interference; Proc. Natl. Acad. Sci. USA; 101; 8676; 2004. 77. Grzelinski M et al.; RNA interference-mediated gene silencing of pleiotrophin through polyethyleniminecomplexed small interfering RNAs in vivo exerts antitumoral effects in glioblastoma xenografts; Hum. Gene Ther.; 17; 751; 2006. 78. Urban-Klein B et al.; RNAi-mediated gene-targeting through systemic application of polyethylenimine (PEI)-complexed siRNA in vivo; Gene Ther.; 12; 461; 2005.

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79. Thomas M et al.; Full deacylation of polyethylenimine dramatically boosts its gene delivery efficiency and specificity to mouse lung; Proc. Natl. Acad. Sci. USA; 102; 5679; 2005. 80. Grayson AC et al.; biophysical and structural characterization of polyethylenimine-mediated siRNA delivery in vitro; Pharm. Res.; 23; 1868; 2006. 81. Werth S et al.; A low molecular weight fraction of polyethylenimine (PEI) displays increased transfection efficiency of DNA and siRNA in fresh or lyophilized complexes; J. Control. Release; 112; 257; 2006. 82. Read ML et al.; A versatile reducible polycation-based system for efficient delivery of a broad range of nucleic acids; Nucleic Acids Res.; 33; e86; 2005. 83. Howard KA et al.; RNA interference in vitro and in vivo using a novel chitosan/siRNA nanoparticle system; Mol. Ther.; 14; 476; 2006. 84. Pille JY et al.; Intravenous delivery of anti-RhoA small interfering RNA loaded in nanoparticles of chitosan in mice: safety and efficacy in xenografted aggressive breast cancer; Hum. Gene Ther.; 17; 1019; 2006. 85. Takei Y et al.; A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics; Cancer Res.; 64; 3365; 2004. 86. Takeshita F and Ochiya T; Therapeutic potential of RNA interference against cancer; Cancer Sci.; 97; 689; 2006. 87. Khan A et al.; Sustained polymeric delivery of gene silencing antisense ODNs, siRNA, DNAzymes and ribozymes: in vitro and in vivo studies; J. Drug Target.; 12; 393; 2004. 88. Kang H et al.; Tat-conjugated PAMAM dendrimers as delivery agents for antisense and siRNA oligonucleotides; Pharm. Res.; 22; 2099; 2005. 89. Li W et al.; GALA: a designed synthetic pH-responsive amphipathic peptide with applications in drug and gene delivery; Adv. Drug Deliv. Rev.; 56; 967; 2004. 90. El-Andaloussi S et al.; Cell-penetrating peptides: mechanism and applications; Curr. Pharm. Des.; 11; 3597; 2005. 91. Zatsepin TS et al.; Conjugates of oligonucleotides and analogues with cell penetrating peptides as gene silencing agents; Curr. Pharm. Des.; 11; 3639; 2005. 92. Simeoni F et al.; Insight into the mechanism of the peptide-based gene delivery system MPG: implications for delivery of siRNA into mammalian cells; Nucleic Acids Res.; 31; 2717; 2003. 93. Davidson TJ et al.; Highly efficient small interfering RNA delivery to primary mammalian neurons induces Micro-RNA-like effects before mRNA degragation; J. Neurosci.; 24; 10,040; 2004. 94. Kim WJ et al.; Cholesteryl oligoarginine delivering vascular endothelial growth factor siRNA effectively inhibits tumor growth in colon adenocarcinoma; Mol. Ther.; 14; 343; 2006. 95. Longmuir KJ et al.; Effective targeting of liposomes to liver and hepatocytes in vivo by incorporation of a Plasmodium amino acid sequence; Pharm. Res.; 23; 759; 2006. 96. Song E et al.; Antibody mediated in vivo delivery of small interfering RNAs via cell-surface receptors; Nat. Biotechnol.; 23; 709; 2005. 97. Jabs DA et al.; Fomivirsen for the treatment of cytomegalovirus retinitis. Am. J. Ophthalmol.; 133; 552; 2002. 98. Gragoudas ES et al.; Pegaptanib for neovascular age-related macular degeneration; New Engl. J. Med.; 351; 2805; 2004. 99. Michels S. et al.; Promising new treatments for neovascular age-related macular degeneration; Expert Opin. Investig. Drugs; 15; 779; 2006. 100. Fire A et al.; Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans; Nature; 391; 806; 1998. 101. Penn JS and Rajaratnam VS; Inhibition of retinal neovascularization by intravitreal injection of human rPAI-1 in a rat model of retinopathy of prematurity; Invest. Ophthalmol. Vis. Sci.; 44; 5423; 2003.

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IV

Other Chemical Classes of Drugs

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CHAPTER

17

Optimization of Second-Generation Antisense Drugs: Going Beyond Generation 2.0 Brett P. Monia, Rosie Z. Yu, Walt Lima, and Andrew Siwkowski

CONTENTS 17.1 17.2 17.3

Introduction .........................................................................................................................487 Gapmers ..............................................................................................................................488 Influence of Gap Size .........................................................................................................490 17.3.1 RNase H1 ..............................................................................................................490 17.3.2 Rodent Studies ......................................................................................................492 17.3.3 Monkey Studies.....................................................................................................500 17.3.4 Caveats ..................................................................................................................500 17.4 Influence of Oligonucleotide Length..................................................................................501 17.5 Future Directions ................................................................................................................503 17.6 Conclusions .........................................................................................................................504 Acknowledgments ..........................................................................................................................504 References ......................................................................................................................................505

17.1 INTRODUCTION Chemically modified oligonucleotide-based drugs that act through an antisense mode of action have been shown to suppress RNA translation through various terminating mechanisms (see Chapter 1 of this volume for a review). For example, translation of target RNAs can be blocked by disrupting pre-mRNA splicing or by blocking the assembly of key protein components on the RNA, which are required for efficient mRNA translation [1–3]. These types of mechanisms, which act by sterically blocking key RNA:protein or RNA:RNA interactions, can be referred to as “occupancy-mediated antisense interference.” A second class of antisense terminating mechanism involves the degradation of the target RNA transcript, thereby preventing translation of mRNA into protein. This type of terminating mechanism typically involves the recruitment of endogenous nucleases in the cell to the double-stranded RNA:oligonucleotide duplex, resulting in the degradation of the RNA sense strand, leaving the antisense oligonucleotide strand intact [4]. The two most commonly employed examples that use this type of mechanism, which can be referred to as “degradation-mediated antisense intereference,” are the siRNA approach, which utilizes endogenous RNase III-like enzymes to degrade the RNA sense strand,

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and the more traditional antisense approach, which utilizes endogenous RNase H enzymes to degrade the target RNA strand [4,5]. Numerous examples exist in which antisense activity that is based on “occupancy-mediated antisense interference” has been demonstrated in cells [1,2,6,7]. Although the majority of these examples have focused on cell-culture systems, antisense action based on this mode of activity has also been demonstrated in animals [8,9]. Clearly, this mode of antisense action offers a number of unique opportunities that may not be possible when utilizing a mechanism based on target RNA degradation. For example, it is possible to redirect pre-mRNA splicing and, thereby, affect the ratio of alternatively spliced protein products when utilizing occupancy interference mechanisms [2,3,7,8]. Additionally, it is possible to utilize oligonucleotide chemistries more effectively that confer unique advantages to antisense drugs when utilizing an occupancy interference mechanism because many oligonucleotide chemistries that offer desirable properties do not support (i.e, serve as substrates) the enzymatic activity of RNase enzymes [5,10,11]. Nevertheless, a large body of experimental evidence exists supporting the conclusion that, in general, antisense mechanisms of action that are based on the degradation of target RNA are the most effective mode of action for antisense drugs in cell culture and in animals. This conclusion is based in part on the superior potency that is generally observed for antisense drugs that utilize RNA degradation mechanisms relative to nondegradation mechanisms, the availability of greater target RNA sequence space for identifying optimized antisense inhibitors for antisense drugs that promote RNA degradation, and the superior versatility and efficiency that is inherent to the identification of optimized antisense inhibitors that utilize a mechanism of action based on RNA degradation. This conclusion is further supported by the fact that the majority of scientific publications in the field and the majority of antisense-based drugs being evaluated in clinical trials today utilize this mechanism of action to interfere with RNA function. As mentioned above, the two most commonly employed antisense mechanisms that act by promoting target RNA degradation utilize either RNase III-like enzymes, as is the case with the siRNA approach, or the RNase H family of enzymes, which is the more common and traditional approach for harnessing antisense activity in cells. To effectively utilize either mechanism in animals,chemical modifications are required to provide sufficient oligonucleotide stability while providing an acceptable pharmacokinetic profile to permit sufficient tissue penetration and distribution. However, RNase enzymes, including RNase III-like enzymes and RNase H enzymes, are exquisitely sensitive to most chemical modifications and do not tolerate many of the preferred oligonucleotide modifications that have been discovered to date. Therefore, strategies have been developed to circumvent these limitations while maintaining some of the benefits that these chemical modifications offer for antisense activity. This chapter will summarize recent progress in the optimization of oligonucleotide design to increase antisense potency in animals by maximizing RNase H activity for oligonucleotides that contain chemical modifications that do not support RNase H activity, but yet offer other benefits for antisense drug technology. Readers are directed to other chapters in this volume to review recent progress in the medicinal chemistry of oligonucleotides that act through siRNA pathways.

17.2 GAPMERS First-generation (Generation 1.0) phosphorothioate oligodeoxynucleotides support RNase H activity, provide acceptable pharmacokinetic properties that confer antisense activity in cell culture and in animals, and are generally well tolerated [12]. However, this chemistry has a number of limitations that can be addressed by employing high-affinity 2⬘-sugar modifications in combination with the phosphorothioate backbone. As mentioned above, 2⬘-sugar modifications in general do not support the highly preferable RNase H terminating mechanism of antisense action. One commonly employed approach for promoting RNase H-dependent antisense activity in cells that allows for the inclusion of beneficial chemical modifications is the chimeric oligonucleotide strategy, which is sometimes referred to as the “Gapmer Strategy,” and the oligonucleotides either as gapmers, gapmer

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oligonucleotides, or as chimeric oligonucleotides [11,13]. Gapmer oligonucleotides typically contain modifications that do not support RNase H activity on their ends (“wings”) while maintaining a central “gap” of 2⬘-deoxynucleotides which serve to support the RNase H mechanism (Figure 17.1). This strategy is particularly useful for the employment of 2⬘-sugar modifications, like 2⬘-O-methyl (2⬘OMe) or 2⬘-O-methoxyethyl (2-MOE), which do not support RNase H activity. The inability of oligonucleotides containing 2⬘-sugar modifications to support RNase H activity is due to the formation

(A) Base

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O OCH3 O O PS O O Base O

O

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Figure 17.1 Chimeric oligonucleotide design (“Gapmers”). (A) Generic design of 2⬘-ribose-modified chimeric oligonucleotides containing uniformly modified phosphorothioate backbones. The number of ribose modifications on the “wings” of the oligonucleotide can vary but are typically 3–5 bases in length. The central RNase H-sensitive 2⬘ deoxy gap has strict minimum requirement of five bases to support RNase H activity, and lengthening this gap further increases RNase H efficiency. (B) Examples of 2⬘-ribose modifications that do not support RNase H activity but have been investigated using the chimeric oligonucleotide design to utilize the RNase H terminating mechanism of antisense action.

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of RNA:RNA-like duplexes, rather than RNA:DNA-like duplexes, which are required for RNase Hcleavage of the target RNA strand. However, oligonucleotides containing 2⬘-sugar modifications are relatively simple to make, and these modifications can offer significant improvements in nuclease stability and /or affinity for target RNA, resulting in improved duration of action and potency in animals [13]. Thus, chimeric oligonucleotides permit a compromise between the desire to employ beneficial oligonucleotide modifications, like 2⬘-MOE, while maintaining the ability to employ the highly effective RNase H terminating mechanism of antisense action. The employment of chimeric oligonucleotides containing 2⬘-sugar modifications in their wings was shown in the early 1990s to provide a number of beneficial effects for antisense activity [11,13,14]. For example, 2⬘-modified oligonucleotides were shown to improve potency in a manner that correlated directly with the increased affinity conferred by the particular 2 modification being utilized. As illustrated in Figure 17.2, deoxy gaps equal to or greater than five bases in length were found to be sufficient to confer RNase H activity under cell-free conditions and RNase H-dependent antisense activity in cells [11]. As a result, gapmer oligonucleotides, typically 20 bases in length and containing central RNase H-dependent gaps five bases or slightly longer in length, surrounded by high-affinity modifications like 2⬘-fluoro and 2⬘-OMe on their ends, were employed and found to improve antisense potency greater than 10-fold in cell-culture systems relative to phosphorothioate oligodeoxyunucleotides (Table 17.1, [11]). Subsequently, the invention of the second-generation (Generation 2.0) 2⬘-MOE modification led to further advancements in antisense action through the utilization of the gapmer approach [13,15]. This chemistry not only conferred improved potency due to increased affinity, but also conferred increased stability against nuclease-mediated degradation, resulting in significant improvements in antisense duration of action in cell culture and in animals (Figure 17.3). Furthermore, 2⬘-MOE modifications reduce plasma protein binding of oligonucleotides and, as a result, can alter the relative distribution to kidney and liver (see Chapter 7 for more detailed discussions). However, most of these early studies were limited to cell-culture systems, and the optimization of gapmer oligonucleotides for in vivo applications had not yet been conducted. This is an important point to consider since the central deoxy gap is more susceptible to nuclease-mediated degradation than the stabilized wings of the oligonucleotide, and therefore, deoxy gaps would eventually need to be optimized for both RNase H activity, affinity, and for stability in animals. Moreover, little was known about the sequence preferences or oligonucleotide structural preferences for RNase H activity, which is another important consideration when attempting to maximize activity of gapmer oligonucleotides. For example, the effects of oligonucleotide gap sequence, the influence of the surrounding “wing” chemistry, the optimization of gap size, and the influence of oligonucleotide length have all now been shown to influence RNase H activity under cell-free systems and in cells [5,16,17]. Thus, although the original studies that led to the development of second-generation antisense drugs resulted in significant improvements in potency and duration of action, advancements made in our understanding of RNase H enzymology and the need to optimize these drugs for in vivo applications resulted in subsequent efforts to optimize chimeric oligonucleotide structure for maximizing RNase H activity while optimizing pharmacokinetics for in vivo applications. The resulting oligonucleotides derived from these efforts that contain 2⬘-MOE modifications are classified as “Generation 2.2,” since they contain the same oligonucleotide chemistry as Generation 2.0 and employ the RNase H terminating mechanism of action, but differ simply in their structural design in order to maximize RNase H activity, target RNA affinity, oligonucleotide stability, and to optimize pharmacokinetic properties.

17.3 INFLUENCE OF GAP SIZE 17.3.1 RNase H1 Two RNase H isoforms (RNase H1 and H2) have been shown to exist in mammalian cells, which differ slightly in their substrate requirements and their pattern of tissue expression [5,18]. The RNase

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Figure 17.2 RNase H activity and antisense activity of 2⬘-O-methyl chimeric phosphorothioate antisense oligonucleotides. (A) 2⬘-O-methyl chimeric phosphorothioate oligonucleotides targeted to mutant Ha-ras codon 12 sequence. 2⬘-O-methyl substituents are shown in boxes. (B) RNase H cleavage of end-labeled, 25-mer RNA containing Ha-ras codon 12 sequences following preannealing with the oligonucleotides shown in panel A. Cleavage reactions were performed in HeLa cell nuclear extracts. (C) Antisense activity of chimeric 2⬘-O-methyl oligonucleotides against Ha-ras in HeLa cells. HeLa cells transfected with a Ha-ras expression plasmid and a ras-responsive reporter gene were treated with oligonucleotides in the presence of cationic lipid for 24 h and reporter gene activity (luciferase assay) was quantitated. (From Monia, B.P., et al. J. Biol. Chem. 268: 14514–14522, 1993. With permission.)

H1 isoform has been shown to be the primary RNase H responsible for mediating antisense action in cells [19]. With the cloning of the human RNase H1 gene, more direct assays became available for characterizing the substrate preferences for this enzyme when acting on RNA:oligonucleotide duplexes [5,18]. These assays were particularly useful for characterizing the influence of oligonucleotide modifications on RNase H1 activity and, therefore, on antisense activity overall [5,16,18]. Some of the first observations that were made using purified RNase H1 enzyme were that the phosphorothioate modification itself inhibited RNase H1 activity at high concentrations, but at lower concentrations were actually superior RNase H1 substrates relative to phosphodiester oligonucleotides [5]. Furthermore, 2⬘ modifications in the gapmer wings were found to inhibit RNase H1 activity

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Table 17.1 Correlation of Tm with Antisense Activity for Chimeric Oligonucleotides in Cell Culture

CCACA

C CGACG G CGCCC O

O

2⬘⬘ Modification (R) Deoxy Pentoxy Propoxy Methoxy Fluoro

B

O

R

Tm(°C) 64.2 68.5 70.4 74.7 76.9

IC50 (nM) 150 150 70 20 10

Note: Tm values for 29-modified chimeric oligonucleotides were determined. IC50 values for inhibition of Ha-ras gene expression in a ras-luciferase cotransfection cell-culture assay in HeLa cells. Sequences contained within box were modified at the 29-sugar position with indicated substituents. Source: From Monia, B.P. et al. J. Biol. Chem. 268: 14514–14522, 1993. With permission.

directly, suggesting that gapmers with shorter wings would serve as better substrates for RNase H relative to oligonucleotides with longer wings. Thus, the typical second-generation “5-10-5” ASO that was commonly employed in antisense studies was actually a relatively poor substrate for RNase H1 activity under cell-free conditions. This latter point is illustrated in Figure 17.4A in which the initial rate of cleavage of RNase H1 enzyme against RNA duplexed to either a uniform 20-base deoxy phosphorothioate or a “5-10-5” MOE gapmer phosphorothioate is measured. As shown, the “5-10-5” gapmer was found to be a substantially poorer substrate for RNase H1 relative to the deoxy phosphorothioate oligonucleotide. In parallel, studies were performed to characterize deoxy gap length requirements for purified RNase H1 in cell-free systems [5]. As shown in Figure 17.4B, RNase H1 has a strict requirement for a minimum deoxy gap length of five residues to cleave duplexed RNA, and lengthening gap size further results in substantial increases in RNase H1 enzyme activity, confirming previous reports on deoxy gap restrictions on RNase H activity using HeLa cell nuclear extracts [11]. Thus, these findings, coupled with the observation that the chemical modifications in the wings of gapmers negatively impact RNase H activity, suggested that ASO potency might be improved by optimizing deoxy gap length and wing content when utilizing an RNase H mechanism of action. 17.3.2 Rodent Studies As described above, increasing 2⬘-deoxy gap length results in increased RNase H activity. This increase in activity is proportional to gap length for gaps five bases in length or larger. Gaps smaller than five bases in length do not support RNase H activity. To test the hypothesis that increasing RNase H activity by increasing gap length results in increased antisense potency in animals, a series of studies were conducted in mice comparing the effects of standard second-generation 2⬘MOE gapmers containing 10-base deoxy gaps with 2⬘ MOE gapmers containing larger gap lengths. A second-generation “5-10-5” MOE gapmer targeted to the tumor suppressor gene PTEN has been fully characterized in mice and shown to produce excellent suppression of PTEN mRNA and

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C-raf mRNA (% control)

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2′-O-propyl

Figure 17.3 Improved antisense potency exhibited by second-generation MOE gapmers in cell culture and in mice. The sequence and design of a first-generation phosphorothioate oligodeoxynucleotide and a second-generation chimeric gapmer targeted to c-raf kinase are shown above with the 2⬘-MOE-modified portion of the molecule indicated with underline. (A) Dose-dependent suppression of c-raf mRNA levels in T24 bladder carcinoma cells comparing second-generation chemistry versus second-generation chemistry. (B) Dose-dependent suppression of c-raf mRNA levels comparing second-generation chemistry versus other 2⬘-ribose modifications. See Ref. 10 for cell-culture methods. (C) Dose-dependent suppression of c-raf mRNA levels in mouse liver following parenteral administration of a second-generation 2⬘ MOE gapmer versus a 2⬘-O-methyl gapmer or a 2⬘-O-propyl MOE gapmer. See Ref. 22 for methods.

protein levels in mouse liver following parenteral administration [20]. To test the influence of gap size on activity for this ASO, a “2-16-2” analog was created and tested for its ability to support RNase H1 activity under cell-free conditions and to suppress PTEN levels in mouse liver. As expected, the “2-16-2” ASO was found to be superior to its “5-10-5” counterpart in supporting RNase H1 activity in a cell-free assay (Table 17.2). To compare the effects of this “2-16-2” gapmer

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION (A) 20

Vo (nM/min)

1.5 1.0 0.5 0.0 20-Base Deoxy P=S

(B)

Figure 17.4

5-10-5 MOE Gapmer

RNase H1 cleavage rate (pmol/liter/min)

Compound

Gap size

Structure

17mer P=S

17

CCACACCGACGGCGCCC

4034 ± 266

4-9-4

9

CCACACCGACGGCGCCC

1081 ± 168

5-7-5

7

CCACACCGACGGCGCCC

605 ± 81

6-5-6

5

CCACACCGACGGCGCCC

7-4-6

4

CCACACCGACGGCGCCC

330 ± 56 N.D.

7-3-7

3

CCACACCGACGGCGCCC

N.D.

Effects of 2⬘-substitution and deoxy-gap size on digestion rates by human RNase H1. Substrate duplexes were hybridized, and initial rates were determined as described in reference 5. (A) Initial rates of RNase H1 cleavage of a first-generation 20-base phosphorothioate (P⫽S) oligodeoxynucleotide (Deoxy) versus a “5-10-5” MOE gapmer duplexed with a complementary 20-base RNA. The RNA sequence corresponds to a segment of the human Bcl-X mRNA. (B) Initial rates of RNase H1 cleavage of a first-generation 17-base phosphorothioate oligodeoxynucleotide versus 2⬘-O-methyl-modified chimeric phosphorothioate oligonucleotides in which the RNase H-sensitive deoxy gap is of varying length. 2⬘-O-methyl modifications are indicated with underline. The RNA substrate sequence corresponds to a segment of the human Ha-ras mRNA. N.D. ⫽ no cleavage detected. (From Wu, H. et al., J. Biol. Chem. 274: 28270–28278, 1999. With permission.)

with its “5-10-5” counterpart on PTEN levels in vivo, mice were treated with increasing dose levels of the two ASOs over a 3-week period and PTEN mRNA levels were evaluated in liver. Treatment with the gap widened ASO resulted in a significant improvement in antisense potency for reducing PTEN mRNA levels (Figure 17.5A; Table 17.2). Approximate ED50 values for the “2-16-2” MOE gapmer versus the “5-10-5” ASO were 4.3 versus 13.7 mg/kg; an improvement in potency based on dose administered of approximately threefold. Oligonucleotide concentrations in liver at the termination of the study were also determined for the two antisense compounds and approximate EC50 values calculated for reduction in PTEN mRNA levels (Figure 17.5B; Table 17.2). Treatment with the gap-widened “2-16-2” MOE gapmer resulted in a significant improvement in potency relative to the “5-10-5” gapmer as reflected by an approximate fivefold reduction in EC50 value for the “2-16-2” gapmer relative to its “5-10-5” counterpart. Tissue half-lives for liver and kidney for the two gapmer configurations were also approximated from this study (Table 17.2). Consistent with previous reports, the “5-10-5” MOE gapmers displayed long tissue half-lives in both liver (t1/2 ⫽ 14.5 days) and kidney (t1/2 ⫽ 12.9 days), which has been shown to be a function of their remarkable resistance to nuclease-mediated cleavage [15,21]. Long kidney and liver tissue half-lives were also observed for the “2-16-2” MOE gapmer (t1/2 ⫽ 8.8 and 11.7 days, respectively). However, these half-lives were significantly shorter relative to the “5-10-5” gapmer. Nevertheless, the tissue half-lives observed for the “2-16-2” gapmer would

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PTEN liver mRNA 120

PTEN RN A (% saline)

495

Generation 2.0 Generation 2.2

100 80

Gen 2.0

60 Gen 2.2

40 20 0 0

2 4 6 ASO dose (µmol/kg)

(B) PTEN mRNA (% control)

120 100 80 Observed Predicted Observed Predicted

60 40 20 0 0

100 200 300 400 Concentration in liver (µg/g)

500

Figure 17.5 Relative potency of a “5-10-5” MOE gapmer (Generation 2.0) and a “2-16-2” MOE gapmer (Generation 2.2) targeted to PTEN in mouse liver. Animals were dosed with equivalent molar doses of MOE gapmer over a 3-week period and PTEN RNA levels were determined in mouse liver by quantitative RT-PCR as described [20]. Oligonucleotide concentrations were determined in mouse liver by capillary gel electrophoresis as described [31].

still be predicted to support infrequent parenteral dosing regimens (e.g., once/week) while reducing excessive tissue accumulation, which could have the added benefit of improving the toxicity profile of MOE gapmer compounds overall. To determine the general applicability of the increased potency observed for gap-widened ASOs targeting PTEN, additional targets were evaluated in which second-generation “5-10-5” gapmers were compared to gapmers with larger gaps in mice. One of these studies involved the target TNF receptor-associated adaptor protein, TRADD, and the evaluation of MOE gapmers 18 nucleotides in length targeted to TRADD in mice [22]. In this series of studies, MOE gapmers containing deoxy gaps between 6–14 bases in length were evaluated for RNase H1 activity and suppression of TRADD mRNA levels in mouse liver. As expected, RNase H1 activity increased in proportion to deoxy gap size (not shown), as was observed for other gap-widened ASOs described above (Figure 17.2, Figure 17.4; Table 17.2). In this study, mice were treated with increasing doses of MOE gapmers that were equivalent on a molar scale (⬃10–40 mg/kg) twice per week over 3 weeks and ED50 values were calculated based on the suppression of TRADD mRNA levels in liver (Figure 17.6). All of the MOE gapmers tested in this study significantly reduced TRADD mRNA levels in liver with the exception of the “6-6-6” MOE gapmer, which was a relatively poor substrate for RNase H1 activity in vitro and did not affect liver TRADD mRNA levels in mice. No significant differences were observed in ED50 for the MOE gapmers containing deoxy gaps between 8 and 12 bases in length. However, a significant improvement in ED50 was observed for the “2-14-2” MOE gapmer relative to the other MOE gapmers tested. Although the improved potency observed for the

8.95

CTGCTAGCCTCTGGATTTGA

SD

1.06

0.414 4.3

13.7

ED50 (mg/kg) SD

2.20

4.60

EC50

47.0

219.0

3

29

SD

8.8

14.5

Liver (days)

11.7

12.9

Kidney (days)

Note: Substrate duplexes were hybridized and initial rates were determined as described in Ref. 5. Animals were dosed with equivalent molar doses of MOE gapmer over a 3-week period and PTEN RNA levels were determined in mouse liver by quantitative RT-PCR as described [20]. Oligonucleotide concentrations were determined in mouse liver by capillary gel electrophoresis as described [31].

6.89


M/min) Rate (


Structure

CTGCTAGCCTCTGGATTTGA

Compound

Tissue Half-Life

496

5-10-5 MOE gapmer 2-16-2 MOE gapmer

Antisense Activity

9:04 AM

RNase H1 Cleavage

5/25/2007

Table 17.2 Relationship between RNase H1 Activity and Antisense Potency for MOE Gapmers Targeted to PTEN in Mouse Liver

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(A)

Effects of gap size on antisense activity against TRADD mRNA in mouse liver Compound

ED50 (mg/kg)

6-6-6

115 ± 13.1

5-8-5

11.1 ± 2.2

4-10-4

13.6 ± 1.4

3-12-3

11.2 ± 0.6

2-14-2

8.2 ± 0.8

(B)

120 TRADD mRNA (% control)

2-14-2 100

4-10-4

80 60 40 20 0 0

10

20 30 Dose (mg /kg /dose)

40

50

Figure 17.6 Relative potency of MOE gapmers targeted to TRADD with variable deoxy gap lengths on TRADD mRNA levels in mouse liver. Animals were dosed with equivalent molar doses of MOE gapmer over a 3-week period and TRADD RNA levels were determined as described in Figure 17.5. (A) Relative ED50 values for TRADD mRNA suppression by various MOE gapmers in mouse liver. (B) Comparison of dose-dependent suppression of liver TRADD mRNA levels in mice dosed with either a “4-10-4” MOE gapmer or a “2-14-2” MOE gapmer. Methods for animal dosing and RNA analysis are as described in Figure 17.5.

“2-14-2” TRADD MOE gapmer is consistent with the improved potency observed for the “2-16-2” MOE gapmer targeted to PTEN, it was surprising that the “5-8-5” MOE gapmer displayed comparable potency to the “4-10-4” and “3-12-3” MOE gapmers. These observations are most likely explained by the fact that a number of factors are known to influence antisense potency in addition to RNase H activity, including RNA structure and the ability of different oligonucleotide chemistries (and designs) to invade such structures, and differences in protein binding conferred by differences in oligonucleotide chemistry and structure, which will influence oligonucleotide pharmacokinetics. Nevertheless, consistent with the effects observed for PTEN, improving RNase H1 activity for the TRADD MOE gapmer was associated with improved antisense potency in mice. ISIS 113715 is a second-generation “5-10-5” gapmer targeted to the tyrosine phosphatase PTP-1B, which is a key regulator of insulin signaling in insulin-sensitive tissues [23]. Administration of ISIS 113715 to diabetic animals has been shown to specifically reduce PTP-1B levels in liver and adipose tissue and to improve insulin sensitivity [24,25]. Based in part on these findings, ISIS 113715 is currently under evaluation for the treatment of type 2 diabetes in Phase 2 clinical trials [26]. To determine whether gap optimization could improve RNase H activity in vitro

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and pharmacological activity in diabetic animals, gap-widened analogs of ISIS 113715 were designed and tested. In addition to the “5-10-5 gapmer, ISIS 113715, two additional analogs were examined; a “4-12-4” MOE gapmer and a “3-14-3” MOE gapmer were examined for their relative effects on RNase H1 activity. In addition, a uniformly 2⬘-MOE-modified and a “gap disabled” MOE gapmer, in which MOE residues were interspersed to disrupt the RNase H-sensitive deoxy gap, were included as a negative control in this assay since neither contains the minimum deoxy gap size required (five nucleotides in length) to support RNaseH activity. Consistent with the findings made against the PTEN target, widening the RNase H-sensitive deoxy gap beyond 10 bases resulted in improved RNase H1 activity, with the “3-14-3 design displaying significantly better activity versus the “4-12-4” design, and the “4-12-4” design showing significantly better activity versus its “5-10-5” counterpart (Figure 17.7). Both negative control oligonucleotides supported RNase H1 activity as predicted, with the uniformly modified oligoucleotide displaying no activity and the “gap disabled” oligonucleotide displaying ⬃10-fold less activity relative to the 2⬘ MOE gapmers. As mentioned above, the “5-10-5” MOE gapmer, ISIS 113715, has shown antidiabetic activity in a range of animal models of diabetes. To determine whether the increased RNase H1 activity conferred by widening the gap of ISIS 113715 would result in improved pharmacological activity, the “3-14-3” MOE gapmer derivative was tested in ZDF diabetic rats at two dose levels (12.5 and 25 mg/kg) over a 4-week period for its effects on glucose levels relative to ISIS 113715 (Figure 17.8A). As expected, treatment with ISIS 113715 resulted in a significant and time-dependent reduction in glucose levels in these hyperglycemic animals. However, treatment

(A)

Compound

RNase H1 cleavage rate (µM/min)

Structure

Uniform 2′MOE

GCTCCTTCCACTGATCCTGC

0.000 ± 000

Gap Disabled

GCTCCTTCCACTGATCCTGC

0.194 ± 0.023

5-10-5

GCTCCTTCCACTGATCCTGC

1.731 ± 0.104

4-12-4

GCTCCTTCCACTGATCCTGC

2.136 ± 0.040

3-14-3

GCTCCTTCCACTGATCCTGC

2.302 ± 0.086

(B)

3.000 Rate (nM/min)

2.500 2.000 1.500 1.000 0.500 0.000 Full MOE

Gap disabled

5-10-5

4-12-4

3-14-3

Figure 17.7 Effects of PTP-1B targeted MOE gapmer design on RNaseH1 digestion rates. MOE gapmers of varying design were evaluated for their ability to support human RNase H1 activity following hybridization to complementary RNA that corresponds to a segment of the human PTP-1B mRNA. (A) Cleavage rates of MOE gapmers. 2⬘-MOE modifications are indicated with underlines. (B) Bar graph representation of RNase H cleavage rates shown in panel A. Methods are as described in Ref. 5.

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with the “3-14-3” analog resulted in significantly improved pharmacological activity relative to ISIS 113715, which was first observed 2 weeks following initiation of dosing and continued throughout the remainder of the study. Because it is known that reducing 2⬘-MOE content in an oligonucleotide can increase plasma protein binding (see Chapter 7 for a more detailed discussion), altered tissue distribution might also be expected for the “3-14-3” MOE gapmer relative to the “5-10-5” gapmer. Therefore, MOE gapmer tissue concentrations were also measured in both liver and kidney (Figure 17.8B). Consistent with the observations made for the PTEN target, oligonucleotide concentrations in the kidney were less for the “3-14-3” MOE gapmer relative to the “5-10-5” MOE gapmer at both doses. Again, this finding most likely reflects differences in plasma protein binding and reduced gapmer stability against endonuclease cleavage for wider gapped oligonucleotides. However, these results were in contrast to the observations made in the liver in which the concentrations of the “5-10-5” MOE gapmer and the “3-14-3” MOE gapmer were equivalent at both doses. This result was quite surprising and suggests that the sequence within the RNase H-sensitive deoxy gap can influence endonuclease degradation of MOE gapmers, and possibly, in a tissue-specific manner. These results demonstrate that gap widening may not only improve

600 500 400 300 200 100

Liver Kidney

4 Week

12.5 mg/kg dose

25 mg/kg dose

al

al To t

ta In

5-10-5 Figure 17.8

3 Week

To t

Oligonucleotide

1400 1200 1000 800 600 400 200 0

2 Week

ct

Concentration in tissue

1400 1200 1000 800 600 400 200 0

1 Week

ct

0 (B)

Saline 5-10-5 12.5mpk 5-10-5 25mpk 3-14-3 12.5mpk 3-14-3 25mpk

In ta

Plasma glucose (mg/dL)

(A)

3-14-3

Glucose-lowering activity and tissue accumulation of PTP-1B targeted 2⬘MOE gapmers in diabetic ZDF rats. MOE gapmers (“5-10-5” versus “3-14-3”) tested in this study are shown in Figure 17.7A. ZDF rats were treated at two dose levels, 12.5 or 25 mg/kg twice a week (i.p.) over a 4-week period. A Effects of MOE gapmer treatment on plasma glucose levels over a 4-week period. (B) Liver and kidney levels of PTP-1B MOE gapmer in ZDF rats following 4 weeks of dosing. MOE gapmer levels were determined as described in Ref. 32.

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pharmacological activity for 2⬘ MOE gapmers in animals, but also demonstrates that they have the potential to improve tolerability as well by reducing tissue (e.g., kidney) exposure. 17.3.3 Monkey Studies Antisense activity has been demonstrated for a number of “5-10-5” MOE gapmers against a variety of liver targets in monkey models [27,28]. These oligoucleotides have displayed potent and specific activity, and are well tolerated. Furthermore, based on the favorable observations made for MOE gapmers containing wider gaps in rodents, a number of studies have now been conducted in monkeys comparing the effects of a “5-10-5” MOE gapmer relative to a gap-widened analog. In the first of two studies conducted, a “5-10-5” MOE gapmer and a “2-16-2” MOE gapmer targeted to the human/monkey glucagon receptor was administered to cynomolgus monkeys at increasing doses (3, 10, and 20 mg/kg) over a 6-week period, glucagon receptor mRNA levels and oligonucleotide concentrations in liver were measured 2 days following the final dose, and EC50 values were calculated. Both MOE gapmers produced specific and significant reductions in glucagon receptor mRNA levels in monkey liver. However, the “2-16-2” MOE gapmer displayed increased potency relative to its “5-10-5” counterpart, as reflected by an improvement in EC50 value between 1.5- and 2-fold (Table 17.3). Both MOE gapmers were well tolerated. In a second study conducted in monkeys, animals were treated with increasing doses (2–10 mg/kg) of MOE gapmers targeted to the acute phase reactant C-Reactive Protein (CRP). In this study, a “5-10-5” MOE gapmer was compared with a “3-14-3” MOE gapmer for activity against IL6-induced CRP mRNA levels in liver and oligonucleotide concentrations were determined and relative EC50 values were calculated (Table 17.3). Treatment with either MOE gapmer resulted in a significant reduction in CRP mRNA levels in liver and CRP protein levels in serum [28]. Furthermore, similar to the observation made for the glucagon receptor, improved potency was again observed for the gap-widened “3-14-3” MOE gapmer relative to the “5-10-5” MOE gapmer, as reflected by an improved EC50 value of 1.5- to 2-fold (Table 17.3). Again, both MOE gapmers were well tolerated. These results demonstrate that optimization of deoxy gap size to improve RNase H activity has the potential to improve antisense potency in both rodents and in primates. 17.3.4 Caveats Numerous examples described above demonstrate that antisense potency can be improved in animals in a manner that is consistent with increased RNase H activity based on optimization of deoxy gap length. These observations have been made for a number of antisense targets, across multiple species, and these improved chimeric oligonucleotide designs appear to be well tolerated. As such, optimizing deoxy gap length for second-generation antisense inhibitors has the potential to improve Table 17.3 Effects of RNase H-Sensitive Gap Size on Antisense Potency in Monkey Liver Compound

Target

Structure


g/g) EC50(


5-10-5 MOE gapmer

Glucagon receptor

GCACTTTGTGGTGCCAAGGC

593 ⫾ 140

2-16-2 MOE gapmer

Glucagon receptor

GCACTTTGTGGTGCCAAGGC

350 ⫾ 269

5-10-5 MOE gapmer

CRP

TCCCATTTCAGGAGACCTGG

612 ⫾ 165

3-14-3 MOE gapmer

CRP

TCCCATTTCAGGAGACCTGG

430 ⫾ 49

Note: Monkeys were dosed s.c. with the indicated MOE gapmer antisense oligonucleotides targeted to either glucagon receptor (GCGR) or C-Reactive Protein (CRP). Glucagon receptor MOE gapmers were dosed with increasing dose levels over a 4-week period as described (ADA ref) and oligonucleotide levels in liver were determined [31]. CRP MOE gapmers were dosed with increasing dose levels prior to CRP induction by IL6 as described (CRP AHA ref if it exists).

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antisense potency in humans, and studies are underway to test this possibility. However, exceptions do exist to these findings. For example, we have attempted to improve antisense potency for “5-10-5” MOE gapmers targeted to some additional mouse liver targets by lengthening deoxy gap length and have not observed improved antisense potency. Some of these findings are illustrated in Table 17.4 for MOE gapmers targeted to either mouse glucagon receptor or mouse glucocorticoid receptor. In these studies, gap-widened ASOs were found to exhibit potency comparable to their “5-10-5” MOE gapmer counterparts. Although definitive explanations for these observations are not yet available, a number of possibilities can be rationalized based on our understanding of antisense drug action. For example, we know that antisense potency is a function of many overlapping contributing factors in addition to RNase H activity, such as the ability to invade complex target RNA structure during the hybridization process, the influence of RNA structure on RNase H enzyme accessibility to the duplex substrate, RNase H sequence preferences, and in protein binding, which can influence tissue and cellular pharmacokinetics. Furthermore, it is also possible that cell-free assays for measuring RNase H1 activity may not be the best predictors of RNase H sensitivity for RNA:oligonucleotide duplexes in cells, due to the influence of a variety of complicating factors like RNA structure and nucleic acid:protein interactions, which will certainly have some influence on RNase H efficiency in a more natural setting. Interestingly, in the case of the glucagon receptor target shown in Table 17.4, the ED50 displayed by the “5-10-5” MOE gapmer is far lower (more potent) than what is typically observed for “5-10-5” gapmers targeted to liver RNAs. Thus, in this case, it is possible that other factors that limit antisense potency have been overcome, including RNase H activity, due to the empirical selection of a “highly preferred” binding site in the structured RNA target. Thus, although improving RNase H activity by lengthening the RNase H-sensitive deoxy gap can improve RNase H activity and antisense potency in animals, the identification of more potent chimeric antisense inhibitors based on this premise must still be determined somewhat empirically.

17.4 INFLUENCE OF OLIGONUCLEOTIDE LENGTH As stated earlier, RNase H activity has not only been shown to be impacted by deoxy gap size, but also by the structure of the non-RNase H supporting gapmer wings, and by the nature of the chemical modifications contained within those wings [10,17,19]. For example, the RNase H1 activity of a 10-base oligodeoxynucleotide has been shown to be equivalent to a 20-base oligodeoxynucleotide under cell-free conditions. However, the RNase H1 activity supported by that same 10-base oligodeoxynucleotide when flanked by 2⬘-modified wings is reduced substantially [17,29]. These results indicate that the wings of a 2⬘-modified gapmer can negatively influence RNase H activity and, therefore, gapmers containing shorter 2⬘-modified wings may improve antisense potency by improving RNase H activity. Table 17.4 Effects of Gap Size on Antisense Activity against GCGR and GCCR in Mouse Liver Compound

Target

ED50 (mg/kg)

95%CI

5-10-5 MOE gapmer

GCGR

4.5 ⫾ 0.7

2.7 – 6.4

3-14-3 MOE gapmer

GCGR

4.2° ⫾ 0.3

3.3 – 5.2

2-16-2 MOE gapmer

GCGR

4.8 ⫾ 0.5

3.4 – 6.3

5-10-5 MOE gapmer

GCGR

28 ⫾ 9.0

14.2 – 33.6

2-16-2 MOE gapmer

GCGR

45 ⫾ 31

29.8 – 56.6

Note: Mice were dosed with increasing doses of MOE gapmer over a 4-week period and glucagon receptor (GCGR) or glucocorticoid receptor (GCCR) RNA levels were determined, as was MOE gapmer levels in liver, as described previously [20,31] and ED50 values calculated.

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The kidney-specific target, SGLT2 (sodium glucose transport protein 2), was chosen to test the effects of a short 2⬘ MOE gapmer relative to a more traditional “5-10-5” MOE gapmer on both RNase H activity and on antisense potency in mice. SGLT2 is a low-affinity, high-capacity, sodium-dependent glucose transporter, which serves as the major reabsorptive mechanism for glucose in the kidney proximal convoluted tubule, and is expressed exclusively in kidney proximal tubule epithelial cells [30]. SGLT2 was chosen for these studies because it is expressed in the kidney and it has been shown that reducing phosphorothioate content in antisense oligonucleotides results in reduced serum protein-binding capacity and, as a result, generally leads to greater oligonucleotide accumulation in the kidney and reduced accumulation in other tissues (e.g., liver) [20,31]. The ability to support RNase H1 activity of a “5-10-5” SGLT2 MOE gapmer was compared with a shortened “1-10-1” MOE gapmer against complementary RNA under cell-free conditions (Figure 17.9). The “1-10-1” gapmer employed in these studies was derived directly from the “5-10-5” gapmer by removing the four 2⬘ MOE residues from each end (5⬘ and 3⬘ ends) of the 20-base gapmer. Thus, the two oligoucleotides are identical except that the 12-base gapmer contains one MOE residue on each end instead of five. As expected, both gapmers supported RNase H activity. However, the “1-10-1”-shortened MOE gapmer was a more efficient substrate for RNase H relative to the “5-10-5” gapmer, displaying an increase in initial rate of cleavage of nearly 10-fold. These two MOE gapmers were next tested in mice for their effects on SGLT2 mRNA levels in kidney. In this study, mice were treated with equivalent molar amounts of “5-10-5” or “1-10-1” MOE gapmer at increasing dose levels, or with a “5-10-5” mismatch control MOE gapmer or a “1-10-1” mismatch control MOE gapmer at the highest dose levels tested. Mice were dosed twice per week over a 3-week period and SGLT2 mRNA levels and oligonucleotide concentrations were determined in kidney 48 h following the final dose. Treatment with either the “5-10-5” or the “1-10-1” MOE gapmer resulted in significant reductions in SGLT2 mRNA levels in kidney whereas neither of the mismatch control MOE gapmers significantly impacted SGLT2 mRNA levels (Figure 17.10). However, the “1-10-1” MOE gapmer was dramatically superior in potency relative to the “5-10-5” MOE gapmer, with remarkable improvements in both ED50 and in EC50

Vo ( nM/min)

SD

20 mer

1.02

0.04

12 mer

8.57

0.22

60

30

15

10

5

0

60

12-mer 30

15

10

5

Time (min)

0

20-mer

Figure 17.9 Effects of SGLT2 targeted 20-base MOE gapmer versus 12-base MOE gapmer on RNase H1 digestion rates. A “5-10-5” or a “1-10-1” MOE gapmer hybridized to a perfectly complementary RNA (20-base or 12-base complement) that corresponds to a segment of the mouse SGLT2 mRNA were tested for their ability to support human RNase H1 cleavage and initial rates were determined as described in Ref. 5. Cleavage patterns and initial rates of cleavage are shown.

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503

120

mSGLT2 expression % saline control

100 80 60 5-10-5 Gapmer 3b MM

40

1-10-1 Gapmer 3b MM 5-10-5 Gapmer

20

1-10-1 Gapmer

0 0

1000 2000 3000 4000 5000 6000 7000 8000 Dose (nM/kg)

(B) Compound

Figure 17.10

Structure

EC50 (µg/g)

5-10-5 Gapmer GAAGTAGCCACCAACTGTGC

> 200

1-10-1 Gapmer TAGCCACCAACT

13.58

Antisense suppression of SGLT2 mRNA levels in mouse kidney by a “5-10-5” or a “1-10-1” MOE gapmer phosphorothioate. (A) Mice were treated (i.p.) with increasing molar doses of “5-10-5” or “1-10-1” MOE gapmer twice per week over a 3-week period and SGLT2 mRNA levels were determined by RT-PCR as described [20]. Doses administered for the “5-10-5” MOE gapmer were 0.875, 1.75, 3.5, and 7.0
mol/kg and for the “1-10-1” MOE gapmer were 0.035, 0.175, 0.875, and 1.75
mol/kg. (B) EC50 values were calculated following determination of MOE gapmers levels in kidney (
g MOE gapmer/g kidney tissue) as described [31].

(Figure 17.10). Although ED50 and EC50 values could not be determined for the “5-10-5” MOE gapmer group, improved potency for the “1-10-1” gapmer could be approximated to be on the order of 100–200-fold. Analysis of oligonucleotide levels in kidney indicated that similar levels of the “1-10-1” and the “5-10-5” MOE gapmer were present in the kidney at the termination of the study (data not shown), supporting the conclusion that the increase in potency observed for the shorter MOE gapmer was due primarily to an increase in RNase H activity. Similar potency has been observed on both RNase H activity and on SGLT2 mRNA suppression using other 12-base MOE gapmers targeted to SGLT2, indicating that these findings have some general applicability to other antisense sequences. These results suggest that the tremendous loss in predicted binding affinity that occurs as a result of shortening a high-affinity “5-10-5” MOE gapmer to a lower affinity “1-10-1” MOE gapmer can be overcome by improving other aspects of antisense action, such as the ability to more efficiently support RNase H activity.

17.5 FUTURE DIRECTIONS As described above, advancements made in the understanding of RNase H enzymology has resulted in improved potency for existing “second-generation” antisense drugs in animals. The magnitude of the increase in potency observed is wide ranging, depending on oligonucleotide sequence, deoxy gap length, and overall oligonucleotide length. Thus, rather simple alterations in chimeric oligonucleotide structure has resulted in the creation of “Generation 2.2” antisense oligonucelotides; drugs that are similar in design and chemistry as Generation 2.0 drugs, but differ slightly in their design to improve RNase H activity and potency. However, many unanswered

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questions still exist that need to be addressed. For example, why does the optimization of some second-generation 2⬘ MOE gapmers result in increased RNase H activity but not increased potency? What will the tolerabilility profile be for Generation 2.2 oligonucleotides relative to Generation 2.0 in the clinic? Afterall, Generation 2.2 oligonucleotides are more “first-generation-like” in their chemical composition and we know that second-generation antisense drugs exhibit an improved tolerability profile relative to first-generation drugs. Third, how do we further achieve improvements in RNase H activity and potency for second-generation antisense drugs? In this regard, it is quite likely that further improvements in antisense potency for 2⬘-modified chimeric oligonucleotides based on further improvements in RNase H activity will require a better understanding of how oligonucleotide modifications, particularly in the wings of the chimeric molecule, influence RNase H activity. With this understanding, modifications and oligonucleotide designs can be utilized to increase RNase H activity while providing additional benefits to oligonucleotide drugs, such as affinity, stability, and tolerability. For example, it has been shown that the nature and position of the 2⬘ modification contained in the wings of a gapmer can have a transmissional influence on the RNase H-sensitive deoxy gap, thereby altering preferred cleavage sites for RNase H, and modulating RNase H efficiency overall [10,17,29]. Thus, insertion of preferred modified nucleotides into appropriate positions within a gapmer may modulate the transmission of the duplex geometry in a manner that increases RNase H activity beyond that observed for Generation 2.2, resulting in further increases in antisense potency. Antisense oligonucleotides that are based on Generation 2.2 design and chemistry that are further optimized for RNase H activity by incorporating subtle changes in chemistry to influence duplex geometry in a beneficial manner are referred to as “Generation 2.5.” 17.6 CONCLUSIONS Second-generation antisense drugs that act through an RNase H-dependent mechanism of action have been developed that demonstrate a number of advantages over first-generation phosphorothioate oligodeoxynucleotides, including better potency, stability, and tolerability. These drugs are designed as chimeric oligonucleotides in which a portion of the molecule contains chemical modifications that provide increased binding affinity and stability, but do not support RNase H activity, is combined with another portion of the molecule that serves to support the RNase H mechanism of action. At the time these oligonucleotides were developed, the key parameters that required optimization to maximize RNase H efficiency were unknown. Thus, although second-generation antisense drugs displayed improved potency over first-generation drugs, further improvements in potency were expected based on advancements in our understanding of RNase H enzymology. Indeed, these predictions have been born out. The development and systematic implementation of assays to quantitatively measure the influence of oligonucleotide chemistry and design on RNase H activity and antisense activity has resulted in further improvements in antisense potency. These increases in potency are expected to have significant implications on the profile of antisense drugs in humans. Most importantly, the need for lower doses in the clinic should translate to an improved therapeutic index, lower cost of goods, and increase the commercial potential for oral routes of administration. Finally, new progress being made in the understanding of oligonucleotide substrate preferences for RNase H enzymes are expected which may result in further advancements in the pharmacology of antisense drugs. ACKNOWLEDGMENTS The authors thank Pamela Black and Tracy Reigle for their administrative assistance and graphical support. The authors also thank Sanjay Bhanot, Rosanne Crooke, and Richard Geary for contributions of unpublished data and to John Matson and Ed Wancewicz for excellent technical assistance.

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REFERENCES 1. B. F. Baker, S. S. Lot, T. P. Condon, S. Cheng-Flournoy, E. A. Lesnik, H. M. Sasmor and C. F. Bennett; 2⬘-O-(2-Methoxy)ethyl-modified anti-intercellular adhesion molecule 1 (ICAM-1) oligonucleotides selectively increase the ICAM-1 mRNA level and inhibit formation of the ICAM-1 translation initiation complex in human umbilical vein endothelial cells; J Biol Chem; 272; 11994–2000; 1997. 2. Z. Dominski and R. Kole; Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides; Proc Natl Acad Sci USA; 90; 8673–8677; 1993. 3. J. G. Karras, R. A. McKay, T. Lu, N. M. Dean and B. P. Monia; Antisense inhibition of membrane-bound human interleukin-5 receptor-alpha chain does not affect soluble receptor expression and induces apoptosis in TF-1 cells; Antisense Nucl Acid Drug Dev; 10; 347–357; 2000. 4. G. Meister and T. Tuschl; Mechanisms of gene silencing by double-stranded RNA; Nature; 431; 343–349; 2004. 5. H. Wu, W. F. Lima and S. T. Crooke; Properties of cloned and expressed human RNase H1; J Biol Chem; 274; 28270–28278; 1999. 6. B. F. Baker, L. Miraglia and C. H. Hagedorn; Modulation of eucaryotic initiation factor-4E binding to 5⬘-capped oligoribonucleotides by modified anti-sense oligonucleotides; J Biol Chem; 267; 11495–11499; 1992. 7. J. K. Taylor, Q. Q. Zhang, J. R. Wyatt and N. M. Dean; Induction of endogenous Bcl-xS through the control of Bcl-x pre-mRNA splicing by antisense oligonucleotides; Nat Biotechnol; 17; 1097–10100; 1999. 8. J. Roberts, E. Palma, P. Sazani, H. Orum, M. Cho and R. Kole; Efficient and persistent splice switching by systemically delivered LNA oligonucleotides in mice; Mol Ther; 14; 471–475; 2006. 9. T. A. Vickers, H. Zhang, M. J. Graham, K. M. Lemonidis, C. Zhao and N. M. Dean; Modification of MyD88 mRNA splicing and inhibition of IL-1beta signaling in cell culture and in mice with a 2⬘-O-methoxyethyl-modified oligonucleotide; J Immunol; 176; 3652–3661; 2006. 10. W. F. Lima, H. Wu and S. T. Crooke; Human RNases H; Methods Enzymol; 341; 430–440; 2001. 11. B. P. Monia, E. A. Lesnik, C. Gonzalez, W. F. Lima, D. McGee, C. J. Guinosso, A. M. Kawasaki, P. D. Cook and S. M. Freier; Evaluation of 2⬘-modified oligonucleotides containing 2⬘-deoxy gaps as antisense inhibitors of gene expression; J Biol Chem; 268; 14514–14522; 1993. 12. C. F. Bennett; General pharmacology of phosphorothioate oligodeoxynucleotides; In Antisense Drug Technology: Principles, Strategies, and Applications; S. T. Crooke; ed. Marcel Dekker, Inc.; New York; 2001; pp. 291–318. 13. K. H. Altman, N. M. Dean, D. Fabbro, S. Freier, T. Geiger, R. Haner, D. Husken, P. Marting, B. P. Monia, M. Muller, F. Natt, P. Nicklin, J. Phillips, U. Pieles, H. Sasmor and H. E. Moser; Second generation of antisense oligonucleotides: from nuclease resistance to biological efficacy in animals; Chimia; 50; 168–176; 1996. 14. B. P. Monia, J. F. Johnston, H. Sasmor and L. L. Cummins; Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras; J Biol Chem; 271; 14533–14540; 1996. 15. N. M. Dean, M. Butler, B. P. Monia and M. Manoharan; Pharmacology of 2⬘-O-(2⬘-Methoxyethoxy) Ethyl Modified Antisense Oligonucleotides; Antisense Drug Technology: Principles, Strategies and Applications; S. T. Crooke; Marcel Dekker, Inc.; New York; 2001; pp. 319–338. 16. W. F. Lima, J. G. Nichols, H. Wu, T. P. Prakash, M. T. Migawa, T. K. Wyrzykiewicz, B. Bhat and S. T. Crooke; Structural requirements at the catalytic site of the heteroduplex substrate for human RNase H1 catalysis; J Biol Chem; 279; 36317–36326; 2004. 17. W. F. Lima, J. B. Rose, J. G. Nichols, H. Wu, M. T. Migawa, T. K. Wyrzykiewicz, G. Vasquez, E. E. Swayze and S. T. Crooke; The positional influence of the helical geometry of the heteroduplex substrate on human RNase H1 catalysis; Mol. Pharmacol; 71; 73–82; 2007. 18. H. Wu, W. F. Lima and S. T. Crooke; Molecular cloning and expression of cDNA for human RNase H; Antisense Nucl Acid Drug Dev; 8; 53–61; 1998. 19. H. Wu, W. F. Lima, H. Zhang, A. Fan, H. Sun and S. T. Crooke; Determination of the role of the human RNase H1 in the pharmacology of DNA-like antisense drugs; J Biol Chem; 279; 17181–17189; 2004. 20. M. Butler, R. A. McKay, I. J. Popoff, W. A. Gaarde, D. Witchell, S. F. Murray, N. M. Dean, S. Bhanot and B. P. Monia; Specific inhibition of PTEN expression reverses hyperglycemia in diabetic mice; Diabetes; 51; 1028–1034; 2002.

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21. R. S. Geary, T. A. Watanabe, L. Truong, S. Freier, E. A. Lesnik, N. B. Sioufi, H. Sasmor, M. Manoharan and A. A. Levin; Pharmacokinetic properties of 2⬘-O-(2-methoxyethyl)-modified oligonucleotide analogs in rats; J Pharmacol Exp Ther; 296; 890–897; 2001. 22. A. M. Siwkowski, L. A. Madge, S. Koo, E. L. McMillan, B. P. Monia, J. S. Pober and B. F. Baker; Effects of antisense oligonucleotide-mediated depletion of tumor necrosis factor (TNF) receptor 1-associated death domain protein on TNF-induced gene expression; Mol Pharmacol; 66; 572–579; 2004. 23. M. Elchebly, P. Payette, E. Michaliszyn, W. Cromlish, S. Collins, A. L. Loy, D. Normandin, A. Cheng, J. Himms-Hagen, C. C. Chan, C. Ramachandran, M. J. Gresser, M. L. Tremblay and B. P. Kennedy; Increased insulin sensitivity and obesity resistance in mice lacking the protein tyrosine phosphatase-1B gene; Science; 283; 1544–1548; 1999. 24. C. M. Rondinone, J. M. Trevillyan, J. Clampit, R. J. Gum, C. Berg, P. Kroeger, L. Frost, B. A. Zinker, R. Reilly, R. Ulrich, M. Butler, B. P. Monia, M. R. Jirousek and J. F. Waring; Protein tyrosine phosphatase 1B reduction regulates adiposity and expression of genes involved in lipogenesis; Diabetes; 51; 2405–2411; 2002. 25. B. A. Zinker, C. M. Rondinone, J. M. Trevillyan, R. J. Gum, J. E. Clampit, J. F. Waring, N. Xie, D. Wilcox, P. Jacobson, L. Frost, P. E. Kroeger, R. M. Reilly, S. Koterski, T. J. Opgenorth, R. G. Ulrich, S. Crosby, M. Butler, S. F. Murray, R. A. McKay, S. Bhanot, B. P. Monia and M. R. Jirousek; PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice; Proc Natl Acad Sci USA; 99; 11357–11362; 2002. 26. L. L. Kjems, S. Bhanot, J. D. Bradley, B. P. Monia, J. Kwoh and M. Wedel; Increased insulin sensitivity in humans by protein tyrosin IB (PTP1B) inhibition—evaluation of ISIS 113715, and antisense inhibition of PTP-1B; Diabetes; 54; A530; 2004. 27. S. Bhanot, L. M. Watts, K. W. Sloop, J. X. C. Cao, A. D. Showalter, M. D. Michael and B. P. Monia; Reduction of hepatic glucagon receptor expression with an optimized antisense oligonucleotide increased active GLP-1 levels in cynomolgus monkeys without pancreatic alpha cell expansion; Diabetes; 55; A326; 2006. 28. T. W. Kim, H. S. Rose, K. Kramer-Strickland, M. J. Graham, K. Subramaniam, R. M. Crooke, P. B. Lappin, G. S. Elliot, A. A. Levin and S. P. Henry; ISIS 326358 an antisense oligonucleotide targeted to ApoB reduces plasma LDL-C in a monkey model of hyperlipidemia; The Toxicologist; 90; 63; 2006. 29. W. F. Lima, J. B. Rose, J. G. Nichols, H. Wu, M. T. Migawa, T. K. Wyrzykiewicz, A. M. Siwkowski and S. T. Crooke; Human RNase H1 discriminates between subtle variations in the structure of the heteroduplex substrate; Mol. Pharmacol; 71; 83–91; 2007. 30. E. M. Wright; Renal Na(+)-glucose cotransporters; Am J Physiol Renal Physiol; 280; F10–F18; 2001. 31. S. T. Crooke, M. J. Graham, J. E. Zuckerman, D. Brooks, B. S. Conklin, L. L. Cummins, M. J. Greig, C. J. Guinosso, D. Kornbrust, M. Manoharan, H. M. Sasmor, T. Schleich, K. L. Tivel and R. H. Griffey; Pharmacokinetic properties of several novel oligonucleotide analogs in mice; J Pharmacol Exp Ther; 277; 923–937; 1996.

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18

Modulating Gene Function with Peptide Nucleic Acids (PNA) Peter E. Nielsen

CONTENTS 18.1 Introduction .........................................................................................................................507 18.2 PNA Chemistry ...................................................................................................................508 18.3 mRNA Targeting .................................................................................................................508 18.4 dsDNA (gene) Targeting .....................................................................................................510 18.5 Anti-Infective Agents ..........................................................................................................512 18.6 Cellular Delivery.................................................................................................................513 18.7 In Vivo Bioavailability of PNA ...........................................................................................514 18.8 Prospects .............................................................................................................................515 References ......................................................................................................................................515 18.1 INTRODUCTION Peptide nucleic acid (PNA) is a pseudo peptide DNA mimic based on an aminoethyl glycine backbone (Figure 18.1). It was introduced 15 years ago [1] and is in contrast to almost all other DNA analogs and mimics uncharged and achiral. Furthermore, PNA oligomers are synthesized via peptide-like solid-phase chemistry rather than DNA-like chemistry. In fact, PNA synthesis is fully and directly compatible with Boc- and Fmoc-based peptide synthesis. Obviously, PNA oligomers are not substrates for nucleases, but due to the nonnatural character of the amide (“peptide”) bond connecting individual PNA units, PNA oligomers are not susceptible to hydrolysis by peptidase or proteases either, and exhibit exquisite stability in serum and cell extracts as well as in vivo. These properties combined with excellent sequence-specific and high-affinity recognition of single-stranded RNA (and DNA) gave very high initial expectations for PNA in an antisense drug discovery context. The finding that homopyrimidine PNA oligomers form exceedingly stable triplex invasion complexes with sequence-complementary targets in duplex DNA further emphasized the gene targeting potential of PNA. Numerous in vitro and cell culture studies have demonstrated that PNA oligomers are indeed very potent modulators of gene expression ranging from gene silencing at the mRNA (antisense) or the dsDNA (antigene) level, and redirection of mRNA splicing to gene activation through transcription bubble mimicking. However, these studies have also revealed that for both cell culture 507

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O -

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PNA Figure 18.1 Chemical structures of PNA compared to DNA (B signifies a nucleobase).

studies and, in particular, for in vivo application, the major challenge to be met is effective cell delivery and sufficient in vivo bioavailability of PNA oligomers (for recent reviews, see [2–5]).

18.2 PNA CHEMISTRY The original aeg (aminoethyl glycine) PNA with the carbonyl methylene (“acetyl”) linker to the nucleobase was based on molecular model building keeping intrabackbone distances as close as possible to that of DNA. Subsequently, numerous derivatives and modifications of the aegPNA structure have been made and it is quite clear that keeping these distances is critical for strong RNA/DNA hybridization. Furthermore, the restricted flexibility imposed by the (two) planar amide groups also appears very important for high-affinity binding [6]. Thus, of the noncyclic derivatives, the original aegPNA still appears to be the most promising candidate for gene targeting. Nonetheless, a variety of cyclic PNA analogs have shown promise as a means of increasing PNA-RNA (and/or DNA) duplex stability [7], but their synthesis is less straightforward than that for aegPNA monomers, and through the ring systems chirality is also introduced. Furthermore, duplex stabilization appears, at least for some of the derivatives, to be more sequence context–dependent than for aegPNA, although for virtually all derivatives too limited data are yet available for drawing more general conclusions in this respect. A large variety of PNA monomers with functionalized backbone have also been developed most commonly by substituting the
-position of the glycine moiety (Figure 18.1) (e.g., [8]), but derivatives having substituents in the ethyl part have also been described [9]. Such derivatives may turn out to be of significant interest for modulating (improving) the bioavailability and pharmacokinetics of PNA oligomers (vide infra, Section 18.7). Finally, a number of nonnatural nucleobases have been exploited in the PNA context for specific purposes, mostly in connection with targeting of duplex DNA (vide infra, Section 18.4).

18.3 mRNA TARGETING It is well established and not surprising that PNA-RNA duplexes are not substrates for RNaseH. Thus, the antisense activity of PNA oligomers must rely on other mechanisms, such as

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direct interference with translation initiation or elongation. Cell-free in vitro translation studies have demonstrated that PNAs targeted to or upstream from the translation initiation site effectively inhibit translation, whereas this is not the case if the PNA is target to sequences within the coding region (unless homopurine regions are targeted with homopyrimidine PNA, or even better, bisPNAs, which form exceedingly stable PNA2-DNA triplexes). Accordingly, in one study the most effective antisense PNA identified by gene walk in a cell culture experiment targets the 5⬘-UTR [10]. Nonetheless, several other less systematic studies have identified active antisense PNAs targeting the coding region [11–13]. The mechanism(s) of action for these PNAs has not been determined, but could involve translation elongation arrest in particularly sensitive regions, changes in RNA secondary structure affecting mRNA translation, stability, or trafficking. Thus, more systematic and mechanistic studies on the effects of mRNA targeting by PNA would be highly desirable. Recently, it was discovered that targeting of RNaseH “negative” oligonucleotides to intron–exon junctions of pre-mRNA can effectively inhibit or redirect mRNA splicing. Likewise, several studies have demonstrated that PNAs effectively can interfere with splicing [14–16]). Blocking of an intron–exon junction may have two fundamentally different outcomes (Figure 18.2). Simply the splicing out of the intron may be inhibited, and a dysfunctional mRNA still containing the intro results and, consequently, synthesis of the protein coded for by the mRNA is disrupted. However, a more biologically interesting consequence arises if the spliceosome utilizes a downstream splice site instead of the one blocked by the PNA as this will result in an mRNA with one (or more) exon(s) missing, thereby producing an alternatively spliced mRNA. The technology may also be exploited for drug discovery of splicing correcting drugs for diseases caused by genes containing aberrant splice sites [15]. Alternative splicing is widely utilized by nature to create multiple products from the same mRNA and antisense agents may thus rationally be used to control this process. Indeed, this principle was recently exploited to convert the apoptosis inhibiting bcl-xL protein isoform into the pro-apoptotic bcl-xS isoform as a means for discovering new anticancer antisense agents [17]. Analogously, it can be envisaged that novel proteins not used by nature may be induced by exon-skipping of functional domains, e.g., active sites, ligand binding sites, membrane spanning domains, etc.

PNA

Intron skipping or

Exon skipping Figure 18.2 Effect of PNA targeting of a pre-mRNA intron–exon splice junction. Thick “lines” signify exons while thin lines signify introns.

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Finally, PNAs targeting the RNA of telomerase have been evaluated in in vitro cell culture studies as potential anticancer agents (e.g., [18,19]), but although very potent inhibitors of telomerase activity have been identified it is still too early to judge the merits of this approach, not least to the uncertainties concerning telomerase as a validated anticancer target.

18.4 dsDNA (GENE) TARGETING It is well established that targeting of double-stranded DNA with PNA in vitro using purified DNA can result in a variety of complexes (Figure 18.3) dependent on target (and PNA) sequence, PNA modification, and experimental conditions. For homopurine targets, and corresponding homopyrimidine PNA oligomers, triplex structures are formed. Initially, traditional external triplexes with the PNA bound by Hoogsteen base-pairing in the major groove take place, and, subsequently, at a much slower rate, a triplex invasion complex having an internal PNA2DNA triplex and an extruded DNA strand is formed (P-loop) [20] (Figure 18.4). Furthermore, the invasion process is dramatically accelerated under conditions that favor opening (denaturation) of the DNA double helix (such as negative supercoiling or active transcription) and severely deaccelerated under conditions that stabilize the double helix, such as elevated ionic strength or the presence of di- or multivalent cations (Mg2⫹, spermidine, etc.). Indeed, triplex invasion binding of simple homopyrimidine PNA oligomers to relaxed duplex DNA hardly takes place under physiologically similar conditions (140 mM K⫹/Na⫹, 2 mM Mg2⫹). Thus, the ratio of external triplex versus triplex invasion products varies dramatically depending on the conditions [21]. However, relative enhancement of invasion can be accomplished by using PNA-intercalator (acridine) conjugates [22], especially in combination with bisPNA clamps, which furthermore allows use of the pseudoisocytosine nucleobase instead of cytosine in the Hoogsteen bound PNA strand, thereby obtaining PNAs that bind strongly to the target independent of pH [23]. Mixed purine/pyrimidine sequence PNAs do not form PNA2DNA triplexes and bind very weakly, if at all, to targets in relaxed double-stranded DNA [24], but binding has been demonstrated to negatively supercoiled targets using PNAs conjugated to cationic peptides [25]. Mixed purine/pyrimidine sequences in duplex DNA can, however, be targeted using pairs of pseudocomplementary PNAs (Figure 18.5). In these PNAs, adenines are replaced with diaminopurines (DAP) and thymines are replaced with thiouracil (Us) [26], resulting in a pair of sequence-complementary PNAs, which bind weakly to each other due to steric clash in the DAP-Us base pair, but which still binds effectively to complementary DNA and therefore can form double duplex invasion complexes with targets in duplex DNA.

Triplex

Triplex invasion Duplex invasion Double duplex invasion

Tail clamp

Figure 18.3 Five different types of PNA-dsDNA complexes. DNA is schematically drawn as a ladder, and the PNA oligomers are in bold.

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H

N

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H

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C N

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G

Figure 18.4 Triplex invasion by homopyrimidine PNA oligomers. One PNA strand binds via Watson–Crick basepairing (preferably in the antiparallel orientation), while the other binds via Hoogsteen base-pairing (preferably in the parallel orientation). It is usually advantageous to connect the two PNA strands covalently via a flexible linker into a bis-PNA, and to substitute all cytosines in the Hoogsteen strand with pseudoisocytosines (
iC), which do not require low pH for protonation at N3.

CH3 O

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H N

N Adenine

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N O

H

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H H

N

N

S

Thymine N

N

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Thiouracil N

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Adenine

Figure 18.5 Double duplex invasion of pseudocomplementary PNAs. To obtain efficient binding, the target (and thus the PNAs) should contain at least 50% AT, and in the PNA oligomers all adenines and thymines are substituted with 2,6-diaminopurine or 2-thiouracil, respectively. Pairing between these base analogs is very unstable due to steric hindrance. Therefore, the two sequence-complementary PNAs will not be able to bind to each other, but they bind to their complementary DNA sequences very well.

In vitro transcription studies using phage- or prokaryotic RNA polymerases or cell extracts have shown that PNA triplex invasion complexes—in particular, when positioned on the template DNA strand—are able to arrest elongating RNA polymerases [27,28]. Very recently, it was demonstrated that the RNA polII open complex can be blocked by PNA binding to the DNA loop [29], in full analogy to previous reports using Escherichia coli RNA polymerase and oligonucleotides [30,31].

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Most interestingly, it was discovered that PNA triplex invasion complexes recruit RNA polymerase for transcription initiation, thereby functioning as an artificial promoter with the PNA as a “transcription factor” [32]. A subsequent study has indicated that such a specific gene-activating effect of PNA can also occur in cells [33], but unfortunately these results haven not been followed up further. The very specific sequence-targeting of triplex-forming (bis)PNAs (and pseudocomplementary PNAs) has been exploited to introduce covalent modification of the target by photocrosslinking or base alkylation using PNA-psoralen [34] or PNA-nitrogen mustard conjugates [35,36]. In view of the recent success in achieving effective cellular gene repair via double-strand-break-provoked homologous recombination using a genetically delivered endonuclease [37], such PNA conjugates could be interesting agents in targeted gene repair studies and drug discovery, as the repair of DNA interstrand crosslinks involves double-strand breaks, which could facilitate sequence-targeted homologous recombination. Unfortunately, no in vivo or even cell culture studies have yet been published in which a biological effect on repair, replication, or transcription has been directly correlated with genomic PNA binding. Therefore, although several reports have implicated or been interpreted in terms of an antigene mechanism, validated studies demonstrating in vivo antigene effects of PNA are still lacking (see also Section 18.7). A series of in vitro cell culture studies have implicated mixed purine/pyrimidine sequence PNA oligomers conjugated to a nuclear localization (NLS) peptide as effective antigene agents, in particular for targeting myc genes [38–41]. This is highly surprising in view of the fact that such PNAs do not efficiently bind duplex DNA targets in vitro; also NLS in comparative studies is an extremely poor cellular delivery peptide (at least in HeLa cells) [42]. Unfortunately, the studies do not directly address the mechanism(s) of the observed effects of the PNAs on mRNA and protein levels, and show no evidence of DNA binding of the PNA in the cells. It is thus premature to conclude that these PNAs elicit bona fide antigene activity.

18.5 ANTI-INFECTIVE AGENTS Infectious agents such as bacteria and viruses have genomes that are dramatically different from the human genome and these pathogens are therefore obvious targets for gene therapeutic drugs. Not surprisingly, several studies have addressed the potential of PNA in this respect. Almost 10 years ago, it was demonstrated that PNA oligomers targeting the
-sarcin loop of bacterial ribosomes were able to inhibit the growth of E. coli [43], and a subsequent study identified a delivery peptide, (KFF)3K, which when conjugated to PNA significantly increased the potency of the PNA [44]. In this study, it was also demonstrated that antisense PNAs targeting the essential acpP gene are bacteriocidal and thus have the potential of being developed into novel antibiotics. Indeed, a recent study has supported these hopes by demonstrating the antibacterial effects of this PNA in a peritonitis mouse infection model [45]. Unfortunately, toxicity studies in dogs have shown that the (KFF)3K peptide induces a severe histamine release and this peptide therefore appears unsuited for further drug development [46]. However, it should be possible to discover other (peptide) carriers that effectively deliver PNA to bacterial cells without causing histamine release as has been accomplished in the field of antibacterial peptides [47], and thereby allow discovery of novel antibacterial PNAs for clinical development. It is encouraging that PNA antisense targeting in bacteria seems a very robust phenomenon [48–50] that is not restricted to E. coli, but has for instance also been demonstrated in Staphylococcus aureus [51]. Interestingly, other unicellular organisms such as amoeba are also susceptible to PNA antisense techniques [52,53]. A number of studies have successfully addressed the discovery of anti-HIV PNAs [54–60]. Reverse transcriptase is extremely sensitive to PNA interference by targeting the template RNA, and by using PNA oligomers conjugated to cell delivery peptides (e.g., transportan), antiviral

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effects of such PNAs have been demonstrated in cell culture at low micromolar concentrations of the PNA-peptide conjugates [58–60]. In vitro data likewise have been reported to support the fact that PNAs against Hepatitis (HBV and HCV) may eventually be developed [61,62].

18.6 CELLULAR DELIVERY It is generally accepted that PNA oligomers, due to their size and hydrophilic nature, are taken up extremely poorly by both pro- and eukaryotic cells. Consequently, a variety of PNA derivatives and delivery methods that enhance the cellular bioavailability of PNA have been developed. These methods include cationic liposome-assisted delivery of PNA oligomers hybridized to an oligonucleotide carrier [63], or of PNAs conjugated to lipophilic ligands, such as fatty acids [64] or (hetero)polyaromatic ligands [65]. Although these methods are adequate for most studies in cell culture, they cannot be directly transferred to in vivo applications, primarily due to toxicity and instability of simple cationic liposomes. Thus, methods based on PNA derivatives that are taken up by cells unaided would be highly advantageous. A variety of such derivatives has been described and almost all rely on conjugation to cationic cell-penetrating peptides (CPPs). The first CPPs were derived from protein domains known to facilitate cellular uptake, such as part of the homeo domain from the antennapedia transcription factor in Drosophila (penetratin) and the Tat-peptide from HIV. These peptides are cationic with a high arginine content and were originally reported to facilitate cellular entry by passive transport over the lipid bilayer membrane [66]. However, supported by more recent studies [67–69], there is now a general consensus that the major route of cellular entry for these and analogous cationic peptides, such as simple oligo arginines and also transportan involves an endosomal pathway, perhaps predominantly macropinocytosis [70]. Therefore, a major challenge is the induction of endosomal escape (at least for such conjugates) rather than achieving cellular uptake per se. A variety of endosome disruptive agents have recently been discovered and their effect on peptide-mediated PNA antisense activity in cells in culture has been studied. The general conclusion from these studies is that all agents (chloroquine [69,71], photochemical internalization (PCI) [72,73], and calcium ions [71]) enhance the potency of all cationic peptides. This effect can be as large as two orders of magnitude as seen for the Tat peptide but differs significantly between the different peptides [71], thereby strongly suggesting that despite an overall similar endosomal uptake mechanism, some peptides may to some extent be taken up by alternative pathways and more or less efficiently escape the endosomal compartment by themselves. Determination of the structure–activity relations for these differences should allow the design of more effective delivery agents. Two studies have attempted to more systematically deduce such structure-activity relations for antisense enhancement of PNA-peptide conjugates for simple cationic [74] as well as amphiphilic peptides [75], but clear-cut conclusions are difficult to draw from these data at this stage except that the presence of multiple lysines or aginines is required. Indeed, other studies have identified hepta- or octaarginines as optimal in this respect [67,72]. However, it is important to note that most cationic peptides exhibit significant general cytotoxicity due to their membrane active/disruptive properties, and that a fine balance between delivery activity and toxicity usually exists [67,72]. A couple of comparative studies of the most common delivery peptides (including Tat, penetratin, transprotan, NLS, and oligoarginines) have been performed exploiting PNA-peptides conjugates in a very sensitive and easily quantifiable HeLa cell luciferase splicing correction assay. The overall conclusion from these studies identifies transportan as the most effective delivery agent followed by oligoarginines [67,72,76,77]. However, the studies also indicate that seemingly very small changes in the peptide (such as changing the C-terminal of transportan from an amide to a free carboxylic acid) can profoundly affect the activity [67]. Furthermore, it appears advantageous to connect the PNA to peptide via a biologically cleavable linker, such as a disulfide or an ester [67]. A variety of other ligands, such as triphenylphosphonium [78] and terpyridyl [79] have been reported to facilitate PNA cellular delivery, but these have not yet been comparatively validated.

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION B O NH

B O

O N

O N

NH

B O NH

O N

NH HN

NH2

Figure 18.6 Structure of PNA containing single arginine derived backbone unit (B signifies a nucleobase).

Also, direct modification of the PNA structure in terms of backbone substituents [8] have yielded compounds with enhanced uptake properties. In particular, exploiting backbones based on lysine [8,64] or arginine (Figure 18.6) [8,80] instead of glycine are of interest as are glycosylated PNAs [81], although there is no evidence that such internal modification is advantageous compared to analogous end-conjugation. Finally, it is important to mention that endosmal uptake and subsequent escape is exploited by Nature as the route of cellular entry for many viruses. Specially evolved proteins are responsible for the endosomal escape process, although the detailed mechanism is still poorly understood. Clearly, low-molecular-weight ligands or small peptides that could mimic the delivery mechanism of these larger (20–40 amino acid) viral peptides (70) would be of major interest for drug discovery of PNA gene-targeting drugs.

18.7 IN VIVO BIOAVAILABILITY OF PNA Although a number of in vivo experiments have been conducted with unmodified PNA oligomers [82–84]), these are poorly validated and all available pharmacokinetic data agree that the bioavailability of unmodified aegPNA in vivo is extremely limited because the PNA is very quickly (t½⬍30 min) excreted through the kidneys [81,85,86]. This conclusion is also supported by the most convincing in vivo study published so far [87]. In this study, a transgenic green fluorescent protein (GFP) mouse in which GFP can be activated by antisense splice correction was used, and the antisense effect of PNA, MOE, and morpholino oligomers were compared and scored as GFP level (fluorescence) as well as mRNA correction (RT-PCR) in several tissues. The results very clearly showed that while no effect could be detected with an unmodified PNA, the same PNA conjugated to four lysines showed significant (comparable to that of the corresponding MOE and morpholino oligomers) antisense effect in liver, muscle, and heart, thereby indicating that simple peptide conjugation can positively affect the in vivo bioavailability of PNA. Accordingly, significantly improved pharmacokinetic behavior of a PNA-(KFF)3K conjugate as compared to an unmodified PNA was found [85]. In a more comprehensive study, a series of cationic peptides have been tested, again identifying several that enhance PNA bioavailability upon conjugation [73]. Using a different approach, the bioavailability in particular organs has been dramatically improved by specifically targeting the PNA to receptors present on the cells of the organ. Specifically, it has been shown that GalNac (N-acetylgalactosamin)-modified PNA up-concentrate very efficiently in the liver [81] due to the presence of asialoglycoprotein receptors, although it is not clear how much of the PNA is actually available for gene targeting inside the hepatocytes or the kupffer cells of the liver. Most likely, in this case most of the PNA will be trapped in endosomes due to the receptor-mediated uptake.

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The experience with the in vivo behavior of PNA oligomers and,in particular, concerning the effects of chemical modification and conjugation is still very limited, but it is fair to conclude that the results so far are encouraging in terms of the very significant effects seen in bioavailability (both pharmacokinetics and tissue distribution) upon chemical modification or conjugation of the PNA. This behavior reflects the fact that aegPNA oligomers are biologically speaking “neutral” molecules whose physicochemical properties can be easily and dramatically changed without severely affecting their affinity toward their nucleic acid cellular target.

18.8 PROSPECTS Clearly, PNA oligomers can modulate cellular gene expression at many levels and via a multitude of mechanisms, and therefore provide a broad opportunity for novel drug discovery. In particular, splice modulation and interference with gene function at the DNA level may open exiting new possibilities such as altering gene function by inducing alternative protein isoforms and targeted sequence-specific somatic gene repair. However, it is also painstakingly clear that the major hurdle to overcome for these opportunities to become reality is in vivo delivery and bioavailability of the PNA oligomers. Whether this challenge will be met by using chemical modification, ligand conjugation, formulation, or some new technology remains to be seen, but if effectively solved a new era in drugs discovery could be dawning.

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40. Cogoi S, Codognotto A, Rapozzi V et al.: Transcription inhibition of oncogenic KRAS by a mutationselective peptide nucleic acid conjugated to the PKKKRKV nuclear localization signal peptide. Biochemistry, 44: 10,510; 2005. 41. Tonelli R, Purgato S, Camerin C et al.: Anti-gene peptide nucleic acid specifically inhibits MYCN expression in human neuroblastoma cells leading to cell growth inhibition and apoptosis. Molecular Cancer Therapy, 4: 779; 2005. 42. Bendifallah N, Rasmussen FW, Zachar V et al.: Evaluation of cell-penetrating peptides (CPPs) as vehicles for intracellular delivery of Antisense peptide nucleic acid (PNA). Bioconjugate Chemistry, 17: 750; 2006. 43. Good L and Nielsen PE: Inhibition of translation and bacterial growth by peptide nucleic acid targeted to ribosomal RNA. Proceedings of the National Academy of Science USA, 95: 2073; 1998. 44. Good L, Awasthi SK, Dryselius R et al.: Bactericidal antisense effects of peptide-PNA conjugates. Nature Biotechnology, 19: 360; 2001. 45. Tan XX, Actor JK, and Chen Y: Peptide nucleic acid antisense oligomer as a therapeutic strategy against bacterial infection: proof of principle using mouse intraperitoneal infection. Antimicrobial Agents and Chemotherapy, 49: 3203; 2005. 46. Frandsen N, Kjærulf S, Nielsen PE et al. (in preparation). 47. Jenssen H, Hamill P, and Hancock RE. Peptide antimicrobial agents. Clinical Microbiology Reviews, 19: 491; 2006. 48. Dryselius R, Aswasti SK, Rajarao GK et al.: The translation start codon region is sensitive to antisense PNA inhibition in Escherichia coli. Oligonucleotides, 13: 427; 2003. 49. Kulyte A, Dryselius R, Karlsson J and Good L: Gene selective suppression of nonsense termination using antisense agents. Biochimica et Biophysica Acta, 1730: 165; 2005. 50. Dryselius R, Nekhotiaeva N, and Good L: Antimicrobial synergy between mRNA- and protein-level inhibitors. Journal of Antimicrobial Chemotherapy, 56: 97; 2005. 51. Nekhotiaeva N, Awasthi SK, Nielsen PE, and Good L: Inhibition of Staphylococcus aureus gene expression and growth using antisense peptide nucleic acids. Molecular Therapy, 10: 652; 2004. 52. Stock RP, Olvera A, Sánchez R et al.: Inhibition of gene expression in Entamoeba histolytica with antisense peptide nucleic acid oligomers. Nature Biotechnology, 19: 231; 2001. 53. Sánchez R, Saralegui A, Olivos-Garcia A et al.: Entamoeba histolytica: intracellular distribution of the sec61alpha subunit of the secretory pathway and down-regulation by antisense peptide nucleic acids. Experimental Parasitology, 109: 241; 2005. 54. Koppelhus U, Zachar V, Nielsen PE et al.: Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA. Nucleic Acids Research, 25: 2167; 1997. 55. Kaushik N, Basu A, and Pandey VN: Inhibition of HIV-1 replication by anti-trans-activation responsive polyamide nucleotide analog. Antiviral Research, 56: 13; 2002. 56. Riguet E, Tripathi S, Chaubey B et al.: A peptide nucleic acid-neamine conjugate that targets and cleaves HIV-1 TAR RNA inhibits viral replication. Journal of Medicinal Chemistry, 47: 4806; 2004. 57. Chaubey B, Tripathi S, Ganguly S et al.: A PNA-transportan conjugate targeted to the TAR region of the HIV-1 genome exhibits both antiviral and virucidal properties. Virology, 331: 418; 2005. 58. Pesce CD, Bolacchi F, Bongiovanni B et al.: Anti-gene peptide nucleic acid targeted to proviral HIV-1 DNA inhibits in vitro HIV-1 replication. Antiviral Research, 66: 13; 2005. 59. Tripathi S, Chaubey B, Ganguly S et al.: Anti-HIV-1 activity of anti-TAR polyamide nucleic acid conjugated with various membrane transducing peptides. Nucleic Acids Research, 33: 4345; 2005. 60. Turner JJ, Ivanova GD, Verbeure B et al.: Cell-penetrating peptide conjugates of peptide nucleic acids (PNA) as inhibitors of HIV-1 Tat-dependent trans-activation in cells. Nucleic Acids Research, 33: 6837; 2005. 61. Nulf CJ and Corey D: Intracellular inhibition of hepatitis C virus (HCV) internal ribosomal entry site (IRES)-dependent translation by peptide nucleic acids (PNAs) and locked nucleic acids (LNAs). Nucleic Acids Research, 32: 3792; 2004. 62. Robaczewska M, Narayan R, Seigneres B et al.: Sequence-specific inhibition of duck hepatitis B virus reverse transcription by peptide nucleic acids (PNA). Journal of Hepatology , 42: 180; 2005. 63. Hamilton SE, Simmons CG, Kathiriya IS, and Corey DR: Cellular delivery of peptide nucleic acids and inhibition of human telomerase. Chemistry & Biology, 6: 343; 1999. 64. Ljungstrøm T, Knudsen H and Nielsen PE: Cellular Uptake of adamantyl conjugated peptide nucleic acids. Bioconjugate Chemistry, 10: 965; 1999.

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65. Shiraishi T, Nadia Bendifallah N, and Nielsen PE: Cellular delivery of polyheteroaromate-peptide nucleic acid (PNA) conjugates mediated by cationic lipids. Bioconjugate Chemistry, 17: 189; 2006. 66. Fotin-Mleczek M, Fischer R, and Brock R: Endocytosis and cationic cell-penetrating peptides—a merger of concepts and methods. Current Pharmaceutical Design, 11: 3613; 2005. 67. Koppelhus U, Awasthi SK, Zachar V et al.: Cell-dependent differential cellular uptake of PNA, peptides, and PNA-peptide conjugates. Antisense and Nucleic Acid Drug Development, 12: 51; 2002. 68. Richard JP, Melikov K, Vives E et al. Cell-penetrating peptides. A reevaluation of the mechanism of cellular uptake. Journal of Biological Chemistry, 278: 585; 2003. 69. Abes S, Williams D, Prevot P et al.: Endosome trapping limits the efficiency of splicing correction by PNA-oligolysine conjugates. Journal of Controlled Release, 110: 595; 2006. 70. Wadia JS, Stan RV, and Dowdy SF: Transducible TAT-HA fusogenic peptide enhances escape of TAT-fusion proteins after lipid raft macropinocytosis. Nature Medicine, 10: 310; 2004. 71. Shiraishi T, Pankratova S, and Nielsen PE: Calcium ions effectively enhance the effect of antisense peptide nucleic acids conjugated to cationic tat and oligoarginine peptides. Chemistry & Biology, 12: 923; 2005. 72. Shiraishi T and Nielsen PE: Photochemically enhanced cellular delivery of cell penetrating peptidePNA conjugates. FEBS Letters, 580: 1451; 2006. 73. Bøe S, Hovig E: Photochemically induced gene silencing using PNA-peptide conjugates. Oligonucleotides, 16: 145; 2006. 74. Albertshofer K, Siwkowski AM, Wancewicz EV et al.: Structure-activity relationship study on a simple cationic peptide motif for cellular delivery of antisense peptide nucleic acid. Journal of Medicinal Chemistry, 48: 6741; 2005. 75. Maier MA, Esau CC, Siwkowski AM et al.: Evaluation of basic amphipathic peptides for cellular delivery of antisense peptide nucleic acids. Journal of Medicinal Chemistry, 49: 2534; 2006. 76. Nelson MH, Stein DA, Kroeker AD et al.: Arginine-rich peptide conjugation to morpholino oligomers: effects on antisense activity and specificity. Bioconjugate Chemistry, 16: 959; 2005. 77. El-Andaloussi S, Johansson H, Lundberg P and Langel Ü: Induction of splice correction by cellpenetrating peptide nucleic acids. The Journal of Gene Medicine, 2006 (in press). 78. Filipovska A, Eccles MR, Smith RAJ, and Murphy MP: Delivery of antisense peptide nucleic acids (PNAs) to the cytosol by disulphide conjugation to a lipophilic cation. FEBS Letters, 556: 180; 2004. 79. Füssl A, Schleifenbaum A, Göritz M et al.: Cellular uptake of PNA-terpyridine conjugates and its enhancement by Zn2⫹ ions. Journal of the American Chemical Society, 128: 5986; 2006. 80. Dragulescu-Andrasi A, Zhou P, He G, and Ly DH: Cell-permeable GPNA with appropriate backbone stereochemistry and spacing binds sequence-specifically to RNA. Chemical Communications, 244; 2005. 81. Hamzavi R, Dolle F, Tavitian B et al.: Modulation of the pharmacokinetic properties of PNA: Preparation of galactosyl, mannosyl, fucosyl, N-acetylgalactosaminyl, and N-acetylglucosaminyl derivatives of aminoethylglycine peptide nucleic acid monomers and their incorporation into PNA oligomers. Bioconjugate Chemistry, 14: 941; 2003. 82. McMahon BM, Stewart JA, Jackson J et al.: Intraperitoneal injection of antisense peptide nucleic acids targeted to the mu receptor decreases response to morphine and receptor protein levels in rat brain. Brain Research, 904: 345; 2001. 83. McMahon BM, Stewart JA, Bitner MD et al.: Peptide nucleic acids specifically cause antigene effects in vivo by systemic injection. Life Sciences, 71: 325; 2002. 84. Boules M, Williams K, Gollatz E et al.: Down-regulation of amyloid precursor protein by peptide nucleic acid in vivo. Journal of Molecular Neuroscience, 24: 123; 2004. 85. Kristensen E: In vitro and in vivo studies on pharmacokinetics and metabolism of PNA constructs in rodents. In Peptide Nucleic Acids: Methods and Protocols, PE Nielsen ed. Copenhagen: Humana Press (Totowa, NJ, United States); 2002, pp. 259–269. 86. McMahon BM, Mays D, Lipsky J et al.: Pharmacokinetics and tissue distribution of a peptide nucleic acid after intravenous administration. Antisense and Nucleic Acid Drug Development, 12: 65; 2002. 87. Sazani P, Gemignani F, Kang S-H et al.: Systemically delivered antisense oligomers upregulate gene expression in mouse tissues. Nature Biotechnology, 20: 1228; 2002.

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Locked Nucleic Acid Troels Koch and Henrik Ørum

CONTENTS 19.1 19.2

Introduction .......................................................................................................................520 Chemistry of LNA and LNA Analogs...............................................................................521 19.2.1 Synthesis of LNA Amidites...............................................................................522 19.2.2 Key Reactions during the Synthesis of Amino- and Thio-LNA Amidites........524 19.2.3 Key Reactions during the Synthesis of LNA-Diastereoisomer Amidites .........526 19.2.4 Base Modifications of LNA ..............................................................................528 19.3 LNA Synthesis...................................................................................................................528 19.4 Structure of LNA and
-L-LNA ........................................................................................530 19.4.1 LNA Hybrid Structure.......................................................................................530 19.4.2
-L-LNA Hybrid Structure ................................................................................531 19.5 Biophysical Properties of LNA and LNA Analogs...........................................................532 19.5.1 Thermal Denaturation of LNA Heteroduplexes................................................532 19.5.2 Thermal Denaturation of LNA Analog Heteroduplexes: Thio- and Amino-LNA.......................................................................................................533 19.5.3 Thermal Denaturation of LNA Diastereoisomer Heteroduplexes.....................534 19.5.4 Thermal Denaturation of LNA Containing Base Modifications.......................535 19.5.5 Thermodynamic Considerations........................................................................536 19.5.6 Hybridization Kinetics ......................................................................................536 19.5.7 Thermal Denaturation of LNA Triplexes ..........................................................537 19.6 Biochemical Properties of LNA and LNA Analogs..........................................................537 19.6.1 General Designs ................................................................................................537 19.6.2 Nuclease Resistance ..........................................................................................537 19.6.3 RNase H Recruitment of LNA and LNA Analogs............................................539 19.7 Inhibition of Coding RNA in Vitro ...................................................................................540 19.8 Inhibition of Micro-RNA in Vitro .....................................................................................544 19.9 Pharmacological Activity in Experimental Animals .........................................................546 19.9.1 Brief Summary ..................................................................................................547 19.10 Pharmacokinetics...............................................................................................................548 19.10.1 Plasma Pharmacokinetics ..................................................................................548 19.10.2 Biodistribution and Tissue Half-Life.................................................................548

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19.10.3 19.10.4

Uptake into Cells ...............................................................................................551 Excretion............................................................................................................552 19.10.4.1 Brief Summary................................................................................552 19.11 Toxicology .........................................................................................................................553 19.11.1 Acute Toxicities.................................................................................................553 19.11.2 Subacute Toxicities............................................................................................554 19.11.2.1 Brief Summary................................................................................556 19.12 LNA Drugs in Development .............................................................................................557 19.13 Conclusions and Future Directions ...................................................................................557 Acknowledgments ..........................................................................................................................558 References ......................................................................................................................................558

19.1 INTRODUCTION The concept of using single-stranded oligonucleotides to therapeutically inactivate RNA (antisense therapy) is enjoying a major renaissance. In part, interest is being driven by the prospect of effectively treating many diseases where the causative proteins have proven difficult or impossible to target by conventional drug approaches. Even greater impetus, however, comes from the explosive growth of information about the human genome and the genetic and molecular basis of disease, which has dramatically expanded the number of potential RNA targets for drug action over the past few years. In addition, enthusiasm is enhanced by the realization that antisense therapy may be the only realistic approach to target noncoding, regulatory RNAs, such as miRNAs, whose role in disease is being increasingly recognized. These regulatory microRNAs exert their function through Watson–Crick pairing to their target messenger RNAs providing strong evidence that effective antisense mechanisms are a natural biological feature of normal cell physiology. Our understanding of the technical, biological, and clinical issues that face antisense therapy has advanced significantly in recent years. Today it is clear that many clinical failures reported for the first-generation antisense drugs made from DNA phosphorothioates can be attributed to shortcomings in their physico-chemical properties. Notably, phosphorothioates exhibit modest metabolic stability and very low affinity for their target mRNA thus necessitating the use of rather large molecules and high doses to achieve adequate binding affinity and pharmacological activity in animals. To aggravate matters, large phosphorothioates are poorly taken up by cells and display size-proportional binding to several plasma proteins, causing potentially serious acute toxicities in animals that limit their therapeutic window. This admittedly simplified description of the clinical challenges that have faced phosphorothioates nevertheless suggests that novel chemistries with improved affinity and metabolic stability are key to the development of antisense oligonucleotides (AONs) with satisfactory clinical performance. Locked nucleic acid (LNA*) is a novel RNA analog characterized by high metabolic stability and truly unprecedented binding affinity to its complementary RNA and DNA sequence. In this chapter we review how these properties have led to the development of shorter-than-usual oligonucleotides that exhibit dramatically improved pharmacological activity and safety compared to oligonucleotides based on other chemistries. The chapter starts with an overview of the chemistry and physical and biochemical properties of LNA oligonucleotides. This is followed by a detailed description of the performance of LNA oligonucleotides against different classes of RNA targets (pre-mRNA, mRNA, and microRNAs), in vitro and in experimental animals. We also describe the pharmacokinetics of LNA oligonucleotides and their toxicity in rodents and monkeys and provide an outline of candidate *

Terminology: (1) LNA is defined as an oligonucleotide comprising one or more 2⬘-O,4⬘-C-methylene--d-ribofuranosyl nucleotide building blocks (⫽ LNA monomeric unit). (2) LNA-analog is defined as an oligonucleotide comprising one or more chemically modified LNA monomeric unit.

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LNA drugs in development. The most advanced of these, SPC2996, directed against the Bcl-2 mRNA, is currently undergoing an international phase IⲐII multicenter trial for the treatment of chronic lymphocytic leukemia (CLL). We end the chapter with a short summation and some thoughts on the future of LNA oligonucleotide-based therapy.

19.2 CHEMISTRY OF LNA AND LNA ANALOGS The name LNA was coined in 1997 by the Wengel group at the University of Copenhagen, and it was introduced in the first publication where the 2⬘-O,4⬘-C-methylene--D-ribofuranosyl nucleotide building block was incorporated into oligonucleotides [1] (Figure 19.1B). The term was selected to signal the structural fact that the bicyclic structure of LNA locks the conformational flexibility of the ribose ring. The design rationale for making the 2⬘-O,4⬘-C-oxymethylene link was to make a high-affinity RNA analog by preorganising—locking—the furanose in a North-type conformation. From a thermodynamic point of view the benefit was intended to be twofold: (1) to reduce the inherent entropy loss during nucleic acid hybridization, and (2) preorganizing the molecule in a high-affinity conformation. LNA showed unprecedented high-affinity binding to nucleic acids [1], but the full thermodynamic explanation for this is significantly more complex than initially anticipated (vide infra). Independently of this work the Imanishi group at the University of Osaka [53] published the synthesis of the 2⬘-O,4⬘-C-methylene--D-ribofuranosyl nucleoside a few months earlier. Obika et al. [2] coined later the name bridged nucleic acid (BNA) for the 2⬘-O,4⬘-C-methylene bicyclic structure that 2 years earlier had been named LNA. The term LNA has been accepted worldwide and is also used across a range of commercial aspects. It is the most frequently used term to cover oligonucleotides comprising one or more 2⬘-O,4⬘-C-methylene--D-ribofuranosyl nucleotides. Despite massive efforts the search for new DNA and RNA analogs with improved therapeutic properties has largely been unsuccessful. In this light the invention of LNA becomes even more remarkable since it was immediately realized by the Wengel group that the bicyclic structure of the LNA-monomer serve as a general template for the development of additional analogs of LNA with further therapeutically useful properties (vide infra). Prominent examples of such LNA-analogs are amino-Ⲑthio- and
-L-LNA where the 2⬘-, 4⬘-bicyclic structure also is composed of five-membered rings (Figure 19.1B). The first LNA-analogs were reported in 1998 by Singh et al. [3–5]. Since then a variety of other chemically derivatized structures of LNA have been prepared which, for the purpose of this review,

(A)

B

O O

O

O O

P

O

−

LNA

(B)

O

B

O

O O

O

Amino-LNA

O O

S

B

O O

O

NH

P

O

B O

O

P

O P O

O

B

O

O

P

Thio-LNA


-L-ribo-LNA

Figure 19.1 Molecular structure of (A) LNA and (B) selected LNA analogs.

-D-xylo-LNA

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will be divided into the following three main categories: (1) amino- and thio-modifications of LNA; (2) diastereoisomers of LNA; and (3) base modifications, which covers LNA having other bases than A, MeC, G, and T. More recently, additional 2⬘-, 4⬘-bicyclic analogs composed of six-membered rings have been synthesized by the Imanishi group. Prominent representatives of this class are 2⬘-,4⬘-BNANC and 2⬘-,4⬘-BNACOC (BNA-NC, BNA-COC) [6,7]. When these conformationally restricted molecules are included in oligonucleotides they show RNA selective binding, have good nuclease resistance and the 2⬘-,4⬘-BNANC thymidine residue increases the Tm against RNA with 5–6°C per modification. These new analogs confirm the high affinity generality of the bicyclic space offered by LNA. Since this review covers only 2⬘-, 4⬘-bicyclic structures composed of five-membered rings, further information of these interesting structures can be appreciated from the literature [6,7]. In the subsequent sections of this chapter the synthesis of LNA-amidites will be described in detail. This is by far the most well-described and optimized synthesis sequence, and it has been performed in multikilogram scales (vide infra). Today, LNA-amidites are commodities and widely used for both academic and commercial purposes. The syntheses of the LNA-analog amidites will also be dealt with but from a more general perspective, where the focus will be on key intermediatesⲐ reactions during the synthesis and on specific synthesis improvements.

19.2.1 Synthesis of LNA Amidites Two different strategies have been used for the LNA amidite synthesis. The Wengel group introduced a convergent strategy using protected glucose as the starting material (Figure 19.2A) [1,8,9]. This strategy employs a common sugar coupling intermediate to which the nucleobases are coupled via a classical Vorbrüggen reaction (Figure 19.2B) [10,11]. The second strategy was introduced by the Imanishi group and here the corresponding RNA nucleosides are used as starting materials [12,53]. Both of these strategies have different pros and cons, but in this review we will focus on the convergent synthesis route that over the past 5 years have proven to be very effective during scale-up and has provided remarkable yields and qualities of the LNA amidites. The starting material is the commercially available 1,2:5,6-di-O-isopropylidene-
-D-glucofuranose (Figure 19.2A, 1). By electrophilic-assisted dimethyl sulfoxide (DMSO) oxidation of the 3⬘-OH and subsequent stereoselective reduction by sodium borohydride, 1,2:5,6-di-O-isopropylidene
-D-allofuranose (2) is formed [13]. Protection of 3⬘-OH is done by alkylation with benzylbromide and the 5,6-isopropylidene group is selectively removed in diluted acetic acid. Oxidative cleavage of the diol with periodate followed by aldol condensation and Cannissaro reaction yields the 4⬘-C-hydroxymethyl furanose (Figure 19.2A, 3). Formation of the bicyclic structure is based on selective reactions of the two alcohols (5⬘-OH and 4⬘-hydroxymethyl). Originally this was done by “selective” 5-O-benzylation followed by tosylation of the 4-C-hydroxymethyl group [1,8,9,14]. However, it turned out that the selectivity was rather poor and the low yield of this crucial step reduced the overall yield of all four monomers significantly. Koshkin et al. [15] reported in 2001 a marked improvement of the synthesis strategy. Instead of using the “selective” benzylation, the alcohol groups in 3 were permesylated (bismesylate, Figure 19.2A, 4). The 5-O-methanesulfonyl group serves as a suitable protecting group of the 5⬘-OH, and the 4-C-methanesulfonoxy-methyl group serves as a suitable activation group for nucleophilic substitution by the 2⬘-OH that is liberated by saponification after the coupling [15–17]. Prior to the coupling and ring closure the diacetylated furanose (Figure 19.2A, 5) is made in situ and the anomeric mixture of 5 is used as a common glycosyl donor for the protected or nonprotected nucleobases. Since all four LNA amidite syntheses follow the same principal reactions only the LNA-T amidite reactions will be described here in detail. The interested reader may consult excellent publications and reviews for further details [8,14,15,18], but a few synthetic highlights and important improvements of these reactions will be touched upon here.

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O

(A)

O

O OH O

O O O

O

3, NaIO4 HO 4, H2CO, NaOH

O

OH

2, NaBH4

Glucofuranose (1) 1, NaH, BnBr 2, 80% AcOH HO

O

1, DMSO-P2O5

Allofuranose (2) MsO

MsCl, Py

O O

MsO

OBn O

(3)

O O OBn O

Bismesylate (4)

MsO

O

MsO

OAc

OBn OAc

Coupling intermediate (5)

(B)

DMTO

5 steps

i Pr2N

MsO

O O P O(C H )-CN 2 4

NH

NH O

CBz

O

O

MsO

Me

O

N

O

5 steps

HO

OBn OAc

N

O HO

DMTO

O 2 steps

O (6)

i Pr2N

BzHN

O

T

O O P O(C H )-CN 2 4

N N MsO MsO

O

OAc

OBn OAc

MsO MsO

N

O

N

DMTO 7 steps

OBn OAc i Pr2N

ABz O O O P O(C H )-CN 2 4

Cl N N MsO MsO

O

N

OBn OAc

N

NH2

DMTO

6 steps

i Pr2N

O

GDMF

O O P O(C H )-CN 2 4

Figure 19.2 Key steps and intermediates in the (A) convergent part of the LNA amidite synthesis and (B) divergent part of the LNA amidite synthesis.

Reaction of 5 (Figure 19.2A) with silylated thymine under classical Vorbrüggen conditions [10,11] provides the glycosylation reaction stereo selectively and the beta-configured nucleoside is obtained in high yield. Liberation of the 2⬘-OH by saponification mediates fast intramolecular 2⬘-C, 4⬘-C-oxymethylene cyclization. The 5-O-methanesulfonyl group is removed in a two-step manner by first substitution with benzoate and second saponification of the benzoate. Catalytic hydrogenation

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of the 3⬘-O-benzyl group affords the LNA-T nucleoside (Figure 19.2B, 6, the “T-Diol”). This latestage intermediate is then 5⬘-OH DMT protected and 3⬘-OH phosphitylated to provide the LNA-T amidite. It is noteworthy that all LNA-DMT-protected nucleosides are phosphitylated according to the same general procedure in which bis-amidite (2-cyanoethyl-N,N,N⬘,N⬘-tetraisopropylphosphorodiamidite) and 0.7 equivalent 4,5-dicyanoimidazole (DCI) is used. This improved procedure produces the amidites in yields higher than 90% after column purification [19]. The LNA-T nucleoside (Figure 19.2B, 6) serves as the starting material for the synthesis of the LNA-MeC nucleoside via the classical reaction of thymine with 1,2,4-triazole and POCl3 to afford methyl-cytosine [15,20]. It is well known that glycosylations performed under Vorbrüggen conditions with protected purines provide both the N-7 and the N-9 isomers, where the N-9 is the wanted isomer [10,11]. The N-9 isomer is the thermodynamically favored product and will gradually be formed at elevated temperature. In the case of protected adenine the N-9 isomer is formed at elevated temperature in high yield, whereas protected guanine (i-Bu) always provide the N-7 isomer in ca. 15% yield [15]. A marked improvement of the LNA guanine synthesis was reported in 2004 by Rosenbohm et al. [21]. Here the guanine nucleoside was obtained regioselectively by coupling with the guanine synthon: 2-amino-6-chloro-purine. The 6-oxo-group of guanine is formed in a subsequent step where the 6-chloro atom is substituted with 3-hydroxy-acrylonitrile that via elimination forms the 6-oxo group. Besides providing the N-9 isomer in high yield, the 6-chloro position of the 2-amino-6-chloro-purine base can be substituted by other nucleophiles to provide a range of naturalⲐunnatural bases like di-amino-purines [16,21]. The overall yields of the LNA amidite syntheses were initially very low. The yields of the LNApurine syntheses were 1–5% and the yields for the LNA-pyrimidine syntheses were 5–10%. Thanks to a further medicinal chemistry effort over the past 6 years the yields are now in range of 25–40%. This is a significant achievement since the total number of synthetic steps of the four amidites is 34, and the total synthetic steps for the individual monomers are ranging from 14 to 17. This work has mainly been done at Exiqon AⲐS, Proligo GmbhⲐInc, Cureon AⲐS, and Santaris Pharma AⲐS, where many talented chemists have made their contributions to making LNA amidites cost-efficient drivers for the commercialization of LNA. Today, LNA amidites are produced in multikilogram scale, and the early intermediates, allofuranose and bis-mesylate are manufactured in scales ranging from 100 to 250 kg. The significant optimization of the entire synthesis sequence combined with the economy of scale has reduced the costs of LNA amidites to a very competitive level. For manufactures larger than 100 kg scale the average cost per step is now around $1, and with the knowledge available today of the LNA amidite synthesis we can predict that this level will be reached for the entire amidite synthesis. 19.2.2 Key Reactions during the Synthesis of Amino- and Thio-LNA Amidites The first LNA-analog to be synthesized was amino-LNA [3,4]. The amino group of the nucleoside was introduced via a double nucleophilic substitution reaction on a di-O-tosylated nucleoside (Figure 19.3, 1) at 130°C in benzylamine to provide the configuration of amino-LNA monomer (Figure 19.3, 3) via a postulated 2,2⬘-anhydro nucleoside (Figure 19.3, 2) intermediate. The benzyl groups were removed by transfer hydrogenation and the resulting fully deprotected amino-LNAnucleoside could selectively be protected on the amine with trifluoroacetyl followed by the standard DMT and phosphitylation reactions. Rosenbohm et al. [22,23] published later an improved synthesis of the amino-LNA nucleoside. This synthesis strategy is convergent to a late stage with the LNA amidite synthesis and can, therefore, benefit from the bulk manufactures of the LNA amidites. After the coupling with thymine the 2⬘-OH is liberated under mild conditions to avoid cyclization. The 2,2⬘-anhydro nucleoside (Figure 19.4, 2) is formed and subsequently hydrolyzed and triflated. The 2,2⬘-anhydro nucleoside is a key intermediate for both strategies that therefore are restricted to syntheses of pyrimidine amino-LNA nucleosides. However, the purines can be made by a transnucleosidation reaction that recently has been published. According to this approach the triflate (Figure 19.4, 3) is reacted with azide by which the configuration at C2⬘ is inverted. The amine is obtained by reduction of the azide and it is subsequently protected with trifluoroacetic anhydride. This compound serves as

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O NH BnO

O

O

O

N

NH

N

O

BnNH

TsO

TsO

OBn OTs

N O O

BnO

1

BnNH

OBn

BnO

O

O

NBn

BnO

2

N

3

Figure 19.3 Key steps and intermediates in the amino-LNA amidite synthesis.

O

O NH

MsO MsO

O

N

OBn OAc

O

O

N

1, NH3 in MeOH 2, MsCl, Py; 3, DBU

N O O

MsO MsO

OBn

NH 1, H2SO4 (dil.)

N O R

MsO

2, Tf2O, DMAP, Py MsO

O

OBn R = OTf

1

2

3

Figure 19.4 Key steps and intermediates of the improved synthesis of the amino-LNA amidite.

a common intermediate for the transnucleosidation reaction with 2-amino-6-chloropurine and N6-benzoyladenine providing the corresponding  purines in 57 and 58% yield, respectively [24]. The trivalent nature of the amino group in amino-LNA offers conjugation opportunities [4,25]. In contrast to a traditional 5⬘-/3⬘-OH end conjugation, ligand linking via an internal position offers new perspectives and the effect of the conjugate can be multiplied as the nucleosides are incorporated into the oligonucleotide [26]. Also, it can be anticipated that the conjugate will exhibit different properties since the location internally, as compared to terminally, of an oligonucleotideⲐhybrid duplex will be different. Sorensen et al. [25] attached a series of ligands to amino-LNA. N-benzoyl and N-aminoethyl showed in particular good hybridization properties, but even a large group as pyrene linked via a methyl group provided good hybridization to complementary DNAⲐRNA. The excited complex of two pyrenes is known to give rise to a strong excimer band at 430–530 nm. This was used to demonstrate interstrand “communication” in duplexes comprising amino-LNA-nucleosides on opposing strands [27,28]. The hybridization can be followed by spectroscopy, since the pyrene groups are structuredⲐstacked during duplex formation. An analogous excimer effect of pyrenes on opposing strands is seen when pyrene is linked to amino-
-L-LNA [29]. Synthesis of the thio-LNA nucleoside posed a new synthetic challenge (Figure 19.1B) [3,30]. The synthetic strategy for the formation of the bicyclic structures of LNA and LNA analog nucleosides is based on the reductive removal of the 3⬘-O-Bn-protection group after cyclization, but when sulfur is present catalytic reductions are not possible. The original synthesis used bis-protection of the 5⬘- and 3⬘-alcohols with TIPS to avoid the problem, but the overall yield was rather low [3]. Pedersen and Koch [31] reported an improved procedure for the synthesis of the thio-LNA-T and Me C nucleoside. The synthesis was convergent with the improved amino-LNA procedure (vide supra) and uses the same late stage 2,2⬘-anhydro nucleoside intermediate (Figure 19.4, 2). The key step in the synthesis is the removal of the 3⬘-O-Bn protection group without formation of the 4⬘-C,3⬘-O oxetane. The rigid structure of the 2,2⬘-anhydro nucleoside intermediate prevents that and the protection group on the 3⬘-OH can be exchanged with benzoyl that after cyclization with Na2S can be removed by hydrolysis (Figure 19.5). As for the amino-LNA nucleoside synthesis only the pyrimidine nucleosides can be made by this procedure.

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION O

O

MsO MsO

N

N

N O O

N O O

OBn

1

1, Pd/C, H2 2, BzCl, Py

MsO MsO

OBz

O NH 1, H2SO4 (dil.) 2, Tf2O,DMAP 3, Na2S, DMF

MsO

O

S

BzO

2

N

O

3

Figure 19.5 Key steps and intermediates of the improved synthesis of the thio-LNA amidite.

19.2.3 Key Reactions during the Synthesis of LNA-Diastereoisomer Amidites Rajwanshi et al. [32–34] reported in 1999 the synthesis and basic properties of thymine xylo-LNA (Figure 19.1B). The xylo-LNA nucleoside is made from 4-C-hydroxymethyl--Lthreo-pentofuranose (Figure 19.6A, 1). Benzoylation of 4-C-hydroxymethyl--L-threo-pentofuranose and subsequent removal of the isopropylidene protection group afforded the xylo-coupling sugar 2 (Figure 19.6A) that is coupled to thymine under Vorbrüggen conditions (Figure 19.6A, 3). The 4⬘-C-hydroxymethyl group is—in a moderate yield—tosylated but cyclization can only proceed if the 5⬘-OH was DMT protected [32]. Reductive debenzylation removes also the DMT protecting group providing the fully deprotected xylo-LNA-T nucleoside. DMT protection and phosphitylation is performed according to the traditional procedures [32]. Xylo-LNA can only give rise to increased Tm when fully modified (3–4°C per modification). Therefore, it does not have the important property of cooperative binding as LNA and
-L-LNA (vide infra) [33–35]. Håkansson et al. [36] has reported several syntheses of thymine
-L-LNA amidites. The most attractive procedure converts the intermediate 3 derived for the xylo-LNA nucleoside synthesis (Figure 19.6A, 3) into the corresponding trimesylate (Figure 19.6B, 1). Treatment of the trimesylate with aqueous base affords the
-L-LNA bicyclic structure and hydrolyzes the 5⬘-O-Ms. Reverting the configuration at C2⬘ is proposed to proceed via the 2,2⬘-anhydro nucleoside intermediate. The 3⬘-O-Bn group is removed by catalytic hydrogenation and the 5⬘-OH is Dmt protected according to the traditional procedures to provide 4 (Figure 19.6B). The MeC
-L-LNA nucleoside is prepared from the thymine nucleoside according to the same procedures as described for LNA (vide supra). The synthesis of adenine
-L-LNA was reported in 2001 [37]. The synthesis of the diastereoisomer
-L-xylo is performed to a procedure closely resembling the one described here for
-L-LNA. The details can be appreciated from Håkansson et al. [36]. Recently, a new chemical modification has been synthesized where the oxygen of the 2⬘-O, 4⬘-C-methylene biradical of the
-L-LNA nucleoside is replaced with nitrogen, amino-
-L-LNA [38]. In the preferred synthesis route the amino functionality is introduced—as in the improved synthesis of the amino-LNA nucleoside [22] via the azide (Figure 19.6C, 3). Surprisingly, both anomers of the triflated nucleoside underwent smoothly nucleophilic substitution with azide to provide 3 (Figure 19.6C) in high yields. Compound 3 was acetolyzed (Figure 19.6C) and the coupling of thymine provided the
Ⲑ-anomer mixture in 80% yield. Subsequent reduction of the azide under Staudinger conditions [22] provided a separable mixture of the nucleosides. The final phosphoramidite was obtained over 15 steps in an overall yield of 4% (Figure 19.6C, 1). When this molecule was incorpotated into oligonucleotides a significant increase in Tm against RNA was observed (vide infra). Amino-
-L-LNA is very interesting since it combines the structure and configuration of
-L-LNA with the changed functionalities that the trivalent amine offers. The
-configured isomer of LNA—
-D-LNA or just
-LNA—has also been synthesized [39,40]. In contrast to the -isomer incorporation of
-LNA monomeric units leads to significant

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(A) HO

OBn O O

HO

BzO

1, BzCl, Py

OBn O

2, 80%, AcOH BzO

O

OAc OAc

1

2 O

O NH

1, BSA, thymine, TMS-Tf HO 2, NaOMe, MeOH

OBn O

HO

N

NH

O

1, TsCl, Py

OH O

HO

2, DmtCl, Py 3,H2, Pd/C

OH

N

O

O

4

3

(B) O

O

NH

NH HO

OBn O

HO

O

N

MsCl, Py

OBn O

MsO MsO

OH

N

O

OMs

1

2 O

O NH

N OBn O O

NaOH

NH

O

HO

N OH O O

1, H2, Pd/C 2, DmtCl, Py

O

DmtO

3

4

(C) HO

OBn O

1, MsCl, Py O

HO

O

OBn O

2, HCl (2.5%) MsO MeOH

1

OMe OH

2

1, Tf2O, Py MsO 2, NaN3

MsO

MsO

1, H2SO4 MsO OBn N3 O OMe 2, T, BSA, MsO MSOTf

3

OBn N3 O N

O NH

O

4

Figure 19.6 Key steps and intermediates of the (A) xylo-LNA amidite synthesis, (B)
-L-LNA amidite synthesis, and (C) amino
-L-LNA amidite synthesis.

decreased affinities in most oligonucleotide designs against both DNA and RNA [41,42]. But fully modified
-LNA oligonucleotides display significantly increased affinities towards RNA when hybridized in the parallel orientation [42,43]. However,
-LNA has not been used in antisense technology.

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19.2.4 Base Modifications of LNA In the improved synthesis procedure of the LNA-G amidite, 2-amino-6-chloropurine is used as guanine synthon (Figure 19.2B). Rosenbohm et al. [21] used this synthon to prepare diaminopurine nucleosides by treating the protected LNA-2-amino-6-chloropurine nucleoside with saturated ammonia. This procedure is convergent with the LNA-G synthesis and affords the final diaminopurine phosphoramidite in (42%) yield counted from the cyclized 5⬘-O-MsⲐ3⬘-O-Bn-protected 2-amino-6-chloro derivative [21]. The 6-chloro atom can be removed reductively by treatment with ammonium formiate and 20% Pd(OH)2ⲐC affording 2-amino purine LNA nucleoside [16]. In the synthesis of the hypoxanthine LNA nucleoside the coupling affords a ratio of N-9ⲐN-7 isomers of 4:1. The isomers are separable chromatographically at high pH [16]. A variety of LNA C-nucleosides have been synthesized. Among these are Pyrene, phenyl, pyrrole, imidazole, and pyridine just to mention a few. The interested reader can consult the literature for further syntheses information of these modifications [44–51].

19.3 LNA SYNTHESIS Synthesis of LNA oligonucleotides follows the well-known solid-phase principles of the phosphoramidite approach [69,70], and it has been performed on most commercial available automated synthesizers [71]. LNA-amidites [19] have a close structural resemblance to DNAamidites and use standard DNA protecting groups: DMT for the 5⬘-OH, Bz for adenine and 5methylcytosine, and DMF or i-Bu for guanine. In the early publications no particular emphasis was made on LNA synthesis, except the need for prolonged coupling times, and average cycle yields of ⬎98% were reported [1,55,60]. However, it was soon realized that modifications of the standard DNA protocols were needed for robust LNA synthesis. As mentioned, it was early observed that the LNA-amidite coupling kinetics were slower when compared to DNA-amidites, and that the standard activator, tetrazole, did not provide satisfactory coupling yields. In contemporary optimized protocols, tetrazole is replaced with 4,5-dicyanoimidazole (DCI), which is used in molar excess to the amidites (1.2–2 times). For small-scale syntheses (1–15
mol) a molar excess of the amidites of four to eight times is used. The recommended coupling time depends on the scale but ranges from 6 to 15 min [72]. In DNA synthesis the phosphite is flush oxidized with aqueous iodine. The reaction time is only ca. 15 s and very high yields are obtained (⬎99%). For LNA syntheses flush oxidation is not adequate and the oxidation time has to be prolonged to 1–2 min [73,74]. When LNA-phosphorothioates (PS) are made the classical oxidation reagent—Beaucage reagent—can be used, but more powerful reagents such as dimethylthioramdisulfideⲐxanthanhydrideⲐphenylacetyldisulfide (PADS)Ⲑ3-ethoxy1,2,4-dithiazoline-5-one(EDITH)Ⲑ1,2,4-dithiazoline-3,5-dione(DtsNH) are recommended. As with iodine oxidation, prolonged oxidation is used during thiolation (2–3 min) [74]. It is clear from the LNA synthesis optimizations, not surprisingly, that locking the conformation by introducing the additional ring in LNA changes the chemical properties of the 3⬘-OHⲐ phosphoramiditeⲐ phosphite. However, by using these minor changes to standard DNA synthesis protocols, LNA synthesis with phosphordiester or phosphorothioate internucleoside linkages can be performed in yields comparable to the corresponding DNA syntheses [73,74]. All subsequent manipulations, work-up, and characterization of LNA, follow the identical procedures developed for DNA synthesis [74]. Synthesis of LNA analogs follows essentially the same protocol as for LNA. For amino- and thio-LNA synthesis coupling times of 12 min are employed but otherwise standard DNA cycles are used [4,30]. For
-L-LNA and xylo-LNA synthesis slightly shorter coupling times are used (10 min) and the amidites are activated with either tetrazole or pyridinium hydrochloride [33,75]. Stereo-controlled synthesis of LNA with PS internucleoside linkages has been reported using the exact analogous procedure reported for deoxyoligonucleotides [76]. The stereo selectivity for

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making an LNA T-MeC dimer was over 96% and the absolute configuration of the isomers—SpⲐRp—is assigned. The Sp dimer is resistant against snake venom diesterase digestion for more that 24 h. Besides phosphordiester and PS internucleoside linkages, methylphosphonate, N5⬘- phosphoramidate and N3⬘-phosphoramidate linkages have been reported for LNA (Figure 19.7) [77–79]. The methylphosphonate internucleoside linkage is easily introduced via the corresponding monomeric methylphosphonamidite (Figure 19.7, 1) that is used directly under standard oligosynthesis conditions [77]. The coupling time is increased to 15 min and the reported coupling yield is 83%. LNA with 3⬘-methylphosphonate internucleoside linkages have decreased affinities compared to LNA diesters. N5⬘- phosphoramidate linkages can also be introduced directly during oligosynthesis via the 5⬘-Mmt-NH-protected LNA phosphoramidite (Figure 19.7, 2). The coupling yield for this LNAanalog is reported to be 94% [79]. The affinity increase per modification is 2–5°C against respective DNA and RNA. LNA comprising N3⬘-phosphoramidate is more complicated to prepare. The N3⬘-phosphoramidate is introduced via a dimer composed of 5⬘-ODMT-LNA-T-N3⬘→ P5⬘-DNA-T3⬘-phosphoramidite (Figure 19.7, 3) [78]. LNA phosphoramidate hybrid duplexes have almost the same thermal stability as LNA diesters with affinity increases per modification of 4–11°C against respective DNA and RNA. LNA without phosphate in the internucleoside linkage has also been prepared by Lauritsen and Wengel [80]. The native diester is here substituted with an amide linkage C3⬘-O-CH2-CH2NH-C(⫽O)-C4⬘ (∗). Four dimers comprising this internucleoside linkage is reported: 5⬘-ODMT-dT∗ L L L dT-3⬘-phosphoramidite (A), 5⬘-ODMT-T — ∗dT-3⬘-phosphoramidite (B), 5⬘-ODMT-T — ∗T — -3⬘L phosphoramidite (C), and 5⬘-ODMT-dT∗ T — -3⬘-phosphoramidite (D). Since the dimers are 5⬘-ODMTⲐ3⬘-phosphoramidite protected they can be directly incorporated into oligonucleotides. The coupling times are increased compared to LNA cycles from 10 to 20 min. Oligonucleotides comprising these dimers have significantly decreased hybrid stabilities, but a Tm increase of 2–9°C per modification is observed for (D) against respective DNA and RNA. This can be explained by the fact that the 5⬘-dT can accommodate by its more flexible and dynamic nature of the long amide L linkage, and that the 3⬘-T — can tune the conformation of the downstream dT towards higher Tm [80]. Oligonucleotides with phosphoramidate and amide internucleoside linkages have an interesting perspective in antisense since the nuclease resistance is much increased compared to oligonucleotides comprising native diesters and PS [78,79]. LNA synthesis has over the past years been scaled up from milligram to hundreds of gram scales. LNA synthesis is very “portable” and the improvements obtained in small scale are directly transferable to large scales. Today, LNA oligonucleotides are routinely made in scales of hundreds of grams in yields comparable to DNAⲐPS. O NH DmtO

O

O NH

DmtO

N

O O

(i-Pr)2N

1

P

O

NH MMTrHN

O CH3

O O

(i-Pr)2N

N

P

2

O

N

O

MeO P O O

OCH2CH2CN

O NH

O O

O

N

O

N

O

O (i-Pr)2N

P

OCH2CH2CN

3

Figure 19.7 Chemical structures of the amidites used to make methylphosphonate (1), N5⬘- phosphoramidate (2), and N3⬘-phosphoramidate (3) linkages.

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19.4 STRUCTURE OF LNA AND
-L-LNA Natural dsDNA exists at physiological pH as a B-form helix, whereas dsRNA exists as an A-form helix. This morphological difference is originated in the difference in the preferred sugar conformations of the deoxyriboses and the riboses. The furanose ring of deoxyribose exists at room temperature in an equilibrium between C2⬘-endo (S-type) and C3⬘-endo (N-type) conformation with an energy barrier of only ⬃2 kcalⲐmol (Figure 19.8) [52]. The C2⬘-endo (S-type) conformation gives rise to the B-form helix, whereas the C3⬘-endo (N-type) conformation gives rise to the A-form helix. For deoxyribose the S-type conformation is slightly lowered in energy (⬃0.6 kcalⲐmol) compared to the N-type and explains why DNA is found in the S-type conformation. For ribose the preference is for the N-type and thus, RNA adopts the A-form helix associated with higher hybridization stability. The structure of LNA and
-L-LNA has been comprehensively studied by NMR and x-ray analyses, and it is evident that LNA and
-L-LNA adopt right-handed helixes and hybridize according to the antiparallel Watson–Crick base-pairing pattern. The LNA–nucleoside has a fixed C3⬘-endo sugar pucker (N-type, 3E) [53–55], a sugar pucker—formalistically—also assigned for the
-L-LNA nucleoside, since the nucleoside is L-ribo configurated. The
-L-LNA nucleoside is often referred to as having a C2⬘-endo and thus “S-type” when compared with D-ribo-configured sugars (e.g., LNA nucleosides). 19.4.1 LNA Hybrid Structure A number of NMR, CD, and x-ray studies have been conducted to study the structure of LNA:RNA hybrids [54–59]. The glycosidic angle of the included LNA–nucleosides is in the anti range and upon inclusion of an increasing number of LNA–nucleosides the overall duplex geometry is progressively altered towards A-type. When only three LNA–nucleotides are included in a 9-mer oligonucleotide the hybrid adopts an almost canonical A-type duplex geometry. This correlates with the observed helical thermostability per LNA–nucleoside that reaches a maximum with ⬍50% LNA nucleotides [1,14,54,60]. This feature of “structural saturation” can be explained from the conformational changes of the deoxynucleotides by the flanking LNA–nucleotides. In unmodified deoxyoligonucleotides the preferred conformation is S-type, corresponding to a low-percentage N-type (Table 19.1, column “DNA”). When one LNA–nucleoside is included the percentage N-type of the 5⬘-adjacent and in particular in the 3⬘-adjacent deoxynucleotide is significantly increased (Table 19.1, column “LNA1”). This is further demonstrated when three LNA–nucleosides are included (Table 19.1, column “LNA3”). This phenomenom of “structural saturation” is a very interesting basic property of LNA since only a few inclusions are able to induce a conformational change of the entire oligonucleotide towards a high-affinity structure. Or in other words, the high-affinity conformation of a single LNA inclusion is amplified by its induction of a conformational change of juxtaposed DNA nucleotides. The consequence of “structural saturation” is that a few LNA inclusions give rise to a higher affinity increase per residue than many inclusions. This property of LNA is, in contrast, to

O

B O

B

O O O

O

C2′-endo (S-type) Figure 19.8 Furanose conformations.

C3′-endo (N-type)

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Table 19.1 Sugar Puckers of the Deoxyriboses in Oligonucleotides Containing LNA and
-L-LNA Nucleosides Hybridized to Complementary RNA and Expressed as % of N -type Sugar Conformation

C1 X G3 A4 X A6 X G8 C9

DNA

LNA1

LNA3


-L-LNA3

64 16 23 38 31 27 20 15 36

56 12 20 ⬃50a na ⬎90 21 17 27

47 na ⬎90 ⬎90 na ⬎90 na ⬎90 70

25 na 37 24 na 30 na 46 43

Note: The first column represents the generic oligonucleotide: 5⬘-CXGAXAXGC-3⬘, where X represents (X ⫽ dT, TL, or
LTL). Thus, the four oligonucleotides are DNA, d(CTGATATGC); LNA1, d(CTGATLATGC); LNA3, d(CTL GATL ATL GC);
-L-LNA, d(C
LTL GA
L TL A
L T L GC). na, no analysis of locked structures. a Estimated value.

many classical 2⬘ modifications (2⬘-O-Me and 2⬘-MOE) that show the exact opposite trend where the largest effect per residue is reached when the strand is fully modified [61]. The LNA–nucleotides fit perfectly to the A-type duplex structure with the 2⬘-O, 4⬘-C methylene bridge positioned at the brim of the minor groove of the duplex and with the backbone torsion angles as the standard A-type genus [54]. The complementary RNA strands seems unperturbed by the opposite LNA strand [54–59]. It has recently been demonstrated that the high-frequency dynamics of a nucleic acid duplex is significantly damped by inclusion of LNA on one strand [62]. Thus, the locked structure of the LNA–nucleoside translates into more rigid and less dynamic helixes. When LNA oligonucleotides are hybridized to DNA a general transition toward an A-type helix is seen [56,58,63,64]. With up to three modifications in a nonamer conformational changes are only seen in the LNA strand and the complementary DNA strand retains its native B-type geometry. For fully modified LNA the complementary DNA strand begins to alter its geometry. Thus, the sugars are changed from S-type conformation to an equilibrium of NⲐS-type. For both LNA:RNA and DNA hybrids increased stacking of the nucleobases are observed. 19.4.2
-L-LNA Hybrid Structure The structure of
-L-LNA:RNA hybrids have been studied by NMR and MD [65–68]. As for LNA–nucleosides the glycosidic angle of
-L-LNA nucleosides is in the anti range with the 2⬘-O,4⬘C methylene bridge positioned at the brim of the major groove. Since the
-L-LNA nucleoside is an unnatural stereochemistry it is basically futile to label it as N- or S-type, but the fact is that when the
-L-LNA nucleoside is incorporated into deoxyoligonucleotides it acts as a B-type mimic [66], and in contrast to LNA–nucleotides
-L-LNA-nucleotides do not induce any significant conformational change of neighboring deoxynucleotides (Table 19.1, column “
-L-LNA”). Owing to these structural facts the overall structural features of
-L-LNA:RNA duplexes are the same as for the DNA:RNA. However,
-L-LNA nucleosides alter the sugar–phosphate backbone slightly, so the phosphate groups are rotated into the minor groove compared to the unmodified hybrid. When
-L-LNA is hybridized to DNA the duplex features all the characteristics for B-type duplexes. For both
-L-LNA:RNA and DNA hybrids increased stacking of the nucleobases is observed. Both LNA and
-L-LNA nucleosides fit perfectly within the Watson–Crick framework. This explains why these modifications can be mixed with nucleic acid nucleosides and analogs thereof and act cooperatively. Interestingly, when LNA and
-L-LNA nucleosides are aligned in space the

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three key atoms N-1, O-3⬘, and O-5⬘ overlay [34]. These key positions are of course fully correlated with the Watson–Crick framework and it also explains why LNA and
-L-LNA-nucleosides can be mixed in the same oligonucleotide and act cooperatively (vide infra) representing respectively an A-type mimic and a B-type mimic. 19.5 BIOPHYSICAL PROPERTIES OF LNA AND LNA ANALOGS 19.5.1 Thermal Denaturation of LNA Heteroduplexes The most intriguing property of the LNA–nucleotide is that it fits so perfectly within the Watson–Crick framework. LNA–nucleotides can be incorporated in any combination with DNAⲐRNA nucleotides, and with any analog hereof that also fits within the Watson–Crick framework. LNA oligonucleotides obey the WⲐC-hydrogen bonding rules and form right-handed helices (vide supra). The hybrid stability is determined by classical thermal denaturation (Tm), and since almost all hybrid duplexes show regular cooperative sigmoid melting curves the Tm is conveniently determined by the first derivative of the Tm curves. The extraordinary high affinity of LNA was the immediate property described for LNA [1]. Since then this property has been confirmed in numerous papers [1,5,9,14,55,81]. When LNA monomers are included in either DNA or RNA each residue adds to the affinity in an additive manner (Table 19.2). When a few LNA residues are included a Tm increase of 6–7°C per modification is observed (Table 19.2, entry 2, 5, and 6). When LNA residues are included in PSs the affinity increase per modification is even larger (⬇10°C against RNA; Table 19.2, entry 4–5, and 8–9). For fully modified LNA oligonucleotides the increase per modification is reduced to 4–5°C (Table 19.2, entry 7). This is a general pattern for LNA: the affinity increase per LNA-monomer is larger the fewer incorporated (Table 19.2, entry 1–7) [81]. The reason for this is “structural saturation” of LNA (vide supra). LNA residues are therefore, the most efficient way of affinity gain, and a single modification in an oligonucleotide may be enough to provide enabling properties of the oligonucleotide [82]. When six nucleotides (nts) of 2⬘-O-Me RNA are included in a DNA oligonucleotide the Tm decreases against the DNA complement and increases only 1°C per modification against the RNA complement (Table 19.2, entry 4, 10–11). When the oligonucleotide becomes fully 2⬘-O-Me Table 19.2 Hybridization Studies with 14-ⲐⲐ 9-mer LNA against Complementary DNA and RNA Entry

Sequence

DNA Compound Tm (oC)

RNA Complement Tm (oC)

1: Reference DNA 5⬘-dT14-3⬘ 2: LNA Ⲑ DNA chimera 3: LNA Ⲑ DNA chimera 4: Reference DNA 5: LNA Ⲑ DNA chimera 6: LNA Ⲑ RNA chimera 7: Fully modified LNA 8: Fully PS 9: PS-LNA Ⲑ -DNA chimera 10: 2⬘-O-Me-RNA 11: 2⬘-O-Me-RNA/DNA chimera 12: LNA Ⲑ 2⬘-O-Me-RNA chimera

5⬘-dT14 5⬘-d(T)3(TLT)4T3 5⬘-(TL)13T 5⬘-d(GTGATATGC) 5⬘-d(GTLGATLATLGC) 5⬘-r(GTLGATLATLGC) 5⬘-(GLTLGLALTLALTLGL MeCL) 5⬘-d(GSTSGSASTSASTSGSC) 5⬘-d(GSTLSGSASTLSASTLSGSC) 5⬘-(GTGATATGC) 5⬘-(GTGATATGC) 5⬘-(GTLGATLATLGC)

35 47 ⬎90 28 44 55 64 21 41 33 22 53

32 53 88 28 50 63 74 17 47 49 34 64

Note: A ⫽ nucleotide monomer with an adenin-9-yl base, C ⫽ nucleotide monomer with a cytosin-1-yl base, G ⫽ nucleotide monomer with a guanin-9-yl base, T ⫽ nucleotide monomer with a thymin-1-yl base. MeC ⫽ nucleotide monomer with a 5-methylcytosin-1-yl base. Oligo-2⬘-deoxyribonucleotide sequences are depicted as d(sequence), oligoribonucleotide sequences as r(sequence), and 2⬘-O-Me-oligoribonucleotide residues are underlined. LNA monomers are shown in boldface with superscript “L”. PS designates phosphorthioates and subscript “S” denotes a phosphorothioate linkage.

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RNA-modified the Tm increases ca. 0.5 and 2.2°C against DNA and RNA, respectively. Despite the fact that LNA inclusions always give rise to increases, also against DNA, the 2⬘-O-Me RNA hybridization pattern is the exact opposite of LNA: the Tm increase per modification increases as the number of 2⬘-O-Me RNA residues increases [81,83]. The same pattern has previously been reported for 2⬘-O-MOE [83]. When three LNA residues are included in the oligonucleotide together with six 2⬘-O-Me RNA residues a dramatic increase in the Tm of the RNA duplex from 34 to 64°C is observed (Table 19.2, entry 12). In other words LNA residues restore entirely the affinity lowering the effect by spiking 2⬘-O-Me RNA residues into oligonucleotides. Kierzek et al. [84] studied in detail the influence of LNA in 2⬘-O-Me RNA. Except for a few isolated cases (2 out of 34) the additive increase in Tm when LNA residues substituted 2⬘-O-Me RNA residues was confirmed. The increase in Tm in 2⬘-O-Me RNA (7-mers) ranged from 2.1 to 10.3°C per LNA modification against RNA. The highest increase was seen for internal inclusions and the lowest increase was seen for 3⬘ inclusions. A Tm prediction algorithm was proposed to approximate the stabilities of 2⬘-O-Me RNA/LNA hybrid duplexes. Recently, LNA has also been included in 2⬘-O-MOE RNA [85]. The affinity of 2⬘-O-MOE oligonucleotides is also increased when MOE is substituted with LNA. A chimeric LNAⲐ2⬘-O-MOE oligonucleotide is reported to have the highest Tm of all the tested oligonucleotides. The oligonucleotides were tested in a microRNA inhibition assay and high activity was related to high affinity. Thus, the chimeric LNA/2⬘-O-MOE oligonucleotides was more potent than the 2⬘-O-MOE or 2⬘-O-Me “alone” oligonucleotides. Since 2⬘-O-MOE and 2⬘-O-Me are very similar in their hybridization properties it is reasonable to assume that LNA can cooperate with 2⬘-O-MOE with the same basic principles so carefully studied for 2⬘-O-Me. Despite LNA hybridize with high affinity to its nucleic acid target the binding is highly specific in terms of Watson–Crick (W–C) base pairing. Mismatch studies have shown that a Tm of ⫺21°C for T/C mismatches and Tm of ⫺14°C is found for a TⲐG mismatch [14]. Sequence dependencies are expected and have been reported [82], but the base-pairing specificity of LNA is in general at least as high as that of DNA. The high affinity and sequence specificity of LNA has been used across the entire field of nucleic acid technologies to improve oligonucleotide properties: For allele-specific primers [86], for specific detection of microRNAs [87–89], for genotyping SNPs in apolipoprotein E [90] and for specific detection of the Factor V Leiden [91]. The conclusion is that LNA exerts its affinity increase additively in a very cooperative manner. LNA can be mixed in any way with nucleic acid residues and analogs hereof. LNA inclusions via their high affinity are able to restore the negative affinity effects of PS and certain combinations of nucleic acids and analogs (e.g., 2⬘-O-Me RNA). In classical antisense designs, large blocks of 2⬘ modifications are needed to provide better affinities, but with LNA only a few nucleotides will suffice. 19.5.2 Thermal Denaturation of LNA Analog Heteroduplexes: Thio- and Amino-LNA The Tm values of both the thio- and amino-LNA analog closely resemble the values of LNA itself (Table 19.3, entry 2–3) [30]. This is particularly surprising in the case of amino-LNA where the amino functionality introduces a positive charge at physiological pH (Table 19.3, entry 2, 4–5) [4]. However, the affinity increase of amino-LNA is not as high as for LNA and thio-LNA due to the positive charge [5]. The influence of the charge is illustrated by an increase of Tm when amino-LNA is N-benzoylated (which removes the positive charge) (Table 19.3, entry 6) and a decrease of Tm when amino-LNA is N-benzylated (Table 19.3, entry 7) [25,28]. A fully modified N-benzoylated(Bz)amino-LNA T9 has an impressive Tm of 73°C against RNA. Molecular dynamics simulation shows that the benzoyl groups are located at the brim of the minor groove and they occupy about half of the space of the groove. They do not stack but are likely to exclude water of the groove during hybridization and to interact via van der Waals interactions with the sugars. Increased thermal denaturations temperatures are also observed for amino-LNA containing bulky groups as pyrene (Table 19.3, entry 9) [27]. When pyrene is linked via a rigid amide to amino-LNA only a few derivatized

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Table 19.3 Hybridization Studies with 9-mer Amino- and Thio-LNAs toward Complementary DNA and RNA Entry 1: Reference DNA 2: LNA Ⲑ DNA chimera 3: Thio-LNA Ⲑ DNA chimera 4: Amino-LNA Ⲑ DNA chimera 5: Methylamino-LNA/DNA chimera 6: N-Bz-amino-LNA/DNA chimera 7: N-Bn-amino-LNA/DNA chimera 8: N-Bz-amino-LNA/DNA chimera 9: N-Py-amino-LNA/DNA chimera

Sequence 5⬘-d(GTGATATGC) 5⬘-d(GTLGATLATLGC) 5⬘-d(GULSGAULSAULSGC) 5⬘-d(GTLNHGATLNHATLNHGC) 5⬘-d(GTLNRGATLNRATLNRGC) 5⬘-d(GTLNBzGATLNBzATLNBzGC) 5⬘-d(GTLNBnGATLNBnATLNBnGC) 5⬘-(TLNBz)9-T 5⬘-d(GTGATLNPyATLNPyGC)

DNA Target Tm (oC) 28 44 42 39 39 47 37 75 38a

RNA Target Tm (oC) 28 50 52 47 49 56 50 73 41

Note: LNA monomers are shown in boldface with superscript “L.” A ⫽ nucleotide monomer with an adenin-9-yl base, C ⫽ nucleotide monomer with a cytosin-1-yl base, G ⫽ nucleotide monomer with a guanin-9-yl base, U ⫽ nucleotide monomer with a uracil-1-yl base, T ⫽ nucleotide monomer with a thymin-1-yl base. Oligo2⬘-deoxyribonucleotide are called d(sequence). Superscript “LS” a thio-LNA monomer, superscript “LNH” an amino-LNA monomer, superscript “LNR” a methylamino-LNA monomer, superscript “LNBz” a benzoylamino-LNA monomer, superscript “LNBn” a benzylamino-LNA monomer, and superscript “LNPy” a pyreneamino-LNA monomer. a Tm of 33°C was reported by Hrdlicka et al. [29].

amino-LNAs are allowed, whereas the more flexible N-methyl linkage provide increase in Tm for even fully modified N-Py-amino-LNA nonamers [25]. When the bulky N,N-bis(2-pyridylmethyl)--alanylgroup is linked to amino-LNA the Tm is increased up to 12°C in a mixed sequenced nonamer. The Tm increase was dependent on sequence and on the concentrations of divalent cations [26]. From the data described in this section it can be concluded that the 2⬘-X-CH2-4⬘ bicyclic structure in the -D configuration is a general high-affinity locked structure that allows the presence of other heteroatoms than oxygen. The trivalent nature of the amino functionality of amino-LNA can serve as a conjugation junction for functionalities otherwise not included by LNA. Therefore, aminoLNA can be used to link high-affinity hybridization with a variety of biological ligands with the potential for novel therapeutic functionalities. 19.5.3 Thermal Denaturation of LNA Diastereoisomer Heteroduplexes Alpha-L-LNA is by far the most studied diastereoisomer of LNA, and since it is the analog of most interest for antisense therapeutics it will be the focus of this section [33–35,75,81,92,93]. Alpha-L-LNA monomers can be included in complex-designed oligonucleotide chimerae. Alpha-L-LNA monomers act cooperatively with the non-LNA residues, and the affinity increase is mediated in an additive way (Table 19.4). However, the affinity increase per modification of
-L-LNA is not as high as it is for LNA (Table 19.4, entry 9–10), and
-L-LNA shows affinity preference for RNA (Table 19.4, entry 2–3, 6, 9–10) [75]. RNA “selection” is also seen when
-L-LNA residues are incorporated in 2⬘-O-Me containing oligonucleotides (Table 19.2, entry 12, and Table 19.4, entry 7). The fact that
-L-LNA does not have the same “spiking” power as LNA can be explained by the observation that it does not steer the conformation of nearby DNA-residues to the high affinity N-conformation (vide supra). This is in line with the observation that fully modified hetero duplexes of
-L-LNA and LNA—where conformational steering has no relevance—have almost the same Tm (Table 19.4, entry 11–12). LNA and
-L-LNA can also be mixed and act cooperatively in the same oligonucleotide to give rise to an increase per modification of 4–5°C (Table 19.4, entry 4). The reason for this is likely to be the canonical position in space of the 5⬘-OH, 3⬘-OH groups on the furanose and the N1 of nucleobases (vide supra). The sequence-dependent specificity of
-L-LNA hybridization is good and comparable to that of DNA and LNA [34,75]. For 11-mer
-L-LNA oligonucleotides a thermal denaturation difference

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Table 19.4 Hybridization Studies with 9-mer, 10-mer, and 11-mer LNA, and
-L-LNA toward Complementary DNA and RNA Targets Entry

Sequence

DNA Target Tm (oC)

RNA Target Tm (oC)

1: Reference DNA 2: LNA 3:
-L-LNA 4:
-L-LNA Ⲑ LNA chimera 5: Reference DNA 6:
-L-LNA Ⲑ DNA chimera 7:
-L-LNA Ⲑ 2⬘-OMe-RNA chimera 8: Reference DNA 9: LNA/DNA chimera 10:
-L-LNA Ⲑ DNA chimera 11: Fully modified LNA 12: Fully modified
-L-LNA

5⬘-d(T)14 5⬘-d(TL)9T 5⬘-d(
LTL)9T 5⬘-d(T)3(TL)4(
LTL)4(T)5 5⬘-d(GTGATATGC) 5⬘-d(G
LTL GA
LTL A
LTL GC) 5⬘-(G
LTL GA
LTL A
LTL GC) 5⬘-d(CACACTCAATA)-3⬘ 5⬘-d(CAL CAL CTL CALATLA)-3⬘ 5⬘-d(C
LALC
LALC
LTLC
LALA
LTLA)-3⬘ 5⬘-(MeCAMeCAMeCTMeCAATA)L-3⬘ 5⬘-
L(MeCAMeCAMeCTMeCAATA)L-3⬘

32 80 63 64 28 37 38 36 54 39 69 65

28 71 66 63 28 45 52 30 63 49 77 75

Note:

A ⫽ nucleotide monomer with an adenin-9-yl base, C ⫽ nucleotide monomer with a cytosin-1-yl base, G ⫽ nucleotide monomer with a guanin-9-yl base, T ⫽ nucleotide monomer with a thymin-1-yl base. Me C ⫽ nucleotide monomer with a 5-methylcytosin-1-yl base. Oligo-2⬘-deoxyribonucleotide sequences are depicted as d(sequence), oligoribonucleotide sequences as r(sequence), and 2⬘-O-Me-oligoribonucleotide residues are underlined. LNA monomers are shown in boldface with superscript “L.” Superscript “
L” indicates
-L-LNA residue.

between the fully matched and the single base pair mismatch was in the range of 6–29°C against DNA. The corresponding Tm against RNA was in the range of 17–25°C [75]. An interesting diastereoisomeric variant is 2⬘-amino-
-L-LNA [38]. This molecule can be regarded as a chemical “combination” of amino-LNA and
-L-LNA. A robust Tm increase is seen for 2⬘-amino-
-L-LNA against RNA (Tm per modification: 2.0–4.5°C). Against DNA the Tm is also increased (Tm per modification: 0.5–2.5°C), except for one sequence where a small decrease is found (Tm per modification: ⫺0.5) [38]. Interestingly, when 2⬘-amino-
-L-LNA is conjugated with pyrene a dramatic increased in Tm is seen against DNA (7–15°C per modification), whereas the Tm increase against RNA is more moderate [94]. In this molecule the pyrene points out into the major groove (in contrast to the amino-LNA congener) and the high affinity against DNA is likely due to intercalation which is also supported by the observed reduced mismatch discrimination and the spectroscopic redshift during hybridization. The conclusion is that the 2⬘-X-CH2-4⬘ bicyclic structure fits within Watson–Crick framework no matter whether it points into the major groove (
-L-LNA) or the minor groove (LNA). The generality is nicely confirmed by 2⬘-amino-
-L-LNA that due to its structure is a vehicle for positioning ligands into the major groove [38]. 19.5.4 Thermal Denaturation of LNA Containing Base Modifications Replacement of adenine with diaminopurine in LNA gives rise to an affinity increase. Rosenbohm et al. [21] reported an increase of 2 and 4.5°C per modification against DNA and RNA, respectively, and Koshkin [16] reported an even higher increase against DNA (6.2°C per modification). In both publications a larger discriminative power of single base-pair mismatches is reported. LNA containing inosine shows—as expected—selective binding to cytosine, but at the same time comparable high binding to the other three nucleobases. In contrast, 2-aminopurine LNA showed selective DNA-thymidine binding, and the Tm is reduced by 7, 15, and 17°C against DNAcytosine, -adenosine, and -guanosine, respectively. A variety of LNA and
-L-LNA aryl C-nucleosides have been made [44,45,48,95]. The general pattern is that LNA-aryl nucleosides have significant reduced affinities, however LNA-pyrenyl could be included in oligonucleotides if it was surrounded by regular LNA residues to compensate for the reduced affinity.

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19.5.5 Thermodynamic Considerations One of the design rationales behind locking the conformations of the furanose in LNA was to make hybridization entropy less unfavorable [1,9], and early reports on LNA:RNA hybridization supported this hypothesis [55,63]. However, it was also reported that for fully modified LNA oligonucleotides hybridized to DNA the formation was strongly enthalpy favored [60]. This observation is in line with the marked increased stacking observed by NMR (vide supra) [56]. Christensen et al. [96] reported also that increased affinity as a function of LNA-residue addition was driven—during LNA:DNA hybridizations—by a more favorable enthalpy contribution, although a minor more favorable entropy component also contributed to a larger negative G. Kvaerno et al. [97,98] demonstrated the importance of the enthalpic factor by including one abasic LNA residue in a duplex that resulted in a dramatic decrease in the Tm. A very comprehensive analysis of LNA:DNA hybridizations was undertaken by McTigue et al. [82]. They concluded that LNA pyrimidines contribute more to the stability than the purines, especially AL, and that the contributions are sequence-dependent. They observed enthalpy–entropy compensations across the entire data set and reported that both entropy (preorganization) and enthalpy (stacking) are contributing, but not both for a given sequence. A detailed study was also undertaken by Kaur et al. [99] and they confirmed the higher contribution of LNA pyrimidines to the affinity of LNA:DNA duplexes and that the thermodynamic parameters are sequence-dependent. They found for most sequences that hybridization was driven by enthalpy, but in a few cases driven by entropy. They reported higher counterion uptake of LNA duplexes but lower uptake of water. These are characteristic parameters during A-form helix formation and thus, in accordance with the structural observations of LNA. The full picture of hybridization thermodynamics is not yet clear. Thermodynamically, hybridization is very complex with contribution of many components: stacking, rotational freedom of nucleotidesⲐoligonucleotides, structure of helices, counterion releaseⲐbinding, water releaseⲐbinding, H bonding, etc. With H and S being a part of each of these components an unequivocal assignment of H and S is very complex. However, the bottom line is that the individual importance of H and S during LNA hybridization is sequence-, context-, and target-dependent, but—very importantly—always with a negative G. 19.5.6 Hybridization Kinetics It is generally believed that duplex formation of oligonucleotides is a process involving two main events, initial formation of a nucleation complex followed by annealing of the duplex where IC is the nucleation complex and D the duplex: k1 k2   → IC ←    → D DNA(A) ⫹ LNA(B) ←   k k ⫺1

⫺2

The temperature-dependent overall dissociation constant, KD is given by K D ⫽ ( k⫺ 1 ⫻ k⫺2 ) Ⲑ ( k1 ⫻ k2 ) ⫽ [DNA ⫺ A eq ][LNA ⫺ Beq ]Ⲑ[D] The hybridization kinetics of LNA 9-mer oligonucleotides containing one to three LNA-T residues have been reported [96]. The observed on-rates are high and found to be in the order of 2 ⫻ 107 MⲐs. The overall KD values are calculated from the Tm curves at various temperatures and decreased from 20 to 0.3 nM as one to three LNA residues were included. It is demonstrated that the on-rates were similar for DNA–DNA and DNA–LNA hybridization. Therefore, the very different KD values between DNA and LNA reflect a marked difference of the off-rate. By taking the fast on-rates into account it is estimated that the hybridization is nearly diffusion controlled, meaning that every correct base-to-base encounter leads to hybridization. Thus, the rate-determining step is the association reaction followed by fast annealing of the duplex.

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Hybridization kinetics is also reported for a 12-mer 2⬘-O-MeⲐLNA mixmer. The rate constants are determined by a PAGE mobility shift assay using TAR 39 RNA as target [100]. On- and offrates rates of respectively 2 ⫻106 MⲐs and 2 ⫻10⫺3/ are observed. Compared to isosequential PNA, the 12-mer 2⬘-O-MeⲐLNA mixmer and the fully modified 2⬘-O-Me 12-mer oligonucleotide have five times faster on-rates. 19.5.7 Thermal Denaturation of LNA Triplexes LNA and
-L-LNA oligonucleotides hybridize also in the classical triplex binding motif, but triplex binding is outside the scope of this review. However, a few comments are appropriate. LNA and
-L-LNA triplexes exhibit significant increased thermal stabilities (up to 5°C per modification) compared to the DNA congeners, with the design provison that fully modified locked oligonucleotides are not allowed. Triplex formation is possible even at neutral pH [2,50,101–107]. The improved triplex binding of LNA is obtained by decreasing the dissociation rate constant and is associated with an entropic gain [105,108]. The biological relevance has been illustrated by the inhibition of NF-B binding to its target sequence at 1
M (pH ⫽ 7) [101]. The interested reader can consult an excellent review by S. Obika [109].

19.6 BIOCHEMICAL PROPERTIES OF LNA AND LNA ANALOGS 19.6.1 General Designs LNA and most LNA-analogs can be mixed in any combination with nucleic acids and analogs hereof (vide supra). Depending on the intended application of LNA, or for that matter that of other oligonucleotide analogs, the design may be divided into five categories: mixmer, gapmer, headmer, tailmer and fully modified (Figure 19.9, 1–5). In the mixmer the LNA residues are dispersed along the sequence of the oligonucleotide (1), while in the gapmer two continuous LNA segments in the flanks are separated by a central nucleic acid segment (2). In the headmer a continuous LNA segment is positioned in the 5⬘ end followed by a continuous nucleic acid segment (3), and vice versa for the tailmer (4). Finally, a fully modified LNA oligonucleotide is self-explanatory (5). The length of the various segments will vary and the specific design is dependent on the intended use and how important affinity, nuclease resistance, and RNase HⲐRISC recruitment are for the proposed application. 19.6.2 Nuclease Resistance The incorporation of LNA nucleosides into ODNs increases their stability against nucleases. The degradation rate is very dependent on the number of LNA residues, their position, and incorporation of other stabilizing entities (e.g., PS internucleoside linkages). When one LNA-T residue 1.

5′

3′

2.

5′

3′

3.

5′

3′

4.

5′

3′

5.

5′

3′

Figure 19.9 General designs of LNA antisense oligonucleotides.

LNA/LNA analogs DNA/PS or analogs

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is included in either the ultimate or penultimate 3⬘ position no significant increase in the stability is obtained against the 3⬘ exonuclease snake venom phosphordiesterase (SVPD) [92,110–112]. When 2 LNA-T modifications were positioned in either the ultimate or penultimate 3⬘ positions the oligonucleotide is almost as stable as the fully modified analog that is virtually resistant to SVPD degradation [1,92,110,111]. Interestingly, when one
-L-LNA-T residue is positioned at the penultimate 3⬘ position the oligonucleotide becomes much more stable and ca. 40% full-length product remains after 2 h incubation where the LNA congener is entirely digested after 1 h [92]. The fact that
-L-LNA contributes more to the stability than LNA can be explained by its nonnatural configuration (vide supra). However, 11-mer LNA and
-L-LNA, mixmers, modified identically with five modifications are both essentially resistant to SVPD digestion [75]. The same resistance is seen for 20-mer LNA mixmer and gapmer designs containing 8–11 residues [113]. Introducing artificial internucleoside linkages can significantly increase the nuclease stability. For instance, one methylphosphonate internucleoside linkage (vide supra) in the 3⬘ position to a single LNA-T residue blocks entirely the 3⬘-exonucleolytic activity of SVPD [77]. 3⬘-Exo-nucleases have very different nuclease activities, and SVPD is one of the most aggressive. To understand the subtle positional differences less aggressive exo-nucleases have to be employed. Di Giusto et al. [114] compared the degradation rate of LNA oligonucleotides singly modified in either the ultimate or the penultimate 3⬘ position against five different exo-nucleases. It turned out that a single LNA modification in the penultimate position is almost as stable as the double modified against all five tested exo-nucleases, whereas the single modification in the ultimate position was labile to all enzymes. This positional effect is likely to be due to the structural change of the ultimate phosphordiester bond by the penultimate LNA substitution (vide supra). However, the main conclusion is that a single modification is most stable in the penultimate position, and that at least two LNA modifications is the lower limit for global 3⬘-exo-nuclease protection. The gapmer design is an effective way to secure exo-nucleolytic protection, but the central DNA segment is sensitive to endo-nucleolytic activity. A fully modified LNA is essentially resistant to S1-endonuclease degradation [92], but if the central DNA segment becomes longer than four nucleotides cleavage starts to occur, and the cleavage rate increases as the DNA segment increases. However, the protection of the LNA flanks is still significant even for a 4LNA-7DNA-5LNA diester gapmer since the cleavage rate is slower for this molecule compared to the isosequential PS [92]. The degradation pattern of 18-mer LNA phosphordiester gapmers has also been studied in human serum [115]. The nuclease stability is closely related to the length of the LNA segments. Substituting just one LNA in either end increases the half-life from 1.5—for the DNA oligonucleotide—to 4 h. Increasing the LNA segment from 1 to 4 at either end increases the half-life from 4 to 15 h. The stability of the LNA containing three or four modifications at either end is also better than the corresponding 2⬘-O-Me congener gapmer and the PS with respective half-lifes of 12 and 10 h. The nuclease stability of LNA 15-mers has also been studied in human serum [111]. The half-life of a 15-mer LNA (mixmer w. 9 LNA residues) is ⬃10 times greater in blood serum compared to the isosequential PS, whereas the isosequential LNA gapmer (4LNA-6DNA-5LNA) has decreased stability compared to the PS. This is explained by more endo-nucleolytic digestion of the central DNA segment in the gapmer compared to the PS [111]. This observation is in contrast to the findings of Frieden et al. [92] (vide supra), but since the sequences are very different in the two studies, the discrepancy could be explained by sequence dependent endo-nucleolytic activity. Since the central DNA segment is degraded—albeit rather slowly—it has to be further protected. One way to do this is to use the above-mentioned methylphosphonate internucleoside linkage, but a more obvious choice is to use PS internucleoside linkages. The PS linkage is easy to introduce under LNA synthesis and it serves as substrate for RNase H (vide infra). We have examined a series of gapmers fully PS modified in human and in rat plasma and observed no sign of endo-nucleolytic activity. The gap size ranged from 9 to 11 PS linkages and is entirely protected over 24 h. Some of the tested gapmers had a single 3⬘-DNA residue in the ultimate position. Despite the fact that immediately to the 5⬘ side of the DNA residue is a PS LNA segment of 2–3 residues some

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cleavage is still observed. However, this lability is base dependent where DNA A is most labile (25% is cleaved over 24 h) and DNA C is entirely stable under the same conditions. LNA inclusions can also effectively stabilize double-stranded DNA. Including just one LNA residue in the ends of both strands of a 30-mer DNA duplex (four LNA in total) increases the half-life of the duplex significantly [116]. After 3 h incubation with DNase 1 65% of the LNA modified duplex remains, whereas the unmodified DNA duplex is completely degraded. Including more LNA residues increase the half-life further. RNA oligonucleotides are even more susceptible to nuclease degradation than DNA oligonucleotides, and with the advent of siRNA as a potent antisense technology, stabilization of RNA has gained great importance. LNA is also here an obvious choice, since it is a structural RNA mimic and it brings two kinds of stabilizing parameters: nuclease stability and an increase in siRNA duplex lifetime, via affinity. The latter is important since duplex siRNA is very stable to nuclease activity in cells and in serum [117]. Inclusion of LNA residues in a siRNA duplex increased the Tm 3–4°C per modification, whereas no increase is seen for either 2⬘-F or 2⬘-O-Me inclusions. Inclusion of LNA in the overhangs + one LNA residue in the 5⬘-sense strand increased the half-life of the siRNA from seconds to 24 h [118]. When further six LNA residues are included in the sense strand the half-life was not reached during the time span of the experiment. The conclusion is that LNA and LNA analogs effectively stabilize oligonucleotides against nucleases. The inclusion of just a few LNA modifications in the ends in combination with PS internucleoside linkages provides essential nuclease resistance. 19.6.3 RNase H Recruitment of LNA and LNA Analogs RNase H belongs to a class of ubiquitous enzymes that cleave the RNA strand in RNA:DNA duplexes. It is a well-established fact in antisense that oligonucleotides that recruit RNase H are among, if not, the most potent to inhibit the activity of mRNA. Since a RNA:DNA duplex recruits RNase H and since LNA resembles an RNA structure, LNA:RNA will not recruit RNase H. Therefore, to be effective in degrading RNA, LNA has to be designed with segments of DNA as in gapmers, headmer or tailmers. In the first antisense report with LNA activation of RNase H with 15-mers, designed as a gapmer (4LNA-6DNA-5LNA) or as a mixmer with 9 LNA modifications, were examined. The RNA target was a 24-mer corresponding to the targeted portion of the rat delta opioid receptor (DOR) [111]. Surprisingly, both the gapmer and the mixmer were able to cleave the RNA target sequence. In a comprehensive study using LNA 18-mers the cleavage pattern by RNase H was studied in detail [115]. The target RNA was the full-length transcript of the vanilloid receptor subtype I (VRI) cDNA (2614 nt). With mixmers containing 5 or 6 LNA residues scattered along the 18-mer sequence no activity was seen. However, for one sequence with a gap of 8 DNA residues, ca. 90% of the RNA was cleaved (same as for the DNA control). The isosequential PS recruits RNase H less efficiently. When the DNA segment in LNA-DNA-LNA gapmers is increased from 4 to 8 residues, RNA cleavage initiates at 6 DNAs and is maximal at 8 DNAs. For isosequential gapmers containing 2⬘-O-Me modifications only a stretch of 6 DNA residues is necessary to elicit RNase H cleavage. This difference between LNA and 2⬘-O-Me is likely to be the “structural saturation” of LNA meaning that the DNA residues proximate to LNA attain a higher degree of N-conformation (vide supra). Cleavage of the RNA by the LNA gapmer is very specific and occurs for these 18-mers on the complementary RNA strand at the position opposing DNA nucleosides 8 and 9 (from 5⬘ end). Kinetically, the LNA gapmer is the fastest to cleave RNA followed by the isosequential 2⬘-O-Me. All the DNA and the PS are the slowest [115]. The optimal gapsize has also been studied in a Firefly luciferase inhibition assay. The cell line HeLa X1Ⲑ5 was used and it was stably transfected with a Tet-Off luciferase system [92]. The LNA gapmers were 16-mers with a central PS segment. For the three different target sequences examined, the trend was the same, a gapsize ranging from 7 to 10 DNA nucleotides being the most potent. Interestingly, this study reveals the subtle relationship between affinity and gapsize. For the

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low-affinity 16-mers with Tms from 42 to 52°C against DNA, an increased gapsize results in reduced activity, whereas for the high-affinity 16-mers of the same general design and with Tms of 61–72°C, increased gap size up to 10 PS leads to increased activity. Further increased activity is seen if also the LNA segments are phosphorothiolated—a modification that does not lead to an affinity decrease for LNA (vide supra). The very high affinity of LNA provides the unique opportunity to combine size reduction with high potency, and this is shown in the study. A 14-mer LNA gapmer (3LNA-8DNA3LNA) fully thiolated with a Tm of 57°C, against DNA, shows an impressive IC50 value ⬍2 nM [92]. Head and tail designs have also been examined but the activity is significantly lower than for the gapmers. Finally, the mechanistic relation to RNase H activity of the gapmers is justified, since all active oligonucleotides in the antisense assay also are active in an isolated Escherichia coli RNase H assay. In contrast to LNA
-L-LNA does attain a B-like structure when hybridized to nucleic acids. As a structural DNA mimic
-L-LNA displays different RNase H recruiting properties of that LNA [65]. Fully modified and mixmers of
-L-LNA do recruit E.coli RNase H whereas the isosequential LNA under the same conditions shows no signs of RNase H activity [75]. However, the cleavage rate for
-L-LNA was much slower than for the control DNA. In the luciferase assay mentioned above, isosequential LNA and
-L-LNA gapmers have roughly the same potency. However, if a single
-L-LNA nucleotide is included in the gap the activity is maintained, whereas the activity ceases for the same sequence if a single LNA nucleotide is included [92]. Recruitment of RNase H has been compared between isosequential gapmers of the 3LNA9DNA-3LNA-1DNA design comprised of LNA,
-L-LNA, amino-LNA and thio-LNA modifications (internal communication). Gapmers containing LNA, thio-LNA and
-L-LNA modifications turned out to be the most active in recruiting both E.coli and human RNase H. Compared to isosequential DNA, all four locked oligonucleotides were more potent with relative RNA cleavages ranging from 42 to 80% compared to 15% for DNA. This section underlines the fact that the most potent design for RNase H recruitment by LNA, amino-LNA, thio-LNA and
-L-LNA is the gapmer design. The optimal gapsize in E. Coli assays is ⬎7 DNA nts and slightly longer, ⬎8–9 DNA nts, in antisense assays using human RNase H [92,119,115]. This is related to the longer DNAⲐPS substrate size required for human RNase H. The optimal design is also related to the affinity of the nativeⲐunmodified DNA oligonucleotide sequence. If it is a low-affinity sequence (⬍40°CⲐ16-mer) the LNA load must be relatively high (6–8 nt). If it is a high-affinity sequence the LNA load can be reduced to 4–6 nts. This section also illustrates the importance of the high affinity of LNA. High affinity offers the opportunity to design short (14–16 mers) highly potent antisense agents, and furthermore, in contrast to other chemistries where PS modifications lead to lower affinity, LNA can be phosphorothiolated without affinity loss. Thus, LNA can be used in combination with PS, to take the advantages and the properties that PS offers, into one short AON without potency compromises (vide infra).

19.7 INHIBITION OF CODING RNA IN VITRO Antisense mechanisms are divided in two main categories: (1) Steric block of the nucleic target by the AON; and (2) inhibition of gene expression through mRNA destruction [120]. The steric block mechanism includes prevention of ribosome binding by blocking the 5⬘-UTR, acting as a “road” block during translation by binding to the message, or by redirection of splicing by blocking splice sites to alter the production of splice variants. When AON’s are acting according to these mechanisms the RNA remains intact. The other main category requires enzyme recruitment by the AON either by RISC-mediated RNAi or by RNase H to mediate cleavage of the mRNA. The antisense properties of LNA—and LNA analogs—flow directly from the basic properties of the molecules. When these are considered (high affinity for RNA, nuclease resistance, and high rates of RNase H recruitment for LNA for selected designs) one would expect high potency to be

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the outcome (vide infra). Since LNA is a relative recent AON technology an important part of the scientific studies to date has been to compare its antisense properties with more traditional antisense chemistries. In particular, it is of great interest to verify whether the high affinity of LNA is a differentiating factor that may enable the fulfillment of the large expectations of antisense technology in human clinical therapies. The majority of antisense experiments made with LNA have mechanistically been focused on mRNA inhibition by RNase H recruitment [92,93,111,113,119,121–127] (Table 19.5). The general picture in all these publications is that LNA AONs are potent, and actually more potent than the competing chemistries. Values of IC50s for mRNA inhibition are frequently obtained in the subⲐlow nano molar range (Table 19.5). However, the reason for the rather high IC50 for Bcl-xL targeting shown in Table 19.5 is that the oligonucleotide is designed to target both Bcl-2 and Bcl-xL meaning that the sequence has three mismatches to Bcl-xL [126]. When compared to the isosequential MOE (IC50 ⫽ 253 nM) LNA is more potent, and the authors explain this by the higher affinity of LNA. The potency of the different LNA designs is also related to the target site. When the target site is the 5⬘-UTR the most potent mixmer—designed for steric block in the 5⬘-UTR region—inhibited luciferase activity with 76% at 25 nM, almost as potently as the most potent gapmer that inhibited 82% [121]. When LNA gap- and mixmers are compared for mRNA inhibition in the translation start site the gapmer is found to be slightly more potent [92,121,125]. However, when the target site is in the coding region the gapmer is superior [121] The explanation for this observation is that a high-affinity LNA mixmer can prevent ribosome binding to the mRNA and thereby stop translation, but once the ribosome is bound and initiates translation, the mRNA has to be cleaved to stop its action. Jepsen et al. [119] confirmed this relation between the target site of gapⲐmixmer and potency, in a study on the Estrogen receptor
. For isosequential gapⲐtailⲐheadmers the headmer was almost inactive, the tailmer was more active reducing the expression to 40% of the control level, whereas the gapmer was the most potent and reduced the expression to 10%. However, such a clear potency differentiation between headⲐtailmers was not established in a HIV replication assay where the potency was found to be sequence dependent on the individual AONs [127]. The most potent 18-mer, targeted at the dimerization initiation site was of a headmer like design, and the HIV-1 replication was reported to be inhibited by 64% compared to untreated Jurkat-Tat cells (AON concentration ⫽ 160 nM). LNA has also been used for mRNA inhibition according to non-RNase H mechanisms. LNA mixmers can inhibit telomerase binding to telomeres effectively, and mixmers as short as eight nucleotides inhibit binding with an IC50 value of 25 nM [128]. Compared to PNA, the LNA mixmers are 200-fold more potent [128]. The most potent were 13-mers with IC50 values ranging from 1 to 10 nM. It was concluded that the high potency was directly related to the high affinity of LNA [128]. Another non-RNase H mechanism is the steric block of TAR-tat binding that inhibits HIV transcription and translation [100,129,130]. Arzumanov et al. [131] showed that the cooperative binding of LNA and 2⬘-O-Me in mixmer designs can be used to make highly potent AONs of only 12 nucleotides. A steric block approach was also used for intracellular inhibition of the hepatitis C virus (HCV) internal ribosomal entry site. An LNA mixmer composed of 8 LNA and 9 DNA—with the highest Tm (92ºC) of all the tested LNAs—was found to be the most potent. The estimated IC50 value is 50 nM. A logical application of the steric block approach is to use LNA mixmers to redirect splicing [132]. Fully modified LNA 14-mers induce potent splice skipping of exon 46 in myotubes. In patient samples with Duchenne muscular dystrophy, 85% splice skipping has been observed, whereas a 20-mer 2⬘-O-Me AON shows only 20%. A 22-mer morpholino showed only 6% and a 14-mer PNA showed no skipping at all [132]. Owing to the fact that the LNA was fully modified the Tm was calculated to be ⬎100ºC and the inhibition was associated with some non-specific target binding.

Steric block of telomerase activity

LNA-2⬘-O -RNA mixmers (12-mer)

LNA mixmersⲐfully modified (13- or 8-mers) LNA-DNA gapⲐmixmer (13- and 15-mers) LNA/thio-LNAⲐα-LLNA mixmers with 2⬘-O -Me (8Ⲑ9/10/12/ 16-mers) LNA-PS gapmers (16 & 18-mers) LNAⲐα-L-LNA gap/ mixmers w. DNA/PS (12Ⲑ14/16-mers) LNA-DNA Gap/tail/ headⲐmix-mer (9 and 18-mers)

HIV dimerization initiation site (DIS)

Luciferase

PKC-α

HIV-1 transactivator element (TAR)

Luciferase

RNase H cleavage of target HIV RNA and inhibition of HIV genome dimerization

RNase H cleavage of mRNA RNase H cleavage of mRNA

Steric block & mRNA cleavage by RNase H activation Steric block of TAR-Tat binding

Steric block of TAR-Tat binding

LNA-DNA gapmer (20-mer) LNA-2⬘-O -RNA mixmers (12-mer)

ICAM-1

In vitro HIV genome dimerization, RNase H assay and cell culture (PEI transfection)

Up to ⬃60% inhibition of HIV dimerization and replication

⬍2 nM

Inhibition of HIV genome dimerization, RNase H activation and inhibition of HIV replication

Inhibition of mRNA and protein expression Inhibition of luciferase expression

⬃5 nM

A549 cellular assay (lipofectamine) HeLa cellular assay (lipofectamine)

Inhibition of luciferase expression 120 nM (16-mer mix-mer LNA)

HeLa cellular assay (effectin 12)

Inhibition of luciferase activity

Inhibition of telomerase activity

Inhibition of luciferase expression

Inhibition of spinal antinociceptive response to deltorphin II Inhibition of protein expression Inhibition of full-length transcription

Biological/Functional Effect of LNA

N/A

1–10 nM

N/A

20 nM (estimated) 70–150 nM

Reduction in DOR densities by 35–50%

Potency (IC50)

CV-1 cellular assay (lipofectamine)

HeLa cellular transcription assay (effectin 12) DU145E cellular assay (lipofectamine)

HUVEC cellular assay (lipofectamine) In vitro transcription assay

Injection in rat brain (in vivo)

Model (transfection)

Comparisons

Mismatch

N/A

PS

N/A

PS

PNA/PS

2⬘-O-Me

DNA/PS PNA/2⬘-O-Me/ propynyl cytocine

DNA

[127]

[92]

[123]

[130]

[121]

[128]

[129]

[113] [100]

[111]

Reference

542

HIV-1 transactivator element (TAR) HIV-1 transactivator element (TAR) Telomerase

RNase H cleavage of mRNA RNase H cleavage of mRNA Steric block of TAR-Tat binding

LNA-DNA Gap/ mix-mer (15-mer)

Mechanism

Delta opioid receptor(DOR)

LNA Design

22:16

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Table 19.5 Inhibition of Coding RNA by LNA in vitro

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Survivin

RNase H cleavage of mRNA and steric block RNase H cleavage of mRNA

RNase H cleavage of mRNA

LNA/thio-LNA/ amino-LNA,
-LLNA gapmers with PS (16-mers) LNA-DNA gap/ mix-mers (22-/18mers, respectively) LNA gapmers with DNA or PS (20-mers) Xenopus laevis embryos (microinjection)

Chicken pinealocytes (lipofectamine)

15PC3 cellular assay (lipofectamine)

Cos-7 cellular assay (lipofectamine)

Human myoblasts/ myotube cultures (PEI tranfection)

MDA-MB-231/H125 cellular assay (lipofectamine)

CV-1 cellular assay (lipofectamine)

MCF-7 cellular assay (lipofectamine)

N/A

N/A

LNA,thio/amino-LNA ⫽ 2 nM
-L-LNA ⫽ 0.4 nM

0.4 nM

N/A

100 nM (Bcl-xL) 65 nM (Bcl-2)

50 nM

Inhibition of cBmal & cAanat mRNA & melatonin secretion Inhibition of mRNA

Inhibition of mRNA

Inhibition of mRNA/ protein expression, proapoptotic phenotypes, caspase-3 activity, and chemo-sensitized Exon skipping, restoring dystrophin synthesis in DMD patient myotube cultures Inhibition of mRNA and protein expression

Inhibition of luciferase activity

Inhibition of mRNA and protein expression

[125]

[124]

2⬘-O-Me/PS phosphoramidate

[93]

N/A

N/A

[122]

[132]

2⬘-O -Me, PNA, morpholinos

siRNA/PS/ 2⬘-O -Me

[126]

[131]

[119]

2⬘-MOE

PNA

PS

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RNase H cleavage of mRNA

LNA-DNA gapmers (18-mers)

VR1 (Vanilliod rec. Subtype 1) H-Ras

Exon 46 skipping— restoration of DMD reading frame

Disrupt RNA secondary structure and protein binding RNase H cleavage of mRNA

RNase H cleavage of mRNA

Fully modified 14-mers

LNA-DNA gapmer (20-mer)

LNA-DNAⲐPS gap/ mix/headⲐtail-mers & fully modified (15-mers) LNA-DNA mixmers (17-mers)

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DMD (Duchenne muscular dystrophy)

HCV internal Ribosomal entry site (IRES) Bcl-2 & Bcl-xL

Estrogen receptor
& p21

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The high affinity of LNA makes it possible to design shorter AONs with retained high potency, but it also means that the “hit rate” of LNA AONs designed to complement different sequences along the mRNA are higher compared to traditional antisense chemistries [122]. This means that the lead identification process in drug discovery becomes faster and that fewer LNA AONs are needed for an adequate gene walk. In a lead selection process involving H-Ras, Thioredoxin and Survivin, we prepared respectively a total of 14, 10, and 14 LNA gapmers [133]. The AONs were of the 4LNA-8PS-4LNA design and fully phosphorothiolated. The AONs were screened at 25 NM in 15PC3 cells and 4 LNAs from each group reduced the expression of the respective targets ⱖ80%. Using this limit as definition for the hit rate, 35% of all LNA AON’s were potent against H-Ras and Survivin, and 40% were potent against thioredoxin. When these candidates were subjected to dose response screening, leads with IC50 values in the range of 0.5–2 nM were identified. The high hit rate is a direct evidence for the broad accessibility of LNA, or in other words, nucleic acid target sites that are not accessible to traditional antisense chemistries are to a large extent accessible with LNA. LNA-analog gapmers containing amino-thio and
-L-LNA have been shown to inhibit H-Ras expression [93]. In a study with isosequential gapmers the design used was 3LNA-9PS-3LNA-1PS, where the LNA analog part was composed of either amino-thio or
-L-LNA. The LNA-analog AONs all had similar Tms ranging from 67 to 71°C. The amino- and thio-LNA had almost the same IC50 value of 2 nM as the LNA congener. Interestingly, the
-L-LNA gapmer was the most potent with an IC50 value of 0.4 nM. Owing to the “DNA”-like structure of
-L-LNA (vide supra) the normal optimal gapsize of 8–9 DNAⲐPS for LNA can be reduced to 7 and still be very potent, IC50 ⬍2 nM [92]. The potency was maintained even if one
-L-LNA residue was placed in the center of the 7 DNA/ PS gap. The functional readouts of the LNA antisense studies described above are listed in Table 19.5. It is reported in almost all cases that the functional effects are concentration dependent related to the RNA inhibition. In this context one example is worthwhile emphasizing. In the study of Simões-Wüst et al. [126], downregulation of Bcl-xLⲐBcl-2 proteins results in strong proapoptotic phenotypes of both MDA-MB-231-, and H125 cells. The cell death is correlated with caspase-3 activation and cleavage of its substrate ICAD (caspase-activated DNase). Interestingly, the treated cells also showed marked increases in chemosensitization. The strongest reduction of cell viability was seen in treated MDA-MB231 cells in combination with paclitaxel, and in treated H125 cells, the strongest effect was seen in combination with cisplatin and gemcitabine [126]. These data underline the strong potential for LNA Bcl-LxⲐBcl-2 inhibitors as antisense therapeutics either alone, or augmented, in combination with classical chemotherapy. This section confirms the broad potential of LNA in antisense. A common conclusion from all of the studies presented here is that the high potency of LNA is directly related to its high binding affinity. The high affinity offers the opportunity to reduce the size of AONs and to retain the potency. Size reduction also has many advantages for AONs: Better cellular uptake, better target specificity, more cost effective to produce, and reduced toxicity [111,128,132,133]. The most potent design of LNAⲐLNA-analogs is the gapmer design, and using that design in vitro the potency of short single stranded LNA AONs is comparable to that of siRNA [122].

19.8 INHIBITION OF MICRO-RNA IN VITRO Micro-RNAs (miRNAs) are a family of short noncoding regulatory RNA molecules expressed in a variety of different cell types. The miRNA pathway serves as an important posttranscriptional regulation mechanism and the potential of miRNAs in pathologically significant pathways is increasingly appreciated [134]. Similar to classical AON extensively developed for the inhibition for coding RNA, synthetic oligonucleotides are the only rational approach for specific inhibition of the individual miRNAs, and they have therefore, the potential to progress into an important new class of drugs.

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Mechanistically the inhibition of microRNA activity is based on the specific hybridization between the microRNA and the synthetic oligonucleotide. A stable and high-affinity binding of the synthetic oligonucleotide to the microRNA will outcompete the binding to the mRNA. Such “sequestering” of the microRNA will inhibit its silencing effect. LNA oligonucleotides directed against microRNAs are named LNA-antimiRs. Naguibneva et al. [135] reported that effective sequestering of miR-125 (at 50 nM) could be obtained by 22-mer LNA-antimiRs of either the gapmer or the mixmer design. Remarkably, the duration of action of these was very long. After 4 days the target miR-125b was undetectable and even at day 10, after several cell divisions, expression of miR-125b was still effectively inhibited. Although sequestering of miR-125 was reported to be the likely mechanism of both designs, it was also possible that the microRNA inhibition was effectuated by an RNase H mechanism. The biological function of miR-181 in muscle differentiation and regeneration has also been studied with LNA-antimiRs. MiR-181 was effectively and specifically sequestered by LNAantimiR mixmers, and in C2C12 myoblast the differentiation was dramatically affected, both by myotube formation and expression of MHC [136]. It was furthermore shown that sequestering of miR-181 was related to an increased expression of Hox-A11 protein indicating that Hox-A11 is the target gene for miR-181. Inhibition of the well-characterizsed interaction between the Drosophila melanogaster bantam miR and its target hid gene was effectuated potently with mixmer LNA-antimiRs [137]. This was demonstrated in a model system where a luciferase reporter plasmid containing the Drosophila hid 3⬘-UTR (hid-pGL3⫹) was cotransfected into HEK293 cells with bantam miR. A bantam miR concentration of 30 nM reduced the luciferase expression to ca. 60%. The expression was restored when 30 nM LNA-antimiR was cotransfected, and the expression of the hid protein was increased to 177% in cell lines expressing both hid and bantam when only 10 nM LNA-antimiR was used. Lecellier et al. [138] used LNA-antimiRs to inhibit the human miR-32 that was shown to effectively limit primate foamy virus type 1 (PFV-1) replication. To address the antiviral effect of miR-32 they designed LNA-antimiRs against mir-32, and when the LNA-antimiR was cotransfected with PFV-1 in HeLa- and in BHK-21 cells the translation prevention by mir-32 disappeared specifically. At LNA concentrations of 10 nM the antiviral effect of mir-32 disappeared leading to accumulation of PFV-1. Davis et al. [85] compared a series of known antisense chemistries for their relative potencies as micro-RNA inhibitors. They tested 2⬘-O-Me, 2⬘-O-MOE, 2⬘-F, and 2⬘-O-MOEⲐLNA nucleotides in combination with DNA and PS nucleotides in a series of designs. The anti-miR oligonucleotides were tested in a lucifirase assay in a construct with the full 22 base-pair sequence complementary to the mature miR-21 inserted into the 3⬘-UTR of pGL3-control in HeLA cells. In general, the gapmers were not particularly active, and targeting the pri-miRNA with siRNA did not lead to microRNA inhibition. The most active anti-miR oligonucleotides were the once with the highest affinity, 2⬘-F and 2⬘-O-MOEⲐLNA, of the mixmer design, and the authors concluded that affinity is the single most important parameter for anti-miR activity. The advantage of the high affinity offered by LNA is nicely illustrated by Kloosterman and coworkers [88]. They showed that the size of anti-miR oligonucleotides could be reduced with out compromising the sensitivity and specificity. LNA oligonucleotides used to detect mir-124a and mir-206 in zebrafish embryos were equally effective in sizes ranging from 22 to 16 nucleotides [88]. These data suggest that the optimal size for LNA-antimiRs is likely to be of the same short size as for LNA AONs (vide supra). The data in this section confirm that LNA is a high potency micro-RNA inhibitor. The fact that the mixmer design generally appears to be the most active is even more suited for LNA compared to other chemistries, due to the “structural saturation” by LNA (vide supra). It is clear in the studies presented here that LNA also as a microRNA inhibitor offers the possibility to make “shorter than usual” anti-miRs with high potency. Thus, LNA is a very promising candidate for microRNA inhibition.

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19.9 PHARMACOLOGICAL ACTIVITY IN EXPERIMENTAL ANIMALS As described in the previous sections, incorporation of LNA nucleotides into ODNs leads to a substantial enhancement in affinity that increases with LNA nucleotide load and is independent of ODN design, for example, gapmers or mixmers. This freedom of design has enabled the development of LNA-ODNs with the ability to effectively and specifically target both coding and noncoding RNAs in vivo utilizing different mode-of-actions, such as target destruction, steric block to translation, inactivation by sequestration and interference with splicing. The first evidence of in vivo activity of LNA-ODNs was provided in 2000 by Wahlestedt et al. [111], who used 15-mer LNApo ODNs to knock down the -opioid receptor potently and sequence specifically in the brain of living rats, thus altering their response to pain in the presence of an opiate. Both gap- and mix-mer designs were highly effective and much more so than their unmodified versions. Fluiter et al. [140] demonstrated allele-specific knockdown of xenografted tumors in mice using 16-mer LNAPO ODNs directed against two different alleles of the large subunit of RNA polymerase II, POLR2A. The LNA-ODNs were all fully modified and therefore unable to direct RNaseH mediated target cleavage, suggesting that the observed activity was caused by a steric block to translation. This latter mode-of-action is generally considered inferior to an RNaseH mode-of-action. Nevertheless, the LNA-ODNs were five times more potent that the corresponding RNaseH recruiting PS-ODNs, giving clear inhibition of tumor growth when administered at 1 mgⲐkgⲐday by subcutaneously implanted osmotic minipumps. Potent suppression of tumor growth in xenografted mice at 1 mgⲐkgⲐday was also observed with 16-mer LNAPS gap-mers directed against H-Ras mRNA [93]. Both LNA-ODNs and
-L-LNA-ODNs, were evaluated in this study and shown to be effective and specific. Recently, Roberts et al. [146] used a transgenic mouse, carrying the splice defective EGFP-654 gene, to evaluate the ability of a 16-mer LNAPS ODN to alter mRNA splicing. To achieve such splice modulation, the ODN must be RNaseH inactive and hence the authors used an LNA-ODN comprising alternating LNA and DNA nucleotides. The authors found the LNA-ODN to be uniquely potent in correcting aberrant EGFP mRNA splicing with activity in the liver at doses as low as 0.75 mgⲐkg administered intraperitoneally once daily for 4 days. At 25 mgⲐkg there was major splice switching activity in the liver, colon and small intestine and minor activity in the kidney, lung, and spleen, all of which are organs in which LNA-ODNs accumulate. In liver, colon and intestine the LNA-ODN was about 17-fold more potent than a corresponding, but somewhat larger, fully modified 18-mer PS-ODN comprised entirely of 2⬘-OMe nucleotides. Furthermore, the duration-of-action of the 2⬘-OMe PS-ODN (after a single intraperitoneal injection of 25 mg/kg) was significantly shorter than that of the LNA-ODN, with no activity being detected in the liver at day 15 compared to 50% activity with the LNA-ODN at day 22. Remarkably, administration of a single dose of 50 mg/kg of the LNA-ODN to mice by oral gavage produced activity in the small intestine, colon and liver that was clearly above background. Notably, whereas liver showed the most activity after intraperitoneal administration, small intestine showed the most activity after oral administration. The above reports of in vivo activity of LNA-ODNs are consistent with our own observations that 16-mer LNAPS-ODNs, termed RNA Antagonists, are able to downregulate mRNA targets effectively in mice and monkeys. Dosing by intraperitoneal injection daily for 14 days of SPC2968, an RNA Antagonist of Hif-1
(vide infra), resulted in potent and dose-dependent downregulation of its mRNA target in the liver and kidney of wild-type mice [133]. Of the two tissues examined, the activity was most pronounced in the liver, where a 50% reduction in Hif-1
mRNA levels was achieved by a daily dose of 3.6 mgⲐkg. Consistent with the role of Hif-1 as a transcription factor, treatment with SPC2968 also caused a substantial reduction in the mRNA from the Hif-1-regulated gene, VEGF. In a similar manner, intravenous administration of SPC3197, an RNA Antagonist of ApoB-100, potently and dose dependently reduced ApoB-100 mRNA in both the liver and jejunum of normal mice with 50% target reduction observed at ⬃5 mg/kg [147]. Paralleling the reduction in ApoB-100

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Normalize Bcl-2 protein values in % of saline

Normalize Bcl-2 mRNA values in % of saline

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100 80 60 40 20 0

120 100 80 60 40 20 0

3 mg/kg

6 mg/kg

SPC2996

3 mg/kg

6 mg/kg

G3139

Saline

3 mg/kg SPC2996

3 mg/kg G3139

Figure 19.10 Steady-state levels of Bcl-2 mRNA (normalized to -actin mRNA) and Bcl-2 protein (normalized to tubulin) in the liver of monkeys after every second day intravenous bolus injection of 3 or 6 mg/kg of SPC2996 (hatch bars) or G3139 (Oblimersen sodium) (black bars) for 2 weeks.

mRNA, circulating levels of cholesterol were reduced by ⬃50% at 5 mgⲐkg and ⬃75% at 25 mg/kg. The study comonitored the performance of a corresponding siRNA (conjugated to cholesterol and containing 2⬘-OMe and PS modifications) that had previously been demonstrated to downregulate ApoB-100 in normal mice after systemic administration [148]. At the 50 mgⲐkg dose the modified siRNA showed no significant activity in the liver and only modest activity in the jejunum. Also, no effects were observed on plasma levels of cholesterol. SPC2996, an RNA Antagonist of Bcl-2, has been evaluated for pharmacological activity in Cynolomolgus monkeys using doses (3 and 6 mgⲐkg), an administration route (intravenously), and a schedule (every second day for two weeks) that are clinically relevant. As shown in Figure 19.10, substantial and dose-dependent reduction in both Bcl-2 mRNA and protein was observed in the liver when compared to the saline control (obtained from an earlier monkey study with SPC2996). In contrast, little to no effect on either Bcl-2 mRNA or protein, and no indication of dose-response, was observed in animals treated with the somewhat larger 18-mer PS-ODN, G3139 (Oblimersen sodium)—a Bcl-2 inhibitor that has been tested in several clinical trials. Recent years have witnessed the discovery of a novel class of small, noncoding, regulatory RNAs, termed micro RNA, that are increasingly attracting interest as pharmaceutical intervention points for the treatment of a diversity of human diseases. ODNs are uniquely suited to therapeutically target these miRNAs exploiting a simple sequestration mode-of-action. Ideally, the ODN should be short to reduce unwanted toxicities, yet have very high affinity to cause tight and durable binding to the miRNA suggesting that LNA-ODNs would be well suited for the task. Consistent with this notion, intraperitoneal injection for three days of a 16-mer LNAPS ODN against miR-122a (comprising alternating LNA and DNA nucleotides) potently inhibited the target in mice liver with an IC50 around 4–5 mgⲐkg [149]. The inhibition was dose-dependent, sequencespecific and at the 25 mgⲐkg dose, more than 50% inhibition was evident one week after dosing. Consistent with its role in cholesterol metabolism, inhibition of miR-122a translated into a significant reduction in plasma cholesterol that was reduced by more than 40% one week after dosing. 19.9.1 Brief Summary As expected from their behavior in cell cultures, LNA-ODNs are specific and potent inhibitors of their cognate RNA targets in vivo at very low doses. To date, pharmacological activity has been demonstrated in many different tissues (liver, kidney, colon, jejunum, small intestine, lung, spleen, and brain), and species (mice, rats, and monkeys) using a variety of administration routes (subcutaneous infusion, intravenous injection, intraperitoneal injection and direct injections into brain). Using different designs of LNA-ODNs it has been possible to target effectively both mRNAs and noncoding miRNAs, causing either a reduction in protein synthesis, a shift in the splicing pattern or inactivation by physical sequestration.

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19.10 PHARMACOKINETICS 19.10.1

Plasma Pharmacokinetics

Detailed plasma pharmacokinetic studies have been completed for two development candidates: SPC2996, a 16-mer LNAPS gapmer directed against the Bcl-2 mRNA and SPC2968, a 16-mer LNAPS gapmer directed against the Hif-1
mRNA. After intravenous bolus administration to rodents and monkeys, plasma concentrations of SPC2996 (Figure 19.11) as well as SPC2968 decreased in a biphasic manner with an initial rapid distribution phase during which the LNA-ODN deposits in tissues (vide infra) followed by a slower elimination phase. For both ODNs, the maximum mean plasma concentration (Cmax) was observed at the first sampling time point postdose, as would be expected following intravenous dosing. The calculated, theoretical plasma concentration at time zero (C0) increased linearly with dose in both mice, rats and monkeys, and in monkeys (where both oligos were tested) peak plasma concentrations of the ODNs were quite similar at similar doses. There was no statistically significant evidence of any sex-related differences in any of the species examined. Table 19.6 shows the C0 values for SPC2996 in rats and monkeys. The extent of systemic exposure (characterized by the area under the mean plasma concentration curve up to 24 h, AUC24) increased with increasing dose in all species. Increases, however, were greater than the proportionate dose increment for both SPC2996 (Figure 19.12) and SPC2968, indicating the existence of one or more saturable elements to distribution. In the SPC2996 study, where doses of up to 60 mgⲐkg were given every second day for 2 weeks, the AUC24 values at day 14 showed evidence of accumulation. By contrast, no accumulation was observed in the SPC2968 study that used lower doses (up to 40 mgⲐkg) and intensity (twice weekly for 4 weeks) 19.10.2

Biodistribution and Tissue Half-Life

Whole body biodistribution, tissue accumulation and subsequent clearance have been studied with several LNA-ODNs, using different dosing schedules, routes of administration and types of LNA

Mean plasma concentrations (µg/ml)

Rat

Monkey

1000

1000

100

100

10

10

1

1 0

200

400

600

Time after dosing (min)

0

500

1000

1500

2000

Time after dosing (min)

Figure 19.11 Plasma clearance of SPC2996 in rats and monkey after intravenous bolus injection of 60 mg/kg. Table 19.6 The Calculated, Theoretical Plasma Concentration at Time Zero Values (C 0) in Rats and Monkeys after Intravenous Bolus Injection of SPC2996 at 6, 20 or 60 mg/kg.
g/mL) C0 (
LAN Oligo

Species

SPC2996

Monkey Monkey Rat Rat

Sex Male Female Male Female

6 mg/kg

20 mg/kg

60 mg/kg

131⫾12 138⫾6 104 73.8

388⫾19 431⫾36 271 152

1120⫾96 1350⫾70 1050 929

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AUC24(µg h/ml)

Rat 800 700 600 500 400 300 200 100 0

2000 1500 1000 500 0 6

20 Dose (mg/kg)

60

6

20 Dose (mg/kg)

60

Figure 19.12 Systemic exposure (AUC24) to SP2996, after intravenous bolus injection of 6, 20 and 60 mg/kg to rats and monkey on day 1 and after repeated dosing (every second day for 13 days). Hatched bars indicate the expected AUC24 assuming dose linearity. Black bars show the actual measurement after the first dose and white bars show the actual measurements after repeated dosing. 1.2 1.0 0.8 0.6 0.4 0.2

Brown fat

Myocardium

Skeletal muscle

Thymus

Lung

Pancreas

Adrenal gland

Lymph node

Pituitary gland

Salivary gland

Spleen

Ovary

Thyroid gland

Bone marrow

Liver

Gastric mucosa

Skin

Uvea (eye)

Uterus

Kidney

0.0

Figure 19.13 Biodistribution of titriated SPC 2996 (white bars) and SPC 2968 (black bars) 4 h after intravenous bolus injection of 60 mg Ⲑ kg to mice. Data normalized to kidney.

monomers. Bolus injection of 50 mgⲐkg of tritium-labeled SPC2968 or SPC2996 in mice resulted in rapid distribution to tissues that tracked ODN clearance from plasma, which was completed within 2–4 h. Except for the brain, spinal cord, bone, testis, and lens both ODNs reached all tissues examined and with quite similar distribution profiles (Figure 19.13). Tissue half-lives were estimated on the basis of disappearance of label over 18 days and were found to be remarkably similar (⬃200 h) for the two ODNs across tissues. This is a much longer tissue half-life than those reported for PS-ODNs [139] and consistent with the much improved metabolic stability of LNA-ODNs. Distribution profiles quite similar to that shown in Figure 19.13 were also reported after intraveneous bolus administration to mice of tritium-labeled 16-mer LNAPO or LNAPS ODNs directed against either the POLR2A or H-Ras gene [93,140]. Moreover, the distribution profile was similar whether the H-Ras LNA-ODN was administered as intravenous bolus or by subcutaneous continuous infusion over 14 days (K. Fluiter, personal communication), suggesting that distribution is independent of the route of systemic administration. The H-Ras study also evaluated the distribution of several LNA analogs, for example, thio-LNA, amino-LNA and
-L-LNA. After subcutaneous continuous dosing of 5 mgⲐkgⲐday for 2 days several significant differences were observed in the distribution profiles of the three analogs. The amino-LNA differed the most with substantially more label accumulating in the heart, skeletal muscles, bones, and liver than observed with either of the other chemistries [93].

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These data show that LNA analogs can be used to influence pharmacokinetics-a potentially useful feature when designing drug candidates against different diseases. Tissue accumulation as a function of dose and schedule has been studied in both mice and monkeys using an HPLC method that enables detection of LNA-ODNs (full-length and n-1 oligonucleotides) with a lower limit of detection of ⬃5
gⲐg of tissue. In mice, intraperitoneal administration of 2, 10 or 50 mgⲐkg on day 0, 3,7,10, and 13 resulted in dose-linear accumulation in liver and kidney with no signs of having reached steady state (Figure 19.14). Different dosing schedules (0.7, 3.6 or 18 mgⲐkg daily for two weeks) that delivered the same total dose resulted in similar final tissue concentrations suggesting that total dose rather than schedule is the major determinant of tissue accumulation. In monkeys, intravenous dosing of 6 to 60 mg/kg of the development candidate SPC3042 (a 16-mer LNAPS gap-mer directed against survivin) twice weekly for 4 weeks also resulted in dose-linear tissue accumulation and a distribution similar to that observed in mice (Figure 19.15). Dose-linear accumulation in liver and kidney was also observed upon repeated intravenous dosing of 6–60 mg/kg of SPC2996 (every second day for 2 weeks) and 6– 40 mgⲐkg of SPC2968 (twice weekly for 4 weeks).

180 160

SPC2968 µg/g tissue

140 120 100 80 60 40 20 0 0.7

3.6

18

mg/kg daily for 14 days

2.0

10

50

mg/kg on days 0,3,7,10, and 13

Figure 19.14 Accumulation of SPC 2968 (intraperitoneal injection) in the liver of mice as a function of dose and dosing schedule. 1800 1600

SPC3042 µg/g tissue

1400 1200 1000 800 600 400 200 0 Kidney

Liver

Bone Pancreas marrow

Colon

Figure 19.15 Accumulation of SPC 3042 in five different tissues in monkeys following intravenous bolus injection of 60 mg/kg.

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The half-lives of LNA-ODNs in monkey livers have been estimated from measurements of ODN present at the end of the treatment phase and after a further recovery period. Assuming monoexponential decay the following half-lives in liver were calculated on the basis of the means of data from six (treatment group) and four animals (recovery group), SPC3042: 19.5 days, SPC2996: 22.6 days, and SPC2968: 29.3 days. Given the quite large standard deviations on the measurements, we infer that half-lives of LNA-ODNs in monkey liver are quite similar and remarkably long (at least several weeks) providing the possibility that pharmacologically active concentrations of LNA-ODNs in humans may be maintained with infrequent and therefore convenient dosing schedules. 19.10.3

Uptake into Cells

Once distributed to tissues, short LNA-ODNs appear to be internalized into the constituent cells in a pharmacologically active form, as evidenced by many examples of potent pharmacological activity in vivo (vide supra). By and large, there seems to be good correlation between pharmacology and pharmacokinetics, that is, good activity is normally observed in highly accumulating tissues. The notable exception identified so far is kidney where pharmacological activity in whole organ homogenates has consistently been much smaller than expected from bulk accumulation data alone. This apparent discrepancy was partly explained by fluorescence microscopy examination of mouse kidney sections after systemic administration of fluorescently labeled LNA-ODNs showing label to be predominantly confined to the kidney cortex with particularly strong staining in the epithelium of the proximal and, to a lesser extend, distal tubules (suggesting that uptake into kidney tissue is primarily through re-absorption into proximal tubule epithelial cells from glomerular filtrate in the lumen of the tubule rather than directly from the blood stream). Uptake into mice hematopoietic cells in vivo has been studied by Flow-Activated Cell Sorting (FACS) analysis, using lineage-specific antibodies and a fluorescently labeled version of SPC2968. After intraveneous bolus injection of either FITC-SPC2968 or FITC alone (control), the ODN was found to be associated with all cell types in the bone marrow, spleen, and circulation (Figure 19.16). The amount of SPC2968 associated with different cell types, however, differed significantly and the ranking between cell types was different in the different compartments analyzed. These differences strongly suggest that SPC2968 was not merely adhering to the surface of the cells but was in fact internalized. Moreover, the data indicate that the ability to internalize LNA-ODNs varies between different cell types and depends on the context in which cells encounter the ODN. As shown in Figure 19.16, SPC2968 was also associated with multipotent stem cells in the bone marrow as defined by the CD34, Lin-stain. That these cells were in fact progenitor cells was subsequently confirmed by their ability to form colonies when plated in soft agar. This finding is crucial to the use of LNA-ODNs in the treatment of leukemia, where the defect is often in the stem cell compartment. More recently, uptake and cytoplasmic compartmentalization of LNA-ODNs has been demonstrated in primary CLL cells in culture, highlighting their applicability to target hematopoietic malignancies (R. Van Oers, personal communication). Uptake of short LNA-ODNs into cultured cells without the use of transfection reagents or carrier molecules has also been reported. Incubation of ACSH cells (⬃106 per well) with 5 nM of a 99mTc labeled 15-mer LNA-ODN against Ri
mRNA led to a continuous accumulation with time that at 24 h corresponded to ⬃5 ⫻ 104 molecules per cell [141]. The ODN was not simply adhering to the cell surface as indicated by the finding that accumulation of the antisense LNA-ODN significantly exceeded that of sense LNA-ODN at all timepoints from 1 h and forward, ending at a fivefold higher concentration at 24 h (i.e., the antisense ODN is assumed to be retained in the cell by binding to its target mRNA whereas the sense ODN, which does not have a target in the cell, is not). Fractionation of the cells showed that the antisense LNA-ODN was significantly present in both the nuclear, cytoplasmic, and membrane fractions lending further support to the conclusion that the ODN was taken up by the cells.

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Median fluorescent intensity (MFI)

(A)

60 50 40 30 20 10 0 CD4

Granulocyte Macrophage

B cells

CD34-Lin/

Dendritic

(B)

50

Median fluorescent intensity (MFI)

Cell lineages

40 30 20 10 0

CD4

CD8

Granulocyte Macrophage

B cells

Cell lineages

(C)

70

Median fluorescent intensity (MFI)

60 50 40 30 20 10 0

CD4

CD8

Granulocyte Macrophage

B cells

Cell lineages

Figure 19.16 Median fluorescent intensity of hematopoitic cell lineages 24 h after intravenous bolus injection of fluorescently labeled SPC 2968s into mice. (A) Bone marrow. (B) Circulation. (C) Spleen. Grey bars indicate the MFI signal obtained with the FITC alone (control) and black bars indicate the MFI signal obtained with FITC labeled SPC 2968.

19.10.4

Excretion

Radioactivity has been detected in the urine and bile of all mice treated with both radiolabeled LNAPS or LNAPO 16-mer gapmers [140] indicated that both are routes of elimination. At pharmacologically active doses plasma clearance of LNAPS ODNs is predominantly by tissue distribution with very little being filtrated by the kidney and even less excreted via the bile [140]. When SPC2968 or SPC2996 were administered intravenously at very high doses (50 mgⲐkg), however, renal excretion was markedly increased suggesting that ODN concentrations had exceeded the capacity of plasma binding that would otherwise prevent renal filtration.

19.10.4.1

Brief Summary

The aggregate data suggest that the pharmacokinetics of short LNAPS ODNs are sequence independent but can be influenced by the choice of LNA chemistry. Cmax increases linearly with dose in

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the concentration range examined (6–60 mgⲐkg) and is observed at the first sampling time point. Plasma clearance is rapid, shows saturation kinetics at very high doses and is largely driven by broad distribution to tissues. With the constraint of limited data, tissue accumulation appears to depend more on total dose than dosing schedule and to be rather independent of the route of systemic administration (continuous subcutaneously, intraperitoneally or intraveneously). Once in the tissues, LNA-ODNs have very long half-lives which in monkeys are measured in several weeks. This is consistent with the good metabolic stability of LNAPS ODNs and suggests that activity in humans may be achieved with relatively infrequent and therefore patient convenient dosing schedules.

19.11 TOXICOLOGY 19.11.1

Acute Toxicities

Acute toxicities of clinically sized phosphorothioates (typically 20-mers) have been characterized in detail in monkeys [142], thus providing a suitable background against which LNA-ODNs can be benchmarked. The principal acute toxicities of PS-ODNs are class related and attributable to complement activation and inhibition of clotting function both of which have been of significant concern in the clinical development of PS-ODN drugs. Complement activation by phosphorothioates consistently occurs at plasma threshold levels of 40–50
gⲐmL in monkeys and manifests as pronounced increases in complement split products such as Bb (which may increase 100-fold above baseline), transient fluctuations in circulating neutrophils and hemodynamic changes. Occasionally, complement activation has resulted in cardiovascular collapse and an anaphylactic-like response, which in some animals have been lethal. The causal mechanism is well understood and involves binding of the PS-ODNs to complement factor H that acts as a break on the constitutively active alternative cascade of complement [142]. Decreasing the size of the ODN has been shown to reduce factor H binding [143,144] suggesting that the 16-mer LNA-ODN development candidates would have a much reduced propensity to activate complement than their larger sized PS-ODN cousins. Consistent with this, intravenous bolus injections of up to 60 mgⲐkg of SPC2996 in primates (C0 values of ⬃1200
gⲐmL; Table 19.6) in the context of formal GLP toxicity studies, did not produce any of the overt signs of acute toxicity reported to accompany PS-ODN induced complement activation. In fact, the dose escalation study preceding the main study reached doses of 300 mgⲐkg (administered as 5 min bolus injection) before encountering signs of reaction to the treatment and then only following the third dose. At the biochemical level, no increase in Bb split products was observed following intravenous bolus injections of SPC3042 until the 15 mgⲐkg dose was reached (expected C0 values ⬃300
g/mL) at which the level of Bb split products was approximately twice that of background. Only at the 60 mg/kg dose (expected C0 values ⬃1200
gⲐmL) did plasma levels of Bb split products clearly increase, approximately five- to sevenfold (Figure 19.17), although this is still far lower than the levels observed with clinical PS-ODNs at much lower doses [142]. Elevated Bb split products in the 60 mg/kg dose group was not associated with any clinical signs or changes in hematological or hemodynamic parameters. In all animals, the increased concentrations of Bb split products were transient in nature, peaking 30 min after dosing and reverting to normal values when measured 24 h after dosing. Also, responses were approximately similar whether measured after the first dose or after twice weekly dosing for 4 weeks. Increases in Bb split product were also observed with SPC2968. At the 40 mg/kg dose the level was about twice that observed at the 60 mgⲐkg dose of SPC3042, indicating some variation between oligonucleotides and complement activation could not be ruled out as a contributing factor to the poor clinical condition of one animal that necessitated sacrifice on day 29 of the 4-week study. Inhibition of clotting function is the other major acute toxicity of PS-ODNs, which manifests as a prolongation of activated partial thromboplastin time (APTT) and prothrombin time (PT), the magnitudes of which have been reported to be linearly proportional to ODN concentrations in

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Bb-fragment (µg/mL)

12 10 8 6 4 2

First dose

24 h

4h

30 min

Predose

24 h

4h

30 min.

Predose

0

Eighth dose

Figure 19.17 Increase in Bb complement split product in monkeys following the first and eighth dose of 60 mg Ⲑkg of SPC 3042 (intravenous bolus injection). Solid circles designate female monkeys and solid squares designate males.

plasma. APTT is significantly more affected than PT and typically doubles in monkeys when plasma concentrations of PS-ODNs reach 150
gⲐmL. Like complement activation, prolongation of bleeding is caused by drug binding to plasma proteins that in the case of APTT have been identified as activated Factor Xa [142]. Prolongation of APTT has been shown to be directly proportional to the length of the phosphorothioate [142], suggesting that the LNA-ODN development candidates would also have a much reduced ability to provoke this acute toxicity. Indeed, dosing of 15 mgⲐkg of SPC3042 had no effect on either APTT or PT at plasma concentrations (expected C0 ⬃300
gⲐmL) that far exceed that at which PS-ODNs will cause a doubling. In fact, a doubling of APTT was only observed at the highest doses tested with SPC3042 (60 mgⲐkg) and SPC2968 (40 mgⲐkg) and even at these doses PT was only marginally affected. Figure 19.18 shows the results for SPC3042. In all cases, APTT and PT values returned to baseline at 24 h postdosing with the LNA-ODN and in only one high dose group animal in the SPC3042 study (sacrificed on the basis of poor clinical condition at day 22 of the 4-week study) were prolongations in APTT associated with pathological bleeding in internal organs. 19.11.2

Subacute Toxicities

Several investigators have reported that LNA-ODNs are well tolerated in rodents at pharmacologically active doses. In a study with 15-mer LNAPO ODNs against the -opioid receptor, no detectable histological toxicity or changes in core body temperature were noted after direct injection of the ODN into the striatum of rat brains [111]. In contrast, the isosequential PS-ODNs caused fever and severe necrosis along the trajectory of the injection site. Continuous subcutaneous administration of up to 5 mgⲐkg for 14 days of various 16-mer LNAPO ODNs against the POL2R mRNA did not result in any histological changes in the liver or kidney of mice [140]. Also, the mixed mononuclear infiltrate in liver that is characteristically found when PS-ODNs are used was absent (vide infra). Minor increases in ALAT and ASAT levels were noted but only at the highest dose, which were well above the therapeutically active dose. Likewise, neither ASAT, ALAT or alkaline phosphatase levels were increased significantly when mice were dosed with pharmacologically active doses of either a 16-mer LNAPS ODN or the isosequential
-L-LNA-ODN against H-Ras [93]. Also, there was no abnormal fluctuation in body temperature during the study. Immune stimulation is a particularly well–studied toxicity of PS-ODNs, which manifests as splenomegaly, lymphoid hyperplasia, hypergammaglobulinemia and infiltration of mixed mononuclear cells in a variety of tisues such as liver, kidney, heart, lung, thymus, pancreas and

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(A) 90 Seconds to thrombus formation

80 70 60 50 40 30 20 Predose

30 min

24 h

Predose

First dose

Seconds to thrombus formation

(B)

30 min

24 h

Eighth dose

14 13 12 11 10 9

Predose

30 min

24 h

First dose

Predose

30 min

24 h

Eighth dose

Figure 19.18 Increase in (A) APTT and (B) PT values after the first and eighth dose of 60 mg/kg of SPC 3042 (intravenous bolus injection). Solid circles designate female monkeys and solid squares designate males.

salivary glands [142]. Stimulation is particularly pronounced with PS-ODNs carrying CpG motifs, but all PS-ODNs, independent of sequence, exhibit some degree of immune stimulation (which is most prominent in rodents). In 2004, Vollmer et al. [145] reported that LNA nucleotides could be used to substantially decrease the immune stimulatory effect of otherwise highly efficient PS-ODNs (scored as cytokine release from human PMBC cells). Increasing the number of LNA nucleotides at the 5⬘ and 3⬘ ends of the highly immunostimulatory PS-ODN, G3139 (Oblimersen sodium), led to progressive loss of IL-10 secretion that dropped to background levels when the ODN contained 4 LNA nucleotides at each end. Likewise, substitution by LNA nucleotides of both the DNA-C and G in the CpG motif of another strongly immunostimulatory PS-ODN led to massive decreases in IL-6, IL-10 and IFN-
production. A substantial reduction in immune stimulation was also observed with single LNA nucleotide substitutions (LNA-C substitutions being more effective than LNA-G). The subacute toxicity of the development candidates SPC2996, SPC2968, and SPC3042 has been investigated in rodents andⲐor nonhuman primates. The first candidate SPC2996 was dosed intravenously to Cynomolgus monkeys every second day for 14 days at dose levels 6, 20, and 60 mg/kg. Recovery after 14 days was evaluated in the 60 mgⲐkg group. Based on experience from the first subacute study with SPC2996 the next two candidates, SPC3042 and SPC2968, were dosed intravenously to Cynomolgus monkeys twice weekly for 4 weeks, with recovery being evaluated after 4 weeks at high- and medium-dose level. The dose levels for SPC3042 were 6, 15, and 60 mgⲐkg, and SPC2968 was dosed at 6, 10, and 40 mgⲐkg.

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Slight degenerativeⲐhypertrophic changes in the liver associated with slight increases in liver weight and liver enzymes (ASTⲐALT) were seen in the medium- and high-dose groups treated with SPC2996 and SPC3042. No liver toxicity was recorded after 4 weeks treatment with SPC2968 (in a preceding MTD study with SPC2968 the monkeys showed slight liver toxicity at 80 mgⲐkg). Slight renal toxicity associated with increased organ weight was recorded in high-dose groups of all three candidates and in the medium groups of SPC2996 and SPC2968. The local response to treatment was identical for the three candidates, slightly increased compared to identical treatment with vehicle but much reduced to that reported for PS-ODNs [142]. Findings at the injections sites were characterized as hemorrhage, inflammatory, and degenerative changes. Sporadic, treatment related, mononuclear infiltration was recorded in spleen, lymph nodes, lung, salivary glands, liver or gall bladder after treatment with SPC2996. Owing to the short recovery period in the SPC2996 study (14 days), full recovery was not obtained, in contrast to the SPC3042 and SPC2968 studies where full recovery of toxicity effect was recorded after 4 weeks. The subacute toxicity of SPC2996 was also estimated in rats in a 14-day intravenous study at 6, 20, and 60 mgⲐkg with 14 days recovery evaluated at high-dose level. Slight dose related liver toxicity, including single cell degeneration, inflammation, and mitotic increase, increased organ weight and slightly increased liver enzymes were recorded at 20 and 60 mgⲐkg. Slight dose-related renal toxicity, including cellular degeneration and inflammation associated with macroscopic changes, increased organ weight and slightly increased urine volumes, were recorded at 20 and 60 mgⲐkg. Inflammatory cell infiltration was recorded in lung and liver and increased cellularity in spleen and lymph nodes associated with increased white blood cell counts were recorded in rats at 20 and 60 mgⲐkg. The local response to treatment was slightly increased compared to identical treatment with vehicle. Owing to the short recovery period in the SPC2996 study in rats full recovery of the treatment-related effects was not obtained. At 6 mgⲐkg, no toxicity or pathological changes were observed. The subacute toxicity of SPC2968 was investigated in mice in a 4-week intravenous study at 2, 8, and 40 mgⲐkg with 4 weeks recovery evaluated at all treatment levels. Slight liver toxicity including cell hypertrophy, degeneration, inflammation, and necrosis associated with macroscopic changes, increased organ weight and slightly increased liver enzymes were recorded at the top dose of 40 mgⲐkg. At 8 mgⲐkg the toxicity was minimal, recorded in only a few animals, and not associated with increases in liver enzymes. Increased extramedullary hematopoiesis andⲐor increased cellularity was recorded in the spleen of mice treated at the 40 mgⲐkg dose. The local reaction to treatment was slightly increased compared to identical treatment with vehicle. Apart from slight inflammatory infiltrate in the livers of mice treated at 40 mgⲐkg, full recovery after treatment was recorded. The immune stimulating effect of the three LNA candidates was minimal in monkeys. The increased immune stimulating effect observed following the treatment with SPC2996 compared to the other two candidates was most likely related to the higher doseⲐmore frequent treatment schedule. In the subacute rat study with SPC2996 evidence of slight immune stimulation was recorded in several organs compared to the mouse study with SPC2968 where the inflammatory infiltrates were restricted to the liver. The difference was most likely related to dose level. In all cases, the immune stimulating effects seen in rodents treated with any of the LNA-ODN development candidates were substantially less than the reported immunostimulatory effects of PS-ODNs.

19.11.2.1

Brief Summary

The available data clearly show that 16-mer LNAPS ODNs have very little—if any ability to activate complement and inhibit clotting at clinically relevant doses. This means that the margin of safety for these molecules is much greater than that of other PS-based ODNs in the antisense field. The available evidence further suggests that the toxicity of LNAPS ODNs observed in rodent and nonhuman primates is a class effect that is independent of sequence and fully reversible upon cessation of treatment. Importantly, the subacute toxicities observed at high doses of the LNAPS ODNs have all been previously described for PS-ODNs and are thought to be due to the

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phosphorothioate backbone suggesting that the addition of LNA monomers to oligonucleotides does not add new class-toxicities.

19.12 LNA DRUGS IN DEVELOPMENT In 2005, the first LNAPS ODN, SPC2996 entered into a phase IⲐII study in patients with CLL. SPC2996 is a potent and selective inhibitor of the Bcl-2 gene that encodes a key sensor protein, which protects cells against apoptosis. The Bcl-2 protein is overexpressed in many cancers, including CLL, and high expression has been firmly correlated with low response rates and resistance to chemotherapy, faster time to relapse and shorter survival times [150]. SPC2996 has the potential to become a broad acting anticancer drug, working through sensitization of cancer cells to naturally produced, or clinically induced, apoptotic instructions. The human phase IⲐII clinical trial is an open label, international multicenter, dose-escalation study being conducted by Santaris Pharma at 13 sites in four countries (Denmark, UK, US, and France). In total, the study will enroll 42 patients. The primary endpoints are to define the maximum tolerated dose, identify clinical side effects, and to investigate changes in Bcl-2 expression during and following treatment. Secondary endpoints include the study of overall tumor response, changes to CLL cell numbers in peripheral blood and time to progression. SPC2996 is given as a 2-h intravenous infusion, three times a week for 2 weeks. Two other LNAPS ODNs, SPC2968 and SPC3042, have completed preclinical toxicology and are ready for clinical trials in patients with cancers. SPC2968 is a potent and selective inhibitor of Hif-1
that encodes the oxygen sensitive
-subunit of the HIF-1 transcription factor. Under low oxygen tension HIF-1 transcriptionally activates more than 60 genes that facilitate cell survival under anaerobic conditions, upregulate neovascularization, increase the oxygen transporting capacity of the blood, facilitate cell proliferation, and enhance the ability of cells to transgress tissue boundaries and metastazise [151]. High levels of Hif-1
are common in human tumors and have in many cases been reported to correlate with increased tumor vascularization, aggressive behavior and overall poor clinical outcome [152]. SPC2968 is being codeveloped by Santaris Pharma and Enzon Pharmaceuticals and has the potential to become an innovative new anticancer drug working by interference with several metabolic pathways important for the malignant phenotype. SPC3042 is a potent and selective inhibitor of survivin that plays a vital regulatory role in apoptosis by inhibiting activation of lethal caspases [153]. In addition, survivin plays a pivotal role in normal mitotic progression and cell division. Survivin is expressed in many cancers, but almost absent in normal, adult, differentiated tissues. Clinically, survivin expression is generally associated with poor prognosis, resistance to therapy and increased risk of relapse [153]. SPC3042 is also the subject of a codevelopment agreement between Santaris Pharma and Enzon Pharmaceuticals and is expected to enter clinical studies in 2007.

19.13 CONCLUSIONS AND FUTURE DIRECTIONS So far, efforts to develop LNA-ODNs into therapeutics have focused on fully phosphorothiolated 16-mers. As reviewed in this chapter such short, single-stranded, LNA-ODNs, which we term RNA Antagonists, combine unprecedented potency in vitro and in vivo with improved safety profiles compared to longer PS-ODNs comprising other chemistries. As we further explore the potential of the LNA chemistry, it is conceivable that the size and PS load of these molecules can be further reduced without compromising potency and specificity, and that such shorter molecules will offer additional advances in regard to for instance cellular uptake and reduced toxicity. We are also just at the beginning of understanding how the different LNA analogs can be used to alter biodistribution

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andⲐor improve uptake, thereby broadening the applicability of the LNA drug platform to human diseases. In conclusion, the advent of LNA has underpinned the importance of affinity and metabolic stability in the development of effective single-stranded oligonucleotide drugs. Over the next few years, LNA-ODNs of a variety of designs will enter and complete human clinical trials, in a number of different disease settings. The data from these trials will be pivotal in confirming whether short LNA-ODNs (RNA Antagonists) represent a successful new class of drugs or only a step along the way to effective RNA targeting therapeutics. ACKNOWLEDGMENTS We are grateful to Keith McCullagh for his valuable suggestions and comments during the review process of the manuscript. Part of the work described in this chapter has been performed by our colleagues at Santaris Pharma and have yet to be published. We thank them for their contributions and assistance all of which are greatly appreciated. In particular, we would like to recognize the contributions of Lisa Eriksen, Vibeke Aarup, Phil Kearney, Christoph Rosenbohm and Sakari Kauppinen. REFERENCES 1. Singh, S.K., Nielsen, P., Koshkin, A. et al., LNA (locked nucleic acids): synthesis and high-affinity nucleic acid recognition, Chem. Commun., 455, 1998. 2. Obika, S., Hari, Y., Sugimoto, T. et al., Triplex-forming enhancement with high sequence selectivity by single 2⬘-O,4⬘-C-methylene bridged nucleic acid (2⬘,4⬘-BNA) modification, Tetrahedron Lett., 41, 8923, 2000. 3. Singh, S.K., Kumar, R., and Wengel, J., Synthesis of novel bicyclo [2.2.1] ribonucleosides: 2⬘-amino-and 2⬘-thio-LNA monomeric nucleosides, J. Org. Chem., 63, 6078, 1998. 4. Singh, S.K., Kumar, R., and Wengel, J., Synthesis of 2⬘-amino-LNA: a novel conformationally restricted high-affinity oligonucleotide analogue with a handle, J. Org. Chem., 63, 10035, 1998. 5. Wengel, J., Koshkin, A., Singh, S.K. et al., LNA (Locked Nucleic Acid), Nucleosides Nucleotides Nucl. Acids, 18, 1365, 1999. 6. Rahman, S.M.A., Seli, S., Utsiki, K. et al., Synthesis and properties of 2⬘,4⬘-BNANC, a second generation BNA, Nucl. Acids Symp. Series, 49, 5, 2005. 7. Hari, Y., Osaki, T., Eguchi, K. et al., Synthesis and properties of oligonucleotiodes containing novel 2⬘,4⬘-BNA analogues (2⬘,4⬘-BNACOC), Nucl. Acids Res. Suppl., 2, 147, 2002. 8. Koshkin, A., Rajwanshi, V.K., and Wengel, J., Novel convenient syntheses of LNA [2.2.1] bicyclo nucleosides, Tetrahedron Lett., 39, 4381, 1998. 9. Singh, S.K. and Wengel, J., Universality of LNA-mediated high-affinity nucleic acid recognition, Chem. Commun., 1247, 1998. 10. Vorbrüggen, H. and Höfle, G., On the mechanism of nucleoside synthesis, Chem. Ber., 114, 1256, 1981. 11. Vorbrüggen, H. and Bennua, B., A new simplified nucleoside synthesis, Chem. Ber., 114, 1279, 1981. 12. Takashi, I. and Satoshi, O., Synthesis and properties of novel conformationally restrained nucleoside analogues, J. Synth. Org. Chem., Jpn., 57, 969, 1999. 13. Christensen, S.M., Hansen, H.F., and Koch, T., Molar-scale synthesis of 1,2:5, 6-Di-0-isopropylidenealpha-D-allofuranose: DMSO oxidation of 1,2:5,6-Di-O-isopropylidene-alpha-D-glucofuranose and subsequent sodium borohydride reduction, Organic Proc. Res. Dev., 8, 777, 2004. 14. Koshkin, A., Singh, S.K., Nielsen, P. et al., LNA (Locked Nucleic Acids): synthesis of the adenine, cytosine, guanine, 5-methylcytosine, thymine and uracil bicyclonucleoside monomers, oligomerisation, and unprecedented nucleic acid recognition, Tetrahedron Lett., 54, 3607, 1998. 15. Koshkin, A.A., Fensholdt, J., Pfundheller, H.M. et al., A simplified and efficient route to 2⬘-O, 4⬘-Cmethylene-linked bicyclic ribonucleosides (locked nucleic acid), J. Org. Chem., 66, 8504, 2001.

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20

Morpholinos Patrick L. Iversen

CONTENTS 20.1 20.2 20.3 20.4

Introduction .........................................................................................................................565 Safety Profile.......................................................................................................................567 Pharmacokinetic Profile ......................................................................................................569 Antiviral ..............................................................................................................................570 20.4.1 Exploration............................................................................................................570 20.4.2 Rapid Response to Emerging Infectious Disease .................................................571 20.4.3 Clinical Trials........................................................................................................571 20.5 Antibacterial........................................................................................................................572 20.6 Cardiovascular.....................................................................................................................572 20.7 Cancer .................................................................................................................................574 20.8 Metabolic Redirection.........................................................................................................575 20.9 Altered RNA Splicing for Duchenne Muscular Dystrophy (DMD)...................................576 20.10 Formulations .......................................................................................................................577 20.11 Summary .............................................................................................................................577 Acknowledgments ..........................................................................................................................577 References ......................................................................................................................................578

20.1 INTRODUCTION The phosphorodiamindate morpholino oligomers (PMO) are comprised of (dimethylamino) phosphinylideneoxy–linked morpholino backbone moieties (Figure 20.1). These morpholino moieties contain a heterocyclic base recognition moiety of DNA (A,C,G,T) attached to a substituted morpholine ring system. When linked to each other via the (dimethylamino) phosphinylideneoxy function, the functional group formed by the intersubunit linkage is commonly referred to as a phosphorodiamidate. Some improved profiles of PMOs include: (1) PMOs mechanism of action does not utilize oligomer as a cofactor for enzymatic cleavage of RNA [1,2]; (2) PMOs do not form G-quartet structures capable of off-target gene regulation [3,4]; (3) PMOs do not interact with traditional drugs such as acetaminophen (Tylenol) [5]; (4) PMOs do not cause severe and occasionally lethal hypotension in primates following bolus intravenous injections [6,7]; (5) PMOs do not chelate

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5′ O

O

HO

O

O N

N NMe2 P O

O

Base1-21

O

N

21

Me2N

P

O O

Base22

O

3′

N H

NH2 N

N

A= N

N

N

C= N

O

NH2

N

NH

G= O

O Me

NH

T= N

N

NH2

N

O

Position is stereochemically homogeneous, with same configuration as D-ribose. Position is not stereochemically homogeneous. Figure 20.1 Chemical structure of phosphorodiamidate morpholino oligomers.

metal ions including zinc, which may interfere with apoptotic mechanisms [8]; (6) PMOs do not alter blood coagulation times [9–12]; and (7) PMOs do not bind to cellular and extracellular proteins [13–15]. A critical advantageous character is that PMO oligomers are highly resistant to degradation [16]. PMOs have been evaluated in animal models and many publications have already documented PMO efficacy for a number of diseases. They include c-myc PMO (AVI-4126) to ameliorate murine infantile polycystic kidney disease [17], CYP3A2 PMO (AVI-4457) to modulate cytochrome P-450 activity in the rat [18], and AVI-4126 to prevent myointimal hyperplasia in a rabbit balloon injury model [19], and in pig model [20]. Further, we have observed efficacy in animal models following a variety of routes of administration including intravenous administration in a prostate model [21], topical application for cytochrome P450 3A2 rat model [22], inhalation for inhibition of TNF [23], and oral application for inhibiting cytochrome P450 3A2 in rats [24].

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20.2 SAFETY PROFILE In brief, there have been no observed clinical, laboratory, or histological abnormalities or toxicity in mice, rats, and nonhuman primates that have received sufficient PMO to cause a desired biological effect and at doses that are assumed to exceed 5–10 times the desired effect (Table 20.1). Toxicology studies have involved up to 150 mg/kg single intravenous doses and 28 daily doses of 140 mg/kg administered subcutaneously. The observation that has established the no effect level has been the appearance of basophilic granules within the cytoplasm of renal tubular epithelial cells, lymph node macrophages, and hepatic Kupfer cells, which was dose-dependent and reversible after discontinuation of administration. PMOs are devoid of teratogenic potential in the zebrafish embryogenesis model. Exposure of zebrafish embryos at early blastula stage to increasing concentrations of AVI-4126 did not cause any developmental abnormality. Long-term survivability of antisense-treated embryos was also not affected [25]. Further, the study was validated when zebrafish embryos were incubated with the PMO targeted to ntl gene, a distinct phenotype similar to ntl null mutation was observed in 3 of 100 embryos [25]. The direct injection of embryos with PMOs is very popular among investigators in the zebrafish developmental biology community. A review of 203 peer-reviewed publications revealed that the teratogenesis rate is 0.3% for PMO. This represents over 500 different PMO sequences in over 47,000 embryos [26]. Finally, no mutational events were observed in the battery of mutagenicity studies that have been conducted for three different PMOs to satisfy FDA preclinical recommendations. Four PMO compounds have been employed in 15 clinical trials, inclusive of Phase 1 and Phase 2 studies (see Table 20.2). A total of approximately 350 individuals have received the PMOs by oral, subcutaneous, or intravenous administration. There have been no observed serious drug-related adverse events. In addition, there is no evidence of any adverse clinically significant trends among Table 20.1 Toxicology and Safety Pharmacology GLP Studies with PMOs Target AVI-4126 c-myc

AVI-4020 WNV

AVI-4065 HCV

a b

Type

Species

N

Dose mg/kg

Route

Frequency

Study

NHP

Cyno

4 Tx 2 cont

Rat

S-D

20/dose 80 total 6/dose 30 total

0, 0.3, 3.0, 30 5, 15, 50, 100, 150

IV

Daily ⫻ 14

Tox

IV

Single

Tox

6/dose 12 total 6/dose 24 total

0, 15

IP

Daily ⫻ 28

Tox

0, 1.5, 7.5, 30

IV

Q 12H ⫻ 15

Tox

0, 4, 12, 40

SCb

Single

Safety

Single 4-way crossover ⫹ 5-day recovery Daily ⫻ 28 ⫹ 14-day recovery Daily ⫻ 28 ⫹ 14-day recovery

Safety

Rat

S-D

NHP

Cyno

Rat

S-D

0, 10

IVa

Single

Tox

Safe, well tolerated Safe, well tolerated Safe, well tolerated

6/dose 24 total 8/dose 32 total 6/dose 24 total

0, 4, 12, 40

SC

0, 4, 12, 40

SC

NHP

Cyno

Rat

S-D

12/group 72 total

0, 14, 70,140

SC

NHP

Cyno

6/group 4/group recovery 32 total

0, 4, 12, 40

SC

Intravenous. Subcutaneous.

Conclusions

Safety

Safe, well tolerated Safe, well tolerated NOAEL ⫽ 40 mg/kg NOAEL ⫽ 40 mg/kg NOAEL ⫽ 40 mg/kg

Tox

NOAEL ⫽ 70 mg/kg

Tox

NOAEL ⫽ 40 mg/kg

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Table 20.2 Survey of PMO Clinical Studies and Safety Total Related Drug

Study Description

AVI-4126 Safety and PK of a single, IV dose of AVI-4126 in healthy adults Safety and PK of oral AVI-4126 administration in healthy adults Safety and PK of single, IV dose of AVI-4126 in patients with polycystic kidney disease (PKD) Exploratory safety and PK of single, IV dose of AVI-4126 in patients with breast and prostate cancer Intramural delivery of AVI-4126 to the coronary artery Safety and efficacy of RESTEN-MP™ to prevent restenosis Safety and efficacy of RESTEN-MP™ to prevent restenosis AVI-4020

1-, 3-, 10-, 30-, 90-mg single oral dose

29 healthy adults

10-, 30-, 90-mg single IV injection

17 adult PKD patients

90-mg single IV injection

2 adult cancer patients

0, 3, 10 mg via intramural injection via the Infiltrator® catheter during angiography 8 mg RESTEN-MP (AVI-4126 ⫹PESDA) via IV injection at time of stent placement 16 mg RESTEN-MP via IV injection at time of stent placement and 24 hours thereafter

46 adult patients (30-AVI-4126; 16 placebo)

Compassionate use of AVI-4020 in patient with sever neuroinvasive disease

30 mg every 12 h for 5 days via IV injection

Safety study of AVI-4065 in healthy adults Exploratory study of AVI-4065 in patients with active HCV infection. Patients refractory to INF and ribavarin and treatment naïve patients

a

30 healthy adults

15 mg every 12 h for 5 days via IV injection

Safety, PK/PD and efficacy of AVI-4557 (based on impact on midazolam metabolism) Safety, PK/PD and efficacy of AVI-4557 (based on impact on midazolam metabolism) Safety, PD/PK and efficacy of AVI-4557 administered orally (based on impact on midazolam metabolism)

Ongoing study.

Subjects

1-, 3-, 10-, 30-, and 90-mg single IV injection

AVI-4020 for West Nile virus neuroinvasive disease

AVI-4557 Safety, PK/PD of AVI-4557; impact on buspirone metabolism (CYP3A4)

AVI-4065

Dosage, Route, etc.

AE

SAE

17

0

6

0

2

0

0

0

0

0

0

0

0a

0a

21

0

0

0

6

0

2

0

3

0

2

0

0

0

10 adult patients

50 patients a

10 patients (9 active; 1 placebo) 1 patient

Single dose of 10, 30, or 90 mg AVI-4557 via SC or IV routes 10 mg oral buspirone 5 daily IV doses of 90 mg 10 mg oral midazolam

96 healthy adults

Single 300-mg IV dose 10 mg oral midazolam

8 healthy adults (6 AVI-4557; 2 placebo)

Day 3 or days 3 and 7: 300 mg AVI-4557 ⫹ deoxycholate in Enterion™ capsule 10 mg midazolam on days 1, 4, and 6 or days 1 and 8

16 healthy adults (2 cohorts; 6 AVI-4557, 2 placebo)

8 healthy adults (6 AVI-4557; 2 placebo)

14 daily SC dose escalation 30 healthy (50, 100, 300 mg); single volunteers dose level for HCV patients 100 mg every 12 h for 12 patients 14 days via SC injections

0a

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these treated volunteers. Furthermore, there is early evidence of efficacy, which is statistically significant at p ⱕ 0.05 that AVI-4126 prevents restenosis of coronary artery stents and P4503A4 PMO modulates drug metabolism that is linked to the corresponding cytochrome system.

20.3 PHARMACOKINETIC PROFILE The lack of iterated charge appears to eliminate non-targeted binding to cellular components other than RNA. PMO–protein binding studies to date indicate affinities of greater than 1 mM (unpublished data). Thus, protein binding would not occur at concentrations utilized in experimental protocols in cell culture or in in vivo efficacy studies. The limited protein binding and weak interactions with cell membranes imply that PMO available for hybridization with RNA will be minimally competed for by nonspecific binding. This difference represents the basis for speculation that the sequence-dependent pharmacokinetics for PMOs is unique relative to the iterated ionic oligomers chemistries [27]. Current studies tend to indicate that in vitro potency determined by the rabbit reticulocyte in vitro translation methods accurately predicts required concentrations for in vivo efficacy (Figure 20.2) and the pharmacokinetic behavior observed in animal models accurately predicts human pharmacokinetic behavior. A database of pharmacokinetic parameters from over 50 different PMOs that have been evaluated in the rat has been compiled. PMOs administered by an extravascular route (intraperitoneal and subcutaneous) are rapidly absorbed into circulation with an absorption half-life between 0.7 and 1 h and time to maximal plasma concentration between 0.5 and 2 h. The distribution is variable with plasma clearance range between 1 and 33 mL/min and the volume of distribution between 0.4 and 56 L/kg. Distribution appears to vary with the sequence composition and existing disease state. The kidney and liver are the primary sites of PMO accumulation and brain; muscle and T-lymphocytes represent the tissues of poorest accumulation. No degradative metabolism has ever been observed and hence no metabolites have ever been recovered. The plasma elimination half-life varies from 1.8 to 15 h but this appears to be an underestimate of the tissue residence time, which tends to range from 7 to 14 days in kidney and liver. Finally, the primary route of excretion is renal with between 15% and 30% of the administered dose in the urine in the first day postadministration. Renal excretion increases with dose. Fecal elimination is less than 2% of an administered dose. Mass balance studies have been conducted with recovery of over 95% of administered dose entirely as unchanged PMO.

Tissue concentration (nM)

600 500

Androgen receptor

400

AVI-4472

300 AVI-4065 200 100 AVI-4126

αV

0 0

100

200

300

400

500

600

700

RRL EC50 (nM) Figure 20.2 In vitro potency predicts in vivo potency. PMO inhibition in rabbit reticulocyte lysate in vitro translation accurately predicts potency in vivo. The potency (EC50) for five different PMOs targeting
V integrin, c-myc (AVI-4126), Cyp3A2 (AVI-4472), HCV (AVI-4065), and the Androgen receptor was determined from in vitro translation indicated on the abscissa, and target tissue concentrations from effective in vivo studies are indicated on the ordinate. The correlation coefficient for in vitro potency versus in vivo efficacy was significant (r ⫽ 0.95) and the slope of the line is significantly different from zero (p ⫽ 0.0078).

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20.4 ANTIVIRAL 20.4.1 Exploration The impetus for AVI BioPharma to seek antiviral exploration is based on our extensive opus of empiric studies that have demonstrated the plausibility that PMOs have a broad application and role in the prevention or treatment of microbial pathogens that are life-threatening or disabling biowarfare pathogens or emerging infectious diseases that would adversely affect general public health, inclusive of NIAID category A, B, and C priority pathogens. We have successfully employed PMOs to inhibit a number of viral infections including replicon and plasmid constructs, infections in cell culture, and infections in animal models (Table 20.2). These studies have been conducted independently by nationally or internationally recognized investigators. The exploration of antisense antiviral PMO agents has now led to successful targeting strategies for all seven viral families with single-stranded positive sense RNA genomes that infect humans (Table 20.3). The positive-sense genome represents the simplest targeting strategy and successful genome targets have included inhibition of viral protein translation, interference with viral replication, and disruption of double-stranded RNA structures including IRES structures. The project has led to

Table 20.3 Antiviral Studies Exploration Viral Family, Virus

Activity

Reference

⫹) Strand Viruses Single-Stranded RNA (⫹ Astroviridae Human Astrovirus Caliciviridae Vesivirus Flaviviridae West Nile virus Dengue Arteriviridae EAV PRRSV Coronaviridae MHV SARS Picornaviridae CVB3 Togaviridae Alphavirus-VEE

Efficacy in cell culture

Manuscript in prep.

In vivo efficacy Efficacy in cell culture

[28] [29] [30] [31]

Efficacy in cell culture [32] [33] Efficacy in cell culture [34] [35] Efficacy in cell culture

Manuscript submitted

Efficacy in cell culture

Manuscript in prep.

⫺) Strand Viruses Single-Stranded RNA (⫺ Arenaviridae Junin Bunyaviridae Rift Valley fever Filoviridae Ebola Zaire Paramyxoviridae measles Rhabdoviridae IHNV Orthomyxoviridae Influenza A

Efficacy in cell culture

Manuscript in prep.

Efficacy in cell culture In vivo efficacy: mouse, guinea pig and nonhuman primate

Manuscript in prep.

Efficacy in cell culture

Manuscript in prep.

Efficacy in cell culture

[38]

In vivo efficacy

[39]

[36] [37]

DNA Viral Genomes Herpesviridae HHV8

Efficacy in cell culture

[40]

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successful inhibition of all six families of single-stranded negative sense RNA genome viruses that infect humans (Table 20.3). The strategies for targeting the negative sense RNA genome viruses have been significantly more complex and less common to all members of the group. The single-stranded viral RNA genome represents an exceptionally mutation-prone system and these viruses are described as a quasispecies due to genome instability. This presents a challenge to a nucleic acid–based therapy but those genome regions of high conservation appear to represent sites of critical function. Viral resistance to the antisense inhibitor through target site mutation was observed but the resultant mutant virus exhibited attenuated viral products in the case of SARS [35], West Nile virus (WNV), and foot and mouth disease virus (data not published). 20.4.2 Rapid Response to Emerging Infectious Disease The PMO technology offers a unique advantage to rapidly respond to one or many identified biowarfare threats. It is clear that PMO technology can readily be applied to emerging infectious disease or newly created bioterrorism threats. For example, an outbreak of WNV infection in Humbolt penguins initiated our WNV program. The therapeutic PMO was designed within two hours of our decision to treat the penguins. The PMOs were synthesized, purified, evaluated for quality, and shipped to the Milwaukee County Zoo within six days. This enabled the zoo to afford significant survival benefit to PMO-treated penguins within 8 days of being notified of this problem. This successful experience led to the treatment of WNV infections in humans with this prototype PMO. An accelerated WNV program was initiated and completed within 6 months to enter Phase 1 testing with a c-GMP WNV PMO among clinically suspected cases of WNV in Colorado. Similarly, the PMO for treating SARS was designed within days of public availability of viral genome sequence. Finally, an emergency response to make Ebola PMOs under an emergency IND was coordinated among AVI, USAMRIID, and FDA because of an accidental Ebola infection at USAMRIID. Within 5 days of the accidental injection exposure, a cocktail of two clinical-grade Ebola PMOs was delivered from AVI facilities in Oregon to USAMRIID in Maryland. Furthermore, due to the inherent safety and tolerability of PMOs, it should be possible to sponsor a program to produce treatment or prevention formulations against a variety of microbes within individual vials. 20.4.3 Clinical Trials Two clinical trials have been initiated for PMO treatment of viral infection. Both involve singlestranded positive-sense RNA genome viruses, WNV, and Hepatitis C virus (HCV). First, AVI-4020 was investigated in WNV patients with neuroinvasive disease. The primary endpoint of the study was safety and due to small numbers of patients, no significant efficacy data have been accumulated. However, headache and rash subsided in most patients. Motion was restored in at least two patients with paralysis at time of entry and rapid, greater than 1 log reduction in white blood cell counts in cerebrospinal fluid was observed within 3 days of initiating treatment. A critical limitation in this study was the difficulty in IRB approval in centers with active WNV infection. Little progress in this trial has been made in the past 2 years due to less active infection, as the spread of WNV came to the west coast of the United States. One explanation has been the effective mosquito abatement programs in western states. A clinical trial with AVI-4065 to treat HCV began late in 2005. The initial trials involved healthy volunteers administered AVI-4065 subcutaneously daily with doses of 50, 100, and 300 mg per day for 14 consecutive days. There were no serious adverse events (Table 20.2), the pharmacokinetic data indicate mean residence time of 7–10 days, renal excretion of unchanged AVI-4065 and the drug was well tolerated. Once these studies were completed, a cohort of active HCV-infected patients was treated with 100 mg administered subcutaneously twice a day for 14 consecutive days. Preliminary observations from AVI-4065 were presented at the 2006 International Congress for Antiviral Research in Puerto Rico. A total of nine patients with chronic active HCV hepatitis had

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received between 4 and 14 days of AVI-4065 at 100 mg twice a day by a subcutaneous route. Among these subjects, there were no clinically significant findings based on physical examinations and conventional laboratory tests (hematology, serum chemistry, coagulation, and urinalysis) to indicate any safety concerns. It should be noted that for all laboratory tests, there were no clinically significant differences between baseline and subsequent days after treatment, up to 28 days for a subset of patients. Furthermore, there have been no drug-related adverse events or serious adverse events, inclusive of site-injection reactions. Among a subset of these patients, five subjects who received at least 14 days of treatment have been monitored to assess preliminary efficacy of AVI-4065. This has been evaluated by comparing the plasma HCV-RNA levels as determined by a branched DNA PCR-based assay on aliquots taken at baseline and sequentially after treatment. Preliminary evaluation suggests AVI-4065 subcutaneous administration at 100 mg twice a day did not lead to a robust reduction of plasma HCV-RNA levels by 14 days of treatment in chronic active HCV hepatitis patients.

20.5 ANTIBACTERIAL In addition to PMO approaches to viral infections, studies have also been initiated to investigate preventing pathology following exposure to various toxins and bacterial pathogens [41]. PMOs have already been made and demonstrated to reduce viable bacterial pathogens in a mouse model by ⬃2 logs within 2 h of administration of a PMO dose. We have recently determined the feasibility of using PMOs to block antibiotic resistance mechanisms. AVI has plans to prevent or treat a number of bacterial diseases and toxin-mediated disease. PMOs with lengths of 9–12 were observed to be effective in inhibiting bacterial gene expression [42]. Longer PMOs will inhibit prokaryotic gene expression in in vitro translation systems but are less effective in intact bacteria, most likely due to penetration of the bacterial cell wall. The short 9–12-mer PMOs are not effective inhibitors of eukaryotic translation either in vitro or in vivo. This factor may add significantly to the potential therapeutic margin of safety of PMO antibiotics. An 11-mer PMO targeting the acyl carrier protein (AcpP) in Escherichia coli was effective in reducing infection in a mouse peritonitis model [43]. The E. coli with a leaky cell wall were more effectively inhibited than strains with intact cell wall, indicating transport across the cell wall as a critical barrier to potential clinical use. A peptide-conjugated PMO was employed to overcome the slow transport across the bacterial cell wall. The peptide conjugates were substantially more potent inhibitors of bacterial gene expression [44]. Multiple gram negative bacteria have now been evaluated successfully. These peptide conjugates represent a feasible approach of development of a PMO antibiotic for antibiotic resistant gram negative bacteria.

20.6 CARDIOVASCULAR Medical therapy of coronary artery disease (CAD) has changed considerably in recent years. It is characterized by expanding use of PCI and continued conversion to minimally invasive percutaneous transluminal coronary angioplasty (PTCA) and stent therapy despite significant advances in pharmacological treatment and implementation of novel surgical techniques in the treatment of CAD [45]. Introduction of stents showed a significant decrease in vessel remodeling and elastic recoil at the site of intervention and clearly demonstrated the superiority of stent implantation over PTCA alone with respect to restenosis in de novo coronary lesions. Extensive use of coronary stents to prevent restenosis has produced a new disease, in-stent restenosis. Unfortunately, this complication continues to be difficult to prevent; regardless of the treatment strategy, the rate of in-stent restenosis (20–60% after bare metal stent implantation) is still unacceptably high, depending on vessel and

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patient bias [46–48,50]. This is particularly true in patients with diabetes and in some lesion sublets, such as bifurcated lesions, long diffuse lesions, and small vessels [50]. However, it was also evident that neointimal proliferation is not affected by the stenting technique [49]. Thus, despite significant advances in the treatment of cardiovascular disease, intimal hyperplasia remains the most common cause of early failure after revascularization. In addition to mechanical procedures, current treatment strategy on intimal hyperplasia includes two main approaches: (i) inhibiting vascular smooth muscle cell (VSMC) proliferation and growth, and stimulating the pathways that lead to VSMC apoptosis, as well as (ii) promoting re-endothelialization and augmenting endothelial functions. New trends toward stent-based drug delivery explored the potential of antiproliferative drugs in treatment and prevention of the intimal hyperplasia. Several completed studies on Sirolimus and Paclitaxel eluting stents showed great capacity of this approach in the prevention and treatment of in-stent restenosis. However, recent advances in vascular gene transfer have shown potential new treatment modalities for cardiovascular disease, particularly in the treatment of vascular restenosis. AVI-4126 is a PMO antisense to c-myc developed to prevent intimal VSMC hyperplasia while sparing endothelial cell function. Initial studies involved New Zealand white, atherosclerotic rabbits maintained on a diet of 0.25% cholesterol. A weeping balloon, transport catheter, delivered approximately 500 g of AVI-4126 into the endoluminal site of PTCA in the internal iliac artery of the rabbit. Table 20.4 shows the 60-day follow-up with significant prevention in late loss of lumen diameter and reduced intimal thickening [51]. A 6-month follow-up of these rabbits confirmed the long-lasting influence of the 30-s delivery of AVI-4126 by significant reduction in intimal thickening [52]. These observations in the rabbit model did not utilize the coronary artery, so subsequent studies investigated the pig restenosis model and coronary artery evaluation. An Infiltrator catheter delivered 1–10 g of AVI-4126 into the PTCA site of pig coronary vessels. Western blot analysis demonstrated a dose-dependent reduction in MYC expression [53]. Further, 28-day follow-up studies showed a significant reduction in the intimal area of the pig coronaries (Table 20.4). The emergence of stent coating technologies led to the examination of this alternative method of AVI-4126 delivery to the PTCA site. A commercially available phosphorylcholine-coated stent (BiodivYsio; Biocompatibles, Surrey, UK) designed to trap drug molecules in the phospholipid layer was utilized to delivery AVI-4126 into pig coronary arteries [54]. These studies show significant protection of lumen diameter and reduction in intimal area (Table 20.4). The polymers from many coated stents appear to obligate long-term use of anticoagulants. Further, the delivery of drug is limited to the site where stents are placed, limiting use to focal lesions and failure due to the systemic nature of acute coronary syndrome. This led to the development of a microbubble delivery system for AVI-4126. The microbubbles are composed of perfluorobutane, which promotes cell penetration of AVI-4126. This noninvasive method of drug delivery

Table 20.4 Summary of in Vivo Restenosis Efficacy Studies with AVI-4126 Intima (mm2)

Late Loss (mm) Study Transport catheter in rabbit iliac Transport catheter in rabbit iliac (6 months) Infiltrator catheter in pig coronary PC-coated stent in pig coronary IV microbubble in pig coronary

Control

AVI-4126

p-value

Control

AVI-4126

p-value Reference

1.8 ⫾ 0.3

0.9 ⫾ 0.2

0.001

1.67 ⫾ 0.44

0.82 ⫾ 0.32

0.002

[51]

–

–

–

1.43 ⫾ 0.25

0.65 ⫾ 0.26

⬍0.05

[52]

–

–

–

3.88 ⫾ 1.04

1.95 ⫾ 0.91

⬍0.001

[53]

1.14 ⫾ 0.16

0.40 ⫾ 0.53

0.04

3.9 ⫾ 0.8

2.3 ⫾ 0.7

0.0077

[54]

–

–

–

3.6 ⫾ 1.4

2.6 ⫾ 1.4

⬍0.05

[55]

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relies on the microbubble retention at sites of vascuolar endothelial dysfunction. Thus, AVI-4126 is delivered on a film at the surface of perfluorobutane gas microbubbles. The microbubble delivery strategy has proven successful in the pig PTCA model [55]. A significant reduction in intimal area was observed in pig coronaries (Table 20.4). The studies have utilized three different delivery technologies each with significant efficacy in the pig coronary model. Phase I studies with AVI-4126 involved simple intravenous and oral routes of administration of 1–90 mg as a single bolus in healthy volunteers. This collection of robust observations and excellent safety profile has led to a phase II study, AVAIL, in which patients with de novo lesions or in stent restenosis were treated with AVI-4126 delivered with Infiltrator catheter [56]. The AVAIL trial was a prospective, evaluator-blinded, randomized study including clinical follow-up at 30 days and 6 months after intervention and 6-month angiographic and IVUS follow-up. Primary endpoints included major adverse cardiac events (MACE), TVR, angiographic restenosis, and IVUS at 6 months. Forty six patients with either de novo lesions or restenosis were randomized into three groups: low dose (3 mg, 15 patients); high dose (10 mg, 15 patients); and control (16 patients). Baseline angiographic characteristics did not differ between the groups (reference vessel diameter: 2.5–4 mm, lesion length ⬍16mm). No MACE was recorded in any group, either in the hospital or within 30 days of treatment. At 6 months, three patients (33.3%) from the control group (n ⫽ 9) and six (46.1%) from the low-dose group (n ⫽ 13) required TVR. In contrast, in the high-dose group (n ⫽ 14) only one patient (7.1%) needed TVR. Angiographic follow-up demonstrated significant reduction in late loss ( p⫽0.025). Binary restenosis was 33.3% in the control group, 33.3% in the low-dose group, and 0.0% in the high-dose group. These preliminary findings from the small cohort of patients require confirmation in a larger trial utilizing more sophisticated drug eluting technologies.

20.7 CANCER Cancer therapy remains an area of unmet medical need. The development of anticancer agents is enticing due to the likelihood of low risk and great potential benefit. However, development is difficult due to lengthy and costly clinical trials in patients with advanced disease. An antisense therapeutic agent should have outstanding antitumor properties to move from preclinical evaluation into clinical development. The PMO AVI-4126 was found to have poor to moderate activity in a lung tumor model but was very good when used in combination with cisplatin [57]. The c-myc target appeared to be significantly more active than both RAD 51 and p21 in the Lewis lung tumor model (Table 20.5). AVI-4126 was found to be more active in a prostate tumor model [58], which provided the rationale Table 20.5 Summary of PMO Antisense Anticancer Efficacy Studies mRNA Target

Tumor Type

Model

Observation

c-myc

Lung

LLC1 Lewis lung syngeneic tumor in C57BL/6 mice

o MYC protein (Western blot) o tumor growth in

c-myc

Prostate

o tumor volume

hCG

Prostate

MMP-9

Prostate

SNAIL

Colorectal

PC-3 xenograft in nu/nu mouse DU145 xenograft in nu/nu mouse DU145 xenografts in athymic male mouse MIN mouse model

XIAP

Prostate

DU145 cell culture

Androgen Rec

Prostate

LAPC-4 xenograft in nu/nu mouse

combo w/ cisplatin Combo of hCG ⫹ c-myc show antitumor synergy m Tumor growth

o SNAIL protein, m E-cadherin exp. o tumor number and incidence o XIAP Expression, o cell viability o hAR protein, o PSA expression

Reference

[57] [58] [59] [60] [61] [62] [63]

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for pilot clinical studies. Combination of AVI-4126 with another PMO was investigated [59]. Antitumor synergy was observed when both AVI-4126 and a PMO targeting human chorionic gonadotropin were administered together (Table 20.5). A combination therapy approach to antisense for cancer chemotherapy is feasible given the outstanding safety data for PMOs in tumor models. Multiple targets have been evaluated to investigate antitumor strategies. An antisense PMO targeting MMP-9 was shown to inhibit protein expression, reduce tumor cell invasion and increase tumor cell apoptosis [60]. However, tumor mass in a xenograft model failed to demonstrate meaningful reduction in tumor mass. A transcriptional repressor, SNAIL, was targeted in the MIN mouse model and a significant antitumor effect was observed [61]. These data are particularly meaningful given the spontaneous tumor model and the intact immune system of the mouse. An anti-apoptitic gene, XIAP, was targeted in prostate cancer cells (Table 20.5) with antisense-linked reduction in protein correlating with reduced cell viability [62]. While encouraging, these observations were not considered sufficiently robust for further development. Finally, targeting the androgen receptor (AR) in prostate cancer cells has generated significant observations [63]. A LAPC-4 xenograft mouse model showed decreased AR protein expression as well as signal transduction, leading to diminished PSA expression. This may represent a feasible anticancer strategy as the patient population of hormone refractory tumor with low PSA would be well suited to the treatment strategy, but this would be a lengthy clinical trial. A pilot clinical trial to evaluate tumor accumulation of AVI-4126 established the feasibility of using PMO in human cancer trials [64]. These studies investigated a single 90-mg intravenous dose of AVI-4126 in patients with breast and prostate cancer. The AVI-4126 was administered one day prior to surgical removal of tumors. The excised tumor was evaluated for AVI-4126 concentrations, which were between 50 and 100 nM, near the 110 nM EC50 for AVI-4126. 20.8 METABOLIC REDIRECTION The cytochrome P450 (CYP) enzymes are heme-containing proteins involved in oxidative metabolism of thousands of compounds. Twelve CYP families have been identified in human beings, with the CYP3A4 being involved in the biotransformation of a majority of all drugs. The most abundant CYP in the human liver is the CYP3A family with specific content of 96 ⫾ 51 nmol/mg microsomal protein, which represents 40% of the total liver CYP content. CYP3A4 is expressed at significant levels extrahepatically with extensive activity in the gastrointestinal tract. This is a significant factor contributing to the poor oral bioavailability of many drugs [65]. The CYP enzymes display relatively low substrate specificity such that two or more individual enzymes often catalyze a given biotransformation reaction. Further, a given cell may express more than one CYP enzyme. Finally, the human CYP3A5 and CYP3A7 enzymes are known, but CYP3A5 is expressed in the liver of 10–30% of individuals and CYP3A7 is expressed in the endometrium and placenta but is not observed in the liver after birth. The CYP3A family metabolizes a number of clinically important drugs such as midazolam, nifedipine, erythromycin, tamoxefin, cyclosporine, phenytoin, digoxin, and 17 -ethylestradiol. Enormous interindividual variation in enzyme content and activity has been reported in the liver, 10- to 20-fold, and intestines, 10- to 49-fold. These differences are responsible for variations in efficacy and disposition of a variety of drugs. The potential to inhibit CYP3A expression is expected to result in a more narrow range of metabolic capacity for individuals. Midazolam metabolism is considered a specific in vivo pharmacological marker of CYP3A enzymatic activity. Early antisense studies with phosphorothioate oligodeoxynucleotides (PSO) demonstrated that a dose of 5 mg/kg administered once a day for 2 days will increase midazolaminduced sleep time in rats from 22 ⫾ 0.4 min to 35 ⫾ 1.5 min (Figure 20.3). This was accompanied by a reduction in CYP3A protein in the liver measured by western blot and reduction in erythromycin demethylase activity in the liver from 124 ⫾ 13 mol formaldehyde/mg microsomal

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION PSO EC50 = 4.2 mg/kg PMO EC50 =1.6 mg/kg

Percent inhibition

75

50

25

0 -0.50

-0.25

0.00

0.25

0.50

0.75

Dose (mg/kg)

Dose (mg/kg)

Route

Substrate

Percent Inhibition

Reference

5.0 2.5 2.5 2.0 1.5

IP IP IP PO TD

Midazolam Paclitaxel Tamoxefin Erythromycin 7-BC

29 62 59 69 51

[66] [67] [68] [69] [70]

Figure 20.3 In vivo inhibition of rat CYP3A2. Inhibition of cytochrome P450 3A2 in a rat model. The compounds were administered by several routes of administration: IP is intraperitoneal; PO, oral; and TD, transdermal. Different substrates were evaluated as endpoints, 7-BC is 7-benzyloxy-4-(trifluoromethyl)coumarin. PSO indicates a phosphorothioate oligodeoxynucleotide; PMO indicates a phosphorodiamidate morpholino oligomer.

protein/min to 64 ⫾ 8, a reduction of 52% [66]. The potency (EC50) of the uniform phosphorothioate olidodeoxynucleotide was 4.6 mg/kg as shown in Figure 20.3. The PMO chemistry represents a significant difference in mechanism of action and molecular properties from these earlier efforts with the PSO chemistry. A PMO, AVI-4472, targeting the rat CYP3A2 enzyme was effective in inhibiting the metabolism of paclitaxel with a single intravenous dose of 2.5 mg/kg resulting in extended half-life, 12 ⫾ 1 min in control versus 32 ⫾ 2 min in the AVI-4472-treated group (Figure 20.2). Significant changes were also observed in reduced plasma clearance, 8 ⫾ 2 versus 2 ⫾ 0 mL/min, and enhanced plasma AUC, 266 ⫾ 48 versus 900 ⫾ 36 g min/mL [67]. More recently, a dose of 2.5 mg/kg/day of AVI-4472 administered intraperitoneally for 8 days was employed to inhibit the rat CYP3A2 and reduce tamoxefin-related DNA adducts from 5.06 to 2.08 TAM-DNA adducts per 108 nucleotides [68]. AVI-4472 was also found to be effective after transdermal delivery with 0.3 mg (1.5 mg/kg) AVI-4472 applied topically to a 2-cm2 area of skin [69]. AVI-4472 was also effective following oral administration of 2.0 mg/kg to rats [70]. These observations were compiled in a single dose-response curve shown in Figure 20.2 and indicate the EC50 for the PMO is 1.6 mg/kg. The human ortholgous PMO, AVI-4557, targets CYP3A4 and confirmed activity in primary human hepatocytes from 11 donors and in Caco-2 cells stably transfected with CYP3A4 cDNA [71].

20.9 ALTERED RNA SPLICING FOR DUCHENNE MUSCULAR DYSTROPHY (DMD) Duchenne and Becker muscular dystrophies are allelic disorders arising from mutations in the dystrophin gene. Disease-causing mutations in the dystrophin gene can be eliminated by removal of exons bearing non-sense mutations or exons that flank frame-shifting deletions to produce an in-frame

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transcript [72]. Reverse transcriptase polymerase chain reaction (RT-PCR), analysis of transcripts from RNA prepared from mdx mouse tibialis anterior muscle after a single injection of 5 g of 2⬘-Omethylphosphorothioate, PMO, and peptide nucleic acid oligomers showed effective skipping of exon 21 in only the PMO treatment group [73]. Studies have been extended with PMO to restore dystrophin expression in the golden retriever muscular dystrophy (GRMD) animal model [74] and normal and DMD human tissue [75]. Recently, PMO-induced skipping of multiple exons was accomplished in the mdx mouse [76]. Maximal in vivo efficacy is observed following 2–10 g dose injected directly into the muscle 4 weeks postinjection and robust exon-skipped dystrophin expression was observed 8 weeks postinjection. These observations indicate that PMO-induced exon skipping is effective in multiple species and that prolonged dystrophin expression can be accomplished. Plans are currently in progress to initiate human clinical trials. 20.10 FORMULATIONS Enhanced potency can be achieved through the creation of a PMO covalently conjugated to arginine-rich peptides at the 5⬘ end, which was determined in an in vitro translation assay [77]. This is likely to be due to ionic interactions between the net positive charge of arginine and the net negative charge in the target complementary RNA. This has been verified through the use of ornithine and lysine peptide conjugates. The addition of a few positive charges also influences the interactions between the PMO and cells by enhanced adsorption to the cell surface. The enhanced adsorption leads to greater cellular internalization of the PMO [78,79]. Recently, we have determined that these conjugated PMOs are different with respect to tissue distribution, more toxic than unmodified PMOs, and have altered pharmacokinetic parameters including enhanced secretion in the renal tubule. Hence, the benefit of added potency and potential for improved delivery to tissue must be balanced with the potential for toxicity. 20.11 SUMMARY The PMO chemistry is an outstanding platform for the development of gene-specific therapeutics. The mechanism of action allows a small and focused search for active agents. The stability, lack of net charge, and resistance to metabolic degradation simplify the process of drug development. A growing database of safety data indicates that the PMO can be used in settings where risk must be minimal. The observations reported here indicate that PMOs are effective in a variety of animal models and humans. They can be administered via most routes of administration with favorable pharmacokinetic properties. The antiviral uses have exploited virtually all single-strand RNA viral families capable of producing pathology. The antiviral capabilities continue to unfold as activity against DNA viral genomes has been reported. The PMOs represent a potential new class of antibiotic compound with outstanding safety characteristics and excellent in vivo efficacy. Studies in areas of cardiovascular, cancer, metabolism, and neuromuscular diseases confirm the broad utility of the PMO antisense platform. ACKNOWLEDGMENTS I would like to thank a large number of collaborators including P-Y Shi, Sina Bavari, Kelly Warfield, Ben Neuman, Mike Buchmeier, Richard Kinney, Eva Harris, Eric Snijder, E van den Born, Yanjin Zhang, David Matson, Al Smith, Jonzhu Chen, Elke Muhlberger, Nick Kipshidze, Marty Leon, Jeff Moses, Tom Porter, Vic Arora, Hemant Roy, Rhonda Brand, and Gayathri Devi. I would also like to thank Dave Stein, Hong Moulton, Bruce Geller, Luke Tilley, Janet Christensen, Peter O’Hanley, and Dwight Weller for their contributions to this chapter. Finally, I would like to express thanks to Stan Crooke for the careful review of this chapter.

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REFERENCES 1. Giles RV, Spiller DG, and Tidd DM (1993) Chimeric oligodeoxynucleotide analogues: Enhanced cell uptake of structures which direct RNase H with high specificity. Anti-Cancer Drug Des. 8: 3–51. 2. Stein D, Foster E, Huang SB, Weller D, and Summerton J (1997) A specificity comparison of four antisense types: morpholino, 2⬘-O-methyl RNA, DNA, and phosphorothioate DNA. Antisense Nucleic Acid Drug Dev. 7: 151–157. 3. Burgess TL, Fisher EF, Ross SL, Bready JV, Qian YX, Bayewitch LA, Cohen AM, Herrera CJ, Kramer TB, Lott FD, Martin FH, Pierce GF, Simoner L, and Farrell CL (1993) The antiproliferative activity of c-myb and c-myc antisense oligonucleotides is smooth muscle cells iscaused by a nonantisense mechanism. Proc. Natl. Acad. Sci. USA 92: 4051– 4055. 4. Hudziak RM, Summerton J, Weller, DD, and Iversen PL (2000) Antiproliferative effects of steric blocking phosphorodiamidate morpholino antisense agents directed against c-myc. Antisense Nucleic Acid Drug Dev. 10: 163–176. 5. Copple BL, Gmeiner WM, and Iversen PL (1995) Reaction between metabolically activated acetaminophen and phosphorothioate oligonucleotides. Toxicol. Appl. Pharmacol. 133: 53–63. 6. Cornish KG, Iversen PL, Smith LJ, Arneson MA, and Bayever E (1993) Cardiovascular effects of a phosphorothioate oligonucleotide with sequence antisense to p53 in the conscious rhesus monkey. Pharmacol. Commun. 3: 239–247. 7. Iversen PL, Cornish KG, Iversen LJ, Mata JE, and Bylund DB (1999) Bolus intravenous injection of phosphorothioate oligonucleotides causes severe hypotension by acting as ␣1-adrenergic receptor antagonists. Toxicol. Appl. Pharmacol. 160: 289–296. 8. Mata JE, Bishop MR, Tarantolo SR, Angle CR, Swanson SA, and Iversen PL (1999) Evidence of enhanced iron excretion during systemic phosphorothioate oligodeoxynucleotide treatment. J. Toxicol. Clin. Toxicol. 38(4): 383–387. 9. Farman CA and Kornbrust DJ (2003) Oligodeoxynucleotide studies in primates: antisense and immune stimulatory indications. Toxicol. Pathol. (Jan–Feb) 31(suppl.): 119–122. 10. Webb MS, Tortora N, Cremese M, Kozlowska H, Blaquiere M, Devine DV, and Kornbrust DJ (2001) Toxicity and toxicokinetics of a phosphorothioate oligonucleotide against the c-myc oncogene in cynomolgus monkeys. Antisense Nucleic Acid Drug Dev. (Jun) 11(3): 155–163. 11. Levin AA (1999) A review of the issues in the pharmacokinetics and toxicology of phosphorothioate antisense oligonucleotides. Biochim. Biophys. Acta 1489(1): 69–84. 12. Henry SP, Novotny W, Leeds J, Auletta C, and Kornbrust DJ (1997) Inhibition of coagulation by a phosphorothioate oligonucleotide. Antisense Nucleic Acid Drug Dev. (Oct) 7(5): 503–510. 13. Stein CA and Cheng YC (1993) Antisene oligonucleotides as therapeutic agents – Is the bullet really magical? Science 261: 1004–1012. 14. Stein CA (1995) Does antisense exist? Nat. Med. 1: 1119–1121. 15. Shoeman RL, Hartig SB, Huang Y, Grub S, and Traub P (1997) Fluorescence microscopic comparison of the binding of phosphodiester and phosphorothioate (antisense) oligodeoxyribonucleotides to subcellular structures, including intermediate filaments, the endoplasmic reticulum, and the nuclear interior. Antisense Nucleic Acid Drug Dev. 7: 291–298. 16. Hudziak RM, Barofsky E, Barofsky DF, Weller DL, Huang SB, and Weller DD (1996) Resistance of Morpholino phosphorodiamidate oligomers to enzymatic degradation. Antisense Nucleic Acids Drug Dev. 6: 267–272. 17. Ricker JL, Mata JE, Iversen PL, and Gattone VH (2002) c-myc Antisense oligonucleotide treatment ameliorates murine infantile polycystic kidney disease. Kidney Int. 61: S125–S131. 18. Arora V, Knapp DC, Smith BL, Statdfield ML, Stein DA, Reddy MT, Weller DD, and Iversen PL (2000) c-Myc antisense limits rat liver regeneration and indicates role for c-myc in regulating cytochrome P-450 3A activity. J. Pharmacol. Exp. Ther. 292: 921–928. 19. Kipshidze N, Keane E, Stein D, Chawla P, Skrinska V, Shankar LR, Khanna A, Komorowski R, Haudenschild C, Iversen P, Leon M, Keelan MH, and Moses J (2001) Local delivery of c-myc neutrally charged antisense oligonucleotides with transport catheter inhibits myointimal hyperplasia and positively affects vascular remodeling in the rabbit balloon injury model. Catheterization Cardiovasc. Interventions 54: 247–256.

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20. Kipshidze NN, Kim H-S, Iversen PL, Yazdi HA, Bhargava B, Mehran R, Haundenschild C, Dangas G, Stone GW, Roubin GS, Leon MB, and Moses JW (2002) Intramural delivery of advanced antisense oligonucleotides with infiltrator cathetor inhibits c-myc expression and intimal hyperplasia in the porcine. J. Am. Coll. Cardiol. 39(10): 1686–1691. 21. London CA, Sekhon HS, Arora V, Stein DA, Iversen PL, and Devi GR (2003) A novel antisense inhibitor of MMP-9 attenuates angiogenesis, human prostate cancer cell cinvasion and tumorigenicity. Cancer Gene Ther. 10: 823–832. 22. Arora V, Hannah TL, Iversen PL, and Brand RM (2002) Transdermal use of phosphorodiamidate morpholino oligomer AVI-4472 inhibits cytochrome P450 3A2 activity in male rats. Pharmaceut. Res. 19(10): 1465–1470. 23. Qin G, Taylor M, Ning YY, Iversen P, and Kobzik L (2000) In vivo evaluation of a morpholino antisense oligomer directed against TNF. Antisense Nucleic Acid Drug Dev. 10: 11–16. 24. Arora V, Knapp DC, Reddy MT, Weller DD, and Iversen PL (2002) Phosphorodiamidate morpholino oligomers demonstrate antisense activity in rat liver following oral administration. J. Pharm. Sci. 91(4): 1009–1018. 25. Ghosh C, Reddy M, and Iversen PL (2003) Analysis of antisense phosphorodiamidate morphino oligomer teratogenicity in the zebrafish embryogenesis model. Anal. Pharmacol. 1(3): 80–89. 26. Iversen PL and Newbury S (2005) Manipulation of zebrafish embryogenesis by phosphorodiamidate morpholino oligomers indicates minimal non-specific teratogenesis. Curr. Opin. Mol. Ther. 7(2): 104–108. 27. Arora V, Devi GR, and Iversen PL (2004) Neutrally charged phosphorodiamidate morpholino oligomers: uptake, efficacy and pharmacokinetics. Curr. Pharm. Biotech. 5(5): 431–439. 28. Stein D, Skilling D, Iversen PL, and Smith AO (2001) Inhibition of Vesivirus infections in mammalian tissue culture with antisense morpholino oligomers. Antisense Nucleic Acid Drug Dev. 11: 317–325. 29. Deas TS, Binduga-Gajewska I, Tilgner M, Ren P, Stein DA, Moulton HM, Iversen PL, Kauffman EB, Kramer LD, and Shi P-Y (2005) Inhibition of Flavivirus infections by antisense oligomers specifically suppressing viral translation and RNA replication. J. Virol. 79(8): 4599–4609. 30. Kinney RM, Huang CY-H, Rose BC, Kroeker AD, Dreher TW, Iversen PL, and Stein DA (2005) Inhibition of dengue virus serotypes 1 to 4 in cell culture with morpholino oligomers. J. Virol. 79(8): 5116–5128. 31. Holden KL, Stein DA, Pierson TC, Ahmed AA, Clyde K, Iversen PL, and Harris E (2005) Inhibition of dengue virus translation and RNA synthesis by a morpholino oligomer targeted to the terminal 3⬘ stem-loop structure. Virology 344: 439–452. 32. van den Born E, Stein DA, Iversen PL, and Snijder EJ (2005) Antiviral activity of morpholino oligomers designed to block various aspects of Equine arteritis virus amplification in cell culture. J. Gen. Virol. 86(11): 3081–3090. 33. Zhang YJ, Stein DA, Fan SM, Wang KY, Kroeker AD, Meng XJ, Iversen PL, and Matson DO (2006) Suppression of porcine reproductive and respiratory syndrome virus replication by morpholino antisense oligomers. Vet. Microbiol. (in press). 34. Neuman BW, Stein DA, Kroeker AD, Paulino AD, Moulton HM, Iversen PL, and Buchmeier MJ (2004) Antisense morpholino oligomers directed against the 5⬘-end of the genome inhibit coronavirus proliferation and growth. J. Virol. 78(11): 5891–5899. 35. Neuman BW, Stein DA, Kroeker AD, Churchill MJ, Kim AM, Dawson P, Moulton HM, Bestwick RK, Iversen PL, and Buchmeier MJ (2005) Inhibition, escape and attenuation of SARS coronavirus treated with antisense morpholino oligomers. J. Virol. 79: 9665–9676. 36. Warfield KL, Swenson DL, Olinger GG, Nichols DK, Pratt WD, Blouch R, Stein DA, Aman MJ, Iversen PL, and Bavari S (2006) Gene-specific countermeasures against Ebola virus based on antisense phosphorodiamidate morpholino oligomers. PLoS Pathogens 2(1): 1–9. 37. Enterlein S, Warfield KL, Swenson DL, Stein DA, Smith JL, Gamble CS, Kroeker AD, Iversen PL, Bavari S, and Muhlberger K (2006) VP35 knockdown inhibits Ebola virus amplification and protects against lethal infection in mice. Antimicrob. Agents Chemother. 50(3): 984–993. 38. Alonso M, Stein DA, Thomann E, Moulton H, Leong J-AC, Iversen P, and Mourich D (2005) Inhibition of infectious hematopoietic necrosis virus in cell cultures with peptide-conjugated morpholino oligomers J. Fish Disease 28: 399–410.

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39. Ge Q, Kroeker AD, Bestwick RK, Iversen PL, Chen J, and Stein D (2006) Inhibition of influenza A virus in Vero cell culture with morpholino oligomers. J. Virol. (in press). 40. Zhang YJ, Wang KY, Stein DA, Patel D, Watkins R, Moulton HM, Iversen PL, and Matson DO (2006) Inhibition of replication and transcription activator and latency-associated nuclear antigen of Kaposi’s sarcoma-associated herpesvirus by morpholino oligomers. Antivir. Res. 73: 12–23. 41. Geller BL, Deere JD, Stein DA, Kroeker AD, Moulton HM, and Iversen PL (2003) Inhibition of gene expression in Escherichia coli by antisense phosphorodiamidate morpholino oligomers. Antimicrob. Agents Chemother. 47: 3233–3239. 42. Deere J, Iversen P, and Geller BL (2005) Antisense phosphorodiamidate morpholino oligomer length and target position effects on gene-specific inhibition in Escherichia coli. Antimicrob. Agents Chemother. 49: 249–255. 43. Geller BL, Deere J, Tilley L, and Iversen PL (2005) Antisense phosphorodiamidate morpholino oligomer inhibits viability of Escherichia coli in pure culture and in mouse peritonitis. J. Antimicrob. Chemother. 10: 1093. 44. Tilley LD, Hine OS, Kellogg JA, Hassinger JN, Weller DD, Iversen PL, and Geller BL (2006) Genespecific effects of antisense phosphorodiamidate morpholino oligomer-peptide conjugates on Escherichia coli and Salmonella enterica Serovar Typhimurium in pure culture and in tissue culture. Antimicrob. Agents Chemother. 50(8): 2789–2796. 45. Simonsen M (2003) Changing role for cardiac surgery as use of stents continues growth. Cardiovasc. Device Update 9: 1–7. 46. Topol EJ and Serruys PW (1998) Frontiers in interventional cardiology. Circulation 98: 1802–1820. 47. Serruys PW, Foley DP, Suttorp M-J, Rensing BJ, Suryapranta H, Materne P, van den Bos A, Benit E, Anzuini A, Rutsch W, Legrand V, Dawkins K, Cobaugh M, Bressers M, Backx B, Wijns W, and Colombo A (2002) A randomized comparison of the value of additional stenting after optimal balloon angioplasty for long coronary lesions. J. Am. Coll. Cardiol. 39: 393–399. 48. van den Brand M, Rensing J, Morel MM, Foley DP, de Valk V, Breeman A, Suryapranata H, Haalebos MM, Wijns W, Wellens F, Balcon R, Magee P, Rigeiro E, Buffolo E, Unger F, and Serruys PW (2002) The effect of completeness of revascularization on event-free survival at one year in the ARTS trial. J. Am. Coll. Cardiol. 39: 559–564. 49. Nakatani M, Takeyama Y, Shibata M, Yorozuya M, Suzuki M, Koba S, and Katagirl T (2003) Mechanisms of restenosis after coronary intervention. Difference between plain old balloon angioplasty and stenting. Cardiovasc. Pathol. 12: 40–48. 50. Goldberg SL, Loussararian A, De Gregorio J, Di Mario C, Albierro R, Colombo A (2001) Predictors of diffuse and aggressive intrastent restenosis. J. Am. Coll. Cardiol. 37: 1019–1025 51. Kipshidze N, Keane E, Stein D, Chawla P, Skrinska V, Shankar LR, Khanna A, Komorowski R, Haudenschild C, Iversen P, Leon M, Keelan MH, and Moses J (2001) Local delivery of c-myc neutrally charged antisense oligonucleotides with transport catheter inhibits myointimal hyperplasia and positively affects vascular remodeling in the rabbit balloon injury model. Catheter. Cardiovasc. Interventions 54: 247–256. 52. Kipshidze N, Iversen P, Keane E, Stein D, Chawla P, Skrinska V, Shankar LR, Mehran R, Chekanov V, Dangas G, Komorowski R, Haudenschild C, Khanna A, Leon M, Keelan MH, and Moses J (2002) Complete vascular healing and sustained suppression of neointimal thickening after local delivery of advanced c-myc antisense at six months follow-up in a rabbit balloon injury model. Cardiovasc. Radiation Med. 3: 26–30. 53. Kipshidze NN, Kim H-S, Iversen PL, Yazdi HA, Bhargava B, Mehran R, Haundenschild C, Dangas G, Stone GW, Roubin GS, Leon MB, and Moses JW (2002) Intramural delivery of advanced antisense oligonucleotides with infiltrator cathetor inhibits c-myc expression and intimal hyperplasia in the porcine. J. Am. Coll. Cardiol. 39(10): 1686–1691. 54. Kipshidze NN, Iversen PL, Kim H-S, Yazdi HA, Dangas G, Seaborn R, New G, Tio FB, Waxman R, Mehran R, Tsapenko M, Stone GW, Roubin GS, Iyer S, Leon MB, and Moses JW (2004) Advanced c-myc antisense (AVI-4126)-eluting phosphrylcholine-coated stent implantation is associated with complete vascular healing and reduced neointimal formation in the porcine coronary restenosis model. Catheter. Cardiovasc. Interventions 61: 518–527. 55. Porter TR, Xie F, Knapp D, Iversen P, Markey LA, Tsutsui JM, Maiti S, Lof J, Radio SJ, and Kipshidze N (2006) Targeted vascular delivery of antisense molecules using intravenous microbubbles. Cardiovasc. Revasc. Med. 7(1): 25–33.

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77. Nelson MH, Stein DA, Kroeker AD, Hatlevig SA, Iversen PL, and Moulton HM (2005) Arginine-rich peptide conjugation of morpholino oligomers: effects on antisense activity and specificity. Bioconjugate Chem. 16: 959–966. 78. Moulton, HM, Hase HC, Smith KH, and Iversen PL (2003) HIV Tat peptide enhances cellular delivery of antisense morpholino oligomers. Antisense Nucleic Acid Drug Dev. 13: 32–37. 79. Moulton HM, Nelson MH, Hatlevig SA, Reddy MT, and Iversen PL (2004) Cellular uptake of antisense morpholino oligomers conjugated to arginine-rich peptides. BioConjugate Chem. 15(2): 290–299.

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Potential Therapeutic Applications of Antisense Oligonucleotides in Ophthalmology Lisa R. Grillone and Scott P. Henry

CONTENTS 21.1 Background ..........................................................................................................................585 21.1.1 Classes of Therapeutic Oligonucleotides ...............................................................586 21.1.2 Pharmacokinetics ...................................................................................................587 21.1.3 Tolerability .............................................................................................................588 21.1.4 Pharmacodynamics ................................................................................................589 21.2 Ocular Therapeutic Areas .....................................................................................................591 21.2.1 Antivirals ...............................................................................................................591 21.2.2 Angiogenesis (Neovascular Age-Related Macular Degeneration and Diabetic Retinopathy) .....................................................................................592 21.2.3 Intraocular Inflammation .......................................................................................593 21.2.4 Glaucoma ...............................................................................................................594 21.3 Drug Delivery Options ........................................................................................................595 21.3.1 Formulations (Liposomes, Nanosized Particles) ...................................................595 21.4 Conclusions...........................................................................................................................595 References .....................................................................................................................................596

21.1 BACKGROUND In the past several decades tremendous strides have been made toward understanding the molecular basis of various ocular diseases. Modern day ocular pharmacology provides the basis for utilization of rational drug design targeted toward molecular mechanisms in a plethora of ocular conditions with the ultimate goal of developing effective therapeutics [1]. The application of antisense oligonucleotide (ASO) therapeutics for the treatment of ocular diseases and conditions has tremendous opportunity to add treatment option in ophthalmology. This class of pharmacologic agent has several advantages that exemplify rational drug design principles. There is the potential to target a vast array of genes that specifically inhibit cellular processes. The use of ASOs to treat ocular diseases is limited only by our knowledge of the underlying molecular pharmacology. With advancements in the understanding of the molecular basis for ocular diseases, spawned by new

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animal and cell-culture models that mimic specific diseases, oligonucleotide therapeutics are well positioned to take advantage of new opportunities in ophthalmology and to address unmet needs. Initial reviews of the state of phosphorothioate oligodeoxynucleotides (PS ODNs), ASOs as ocular therapeutics noted the opportunity for treatments of serious and sight-threatening diseases such as diabetic retinopathy, macular degeneration, glaucoma, uveitis, and retinitis pigmentosa [2,3]. Since that time, research in oligonucleotide therapeutics has expanded and now includes small interfering RNA (siRNA) inhibitors and oligonucleotide aptamers (Chapter 1). The culmination of the last 10 years of research in this area has resulted in the approval of two therapeutic agents for the treatment of ocular conditions, an antisense oligonucleotide and an aptamer [4–6]. The opportunity for the development of ASOs in the treatment of ocular conditions is fortified by the current interest in ophthalmology to treat many “back of the eye” conditions previously untreatable with any therapeutic agent. Although ocular conditions are frequently manifestations of systemic diseases and, as such, may best be treated with systemic administration of therapeutic agents, there are advantages to local administration directly to the eye with a specific tissue as the target (e.g., by intravitreous administration). Nevertheless, there remain hurdles to the use of ASOs to treat ocular diseases. This review will summarize the state of the art in the use of oligonucleotides in ophthalmology, the pharmacokinetics, local tolerability, and will summarize the various classes of oligonucleotide agents along with the fundamental properties that make them good drug candidates. Lastly, a look to the future and the opportunities for targeting a variety of ocular diseases will be presented. 21.1.1 Classes of Therapeutic Oligonucleotides Oligonucleotide therapeutic agents fall into two broad categories. The first includes oligonucleotides that work through an antisense mechanism to inhibit the expression of a specific protein. The inhibition of the production of a targeted protein involved in a disease process results from the hybridization of an oligonucleotide to a single-stranded mRNA molecule and activation of enzyme systems that subsequently degrade the mRNA, inhibit the translation process, or modulate RNA processing [7–10]. The single-stranded ASOs utilize the RNase H enzyme mechanism and are further divided into categories based on chemical modification as described below. To date this first broad category is represented by fomivirsen sodium (Vitravene, Isis Pharmaceuticals, Inc.) approved in the United States and Europe for the treatment of cytomegalovirus (CMV) retinitis in patients with AIDS [4,11–13]. The development of the first antisense oligonucleotide to be approved is covered in more detail in the section of this chapter on antivirals. Also included in this category of antisense oligonucleotides are siRNA inhibitors that function through the RISC-complex mechanism [5]. The second broad category is a class referred to as oligonucleotide aptamers which function through the selection of an oligonucleotide that will interact with a specific soluble protein or cell-surface receptor rather than through hybridization with mRNA. In this case the interaction between aptamer and target is dependent on the conformational structure of the oligonucleotide [14]. The only approved drug in this category is pegaptanib (Macugen, Pfizer, Inc.), a pharmacologic agent for the treatment of neovascular age-related macular degeneration (AMD) [15–17]. Pegaptanib is a 28-base ribonucleic acid aptamer covalently bound to polyethylene glycol, that blocks the activity of the vascular endothelial growth factor (VEGF), specifically VEGF165 [6]. The approval of pegaptanib, ⬃6 years after the approval of fomivirsen, supports the use of oligonucleotides in the treatment of retinal conditions through an intravitreous route of administration. While there are other aptamers with the potential for use as ocular therapeutics, there are not many examples in this category in the published literature. Therefore, this review will focus on the application of antisense inhibitors. The bulk of the experience with antisense inhibitors has been single-stranded agents, specifically PS ODN (Table 21.1). Fomivirsen sodium approved for the treatment of CMV retinitis is representative of this class of compound. Since the time of the approval of fomivirsen, additional

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Table 21.1 Structure of Typical PS ODN and 2⬘⬘-MOE ASO Compound

Molecular Target

ISIS 2922 (Fomivirsen) ISIS 13312

Human CMV Human CMV

Sequence (5⬘⬘→ 3⬘⬘) GsCsGsTsTsTsGsCsTsCsTsTsCsTsTsCsTsTsGsCsG GsC*sGsTsTsTsGsC*sTsC*sTsTsC*sTsTsC*sTsTsGsC*sG

Notes: The inhibitors of human CMV (Fomivirsen and ISIS 13312) are presented as examples of the class. C, deoxynucleotide; C*, 5 methyl cytosine; T, 2⬘-methoxyethyl; s, phosphorothioate linkage.

chemical modifications to PS ODN have greatly improved their potency, pharmacokinetic properties, and tolerability profile (Chapter 1). Modified oligonucleotides of this type contain phosphorothioate linkages, but also contain alkoxy substituents on the 2⬘-position of ribose. The most common substituents are either methoxyethoxy (i.e., 2⬘-MOE ASO), and are typically placed on several terminal residues on the 3⬘ and 5⬘ ends of the oligonucleotide, leaving the center residues as deoxyribose sugars (Table 21.1). The modified oligonucleotides that have progressed the most toward therapeutic application are the 2⬘-MOE ASO. Incorporation of the 2⬘-MOE modifications resulted in an increased hybridization affinity and a concomitant increase in potency [18]. In addition, the 2⬘-MOE substituents provide another improvement to these molecules, that is, increased resistance of phosphorothioate linkages to nuclease cleavage [19]. Since the primary route of clearance for PS ODN is exonuclease-mediated degradation, these modifications effectively extend the residence time of oligonucleotides in tissue as described below. The chemical modifications that result in increased residence time in tissue are significant in that there is a clinical benefit to less frequent treatment administration. The final change imparted by the 2⬘-MOE substituents, in combination with 5-methyl cytosine substitution, is to improve the tolerability profile by decreasing the proinflammatory effects, resulting in less toxicity. The consequence of these changes is demonstrated in the ocular pharmacokinetic and tolerability properties discussed below. The siRNA molecules used to date are primarily the typical double-stranded DNA inhibitors that are not stabilized by phosphorothioate linkages or are partially stabilized with the mixture of phosphorothioate and 2⬘-alkoxy modifications, such as 2⬘-methoxy. The successful in vivo application of these inhibitors has been limited to date, because of issues with metabolic stability and cell uptake. Most in vivo applications have used some sort of formulation to protect and facilitate cellular uptake [20]. However, the activity of these siRNA inhibitors in vitro is impressive and local application to the eye in sufficient amounts to produce pharmacological effects appear possible. Further, advances in medicinal chemistry will likely enable this very important class of antisense inhibitor to become viable therapeutic agents. Additional research into oligonucleotide modification that replace the carbohydrate backbone all together using peptide nucleic acid or morpholino chemistry are also being performed, but will not be reviewed herein [21,22]. 21.1.2 Pharmacokinetics While the ability of ASO or siRNA to inhibit the specific target and affect a disease process is important, it is equally important, especially for platform technologies such as antisense inhibitors, that the compounds have favorable pharmacokinetic and tolerability profiles to be effective therapeutic agents. Delivery of the oligonucleotides to the necessary site of action must also be considered, both with respect to tissue and cell type. The majority of the ocular experience with these compounds has targeted treatment of the posterior ocular segment using intravitreous administration. However, by varying the method of application or route of administration of oligonucleotides to the eye, it is possible to target other ocular tissues and regions. Thus, one must characterize the distribution and pharmacokinetic properties with a specific focus on the unique aspects of each application. For the antisense mechanism to work, the oligonucleotide must be taken up into cells that express the target mRNA and protein [23]. Documentation of tissue distribution and cellular uptake are readily addressed for PS ODN and 2⬘-MOE ASO using available analytical techniques [24,25].

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This is more difficult, but no less important, for siRNA inhibitors because of their lower degree of stability. For PS ODN and 2⬘-MOE ASO, the mechanism of cellular uptake is not well defined, but internalization of oligonucleotides does occur [26–28]. Studies in rabbits, monkeys, and pigs have shown that intravitreous injection of both PS ODN and 2⬘-MOE ASO have very favorable distribution and clearance patterns for treating ocular disease [29–32]. The kinetics of fomivirsen in rabbits, monkeys, and humans was characterized by slow clearance from vitreous and distribution to retina [29,30,33]. The pharmacokinetic properties of 2⬘-MOE ASO have been shown to be very similar to PS ODN [34]. In the eye specifically, immunohistochemical staining of retina following a single intravitreous administration of PS ODN and 2⬘-MOE ASO revealed extensive and similar distribution to most ocular tissues in proximity to the vitreous [31]. Cell types in the eye that contained 2⬘-MOE ASO following intravitreous injection included outer plexiform layer, outer limiting membrane, inner plexiform layer, ganglion cells, ciliary body, iris, retinal-pigmented epithelium, and optic nerve. Similar results are reported for fluorescent-labeled oligonucleotides [35]. This distribution profile potentially supports treatment of many types of posterior segment ocular diseases, including ocular infection, inflammation, neovascular disease, or other metabolic and functional abnormalities. Once in retinal cells, PS ODN or 2⬘-MOE ASO are cleared slowly relative to clearance from vitreous. The half-life of PS ODN in monkey retina is ⬃3–7 days [30]. This slow clearance was the basis of the once monthly dose regimen approved for the use of fomivirsen in the treatment of CMV retinitis. By comparison, the clearance of 2⬘-MOE-modified oligonucleotides is much slower than for phosphorothioate oligodeoxynucleotides. The half-life for ISIS 13312 (a 2⬘-MOE-modified oligonucleotide with the same sequence as fomivirsen, Table 21.1) in monkey retina was on the order of 2 months compared to 3–7 days [31]. This resulted in significant accumulation in retina with once-monthly dosing, and retinal concentrations that were considerably higher than those achieved with PS ODN. The slower clearance from retina of the 2⬘-MOE ASO may be exploited to extend the dose frequency to once every 3–6 months. The durability of effect in the retina still needs to be investigated for 2⬘-MOE ASO, but a close pharmacodynamic correlation between activity and oligonucleotide concentration in tissue has been established for systemic target organs [36,37]. Infrequent intravitreal administration would allow for the consideration of treatment of chronic ocular diseases. The ocular pharmacokinetics for siRNA are not as well characterized to date. The half-life appears to be far shorter than for 2⬘-MOE ASO because the siRNA molecules are not stabilized against nuclease degradation to the same degree. However, while the residence time in cells will be shorter, an impressively long duration of action has been reported for siRNA in cells [38]. Exactly how this will translate into duration of action in the clinical setting is not known but will be revealed overtime in nonclinical and clinical studies. The pharmacokinetic considerations for oligonucleotide aptamers, such as Macugen, are quite different from antisense inhibitors. Most notable is that the aptamer oligonucleotides do not need to enter the cells, and instead interact outside of the cells, binding to receptors or soluble factors such as cytokines, or growth factors. Obviously the vitreal kinetics are more important for the ocular therapeutic application of aptamers. In the case of Macugen the dose interval is once every 6 weeks, which likely reflects the clearance rate from vitreous. 21.1.3 Tolerability In addition to optimizing the ocular kinetics for therapy, the tolerability of agents administered directly into the eye is very important. The experience gained thus far from the approval of Vitravene (fomivirsen) and Macugen (pegaptanib) and the effect of pegaptanib on macular edema and retinal neovascularization in patients with diabetes clearly demonstrates that oligonucleotide therapy administered by intravitreous injection can be well tolerated [4,6,11–13,15,39,40]. With the

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publication of specific guidelines for the delivery of drugs via intravitreous injection to reduce risks [41] and with the approval of the third therapeutic agent, Lucentis (ranibizumab), administered via this route [42], there is sufficient evidence that this is a reasonable route for administration of ocular therapeutics. Modifications made to 2⬘-MOE ASO have also improved the tolerability of oligonucleotides in animals relative to PS ODN (for review, see Chapter 12). The primary source of ocular toxicity for PS ODN is a proinflammatory reaction [31,43]. Ocular inflammation is particularly evident in rabbits, and best characterized by infiltrating cells in the vitreous and the anterior chamber. This proinflammatory reaction to PS ODN is an effect common to most oligonucleotides in this class, and is also observed with systemic administration in mice, rats, and rabbits [44]. Although proinflammatory effects are not an antisense effect of the oligonucleotide, specific sequence motifs that contain cytosine–guanosine dinucleotide motifs (CpG) contribute to the inflammatory response [45]. There is a difference in species sensitivity to this reaction, which is greatest in rodents and seen to a lesser degree in primates. While some proinflammatory effect was seen in clinical studies of fomivirsen, the effect was relatively mild, transient, and well managed by the use of topical corticosteroids [4,12,13]. The compound selection and chemical modifications made to 2⬘-MOE ASO have greatly reduced the proinflammatory activity of these so-called second-generation oligonucleotides. Avoiding the CpG dinucleotide motifs and incorporation of the 2⬘-MOE modifications have combined to reduce the interaction with the primary receptor on monocytes and dendritic cells responsible for triggering the proinflammatory effects. Substitution of cytosine with 5-methyl cytosine residues also reduces the proinflammatory effects of oligonucleotides [46]. These modifications appear to combine to decrease the potency of immune stimulation relative to PS ODN [43,47]. These modifications result in an improved ocular tolerability profile for 2⬘-MOE ASO, despite the higher retinal concentrations. The primary evidence of increased ocular tolerability of 2⬘-MOE ASO has been the observation of little or no ocular inflammation in rabbit following intravitreal injection. The primary effect in the eye associated with repeated intravitreous injections of 2⬘-MOE ASO appears to be the development of faint lens opacities at high doses or with frequent administration. These occur at the posterior surface of the lens in a dose- and time-dependent fashion consistent with accumulation of the oligonucleotide. The histologic appearance of the opacities on the lens surface supports the hypothesis that these are related to oligonucleotide accumulation. This effect has only been observed at high doses when the oligonucleotide is administered on a monthly dose regimen, far more frequent than would be anticipated in the clinic based on pharmacokinetic properties, and therefore accumulation is occurring. Lens opacities were avoided by using lower doses or less frequent administration in pharmacology and toxicology studies. Thus, 2⬘-MOE ASO appear to have favorable ocular safety and pharmacokinetic profiles for the treatment of a number of ocular diseases. There appears to be no information in the published literature on the ocular tolerability of siRNA inhibitors, however, Phase 1 and 2 clinical studies were recently completed for the two compounds currently in development. There have been no reports of issues with ocular irritation or functional abnormalities. It is expected that siRNA would be well tolerated in the eye since there is little opportunity for accumulation and the oligonucleotide will be readily cleared. However, siRNA compounds have been associated with proinflammatory effects, much like PS ODN and 2⬘-MOE ASO [48]. As a result, one must be vigilant to the possibility of proinflammatory effects, and each sequence will need to be thoroughly characterized. 21.1.4 Pharmacodynamics To efficiently utilize a platform technology for either mechanistic or therapeutic purposes, it is important to understand the dose, pharmacokinetic, and activity correlations. By and large, the tissue distribution and kinetic behavior of ASO have proved to be independent of sequence. If these

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properties are well understood for the desired application, then this knowledge can be used to greatly improve the efficiency of properly designed experiments. For PS ODN and 2⬘-MOE ASO it is relatively straightforward to measure concentrations of oligonucleotide in dissected tissues. There are also approaches that can be used to reveal what cell types take up the oligonucleotide. The characterization of cellular uptake and elimination half-lives following intravitreous injection of both PS ODN and 2⬘-MOE ASO was presented above. For pharmacologic application, the subsequent characterization of a dose and tissue concentration that will produce efficacy is important. These pharmacodynamic relationships have been defined in liver for several specific targets following systemic administration. While there is obviously some variability, the general pharmacodynamic relationships are reasonably consistent even with respect to the relationship between tissue clearance and the durability of activity. This is important with a compound that has a long half-life in tissue. In cases where oligonucleotide concentration in liver has been correlated with target mRNA levels, the rate of clearance is very close to the reversibility of pharmacologic effect. In the eye, these types of relationships can also be studied. Preliminary characterization of a 2⬘-MOE ASO inhibitor of ERK-6 in mice suggests that there is a correlation between exposure and effect that was time- and dose-dependent (Figure 21.1).

ERK6 mRNA in retina PBS 140

ERK6

ERK6 mRNA (% PBS treatment)

120 100 80 60 40 20 0

0.3

1

3

10

ASO (µg)

Oligonucleotide concentration in retina

Concentration (µg/g)

100 80 60 40 20 0 10

1

3

0.3

ASO (µg) Figure 21.1 Dose-dependent reduction of ERK-6 mRNA is correlated with increase in retina oligonucleotide concentration.

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21.2 OCULAR THERAPEUTIC AREAS 21.2.1 Antivirals The local application of ASO inhibitors for the treatment of viral infections in the eye has proved beneficial. The eye and surrounding structures are potential targets for viruses, either by direct infection at the cellular level or by ocular manifestations of systemic diseases as in the case of CMV retinitis. Because viruses must depend on the machinery of the host cell for replication they are virtually the ideal targets for the development of ASO therapeutic agents, particularly in the eye. Virally encoded proteins make excellent targets for ASOs and have been considered as viable potential targets in the development of ASOs as antiviral therapeutic agents in general [49]. Importantly, viral diseases of the posterior and anterior segments of the eye remain prevalent, often sight threatening, and in need of new treatments. The number of antiviral agents available to treat ocular infections is somewhat limited and even for those agents in use, the development of resistance remains a challenge. As an example, the prevalence of and growth in the number of patients with the acquired immune deficiency syndrome (AIDS) in the late 1980s and 1990s leads to the increasing prevalence of opportunistic infections, such as, CMV retinitis. The development of ASOs to target specific viral proteins provides several advantages over traditional antiviral drugs. First, unlike traditional drugs that bind not only to the targeted protein but also to other proteins, ASOs are highly selective and specific [50]. Thus, selection of a unique target in combination with a unique mechanism of action is likely to result in a therapeutic agent with significantly less likelihood of inducing resistance. The development of fomivirsen exemplifies the therapeutic utility of an ASO and demonstrates that an ASO can be delivered directly into the eye with a good benefit to risk profile even in the face of repeated intravitreous injections over the course of several months to years [12,13]. Fomivirsen is a first-generation ASO, a PS ODN that is specific for the immediate early region 2 (IE2) of the human CMV virus [51]. The reduction of the expression of the immediate early viral proteins by fomivirsen demonstrates a mechanism of action consistent with the antisense theory and mechanism of action. The targeted region is unique to CMV and selective inhibition of the IE proteins results in a highly effective antiviral agent. A second-generation ASO with the identical sequence as fomivirsen, ISIS 13312, is a 2⬘-MOE ASO that has been shown to have antiviral activity comparable to fomivirsen in fibroblasts and retinal pigment epithelial cells [31,52]. Both fomivirsen and ISIS 13312 demonstrated comparable and consistent antiviral activity with the IC50 between 0.1 and 1.0
M. The advantage derived from the 2⬘-MOE-modified ASO ISIS 13312 was evident following single and multiple intravitreous injections in rabbit and monkey eyes. The modification, as described earlier in this chapter, resulted in a longer residence time and better local ocular tolerability. While a Phase 2a clinical study to investigate the efficacy of ISIS 13312 was initiated, the program was discontinued following a significant decrease in the number of patients with CMV retinitis as a result of the introduction of the “triple cocktail” antiretroviral drugs [53]. Other viruses that manifest as an ocular condition in the anterior segment include herpes simplex (HSV), Varicella–Zoster (VZV), Epstein–Barr (EBV) and adenovirus. Among these, HSV-1 is commonly associated with ocular infections and is the leading cause of corneal blindness. Targeting HSV-1 is yet another opportunity for ASOs as therapeutic agents that has not been fully developed for ophthalmology. There are a variety of sites on the HSV-1 genome that are likely to play a critical role in viral replication including IE and early genes. As well there are a variety of ASO chemical modifications that have been designed to specifically inhibit a variety of targets and which have resulted in varying degrees of success discussed earlier in this chapter. All of the published information to date is based on in vitro cell assays and animal studies with no application to date in clinical studies. Nevertheless, the data strongly suggest that there is an opportunity to target both single and multiple genes, in some instances with synergistic effects.

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ASOs modified as methylphosphonates designed to target the splice junction and splice donor site of the HSV-1 IE-pre-mRNA 4 and 5 and to the translation initiation site of IE4 mRNA [54–57] used in cell assays as well as in a mouse ear and footpad model of HSV infection [56]. The antiviral effect when ASO-methylphosphonates are targeted to multiple sites (e.g., IE1 and IE4) has been studied in cell-culture assays and the findings suggest that there is the potential for synergistic antiviral activity [58]. Other ASO modifications designed to target specific regions of the HSV-1 gene have been evaluated, including a second-generation 2⬘-O-methylribonucleoside containing alternating methylphosphate/phosphodiester linkages. The in vitro activity in rabbit corneal epithelial cells and keratocytes infected with HSV-1 has been investigated with just such a modified ASO [59]. This second-generation ASO has a chimeric backbone with alternating methylphosphate/phosphodiester linkages and is targeted to a splice junction region of the HSV IE68 and IE12 gene. The results suggest potent antiviral activity in vitro in a dose-dependent manner that was sequence-specific. Thus, careful selection of both sequence-specific target, perhaps even including multiple targets, and the appropriate modification that may reduce cytotoxicity and enhance cellular uptake combine to play a significant role in the control of antiviral replication and open the way as a novel approach to treating viral infections in the eye. 21.2.2 Angiogenesis (Neovascular Age-Related Macular Degeneration and Diabetic Retinopathy) A good deal of the research into molecular mechanisms of ocular disease is focused on ocular neovascularization since diabetic retinopathy (retinal neovascularization) and neovascular AMD (choroidal neovascularization) are among the most prevalent causes of blindness [60]. Although all neovascular diseases are complex processes, involving multiple growth factors, proliferation of vascular endothelium, and differentiation, much of the attention of ocular disease is focused on the role of VEGF in this process [61,62]. It has been shown that VEGF expression is increased in ocular angiogenesis [62,63], and inhibition of VEGF signaling using a soluble oligonucleotide aptamer (e.g., Macugen) or a monoclonal antibody fragment (Lucentis) have recently been approved for treatment of neovascular AMD [6,15,17,64]. This recent experience both demonstrated the utility and tolerability of oligonucleotide-based therapeutics in the eye, and also confirmed the acceptability of intravitreous injection as a viable route of administration for chronic ocular disease. Inhibition of VEGF or VEGF-receptor expression have been targeted using antisense therapy as well, among other potential targets in the angiogenesis cascade. Based on the pharmacokinetic properties described above, they have the opportunity to be administered less frequently than current therapies. This durability of activity for PS ODN inhibitors targeting VEGF (i.e., more than 2 months) has been demonstrated in rat models of choroidal neovascularization when administered with a dendrimer formulation [65,66]. While single-stranded antisense inhibitors of VEGF expression have been reported, the most progress in the last few years has been with siRNA inhibitors [67]. Of the two siRNA compounds currently in development one is targeted to the VEGF receptor while the other is targeted to VEGF expression itself [68,69]. The siRNA targeting VEGF-R1 clearly demonstrated inhibition of target mRNA by 50% that corresponded with a 50% decrease in neovascular area in a mouse model of laser-induced CNV [68]. This work also demonstrated the presence of inhibitor in the retina for 5 days. The Phase 1 clinical trial for the VEGF-R1 siRNA (Sirna-027; Sirna Therapeutics, Inc.) was initiated in November 2004. Subjects were treated with a single intravitreous injection at doses between 100 and 1600
g with safety evaluations up to 15 months. Phase 1 trial has also been conducted for the siRNA targeting VEGF (Cand5; Acuity Pharmaceuticals, Inc.). In this trial, Cand5 was administered to 15 subjects as an intravitreous injection with doses ranging from 100 to 3,000
g per eye. Tolerability was good in Phase 1 studies, and repeat-dose-randomized, masked, controlled Phase 2 studies are being conducted for both these siRNA inhibitors (from presentation by P. Kaiser, Retinal Physician 2006 Symposium, Bahamas).

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In addition to diabetic retinopathy and neovascular AMD, these VEGF inhibitors may also be studied for other types of ocular angiogenesis. For example, a PS ODN targeted against VEGF has shown efficacy in a monkey model of iris neovascularization, and both PS ODN and siRNA have shown efficacy in rodent models of corneal neovascularization [68,70]. Another approach to inhibiting VEGF action using ASO is to inhibit a downstream component of the signaling cascade. As it turns out, both bFGF and VEGF signal through the MAP kinase cascade, which includes raf kinase [71,72]. Raf kinase activation following VEGF stimulation was confirmed in human endothelial cells [73]. More recently it has been reported that other factors such as erythropoietin [74] and hepatocyte growth factor [75] may play a major role in the etiology of diabetic macular edema and diabetic retinopathy. Thus, affecting a downstream target such as C-raf kinase may be a more effective strategy in that targeting multiple growth factors (much like a combination drug in one product approach) rather than targeting the individual growth factors themselves. A 2⬘-MOE antisense inhibitor of C-raf kinase has been investigated in a pig model of venous-occlusion retinal neovascularization and shown to decrease retinal neovascularization with good tolerability [32]. This compound is expected to enter Phase 1 clinical trials in 2007 (iCo-007; iCo Therapeutics, Inc.), initially targeting diffuse diabetic macular edema. Other ASO targeting MAP kinase pathways (e.g., B-raf kinase and FAK) have also shown promising activity in a rabbit model of angiogenesis initiated by VEGF [76]. Inhibiting a common component of the signaling cascade for both bFGF and VEGF in this manner has proved effective for small molecule inhibitors as well, and may increase the likelihood of antiangiogenic activity by eliminating the possible signaling through redundant pathways [77,78]. 21.2.3 Intraocular Inflammation Inflammation of the uveal tract (iris, ciliary body, and choroid) is termed uveitis and is a potentially blinding condition that may result from trauma or injury to the eye, invasive infectious agents (e.g., viral infection) or autoimmune reactivity and can affect the anterior chamber and/or vitreous cavity. Although several animal models exist and have been key in our understanding of uveitis there have been relatively few instances where the effects of ASO have been evaluated in these models. Still the opportunity for ASO therapy is significant given the number of antiinflammatory strategies that have been studied (see Chapter 24). For example, within the eye the expression of intracellular adhesion molecules (e.g., ICAM-1, ICAM-2), P-selectin, vascular adhesion molecule (VCAM-1) and the subsequent induction of cytokines play a critical role in the ocular immune response [79,80]. Blocking the expression or function of these adhesion molecules with either local or systemic administration could interfere with the inflammatory process. HSV infection was discussed above in the context of ocular antiviral targets; however, another aspect of viral infections of the eye is the resultant devastating ocular inflammation. A discussion of the inflammatory component of HSV infection includes the diagnoses of blepharitis, scleritis, keratitis, and anterior uveitis to name a few. The cytokines that play a role in the inflammatory response to viral infection include IL-2 and tumor necrosis factor (TNF)-. Topical application of a specific ASO targeted at TNF- mRNA may provide an advantageous local treatment for corneal inflammation in this scenario. Demonstration of the effectiveness of an anti-TNF- ASO in the downregulation of this proinflammatory cytokine in cell culture and an infected mouse corneal model supports the potential of ASO as antiinflammatory agents. Not only were these authors able to demonstrate significant decrease in the secretion of TNF- from the cells in culture and mouse cornea, but this may be the first evidence that administration of an ASO via a subepithelial injection to the cornea is a safe method which also allows for multiple injections [81]. Others have evaluated the potential of ASO to downregulate NOSII in a rat model of endotoxininduced uveitis [82]. To enhance intraocular delivery to the appropriate target cell type these investigators used iontophoresis. In this model significant levels of ASO were observed in the iris and ciliary body within 1 h after delivery of the ASO via iontophoresis and with time, levels in the retina and

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choroid were also observed. NOSII mRNA levels were decreased in iris and ciliary body compared with saline control and control oligonucleotides, and there was a concomitant decrease in nitrate production that was sequence-specific [82]. Thus, ASO treatment was associated with antiinflammatory effect in rats, but whether this method of delivery is viable in the clinic remains to be seen. The opportunity for treatment of ocular inflammation has had only limited investigation. It would seem that ocular manifestations of infection and immune-mediated inflammation are in need of intense study with ASO therapeutics targeted to any number of proteins. 21.2.4 Glaucoma Glaucoma is another leading cause of blindness, but very little work with antisense inhibitors has been focused on this particular ocular condition [2]. Glaucoma is a complex pathology with several different approaches to treatment that may be amenable to antisense therapy. The greatest areas of need in the management of glaucoma are treatments that limit the damage to the neurosensory retina caused by elevated pressure that ultimately leads to blindness, [83] an approach called neuroprotective therapy. Nueroprotection in the management of glaucoma is a complex multifactorial process making investigative studies difficult. To date the only clinical studies in this area are the memantine trials that are nearing completion [83,84]. One proposed mechanism of blindness is retinal ganglion cell death caused by chronic increased ocular pressure. Regulating expression of genes in retinal ganglion cells are thus of interest, and there are reports of in vitro and in vivo uptake and activity of antisense inhibitors (both single strand ASO and siRNA) in this cell type. Although not specific to the treatment of glaucoma, the examples of antisense activity in vitro in retinal ganglion cells include the demonstration of uptake and decreased expression of targets such as optinuerin and OPA1 in RCG-5 cells treated with siRNA inhibitors [85,86]. The ability to deliver PS ODN by intravitreous injection to retinal ganglion cells in vivo, and affect target mRNA level and ocular processes has also been demonstrated in rabbit targeting kinesin and in rats targeting kynurenine aminotransferase II [87–89]. These data demonstrate the opportunity to affect retinal ganglion cell physiology with ASO inhibitors. With advances in the understanding into the mechanism of apoptosis, ASO may be used to inhibit cell death. For example, intravitreous injection of a PS ODN ASO inhibitor of bax, a pro-apoptotic protein, did reduce ganglion cell death in a rodent model of neurodegeneration [90]. The control of ocular pressure using ASO is another potential approach to treat glaucoma. The opportunity exists because of the uptake of oligonucleotide in ciliary epithelium and trabecular meshwork cells. The issue with IOP control is whether there is the medical need for additional therapies given the efficacy of topically applied medicines. Nonetheless, recently investigators have provided preliminary reports of successful use of siRNA inhibitors to regulate ocular pressure by targeting either - or -adrenoreceptors, or carbonic anhydrase [91,92]. ASO inhibitors might also prove useful as an adjuvant to glaucoma filtration surgery. One of the methods of glaucoma therapy is to provide a shunt that drains the anterior chamber, and thus, controls ocular pressure. The primary limitation of this surgery is scarring and fibrous buildup in subconjunctival and tenon membranes that blocks aqueous flow and leads to failure of the shunt. Scar formation in the eye is thought to be mediated in large part by TGF-, and a monoclonal antibody inhibitor of TGF-2 has been shown to inhibit proliferation and migration of fibroblasts [93]. Experiments using an ASO inhibitor of TGF-1 and 2 in a mouse model of glaucoma surgery demonstrated a significant reduction in the conjunctival scar formation and increased survival of filtration bleb compared to a control oligonucleotide [94]. In this model, the ASO was injected subconjunctivally directly into the filtration bleb, and oligonucleotide uptake into fibroblasts, epithelial cell, and macrophages was confirmed by immunohistochemical staining. These results were confirmed along with improvement in efficacy and duration of activity when anti-TGF-2 PS ODNs were formulated with polyethylenamine [95]. In this case the PS ODN alone was effective, but the formulated material appeared to be taken up in target cell types to a greater degree. The opportunity

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to replace the use of mitomycin C in this surgical procedure is significant, and is an interesting application of antisense inhibitors in that it uses a method of administration that is less invasive even than intravitreous injection and illustrates the opportunity for topical application for anterior ocular indications.

21.3 DRUG DELIVERY OPTIONS Antisense inhibitors and other oligonucleotide therapeutics are showing promise in the treatment of ocular diseases and conditions, but in order for them to reach their full potential and optimal safety profile, alternatives to intravitreous injections must be developed, particularly for those agents expected to treat chronic conditions that affect the retina. Preliminary work has been done with nonintravitreal routes of administration, which indicate that periorbital administration can achieve the necessary retinal concentrations for inhibition of target gene expression. The sclera is a relatively porous structure composed primarily of collagen and glycosaminoglycans, and is largely acellular [96]. Thus, transscleral delivery of drugs is a viable alternative to intravitreal injection [97]. In fact, transscleral absorption of macromolecules such as a 70-kDa dextran and a monoclonal antibody for ICAM-1 has been demonstrated using subconjunctival injection or infusion [98,99]. The permeability of sclera to ASO has been studied using iontophoresis as a way to direct the charged molecules across various ocular barriers. Iontophoresis has been shown to facilitate penetration into all layers of the cornea, and into both epithelial and endothelial cells [100]. Iontophoresis of PS ODN targeted against NOSII was also used to penetrate the corneoscleral barrier to effectively reduce expression and inflammation in endotoxin-induced uveitis model [82]. Development of a convenient and reliable nonintravitreal route of administration would greatly increase the therapeutic utility of ASO and appears possible. 21.3.1 Formulations (Liposomes, Nanosized Particles) The need for formulations in the application of oligonucleotides will depend on the type of inhibitor and the nature of the application. Certainly less frequent administration to the eye is preferable regardless of whether the route is intravitreous injection or subtenon injection. Still for some oligonucleotide therapeutics, such as 2⬘-MOE ASO where the site of action of the drug is in cells and the half-life in tissues is on the order of 2 months, sustained release formulations may not be necessary for the therapeutic application. In other contexts such as siRNA that are rapidly degraded or oligonucleotide aptamers that operate outside the cells, a formulation approach might be beneficial. There are a number of formulation strategies that have been used in ocular administration of ASO in the eye, including dendrimers, PLGA microspheres, liposomes, and polyethylenimine [66,95,101–103]. In most cases, the ASO studied were PS ODN and formulations enhanced the retinal absorption of ASO. In the case of a VEGF ASO formulated with dendrimer, the duration of action was reported to be greater than 2 months in a rat model of choroidal neovascularization [65]. These investigators also report good ocular tolerability in all cases. Still, it has already been demonstrated that formulations of oligonucleotide therapeutics are not necessarily needed [4,15]. However, in applications where it provides some advantage to the controlled delivery, these technologies appear to be compatible with ocular pharmacology.

21.4 CONCLUSIONS Much progress has been made in the last few years in the application of oligonucleotide therapeutics to the eye. This approach to treating ocular disease is attractive in that the local delivery of oligonucleotides results in distribution to numerous ocular cell types. The tolerability of these

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molecules is good, and the opportunity for long residence time, and thus infrequent administration, affords the desirable properties for treating ocular disease. With continued success of the VEGF siRNA development programs, it is expected that application of antisense inhibitors will expand in scope over the next few years.

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83. Weinreb, R. N., Clinical trials of neuroprotective agents in glaucoma, Retina 25 (8 Suppl), S78, 2005. 84. Levin, L. A., Neuroprotection and regeneration in glaucoma, Ophthalmol. Clin. North Am. 18 (4), 585, 2005. 85. Davies, V. J. and Boulton, M., Expression profile of rat primary retinal ganglion cells and retinal cell lines, RGC-5 and R28 and mitochondrial morphological changes following OPA1 knockdown mediated siRNA, in ARVO, Florida, 2006. 86. Duong, M. and Ball, A. K., siRNA-mediated knowckdown of optineurin in Rgc-5 cells, in ARVO, Florida, 2006. 87. Amaratunga, A. P., Kosik, K. S., Rittenhouse, P. A., Leeman, S. E., and Fine, R. E., Antisense inhibition of protein synthesis and function—Rabbit retinal protein, in Antisense Therapeutics, Agrawal, S., ed., Humana Press, Inc., Tatowa, NJ, 1996, p. 109. 88. Amaratunga, A., Morin, P. J., Kosik, K. S., and Fine, R. E., Inhibition of kinesin synthesis and rapid anterograde axonal transport in vivo by an antisense oligonucleotide, J. Biol. Chem. 268 (23), 17427, 1993. 89. Thaler, S., Rejdak, R., Dietrich, K., Ladewig, T., Okuno, E., Kocki, T., Turski, W. A., Junemann, A., Zrenner, E., and Schuettauf, F., A selective method for transfection of retinal ganglion cells by retrograde transfer of antisense oligonucleotides against kynurenine aminotransferase II, Mol. Vision 12, 100, 2006. 90. Isenmann, S., Engel, S., Gillardon, F., and Bahr, M., Bax antisense oligonucleotides reduce axotomy-induced retinal ganglion cell death in vivo by reduction of Bax protein expression, Cell Death Differ. 6 (7), 673, 1999. 91. Pintor, J. J., Mediero, A., Jimenez, A., Sesto, A., Gonzalez de Buitrago, G., and Peral, A., SiRNA in the treatment of ocular hypertension targeting alpha and beta adrenoceptors, Invest. Ophthalmol. Vis. Sci. 47 (5), 403, 2006. 92. Jimenez, A., Sesto, A., Pintor, J., Mediero, A., Peral, A., and Gonzalez de Buitrago, G., RNAi: a new strategy for treating ocular hypertension silencing carbonic anhydrases, Invest. Ophthalmol. Vis. Sci. 47 (5), 405, 2006. 93. Corderio, M., Gay, J., and Khaw, P., A new glaucoma anti-scarring agent, Invest. Ophthalmol. Vis. Sci. 40, 2225, 1999. 94. Cordeiro, M. F., Mead, A., Ali, R. R., Alexander, R. A., Murray, S., Chen, C., York-Defalco, C., Dean, N. M., Schultz, G. S., and Khaw, P. T., Novel antisense oligonucleotides targeting TGF-beta inhibit in vivo scarring and improve surgical outcome, Gene Ther. 10 (1), 59, 2003. 95. Gomes dos Santos, A. L., Bochot, A., Doyle, A., Tsapis, N., Siepmann, J., Siepmann, F., Schmaler, J., Besnard, M., Behar-Cohen, F., and Fattal, E., Sustained release of nanosized complexes of polyethylenimine and anti-TGF-beta 2 oligonucleotide improves the outcome of glaucoma surgery, J. Controlled Rel. 112 (3), 369, 2006. 96. Ahmed, I., Gokhale, R. D., Shah, M. V., and Patton, T. F., Physicochemical determinants of drug diffusion across the conjunctiva, sclera, and cornea, J. Pharm. Sci. 76 (8), 583, 1987. 97. Ambati, J. and Adamis, A. P., Transscleral drug delivery to the retina and choroid, Prog. Retin. Eye Res. 21 (2), 145, 2002. 98. Kim, T. W., Lindsey, J. D., Aihara, M., Anthony, T. L., and Weinreb, R. N., Intraocular distribution of 70-kDa dextran after subconjunctival injection in mice, Invest. Ophthalmol. Vis. Sci. 43 (6), 1809, 2002. 99. Ambati, J., Gragoudas, E. S., Miller, J. W., You, T. T., Miyamoto, K., Delori, F. C., and Adamis, A. P., Transscleral delivery of bioactive protein to the choroid and retina, Invest. Ophthalmol. Vis. Sci. 41 (5), 1186, 2000. 100. Berdugo, M., Valamanesh, F., Andrieu, C., Klein, C., Benezra, D., Courtois, Y., and Behar-Cohen, F., Delivery of antisense oligonucleotide to the cornea by iontophoresis, Antisense Nucl. Acid Drug Dev. 13 (2), 107, 2003. 101. Roy, S., Zhang, K., Roth, T., Vinogradov, S., Kao, R. S., and Kabanov, A., Reduction of fibronectin expression by intravitreal administration of antisense oligonucleotides, Nat. Biotechnol. 17, 476, 1999. 102. Bourges, J. L., Gautier, S. E., Delie, F., Bejjani, R. A., Jeanny, J. C., Gurny, R., BenEzra, D., and Behar-Cohen, F. F., Ocular drug delivery targeting the retina and retinal pigment epithelium using polylactide nanoparticles, Invest. Ophthalmol. Vis. Sci. 44 (8), 3562, 2003. 103. Bochot, A., Fattal, E., Boutet, V., Deverre, J. R., Jeanny, J. C., Chacun, H., and Couvreur, P., Intravitreal delivery of oligonucleotides by sterically stabilized liposomes, Invest. Ophthalmol. Vis. Sci. 43 (1), 253, 2002.

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22

Cardiovascular Therapeutic Applications Rosanne Crooke, Brenda Baker, and Mark Wedel

CONTENTS 22.1 Introduction .........................................................................................................................601 22.1.1 Background............................................................................................................601 22.1.2 Current Treatment Paradigm..................................................................................602 22.1.3 The Unmet Medical Need .....................................................................................602 22.1.4 Antisense Oligonucleotides as Novel Cardiovascular Therapeutics .....................603 22.2 Application of Antisense Compounds to Cardiovascular Disease......................................605 22.2.1 Liver Targets Involved in Dyslipidemias...............................................................605 22.2.1.1 Inhibition of Cholesteryl Ester Transfer Protein to Increase Low HDL-C Levels................................................................605 22.2.1.2 Inhibition of Lp(a) ................................................................................605 22.2.1.3 Inhibition of Apolipoprotein C-III........................................................606 22.2.1.4 Inhibition of Acyl-Coenzyme A: Cholesterol Acyltransferase 2..........608 22.2.1.5 Antisense Inhibition of ApoB-100........................................................611 22.2.2 Antisense Inhibition of CRP, a Nonlipid Hepatic Target Involved in CHD..........626 22.2.3 Antisense Inhibitors for the Treatment of Hypertension.......................................628 22.2.4 Antisense Inhibitors Affecting Restenosis.............................................................628 22.3 Summary and Future Perspectives ......................................................................................629 Acknowledgments ..........................................................................................................................630 References ......................................................................................................................................630

22.1 INTRODUCTION 22.1.1 Background Coronary heart disease (CHD), despite tremendous medical and technological advances and clearer insights into the pathogenesis of atherosclerosis, has been the leading cause of death in the United States for over a century [1–4]. Complications from atherosclerotic heart disease are the most common causes of morbidity and mortality in industrialized nations. In fact, the World Health Organization and others project that CHD will become the primary cause of death worldwide by the

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year 2020 [5,6]. Several factors have been identified that increase the probability that an individual might develop atherosclerosis [1,7,8]. The major independent risk factors include elevated total and low-density lipoprotein cholesterol (LDL-C) levels, low serum high-density cholesterol (HDL-C) levels, cigarette smoking, hypertension, diabetes mellitus, insulin resistance, and advancing age. Other factors include obesity, physical inactivity, family history of premature cardiovascular disease, ethnicity, elevated serum triglyceride, homocysteine, and lipoprotein(a) [Lp(a)] levels, and the presence of prothrombotic factors such as fibrinogen and inflammatory markers such as C-reactive protein (CRP). These categories can be further subdivided into those that can be modified through various interventions (alterations in lifestyle and diet, or pharmacological agents) and those that cannot. Unmodifiable factors include advancing age, gender, ethnicity, and genetic predisposition. Of those factors that can be modulated, the abnormalities associated with lipid metabolism have been the most extensively studied and therefore, the best understood. 22.1.2 Current Treatment Paradigm Experimental and epidemiological studies have strongly implicated elevated cholesterol and LDL-C in the development of atherosclerosis [9–11]. Several large interventional clinical studies have clearly demonstrated that lowering LDL-C levels reduced not only the incidence of CHD, but also the associated morbidity and mortality of the disease [12–14]. Due to the strong correlation between elevated LDL-C plasma levels, the atherogenic process, and the log-linear relationship that exists between LDL-C and cardiovascular event rates [15,16], its reduction has been the principal goal for CHD treatment and prevention. The lipid management guidelines have been established since 1988 by the National Cholesterol Education Program (NCEP) Adult Treatment Panel (ATP) [10]. The most recent recommendations suggest that patients at greatest risk should aggressively reduce LDL-C below 70 mgⲐdL. This value was chosen as a result of an increasing number of studies in the past several years demonstrating that lower levels are highly beneficial. For example, a metaanalysis of 14 outcome trials suggested that an approximate 2 mg/dL reduction in LDL-C would produce a 1% reduction in CHD rate per year [17]. More compelling data from the REVERSAL and ASTEROID trials revealed that the greatest reduction in LDL-C levels resulted in significant regression in atherosclerotic plaque burden [18,19]. While exercise and sensible dietary changes may lower LDL-C and triglyceride levels, many patients require further therapeutic interventions. The five classes of approved antihyperlipidemic therapeutic agents are the fibrates, niacin, bile acid sequestrants, cholesterol absorption inhibitors, and competitive inhibitors of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase), or the statins [7,11,20]. The choice of therapy is individualized to meet the needs of patients; however, the most widely prescribed and efficacious therapeutic LDL-C lowering agents are the statins. Administration of these drugs has been shown to reduce mortality rates in patients with existing CHD, prevent the onset of a first coronary event, reduce the risk of progressive atherosclerotic disease and, as described above, cause regression of atheromatous plaque volume [19,21–23]. Other lipid or lipoprotein risk factors may also be altered by some of the agents listed above. For example, fibrates can reduce high serum triglyceride levels, but in some refractory patients, a combination of fibrates, statins, and niacin are required to avoid pancreatitis [8,9,24]. Niacin, or nicotinic acid is currently the most effective agent to raise HDL-C levels [25,26]. High Lp(a) levels may also be modestly reduced by niacin as well [7,27]. However, dermatological, gastrointestinal, and hepatic side effects have limited its use [7,28]. 22.1.3 The Unmet Medical Need Even with the availability of statins and the second tier agents, several studies have shown that the attainment of the ATP-III recommended LDL-C goal is low and cardiovascular mortality

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rates are still high, especially in patients with CHD with multiple risk factors [29–32]. A number of potential factors can contribute to the failure to reach target LDL-C levels and include patient noncompliance (not taking prescribed medications/expense of therapies), inadequate treatment by physicians (e.g., inappropriate choice of drugs, use of ineffective dose of drugs, and unwillingness to escalate appropriately), and ineffectiveness or intolerance to single/combination therapies [29,33]. There are also data suggesting that maximal doses of statins, either alone or in combination with other agents, are ineffective in some high-risk patients [34]. Additionally, when combination interventions are employed, many of the drugs are metabolized by common enzymatic machinery, and thus possess significant potential for untoward drug-drug interactions [7,33]. Increasingly stringent LDL-C goals, coupled with the inadequacy of existing drugs and a clearer understanding of the molecular basis of various types of dyslipidemias and cardiovascular disease in general, underscore the need for novel therapeutics that can be safely administered either alone or in combination with statins and other existing agents. One class of novel cardiovascular drugs showing great promise, both preclinically and in humans, exploits antisense technology. 22.1.4 Antisense Oligonucleotides as Novel Cardiovascular Therapeutics The tremendous advances in antisense technology over the past 17 years have facilitated the development of novel therapeutics that target many of the risk factors that predispose individuals to cardiovascular disease. Many aberrantly expressed genes contributing to various dyslipidemic states, or thought to be involved in the pathogenesis of atherosclerosis can be rapidly validated both in vitro and in vivo with exquisite specificity and isoform selectivity. Theoretically, any region within an mRNA sequence can be targeted and full-length gene sequences are not necessary to obtain effective antisense inhibitors. The opportunity, therefore, exists to selectively inhibit classically undruggable targets, such as lipid phosphatases, transcription factors, and macromolecules with no intrinsic enzymatic activity such as transport, structural proteins, and lipoproteins. Another advantage of this technology is the predictable chemical class-related toxicological properties of the drugs. As others will describe in this book (Chapters 12 and 13), [35], first- and second-generation antisense oligonucleotides (ASOs) have attractive safety profiles, both preclinically and in man. Key safety issues have been identified, and tremendous progress has been made toward understanding potential adverse events. It is also known that these agents are cleared from tissue via slow endonucleolytic degradation and not through interaction with the CYP3A4 and CYP3A5 isoforms of the cytochrome P450 system [36], the common pathway for the metabolism of statins and a multitude of other agents [37]. Antisense compounds can, therefore, be used safely in combination with known cardiovascular therapeutics with different mechanisms of action. The pharmacokinetic properties and tissue distribution of ASOs have also been extensively characterized in preclinical models and humans (Chapter 11) [38–41]. The second-generation 2⬘-O-(2-methoxy)ethyl (2⬘MOE)–modified gapmer analogs, which represent the most advanced antisense therapeutics in the clinic, are localized in tissues such as kidney, liver, adipose tissue, spleen, and lymph nodes after parenteral administration. More recent data suggest that antisense oligonucleotides also distribute to macrophages and endothelial cells within atheromas (Figure 22.1) [42]. Vascular endothelial cells provide substances that help regulate vascular tone, coagulation, thrombosis, and inflammation and their dysfunction is thought to be integral to the development and progression of atherosclerosis [43–46]. Accumulation of LDL-C within monocytes/macrophages, and its subsequent oxidation results in the formation of foam cells and the release of various cytokines and chemokines that are critical to the initiation and propagation of the inflammatory process within the plaque.

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Figure 22.1 (See color insert following page 270.) Localization of a representative 20-mer phosphorothioate within an atherosclerotic lesion of a Watanabe Heritable Hyperlipidemic rabbit. Animals were administered 25 mg/kg ASO twice weekly for 3 weeks. Top left panel: H&E staining of the aortic plaque indicating the presence of foamy macrophages in the lesion. Bottom left panel: Masson’s Trichrome staining of the lesion to highlight excess collagen deposition. Top right panel: Localization of the ASO using immunostaining. Bottom right panel: Localization of macrophages using a monoclonal mouse anti-macrophage antibody (RAM 11).

The highest concentrations of second-generation antisense drugs are found in kidney and liver [39–41,47–49], organs whose interactions and dysregulation contribute to various aspects of cardiovascular disease. The kidney plays an important role in maintaining blood pressure levels and is also the site of action of many of the marketed antihypertensive agents [50]. The liver, most importantly, regulates several key metabolic pathways that directly influence the development of CHD [3,51]. It is the major site of cholesterol and fatty acid/triglyceride synthesis in mammals and is also responsible for the production and degradation of all atherogenic apolipoprotein B-100 containing lipoproteins such as very low-density lipoprotein cholesterol (VLDL-C), LDL-C, and Lp(a). Because of its vital role in cholesterol and lipid homeostasis and since most of the lipid-lowering drugs ultimately target hepatic lipid metabolism (statins, fibrates, bile acid resins), perhaps as Davis and Hui suggested [3], atherosclerosis may, indeed, be a liver disease of the heart. In this chapter, we will summarize preclinical data and, where possible, the clinical activity of ASOs directed against several risk factors that contribute to CHD. The majority of the ASOs to be discussed represent the most advanced, second-generation 2⬘MOE chemistries and have been designed to inhibit the production of novel proteins produced in the liver that contribute to various dyslipidemic states (elevated serum LDL-C, Lp(a) and triglycerides and low levels of HDL-C). A nonlipid-related hepatic target, CRP, which is thought to contribute to the initiation and progression of the atherosclerotic plaque, will also be described. Finally, the last two sections will discuss experiments using first- and second-generation antisense inhibitors that affect hypertension and the development of restenosis.

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22.2.1.1 Inhibition of Cholesteryl Ester Transfer Protein to Increase Low HDL-C Levels As described in the introduction, low HDL-C levels are now recognized as a risk factor for the development of CHD. The metabolism of HDL-C is a complex process and involves the interplay of a number of lipoproteins, receptors, and enzymes that regulate its synthesis, intravascular remodeling, and catabolism [25,26,52]. More importantly, HDL’s atheroprotective mechanisms are not completely understood, but may include reverse cholesterol transport, whereby excess cholesterol is removed from plaque sites and other tissues for redistribution to liver, and various antioxidant and anti-inflammatory effects. Cholesteryl ester transfer protein (CETP) is the enzyme that facilitates the transfer of cholesteryl esters from HDL-C to apolipoprotein B (apoB) containing lipoproteins [25,52–54]. Some humans who are deficient in CETP have increased HDL-C levels and decreased LDL-C levels. Additionally, CETP deficiency has been associated with decreased coronary artery disease and predisposition to ischemic heart disease [55–57]. Sugano used a rabbit-specific first-generation phosphodiester antisense inhibitor to CETP to determine the importance of that enzyme in determining serum HDL-C levels and its role in the development of atherosclerosis in cholesterol-fed Japanese white rabbits [58,59]. Since the liver is the principal site of CETP synthesis, the authors believed that reduction of CETP in that organ could favorably alter the HDL/LDL ratio and reduce the risk of atherosclerosis. The first-generation phosphodiester CETP antisense inhibitor used in their studies was complexed to asialoglycoprotein-poly(L-lysine) to enhance the uptake of the drug in liver and protect the oligonucleotide from degradation by nucleases [60]. Rabbits were systemically administered 30
g/kg oligonucleotide complex twice weekly for 8 weeks. At that time, reductions in CETP liver mRNA, plasma CETP, and total cholesterol, principally VLDL and LDL-C, were observed in the antisense but not saline or oligonucleotide control rabbits. Surprisingly, HDL-C levels were not significantly affected. Nonetheless, the aortic cholesterol contents and total atherosclerotic lesion surface area were significantly lower in treated animals. These data suggest that reduction of liver CETP may be beneficial for reducing plasma LDL-C, possibly by enhancing the catabolism of LDL-C through up-regulation of LDL receptors and decreasing the transfer of cholesteryl esters from HDL-C to atherogenic apoB-containing lipoproteins. While the plaque data are encouraging, no further information regarding the use of firstgeneration antisense compounds has been published, possibly due to potential issues related to using nuclease-labile, phosphodiester oligonucleotides and long-term toxicities related to the poly(L-lysine) moiety on the carrier protein [60]. Nonetheless, given the positive clinical data using small-molecule inhibitors of CETP [55–57,61], second-generation antisense inhibitors have been generated and are being tested in vivo in our laboratories [62].

22.2.1.2 Inhibition of Lp(a) Lp(a) is a heterogeneous family of lipoprotein particles synthesized exclusively in the liver of humans, Old World nonhuman primates, and the European hedgehog that was discovered in 1963 by Kare Berg [28,63,64]. It is a complex molecule that consists of a single copy of apoB-100 linked by a disulfide bond to a single copy of a glycoprotein, apolipoprotein (a) or apo(a), at the C-terminal regions of both molecules [28,64]. The human apo(a) gene is thought to have evolved from the plasminogen gene on chromosome 6q27. Lp(a), similar to plasminogen, has coding

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sequences for loop structures, or modules, called kringles (multiple K4 and one KV domains) that contain 80–85 amino acids each. The size of the apo(a) gene varies from 300–800 kDa and these differences arise principally from variable copies of K4. Generally, there is an inverse relation between apo(a) size and Lp(a) levels, with smaller apo(a) sizes resulting in higher plasma Lp(a) levels. In recent years, elevated Lp(a) levels (⬎30 mg/dL) have become recognized as an independent risk factor for the development of CHD, cerebrovascular, and peripheral vascular disease [28,65–67]. The exact mechanisms by which this molecule contributes to the pathogenesis of atherosclerosis are unknown. However, Lp(a) has pleotropic effects that include inhibiting the conversion of plasminogen to plasmin, attenuation of fibrinolysis and promotion of the prothrombotic state, enhancing proliferation and migration of smooth muscle cells and up-regulation of cell adhesion molecules [28,64]. More recently, it has been suggested that Lp(a) might act as a preferential acceptor of proinflammatory oxidized phospholipids and it is the modified molecule that is taken up into vessel walls, ultimately contributing to foam cell formation within the plaque [67–71]. Apo(a) has also been localized in atherosclerotic plaques using immunohistochemistry [72] and found to be deposited in vein grafts following coronary artery bypass [73]. As mentioned above, with the exception of niacin, conventional therapeutic options for lowering elevated Lp(a) levels are limited. In some cases, LDL-apheresis has even been used to lower significantly elevated LDL and Lp(a) levels in patients with familial hypercholesterolemia [74]. Our laboratory has taken two approaches in an attempt to modulate Lp(a)—first, by using second-generation antisense drugs that target hepatic apo(a) and second, by inhibiting apoB-100. The latter approach will be discussed later in this chapter (Section 22.1.5, High Fat–Fed Monkey Studies). The principal challenge to the design of apo(a) antisense oligonucleotides has been the close homology of the apo(a) gene to plasminogen. Nonetheless, we have successfully identified an exon 2–specific human apo(a) ASO that does not affect plasminogen expression in vitro or in vivo. As shown in Figure 22.2a, this inhibitor selectively inhibits human apo(a), not plasminogen, mRNA expression in a dose-responsive fashion in primary human hepatocytes. Pharmacological evaluation of potential inhibitors to Lp(a) is also problematic because only humans and Old World monkeys express the apo(a) gene. However, our collaborators and we validated the activity of the human antisense inhibitor in transgenic mice expressing human apo(a) [75]. The hLPA-YAC transgenic mice used in this study had previously been shown to develop significant atherosclerosis on a normal chow diet [76]. Mice were administered 50 mg/kg/week via intraperitoneal (i.p.) injection of ISIS 144367 and a control ASO. After completion of the experiment, liver apo(a) mRNA and plasma apo(a) protein levels were determined by quantitative RT-PCR and ELISA, respectively. After 4 weeks of treatment with the antisense inhibitor, apo(a) mRNA expression levels were reduced by 85% (Figure 22.2b) and serum apo(a) by 70% compared to the saline control animals (Figure 22.2c). As expected, the control ASO had no effect on any measured parameter. Administration of the apo(a) ASO also had no effect on triglyceride, LDL-C, HDL-C, and total cholesterol levels. Plasma from mice was used to assess potential toxic effects of the drugs. Alanine transaminase (ALT), aspartate transaminase (AST), and blood urea nitrogen (BUN) levels were unchanged. Interestingly, the hepatosteatosis that is characteristic of this model was ameliorated as judged from Oil Red O staining of liver sections. The mechanisms for this effect are not known. These preliminary data suggest that specific inhibition of apo(a) with antisense technology could be a useful strategy for the treatment of high Lp(a) levels in man. Several studies are planned in transgenic mice to address the pharmacological effects of this compound on the development of atherosclerosis and to help dissect the role of Lp(a) and its relationship to oxidized phospholipids [77].

22.2.1.3 Inhibition of Apolipoprotein C-III Apolipoprotein C-III (apoC-III) is another hepatically derived lipoprotein that is thought to contribute to the development of elevated triglyceride levels, or hypertriglyceridemia (HTG).

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Figure 22.2 Second-generation ASOs targeting apo(a) inhibit apo(a) expression in vitro and in vivo. (a) Dosedependent reduction of apo(a) mRNA in human primary hepatocytes. The apo(a) ASO has no effect on plasminogen mRNA levels. (b) Reduction in hepatic human apo(a) expression in transgenic mice after 4 weeks of treatment with ISIS 144367, the human apo(a) inhibitor, or control ASO, ISIS 299705 (50 mg/kg/week). Each bar represents the mean and SEM of six replicate animals. After qRT-PCR using two human apo(a) primer probe sets positioned either on the 5⬘ or 3⬘ region of apo(a), the samples were normalized using mouse-specific G3PDH. (c) Reduction in apo(a) serum protein secretion from transgenic mouse liver after 4 weeks of treatment as described above. Each bar represents the mean and SEM of six replicate animals. Serum apo(a) levels were determined using a human apo(a) elisa assay.

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HTG, with or without concomitantly low levels of HDL-C, is now recognized as contributing to the risk of CHD [21,78,79]. Statins lower serum triglyceride levels in some dyslipidemic patients, but the fibrates are still the preferred therapeutic modality for the treatment of HTG [7,80–83]. Recent data suggest that these agents increase lipolysis and clearance of triglycerides through a reduction of hepatic apoC-III gene expression mediated by peroxisome proliferator–activated receptor ␣ [7,80–83]. ApoC-III associates with triglyceride-rich lipoproteins (VLDL and remnants) and is thought to inhibit the clearance of these particles by noncompetitively inhibiting lipoprotein lipase, an enzyme localized in the capillary endothelium [84]. The importance of apoC-III in triglyceride homeostasis has been demonstrated in vivo using genetically engineered mice. For example, transgenic mice overexpressing apoC-III have high levels of serum triglycerides as a result of their inability to catabolize triglyceride-rich lipoproteins [81,82,85]. Conversely, apoCIII-deficient mice rapidly clear VLDL [86]. More importantly, humans with HTG have increased levels of apoC-III and, in contrast, those with genetic deficiencies of that lipoprotein have low levels of VLDL enriched with TG, and rapidly convert VLDL to LDL-C [85,87]. There is also an association between increased apoC-III levels and susceptibility to type I diabetes in mice and humans [88–90]. In an insulin-dependent murine model of diabetes, mice have elevated glucose, triglyceride, and hepatic apoC-III mRNA. Administration of insulin resulted in a dosedependent down-regulation of apoC-III transcriptional activity [91], and in more recent studies, Altomonte [92] demonstrated that forkhead box O1, a nuclear transcription factor, mediated insulin’s effects on apoC-III. Takahashi [93] also suggested that apoC-III deficiency in streptozotocininduced diabetic mice prevented the development of hypertriglyceridemia. These data and clinical studies implicating total apoC-III and apoB-associated apoC-III levels as increased risks for CHD [94,95] suggest that apoC-III would be an ideal liver-derived target for antisense inhibition. Evaluation of several second-generation mouse and rat-specific apoC-III ASOs suggest that direct inhibition of that target has the predicted beneficial effect of lowering serum triglyceride levels in rodents [96,97]. As demonstrated in Figure 22.3, administration of two murine apoC-III ASOs for 6 weeks (50 mg/kg/week, i.p.) decreased hepatic apoC-III mRNA levels by 60–80% (Figure 22.3a), with concomitant reductions in serum triglyceride levels (40–60%; Figure 22.3b). Interestingly, both ASOs appeared to reduce hepatic steatosis in these C57BL/6 mice fed a high-fat Western diet, as determined by Oil Red O staining and quantitation of liver triglyceride levels (Figures 22.3c and 22.3d). More detailed studies in rat HTG models with rat-specific ASOs demonstrated similar pharmacological effects [96]. For example, administration of apoC-III compounds to Zucker fa/fa and fructose-fed Sprague-Dawley rats resulted in dose- and time-dependent reductions of apoC-III mRNA and serum triglycerides. Liver and serum apoC-III proteins and circulating free fatty acids were commensurately reduced. ASO treatment did not produce hepatotoxicity in mice or rats, as defined by increases in serum transaminases or alter any other metabolic parameter.

22.2.1.4 Inhibition of Acyl-Coenzyme A: Cholesterol Acyltransferase 2 Acyl-coenzyme A: cholesterol acyltransferase (ACAT) is an integral endoplasmic reticulum protein that plays a major role in lipoprotein particle secretion, dietary fat absorption, and intracellular cholesterol homeostasis [98–100]. Six years after the cloning of the ACAT gene in 1993 [101], two isoforms of the enzyme, ACAT1 and ACAT2, with different tissue localizations and functions, were identified [100,102–105]. ACAT1 is found in at least one cell type within most tissues of the body, with the highest levels in mice observed in macrophages and foam cells within atherosclerotic lesions, adrenal glands, and the dermis. ACAT2 is localized primarily in hepatocytes and the apical region of enterocytes. Data derived from experiments in ACAT-deficient mice helped define the potential roles of the isoforms in CHD and suggested that selective inhibition of ACAT2, not ACAT1, would be therapeutically beneficial. For example, mice lacking ACAT1 accumulated toxic levels of unesterified cholesterol in dermis and brain [106,107] and developed severe atherosclerotic lesions as a result

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ISIS 167880 50 mg/kg/week

Figure 22.3 (See color insert following page 270.) Pharmacological effect of two ASOs targeting murine apoC-III in high fat–fed C57BL/6 mice. In this representative experiment, mice were administered 50 mg/kg/week ISIS 167878 and ISIS 167880 for 6 weeks. (a) Reduction in hepatic apoC-III mRNA analyzed by qRT-PCR. Data are expressed as the mean percentage of mRNA levels in saline treated animals. (b) Reduction in serum triglyceride levels of treated animals. (c) ApoC-III ASOs reduced hepatic steatosis as assessed by Oil Red O staining of livers. (d) Quantitation of liver triglyceride levels.

of cholesterol-induced macrophage cell apoptosis [108,109]. In contrast, a deficiency in ACAT2 appeared to be beneficial, as mice lacking that isoform were protected against diet-induced hypercholesterolemia and gallstone formation [110–113]. Most importantly, mice lacking ACAT2 bred onto an apolipoprotein E–deficient (Apoe) or LDL receptor–deficient (Ldlr) background, lacked cholesterol esters in apoB-containing lipoproteins, and were protected against the development of atherosclerosis. Over the past 30 years, the pharmaceutical industry has been keenly interested in inhibiting ACAT as a way of modulating elevated lipids and preventing the development of atherosclerosis. However, this drug discovery path was initiated well before the identification of the two forms of the enzyme [114,115]. While a multitude of nonselective ACAT inhibitors of varying chemical classes (fatty acid amides, urea-derived compounds, natural products) showed promising results in vitro and in vivo in multiple models of atherosclerosis, recent clinical trial failures of two ACAT inhibitors, Avisimibe [116,117] and Pactimibe [118] have engendered skepticism as to the feasibility of this therapeutic approach to atherosclerotic heart disease. Clearly, inhibition of ACAT by antisense drugs offers a distinct advantage over the current nonselective agents as the ACAT2 isoform can be specifically targeted. Additionally, based on the well-defined pharmacokinetic properties of ASOs, liverlocalized ACAT2 can be preferentially modulated without altering its activity in the enterocytes or the availability/activity of ACAT1 in other tissues, e.g., brain, macrophages, and adrenals.

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Activity (nmol/mg/min)

Protein (AU X 1000)

mRNA (A2/gapdh)

Second-generation antisense inhibitors targeting ACAT2 and a control ASO were administered 50 mg/kg/week, i.p. to apoB-100-only Ldlr–deficient mice for 16 weeks to determine the effects of liver-specific inhibition on the development of hypercholesterolemia and atherosclerosis [119]. The ASOs selectively reduced hepatic, not intestinal, ACAT2 mRNA (80%), protein, and ACAT2 enzymatic activity. As predicted from pharmacokinetic studies, intestinal cholesterol absorption was unaffected. ACAT1 mRNA was not altered in liver or in other tissues. Hepatic cholesteryl ester (CE) was reduced, and plasma CE content was shifted to a predominantly polyunsaturated form. Plasma lipid concentrations of treated mice were similar to those found in ACAT2-deficient mice. LDL-C size was altered by ASO treatment and most likely reflected the loss of ACAT2-derived CE in the core of the LDL particle. These cumulative lipid changes resulted in a reduction in the severity of atherosclerosis in mice, as quantified by aortic CE concentration, with the most potent ASO reducing CE content by 67%. While the effect on plaque progression was not as pronounced as that observed in ACAT2-deficient mice, it was more than sufficient to suggest that pharmacological modulation of hepatic ACAT2 was atheroprotective in this model. While these studies provide convincing evidence that selective inhibition of ACAT2 reduced lipids and limited the development of atherosclerosis, additional studies in nonhuman primates are in progress to determine if these results can be reproduced in a species whose lipid and lipoprotein metabolism more closely resembles that of humans. Initial data derived from a pilot study in cynomolgus monkeys administered a monkey-specific ACAT2 ASO (30 mg/kg/week for 8 weeks) corroborate the murine results described above [120]. For example, as seen in Figure 22.4, inhibition of the monkey ACAT2 ASO significantly reduced hepatic ACAT2 mRNA (⬎70%), protein and enzymatic activity. Lipid parameters were affected as well, with 30% and 40% reductions, respectively, in total and LDL-C. As observed in mice, the CE content of the LDL-C particle decreased by approximately 50%. While preliminary, these results are encouraging and suggest that antisense inhibition of ACAT2 may be useful as an antiatherogenic therapeutic in humans.

100 80 60 40 20 0 40 35 30 25 20 15 10 5 0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0

ACAT2 mRNA Baseline 8 weeks

ACAT2 protein

ACAT2 activity

Control

ACAT2 ASO

Figure 22.4 An ACAT2 ASO specifically reduces ACAT2 mRNA, protein, and ACAT2 enzymatic activity in cynomolgus monkeys compared to baseline values. Animals, on a high-cholesterol, saturated-fat diet, were administered 30 mg/kg/week ASO for 8 weeks.

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22.2.1.5 Antisense Inhibition of ApoB-100 The most advanced cardiovascular antisense therapeutic agent, ISIS 301012, a second-generation 2⬘MOE gapmer targeted to human apoB-100, entered clinical trials in December 2003 [121,122]. The following sections will, first, describe the rationale for the selection of this target; second, review data from extensive preclinical pharmacological and toxicological studies using speciesspecific apoB-100 ASOs; and third, provide efficacy and safety data from phase 1 and 2 clinical trials.

Rationale for Inhibition of ApoB-100 The atherogenic apoB-containing lipoproteins play essential roles in the secretion and transport of dietary and endogenously produced lipids and the transport and uptake of several classes of lipoproteins [123–126]. The human apoB gene codes for two related proteins, apoB-100 and apoB-48. ApoB-100 is the principal apolipoprotein of VLDL, intermediate density lipoprotein (IDL), and LDL-C, and, as described in Section 22.2.1.2, is a key component of Lp(a), an independent risk factor for CHD. It is primarily synthesized in the liver and serves as the ligand for the removal of LDL-C by the LDL receptor and is required for the assembly and secretion of VLDL from the liver. By comparison, apoB-48, which corresponds to the N-terminal 48% of apoB-100, is the integral structural component for chylomicrons, which play a key role in dietary fat absorption. It is encoded by the same mRNA as apoB-100, but is edited by the multicomponent enzyme complex, apobec-1, in the human small intestine [123,124,126–129]. In contrast to man, mice edit apoB-100 to apoB-48 in liver as well as small intestine. The production of apoB-100-containing particles in the liver is a complex, highly regulated, and well delineated process that requires the coordinated addition of triglycerides, cholesteryl esters, free cholesterol, and the apolipoprotein itself, facilitated by the rough endoplasmic reticulum localized protein, microsomal triglyceride transfer protein (MTP) [126,127,130–133]. The expression of apoB mRNA is constant but secretion of the protein is controlled at the posttranscriptional level and varies as a function of the rate lipogenesis [124,134–136]. ApoB-100 is considered to be an atypical secretory protein in that its translocation across the endoplasmic reticulum is inefficient. This results in an increased susceptibility to ubiquitin-dependent proteasomal degradation within the hepatocyte under certain physiological conditions, i.e., the lack of MTP or the core lipids themselves [135,137]. Conversely, when lipid is in excess, the liver responds by rapidly incorporating lipids onto apoB-100, resulting in increased VLDL secretion [138,139] and, ultimately, higher levels of circulating atherogenic apoB-100-containing particles [125,126,128]. ApoB-100 has been shown to play a critical role in cholesterol homeostasis and its overproduction has been associated with various diseases. Elevated apoB and LDL-C levels are observed in several inherited diseases and correlate with premature atherosclerosis [140]. These include familial hypercholesterolemia (FH), familial defective apoB, and familial combined hypercholesterolemia. The latter is the most common of the inherited lipid disorders, affecting 1–2% of the general population [141–145] and results from the hepatic overproduction of VLDL. It is believed to be responsible for 10–20% of premature CHD [144,145]. Abnormalities in apoB-100 metabolism that increase the risk of CHD are also observed in diabetes mellitus and obesity [139,146,147]. In addition to the clear relationship of apoB-100 to LDL-C and cholesterol homeostasis, there are emerging data suggesting that apoB-100 itself is atherogenic through its ability to bind at specific sites to the proteoglycan matrix components of the vascular subendothelium [126]. Eight proteoglycan/apoB-100 binding sites have been identified to date. Interestingly, transgenic mice expressing proteoglycan binding–deficient human LDL-C exhibited far less atherosclerosis than those with the wild-type LDL-C. Conversely, reduction of apoB in vivo has been shown to be beneficial. For example, heterozygous apoB-deficient mice, whose cholesterol and apoB levels are reduced, are protected from diet-induced

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hypercholesterolemia [148,149]. Similarly, the benefits of indirectly reducing apoB levels in vivo have been suggested by using chemical inhibitors of MTP [150–152], MTP knockout mice [153], and with Reversa mice whose hypercholesterolemia can be reversed by transient induction of an Mx-I-Cre transgene [151]. Enhancement of hepatic apoB editing by providing apobec-1 to transgenic mice and rabbits reduced plasma apoB-100 [151,154] but resulted in hepatocellular dysplasia and carcinoma, which was thought to be the result of a nonspecific, aberrant editing of unrelated mRNAs involved in cell growth and regulation [155]. More recent data, however, suggest that all members of the apobec gene family might have DNA mutator activity [123]. The most relevant data suggesting that inhibition of human apoB-100 would have beneficial therapeutic effects come from studies in humans with familial hypobetalipoproteinemia, or FHBL [140,156–158]. The majority of individuals with FHBL are clinically asymptomatic and usually detected through routine cholesterol screening programs. These patients are hypolipidemic, with plasma concentrations of apoB and LDL-C typically 25–50% of that observed in normocholesterolemic individuals. In some subjects, triglyceride levels are also significantly reduced [140,156–159]. More importantly, some of these individuals have a low incidence of CHD [156,160,161] and in a recent study, have been shown to have a decreased level of arterial wall stiffness in the presence of nonlipid risk factors, suggesting that low levels of apoB-100 afford some form of cardiovascular protection [159]. Although the genetic defect in many FHBL subjects is not known, three genetic subclasses of this disease have been characterized [140,157–159]. The best-characterized defect in this autosomal dominant condition is linked to chromosome 2, and results in the inability to translate the full-length apoB-100 protein. ApoB/LDL-C levels are ⬍30% compared to age- and gender-matched controls. To date, 45 truncated apoB proteins, ranging in size from apoB-2 to apoB-89 have been identified, with metabolic consequences ranging from reduced VLDL apoB pool size and production rates, to enhanced clearance. Although the subjects with identified truncation mutations are usually clinically asymptomatic, one commonly identified consequence of impairing hepatic VLDL/triglyceride export is a mild hepatosteatosis with concomitant three- to five-fold increases in transaminases. The long-term consequences of these transaminase elevations have not been established [140,157–159]. The second subtype of FHBL has been linked to a susceptibility locus on chromosome 3p21. The genes involved at this locus have not been identified. Subjects with this defect have apoB/LDL-C levels that are approximately 50% of normal controls, are clinically asymptomatic, and have similar metabolic consequences to those with truncation mutations. Interestingly, hepatic steatosis is absent. A third FHBL group that is not linked to chromosome 2 or 3p21, but with similar characteristics to the 3p21 locus, has also been identified. Once again, these subjects lack hepatic steatosis [140,157,158].

In vivo Pharmacology of Species-Specific ApoB Antisense Inhibitors Inhibition of apoB expression by 2⬘MOE ASOs has been shown to significantly reduce hepatic mRNA and protein, serum apoB-100, LDL-C, and total cholesterol in a dose-, drug concentration–, and time-dependent fashion in several species, including mouse, hamster, rabbit, and monkey (Table 22.1). The most extensive studies have been performed in mouse and monkey and key results of these experiments will be described below. Additionally, data from a hamster-specific-apoB ASO and statin combination study will be discussed.

Murine Pharmacology The pharmacology of an optimized murine-specific apoB antisense inhibitor, ISIS 147764, was studied in various models of hyperlipidemia including C57BL/6 mice fed a high-fat diet (diet-induced obesity model), Apoe-deficient mice, and Ldlr-deficient mice. Other studies were performed in C57BL/6 mice on a normal chow diet. In each case, reductions in apoB, LDL-C, and total cholesterol were observed. A majority of these experiments was performed using the high

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Table 22.1 Evaluation of ApoB-100 Antisense Oligonucleotides in Various Animal Models Model Lean C57/BL/6 mouse High fat–fed C57/BL/6 mouse Apoe-deficient mouse Ldlr-deficient mouse High fat/chol–fed ob/ob mouse hApoB trangenic mouse Lean New Zealand white rabbit Lean hamster High fat–fed monkey

Liver ApoB mRNA (% Reduction)

LDL-Cholesterol (% Reduction)

Total Cholesterol (% Reduction)

73 87 75 74 57 75 70 80 50

64 88 40 62 52 58 67 66 71

43 55 25 40 43 30 44 52 46

fat–fed model because these mice had significantly elevated LDL-C and total cholesterol levels compared to normal chow–fed littermates [121]. Significant reductions in apoB mRNA levels in liver and serum apoB-100 levels (⬎80%) were observed as a function of dose and time in mice administered the ASO via i.p. injection. None of the multiple control ASOs—including an antisense inhibitor of MTP, scrambled and mismatch compounds—nor atorvastatin, affected apoB mRNA levels. ISIS 147764 did not alter mRNA expression of nontargeted genes, including ACAT, MTP, and HMG-CoA reductase, indicating that inhibition of apoB gene expression was both target- and sequence-dependent. Consistent with those findings, total cholesterol and LDL-C decreased by 25–55% and 40–88%, respectively. Serum triglycerides were also reduced by approximately 25%. The absolute particle numbers of all apoBcontaining lipoproteins, as assessed by NMR, were significantly suppressed as well. However, chylomicrons were unchanged. Loss of apoB mRNA and protein occurred as early as 48 h after dosing and reached maximal reductions 4–6 weeks after treatment was initiated. The lipid-lowering effects of the drug were prolonged (50% reduction 6 weeks after cessation of dosing) and correlated closely with the parent drug elimination half-life of 21 days. These results were also consistent with the extended half-life of 2⬘MOE ASOs in the liver (Chapter 11) [39,41]. Administration of drug in high fat–fed mice for as long as 5 months was not associated with any adverse effects. Levels of ALT and AST, and other metabolic parameters (glucose, ketones, and liver and spleen weights) in apoB-100 ASO–treated mice were similar to those observed in saline and ASO control animals, regardless of the model being evaluated. A key finding was that the murine apoB-100 drug did not produce hepatic or intestinal steatosis. Dietary fat absorption was not altered principally because apoB-100 ASO treatment did not affect intestinal apoB-48 protein or chylomicron formation. Histological evaluation of the small intestine from multiple experiments has demonstrated that there were no differences between saline- and apoB-treated mice.

High Fat–Fed Monkey Studies A primate study was also performed using a monkey-specific apoB ASO, ISIS 326358, to demonstrate that an antisense inhibitor targeting apoB could effectively reduce total-C and LDL-C in high fat–fed cynomolgus monkeys [162]. ISIS 326358 was administered subcutaneously to cynomolgus monkeys (5, 10, or 33 mg/kg/week) for 5 weeks. Treatment with the apoB ASO produced dose- and time-dependent decreases in hepatic apoB mRNA and serum apoB-100 protein (苲50%). Statistically significant ( p ⬍ 0.01) reductions in total cholesterol (up to 50%) and LDL-C (苲70%) were observed as well. In addition to apoB reductions, treatment with the monkey-specific ASO also reduced serum Lp(a) levels by 35%. This finding may have been anticipated because of the nature of the Lp(a) particle described in Section 22.2.1.2. Interestingly, ASO treatment also reduced apo(a) mRNA by 77% through an as yet undefined mechanism. The reduction apoB mRNA and subsequent reduction in serum lipids did not produce hepatotoxicity, as assessed by serum transaminases, nor produce hepatic or intestinal steatosis.

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ApoB ASO Treatment Does Not Produce Hepatic Steatosis in Mouse or Monkey A potential toxicity that could result from a reduction in apoB expression might be an accumulation of fat or triglycerides in the liver. Suppression of formation of hepatic apoB-100 protein, could, in theory, reduce the export of triglyceride rich-VLDL, resulting in hepatosteatosis. This phenomenon has been observed in some FHBL patients (described in Section 22.2.1.5, Rationale for Inhibition of ApoB-100), in mice with known truncation mutations of apoB-100 [159,163,164], and after administration of small-molecule inhibitors of MTP [165]. However, as mentioned above, no hepatic steatosis has been observed following apoB antisense–mediated suppression of apoB/LDL-C in either normal or hyperlipidemic mice or monkeys. Key experimental data will be described below. A study was performed in high fat–fed mice over a 20-week treatment period to determine if reductions of apoB mRNA and protein after weekly administration of 50 mg/kg ISIS 147764 would result in accumulation of triglycerides in liver. During this 20-week period, apoB mRNA, total-C, and LDL-C were reduced significantly to 70–85%, 50–70%, and 50–80% of control values, respectively. These effects on apoB-100 expression, total-C, and LDL-C were sustained over the duration of treatment. No adverse effects were observed after this prolonged treatment of mice with ISIS 147764. When liver triglyceride levels were measured, there appeared to be a slight, though not statistically significant, increase in hepatic triglyceride levels after 6 weeks of treatment with ISIS 147764. However, by 12 and 20 weeks, liver triglycerides were significantly lower (⬎60%; p ⬍ 0.01) than those observed in saline control animals. These reduced levels of hepatic triglycerides were also confirmed in Oil Red O–stained livers of ISIS 147764–treated mice. A number of experiments performed using transcriptional profiling (microarray analysis) of liver mRNA derived from apoB-100 ASO–treated mice, as a function of both time and dose, provided possible insight into the absence of hepatic steatosis associated with inhibition of apoB-100 expression. These analyses suggested that a number of key genes involved in cholesterol and fatty acid biosynthesis, as well as transport, were down-regulated as a result of the reduction in apoB. These effects were specific to apoB-100 reduction and were not observed with control ASOs or atorvastatin in high fat–fed mice. Examples of the affected genes include fatty acid synthase (FASN), stearoyl CoA desaturase (SCD), hepatic lipase (LIPC), fatty acid binding protein 2 (FABP-2), and the transcriptional activator sterol responsive element binding protein-1 (SREBP-1). Concomitant with these effects on SREBP-1, fatty acid synthase protein levels were reduced as early as 1 week after ASO administration. Fatty acid synthase is the ratelimiting enzyme responsible for the biosynthesis of saturated long-chain fatty acids. This down-regulation of the fatty acid synthetic pathway is consistent with metabolic adaptations previously described with a targeted apoB-38.9 mutation [163,164]. Up-regulation of AMP-activated protein kinase-␣ (AMPK-␣), a vital sensor of cellular energy levels that affects multiple catabolic and anabolic metabolic pathways, was also observed and is thought to be a key mechanism for promoting fatty acid oxidation [51,166]. AMPK-␣ mRNA levels were doubled after 6 weeks of treatment with the apoB ASO. Consistent with these alterations in lipogenesis/energy balance, while apoB ASO–treated mice do gain weight, they appear to be protected over time from the incremental increases in body mass typically seen in high fat–fed control mice. The resolution of hepatic steatosis via up-regulation of AMPK-␣ observed with ISIS 147764 is consistent with metabolic effects, including stabilization of weight gain without fat malabsorption, observed in ob/ob mice treated with metformin, a drug used for the treatment of Type 2 diabetes [167,168]. Inhibitors of MTP in vivo generally cause significant hepatic and intestinal steatosis, elevations in liver and intestinal triglycerides, inhibition of dietary fat absorption, and elevations in transaminases [165]. Treatment of high fat–fed mice with small-molecule and antisense inhibitors to MTP caused similar effects [169], i.e., increases in transminases and significant hepatic steatosis (Figure 22.5). Secondary transcriptional effects differed as well. For example, AMPK-␣ is not activated after MTP ASO treatment, nor is FABP2 mRNA reduced. These data suggest that pharmacological inhibition

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Saline

SMI

615

ISIS 147764

ISIS 158661

ISIS 144477

Figure 22.5 (See color insert following page 270.) Microsomal triglyceride transfer protein (MTP) inhibitors exacerbate hepatic steatosis in high fat–fed C57BL/6 mice. In this representative experiment, mice were administered 50 mg/kg/week ASOs for 6 weeks and 1 mg/kg daily of a small-molecule MTP inhibitor. Oil Red O–stained liver sections of high fat–fed C67BL/6 mice administered either saline (top left panel), ISIS 147764, the apoB inhibitor (top right panel), small-molecule MTP inhibitor (bottom left panel), ISIS 158661 and ISIS 144477, antisense inhibitors to MTP (bottom middle and right panel, respectively).

of apoB-100 with antisense therapeutics is clearly different from that of MTP using small-molecule or antisense inhibitors and that at least one factor in the toxicities of MTP inhibitors may be the failure to induce secondary liver-protective effects on the transcription of AMPK-␣ and SREBP-1 regulated genes. As described, antisense inhibition of apoB in normal and high fat–fed monkeys is not associated with hepatic steatosis. To further assess the mechanism of this effect, a subset of lipogenic genes that were altered after apoB treatment in mice were examined using monkey hepatic mRNA isolates. A similar expression pattern was observed in monkey, in that the transcription of a number of genes was reduced as a result of apoB-100 suppression, e.g., FASN, FABP2, LIPC, and SCD-1. The key finding is that apoB inhibition appears to result in reduced transcription of SREBP-1 in both species and this, in turn, reduces lipid and fatty acid synthesis. The fact that this relationship is present in rodent and monkey suggests that these compensatory mechanisms are highly conserved and likely to be operant in man. In summary, the data derived from mouse and monkey models of hyperlipidemia using speciesspecific analogs indicate that steatosis is not associated with antisense inhibition of apoB and reduction in total cholesterol and LDL-C. The apparent absence of hepatic and intestinal steatosis may be attributed to several factors. First, while humans and mice with truncation mutations of apoB have deficient export of triglycerides from the liver, antisense inhibition of apoB does not completely abolish apoB-100 protein production; therefore, the export of triglyceride-rich VLDL into the plasma still occurs, although at a diminished rate. Nor does antisense inhibition of apoB produce protein truncation mutations—full-length apoB-100 is still present, albeit at lower levels.

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Those characteristics, combined with the down-regulation of a significant number of lipogenic genes and the induction of fatty acid oxidation, appears to ameliorate any potential steatotic progression coincident with apoB suppression.

Hamster Combination Studies In the clinic, it is very likely that the human apoB antisense inhibitor will be co-administered with statins. Because both drugs lower LDL-C by distinctly different mechanisms, it might be predicted that their effects would be additive. To address this question, combination studies were performed in hamsters, a species whose lipoprotein metabolism more closely resembles that of humans [170]. In chow-fed hamsters, administration of simvastatin (10 mg/kg/day) alone reduced total cholesterol (5%) and LDL-C (16%) levels. Administration of the hamster-specific apoB-100 ASO alone for 6 weeks (50 mg/kg/week) reduced apoB-100 hepatic mRNA (Figure 22.6a) and serum protein expression by 70% and reduced serum total cholesterol (27%) and LDL-C (18%) levels, with no overt hepatotoxicity, as defined by increases in serum transaminases. A significant additive reduction in plasma LDL-C was observed in animals treated with an apoB-100 inhibitor (50 mgⲐkgⲐweek) in combination with simvastatin (10 mgⲐkgⲐday). When co-administered for 6 weeks, a 33% reduction in plasma LDL-C levels was observed when compared to saline-treated animals (from 50.8 mg/dL to 33.6 mg/dL, p ⬍ 0.01; Figure 22.6b). These data suggest that combining an apoB-100 antisense inhibitor with statins could produce additive cholesterol-lowering effects in man. ISIS 301012—The Human Antisense Inhibitor to ApoB ISIS 301012 is a second-generation antisense inhibitor that is complementary to a sequence (3249-3269 bp) within the coding region of human apoB mRNA. It is a 20-mer phosphorothioate oligonucleotide comprised of five 2⬘MOE-modified ribonucleosides at the 3⬘ and 5⬘ ends with ten 2⬘-deoxy nucleosides in between. The sequence of ISIS 301012 is 5⬘GCCTCAGTCTGCTTCGCACC-3⬘, where the underlined bases are 2⬘MOE-modified ribonucleosides and all cytosines are methylated at the C5 position.

Preclinical Evaluation The in vitro pharmacological activity of ISIS 301012 was characterized in human cell lines (HepG2, Hep3B) and in human and cynomolgus monkey primary hepatocytes. In these experiments, ISIS 301012 selectively reduced apoB mRNA, protein, and secreted protein in a concentrationand time-dependent manner, with the IC50 for mRNA reduction in primary hepatocytes ⬍10 nM. The effects of ISIS 301012 were shown to be highly sequence-specific. When a series of mismatches were introduced into the ISIS 301012 sequence and tested in HepG2 cells, a single mismatch abolished pharmacological activity. The in vivo activity of ISIS 301012 was also assessed in human apoB–expressing transgenic mice. In multiple experiments, the human apoB inhibitor specifically reduced human, not murine, apoB mRNA, hepatic and serum apoB-100 by more than 80%. Additionally, administration of ISIS 301012 to apoB transgenic/Ldlr–deficient mice significantly reduced aortic sinus plaque volume in animals with advanced atherosclerotic lesions (Figure 22.7) [171]. The potential adverse effects of treatment with ISIS 301012 have been assessed in a number of experiments. Genetic toxicity studies (in vitro bacterial cell and mouse lymphoma gene mutation) and human ether-a-go-go related gene assays have been performed, and results were all negative. Pharmacology studies to assess safety were conducted in accordance with ICH Guidelines and cardiovascular, CNS, renal, and pulmonary systems were unaffected. Fertility and reproductive toxicity studies in mice (Segment I/II) and rabbits (Segment II) were also conducted and no effects on fertility and fetal development were observed. To support clinical trials of longer duration, chronic toxicity studies are currently in progress in mice (6 months) and monkeys (1 year). To date, the toxicity of ISIS 301012 has been evaluated after

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(a) 120

ApoB mRNA (% saline control)

100

80

60

40

141923 50 mg/kg/week

349811 50 mg/kg/week

349811 25 mg/kg/week

349811 12.5 mg/kg/week

Simvastatin 10 mg/kg/day

349811 50 mg/kg/week

Saline

349811 25 mg/kg/week

0

349811 12.5 mg/kg/week

20

Simvastatin (10 mg/kg/day)

(b) 75

33% 16%

50

141923 50 mg/kg/week

Saline

349811 50 mg/kg/week + Simvastatin 10 mg/kg/day

0

Simvastatin 10 mg/kg

25

349811 50 mg/kg/week

LDL-C (mg/dL)

20%

Figure 22.6 Administration of an apoB ASO in combination with simvastatin, an HMGCoA reductase inhibitor, lowers hepatic apoB and results in additive reductions in LDL-C in chow-fed Golden Syrian hamsters. Hamsters received ISIS 349811, the apoB ASO, and ISIS 141923, a control ASO, at the indicated doses for 6 weeks, and orally administered simvastatin (10 mg/kg/day). (a) Total mRNA was prepared from liver and apoB mRNA expression analyzed by qRT-PCR. Data are expressed as the mean percentage of mRNA levels in saline-treated hamsters. (b) Additive effects of combination therapy on LDL-C levels.

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P 0.6 0.5

P P Saline

0.4 0.3 0.2

ISIS 301012 50 mg/kg/week

301012 20 mg/kg/week

Volume mm3

Normal intima

Saline

0

301012 50 mg/kg/week

*

0.1

*P = 0.033 (one-tailed t test) Figure 22.7 (See color insert following page 270.) ISIS 301012, the human apoB antisense inhibitor, reduces aortic sinus plaque volume in human apoB transgenic/Ldlr–deficient mice. Transgenic mice were administered 20 or 50 mg/kg/week ASO for 14 weeks. Top left panel: Aortic sinus region of saline treated mice. P indicates the plaque that is characterized by neointimal hyperplasia, macrophage foam cells, intracellular lipid, and fibrous caps. Lower left panel: Aortic sinus region of ISIS 301012–treated animals indicating the decrease in plaque volume. Right panel: Quantitative imaging analysis of total plaque volume within the aortic sinus. Administration of ISIS 301012 reduced total aortic sinus plaque volume in a dose-dependent fashion, with the highest dose group reducing plaque by approximately 60%.

4 and 13 weeks of treatment in mice (3.5–88 mg/kg/week) and monkeys (3.5–33 mg/kg/week). A dose-dependent exposure in plasma and tissues was observed with both ISIS 301012 and its metabolites in each of these studies. Repeated administration up to 88 mg/kg/week in mice and 33 mg/kg/week in monkeys produced no treatment-related signs of clinical toxicity, mortality, body weight change, change in food consumption, ophthalmic abnormalities, or changes in renal and cardiovascular function. Mild to moderate dose-related signs of toxicities were observed and can be attributed to the generic chemical class-specific effects of second-generation ASOs rather than specific pharmacological inhibition of apoB expression. These effects included acute and transient changes in hemostasis (primarily in monkeys), or more sustained alterations in tissue morphology (hyperplasia in liver Kupffer cells, kidney tubule epithelial vacuolation). In mice, some of the changes in tissue morphology were associated with modest alterations of hematological and serum chemistry parameters. These effects were generally observed at the high doses levels, correlated with high concentrations of ISIS 301012 in plasma and tissues, and were reversible following discontinuation of treatment. Although the tissue distribution and plasma pharmacokinetic parameters were similar between mice and monkeys, the toxicity profiles were distinct and reflect the sensitivity of mice to the immunostimulatory effects of the drugs [38,172]. Oral tolerability and intestinal absorption of ISIS 301012 with a penetration enhancer (sodium caprate, C10) have also been characterized in mice and dogs after 4 and 13 weeks of treatment. ISIS 301012 in an oral formulation with C10 was well tolerated at doses up to 2100 mg/kg/week in mice and 700 mg/kg/week in dogs. There was no histologic evidence of intestinal toxicity with oral administration of up to 2100 mg/kg/week and 700 mg/kg/week ISIS 301012 with C10 in mice and dogs, respectively.

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Clinical Trials ISIS 301012 has been extensively studied in the clinic, both in healthy volunteers and in hypercholesterolemic subjects. To date, it has been evaluated for safety, tolerability, and pharmacokinetics in four phase 1 clinical trials, both as monotherapy and in combination with other lipid-lowering agents. The first-in-man study of ISIS 301012 demonstrated significant statin-like reductions in serum apoB, LDL-C, and non-HDL cholesterol. Those results have now been confirmed in two additional phase 1 studies, providing compelling evidence for its efficacy as a lipid-lowering agent in healthy volunteers. In addition to suppression of all atherogenic lipoprotein levels, clinically significant reductions in serum triglycerides were observed. These data are consistent with the pharmacodynamic/pharmacokinetic effects observed in preclinical experiments in multiple species. Data from the completed phase 1 clinical trials are summarized below, along with a brief summary of initial results from the initial phase 2 dose escalation study in hypercholesterolemic subjects failing to reach lipid target levels with diet and exercise alone. Safety and Efficacy of ISIS 301012 in Humans First-in-Human Clinical Trial (ISIS 301012-CS1) The first-in-man clinical trial was conducted as a double-blind, randomized, placebo-controlled, dose-escalation study in 36 healthy volunteers [122]. Eighteen men and 18 women ranging from 30 to 64 years of age were enrolled. Mean fasting total cholesterol and LDL-C levels were 219 (⫾27) mg/dL and 128 (⫾22) mg/dL, respectively. ISIS 301012 was administered at doses of 50, 100, 200, or 400 mg. Following an initial single dose for safety evaluation, subjects entered a multiple dosing regimen comprised of three intravenous (i.v.) loading doses in the initial week (designed to achieve approximately 65% hepatic tissue steady state) followed by once-weekly subcutaneous (s.c.) dosing for three weeks at the same assigned dose. A statistically significant, dose-dependent and prolonged reduction of apoB and LDL-C was observed in subjects administered ISIS 301012 (Figure 22.8). Mean percent reduction in serum apoB and LDL-C levels ranged from 14% to 50% and 4% to 44% relative to baseline shortly after the end of the treatment period (Table 22.2). Significant reductions in apoB and LDL-C were observed in the 200-mg group by day 15 of the multiple dose regimen and levels of these atherogenic lipoproteins remained below baseline ( p ⬍ 0.05) up to 3 months after the last dose. This extended pharmacology was the result of a dose-dependent terminal elimination half-life of 23 days in the 50-mg group and 31 days in the 200-mg group. The prolonged duration of the effect of ISIS 301012 is consistent with (1) preclinical data with species-specific apoB ASOs and (2) the general pharmacokinetic behavior of second-generation antisense inhibitors (Chapter 11). Dose-dependent reductions were also observed in total cholesterol and triglycerides, with maximum reductions of 39% and 43% in the highest dose group of 400 mg (Table 22.2). No statistically significant changes were observed in HDL-C. NMR analysis of lipoprotein particle subclass levels demonstrated preferential reduction of small dense LDL particles. A maximum of 63% and 88% reduction in small LDL particle number was observed in the higher dose groups of 200 mg (Figure 22.9) and 400 mg (data not shown), respectively. The safety profile of ISIS 301012 in this study was satisfactory as no drug-related serious adverse events were reported. Injection-site reactions were the most common adverse event, occurring in 72% of all treated subjects. These responses typically consisted of mild, painless erythema, with spontaneous resolution over a median period of 5 days. Mild elevations in serum ALT levels, with an incidence less than or equal to currently marketed statins [173], were also observed during the course of study. These elevations were asymptomatic and never accompanied by abnormalities in liver function. This seminal phase 1 study with ISIS 301012 provides the first demonstration of potent apoB and LDL-C lowering by an antisense mechanism in humans. The outcome from this study also provided precedent for an investigation of the safety and pharmacokinetics of ISIS 301012 in combination treatment with existing oral hypolipidemic agents as well as the first phase 2 monotherapy study in subjects failing to meet target cholesterol levels with diet and exercise alone.

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(a)

(b) Placebo

50 mg

100 mg

200 mg

Placebo

400 mg

LDL-C mean % change

ApoB mean % change

−10 −20 −30 −40

200 mg

400 mg

−10 −20 −30 −40 −50

−50

−60 0

10

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0 −10 −20

*

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†

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400 mg

LDL-C mean % change ±SEM

ApoB mean % change ± SEM

100 mg

0

0

−60

50 mg

10

10

0

Placebo

50 mg

100 mg

200 mg

400 mg

−10 −20 −30 −40

†

−50

Figure 22.8 Dose-dependent effect of ISIS 301012 on (a) apoB and (b) LDL C levels, presented as mean percent change from baseline. The multiple dosing period was from day 0 to day 22. Baseline is defined by measure prior to initial treatment of each subject. Significant reductions in (c) ApoB and (d) LDL-C were observed in the 200-mg group in the posttreatment period (day 39). Data presented as percent change from baseline, bars represent mean ⫾ standard error. P values determined from the Wilcoxon Rank-Sum test, where “*” indicates ⱕ0.05 and “†,” ⱕ0.01.

Table 22.2 ApoB and Lipids Levels in Humans after Administration of ISIS 301012 (ISIS 301012-CS1) Characteristic ApoB

LDL-C

HDL-C

Cholesterol

Triglyceride

Mean ⫾ SD Median IQR Mean ⫾ SD Median IQR Mean ⫾ SD Median IQR Mean ⫾ SD Median IQR Mean ⫾ SD Median IQR

Placebo (7)

50 mg (8)

100 mg (8)

2.3 ⫾ 20.9 ⫺13.5 ⫾ 9.0 ⫺22.2 ⫾ 15.0* 4.4 ⫺15.9 ⫺19.2 ⫺20.4 to 12.2 ⫺19.0 to ⫺ 7.6 ⫺23.2 to ⫺12.7 ⫺0.8 ⫾ 13.0 ⫺3.7 ⫾ 15.8 ⫺18.6 ⫾ 13.5 –2.6 ⫺7.8 –17.8 ⫺8.6 to 12.6 ⫺13.5 to 2.1 ⫺31.1 to ⫺7.1 7.4 ⫾ 19.7 5.8 ⫾ 9.5 1.2 ⫾ 13.2 ⫺1.6 6.1 ⫺0.8 ⫺3.6 to 29.2 1.6 to 12.4 ⫺8.3 to 12.2 7.2 ⫾ 9.6 ⫺1.2 ⫾ 8.7 ⫺12.4 ⫾ 8.2† 8.5 ⫺0.1 ⫺12.9 ⫺2.9 to 14.9 ⫺10.0 to 6.9 ⫺18.5 to –5.2 12.0 ⫾ 43.1 23.4 ⫾ 34.5 9.9 ⫾ 44.1 ⫺3.3 15.6 3.7 ⫺17.1 to 39.2 ⫺1.7 to 40.2 ⫺12.7 to 17.5

200 mg (8) ⫺38.5 ⫾ 18.0† ⫺36.6 ⫺57.2 to ⫺21.3 ⫺35.2 ⫾ 19.3† ⫺35.2 ⫺47.1 to ⫺21.6 0.9 ⫾ 9.5 0.6 ⫺4.9 to 4.5 ⫺26.8 ⫾ 13.9‡ ⫺28.0 –35.7 to ⫺12.9 ⫺15.1 ⫾ 23.1 ⫺19.9 ⫺30.0 to ⫺3.2

400 mg (2) ⫺49.5 ⫺49.5 ⫺57.9 to ⫺41.1 ⫺44.2 ⫺44.2 ⫺51.2 to ⫺37.1 5.2 5.2 ⫺0.4 to 10.8 ⫺38.6 ⫺38.6 ⫺46.5 to ⫺30.7 ⫺42.8 ⫺42.8 ⫺51.7 to ⫺34.0

Note : IQR stands for interquartile range and SD, standard deviation. Percent change relative to baseline 2 weeks after cessation of the treatment period (day 39). P values were determined by the Wilcoxon Rank-Sum Test. ∗ ⱕ0.05. † ⱕ0.01. ‡ ⱕ0.001.

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(a)

Large

621

Small

1600

Mean particle conc. (nM)

1400 1200 1000 800 600 400 200 0 0

8

15

22

25

39

55

69

83

Day (b)

Day 55 (200 mg)

LDL (nmol/L)

% change (baseline)

Total

684 ± 87

− 51 ± 7‡

Large

385 ± 41

− 27 ± 6*

Small

289 ± 82

− 63 ±11†

Figure 22.9 Preferential and prolonged reduction of small LDL-C after administration of ISIS 301012 in the 200-mg dose group. (a) Mean LDL particle subclass levels from day 0 to day 83. (b) Maximum mean percent change from baseline in small LDL particle levels was observed on day 55. P values were determined by the Wilcoxon Rank-Sum test, where “*” indicates ⱕ0.05; “†,” ⱕ0.01; and “‡,” ⱕ0.001.

Confirmation of Safety and Efficacy (ISIS 301012-CS101) In a second phase 1 study designed to evaluate oral dosage forms of ISIS 301012 (Chapter 8), six subjects were administered an equivalent 350 mg/week of ISIS 301012 by i.v. infusion over a 6-week treatment period. Three men and 3 women ranging from 21 to 55 years of age, with a mean fasting total cholesterol level of 228 (⫾ 50) mg/dL and LDL-C level of 160 (⫾ 38) mg/dL were randomized into this cohort. This “positive control” group (the cohort receiving the parenteral equivalent of 350 mg/week) achieved a median reduction in apoB and LDL-C of 60% and 54%, relative to baseline (Figure 22.10). Serum triglycerides were also significantly reduced by 46% ( p ⫽ 0.005) in this cohort. The long duration of effect was similar to that observed in the initial phase 1 study (Figure 22.8). Two subjects experienced minor elevations in liver transaminases that might be related to study drug, however, this relationship is unclear. No other unexpected toxicities or safety issues were reported. Combination Treatment with Other Lipid-Lowering Agents (ISIS 301012-CS2) Statins and ezetimibe are two of the most commonly prescribed cholesterol-lowering agents today. Statins act by inhibiting HMG CoA reductase in the liver and subsequently increasing hepatic LDL receptor expression, while ezetimibe inhibits absorption of dietary cholesterol from the small intestine. A phase 1 study was performed to determine the safety and pharmacokinetics of ISIS 301012 as an add-on therapy to each of these two cholesterol-lowering agents and to specifically provide evidence for the lack of a drug-drug interaction with the antisense inhibitor to apoB. This study specifically evaluated the pharmacokinetics of combinations of simvastatin/ISIS 301012 and ezetimibe/ISIS 301012 and was designed as a one-sequence crossover of the oral hypolipidemic agent, initially administered alone, and subsequently, in combination with 200 mg of ISIS 301012. The dose regimen for ISIS 301012 was designed to achieve near steady-state tissue accumulation by the end of dosing, equivalent to once-weekly dosing of 200 mg. Twenty healthy male volunteers ranging in age from18 to 64 years and with a mean fasting total cholesterol level of 183 (⫾ 40) mg/dL and LDL-C level of 129 (⫾ 37) mg/dL were enrolled in this

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20

Triglyceride mean % change

10 0 −10 −20 −30 −40 −50 −60

0

10

20

30

40

50

60

70

80

90

Days

Figure 22.10 Confirmation of ISIS 301012 efficacy after administration of 350 mg/week to subjects in a second phase I study (ISIS 301012-CS101). Mean percent change from baseline in (a) apoB, (b) LDL-C, and (c) triglycerides. Treatment period was for 6 weeks starting on day 0. Placebo is pooled data from two cohorts receiving oral dosage forms without drug (n ⫽ 12). Data are presented as percent change from baseline, where bars represent mean ⫹ standard error.

open-label study. Subjects received either a single oral dose of 40 mg of simvastatin (n ⫽ 10) or 10 mg of ezetimibe (n ⫽ 10) on day 1. Following clearance of the initial dosed drug, subjects received four i.v. infusions of 200 mg of ISIS 301012 over an 8-day period. The combination agents were then administered again with the last dose of ISIS 301012. The pharmacokinetics of ISIS 301012 was not significantly affected by concomitant administration of either simvastatin or ezetimibe. Consistent with the well-established fact that antisense compounds are not metabolized via the cytochrome P450 system, plasma concentration curves and plasma half-lives of simvastatin and ezetimibe were also unaffected when administered in combination with ISIS 301012. A moderate lowering in the Cmax of simvastatin and free ezetimibe was

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observed upon co-administration with ISIS 301012. These values, however, were consistent with those reported in the literature for each agent alone. The safety profile of ISIS 301012 in combination with each of these drugs was satisfactory. No abnormal laboratory parameters, including elevations in transaminases, were present in this study and no serious adverse events were reported.

Treatment of Hypercholesterolemia (ISIS 301012-CS3; NCT00216463) Following demonstration of substantial apoB and LDL-C lowering in healthy volunteers, a phase 2 clinical study was designed to examine dose and dose frequency of ISIS 301012 as a single agent in hypercholesterolemic subjects unable to reach target cholesterol levels by lifestyle changes alone. Treatment duration was extended to 13 weeks. Pharmacological outcomes from the first three dose cohorts are summarized below. Twenty-five men and 5 women, 28 to 64 years of age, were enrolled in this placebo-controlled, double-blind study. Fasting baseline total cholesterol ranged from 195 to 340 mg/dL, with a mean of 251 (⫾ 36) mg/dL, while baseline LDL-C levels ranged from 127 to 266 mg/dL, with a mean of 168 (⫾ 32) mg/dL. Subjects were randomly assigned to receive one of three ISIS 301012 dose regimens or placebo (4:1 ratio of active:placebo) for 3 months. Study drug was administered by s.c. injection at a dose of 200 mg twice weekly for two weeks followed by 100 (50 mg/week group) or 200 mg (100 mg/week group) every other week for 11 weeks, or 200 mg once per week for 13 weeks (200 mg/week group) without an initial loading dose. Weekly dosing with 200 mg ISIS 301012 for 13 weeks resulted in a 42% reduction in LDL-C from baseline ( p ⬍ 0.001 to placebo) two weeks after treatment, with concomitant reductions in apoB (47%, p ⬍ 0.001) and non-HDL cholesterol (44%, p ⬍ 0.001—data not shown). Significant reductions in triglycerides and VLDL were also observed with median reductions of 46% ( p ⫽ 0.04) and 54% ( p ⫽ 0.02), respectively. ApoB and LDL-C levels were reduced by 22–23% and 12–22%, respectively, in the 50- and 100-mg/week dose groups. No significant changes in HDL-C levels were observed. As monotherapy, ISIS 301012 has now demonstrated significant lowering of apoB, LDL-C, VLDL, total cholesterol, and triglycerides in healthy volunteers and subjects who are unable to control elevated cholesterol by diet and exercise alone. Treatment has been well tolerated in all dosing cohorts. Tolerability and Safety Profile Subcutaneous Injections Local injection-site reactions (ISRs) are the most common adverse event observed in subjects after s.c. administration of ISIS 301012 (Table 22.3). These reactions are a common side effect in humans of subcutaneous administration of both first- and second-generation ASOs (Chapter 13), as well as other pharmacologic agents [174]. ISRs, which appear to be dosedependent, are most accurately characterized as painless local erythema that subsides spontaneously. Two approaches have been evaluated to mitigate the incidence and severity of skin reactions in an open-label, observer-blind, dose-escalation phase 1 study (ISIS 301012-CS301). The first was the co-administration of ISIS 301012 with local corticosteroid. The second approach was to divide a single dose into multiple simultaneous, smaller, s.c. injections. Local skin responses were assessed clinically and by microscopic analysis of skin biopsies. In cohorts that evaluated the effects of concomitantly administered corticosteroids, the overall incidence of erythema was 42% (21 of 50 subjects) at the ISIS 301012–alone injection site, and 36% (18 of 50 subjects) at the steroid co-administered site. A notable reduction in the duration of erythema was observed in subjects who received a 200-mg dose of ISIS 301012 admixed with 1 mg dexamethasone sodium phosphate (n ⫽ 10), where a median duration of 2 days was observed compared to 4 days at the ISIS 301012–alone injection site. Usage of multiple injection sites for a single dose had no clinically relevant effect on the incidence or duration of erythema. Histologically, ISRs were characterized by prominent macrophage and neutrophil infiltration, whereas negative clinical findings were associated with minimal infiltration. The onset of microscopic changes was generally seen at 24–48 h postinjection. No significant microscopic findings were

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ANTISENSE DRUG TECHNOLOGY, SECOND EDITION Table 22.3 Adverse Events Reported in ⱖ5.0% of ISIS 301012–Treated Subjects in ISIS 301012-CS1, CS2, CS3, CS101 (Positive Control Cohort), and CS301 ISIS 301012 (N ⫽ 139) Adverse Event

n

Erythema Swelling Bruising Induration Inflammation Hemorrhage

76 46 30 23 21 8

Headache Fatigue Discomfort Pyrexia

33 17 12 9

Placebo (N ⫽ 13) %

n

%

Injection Site Reactions* 67 41 27 20 19 7.1

0 0 0 0 0 0

0 0 0 0 0 0

24 12 8.6 6.5

7 1 0 0

54 7.7 0 0

8.6

1

7.7

7.2 5.8

1 1

7.7 7.7

5.0

1

7.7

General

Infection Nasopharyngitis

12 Gastrointestinal

Nausea Constipation

10 8

Elevated ALT

7

Liver Function

* Percent of subjects are based on a total of 113 subjects who received ISIS 301012 subcutaneously. No ISRs were reported for subjects who were administered ISIS 301012 by intravenous infusion only (CS2, CS101).

observed in biopsies from the steroid co-administered site at these time points. The degree of histologic change within the biopsy sites correlated directly with the amount of stainable (and measured) drug present. Methods to determine whether more rapid systemic absorption may mitigate this histology are currently under investigation.

Other Adverse Events Asymptomatic, transient, and, in most cases, mild elevations in hepatic transaminases (⬍3 X upper limit of normal) have also been observed in ISIS 301012–treated subjects. In each incidence, transaminase levels returned to normal without the need for additional interventions. No associated abnormalities in bilirubin or evidence for liver dysfunction have been observed. Two reports of hepatic steatosis in subjects with elevated ALTs have been received, but in each instance the relationship to study drug is uncertain. No clinically relevant changes in renal function, as determined by urinalysis, glomerular filtration rate or serum creatinine, have been observed. Additionally, in contrast to MTP inhibitors [165], there has been no evidence of steatorrhea in any ISIS 301012 study to date, nor of changes in serum vitamin A levels that would indicate an adverse affect on fat-soluble vitamin absorption. Hybridization-independent toxicities such as aPTT prolongation have occurred within the expected ranges, been rapidly reversible, and have not been clinically significant. No other clinically significant adverse events have been observed and no treatment-related serious adverse events (SAEs) have been reported to date. Combination Treatment Combination dosing of ISIS 301012 with simvastatin or ezetimibe yielded no adverse events in healthy volunteers. This result sets the stage for further evaluation of ISIS 301012 in patients on stable doses of these widely prescribed lipid-lowering agents. These studies are currently in progress.

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Table 22.4 Efficacy of ISIS 301012 after 3 Months of Dosing at 200 mg/Week in Comparison to Efficacy of Statins after Intensive Treatment for 4 (Rosuvastatin) and 18 Months (Atorvastatin) Intervention

Antisense ApoB

Rosuvastatin

Atorvastatin

Clinical Trial Dose Treatment Period

ISIS 301012-CS33 200 mg/Week 3 Months

ASTEROID1 40 mg/Day 24 Months

REVERSAL2 80 mg/Day 24 Months

125 69 ⫺47

128 75 ⫺42

152 92 ⫺39

171 96 ⫺42

130 61 ⫺53

150 79 ⫺46

ApoB Baseline Endpoint % Change LDL-C Baseline Endpoint % Change

Note : Mean baseline and endpoint values are in mg/dL. 1 Nissen, S.E. et al., JAMA, 295, 1556–1565, 2006. 2 Nissen, S.E. et al., JAMA, 291, 1071–1080, 2004. 3 Percent change from baseline is presented as the median value.

Efficacy Profile Antisense inhibition of apoB in humans results in statin-like reductions in apoB, LDL-C (Table 22.4), and all atherogenic lipoproteins and lipids, including triglycerides. The favorable impact on LDL-C subclass distribution is encouraging. Significant changes in HDL-C have not yet been observed in these studies with small numbers of subjects [175]. The relatively long, dose-dependent tissue half-life of ISIS 301012 yields persistent and prolonged reductions in atherogenic lipoproteins. This attribute can be expected to eventually facilitate less frequent dosing, perhaps monthly or even quarterly drug administration. Ongoing and Future Studies With positive outcomes in the initial clinical trials, phase 2 studies are now in progress to (1) assess pharmacology when administered in combination with statin therapy, (2) evaluate safety and pharmacology of higher doses, (3) define dosing intervals, (4) investigate safety and pharmacology in subjects with familial hypercholesterolemia (FH), and (5) determine if pharmacological reduction in apoB results in hepatic steatosis (as assessed by serial liver triglyceride content) in subjects after administration of ISIS 301012. Inhibition of ApoB Using siRNA in Preclinical Animal Models Inhibition of apoB expression was also observed in mice after short-term treatment with a cholesterol-conjugated apoB siRNA [176]. In these studies, intravenous administration of the conjugate to C57BL/6 and human apoB transgenic mice delivered drug into several organs, including liver, intestine, heart, kidney, lung, and adipose tissue. ApoB mRNA was reduced by 苲70% in the jejunum and 50% in liver. Serum apoB-100 protein, total cholesterol, LDL-C, and chylomicron levels were also reduced (68%, 37%, 40%, and 50%, respectively). Unconjugated apoB siRNAs were not taken up into cells and were, therefore, pharmacologically inactive. The authors did not show pharmacological effects and stability of the drug in long-term studies in the chow-fed, transgenic or, importantly, hyperlipidemic mice. The potential toxicities of this compound and the cholesterol adduct alone, including elevations in transaminases and effects on lipid accumulation in liver and intestine, were not addressed. This is particularly important given that our laboratories have demonstrated that cholesterol-conjugated oligonucleotides are hepatotoxic [177]. Finally, given the significant reductions in chylomicrons observed after drug treatment, dietary fat and fat-soluble vitamin malabsorption could be a serious side effect of the cholesterol-conjugated apoB siRNA in vivo. An apoB specific-siRNA that was encapsulated in a liposomal preparation (stable nucleic acid lipid particles-SNALP) also inhibited apoB mRNA and protein and lowered total and LDL-C in

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monkeys after single i.v. injections of 1 or 2.5 mg/kg of the formulated drug [178]. Two days after administration of the compounds, liver apoB mRNA was reduced from 68% to 90% (1 and 1.5 mg/kg doses, respectively) and correlated with tissue levels of the drug. The reduction in apoB message was prolonged, as 2 monkeys, 11 days after receiving the higher dose of drug, still had an approximate 91% reduction of apoB message. There was no effect of the siRNA-SNALP on apoB mRNA levels in the jejunum, in contrast to the effects of the cholesterol-siRNA conjugate described. Maximal reductions of LDL-C and total cholesterol were 82% and 62%, respectively in the highest dose group. Tolerability of the SNALP-siRNA was evaluated using various parameters and while there was no evidence of complement activation, delayed coagulation, or the induction of proinflammatory cytokines, there was, however, a significant and prolonged increase in transaminase levels that peaked 48 h after injection of the highest dose. Both AST and ALT levels were approximately 25 times greater than predose values at this time. The transaminitis appeared to resolve modestly over time. For example, at days 6 and 11, ALT levels day values were still 12 and 3 times greater, respectively, than those observed before administration of the drug. The long-term effects of SNALP-apoB siRNA were not addressed in lean or hyperlipidemic monkeys and, most importantly, the authors did not determine what factor or factors (e.g., liposomes) were responsible for the transaminitis. As others have mentioned in this volume (Chapters 1 and 3), while siRNA is a great target validation tool in vitro, many hurdles, including delivery, safety (off-target effects, toxicities of delivery vehicles), and development issues, remain before siRNA therapeutics can be used for the chronic treatment of a variety of diseases. 22.2.2 Antisense Inhibition of CRP, a Nonlipid Hepatic Target Involved in CHD CRP, the prototypic positive acute phase protein, is produced primarily by the liver as part of the body’s homeostatic mechanism to restrict injury and promote repair after an acute inflammatory stimulus [179–183]. Elevated levels of CRP are often observed in a variety of medical conditions including metabolic syndrome, various inflammatory diseases and malignancies, diabetes mellitus, and end-stage renal disease. However, in recent years, the relationship of CRP to the inflammatory aspects of atherosclerosis has been an area of intense interest [184–186]. On the basis of multiple prospective epidemiological studies, CRP has become recognized as an independent marker and powerful predictor for future risks of myocardial infarction (MI), stroke, and deaths from CHD in individuals apparently free of known cardiovascular disease [181,185,187]. The precise function of CRP in CHD is extremely controversial and many in the scientific community believe that it is solely a biomarker, and not a participant in the pathogenesis of atherosclerosis [188,189]. Nonetheless, there is increasing evidence in vitro that CRP promotes vascular damage, increases thrombosis, and inhibits fibrinolysis within plaques. In vivo studies have demonstrated that CRP can activate complement [190], was detected in early atherosclerotic lesions [191], and was colocalized with activated complement components and enzymatically degraded LDL in human atherectomy lesions [192,193]. Even more compelling data suggestive of CRP’s role as a mediator of atherothrombotic events emerged from a study by Bisoendial [194] where administration of recombinant human CRP to human volunteers was shown to induce endothelial cell activation, activate the coagulation cascade, and elicit an acute systemic inflammatory response. What is needed to address (1) the epiphenomenon versus causal role of CRP in the pathogenesis of atherosclerosis and (2) whether reduction of that acute phase reactant results in a meaningful decrease in adverse clinical outcomes in CHD is a pharmacological inhibitor of CRP. To date, the only small-molecule inhibitor of CRP is 1,6-bis(phosphocholine)-hexane, which binds circulating CRP [195]. Preliminary data suggested that daily, intravenous infusion of this compound was cardioprotective in rats. However, a more stable and direct inhibitor of human CRP would be more useful to delineate the role of that acute phase reactant in CHD. Several monkey/human cross-reactive antisense inhibitors targeting hepatic CRP have been characterized and tested in vitro and in vivo in our laboratories [196]. The potency for reduction of CRP mRNA and protein was confirmed in both monkey and human primary hepatocytes

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(IC50 ranged from 5 to 25 nM). Two sequences were compared in a dose escalation study where animals were administered these second-generation 2⬘MOE compounds, using either the 5-10-5 or 3-14-3 configuration, in four dosing cycles over an 8-week period and then challenged with IL-6. Several of the compounds were pharmacologically active as defined by their ability to reduce IL-6-induced hepatic CRP mRNA (60–85%) and serum CRP (30–66%) levels. Standard toxicological endpoints, including clinical signs, serum chemistry, hematology, and body weights, were evaluated as well. Our data suggest that the antisense pharmacological reduction of CRP was safe. All four ASOs produced a similar spectrum of changes, although subtle differences in tissue distribution and concentration were observed in the kidney with the 3-14-3 gap-widened compounds. Several human/monkey CRP inhibitors were also evaluated in mice expressing the human CRP transgene [197–199]. Human CRP has been shown to accelerate the rate of thrombotic occlusion after vascular injury and potentially contribute to the progression of atherosclerosis in apolipoprotein E–deficient mice in one laboratory [200]. In a preliminary experiment from our laboratories, serum CRP was reduced (75–99%) in transgenic mice after 2 weeks of treatment with 50 mg/kg/week CRP ASOs. In another study, 2-week systemic administration of a human CRP antisense inhibitor (50 mg/kg/week) significantly reduced serum CRP levels (⬎90%; Figure 22.11a) and improved time to thrombotic occlusion in animals after photochemical injury (Figure 22.11b). More extensive (a) 6

CRP (mg/L)

4

2

0 Baseline

Pre-injury

Post-injury

(b)

Occlusion time (min)

160

120

80

40

0 WT

ASO-tg

CRPtg

Figure 22.11 Effect of a human CRP ASO in CRP transgenic mice administered drug (50 mg/kg/week) for 2 weeks. (a) Reduction in serum CRP levels pre- and post-photochemical vascular injury. (b) CRP ASO treatment improves time to occlusion after vascular injury.

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studies are in progress to determine the therapeutic effectiveness of these inhibitors in models of carotid artery ligation (transgenic mouse and normal rat) [201]. 22.2.3 Antisense Inhibitors for the Treatment of Hypertension Hypertension has been established as a causal, independent risk factor for the development of CHD and the benefits of antihypertensive therapy have been demonstrated in several large, interventional clinical trials [50,201–203]. The factors that contribute to the normal and hypertensive states are complex and multifactorial. Essential hypertension, or systemic arterial hypertension, occurs in 90% of patients with elevated blood pressure, while the remaining 10% of patients develop hypertension that is secondary to renal disease [203]. Even with an extensive antihypertensive therapeutic armamentarium (diuretics, Ca2+ channel blockers, vasodilators, sympatholytic drugs, angiotensin-converting enzyme inhibitors, and angiotensin II- receptor antagonists), only 30% of hypertensive patients can adequately control their blood pressure. In fact, the incidence of hypertension has increased over the past decade [201–204]. The renin-angiotensin system (RAS) is vital to the regulation of arterial blood pressure and components of the system are localized in the CNS, kidney, pituitary, adrenal glands, liver, heart, lung, fat, and the vasculature [205,206]. Key mediators of the RAS include angiotensin I and II, (Ang I and II), angiotensin II type 1 and type 2 receptors (AT1R, AT2R), angiotensinogen (AGT), angiotensin-converting enzyme (ACE), and renin. Data from human genetic studies and a variety of rodent transgenic and knockout models suggest that dysregulation of this complex pathway contributes to the hypertensive state [203,206,207]. First-generation (phosphodiester and phosphorothioate) ASOs designed to inhibit components of RAS have been tested in rodent models [203,206,208,209], including the spontaneously hypertensive rat (SHR), a model of essential hypertension [210], the two kidney one clip surgical [211], and the cold-induced hypertension model [212]. AT1R, AGT, and renin ASOs administered by intracerebroventricular (i.c.v) injection into the 3 models described above resulted in transient 16–45% reductions in blood pressure [203,206]. In some experiments, in addition to reduction in blood pressure, drinking responses to renin and isoproteronol were attenuated. The extent and duration of the effects varied (2 to more than 5 days) as a function of the model and the chemical modification of the drug. Systemic administration of those ASOs has also been reported to modestly reduce elevated blood pressure (20–30%) [206]. More recently, viral vectors have been used to deliver these first-generation RAS ASOs in the brain and systemically [203,206,208], with several groups demonstrating long-lasting antihypertensive effects in normo- and hypertensive animal with no evidence of toxicities. An enormous amount of work must be completed before RAS or other antihypertensive-specific ASOs can approach the clinic. For example, while reductions in hypertension were observed after ASO treatment, the effects were modest and not superior to those observed with existing antihypertensive therapeutics. Additionally, the routes of administration of these ASOs for this indication are inconvenient (i.c.v) and have the potential to produce serious toxicities (viral vectors) [206,213,214]. The use of the retroviral vectors for delivery of ASOs is also not necessary given that first and, most importantly, second-generation compounds have been shown to be widely distributed and pharmacologically active in vivo after multiple routes of administration. Nevertheless, these proof-of-principle studies suggest that antisense inhibitors may be a useful therapeutic approach for the treatment of another unmet cardiovascular medical need. 22.2.4 Antisense Inhibitors Affecting Restenosis Percutaneous coronary intervention (PCI) is the primary revascularization procedure for the treatment of coronary artery disease. Drug eluting stents coated with sirolimus or paclitaxel have proven to limit the incidence of in-stent restenosis by local delivery of these antiproliferative

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agents to the site of angioplasty [215,216]. Consistent with this approach, ASOs targeted to a variety of regulatory and signaling molecules involved in excessive vascular smooth muscle proliferation have also been investigated for prevention of neointimal hyperplasia [217–224]. Some of the targets that have been investigated both in vitro and in vivo include cyclin-dependent kinase 2 (cdk2), cell division cycle 2 kinase (cdc2), cyclin B1, proliferating cell nuclear antigen (PCNA), NF-B, platelet-derived growth factor (PDGF), c-raf, basic fibroblast growth factor (bFGF), c-myb, and c-myc. Antisense inhibitors for c-myc, a proto-oncogene that regulates the growth of vascular cells within atherosclerotic lesions, have been evaluated in the clinic for their ability to prevent PCIinduced intima cell proliferation. Two types of antisense chemistries have been evaluated—a firstgeneration phosphorothioate ASO [225] and a neutral (dimethylamino) phosphinylideneoxy-linked morpholino ASO (Chapter 20) [218,226]—both based on the sequence complementary to the translation initiation region of the c-myc transcript. In the ITALICS trial, a 15-mer phosphorothioate ASO was evaluated for its ability to inhibit restenosis in patients who received a coronary stent implant [225]. A single dose of 10 mg of ASO was delivered locally by catheter to 85 patients immediately after stent placement. The primary end point was percent in-stent volume obstruction as measured by intravascular ultrasound (IVUS) 6 months following catheterization. Unfortunately, the investigators were unable to distinguish differences between the in-stent volume in placebo and antisense-treated patients. Additionally, there were no significant differences in secondary angiographic parameters or in the clinical event rate between treatment groups at the 6-month follow-up visit. Lack of efficacy was attributed to insufficient absorption and uptake of oligonucleotide by the intima tissue, and, possibly, insufficient local drug retention time using the catheter-based delivery system. The c-myc ASO, AVI-4126, or Resten-NG, used in the AVAIL trial had been previously reported to inhibit c-myc mRNA expression and reduce neo-intimal formation after endoluminal delivery in treated vessels in rabbit and pig models of restenosis (Chapter 20) [225,226]. The investigators concluded that high-dose Resten-NG, 6 months after local delivery (10 mg) via infiltrator catheter during PCI to a small cohort of patients (n ⫽ 12), safely reduced neointimal formation. A larger phase 3 trial has been initiated to confirm and extend these results and to show superiority over existing drug-eluting stents. To date, no further data have been reported.

22.3 SUMMARY AND FUTURE PERSPECTIVES Cardiovascular disease is currently the leading cause of morbidity and mortality in the United States and all industrialized nations, with total healthcare costs approaching hundreds of billions of dollars. While multiple risk factors have been identified that predispose individuals to this disease, it has become fairly clear that there is a direct and causal relationship between elevated LDL-C and CHD. Despite the availability of existing therapies, only a small fraction of high-risk patients achieve their LDL-C goals. The recent revision of the NCEP guidelines recommending even lower LDL-C levels, coupled with the growing recognition of the involvement of other lipid (Lp(a), triglycerides, HDL-C) and nonlipid factors (CRP) in cardiovascular disease, suggests the need for novel and more effective pharmacological agents to be used as monotherapy or in combination with existing drugs. The (1) specificity of antisense technology, (2) optimal distribution within liver, kidney, and atherosclerotic plaques, (3) ability to inhibit unique targets involved in dylipidemias and cardiovascular disease, and (4) their lack of interaction with the cytochrome P450 system suggest that antisense drugs represent a valuable, novel therapeutic modality. Efficacy and safety data derived from extensive preclinical and clinical studies, especially with the newer generation 2⬘MOE drugs, suggest that these compounds have great potential to fulfill the unmet medical needs in cardiovascular medicine.

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The antisense agents described in this chapter, many representing previously unapproachable targets, have shown impressive effects in vivo, and may, with more extensive evaluation, prove efficacious in the clinic. The collective data provide an excellent demonstration of the strengths of the technology platform and, importantly, help delineate the role of various unique genetic factors contributing to dyslipidemias and the pathogenesis of atherosclerotic heart disease. ISIS 301012, the second-generation 2⬘MOE drug to human apoB-100, is the most advanced of the antisense inhibitors for the treatment of cardiovascular disease. It is an exemplar antisense drug and highlights the efficiencies of the technology. ApoB-100 has been therapeutically unapproachable by small-molecule inhibitors or antibodies but has proven to be an excellent candidate for antisense inhibition. On the basis of data from extensive preclinical studies, our hypothesis that suppressing apoB mRNA in the liver would ultimately result in concomitant reductions in total cholesterol and LDL-C was proven correct. Consistent with the preclinical data, ISIS 301012 has shown impressive efficacy in four phase 1 studies and one phase 2 trial, with statistically significant and prolonged statin-like reductions in serum apoB, LDL-C, and non-HDL cholesterol. Serum triglycerides were reduced as well, a predicted consequence of reducing the export of triglyceriderich VLDL from the liver. While the efficacy and safety data are encouraging, ISIS 301012 is still early in development. The studies to date have been small in size and limited in duration and are probably not sufficient to demonstrate all possible side effects. Areas of continued research for ISIS 301012 (and the other compounds described above) include evaluating toxicities that are (1) specific to inhibition of the target, (2) that might arise when ISIS 301012 is given in combination with existing antihyperlipidemic agents, and (3) after chronic administration. Assuming the safety and efficacy profile continues to be positive, we believe this drug will not only be an important addition to the cardiovascular therapeutic armamentarium but also a significant proof of principle for the antisense platform.

ACKNOWLEDGMENTS The authors would like to thank Tracy Reigle for her expertise in graphics, Donna Parrett for her help in finalizing the manuscript, and Kristina Lemonidis, Mark Graham, and Kannan Subramaniam for their keen laboratory skills and review of the chapter.

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Developing Antisense Drugs for Metabolic Diseases: A Novel Therapeutic Approach Sanjay Bhanot

CONTENTS 23.1 23.2 23.3

Introduction .........................................................................................................................642 Rationale .............................................................................................................................642 Antisense Drug Discovery ..................................................................................................642 23.3.1 Drug Discovery for Type 2 Diabetes ....................................................................644 23.3.1.1 Antisense Targeting of Protein Phosphatases ......................................644 23.3.1.2 Targeting Transcription Factors with Antisense Drugs........................647 23.3.1.3 Antisense Strategies to Inhibit Hepatic Glucose Output .....................648 23.3.1.4 Exploiting Tissue Selectivity and Pharmacokinetic Properties of Antisense Drugs ...................................................................................649 23.3.1.5 Targeting the Kidney Using Novel Antisense Chemistries .................651 23.3.2 Antisense Drug Discovery for Obesity.................................................................652 23.3.2.1 Antiobesity Effects of ISIS 113715, a PTP-1B Antisense Inhibitor ...652 23.3.2.2 Antisensing Additional Peripheral Targets for Obesity .......................654 23.3.3 Discovery of Antisense Drugs for Nonalcoholic Steatohepatitis .........................655 23.3.3.1 Acyl-CoA : Diacylglycerol Acyltransferase 2 (DGAT2).....................655 23.3.3.2 Antisense Reduction of Stearoyl-CoA Desaturase Expression ...........656 23.3.3.3 Antisense Reduction of Acetyl-CoA Carboxylases 1 and 2 Expression ............................................................................................656 23.4 Antisense Drug Development .............................................................................................656 23.4.1 Phase 1/2 Clinical Program Overview..................................................................657 23.4.1.1 Clinical Safety Summary .....................................................................657 23.4.1.2 ISIS 113715 Clinical Pharmacokinetics ..............................................657 23.4.1.3 ISIS 113715 Clinical Pharmacology....................................................658 23.5 Conclusion ..........................................................................................................................659 Acknowledgments ..........................................................................................................................659 References ......................................................................................................................................659

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23.1 INTRODUCTION The incidence of several metabolic disorders such as type 2 diabetes, obesity, and fatty liver disease has increased to epidemic proportions in the past decade, primarily due to indulgence in a diet rich in fats, coupled with a sedentary lifestyle [1]. While significant progress has been made in understanding the mechanisms underlying the pathophysiology of these disorders, the development of novel therapeutics for these diseases has been a slow and arduous process. Two key factors that have hampered the development of traditional small molecule approaches are lack of target specificity and limited versatility. In contrast, antisense technology represents an innovative approach for the discovery and development of target-specific inhibitors against a wide variety of targets, many of which are undrugable with traditional approaches [2]. The use of advanced and proprietary chemistries has generated antisense drugs that are not only highly selective, but that also have a high therapeutic index [2]. Furthermore, since the pharmacokinetics and tissue distribution are largely similar and predictable between different antisense sequences with the same chemistry backbone, it has facilitated rapid evaluation of hundreds of gene targets in animal models, thereby generating a very efficient in vivo drug-discovery process. In addition, direct comparison of the pharmacology of different targets in vivo has paved the way for selection of the best preclinical targets for clinical development. The antisense drugs that have been the most extensively studied in vivo are 20-base chimeric oligonucleotides, where the first five bases and last five bases have a 2⬘-O-(2-methoxy)-ethyl (2⬘-MOE) modification [3]. This chimeric strategy enhances the binding affinity of the ASOs to complimentary sequences and their resistance to the action of nucleases, thereby increasing the potency and half-life of these compounds. The term “antisense drugs” in this chapter refers to this advanced MOE gapmer chemistry, unless specifically stated otherwise.

23.2 RATIONALE The rationale for evaluating the therapeutic potential of antisense drugs for metabolic disorders stems from observations that these drugs distribute very well to liver and adipose tissue [4], two tissues that play a key role in the pathology of type 2 diabetes, obesity, and fatty liver disease. Furthermore, since these drugs display long tissue half-lives, it allows infrequent subcutaneous administration (once a week or less frequent), which confers important advantages in terms of patient compliance and cost-competitiveness for chronic therapy [4,5]. These drugs also have the potential for oral delivery, which could make them very attractive for the treatment of metabolic disorders. Importantly, growing clinical experience after systemic administration of these compounds indicates that these drugs demonstrate a favorable safety and tolerability profile [5], which is essential for treating chronic diseases such as type 2 diabetes and obesity.

23.3 ANTISENSE DRUG DISCOVERY Over the past 4 years, antisense inhibitors against ⬎125 cellular targets have been evaluated in a variety of animal models of diabetes and obesity (Figure 23.1). More than 65 gene targets have been invalidated and ⬎15 targets have demonstrated robust pharmacology. Antisense drugs against several of these targets are currently under development. The most advanced compound, ISIS 113715, is an antisense inhibitor of the molecular target protein tyrosine phosphatase 1B (PTP-1B) and is currently under evaluation in Phase 2 trials for type 2 diabetes [6–8]. Since antisense inhibitors cause profound and specific target reduction in adipose tissue, peripheral targeting of antisense compounds to treat obesity is also being explored. In addition,

Miscellaneous [27] Miscellaneous [28] Miscellaneous [29] Miscellaneous [30] Transcription factor [6] Transcription factor [7] Miscellaneous [22] Miscellaneous [23] Miscellaneous [24] Kinase [20] Phosphatase [18] Kinase [18] Kinase [19] Miscellaneous [25] Miscellaneous [26] Phosphatase [14] Miscellaneous [11] Miscellaneous [12] Receptor [4] Transcription factor [3] Miscellaneous [13] Transcription factor [4] Miscellaneous [14] Kinase [12] Enzyme [6] Kinase [13] Miscellaneous [8] Phosphatase [6] Phosphatase [7] Kinase [3] Kinase [4] Phosphatase [4] Phosphatase [5] Enzyme [3] Enzyme[4] Phosphatase [6] Phosphatase [7] Miscellaneous [4] PTFN Enzyme Enzyme [2] Kinase Receptor

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Figure 23.1 Schematic depicting the in vivo antisense drug discovery paradigm. Potent and selective antisense inhibitors were identified in vitro and subsequently evaluated in tier 1 and tier 2 in vivo pharmacology screens in ob/ob and db/db diabetic mice, respectively. Targets that were evaluated included phosphatases, kinases, transcription factors, adaptor proteins, and metabolic enzymes (⬎125 targets have been evaluated to date). Targets that were validated in mouse screens (depicted in gray) were then evaluated for second species efficacy in Zucker Diabetic Fatty rats by employing specific antisense inhibitors against the specific rat gene/s. Similarly, targets with robust pharmacology in rats were subsequently tested in monkeys. Antisense drugs against three of these targets (PTP-1B, glucagon receptor and glucocorticoid receptor) are currently being pursued for clinical development.

Human

Primate

Second species rat

Tier 2 (db / db)

Tier 1 (ob /ob)

Leads identified

Glucagon receptor PTP-1B Glucocorticoid receptor

Transcription factor [2] Kinase [2] Miscellaneous Phosphatase [2] Miscellaneous [5] Miscellaneous [6] Kinase [5] Kinase [6] Phosphatase [8] Miscellaneous [7] Enzyme [5] Miscellaneous [2] Transcription factor [2] Ligand Receptor [3] Phosphatase [10] Phosphatase [11] Phosphatase [12] Ligand [2] Miscellaneous [9] Kinase [7] Kinase [8] Kinase [9] Ligand [3] Miscellaneous [10] Kinase [10] Kinase [11] Phosphatase [13] Phosphatase [15] Phosphatase [16] Miscellaneous [15] Miscellaneous [16] Kinase [14] Kinase [15] Phosphatase [17] Transcription factor [5] Miscellaneous [17] Kinase [16] Kinase [17] Miscellaneous [18] Miscellaneous [19] Miscellaneous [20] Miscellaneous [21]

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antisense compounds that improve hepatic steatosis in preclinical models have been discovered [9]. The improvement in hepatic steatosis is marked, rapid and is accompanied by improved liver function. These drugs are being evaluated for the treatment of fatty liver disorders including nonalcoholic steatohepatitis (NASH), a disease that affects up to 2–3% of the adult population and for which no effective drug therapies are available today [10]. In the following sections, an attempt has been made to summarize some of the key observations from this extensive drug-discovery effort. Since a discussion of data with antisense drugs against individual targets is beyond the scope of this chapter, findings are summarized in the context of specific pathways or target classes, with key examples from each class highlighted to reflect the scope and nature of the findings. 23.3.1 Drug Discovery for Type 2 Diabetes Diabetes mellitus is an endocrine disorder in which the body’s ability to regulate blood sugar is impaired, resulting in high blood sugar levels. The incidence of diabetes has increased by ⬎250% in the past decade and is predicted to rise to epidemic proportions, with the global figure of diabetic patients reaching 300 million by 2025 (World Health Organization). Type 2 diabetes, which represents ⬃90% of the diabetic population, is characterized by relative insulin deficiency coupled with the inability of the body to respond to circulating insulin levels (also known as insulin resistance). The inability of the body to respond to circulating insulin levels is a consequence of abnormal regulation of intracellular signaling mechanisms. Intracellular protein phosphatases function as key negative regulators of insulin signaling and several phosphatases, including PTEN (Phosphate and Tensin Homolog on Chromosome Ten), SHIP2 (SH2-domain containing inositol 5-phosphatase 2) and PTP-1B have been shown to regulate insulin action by dephosphorylating key intracellular enzymes involved in insulin signal transduction [11–13]. Inhibition of these phosphatases would be expected to result in enhanced insulin action, resulting in an improvement in insulin sensitivity. However, protein phosphatases are difficult targets to approach with small molecules due to the lack of target specificity of such approaches and therefore, elucidation of the pharmacological roles of these enzymes has been challenging.

23.3.1.1 Antisense Targeting of Protein Phosphatases By using potent and specific antisense inhibitors, we have explored the roles of ⬎25 phosphatases in animal models of diabesity (Figure 23.2). In studies conducted several years ago, we demonstrated that antisense reduction of PTEN, a lipid phosphatase, normalized blood glucose concentration, attenuated hyperinsulinemia and improved insulin sensitivity in diabetic rodents [11]. These findings have since been confirmed and extended by several investigators by using a variety of different approaches [14,15]. Another phosphatase that has been investigated extensively using an antisense approach is PTP-1B [6–8]. Interest in PTP-1B as a potential therapeutic target for type 2 diabetes and obesity was sparked by the observation that targeted disruption of the PTP-1B gene in mice resulted in improved insulin sensitivity and resistance to diet-induced obesity [16,17]. These findings, coupled with observations that the expression of PTP-1B was increased in rodent models of type 2 diabetes and obesity [18], raised the possibility that inhibition of PTP-1B could be an attractive approach for treating these metabolic disorders. Developing small molecule PTP-1B inhibitors has been challenging and many pharmaceutical companies have unsuccessfully pursued this target for almost a decade. While all reported small molecule agents against PTP-1B require improved pharmacokinetic properties, the overriding concern is selectivity for the target [19]. Adequate selectivity with a small molecule inhibitor may not be achievable due to active site homology among closely related phosphatases.

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Isis phosphatase program Relative liver expression

Phosphatase class

Common name

Class name

Receptor-type

P TP 1B

P TP N1

tyrosine phosphatases

TCP TP

P TP N2

++

ME G2

P TP N9

+++

S HP 2

P TP N11

++

PEST

P TP N12

+++

+++

P TP 36

P TP N14

+

B DP

P TP N18

+

P TP RL10

P TP N21

+++

Nonreceptor-type

LA R

P TP RF

++

tyrosine phosphatases

P TP del ta

P TP RD

+++

P TP s i gma

P TP RS

++

P TP l amda

P TP RU

+

P TP k appa

P TP RK

+++

P TP beta

P TP RB

++

DE P 1

P TP RJ

+++

P TP al pha

P TP RA

+++

P TP S L

P TP RR

+

IA 2

P TP RN

+++

Dual specificity

MK P 1

DUS P 1

+++

Tyrosine phosphatases

MK P 3

DUS P 6

++

PKP4

DUS P 9

+

Other classes of

P TE N

P TE N

tyrosine phosphatases

LMW-PTPase

LMW-PTPase

PTP-4A2

+++ +++ +++

Figure 23.2 List of protein tyrosine phosphatases that have been evaluated in animal models using antisense drugs. The expression of each of these phosphatases relative to the expression of protein tyrosine phosphatase-1B (PTP-1B) in the liver of diabetic rodents is also shown. PTP-1B is depicted as ⫹⫹⫹ (highly expressed). Five phosphatases with novel roles in glucose and lipid metabolism were discovered and are presently under evaluation in late-stage pharmacology studies.

ISIS 113715: A PTP-1B Antisense Inhibitor ISIS 113715 is an antisense inhibitor of PTP-1B that reduces PTP-1B expression in a highly target-specific manner without affecting the expression of other phosphatases including T-cell phosphatase, a phosphatase that has ⬃80% homology with PTP-1B in the catalytic domain [8]. The binding site of ISIS 113715 is conserved across all species studied to date including rodents, dog, monkey, and man. Multiple studies in our laboratory have demonstrated remarkably consistent, specific, and significant reduction of PTP-1B mRNA and protein levels in liver and adipose tissue after ISIS 113715 treatment [6–8]. In diabetic rodents, ISIS 113715 treatment normalized blood glucose levels without producing hypoglycemia or body weight gain [8]. ISIS 113715 treatment in diabetic mice was accompanied by enhanced insulin receptor activity as well as enhanced postreceptor signaling through key signaling intermediates, including insulin receptor substrates 1 and 2, phosphatidylinositol 3-kinase and Akt [8]. These findings mirrored those observed in PTP-1B null mice and provided further validation for PTP-1B as an attractive therapeutic target for type 2 diabetes. The effects of ISIS 113715 were also evaluated in obese, insulin-resistant, hyperinsulinemic monkeys [20]. ISIS 113715 improved insulin sensitivity and attenuated hyperinsulinemia in glucose intolerant, obese monkeys without causing hypoglycemia (Figure 23.3A and Figure 23.3B). The effects in obese monkeys were accompanied by an increase in plasma adiponectin levels

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Figure 23.3 Effects of ISIS 113715 in obese, insulin-resistant, hyperinsulinemic Rhesus monkeys. Following the baseline data collection, animals (n ⫽ 5) received ISIS 113715 subcutaneously at a dose of 20 mg/kg on three alternate days during the first week and once each week as a subcutaneous injection for the next three weeks. 16-h-fasted plasma samples were collected 48 h after dosing and were assayed for insulin (A), glucose (B), triglycerides (C), and adiponectin (D). Data are expressed as means ⫾SEM. *p ⬍ 0.05 versus baseline, paired t-test.

(Figure 23.3D), which, in turn, can lead to secondary improvements in peripheral insulin sensitivity. In addition, a reduction in circulating triglycerides (Figure 23.3C) as well as a 20% reduction in serum ApoB100, LDL, and total cholesterol levels was observed, which is consistent with recent reports demonstrating indirect regulation of ApoB100 degradation and assembly by PTP-1B [21]. Hypoglycemia was not observed even in 16-h-fasted animals at doses that were ⬎10 times the anticipated efficacious dose in humans. These results were the first demonstration of positive pharmacology of any PTP-1B inhibitor in a nonrodent species and further supported the clinical development of ISIS 113715. Collectively, the preclinical data obtained indicate that ISIS 113715 may offer a substantial improvement over currently available diabetes drugs. ISIS 113715 may control blood glucose without the risk of hypoglycemia and weight gain and may promote weight loss, a significant benefit in this patient population. In addition, ISIS 113715 has been shown to be additive to most currently available therapies, without increasing any of the adverse effects associated with those drugs [22].

Discovery of Additional Phosphatases as Novel Antidiabetic Targets In addition to PTP-1B, several additional phosphatases that play novel roles in insulin signaling have been identified. One such phosphatase is low-molecular-weight phosphatase (LMW-PTP), also known as acid phosphatase locus 1 [23]. Although discovered more than a decade ago, no data were available describing its role in glucose metabolism. Antisense reduction of this phosphatase resulted in improved hepatic insulin signaling in diabetic animals [24]. In contrast to other phosphatases such as PTP-1B, PTEN and SHIP2, enhanced insulin signal transduction was also observed in adipose tissue after LMW-PTP antisense treatment, suggesting a distinct and broader role in regulation of metabolic pathways [24]. Treatment with LMW-PTP antisense inhibitors normalized hyperglycemia, improved insulin sensitivity and glucose tolerance and decreased hepatic steatosis in diabetic mice.

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Another phosphatase that appears to play an important role in glucose and lipid metabolism is protein tyrosine phosphatase R alpha (PTPR
). Antisense reduction of hepatic PTPR
mRNA expression in diabetic mice resulted in decreased plasma glucose and triglyceride levels [25]. In addition, antisense treatment decreased hepatic triglyceride levels and improved hepatic function, as assessed by a reduction in plasma ALT and AST levels. Thus, several phosphatases with novel roles in the regulation of glucose and lipid metabolism have been identified, some of which are under further evaluation as insulin sensitizers for type 2 diabetes. Future discovery efforts will be aimed at exploring the specific mechanism of action of these phosphatases, exploring intracellular cross talk between them as well as exploring the pharmacological effects of the combined reduction of these phosphatases in diabetic and obese animals.

23.3.1.2 Targeting Transcription Factors with Antisense Drugs Antisense inhibitors against ⬎12 transcription factors have been studied in preclinical models. Several transcription factors appear to play broad and critical roles in the regulation of glucose and lipid metabolism. These include eukaryotic initiation factor binding protein 2 (eIF4E-BP2) and forkhead transcription factor (FKHR). eIF4E-BP2 belongs to a family of three inhibitors (eIF4E-BP1, 2, 3) that inhibit the 5⬘ cap-dependent translation initiation by sequestering eIF4E from the eIF4F complex [26]. eIF4E-BP1 gene knockout mice show decreased body weight and adiposity, and increased metabolic rate [26]. However, no data are available for the other two eIF4E-BP gene knockouts. We recently reported that treatment of mice fed a high-fat diet with an eIF4E-BP2 antisense drug caused reduction of eIF4E-BP2 gene expression by 84% in fat and 74% in liver, which was accompanied by decreased body fat percentage content and liver triglyceridecontent [27]. Treatment also lowered plasma glucose and insulin levels and improved glucose tolerance. Western blot analysis revealed that treatment with the eIF4E-BP2 antisense drug increased phosphorylation levels of Akt (Ser 473) but not insulin receptor beta subunit (Tyr1163/1164) or IRS-1 in both liver and fat in response to an insulin challenge, indicating a postreceptor mechanism of action. Importantly, treatment did not affect the protein levels of several proteins involved in mitogenesis, including eIF4E, eIF4E-BP1, ERK1/2, p70s6k or GSK3b. Thus, eIF4E-BP2 appears to play a very specific role in regulating metabolic pathways. Robust data were also reported with the winged helix transcription factor FKHR [28]. It has been demonstrated that FKHR regulates the transcription of key gluconeogenic enzymes and can thereby modulate hepatic gluconeogenesis [28]. We recently demonstrated that in high-fat diet fed mice, FKHR antisense therapy lowered the rate of hepatic glucose production, hepatic triglyceride and diacylglycerol content, and caused a significant improvement in hepatic insulin sensitivity [29]. Similarly, reduction of hepatic FKHR expression by ⬎50% caused significant attenuation of hyperglycemia in diabetic mice [30]. Reduction of FKHR expression in adipose tissue resulted in improved insulin action, which was reflected as an increase in glucose uptake and an improved suppression of lipolysis. The net result was improved glucose clearance after an intraperitoneal glucose load and increased whole body glucose disposal in response to insulin. Thus, hepatic and adipose reduction of FKHR expression caused an improvement in both hepatic and peripheral insulin action, indicating that inhibition of FKHR with antisense drugs may provide a useful intervention point in the treatment of type 2 diabetes. While transcription factors regulate a multitude of transcriptional events inside cells, what was surprising was that pharmacological reduction of most of these targets in the liver by ⬎75% did not result in either overt toxicity or abnormalities in hepatic function (unpublished data). This suggests that evolution has created a lot of redundancy in these pathways and that alternate mechanisms exist that can potentially compensate for the selective reduction of these regulatory switches in a very specific manner. Thus, selective reduction of these targets can provide exciting opportunities for clinical development with antisense compounds, since most of these targets are not approachable with small molecules.

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23.3.1.3 Antisense Strategies to Inhibit Hepatic Glucose Output The inability to suppress excessive hepatic glucose production is a key defect in type 2 diabetes and inhibition of hepatic glucose output has been pursued widely as a therapeutic strategy. Excessive hepatic glucose output is believed to occur due to excessive glycogenolysis as well as increased gluconeogenesis. Attempts have been made to inhibit both these pathways with traditional approaches with limited success [31]. Since liver is one of the key tissues of pharmacological action of antisense drugs, the contribution of these pathways towards diabetic hyperglycemia was evaluated in considerable detail. This was achieved by inhibiting rate-limiting enzymes in these pathways with specific ASOs and observing the resultant effects on plasma glucose levels. Since PEPCK is believed to be the rate-limiting enzyme in gluconeogenesis, it was one of the first targets that was evaluated. Surprisingly, it was found that even a 90% reduction in hepatic PEPCK mRNA expression in several diabetic animal models failed to have any effect on fed or 16-h-fasted glucose levels [32]. These data have been confirmed by using liver-specific PEPCK knockout approaches and have challenged the contribution of hepatic PEPCK toward the fasting hyperglycemia observed in type 2 diabetes [33]. Equally surprising was the observation that hepatic reduction of the enzyme fructose 1, 6 bisphosphatase (FBP-1) by ⬎95% did not reduce fasting blood glucose levels in diabetic animals [34]. Although not a rate-limiting enzyme, near complete reduction of the enzyme would be expected to have an impact on gluconeogenesis, since several gluconeogenic substrates (e.g., fructose and glycerol) have to be processed by FBP-1 to complete subsequent steps in the gluconeogenic cycle. These antisense observations were in contrast to those reported with small molecules [35] as well as findings in humans with the FBP-1 null mutation [36]. Further evaluation revealed that although hepatic FBP-1 activity was almost completely inhibited after antisense treatment, only ⬃50% reduction of FBP-1 activity was achieved in the kidney. Since the kidney also contributes to gluconeogenesis, the antisense data point to a very important contribution of extrahepatic gluconeogenesis toward maintaining circulating glucose levels. Thus, liver-specific pharmacological antagonism of FBP-1 may not yield the postulated therapeutic benefit for ameliorating hyperglycemia in type 2 diabetes. Using a similar approach, the contribution of the glycogenolysis pathway toward hyperglycemia was examined by inhibiting glycogen phosphorylase, the rate-limiting enzyme in that pathway. Antisense reduction of hepatic glycogen phosphorylase levels by ⬎75% resulted in very modest reductions in plasma glucose levels that were accompanied by increased glycogen accumulation in the liver [37]. Although extrapolation of animal data to humans needs to be made with caution, these data suggest that therapeutic approaches being pursued with small molecule glycogen phosphorylase inhibitors [38] may not yield the desired efficacy in patients with type 2 diabetes. In contrast to data obtained after reduction of hepatic PEPCK and FBP-1 expression, attenuation of hepatic glucose-6-phosphatase (G6P) expression resulted in significant glucose lowering effects in the same animal models [39]. (G6P) is a multicomponent enzyme that catalyzes the final step of both the gluconeogenesis and glycogenolysis pathways by hydrolyzing glucose-6-phosphate to D-glucose, which is then released into the plasma. G6P is comprised of a transport protein T1, which permits the entry of glucose-6-phosphate into the endoplasmic reticulum, a catalytic subunit that cleaves the glucose-6-phosphate, transporters T2 and T3 that transport the hydrolysis products to the cytosol, and a stabilizing protein. While studies using small molecule inhibitors targeting the G6PT1 protein have provided some validation for pursuing G6P as a treatment for type 2 diabetes [31], these approaches have been fraught with significant side effects such as increased hepatic and renal glycogen accumulation, hypoglycemia and increased plasma lactate levels. In contrast, partial and tissue selective reduction of hepatic G6PT1 mRNA expression (by ⬃85%) with antisense drugs significantly reduced plasma glucose levels in the fed and fasted states [39] without causing hypoglycemia, kidney enlargement, neutropenia, hyperlipidemia or increased hepatic glycogen content.

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Several factors likely underlie these fascinating findings. First, in contrast to patients and rodents with global G6PT1 deficiency, G6P transport activity in the liver was not completely abolished by G6PT1 antisense therapy (⬃20–30% G6P hydrolysis activity was still present after antisense treatment). Similarly, when ⬃20% hepatic G6Pase activity was restored in G6P knockout animals by G6P gene-bearing adenovirus treatment, most of the side effects associated with global gene knockdown were abolished [40], supporting the notion that 20–30% residual activity of this enzyme may be sufficient to mitigate these adverse effects. Second, reduction of G6PT1 activity resulted in a compensatory increase in hepatic G6P catalytic subunit expression, which preserved some residual capacity of the liver to breakdown glucose despite G6PT1 reduction. Third, since G6PT1 ASOs lowered G6PT1 mRNA levels by only 45% in the kidney, there was likely enough residual activity to allow for glycogen mobilization, thereby avoiding renal glycogen accumulation. Finally, G6PT1 mRNA levels in the small intestine, another extra-hepatic source of gluconeogenesis were unchanged, which likely also helped avoid hypoglycemia in the treated animals. In summary, antisense drugs have provided novel insights into the contribution of hepatic gluconeogenic and glycogenolytic pathways toward increased hepatic glucose production in type 2 diabetes. Surprising data have been obtained that challenge the role of key hepatic gluconeogenic enzymes such as PEPCK towards fasting hyperglycemia in diabetic animals. Finally, this drug discovery effort has provided a new basis to reconsider pursuing strategies to specifically target the hepatic G6P enzyme for type 2 diabetes.

23.3.1.4 Exploiting Tissue Selectivity and Pharmacokinetic Properties of Antisense Drugs The preferential distribution of antisense drugs to tissues such as liver and adipose tissue after systemic administration, coupled with poor distribution to other tissues such as the central nervous system (CNS), has opened up unique therapeutic opportunities for the treatment of metabolic diseases [4]. One such strategy that has been investigated in considerable detail is to explore tissue selective reduction of the glucocorticoid (GC) receptor, as discussed below.

Antisense Reduction of Hepatic and Adipose Tissue Glucocorticoid Receptor Expression Excessive GC action is known to cause a wide spectrum of clinical features such as obesity, insulin resistance, and glucose intolerance [41]. GCs bind to an intracellular GC receptor, which then translocates into the nucleus and binds to GC response elements, resulting in the transcriptional activation of gluconeogenic enzymes and a consequent increase in hepatic glucose output. Furthermore, GCs stimulate lipogenesis and increase secretion of triglycerides from the liver as well as regulate cholesterol biosynthesis by elevating HMG-CoA reductase expression levels [42]. In adipose tissue, GCs promote differentiation of adipocytes from preadipocytes and increase triglyceride storage. Although systemic GC inhibition has been shown to improve hyperglycemia in rodents and man, it leads to adrenal insufficiency and stimulation of the hypothalamic-pituitary-adrenal (HPA) axis. Recent data suggest that increased GC action within liver and white fat (WAT) tissues may cause tissue-specific amplification of GC effects such as increased adipocyte differentiation, increased lipogenesis, and increased gluconeogenesis, without any change in circulating GC levels [43]. Thus, tissue-specific reduction of GC action could be an attractive therapeutic strategy for type 2 diabetes. In fact, GC receptor (GCCR) antagonists have been shown to reduce hyperglycemia in rodent models of diabetes. Despite this positive effect, these agents also caused unfavorable extrahepatic effects, including activation of the HPA axis [44]. Antagonism of tissue-specific GC action was attempted by reducing GCCR expression with antisense drugs. In several mouse and rat models of diabetes, treatment with a GCCR antisense inhibitor caused ⬎60% reduction in GCCR expression in liver and adipose tissue, which led to

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significant attenuation of hyperglycemia and hyperlipidemia [43]. Decreased hepatic steatosis and improved hepatic function was also observed. In addition, reduction of GCCR expression in adipose tissue caused a reduction in adipose mass in obese mice. As expected, the antisense inhibitors did not cause any reduction of GCCR expression in the pituitary and adrenal glands [43]. Circulating levels of corticosterone and adrenocorticotropic hormone (ACTH) remained unchanged, indicating that the HPA axis was not stimulated. Furthermore, when animals treated with the GCCR antisense inhibitor were challenged with dexamethasone, a complete reduction of dexamethasone-induced increase in hepatic gluconeogenesis was observed, without any change in dexamethasone-induced lymphopenia (a marker for systemic effects of dexamethasone). Another target in this pathway that has generated intense pharmaceutical interest in the past couple of years is the enzyme 11-beta hydroxysteroid dehydrogenase 1 (HSD-1). This enzyme catalyzes the conversion of inactive cortisol (or corticosterone in rodents) into its active form. Therefore, inhibition of this enzyme is being pursued as a treatment for type 2 diabetes [31]. Reduction of HSD1 in liver and adipose tissue (⬎80%) with antisense drugs revealed only modest effects on plasma glucose levels as compared to those seen with a GCCR antisense inhibitor, indicating the latter to be a better therapeutic target for pharmacological intervention [45]. Thus, tissue-selective reduction of GCCR expression with antisense drugs presents another unique therapeutic strategy and this approach is being actively pursued for clinical development.

Antisense Reduction of Hepatic and Adipose Tissue Glucagon Receptor Expression While most treatments for type 2 diabetes are focused on increasing insulin secretion or improving insulin sensitivity, it is in fact the disruption of the normal glucagon-insulin ratio that causes diabetes [46]. Not only are basal glucagon levels elevated in type 2 diabetes, but its suppression after meal ingestion is also impaired [47,48]. Glucagon receptor null mice show slightly reduced plasma glucose and insulin levels and improved glucose tolerance [49]. Therefore, it is not surprising that pharmacological antagonism of glucagon action has been investigated as a therapeutic approach for type 2 diabetes. To that aim, peptide antagonists as well as monoclonal antibodies against glucagon receptor have been shown to attenuate hyperglycemia in animal models [50,51]. Development of small molecules against the glucagon receptor has been slow due to issues with pharmacokinetics, selectivity, cross-species differences and lack of sustained effects after noncompetitive blockade [52]. Only a single Phase 1 study has been published that describes the acute effects of a glucagon receptor inhibitor in normal subjects [53]. In a series of studies, we demonstrated that antisense drugs caused a marked reduction of hepatic glucagon receptor expression (⬎75%), which was accompanied by normalization of blood glucose levels in multiple animal models of diabetes [54]. In addition to hepatic effects, glucagon receptor antisense therapy increased the levels of active glucagon-like peptide-1 (GLP-1), an incretin that is also known to improve pancreatic beta cell function [54]. In agreement with observations made in glucagon receptor deficient mice, reduced hepatic glucagon receptor expression with antisense drugs caused pancreatic alpha cell hyperplasia and hyperglucagonemia [54]. However, when the effects of a glucagon receptor antisense inhibitor were evaluated in monkeys, about a sixfold increase in the levels of GLP-1 were observed (Figure 23.4) without any evidence of alpha cell expansion or hyperplasia [55]. GLP-1 levels achieved by antisense treatment were similar to or exceeded those shown to be efficacious in humans by exogenous dosing [56]. In addition, it has been demonstrated that the alpha-cell produced active GLP-1 may have local effects within islets [57], suggesting that high levels of circulating GLP-1 may not be required to achieve efficacy and could ameliorate dose-related side effects associated with exogenous GLP-1 administration. These data in nonhuman primates support earlier observations indicating that differences exist across species in the physiological mechanisms by which islets respond to increases in hormone demand. For example, alpha-cells

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Figure 23.4 Glucagon receptor antisense drug increased plasma-active GLP-1 levels in Cynomolgus monkeys. Following the baseline data collection, animals (n ⫽ 4) received the glucagon receptor antisense drug (ASO) at a dose of 10 mg/kg twice a week subcutaneously for 10 weeks. Plasma samples were collected 48 h after dosing and were assayed for active GLP-1 (A) and glucagon levels (B). (C) Liver tissue from the animals was homogenized and glucagon receptor mRNA expression analyzed by RT-PCR as previously described [37]. Data are expressed as means ⫾SEM. *p ⬍ 0.05 versus saline, ANOVA.

within rodent islets predominantly proliferate to meet the animal’s insulin need, while in humans, neogenesis (and not proliferation) occurs to satisfy an increasing demand [58]. Thus, it is expected that efficacy in humans will be achieved at doses that do not result in alpha-cell expansion. If successful, the development of antisense drugs against the glucagon receptor will likely provide significant glucose control in diabetic patients due to a dual mode of action (antagonism of hepatic glucagon action, combined with an increase in active GLP-1 levels).

23.3.1.5 Targeting the Kidney Using Novel Antisense Chemistries Since antisense drugs distribute very well to the kidney, pharmacological reduction of targets in this organ was attempted as a therapeutic approach for type 2 diabetes. An interesting target in the kidney that is involved in the reabsorption of glucose is the sodium glucose transporter 2 (SGLT2) [59]. SGLT2 is a low-affinity, high-capacity sodium-dependent glucose transporter whose function is to transport glucose into cells against its concentration gradient via a secondary active transport system. SGLT2 is the major reabsorptive mechanism for D-glucose in the kidney proximal convoluted tubule [59]. Inhibition of this target would be expected to decrease renal glucose reabsorption, resulting in increased glycosuria and a consequent attenuation of hyperglycemia. Using a modified version of the second-generation antisense chemistry termed mixed-backbone (MBB) chemistry, we specifically inhibited SGLT2 expression in the kidney and evaluated the resultant effects on plasma glucose levels in diabetic mice. MBB chemistry enhanced the distribution of antisense drugs to the kidney and increased potency by ⬃30-fold as compared to the standard phosphorothioate antisense compounds [60]. Treatment of diabetic mice for 4 weeks at doses as low as 1–2 mgⲐkgⲐweek with an optimized MBB antisense drug resulted in ⬃80% reduction of kidney

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SGLT2 mRNA expression and a marked decrease in plasma glucose levels [60]. Furthermore, the MBB antisense did not affect the expression of SGLT1 and GLUT-2, two other glucose transporters that are coexpressed within the early proximal convoluted tubule. No hepatotoxicity or nephrotoxicity was observed based on blood chemistry, organ/body weight ratio or histology. These results suggest that antisense chemistries optimized to target the kidney may provide a novel therapeutic approach to treat diabetes as well as other renal disorders. As is evident from the preceding sections, using the antisense approach for diabetes drug discovery has led to rapid evaluation of a multitude of exciting drug targets with robust pharmacology in preclinical models. Furthermore, the specificity and versatility of this approach has allowed identification of targets that are difficult to drug with traditional approaches, including novel phosphatases and transcription factors. 23.3.2 Antisense Drug Discovery for Obesity Obesity is a major cause of morbidity and mortality in the Western hemisphere, and is a disease that afflicts one-third of all adults and one in five children [61]. Although there has been an explosion in the prevalence of this disease, current treatment strategies are very limited [61]. While most of the research effort has focused on exploring the orexigenic and anorexigenic pathways in the CNS, recent research has demonstrated that the adipose tissue is a very dynamic endocrine organ and that inhibiting specific targets in that tissue can lead to decreased body weight gain and increased metabolic rate in animals, thus opening a new peripheral approach for the treatment of obesity [61]. Since antisense inhibitors cause profound and specific target reduction in adipose tissue, we have recently started exploring the potential to treat obesity with antisense compounds. Such a drug would be complementary to CNS-based therapeutics and would provide substantial benefit in treating a disease that has reached enormous proportions [61]. One of the best examples validating the antisense approach comes from ISIS 113715, the PTP-1B antisense inhibitor, the glucose lowering effects of which have been described previously in this document.

23.3.2.1 Antiobesity Effects of ISIS 113715, a PTP-1B Antisense Inhibitor PTP-1B knockout mouse studies demonstrated that, in addition to improved insulin sensitivity, the animals were also resistant to weight gain when fed a high-fat diet [16]. Subsequent studies revealed that the lean phenotype in PTP-1B deficient mice was accompanied by an increase in metabolic rate and energy expenditure [17]. PTP-1B-deficient mice demonstrate leptin hypersensitivity, have an exaggerated response to leptin-mediated weight loss and suppression of feeding, and display enhanced leptin-induced hypothalamic STAT3 tyrosyl phosphorylation [17]. To investigate the role of pharmacological reduction of PTP-1B, the antiobesity effects of ISIS 113715 were explored in several preclinical models of obesity. In diabetic and obese leptin deficient obⲐob mice, ISIS 113715 treatment resulted in a decrease in fat pad weight, which was accompanied by a 10–15% reduction in body weight after 6 weeks of treatment [7]. Microarray studies revealed that ISIS 113715 caused a decrease in adipose tissue triglyceride levels and affected several key lipogenic genes including sterol regulatory element-binding protein, fatty acid synthase as well as genes involved in adipogenesis such as lipoprotein lipase and PPAR-gamma [7]. These data suggested a role of PTP-1B in adipocyte hypertrophy and energy storage. When ISIS 113715 was administered to mice fed a high-fat diet, significantly reduced weight gain was observed (Figure 23.5A). At the end of 6 weeks, ISIS 113715-treated mice had gained 45% less body weight versus saline or control antisense oligonucleotide-treated mice and had a similar reduction in epididymal fat pad weights. The decrease in body weight gain was accompanied by an increase in metabolic rate, as indicated by an increase in oxygen consumption without any change in the respiratory quotient

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Figure 23.5

Light

ISIS 113715 increased metabolic rate and reduced body-weight gain in mice fed a high-fat diet. Four-week-old male C57BL/6J mice were placed on a high-fat diet for 4 weeks and then treated with either saline, control ASO or ISIS 113715 at a dose of 50 mg/kg once a week i.p. for 6 weeks (n ⫽ 7/group). ISIS 113715 reduced body weight gain (A) and caused an increase in metabolic rate, as reflected in increased oxygen consumption (C) without any change in the respiratory quotient (B).

(Figure 23.5B). In these studies, PTP-1B protein expression was reduced by ⬃50% in the liver and adipose tissues. ISIS 113715 treatment also decreased subcutaneous and abdominal adipose tissue mass in prediabetic, obese, faⲐfa Zucker rats [7]. These effects were observed after only 4 weeks of treatment and were not accompanied by any change in food intake. ISIS 113715 administration to obese monkeys for only 4 weeks caused a fourfold increase in plasma adiponectin levels, a cytokine that has been shown to increase fat oxidation. If the preclinical data gets translated to human diabetic patients in the ongoing clinical studies with this drug, it will make this compound very attractive for the treatment of type 2 diabetes, since ⬎60% of diabetic patients are also obese.

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23.3.2.2 Antisensing Additional Peripheral Targets for Obesity Several additional targets have demonstrated attractive antiobesity effects in rodent models, one of which is described below to further demonstrate the utility of the antisense approach for this indication. Recent data suggest that c-Jun N-terminal kinases (JNKs) may act as a key mediator between obesity and insulin resistance. There are three JNKs (JNK1, 2, and 3) present in mammals each encoded by a distinct gene [62]. In contrast to JNK2 knockout mice, JNK1 null mice demonstrate lower body weight gain and improved insulin sensitivity when fed a high-fat diet as compared to the wild-type controls [62]. To investigate the role of peripheral JNK1 reduction in metabolism, we employed antisense drugs to reduce its expression in liver and fat in a genetic model of obesity. Treatment of obese, diabetic mice with a JNK1 antisense inhibitor for 6 weeks reduced JNK1 mRNA by ⬎80% in both liver and WAT and by 78% in brown fat (BAT), but did not change JNK2 mRNA levels in any of these tissues (Figure 23.6A). Treatment with JNK1 ASO did not change food intake but decreased BW gain, epididymal fat pad weight and whole body fat content (unpublished data), and increased metabolic rate (Figure 23.6B). Furthermore, treatment markedly lowered both fed and fasting plasma glucose and insulin levels, improved glucose and insulin tolerance and improved liver steatosis (decreased triglyceride content by ⬎40%). This positive phenotype was accompanied by increased mRNA levels of adrenoceptor 3 and UCP1 mRNA in BAT, and increased mRNA levels of both UCP2 and PPAR
in liver. These data indicate that specific reduction of JNK1 expression results in increased fuel combustion and that robust antiobesity effects can be achieved by targeting peripheral tissues

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Figure 23.6 JNK1 antisense drug reduced JNK1 expression and increased metabolic rate in ob/ob mice. Male, 5-week-old ob/ob mice (n ⫽ 6/group) were treated with a JNK1 antisense oligonucleotide (ASO) or control ASO at a dose of 25 mg/kg BW twice a week (subcutaneously, dissolved in saline) or with saline for 6 weeks. JNK1 ASO caused significant reductions in JNK1 mRNA expression in liver, white and brown adipose tissue (A) but did not alter JNK2 mRNA expression. JNK1 ASO also caused an increase in metabolic rate, as reflected by increased oxygen consumption (B). *p ⬍ 0.05 versus saline, **p ⬍ 0.05 versus saline, #p ⬍ 0.001 versus control ASO, ##p ⬍ 0.001 versus control ASO, ANOVA.

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such as liver and fat. Several additional targets are currently under evaluation, most of which are undrugable with small molecules. 23.3.3 Discovery of Antisense Drugs for Nonalcoholic Steatohepatitis Nonalcoholic fatty liver disease now afflicts a significant percentage of people in the Western world, with an estimated prevalence of ⬎14% in the entire population [63]. It is caused by an excess buildup of triglycerides in liver and is often associated with obesity. NASH is a form of nonalcoholic fatty liver disease that presents itself as significant steatohepatitis that cannot be ascribed to the use of alcohol, drugs or any other single identifiable cause [64]. It is a form of metabolic liver disease in which hepatic steatosis is complicated by chronic inflammatory changes that result in steatohepatitis and progressive fibrosis with subsequent progression to cirrhosis, end-stage liver disease, and even hepatocellular carcinoma. The prevalence of NASH has also increased considerably due to an increase in the incidence of obesity and insulin resistance in recent years [64]. The incidence is believed to range from 2 to 3% in normal subjects to ⬃20% in subjects who are obese or have type 2 diabetes. Although equivocal, the pathogenesis of NASH is believed to involve an initial metabolic disturbance that results in steatosis, followed by a second insult resulting in oxidative stress, lipid peroxidation, and a resultant steatohepatitis [64]. The role of inflammatory mediators and other environmental mechanisms in the pathogenesis of NASH is poorly understood. Current management strategies include preventive measures such as lifestyle changes, diet and exercise as well as the treatment of concomitant disorders such as hyperlipidemia and diabetes. There are no therapies that have shown to be successful in treating NASH, although limited studies have shown minimal benefit with antioxidants and thiazolidinediones [65,66]. However, treatment with thiazolidinediones also resulted in weight gain, which is highly undesirable in this population. Thus, there is an immense need for developing drugs that would reduce the steatosis and consequently prevent the fibrosis observed in NASH. The antisense drug discovery effort involved evaluation of ⬎15 targets in intermediary lipid metabolism, with the goal of directly comparing the effects of these targets on hepatic steatosis in rodent models. Antisense drugs against several targets caused marked improvements in hepatic steatosis that were also accompanied by beneficial effects on serum lipids, some of which are discussed below.

23.3.3.1 Acyl-CoA : Diacylglycerol Acyltransferase 2 (DGAT2) Acyl coenzyme A:diacylglycerol acyltransferase (DGAT), is an enzyme that catalyzes the last step in mammalian triglyceride synthesis via the covalent binding of the acyl moiety with diacylglycerol [67]. Two DGATs (DGAT1 and DGAT2), which are encoded for by two different gene families, have been identified [67,68]. While DGAT1 gene knockout mice were found to be resistant to high-fat diet-induced obesity due to an increased metabolic rate, DGAT2 null mice were lipopenic and died soon after birth due to profound reductions in substrates for energy metabolism and impaired skin permeability [69]. Therefore, the consequences of pharmacological reduction of DGAT2 expression in adult tissues remained undetermined. In a series of studies, we explored the effects of specific antisense reduction of each of these isoforms on hepatic steatosis. While reduction of DGAT1 expression by ⬎80% in the liver did not result in a significant change in hepatic steatosis, a similar reduction of DGAT2 expression caused profound improvements in hepatic steatosis in obese rodent models of diabetes that also display marked hepatic steatosis [9]. There was a decrease in hepatic lipid export and an attenuation of circulating triglyceride and cholesterol levels. Reduction of hepatic DGAT2 expression also initiated a

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secondary inhibitory feedback loop that inhibited key enzymes involved in lipogenesis, including acetyl CoA carboxylase 1 and 2, stearoyl CoA desaturase, and fatty acid synthase. This decrease in lipogenesis, coupled with an increase in fatty acid oxidation, resulted in clearing of hepatic fat and a paradoxical decrease in hepatic diacylglycerol levels. In contrast to findings reported in the DGAT2-deficient mice, no changes in hepatic energy substrates or any abnormalities in skin microstructure were observed [9]. These findings were further extended by evaluating the effects of a DGAT2 antisense inhibitor in rats fed a high-fat diet. In agreement with data obtained in murine models, DGAT2 antisense treatment caused a decrease in hepatic steatosis, which was accompanied by a marked improvement in hepatic and peripheral insulin sensitivity [70].

23.3.3.2 Antisense Reduction of Stearoyl-CoA Desaturase Expression Stearoyl-CoA desaturase (SCD) is a key enzyme involved in the synthesis of monounsaturated from saturated fatty acids. Mice with a targeted disruption of SCD1 gene are lean and display increased insulin sensitivity [71]. Treatment of obese, diabetic mice with an optimized SCD1 antisense inhibitor (in rodents, this isoform constitutes ⬎99% expression of total SCD activity in the liver) caused an improvement in hepatic steatosis [72]. In rats fed a high-fat diet, antisense reduction of hepatic SCD1 expression caused a significant increase in insulin sensitivity and a reduction in hepatic glucose production. The effects obtained after antisense reduction of SCD1 were less robust than those seen after DGAT2 antisense treatment.

23.3.3.3 Antisense Reduction of Acetyl-CoA Carboxylases 1 and 2 Expression Acetyl-CoA carboxylases (ACCs) catalyze the synthesis of malonyl-CoA, which is both an intermediate in fatty acid synthesis and an allosteric inhibitor of carnitine palmitoyl transferase 1, a key enzyme involved in fatty acid oxidation [73,74]. Of the 2 ACC isoforms, ACC1 is highly expressed in liver and fat whereas ACC2 is largely expressed in heart and skeletal muscle [73]. While it has been suggested that cytosolic ACC1 regulates malonyl-CoA synthesis for incorporation into fatty acids and that ACC2 regulates mitochondrial fatty acid oxidation [75], this postulation has not been directly evaluated. We evaluated the role of individual ACC isoforms by reducing their expression in the liver with antisense inhibitors. While reduction of ACC1 caused a slight decrease in hepatic steatosis, ACC2 reduction did not have any meaningful effect on hepatic lipids [76]. Interestingly, reduction of both ACC1 and ACC2 together caused marked reductions in hepatic malonyl-CoA levels, hepatic lipids, and improved hepatic insulin sensitivity, indicating that combined reduction of the ACC isoforms may be a novel approach for the treatment of disorders such as NASH and the metabolic syndrome. Thus, antisense inhibitors against several lipid targets have demonstrated robust effects on hepatic steatosis that are also accompanied by significant improvements in circulating lipids and hepatic insulin sensitivity. Of all the targets studied to date, antisense reduction of hepatic DGAT2 expression has demonstrated the most profound effects on these parameters. This is not unexpected, since reduction of DGAT2 expression caused secondary inhibition of several other enzymes, including ⬎80% reduction of SCD1, ACC1, and ACC2 [9]. Therefore, the net effect of DGAT2 inhibition exceeds the effect of reducing any single enzyme in the intermediary lipid metabolism pathway, making DGAT2 a promising therapeutic target for NASH and metabolic syndrome.

23.4 ANTISENSE DRUG DEVELOPMENT Several antisense drugs, including those against the glucagon receptor and GC receptor, are expected to enter Phase 1 clinical trials within the next 18–24 months. The most advanced

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drug, ISIS 113715, is under evaluation in Phase 2 trials and will be discussed in the following section. 23.4.1 Phase 1/2 Clinical Program Overview ISIS 113715 is being developed as combination therapy in patients who do not achieve adequate glycemic control (HbA1c ⬍7%, ADA criteria) despite maximal effective or maximal tolerated therapy with oral antidiabetic agents such as sulfonylureas, metformin, and thiazolidinediones. In addition, since ISIS 113715 is an insulin sensitizer, it is also being developed as combination therapy in patients inadequately controlled on insulin. To date, ISIS 113715 has been evaluated in normal subjects at doses ranging from 0.5–7.5 mgⲐkg for short durations (7–10 days). ISIS 113715 has also been examined in type 2 diabetic subjects as a single agent at doses of 100, 200, 400, and 600 mgⲐweek for 6 weeks and at a dose of 200 mgⲐweek for 12 weeks. While the ISIS 113715 single-agent study in type 2 diabetic patients was designed primarily to assess the safety, tolerability, and pharmacokinetics of the drug (to support the intended combination studies), encouraging pharmacology was observed in that short-term trial. Key findings obtained in the clinical program are summarized below.

23.4.1.1 Clinical Safety Summary ISIS 113715 has been administered to 169 subjects, 89 healthy volunteers, and 80 patients with type 2 diabetes. Treatment with ISIS 113715, at the doses and regimens examined, did not cause any clinically remarkable changes in markers of glomerular or renal function, nor did it cause any changes in estimated glomerular filtration rates. There were no cases of renal deterioration, renal insufficiency or renal failure in any trial. In addition, no clinically significant changes in hepatic function, no weight gain and no clinically remarkable changes in other laboratory parameters (hematology, coagulation, and complement split product) were observed. Importantly, ISIS 113715 did not induce hypoglycemia when administered as a single agent either in normal or diabetic subjects. There were no drug-related serious adverse events in any clinical trial. Asymptomatic, transient prolongation of aPTT during intravenous infusion of phosphorothioate oligonucleotides has been described previously and was observed in preclinical studies with ISIS 113715. In agreement with these findings, intravenous infusion of the drug caused mild, transient, dose-related prolongations of aPTT (⬃37 and 66% increases for the 400 and 600 mg doses, respectively). These prolongations reversed within minutes after the end of infusion and were not associated with any clinically significant manifestations. Since the intravenous route is not the intended route of administration of the drug upon approval and is primarily used to explore pharmacokinetics in early trials, the aPTT changes become a nonissue for late-stage development of the compound. It is also worth noting that nonclinical and clinical experience indicate that ISIS 113715 treatment, at the doses selected for clinical evaluation, is not expected to have CNS, cardiovascular system, bone marrow, skeletal muscle, gastrointestinal, respiratory, immunologic (i.e., antibody formation) or genotoxic effects.

23.4.1.2 ISIS 113715 Clinical Pharmacokinetics Dose-dependent pharmacokinetics were observed in Phase I studies in healthy volunteers. Subcutaneous administration resulted in complete absorption of ISIS 113715 from the injection site and resulted in similar tissue distribution and elimination as that produced after intravenous infusion. No pharmacokinetic interactions were seen following coadministration of ISIS 113715 with glipizide, rosiglitazone or metformin in healthy volunteers [77].

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Plasma pharmacokinetics of ISIS 113715 following single- and multiple-dose administration in normal volunteers and diabetics were remarkably similar. Terminal elimination half-life for ISIS 113715 is preliminarily estimated to be 16 days in type 2 diabetic patients (following treatment at 400 mgⲐweek). Once weekly maintenance dosing did not result in substantial additional accumulation beyond the first week for up to 12 weeks of dosing. In addition, pharmacokinetics were not altered after repeated dose administration up to 12 weeks in type 2 diabetic subjects.

23.4.1.3 ISIS 113715 Clinical Pharmacology Studies in Normal Subjects The pharmacology of ISIS 113715 was examined after administration of the drug at doses of 5.0 and 7.5 mgⲐkg, each administered three times over a course of 1 week [78]. As expected in nondiabetic subjects, there were no changes in glucose excursion during the intravenous glucose tolerance tests (AUC % change from baseline: 5.9, 0.3, 1.3% in the placebo, 5.0 mgⲐkg, and 7.5 mgⲐkg groups, respectively). Pharmacology with ISIS 113715 was reflected as reductions in insulin AUC of 27 and 32% relative to baseline in the 5.0 and 7.5 mgⲐkg cohorts, respectively, a finding not seen in the placebo-treated subjects. A glucose tolerance test in normal volunteers is a particularly rigorous test, since the subjects already have normal insulin sensitivity. Further improvements in insulin sensitivity result in reduced insulin secretion by the pancreas, since the treated subjects require less insulin to maintain normal glucose utilization. This, in turn, is reflected as a reduction in postprandial insulin levels after a glucose challenge, as was seen after just 1 week of ISIS 113715 treatment. The data obtained with ISIS 113715 are consistent with results of preclinical studies in normal and prediabetic animals [8].

Studies in Treatment Naïve Diabetic Subjects The longest duration of ISIS 113715 treatment in type 2 diabetic patients has been 12 weeks at a dose of 200 mgⲐweek (n ⫽ 30). The subjects were newly diagnosed, treatment naive, and had moderate diabetes, as reflected in the average baseline HbA1c of ⬃8.0% and fasting plasma glucose levels of ⬃160 mgⲐdL. Each subject received three doses of ISIS 113715 or placebo during the first week, followed by weekly doses for the remaining treatment weeks. After 3 months of treatment, subjects treated with 200 mgⲐweek ISIS 113715 demonstrated significant improvement in several measurements of glucose homeostasis as compared to the placebo group. Specifically, reductions were seen in both plasma fasted (⬃20 mgⲐdL) and postprandial (⬃30 mgⲐdL) glucose levels [79]. In addition, by the end of treatment, patients treated with ISIS 113715 achieved significant reductions in total and LDL cholesterol levels (⬃35 and ⬃20 mgⲐdL, respectively) as compared to the placebo group. Body weight decreased (⬃2 kg) in both placebo and 113715-treated groups, reflecting compliance to diet and exercise in this drug-naive population. Dietary compliance also resulted in an average decrease in plasma glucose levels of ⬃15 mgⲐdL during the 3-week baseline period in both the placebo- and ISIS 113715-treated groups. As expected, this reduction in plasma glucose levels during the baseline period contributed towards the observed reduction in HbA1c at the end of the subsequent 12-week treatment period, resulting in a significant placebo effect of 0.7% on HbA1c. ISIS 113715 treatment caused a reduction of 1.1% versus baseline, indicating a placebo-subtracted reduction of 0.4%. Importantly, while HbA1c reductions in the placebo group reached a plateau at 9 weeks, those in the ISIS 113715-treated group continued to decline at the end of treatment. Thus, whereas the primary goal of this small, short-term study was to evaluate safety of the drug in a “pure” type 2 diabetes population, encouraging pharmacology was observed in several measures of glucose and lipid control. Evaluation of ISIS 113715 as combination therapy with oral antidiabetic agents is in progress to assess its therapeutic potential for type 2 diabetes.

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These clinical findings provide the first demonstration of pharmacology in type 2 diabetic patients with any specific PTP-1B inhibitor and validate PTP-1B as a therapeutic target for type 2 diabetes. The data suggest that ISIS 113715 may offer a substantial improvement over currently available diabetes drugs. ISIS 113715 could control blood glucose without the risk of hypoglycemia and weight gain. In addition, ISIS 113715 could also be useful for the treatment of dyslipidemia that is often observed in diabetic patients. 23.5 CONCLUSION As is evident from the preceding sections, the extensive in vivo drug-discovery effort conducted in recent years has identified multiple, exciting targets for the treatment of metabolic disorders. Reduction of these targets with antisense drugs has produced pharmacological effects that not only provide the desired glucose control, but also encompass reductions in hyperlipidemia and body weight gain, thereby offering a multipronged approach for the treatment of diabetic patients. Furthermore, several drug-development opportunities have been identified that have unique antisense advantages, making them very compelling therapeutic strategies. During the next few years, multiple antisense drugs are poised to enter the clinic. These drugs display unique therapeutic profiles that are unmatched by existing therapies. While long-term efficacy and safety of antisense drugs needs to be carefully evaluated in the coming years, the data obtained to date suggest that these drugs may add a new dimension to our therapeutic arsenal for metabolic diseases such as type 2 diabetes and NASH. ACKNOWLEDGMENTS The author thanks Pamela Black, Tracy Reigle, and Susan F. Murray for assistance in preparation of this chapter. The author is also indebted to Drs. Brett P. Monia, Frank Bennett, Mark Wedel, and Stan Crooke for their guidance and support and to members of the Metabolic Research & Development teams at Isis for their contribution to the unpublished data discussed in this chapter. REFERENCES 1. B. B. Kahn and L. Rossetti; Type 2 diabetes—who is conducting the orchestra?; Nat Genet; 20; 223–225; 1998. 2. S. T. Crooke; Basic principles of antisense technology; Antisense Drug Technology: Principles, Strategies, and Applications; S. T. Crooke, ed.; Marcel Dekker, Inc., New York, USA; 2001. 3. S. T. Crooke; Progress in antisense technology; Annu Rev Med; 55; 61–95; 2004. 4. R. S. Geary, R. Z. Yu and A. A. Levin; Pharmacokinetics of phosphorothioate antisense oligodeoxynucleotides; Curr Opin Invest Drugs; 2; 562–573; 2001. 5. S. T. Crooke; Antisense strategies; Curr Mol Med; 4; 465–487; 2004. 6. R. J. Gum, L. L. Gaede, S. L. Koterski, M. Heindel, J. E. Clampit, B. A. Zinker, J. M. Trevillyan, R. G. Ulrich, M. R. Jirousek and C. M. Rondinone; Reduction of protein tyrosine phosphatase 1B increases insulin-dependent signaling in obⲐob mice; Diabetes; 52; 21–28; 2003. 7. C. M. Rondinone, J. M. Trevillyan, J. Clampit, R. J. Gum, C. Berg, P. Kroeger, L. Frost, B. A. Zinker, R. Reilly, R. Ulrich, M. Butler, B. P. Monia, M. R. Jirousek and J. F. Waring; Protein tyrosine phosphatase 1B reduction regulates adiposity and expression of genes involved in lipogenesis; Diabetes; 51; 2405–2411; 2002. 8. B. A. Zinker, C. M. Rondinone, J. M. Trevillyan, R. J. Gum, J. E. Clampit, J. F. Waring, N. Xie, D. Wilcox, P. Jacobson, L. Frost, P. E. Kroeger, R. M. Reilly, S. Koterski, T. J. Opgenorth, R. G. Ulrich, S. Crosby, M. Butler, S. F. Murray, R. A. McKay, S. Bhanot, B. P. Monia and M. R. Jirousek; PTP1B antisense oligonucleotide lowers PTP1B protein, normalizes blood glucose, and improves insulin sensitivity in diabetic mice; Proc Natl Acad Sci USA; 99; 11357–11362; 2002.

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26. K. Tsukiyama-Kohara, F. Poulin, M. Kohara, C. T. DeMaria, A. Cheng, Z. Wu, A. C. Gingras, A. Katsume, M. Elchebly, B. M. Spiegelman, M. E. Harper, M. L. Tremblay and N. Sonenberg; Adipose tissue reduction in mice lacking the translational inhibitor 4E-BP1; Nat Med; 7; 1128–1132; 2001. 27. X. X. Yu, S. L. Booten, S. K. Pandey, B. P. Monia and S. Bhanot; Antisense reduction of eukaryotic initiation factor 4E-binding protein 2 expression improves adiposity and insulin sensitivity in diet-induced obese mice; Keystone Symposia—Diabetes Mellitus and the Control of Cellular Energy; 183; 2006. 28. A. Barthel, D. Schmoll, K. D. Kruger, G. Bahrenberg, R. Walther, R. A. Roth and H. G. Joost; Differential regulation of endogenous glucose-6-phosphatase and phosphoenolpyruvate carboxykinase gene expression by the forkhead transcription factor FKHR in H4IIE-hepatoma cells; Biochem Biophys Res Commun; 285; 897–902; 2001. 29. V. T. Samuel, C. S. Choi, T. G. Phillips, A. J. Romanelli, J. G. Geisler, S. Bhanot, R. McKay, B. Monia, J. R. Shutter, R. A. Lindberg, G. I. Shulman and M. M. Veniant; Targeting foxo1 in mice using antisense oligonucleotide improves hepatic and peripheral insulin action; Diabetes; 55; 2042–2050; 2006. 30. J. G. Geisler, S. Bhanot, R. A. McKay, J. R. Shutter, B. P. Monia, R. A. Lindberg and M. M. Veniant; Effect of Foxo1 antisense oligonucleotide (ASO) therapy in mice; Keystone Symposia–Diabetes Mellitus: Molecular Signaling, Genes and Therapeutics; 224; 2004. 31. J. T. Link; Pharmacological regulation of hepatic glucose production; Curr Opin Invest Drugs; 4; 421–429; 2003. 32. S. Bhanot, S. F. Murray, S. L. Booten, R. A. McKay, S. J. Jacobs, M. D. Michael, A. Nestorowicz, K. W. Sloop and B. P. Monia; Inhibition of hepatic PEPCK (cytosolic) mRNA expression with an antisense oligonucleotide does not attenuate hyperglycemia in diabetic ob/ob and db/db mice; Diabetes; 53 (Suppl. 2); A574; 2004. 33. P. She, S. C. Burgess, M. Shiota, P. Flakoll, E. P. Donahue, C. R. Malloy, A. D. Sherry and M. A. Magnuson; Mechanisms by which liver-specific PEPCK knockout mice preserve euglycemia during starvation; Diabetes; 52; 1649–1654; 2003. 34. J. G. Geisler, M. Veniant-Ellison, S. Bhanot, B. Monia, R. Lindberg, R. McKay and J. Shutter; 98% reduction in liver FBP1 is insufficient to lower fasting blood glucose levels in mice; Diabetes; 54 (Suppl. 1); A373; 2005. 35. M. D. Erion, P. D. van Poelje, Q. Dang, S. R. Kasibhatla, S. C. Potter, M. R. Reddy, K. R. Reddy, T. Jiang and W. N. Lipscomb; MB06322 (CS-917): A potent and selective inhibitor of fructose 1,6-bisphosphatase for controlling gluconeogenesis in type 2 diabetes; Proc Natl Acad Sci USA; 102; 7970–7975; 2005. 36. B. Herzog, U. Wendel, A. A. Morris and K. Eschrich; Novel mutations in patients with fructose-1,6bisphosphatase deficiency; J Inherit Metab Dis; 22; 132–138; 1999. 37. M. Butler, R. Valley, L. M. Watts, S. F. Murray, S. L. Booten, B. P. Monia, M. D. Michael, K. W. Sloop, S. I. Taylor and S. Bhanot; Inhibition of liver glycogen phosphorylase expression using an antisense oligonucleotide lowers blood glucose levels in diabetic mice; Diabetes; 51 (Suppl. 2); A43; 2002. 38. J. L. Treadway, P. Mendys and D. J. Hoover; Glycogen phosphorylase inhibitors for treatment of type 2 diabetes mellitus; Expert Opin Investig Drugs; 10; 439–454; 2001. 39. S. F. Murray, S. L. Booten, E. N. Finger, R. A. McKay, B. P. Monia, A. M. Siesky, J. X. Cao, A. Nestorowicz, S. J. Jacobs, M. D. Michael, K. W. Sloop and S. Bhanot; Antisense inhibition of glucose-6 phosphatase (G6Pase) transport protein T1 expression lowers plasma glucose in diabetic mice; Diabetes; 53 (Suppl. 2); A70; 2004. 40. J. Y. Chou, A. Zingone and C. J. Pan; Adenovirus-mediated gene therapy in a mouse model of glycogen storage disease type 1a; Eur J Pediatr; 161 Suppl 1; S56–S61; 2002. 41. R. C. Andrews and B. R. Walker; Glucocorticoids and insulin resistance: old hormones, new targets; Clin Sci (Lond); 96; 513–523; 1999. 42. R. C. Lin and P. J. Snodgrass; Effect of dexamethasone on 3-hydroxy-3-methylglutaryl-coenzyme A reductase activity and cholesterol synthesis in rat liver; Biochim Biophys Acta; 713; 240–250; 1982. 43. L. M. Watts, V. P. Manchem, T. A. Leedom, A. L. Rivard, R. A. McKay, D. Bao, T. Neroladakis, B. P. Monia, D. M. Bodenmiller, J. X. Cao, H. Y. Zhang, A. L. Cox, S. J. Jacobs, M. D. Michael, K. W. Sloop and S. Bhanot; Reduction of hepatic and adipose tissue glucocorticoid receptor expression with antisense oligonucleotides improves hyperglycemia and hyperlipidemia in diabetic rodents without causing systemic glucocorticoid antagonism; Diabetes; 54; 1846–1853; 2005.

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44. J. E. Friedman, Y. Sun, T. Ishizuka, C. J. Farrell, S. E. McCormack, L. M. Herron, P. Hakimi, P. Lechner and J. S. Yun; Phosphoenolpyruvate carboxykinase (GTP) gene transcription and hyperglycemia are regulated by glucocorticoids in genetically obese db/db transgenic mice; J Biol Chem; 272; 31475–31481; 1997. 45. S. F. Murray, S. Booten, R. A. McKay, M. Butler, B. P. Monia and S. Bhanot; Liver and fat specific reduction of 11-beta-hydroxysteroid dehydrogenase type 1 expression does not cause significant glucose or lipid lowering effects in db/db mice; Keystone Symposia–Diabetes Mellitus and the Control of Cellular Energy; 74; 2006. 46. R. H. Unger; Letter: Glucagon in pathogenesis of diabetes; Lancet; 1; 1036–1042; 1975. 47. G. M. Reaven, Y. D. Chen, A. Golay, A. L. Swislocki and J. B. Jaspan; Documentation of hyperglucagonemia throughout the day in nonobese and obese patients with noninsulin-dependent diabetes mellitus; J Clin Endocrinol Metab; 64; 106–110; 1987. 48. P. Shah, A. Vella, A. Basu, R. Basu, W. F. Schwenk and R. A. Rizza; Lack of suppression of glucagon contributes to postprandial hyperglycemia in subjects with type 2 diabetes mellitus; J Clin Endocrinol Metab; 85; 4053–4059; 2000. 49. J. C. Parker, K. M. Andrews, M. R. Allen, J. L. Stock and J. D. McNeish; Glycemic control in mice with targeted disruption of the glucagon receptor gene; Biochem Biophys Res Commun; 290; 839–843; 2002. 50. C. L. Brand, B. Hansen, S. Groneman, M. Boysen and J. J. Holst; Sub-chronic glucagon neutralisation improves diabetes in ob/ob mice [abstract]; Diabetes; 49; A81; 2000. 51. C. L. Brand, B. Rolin, P. N. Jorgensen, I. Svendsen, J. S. Kristensen and J. J. Holst; Immunoneutralization of endogenous glucagon with monoclonal glucagon antibody normalizes hyperglycaemia in moderately streptozotocin-diabetic rats; Diabetologia; 37; 985–993; 1994. 52. J. G. McCormack, N. Westergaard, M. Kristiansen, C. L. Brand and J. Lau; Pharmacological approaches to inhibit endogenous glucose production as a means of anti-diabetic therapy; Curr Pharm Dev; 7; 1451–1474; 2001. 53. K. F. Petersen and J. T. Sullivan; Effects of a novel glucagon receptor antagonist (Bay 27-9955) on glucagon-stimulated glucose production in humans; Diabetologia; 44; 2018–2024; 2001. 54. K. W. Sloop, J. X. Cao, A. M. Siesky, H. Y. Zhang, D. M. Bodenmiller, A. L. Cox, S. J. Jacobs, J. S. Moyers, R. A. Owens, A. D. Showalter, M. B. Brenner, A. Raap, J. Gromada, B. R. Berridge, D. K. Monteith, N. Porksen, R. A. McKay, B. P. Monia, S. Bhanot, L. M. Watts and M. D. Michael; Hepatic and glucagon-like peptide-1-mediated reversal of diabetes by glucagon receptor antisense oligonucleotide inhibitors; J Clin Invest; 113; 1571–1581; 2004. 55. S. Bhanot, L. M. Watts, K. W. Sloop, J. X. Cao, A. D. Showalter, M. D. Michael and B. P. Monia; Reduction of hepatic glucagon receptor expression with an optimized antisense oligonucleotide increased active GLP-1 levels in cynomolgus monkeys without pancreatic alpha cell expansion or hyperplasia.; Diabetes; 55 (Suppl. 1); A326; 2006. 56. R. Ritzel, M. Schulte, N. Porksen, M. S. Nauck, J. J. Holst, C. Juhl, W. Marz, O. Schmitz, W. H. Schmiegel and M. A. Nauck; Glucagon-like peptide 1 increases secretory burst mass of pulsatile insulin secretion in patients with type 2 diabetes and impaired glucose tolerance; Diabetes; 50; 776–784; 2001. 57. K. Cejvan, D. H. Coy and S. Efendic; Intra-islet somatostatin regulates glucagon release via type 2 somatostatin receptors in rats; Diabetes; 52; 1176–1181; 2003. 58. A. E. Butler, J. Janson, S. Bonner-Weir, R. Ritzel, R. A. Rizza and P. C. Butler; Beta-cell deficit and increased beta-cell apoptosis in humans with type 2 diabetes; Diabetes; 52; 102–110; 2003. 59. Y. Kanai, W. S. Lee, G. You, D. Brown and M. A. Hediger; The human kidney low affinity Na⫹/glucose cotransporter SGLT2. Delineation of the major renal reabsorptive mechanism for D-glucose; J Clin Invest; 93; 397–404; 1994. 60. L. M. Watts, T. A. Leedom, E. V. Wancewicz, A. M. Siwkowski, S. Bhanot and B. P. Monia; Reduction of sodium dependent glucose transporter SGLT2 expression with an antisense oligonucleotide (ASO) optimized to target the kidney results in significant glucose lowering effects in diabetic mice; Diabetes; 54 (Suppl. 1); A386; 2005. 61. D. M. Schnee, K. Zaiken and W. W. McCloskey; An update on the pharmacological treatment of obesity; Curr Med Res Opin; 22; 1463–1474; 2006. 62. J. Hirosumi, G. Tuncman, L. Chang, C. Z. Gorgun, K. T. Uysal, K. Maeda, M. Karin and G. S. Hotamisligil; A central role for JNK in obesity and insulin resistance; Nature; 420; 333–336; 2002.

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63. J. D. Browning and J. D. Horton; Molecular mediators of hepatic steatosis and liver injury; J Clin Invest; 114; 147–152; 2004. 64. G. Marchesini, E. Bugianesi, G. Forlani, F. Cerrelli, M. Lenzi, R. Manini, S. Natale, E. Vanni, N. Villanova, N. Melchionda and M. Rizzetto; Nonalcoholic fatty liver, steatohepatitis, and the metabolic syndrome; Hepatology; 37; 917–923; 2003. 65. S. H. Caldwell, E. E. Hespenheide, J. A. Redick, J. C. Iezzoni, E. H. Battle and B. L. Sheppard; A pilot study of a thiazolidinedione, troglitazone, in nonalcoholic steatohepatitis; Am J Gastroenterol; 96; 519–525; 2001. 66. T. Hasegawa, M. Yoneda, K. Nakamura, I. Makino and A. Terano; Plasma transforming growth factor-beta1 level and efficacy of alpha-tocopherol in patients with non-alcoholic steatohepatitis: a pilot study; Aliment Pharmacol Ther; 15; 1667–1672; 2001. 67. S. Cases, S. J. Smith, Y. W. Zheng, H. M. Myers, S. R. Lear, E. Sande, S. Novak, C. Collins, C. B. Welch, A. J. Lusis, S. K. Erickson and R. V. Farese, Jr.; Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis; Proc Natl Acad Sci USA; 95; 13018–13023; 1998. 68. S. Cases, S. J. Stone, P. Zhou, E. Yen, B. Tow, K. D. Lardizabal, T. Voelker and R. V. Farese, Jr.; Cloning of DGAT2, a second mammalian diacylglycerol acyltransferase, and related family members; J Biol Chem; 276; 38870–38876; 2001. 69. S. J. Stone, H. M. Myers, S. M. Watkins, B. E. Brown, K. R. Feingold, P. M. Elias and R. V. Farese, Jr.; Lipopenia and skin barrier abnormalities in DGAT2-deficient mice; J Biol Chem; 279; 11767–11776; 2004. 70. A. R. Kulkarni, C. S. Choi, D. Savage, K. Morino, V. T. Samuel, S. Kim, A. Wang, J. G. Geisler, S. Bhanot, B. Monia, X. X. Yu, S. Neschen, A. J. Romanelli, G. Cline and G. I. Shulman; Suppression of DGAT2 expression by antisense oligonucleotide improves hepatic steatosis and prevents fat induced insulin resistance in vivo; Diabetes; 54 (Suppl. 1) Late Breaking; 17; 2005. 71. J. M. Ntambi, M. Miyazaki, J. P. Stoehr, H. Lan, C. M. Kendziorski, B. S. Yandell, Y. Song, P. Cohen, J. M. Friedman and A. D. Attie; Loss of stearoyl-CoA desaturase-1 function protects mice against adiposity; Proc Natl Acad Sci USA; 99; 11482–11486; 2002. 72. G. Jiang, Z. Li, F. Liu, K. Ellsworth, Q. Dallas-Yang, M. Wu, J. Ronan, C. Esau, C. Murphy, D. Szalkowski, R. Bergeron, T. Doebber and B. B. Zhang; Prevention of obesity in mice by antisense oligonucleotide inhibitors of stearoyl-CoA desaturase-1; J Clin Invest; 115; 1030–1038; 2005. 73. M. R. Munday; Regulation of mammalian acetyl-CoA carboxylase; Biochem Soc Trans; 30; 1059–1064; 2002. 74. D. Zhang, Z. X. Liu, A. Wang, D. Savage, J. Dong, V. Samuel, B. Monia, S. Bhanot, J. Geisler and G. I. Shulman; Reversal of high-fat diet induced liver insulin resistance in rats treated with stearoyl-CoA desaturase 1 anti-sense oligonucleotide; Diabetes; 54 (Suppl. 1); A360; 2005. 75. L. Abu-Elheiga, M. M. Matzuk, K. A. Abo-Hashema and S. J. Wakil; Continuous fatty acid oxidation and reduced fat storage in mice lacking acetyl-CoA carboxylase 2; Science; 291; 2613–2616; 2001. 76. D. B. Savage, C. S. Choi, V. T. Samuel, Z. X. Liu, D. Zhang, A. Wang, X. M. Zhang, G. W. Cline, X. X. Yu, J. G. Geisler, S. Bhanot, B. P. Monia and G. I. Shulman; Reversal of diet-induced hepatic steatosis and hepatic insulin resistance following suppression of both ACC1 and ACC2 with antisense oligonucleotides; Keystone Symposia—Diabetes Mellitus and the Control of Cellular Energy Metabolism; 74; 2006. 77. R. S. Geary, J. D. Bradley, T. Watanabe, Y. Kwon, M. Wedel, J. J. van Lier and A. A. VanVliet; Lack of pharmacokinetic interaction for ISIS 113715, a 2⬘-o-methoxyethyl modified antisense oligonucleotide targeting protein tyrosine phosphatase 1B messenger RNA, with oral antidiabetic compounds metformin, glipizide or rosiglitazone; Clin Pharmacokinet; 45; 789–801; 2006. 78. G. Liu; Technology evaluation: ISIS-113715, Isis; Curr Opin Mol Ther; 6; 331–336; 2004. 79. Isis Pharmaceuticals; Isis Pharmaceuticals reports positive phase 2 data: ISIS 113715 Improves glucose control in patients with type 2 diabetes; Press Release; June 13, 2006.

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24

Inflammatory Diseases Susan A. Gregory and James G. Karras

CONTENTS 24.1

24.2

24.3

24.4

24.5

24.6

24.7

Introduction .........................................................................................................................666 24.1.1 The Inflammatory Disease Process.......................................................................666 24.1.2 Advantages and Challenges in Antisense Therapy Development.........................666 Inflammatory Bowel Disease (IBD) ...................................................................................667 24.2.1 Pathology and Current Therapy of Inflammatory Bowel Disease........................667 24.2.2 Clinical Evaluation of Intercellular Adhesion Molecule (ICAM)-1 Antisense (Alicaforsen) in Crohn’s Disease..........................................................................667 24.2.3 Clinical Evaluation of Alicaforsen in Ulcerative Colitis ......................................668 24.2.4 Preclinical Application of Antisense in Models of Inflammatory Bowel Disease................................................................................671 Rheumatoid Arthritis...........................................................................................................672 24.3.1 Pathology and Current Therapy of Rheumatoid Arthritis.....................................672 24.3.2 Clinical Evaluation of ICAM-1 and TNF-
Antisense in Rheumatoid Arthritis.........................................................................................672 24.3.3 Preclinical Applications of Antisense in Models of RA.......................................674 Multiple Sclerosis (MS)......................................................................................................675 24.4.1 Disease Pathology and Current Therapy of Multiple Sclerosis............................675 24.4.2 Clinical Evaluation of Very Late Activation Antigen (VLA)-4 Antisense in Multiple Sclerosis .................................................................................................675 24.4.3 Preclinical Application of Antisense in Models of Multiple Sclerosis.................676 Asthma ................................................................................................................................678 24.5.1 Disease Pathology and Current Therapy of Asthma.............................................678 24.5.2 Clinical Evaluation of Antisense Oligonucleotides in Asthma.............................678 24.5.3 Preclinical Application of Antisense in Models of Asthma..................................679 Additional Preclinical in Vivo Pharmacology in Models of Inflammation ........................682 24.6.1 Immunomodulation and Transplantation ..............................................................682 24.6.2 Hyperalgesia..........................................................................................................684 Cellular and Molecular Pharmacology ...............................................................................685 24.7.1 Cell Proliferation, Maturation, and Survival.........................................................685 24.7.2 Cell Activation ......................................................................................................686 24.7.3 Cell Migration and Adhesion................................................................................686 665

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24.7.4 Gene Expression and Receptor Signaling.............................................................686 24.7.5 Mediator Release...................................................................................................687 24.7.6 Immunomodulation and Immune Surveillance.....................................................688 24.8 Future Directions ................................................................................................................689 References ......................................................................................................................................690 24.1 INTRODUCTION 24.1.1 The Inflammatory Disease Process Inflammation is normally a localized, protective host response to trauma or infectious agents. Acute inflammation confines the area of injury, destroys or dilutes the injurious agent, and contributes to the restoration of tissue integrity [1]. Chronic inflammation, resulting from repeated exposure, persistent injury, or a failure to appropriately terminate the immune or inflammatory response, may lead to organ injury and morbidity. Current therapeutic strategies aim to suppress or redirect aberrant cellular responses to environmental and infectious stimuli. 24.1.2 Advantages and Challenges in Antisense Therapy Development Antisense provides a novel pharmacological approach to the modification of the inflammatory response. Advantages of antisense strategies include the ability to achieve hybridization-based target specificity and the ability to reduce expression of a target protein in the nuclear, cytoplasmic, or membrane compartment(s) of cells. This combination of properties distinguishes antisense oligonucleotides (ASOs) from small-molecule and protein-based technologies. In addition, the pharmacokinetic properties of first- and second-generation phosphorothioate oligodeoxyribonucleotides (PS-ODNs) are compatible with more convenient, once daily, or less frequent administration of antisense therapeutics in patients. Polygenetic inflammatory diseases pose formidable challenges for antisense drug development, however, including dose, dose interval, and delivery optimization. The involvement of aberrant local and systemic host responses is apparent in syndromes such as Crohn’s disease, asthma, and rheumatoid arthritis (RA). There is experimental evidence for heterogeneity in cell responsiveness to ASOs [2], which may reflect differences in oligonucleotide uptake efficiency or intracellular distribution. The evaluation of antisense therapeutics in inflammatory diseases can also be complicated by off-target proinflammatory effects of oligonucleotides. Both first- and second-generation PS-ODNs are capable of activating complement and eliciting release of cytokines and chemokines [3]. Cells of the innate immune system may recognize internalized oligonucleotides via Toll-like receptor (TLR) 9 [4]. The existence of non-TLR-based mechanisms for recognition of single- and doublestranded DNA have also been documented in human neutrophils and may also be involved in the acute inflammatory response to specific sequence motifs [5]. Second-generation, 2
-O-methoxyethyl (2
-MOE)-modified PS-ODNs have demonstrated increased pharmacological potency relative to first-generation antisense inhibitors [6]. Consequently, fewer proinflammatory effects of 2
-MOE PS-ODNs are observed at clinically relevant doses [7]. Several lines of evidence can be pursued to distinguish antisense effects of ODNs from off-target effects in vivo, including demonstrations of target mRNA or protein reduction concomitant with pharmacology and activity of multiple ASOs directed to nonoverlapping target mRNA hybridization sites. Mismatched oligonucleotides can also be evaluated in preclinical studies to explore chemical-class-related effects of ODNs. Collectively, these analyses can provide insight into the mechanism(s) underlying oligonucleotide drug activity. While significant challenges lie ahead for clinical development of antisense therapies for inflammatory diseases, important achievements have recently been made. The clinical development status of antisense therapies and the molecules that are likely to advance into the clinic for treatment of inflammatory diseases are the focus of this review.

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24.2 INFLAMMATORY BOWEL DISEASE (IBD) 24.2.1 Pathology and Current Therapy of Inflammatory Bowel Disease Ulcerative colitis (UC) and Crohn’s disease (CD) are polygenetic, chronic inflammatory disorders of the gastrointestinal tract of unknown etiologies. Inflammation in UC is usually limited to the large bowel wall while CD involves transmural inflammation that may occur anywhere in the alimentary canal. UC appears to be driven by the local production of interleukin (IL) 13 [8]. In contrast, T helper (Th) 1 cytokines are characteristically produced in CD, and IL 12 and IL 23 have been implicated in polarization of the Th cell response [9]. Constitutional abnormalities in innate immunity have also been described in CD patients [10,11], and these disturbances may predispose CD patients to accumulation of intestinal contents, leading to breach of the bowel wall mucosal barrier and chronic inflammation. Therapy for IBD has traditionally relied upon 5-aminosalicylic acid compounds and corticosteroids [12,13]. Corticosteroids are effective for induction and maintenance of remission, but toxicities and dependency can complicate treatment. Azathioprine, methotrexate, and cyclosporin have limited use in patients with refractory disease because of the potential for toxicities and infections. The IgG1 antitumor necrosis factor (TNF)-
monoclonal antibodies (mAb) infliximab (Remicade®; Centocor, Malvern, PA) [14] and adalimumab (D2E7, Humira®; Abbott Laboratories, Abbott Park, IL) [15] have also demonstrated efficacy for induction of CD remission. In contrast, the soluble TNF receptor fusion protein etanercept (Enbrel®, Amgen, Thousand Oaks, CA and Wyeth, Madison, NJ) [16] and the IgG4 mAb certolizumab pegol (CDP)571 (Humicade®, UCB, Brussels, Belgium,) [17] failed to demonstrate a therapeutic benefit in CD patients. These differences may be explained by the abilities of infliximab [18] and adalimumab [19] to bind cell-bound and soluble TNF-
and induce apoptosis of lamina propria T lymphocytes (LPLs). 24.2.2 Clinical Evaluation of Intercellular Adhesion Molecule (ICAM)-1 Antisense (Alicaforsen) in Crohn’s Disease The first antisense strategy evaluated in CD patients aimed to block T-cell and neutrophil migration and activation by reducing the expression of ICAM-1 (CD54). ICAM-1 mRNA is highly expressed in tissues from patients with active CD or UC [20], and ICAM-1 antisense has been shown to reduce disease severity, neutrophil infiltrates, and epithelial damage in a mouse model of experimental colitis [21]. Alicaforsen (ISIS 2302, Isis Pharmaceuticals, Carlsbad, CA) is a first-generation oligonucleotide inhibitor of ICAM-1 expression that has demonstrated a satisfactory safety profile and trends suggesting effects on mucosal ICAM-1 expression, endoscopic measures of disease severity, and quality of life in subjects with active CD [22,23]. While alicaforsen failed to achieve the primary endpoint of clinical remission at week 12 when administered at doses of 0.5 to 2 mg/kg/d intravenously 3 times per week for 4 weeks, post hoc population pharmacokinetic analysis indicated that patients with the highest drug exposure had consistent improvements in median Crohn’s disease activity index (CDAI) and quality of life scores. Fixed doses of 300 mg and 350 mg were selected for further evaluation based on a pharmacokinetic/pharmacodynamic model developed from phase 2 results [24]. The 300-mg dose was well tolerated and carried forward into phase 3 development. The clinical efficacy of 300 mg of alicaforsen (3 times per week, intravenous [i.v.], for 4 weeks) was evaluated in two identical phase 3 trials in subjects with active, steroid-dependent CD [25]. While alicaforsen was well tolerated in these studies, no significant difference in clinical remission at week 12 was demonstrated for the alicaforsen and placebo treatments. Several factors may have contributed to the clinical failure of alicaforsen in CD. While ICAM-1 may be overexpressed and play an active role in cell recruitment and activation in mucosal tissues, other integrins are also overexpressed in IBD tissues [26] and vascular addressins such as the integrins have been shown to facilitate lymphocyte homing to mucosal tissues [27]. Consistent and

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adequate reduction of ICAM-1 protein expression may have been achieved in only a portion of targeted cells at the alicaforsen doses and regimen tested, resulting in incomplete suppression of the inflammatory response in affected tissues. Finally, the inflammatory status of the subject population may have influenced the clinical response to alicaforsen. No significant differences in baseline subject characteristics, including previous treatment history, current mean steroid dose, and serum levels of C-reactive protein (CRP), were noted between alicaforsen- and placebotreated subjects. A post-hoc analysis of the phase 3 results suggested, however, that the likelihood of achieving a clinical response increased with higher alicaforsen exposure in subjects who had elevated CRP [25]. The presence or absence of elevated levels of CRP in serum has predicted the clinical outcomes of other agents including TNF-
inhibitors [17,28–30] and the integrin antagonist natalizumab (formerly Antegren, Tysabri®, Biogen Idec, Cambridge, MA and Elan Corporation, Dublin, Ireland) [31]. Whether CRP elevation defines a subset of Crohn’s patients is not known, but these results suggest that customized therapies may be required to manage systemic and local manifestation of Crohn’s disease. 24.2.3 Clinical Evaluation of Alicaforsen in Ulcerative Colitis ICAM-1 interacts with leukocyte function antigen (LFA)-1 (
L2) and integrin CD11b/CD18 (Mac-1,
M2) on neutrophils [32] and eosinophils [33] to facilitate granulocyte adhesion and migration into colonic mucosa in response to inflammatory stimuli. The safety, tolerability, and efficacy of alicaforsen enema (6–240 mg administered once daily for 28 days) were demonstrated in patients with mild to moderate, descending UC [34]. A dose-related improvement in the mean absolute DAI was apparent in alicaforsen-treated patients at the end of the treatment period (day 29) and also at the 3- and 6-month assessment time points as shown in Figure 24.1. Statistically significant differences in DAI improvement between the 2- and 4-mg/mL alicaforsen groups and the placebo group were observed up to 3 months postinitiation of treatment. Improvement in disease severity relative to baseline measures was also significantly increased in the 2- and 4-mg/mL alicaforsen enema dose groups as shown in Figure 24.2. Complete normalization of endoscopy at 3 months was noted in 9 of 16 patients receiving 2 mg/mL or 4 mg/mL alicaforsen enema compared to 0 of 8 placebo-treated patients.

12

10

Baseline Month 1 Month 3 Month 6

Mean DAI

8

6

0.377 p = 0.568

0.744 0.125 p = 0.034

4 0.107

2 n= n= n= 8 7 3 3 8 8 5 4 8 8 3 3

n= 8 8 7 7

n= 8 8 8 8

2 mg/mL

4 mg/mL

0 Placebo Figure 24.1

0.1 mg/mL

0.5 mg/mL

Mean absolute value for disease activity index (DAI) for each alicaforsen enema dose group is shown at each time point and compared with placebo. P values are for change from baseline versus placebo. (From van Deventer, S.J., et al., Gut 53, 1646, 2004. With permission.)

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Mean DAI (% change from baseline)

140

669

Month 1 Month 3 Month 6

120

0.021 p = 0.004

0.016

100

0.414

0.628

80 p = 0.201

60 40 20 n= 8 5 4

n= 7 3 3

n= 8 3 3

n= 8 7 7

n= 8 8 8

Placebo

0.1 mg/mL

0.5 mg/mL

2 mg/mL

4 mg/mL

0

Mean percent change in DAI score from baseline for each alicaforsen enema dose group was compared with placebo at each time point. P values versus placebo. (From van Deventer, S.J., et al., Gut 53, 1646, 2004. With permission.)

Mean % change in DAI (± S.E.M)

Figure 24.2

Figure 24.3

4.0 g Mesalamine

120 mg Alicaforsen

240 mg Alicaforsen

0 −20 −40 −60 0

3

6

9

12

15 18 Week

21

24

27

30

33

Mean percent change in DAI score from baseline for each alicaforsen enema dose group was compared with placebo at each time point. (From Miner, P.B., Wedel, M.K. Xia, S., and Baker, B.F., Aliment. Pharmacol. Ther. 23, 1403, 2006. With permission.)

Alicaforsen was well tolerated, and no significant safety effects were reported. In a separate study, alicaforsen enema (240 mg nightly for 6 weeks) demonstrated improvement in DAI scores and clinical remissions in subjects with active UC. This course of exposure resulted in minimal ( 0.6%) systemic drug exposure [35]. These results suggested that alicaforsen acts locally to reduce colonic mucosal inflammation in UC patients. The effects of alicaforsen enema were compared with standard of care mesalazine enema in subjects with mild to moderate active left-sided UC [36]. Subjects received a nightly enema of 120 mg ISIS 2302, 240 mg ISIS 2302, or 4 g mesalazine for 6 weeks and were then followed for 6 months. As shown in Figure 24.3, DAI relative to baseline decreased over time in subjects in all treatment arms. Clinical improvement was reached by a similar proportion of subjects in each cohort by the end of treatment. By week 18 a higher percentage of subjects in the 120 and 240 mg alicaforsen cohorts (33% and 42%, respectively) met the target improvement criteria (a three point reduction in DAI from baseline). DAI at week 6 relative to baseline, the primary endpoint, was

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% Clinical remission

(a) 20

4.0 g Mesalamine 120 mg Alicaforsen 240 mg Alicaforsen

10

0 0

5

10

15

20

25

30 Week

35

40

(b)

50

55

60

4.0 g Mesalamine 120 mg Alicaforsen 240 mg Alicaforsen

30 % Clinical remission

45

20

10

0 0

5

10

15

20

25

30 Week

35

40

45

50

55

60

Figure 24.4 Rate of clinical remission. (a) Percentage remission over time, where remission is defined as disease activity index (DAI): 2, stool frequency: 1, rectal bleeding: 0, endoscopy: 0, and Physicians’ Assessment of Disease (PAD): 1. (b) Percentage remission over time, where remission is defined as an endoscopic score of 0 (normal or inactive disease). (From Miner, P.B., et al., Aliment. Pharmacol. Ther. 23, 1403, 2006. With permission.)

Proportion in remission

4.0 g Mesalamine

120 mg Alicaforsen

240 mg Alicaforsen

1.00 0.75 0.50 0.25 0.00 0

50

100

150 200 Time to relapse (days)

250

300

350

Figure 24.5 Kaplan-Meier curve estimates of the probability of relapse through week 54. Relapse is defined as an endoscopic score 1. Subjects in remission at week 6 had a Mayo Score of 0 (4 g mesalazine [n  9], 120 mg alicaforsen [n  11], and 240 mg alicaforsen [n  10]). (From Miner, P.B., et al., Aliment. Pharmacol. Ther. 23, 1403, 2006. With permission.)

similar in all treatment groups. Dose-dependent effects of alicaforsen on clinical remission and mucosal healing were also demonstrated as shown in Figures 24.4a and 24.4b, respectively. The long-term outcome of treatment, as determined by a Kaplan-Meier analysis of the rate of clinical relapse, is shown in Figure 24.5. The response to alicaforsen enema was 2–3 times more durable than the response to mesalazine enema based on endoscopic appearance over time. This study suggested that local ICAM-1 reduction leads to modification of the inflammatory disease process in affected colonic mucosal tissue. Durable responses to alicaforsen were also demonstrated in

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Table 24.1 Summary of Decreases in Pouchitis Disease Activity Index (PDAI) and Subscores at Weeks 3 and 6 for 12 Patients Treated with Alicaforsen Baseline PDAI Endoscopy subscore Clinical symptom subscore Histology

11.42  1.62 5.25  0.97 3.75  1.06 2.42  0.51

a

Week 3

Week 6

N/A 3.08  1.88 b 2.33  1.07 b N/A

6.83  2.17 b 2.58  1.68 b 2.25  1.36 b 2.00  0.00

Data are presented as the mean  standard deviation. Statistically significant decreases compared to baseline at P  0.05. Source: Miner, P. et al., Aliment. Pharmacol. Ther. 19, 281, 2004. With permission. a b

subjects with an acute exacerbation of mild to moderate left-sided UC [37]. Collectively, these studies demonstrate the safety and durable effects of topical alicaforsen on UC symptoms and endoscopic appearance of the bowel. Alicaforsen enema has also shown promising results in a related syndrome, pouchitis. Following small bowel resection in refractory UC patients, anal anastomosis is performed along with construction of an ileal pouch. The incidence of pouchitis in these patients is as high as 50% several years after surgery. Alicaforsen enema demonstrated significant clinical benefit in patients with pouchitis in a 6-week, open-label trial [38]. Daily treatment with 240 mg alicaforsen enema improved the pouchitis disease activity index (PDAI) at 6 weeks after treatment initiation as shown in Table 24.1. Significant decreases in endoscopy and clinical symptom subscores were noted after 3 weeks of alicaforsen treatment. Alicaforsen, therefore, appears to provide clinical benefit in UC and pouchitis when administered as a topical therapy. 24.2.4 Preclinical Application of Antisense in Models of Inflammatory Bowel Disease Numerous recent studies have documented the efficacy of systemically or locally administered ASOs in rodent models of IBD and gastric inflammation. Antisense strategies aimed at inhibiting cellular activation [39–42], leukocyte recruitment [43–46], and apoptosis [47] have all demonstrated protective effects in these models. Of particular note, systemic administration of TNF-
antisense was shown to improve disease activity scores in both dextran sodium sulfate (DSS)induced and IL-10-deficient mouse colitis models [40]. These results support the clinical success of TNF-
-directed protein biological therapies in IBD. Systemic or local administration of a firstgeneration ICAM-1 ASO significantly reduced colonic mucosal wall thickness and inflammation, as well as the percentage of colon weight per final body weight in the human leukocyte antigen (HLA)-B27/2 microglobulin transgenic rat IBD model [44]. These effects coincided with inhibition of ICAM-1 protein expression in colonic tissue and in peripheral blood lymphocytes and with reduction of TNF-
immunostaining in the colon. Finally, the efficacy of antisense directed at the p65 subunit of nuclear factor (NF)-B has been demonstrated in mouse DSS colitis following a single intrarectal dose of ASO [39] and in a chronic model of trinitrobenzene sulfonic acid (TNBS)-induced colitis [48] using PS antisense inhibitors. A prevalent complication of abdominal surgery is the development of postoperative ileus, a generalized hypomotility of the gastrointestinal tract that is self-limiting but is responsible for increased morbidity and prolonged hospitalization. Recently, it became clear that manipulation of the intestine results in an influx of inflammatory cells, an occurrence thought to play a key role in the disruption of gastrointestinal tract motility. In an experimental model of postoperative ileus, subcutaneous administration of an ICAM-1 ASO improved gastric emptying, reduced manipulation-induced inflammation, and reduced ICAM-1 protein expression [49]. ICAM-1 appears to be an important mediator of inflammation in a model of postoperative ileus, and parenteral administration of ICAM-1 antisense may offer a potential prophylactic treatment for this condition.

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24.3 RHEUMATOID ARTHRITIS 24.3.1 Pathology and Current Therapy of Rheumatoid Arthritis RA is a chronic inflammatory polyarthritis affecting approximately 1% of adults worldwide. The disorder is characterized by inflammatory synovitis with pain, swelling, and stiffness, and progressive bone erosion that leads to significant disability and poor quality of life. A minority of patients have systemic features of the disease including rheumatoid nodules and lung disease. The etiology of RA is unknown, but experimental evidence suggests that both innate and acquired immune responses are involved in disease initiation [50]. Cytokines produced by macrophages and fibroblasts dominate in affected synovial tissues, and the cytokine profile within the macrophage and fibroblast reflects a hierarchy, with IL-1 and TNF-
assuming particular importance [51]. The TNF-
inhibitors infliximab, etanercept, and adalimumab have been approved for treatment of RA based on their ability to induce disease remissions. TNF-
inhibition also improves clinical and laboratory measures, reduces erosive damage, and decreases disability in patients with RA [52]. Serious side effects, including increased risk for serious infection and malignancies, have been documented for these drugs in clinical trials [53] and in clinical practice [54]. Disease-modifying antiinflammatory drugs (DMARD), including leflunomide and methotrexate, have also been documented to increase the risk for infection [55]. Therapies that safely and effectively modify disease progression alone or in combination with other DMARD are still needed for the management of RA.
Antisense in Rheumatoid Arthritis 24.3.2 Clinical Evaluation of ICAM-1 and TNF-
Therapeutic strategies aimed at suppressing lymphocyte activation and trafficking through joint tissues have recently been evaluated in clinical trials. ICAM-1 protein expression is elevated in the rheumatoid nodules and synovium of RA patients [56]. High concentrations of soluble ICAM-1 have been reported in serum and synovial fluid from patients with active juvenile idiopathic polyarthritis, and correlations between ICAM-1 levels and joint counts suggested a synovial origin for the adhesion molecule [57]. The safety of alicaforsen, the first-generation ICAM-1 antisense inhibitor, was evaluated in a 6-month, placebo-controlled study in patients with active RA [58]. Alicaforsen (0.5–2 mg/kg, administered i.v. 13 times over a 4-week period) was well tolerated but failed to demonstrate clinical efficacy based on the day 26 Paulus index. T- and B-cell immunophenotyping, recall antigen skin testing, and serum immunoglobulin levels revealed no significant immunosuppressive effects of alicaforsen. The lack of pharmacological activity may have been related to poor drug exposure, as plasma areas under the curve were lower than those associated with efficacy in a subsequent study of subjects with Crohn’s disease. A murine mAb directed against human ICAM-1 showed initial efficacy for clinical improvement of early or indolent RA [59,60], but repeated treatment with this mAb produced diminished clinical efficacy that may have been related to the development of antibodies to the murine protein [61]. These clinical results are inadequate to draw formal conclusions regarding the efficacy of ICAM-1 antisense or antibody inhibitors in patients with RA. Antisense inhibition of TNF-
expression has also been evaluated as a therapeutic approach for RA. ISIS 104838 is a second-generation PS-ODN that has demonstrated the ability to markedly and specifically reduce the levels of TNF-
mRNA and secreted TNF-
protein in activated human keratinocytes and monocytes [62]. The safety and pharmacokinetics of ISIS 104838 were demonstrated in healthy subjects in two phase 1 studies [62]. Multiple i.v. infusions of ISIS 104838 (0.1–6 mg/kg on days 1, 8, 10, and 12) and s.c. injections of ISIS 104838 (0.1–6 mg/kg on days 1, 3, and 5) were well tolerated, with transient and reversible partial prothrombin time prolongation observed at higher dose levels. Skin injection sites showed some mild tenderness, erythema, or induration that resolved by 4 days postdosing. Infused ISIS 104838 produced a significant, dose-related reduction in the level of TNF-
protein produced ex vivo by stimulated peripheral

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blood mononuclear cells compared to levels produced at baseline with no effect on IL-1 protein production. Dose-dependent plasma pharmacokinetics and synovium distribution of ISIS 104838 (100 or 300 mg for 6 doses over 22 days) were demonstrated in subjects with active RA, and high synovial drug concentrations were achieved using either i.v. or s.c. dosing over a 1-month period [63]. ISIS 104838 was well tolerated in this study with generally mild s.c. injection-site reactions and transient prolongation of partial prothrombin times at higher dose levels. Synovial biopsies of knee or wrist at the end of the treatment showed a 50% decrease in TNF-
mRNA in three of five patients treated with 300 mg ISIS 104838, whereas no biopsies from placebo subjects showed a 50% reduction. The safety and clinical efficacy of ISIS 104838 were further evaluated in a placebo controlled phase 2 study in TNF inhibitor–naïve subjects with active RA [64]. Subjects received at least eight subcutaneous injections of ISIS 104838 (200 mg every other week, 200 mg every week, or 400 mg every week) or placebo (every week) over a 40-day period. ISIS 104838 was well tolerated in this study, with reversible reduction in circulating platelet counts observed by the end of treatment but no adverse clinical sequelae associated with reduced platelet counts. These results are consistent with reversible platelet reductions observed in monkeys treated with ISIS 104838 [65]. Mild injection-site reactions and mild diarrhea were noted in the 400-mg dose cohort, and no serious adverse events were considered by the investigator to be potentially related to study drug. Response to ISIS 104838 was measured at the end of the 3-month treatment period as the percentage of subjects who achieved a 20% decrease in disease activity as defined by the American College of Rheumatology (ACR20). A trend toward improvement in the ACR20 compared with placebo was observed at each of the highest two ISIS 104838 dose levels as shown in Table 24.2. The ACR20 for the 200-mg biweekly cohort (18.6%) was not significantly different from the placebo group (22.5%). ACR20 results for the 200- and 400-mg weekly dose groups at day 85 were similar (41.2 % and 40.5%, respectively). When the 200- and 400-mg weekly dose groups were combined, a significant difference in ACR20 was observed compared with the placebo-treated group (p  0.05 using the chi-square test), and 50% of the responders in the top two dose groups (combined) maintained their response at day 169. A trend toward improvement over baseline in the number of swollen and tender joints was observed in subjects treated with 400 mg/week ISIS 104838 as shown in Table 24.3. These effects were evident up to 2 months after the end of treatment. Response rates and improvements in primary and secondary endpoint measures increased with drug dose as well as drug exposure based on the plasma drug concentration AUC. These results demonstrated durable clinical activity of ISIS 104838 as a single agent in patients with RA and an exposure-response relationship. No significant difference in the ACR50 rate at day 85 was observed, however, for ISIS 104838 at any dose compared to placebo as shown in Table 24.2. While ISIS 104838 was well tolerated and pharmacologically active in RA patients, the clinical response rates (ACR 20 and ACR 50) were lower than the response rates Table 24.2 ACR 20 and ACR 50 Response Rates at the End of the 3-Month ISIS 104838 Treatment Period

a

ACR 20% at 3 Months: Evaluable a Population (%; n  157)

ACR 50% at 3 Months: Evaluable a Population (%; n  157)

22.5 18.6

7.5 4.7

Placebo ISIS 1048383 200 mg every other week 200 mg every week

40.0 (P  0.09)

5

400 mg every week

41.2 (P  0.08)

11.8

ISIS 1048383 200 and 400 mg combined

40.5b (P  0.05)

N/A

Evaluable indicates all patients who received a minimum of 8 injections over 40 days, without relevant protocol deviations. b Statistically significant increase in response rate compared to placebo.

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Table 24.3 ISIS 1048383 Phase 2 Secondary Endpoint Results at Week 12 Dose

Placebo

200 mg Every Other Week

200 mg Every Week

400 mg Every Week

Count Decrease % Change

Week 12 Swollen Joint Count Decrease, % Change 4.6 3.1 4.8 18.5 0.3 8

3.9 22

Count Decrease % Change

Week 12 Tender Joint Count Decrease, % Change 5.4 3.3 6.1 0.2 14 22

13.5 33

reported for etanercept [66] or adalimumab [67] after 3 months of treatment. These results may be attributed to differences in the systemic or synovial distribution of the drugs. Pharmacodynamic analyses suggested that TNF-
protein expression was only partially reduced under the treatment conditions evaluated in the ISIS 104838 trials. Incomplete reduction of TNF-
expression in some tissues or cell populations may have allowed systemic or synovial inflammatory responses to persist during treatment. It is also possible that etanercept and adalimumab have additional pharmacological activities in vivo that are not achieved using an antisense approach to inhibition of cellular responses to TNF in RA. The inferior clinical efficacy of ISIS 104838 suggests that second-generation PS-ODNs lack the biopharmaceutical profile required for effective parenteral treatment of active RA. Progress toward achievement of oral bioavailability for second-generation oligonucleotides has been made using formulations of ISIS 104838 that include the permeation enhancer, sodium caprate, in an enteric-coated solid dosage form. The tolerability and relative plasma bioavailability of single and multiple doses of each of seven tablet formulations of ISIS 104838 were evaluated in healthy subjects [68]. Each of the administered formulations was well tolerated. The average plasma bioavailability was 1.4% relative to i.v. administration with similar tissue distribution profiles. Mean systemic tissue bioavailability ranged from 2% to 4.3%, relative to i.v. tissues, and was dependent on tissue type, with highest drug concentrations observed in kidney followed by liver, lymph nodes, and spleen. These results demonstrate that comparable tissue concentrations of a second-generation oligonucleotide can be achieved using oral or i.v. administration. Oral formulations could provide greater flexibility in oligonucleotide administration, e.g., dose and dose interval, and facilitate the customization of combination antisense inhibitor therapies for the treatment of chronic diseases. 24.3.3 Preclinical Applications of Antisense in Models of RA In rats with arthritis induced by injection of Mycobacterium butyricum, daily intraperitoneal administration of an 18-mer first-generation phosphorothioate cyclooxygenase (COX)-2 ASO (⬃25–30 mg/kg) in a prophylactic treatment regimen reduced synovial tissue COX-2 but not COX-1 mRNA expression and protein immunostaining, hind paw swelling, synovial hyperplasia, mononuclear cell infiltration, and joint destruction [69]. Neither sense nor scrambled-sequence control oligonucleotides demonstrated these effects. The COX-2 ASO failed to demonstrate efficacy when administered to mice with established arthritis, however. The differences in efficacy may be related to unique patterns of oligonucleotide distribution in normal and inflamed tissues or the expression of COX-2 in additional cell populations in synovial tissue with established inflammation. Canonical rodent cytosine-guanosine-dinucleotide (CpG) motifs (Purine-Purine-CpG-Pyrimidine-Pyrimidine) were present in antisense, sense, and scrambled control oligonucleotides, indicating that the investigators controlled for the possible non-hybridization-based effects of this motif in the ASO. Deng et al. utilized intraarticular administration of PS-ODNs targeted to the p65 subunit of the NF-B transcription factor in mice with bacterial CpG deoxyribonucleic acid (DNA)-induced arthritis [70]. p65 ASO treatment reduced the incidence of joint inflammation by 50%, suggesting that local administration of ASO may effectively block the activation of macrophages in the joint or

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inhibit TNF-
-mediated signaling in other synovial inflammatory cells in a p65 NF-B-dependent manner.

24.4 MULTIPLE SCLEROSIS (MS) 24.4.1 Disease Pathology and Current Therapy of Multiple Sclerosis MS is a polygenic disorder of unknown etiology characterized by perivascular T-cell infiltration and disseminated demyelinating white matter disease of the central nervous system (CNS) that usually affects young adults. Considerable debate exists within the clinical community regarding an autoimmune pathogenesis in MS [71]. Recent histopathology studies suggest that there may be considerable heterogeneity in the pathogenesis of MS as evidenced by the identification of four distinct subtypes of MS, all characterized by perivascular and parenchymal T-cell and macrophage inflammation [72]. While no infectious process has been linked to MS, the involvement of viral infection has been suggested by the ability of a variety of viruses to produce acute and chronic demyelinating conditions in experimental animals [73]. The unresolved inflammatory response in MS contributes to neuronal damage and the formation of plaques or lesions that interfere with axon function, resulting in neurocognitive or neuromuscular impairment. Four clinical subtypes of MS have been described based on the clinical course of the disease, with the majority of patients developing a relapsing-remitting lifelong course that is rarely fatal and a minority developing progressive disability from the onset with or without superimposed relapses (so-called primary progressive multiple sclerosis), with reduced life expectancy [74]. The -interferon (IFN-) preparations Avonex® (Biogen Idec), Rebif ® (Merck KGaA, Darmstadt, Germany and Pfizer, New York, NY) and Betaferon/Betaseron® (Chiron, Emeryville, CA and Berlex Laboratories, Montville, NJ) and the immunomodulatory glatiramer acetate Copaxone® (Teva Neuroscience, Petach Tikva, Israel and Aventis Pharmaceuticals, Strasbourg, France) have been approved for the long-term treatment of relapsing-remitting MS. Each of these drugs is generally well tolerated with reported adverse effects including flu-like symptoms and injection-site reactions [75]. These therapies produce moderate effects on disease relapse rate, disease severity, and progression of disability in MS patients [76]. Immunomodulators such as cyclosporin, methotrexate, and azathioprine provide some therapeutic benefit in progressive MS, but the frequent occurrence of adverse reactions to treatment, especially nephrotoxicity, together with the small magnitude of the potential benefit, makes the risk/benefit of this therapeutic approach unacceptable. The immunosuppressant mitoxantron (Ralenova®, Wyeth, Madison, NJ) was recently approved by the European Agency for the Evaluation of Medicinal Products (EMEA) as a second-line therapy for patients with secondary-progressive and progressive-relapsing MS who have failed to respond to immunomodulatory agents [77]. 24.4.2 Clinical Evaluation of Very Late Activation Antigen (VLA)-4 Antisense in Multiple Sclerosis Antagonism of T-cell trafficking has been investigated as a therapeutic approach to MS using antibody and antisense technologies. Clinical evidence of the central role of VLA-4 (
41-integrin) in lymphocyte transmigration into the CNS has recently been confirmed based on the efficacy of the humanized mAb to
4 integrin, natalizumab (Antegren, Tysabri, Elan Pharmaceuticals and Biogen/Idec) in patients with relapsing MS. Long-term administration of natalizumab provided significant benefits in clinical trials, including a marked reduction in the risk of new lesions and a significant reduction in the risk of exacerbations within 2 months of the initiation of therapy [78]. In early 2005, Biogen Idec and Elan Corporation voluntarily suspended marketing of Tysabri in the United States based on two reported cases of progressive multifocal leukoencephalopathy (PML) [79,80],

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a rare and frequently fatal, demyelinating disease of the CNS, in patients who received Tysabri. In June 2006, after an extensive safety review, the FDA approved the reintroduction of Tysabri for the treatment of relapsing forms of MS. Prophylactic and therapeutic treatment with s.c. injections of a second-generation ASO directed to the mouse VLA-4
4 integrin molecule was demonstrated to reduce the incidence and severity of paralytic symptoms in the experimental autoimmune encephalomyelitis (EAE) model of MS in mice [81]. The effect on key disease severity indicators appeared comparable to results reported for a mouse VLA-4 antibody [82] and mouse interferon [83] in similar mouse models of MS. These results suggested that antisense targeting of VLA-4 would be an effective therapeutic approach in MS. A second-generation antisense inhibitor of VLA-4 (ATL1102, Antisense Therapeutics Limited or ATL, Melbourne, Australia) is currently being investigated as a subcutaneous therapy for relapsing-remitting MS. The pharmacokinetics and safety of ATL1102 was investigated in a doubleblind, placebo-controlled study in healthy subjects [84]. Both i.v. and s.c. formulations of ATL1102 were well tolerated. The most frequently reported side effects included mild flu-like symptoms and occasional injection-site reactions, which were generally mild but increased in incidence and severity with escalating dose levels, particularly at 12 and 18 mg/kg/week. A dose of 6 mg/kg/week of ATL1102 appeared well-tolerated and was selected for further clinical evaluation. A phase 2a clinical trial designed to obtain preliminary evidence of the drug’s effectiveness using magnetic resonance imaging (MRI) indices was initiated in December 2004. Although no safety problems had been reported, ATL voluntarily halted its trial in March 2005 in light of safety issues associated with the drug, Tysabri. In August 2005, an independent, international medical advisory board, convened by ATL, unanimously recommended that the company continue the development of ATL1102 in MS, and that the phase 2a trial be restarted with the addition of certain safety parameters to address the potential safety issues reported in the Tysabri trials. The ongoing study is a randomized, double-blinded, placebo-controlled trial in 80 patients with relapsing-remitting MS. Patients receive ATL1102 (200 mg administered twice weekly, s.c., for 8 weeks) or placebo [85]. MRI will be conducted at monthly intervals over the 8-week dosing period and at monthly intervals during the 8-week period following completion of dosing. 24.4.3 Preclinical Application of Antisense in Models of Multiple Sclerosis A series of recent reports indicate that systemic administration of ASO may show promise for treatment of MS. All these studies were conducted in EAE rodent models and aimed to inhibit specific mechanisms of immune cell activation. The p75 low-affinity neurotrophin receptor (p75NTR) is associated with the pathology of MS, CNS inflammation mediated by immune cells, oligodendrocyte demyelination, and death [86]. Intraperitoneal injection of phosphorothioate p75NTR ASO daily for 18 days following immunization of SJL/J (H-2s) mice (with myelin proteolipid protein peptide 139–151) significantly reduced the mean maximal disease score, disease incidence, CNS inflammation, and demyelination, and produced a 30% decrease in p75NTR expression at the blood-brain barrier as determined by immunohistochemistry, compared to control animals injected with PBS or non-sense oligonucleotide [87]. p75NTR expression in neural cells was not different between the three treatment groups. p75NTR protein has been demonstrated on the surface of perivascular macrophages, endothelial cells, and infiltrating monocytes [88,89] and EAE was exacerbated rather than improved in p75NTR-deficient mice [90]. These observations suggest that the role of p75NTR in EAE models may depend on the cell population in which it is expressed, and may mediate a proinflammatory response in immune cells. The role of the Th1 cell transcription factor T-bet was evaluated in a mouse model of EAE by i.v. injection of a T-bet phosphorothioate ASO at the time of immunization [91]. A single injection of T-bet ASO (50 g; ⬃2 mg/kg) prevented the onset of actively induced disease, suppressed disease score in a dose-related manner for up to 45 days and reduced T-bet protein in splenocytes at day 13 by 60%, compared to animals that received non-sense control oligonucleotide injection.

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In this study, both the T-bet antisense and non-sense oligonucleotides contained noncanonical CpG motifs. Injection of T-bet small interfering ribonucleic acid (siRNA) produced similar effects in this model and both T-bet ASO and siRNA inhibited IFN- production in splenocytes and lymph node cells. An
4 integrin ASO approach demonstrated efficacy in mouse EAE using either a prophylactic or therapeutic dosing regimen [81]. In this study, a second-generation 2
-MOE ASO (1 mg/kg) was administered s.c. each day starting either one day before EAE induction or when more than 50% of the mice in the vehicle group exhibited Grade 1 or higher EAE and continued for 15 days. Significant effects on disease severity and relapse rate were observed, coupled with a reduction in immunohistochemical staining for VLA-4 cells, CD4 T cells, and BM8 macrophages in spinal cord sections as shown in Figure 24.6, suggesting that
4 down-regulation inhibits trafficking of immune cells into the CNS during EAE. It has been shown that cholinergic stimulation can mediate suppression of proinflammatory cytokine production in a mouse model of lethal endotoxemia [92]. Nizri and colleagues targeted acetylcholinesterase (AChE) mRNA in mice induced to develop EAE with a 20-mer ASO containing three 2
-O-methyl modifications on the 3
end. Daily treatment with 0.1 mg/kg administered by intraperitoneal injection starting at the day of induction of disease produced a reduction in AChE activity in serum and suppressed disease severity by 90% [93]. Leukocyte infiltration in the CNS was Saline

VLA-4 ASO

(a)

(b)

(c)

(d)

(e)

(f)

VLA4+ cells

CD4+ T cells

BM8+ Mφs

Figure 24.6 (See color insert following page 270.) VLA-4, T cell, and macrophage immunostaining on spinal cords from EAE mice. Saline-treated mice (a, c, e) had a Grade 2 paralysis but were symptom free while receiving treatment with a VLA-4 antisense inhibitor (b, d, f). Antibodies were used to detect VLA-4 (a, b), CD4 T cells (c, d), or BM8 macrophages (e, f). Magnification: 250X. (From Myers, K.J. et al., J. Neuroimmunol., 160, 12, 2005. With permission.)

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reduced in AChE ASO–treated animals, although this study did not compare the ASO results to those of a control oligonucleotide sequence.

24.5 ASTHMA 24.5.1 Disease Pathology and Current Therapy of Asthma Asthma is a polygenetic syndrome characterized by symptoms of wheeze, dyspnoea, and cough. Objective evidence of variable airflow obstruction provides confirmation of the clinical diagnosis. In developed countries the prevalence of asthma has steadily increased over the past 30 years and is strongly but not exclusively linked to allergic sensitization to environmental antigens [94]. Interactions between environmental and genetic factors likely influence disease progression and severity as well as individual risk for development of asthma [95]. Linkage analyses have also identified polymorphic variation in treatment response [96]. Despite the marked heterogeneity in asthma phenotype that has been documented in clinical practice [97], extensive experimental and clinical evidence indicates that persistent asthma is an inflammatory disorder of the airways involving resident pulmonary and inflammatory cells as well as recruited lymphocytes, mast cells, and granulocytes. Chronic inflammation in susceptible individuals causes recurrent episodes of symptoms, variable airflow obstruction, and increased airway hyperresponsiveness (AHR) to a variety of stimuli [98]. In severe disease, the pathology may include airway remodeling and irreversible airflow obstruction. The inflammatory processes in asthma and animal models of airway inflammation are associated with Th 2 cells and cytokines, especially IL-4, IL-5, and IL-13 [99–101]. Other inflammatory mediators, including leukotrienes, prostaglandins, and platelet-aggregating factors, are also produced in response to inhaled antigens and are capable of inducing bronchospasm, mucus overproduction, and cell recruitment to the lung. Leukotriene modifiers and receptor antagonists have been shown to provide clinical benefit to patients with persistent asthma [102–104]. The goals of asthma management currently emphasize environmental control measures and treatment to prevent chronic persistent symptoms and preserve lung function [105]. Corticosteroids are the most effective treatment available for atopic diseases, and inhaled corticosteroids (ICSs) are the firstline treatment for chronic asthma in patients of all ages and severity of disease [106]. These drugs inhibit transcription factors such as activator protein-1 (AP-1), NF-B, and nuclear factor of activated T cells (NF-AT), and thereby suppress the expression of multiple inflammatory genes, including cytokines, enzymes, adhesion molecules, and inflammatory mediator receptors [107,108]. While ICSs have a proven track record of safety and efficacy for symptom control, these drugs do not cure asthma and do not adequately control symptoms in all individuals. Step-up controller therapies include higher dose ICS, long-acting 2-agonists (LABA), leukotriene receptor antagonists, theophylline, cromolyn, and oral costicosteroids. Unmet medical needs in asthma include medications that can modify the progression of asthma, especially in children, or provide more complete and consistent asthma control in combination with low doses of ICS. Novel corticosteroids with very limited systemic exposure and therapies that offer more convenient dosing schedules are also being evaluated in clinical studies. 24.5.2 Clinical Evaluation of Antisense Oligonucleotides in Asthma Respiratory diseases, including asthma, are well suited for inhaled therapies, and present an attractive opportunity for topical antisense strategies. Phosphorothioate oligonucleotides distribute broadly in the lungs of mice [109,110] and rabbits [110] with very limited systemic exposure. Similar results have been observed for second-generation oligonucleotides in mice and monkeys [111]. Pulmonary concentrations of oligonucleotides orders of magnitude higher than the concentrations required for pharmacological activity are readily achieved and well tolerated in mice using aerosolization and nose-only inhalation [112]. These findings suggest that inhaled antisense

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inhibitors could provide a novel therapeutic approach for control of symptoms and disease modification in patients with asthma. EpiGenesis Pharmaceuticals (Cranbury, NJ) was the first to demonstrate clinical efficacy of an aerosolized ASO. EPI-2010, a first-generation ASO targeting the adenosine A1 receptor mRNA, was well tolerated in normal subjects and patients with mild asthma. A single inhaled dose of EPI-2010 (50 mcg/kg) produced a significant decrease in the requirement for a rescue bronchodilator concomitant with reduction in asthma symptom scores in patients with mild asthma [113]. However, EPI-2010 failed to demonstrate efficacy in patients with moderate asthma that was not well controlled by ICS [114], and development of this compound was discontinued. Topigen Pharmaceuticals (Montreal, Quebec, Canada) recently demonstrated the safety, tolerability, and pharmacological activity of TPI-ASM8, a first-in-class antisense drug that combines two oligonucleotides into a single inhaled product, in patients with mild allergic asthma. TPI-ASM8 is a combination of two ASOs targeting the cysteine-cysteine chemokine receptor (CCR)-3 and the common  chain of the IL-3, IL-5, and granulocyte and macrophage colony stimulating factor (GM-CSF) receptors [115]. CCR-3 is an important receptor involved in the differentiation, adhesion, and chemotaxis of eosinophils, mast cells, and macrophages [116], while IL-3, IL-5, and GM-CSF are involved in the survival and activation of eosinophils, mast cells, and macrophages [117]. Preliminary data from a phase 2 study in patients with mild asthma was recently reported showing substantially reduced sputum eosinophil cell number, suppressed target gene expression, and significant physiological effects (on the early and late phase response) in these patients following three daily doses of TPI-ASM8 prior to allergen challenge [118]. The compound combines a phosphorothioate backbone with 2, 6-diaminopurine modifications of adenosines. A second-generation oligonucleotide, ISIS 369645, has advanced into clinical development. ISIS 369645 is a 5-10-5 2
-MOE gapmer oligonucleotide that reduces expression of IL-4 receptor-
(IL-4R
) mRNA and protein in human cells [119]. IL-4R
is shared by the IL-4 and IL-13 receptors, and its presence is required for cellular responses to both cytokines [120]. Inhaled antisense inhibition of this receptor has been shown to reduce antigen-induced airway eosinophilia and neutrophilia, mucus production, and AHR to methacholine in mice concomitant with reduction of IL-4R
protein on pulmonary antigen presenting cells and epithelial cells [121]. The mouse IL-4R
ASO was effective when administered to mice with established asthma and also demonstrated additive effects with inhaled budesonide on airway inflammation and AHR in mice [122]. ISIS 369645 is currently being evaluated in preclinical toxicology studies and is expected to enter phase 1 studies in 2007. 24.5.3 Preclinical Application of Antisense in Models of Asthma In recent years, substantial evidence has been generated supporting the use of ASO for allergic diseases, in particular, by topical delivery to the respiratory tract to treat disorders such as asthma and allergic rhinitis [114,123]. The sufficiency of local targeting of a gene product(s) in the pulmonary environment, in the absence of systemic suppression, remains to be demonstrated in asthma patients, but preclinical models have provided encouraging results to date. Both firstand second-generation ASO administration, either by systemic or local routes, have produced anti-inflammatory effects in rodent asthma models with concomitant pharmacodynamic activity in relevant immune cells, suggesting that current chemical designs appear to be satisfactory for clinical development as aerosolized medications. Systemic antisense administration may be required for targets with significant biological roles outside of the pulmonary tissue, such as the role of the IL-5R
in eosinophil maturation and emigration from the bone marrow. Lach-Trifilieff and colleagues administered a 2
-MOE-modified second-generation IL-5R
ASO by i.v. injection to mice and observed suppression of bone-marrow and blood eosinophilia following treatment with recombinant murine IL-5. IL-5R
ASO treatment also inhibited blood and tissue eosinophilia in a ragweed-induced allergic peritonitis model [124].

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Other investigators evaluated the effects of pretreatment of mice with i.v. administration of a first-generation phosphorothioate ASO targeting the p65 subunit of NF-B in the ovalbumin (OVA) sensitization and challenge model of asthma [125]. Two successive days of treatment with the p65 ASO immediately before OVA challenge significantly reduced lung and airway inflammation, AHR, bronchoaveolar lavage (BAL) Th2 cytokine levels, and antigen-specific IgE and IgG in serum. Parameters of target-dependent toxicity that might be expected with systemic exposure to an NF-B pathway inhibitor were not reported in the study. The broad utility of locally delivered ASO for treatment of allergic respiratory disease is supported by the effective targeting of an array of diverse molecular targets expressed by a variety of different cell types. Intranasal administration of phosphorothioate ASOs targeting stem cell factor (SCF), the c-kit ligand [126] or the Th2 cell transcription factor GATA-3 [127] in mice reduced pulmonary inflammation in both studies following OVA sensitization and challenge. In the GATA-3 ASO experiments, the investigators also monitored AHR and showed significant ASO-mediated suppression following cholinergic stimulation. The treatment effects of the ASOs were correlated with inhibition of protein expression in lung interstitial cells (SCF and GATA-3) and epithelium (SCF). In other supporting studies, rats were treated with a first-generation PS-ODN designed to suppress the expression of the common  chain of the IL-3, IL-5, and GM-CSF receptors by intratracheal administration prior to OVA challenge [128]. Levels of  chain mRNA levels in lung were found to be reduced by 60% compared to mismatch control oligonucleotide treatment, and  chain immunostaining was also decreased in the subepithelial regions of the lung. One dose (⬃1 mg/kg, intratracheal) administered before allergen challenge inhibited airway and lung antigen-induced eosinophilia as well as leukotriene D4–induced AHR. Further, similar anti-inflammatory effects were observed in OVA sensitized and challenged rats treated with an aerosolized stem loop structure Syk ASO consisting of 60 nucleotides formulated in liposomes [129,130]. Excellent potency of inhaled first- and second-generation ASO has been demonstrated in animal models of asthma [131,132]. Reduction of allergen-induced phenotypes in OVA sensitized and challenged mice were observed at lung concentrations of a 2
-MOE p38
mitogen–activated protein kinase (MAPK) ASO from ⬃10 to 300 ng/g of lung tissue (⬃3–300 g/kg estimated inhaled dose; EID) [132]. The specificity of the p38
ASO used in this study for the
isoform and not the other p38 family members was demonstrated in vitro and in vivo as shown in Figure 24.7 [133], suggesting that an inhaled ASO strategy can selectively inhibit expression of individual molecular targets despite a high degree of protein similarity. An inhaled 2
-MOE IL-4R
ASO produced a dose-dependent reduction in the percentage of BAL eosinophils in OVA challenged mice, with activity observed at 10 ng ASO/g lung tissue [111,121,134]. Flow cytometric analyses of lung cells following inhaled ASO treatment showed reduced cell-surface IL-4R
expression in dendritic cells and the mixed alveolar macrophage and eosinophil population (Figure 24.8) compared to vehicle-treated, challenged mice [122]. Since asthma is a chronic disease with ongoing pulmonary inflammation, novel therapies must effectively suppress existing disease or prevent episodic exacerbations. Previous studies have indicated mixed results for Th2 pathway inhibitors in chronic mouse asthma models, with activity demonstrated following intraperitoneal injection of an IL-13 antibody [135] and only modest effect of an intranasally administered IL-4 mutein that blocks IL-4 and IL-13 bioactivity [136]. More recently, therapeutic efficacy in a chronic mouse asthma model was demonstrated following once-weekly inhalation of a 2
-MOE IL-4R
ASO. IL-4R
ASO treatment reduced the percentage of eosinophils and neutrophils in the airways, suppressed perturbation of the animals’ breathing pattern (measured as enhanced pause or Penh) in response to methacholine, and decreased mucus production and lung inflammation. Systemic treatment with dexamethasone had similar effects but did not reduce the percentage of airway neutrophils (Figure 24.9) [122]. Inhaled IL-4R
antisense demonstrated significant effects on Penh and airway neutrophilia at an estimated delivered dose of

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Figure 24.7 Isoform specificity of p38
ASO in cells and in liver tissue. (a) THP-1 cells were electroporated with p38
ASO at the concentrations shown. Cellular mRNA was extracted 24 h later and analyzed for p38 isoform expression by quantitative RT-PCR. Data are normalized to cells that were electroporated in the absence of p38
ASO. (b) Subcutaneous administration of p38
ASO decreased the level of p38
mRNA in livers of Balb-c mice but had no effect on p38 mRNA expression. The mismatched (MM) control oligonucleotide had no effect on either p38
or p38 mRNA expression. (From Karras, J. et al., Strategic Research Institute’s Protein Phosphorylation Drug Discovery World Summit, La Jolla, March 1, 2005. With permission.)

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Control oligo IL 4R α ASO Oligonucleotide concentration (mcg/kg) Figure 24.8 Reduction of cell-surface IL-4R
protein on pulmonary antigen presenting cells from allergic mice following inhalation of IL-4R
ASO. OVA-sensitized and challenged mice were rested for 1 month before treatment with aerosolized oligonucleotides and subsequent rechallenge with nebulized OVA. Lungs were harvested 6 h following the second nebulized OVA rechallenge on day 67 from mice treated with IL-4R
ASO or its seven-base mismatched (MM) control oligonucleotide (10, 100 and 500 g/kg administered on days 59, 61, 61, 66, and 68). Lung cells were recovered after collagenase treatment of the tissue and analyzed by multiparametric flow cytometry. IL-4R
protein expression was measured on a mixed population of eosinophils and macrophages (CD11b-positive, GR-1 negative or low; upper panel) and CD11c-positive and MHC class II-positive dendritic cells (lower panel). Data are expressed as (a) the group mean percentage of cells from vehicle-treated, rechallenged mice or (b) the group mean fluorescence intensity (MFI)  standard error of the mean (SE), n  4 per group. “ ∗ ” indicates p  0.05 using Student’s T-test. NA, naïve; MM, mismatched base control oligonucleotide; VH, saline vehicle. (From Karras, J. et al., Am. J. Respir. Cell Mol. Biol.; published ahead of print on September 21, 2006 as doi:10.1165/rcmb.2005-0456OC. With permission.)

5 g/kg (the lowest dose evaluated) in this model. In separate studies, an inhaled 2
-MOE TNF-
ASO administered in this model reduced subsequent allergen-induced eosinophil and neutrophil recruitment, mucus production, and Penh [137]. Interestingly, the inhaled TNF-
ASO inhibitor did not show pronounced activity in an acute OVA challenge model when administered in a prophylactic regimen. The ability of an inhaled antisense inhibitor to reduce pulmonary inflammation and AHR in chronically challenged mice with established pulmonary inflammation suggests that topical ASO intervention may have clinical utility in chronic asthma.

24.6 ADDITIONAL PRECLINICAL IN VIVO PHARMACOLOGY IN MODELS OF INFLAMMATION 24.6.1 Immunomodulation and Transplantation The activity of systemically administered ICAM-1 PS-ODN and 2
-MOE ASOs in animal models of organ transplantation has previously been reviewed [138]. In addition to preservation of heart and

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Figure 24.9 Suppression of lung inflammation by inhaled IL-4R
ASO using a therapeutic treatment regimen in chronically OVA-challenged mice. (a) Methacholine-induced Penh dose-response curves from Balb/c mice exposed to inhaled IL 4R
ASO (5 and 500 g/kg administered once weekly) following the development of established lung inflammation. Additional groups of mice were exposed to nebulized saline or injected with dexamethasone (2.5 mg/kg i.p. on days 47, 62, 73–75). Responses of naïve mice (open triangles), saline vehicle–treated mice (circles), vehicle- plus dexamethasonetreated mice (filled triangles), IL 4R
ASO (5 g/kg)–treated mice (open squares), and IL 4R
ASO (500 g/kg)–treated mice (filled squares) are shown. (b) Airway cells were recovered by bronchoalveolar lavage and analyzed by multiparametric flow cytometry on day 62 for neutrophils, (c) or immediately following measurement of AHR on Day 76 for eosinophils. Data are presented as group means  SE, n  10 per group for Penh and n  7 per group for cell differentials. “∗ ” indicates p  0.05; Student’s T-test. (From Karras, J. et al., Am. J. Respir. Cell Mol. Biol.; published ahead of print on September 21, 2006 as doi:10.1165/rcmb.2005-0456OC. With permission.)

renal allografts, and reduced severity of ischemia-reperfusion injury shown earlier, recent work has demonstrated that systemic administration of ICAM-1 ASO improves pancreatic islet cell allograft survival and function in mice [139]. Cyclosporin-induced nephrotoxicity, particularly the chronic form of the disorder, can cause decreased kidney function and structural changes including tubulointerstitial fibrosis, tubular atrophy, and glomerulosclerosis. The combination of perfusion of the allograft and systemic administration (10 mg/kg per day, i.v., for 14 days) of the host rat with 2
-MOE ICAM-1 ASO

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was found to completely alleviate Cyclosporin-induced nephrotoxicity, whereas perfusion alone produced a partial protective effect [140]. In this study, renal allograft rejection was significantly delayed by oral administration of ICAM-1 2
-MOE ASO, although 50–100 mg/kg doses were required due to an estimated 10–15% bioavailability. Several other studies have demonstrated delayed allograft rejection following PS-ODN perfusion of the graft tissue before transplant, identifying early growth response (Egr)-1, IL-15, and TNF-
as additional targets of interest in lung, islet, and liver transplantation models, respectively [141–143]. Together, this work supports continued investigation of improved ASO therapies in organ transplantation. Human acute allogeneic graft rejection responses have been recently modeled in severe combined immune deficiency (SCID)/beige mice engrafted with human skin and reconstituted with allogeneic peripheral blood mononuclear cells (PBMC). Intradermal injection of human IL-11 delayed the CD3 T cell–mediated destruction of the graft microvessels and up-regulated survivin gene expression in graft endothelial cells and keratinocytes in a manner consistent with its protective effect [144]. Topical application of survivin 2
-MOE ASO formulated in cream to healed engrafted skin (100 L of a 5% w/v cream 3 times daily from days 5–10 following PBMC inoculation) suppressed both IL-11-mediated survivin expression and protection of microvessel integrity. A role for endocrine hormones, particularly estrogens, in the increased incidence of autoimmune disease in females has long been postulated, although the activities of estrogens in immunity are not well characterized. The immunomodulatory hormone, gonadotropin-releasing hormone (GnRH), is increased by estrogens and pituitary cells from females are more responsive to GnRH than those from males. GnRH has been shown to be immunostimulatory, to exacerbate systemic lupus erythematous (SLE) in female but not male mice, and to signal through the stimulatory G-proteins G
q and G
11 (together termed G
Q/11) [145–147]. In ovariectomized female (NZB NZW) F1 hybrid mice, a model of SLE, subcutaneous injection of G
Q/11 PS-ASO (0.5 mg/kg 3 times weekly) significantly reduced serum anti-DNA antibody levels, and proteinuria compared to treatment with missense control oligonucleotide [147]. These effects were not evident until 20–25 weeks of treatment. At 1 year of treatment, mesangial hypercellularity, sclerosis, and capillary wall thickening were decreased in ASO-treated animals, and G
Q/11 mRNA levels were shown to be decreased in splenic mononuclear cells. The PS-ODN used in this study contained a noncanonical CpG motif and although in vitro IL-6 production by splenocytes was not increased compared to control oligonucleotide not containing a CpG motif, in vivo cytokine measurements were not reported. This study suggests that GnRH may promote autoimmunity by modulating immunocyte responsiveness and that the endocrineimmune axis may be a target for ASO intervention in vivo. 24.6.2 Hyperalgesia Chronic neuropathic pain is a significant clinical problem. There is growing evidence that inflammation contributes to hypersensitivity to subsequent afferent stimuli following tissue injury, termed hyperalgesia. A number of targets associated with inflammation have been implicated in sensitizing peripheral nociceptors to produce a decrease in pain threshold [148]. Inflammatory mediators, such as prostaglandin E2 (PGE2) and carrageenan, cause hyperalgesia in animal models. Therefore, anti-inflammatory strategies that can be safely practiced in the CNS may be of future therapeutic value. In rat models of hyperalgesia, signaling molecules classically associated with inflammation, such as NF-B and p38, have been recently implicated. Intrathecal administration of NF-B p65 subunit PS-ODN suppressed p65 protein expression in spinal cord and significantly attenuated chronic constriction injury-induced pain sensation and thermal hyperalgesia [149]. In rats administered p38 2
-MOE ASO by intrathecal injection, both nocifensive flinching evoked by intraplantar injection of formalin and hyperalgesia produced by intrathecal injection of substance P were prevented [150]. Phosphorylation of p38 in the immediate spinal cord tissue surrounding the ASO injection site was reduced and p38
ASO had no effect on nociception or hyperalgesia.

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Other studies have shown that TNF-
can activate the protein kinase C (PKC)-dependent development of a chronic hyperalgesia susceptible state [151,152]. The intrathecal administration of tumor necrosis factor receptor type-1 or PKC ASOs prevented hyperalgesic priming of sensory neurons and PKC ASO treatment was found to terminate a fully developed state of priming. Inflammatory hyperalgesia induced by PGE2, epinephrine, forskolin, and MAPK or PKC agonists all were found to depend on the function of integrins [153]. Intrathecal administration of integrin subunit ASOs differentially blocked hyperalgesia induced through the PKA and PKC pathways, or by PGE2 or epinephrine, indicating that integrin subunits regulate extracellular matrix-induced signaling in neurons by uniquely integrating second messenger systems. In the case of PGE2, integrity of lipid rafts was essential to maintain a state of hyperalgesia, as detergent treatment of membrane fractions antagonized hyperalgesic signaling induced by PGE2. Since changes in extracellular matrix occur at sites of inflammation, it is likely that inflammatory processes mediate hyperalgesia by activating neuronal second messenger pathways through engagement of specific sets of integrins.

24.7 CELLULAR AND MOLECULAR PHARMACOLOGY 24.7.1 Cell Proliferation, Maturation, and Survival The contribution of resident synovial cells and joint-infiltrating leukocytes to the pathophysiology of RA has been actively investigated in recent years [154]. There is considerable evidence that immune and inflammatory mechanisms drive the abnormal formation of the pannus, an outgrowth of synovial fibroblast-like tissue observed in affected joints, resulting in reduction of the synovial space and damage to the surrounding articular tissue. The use of modern genetic manipulations, including the use of ASO, has helped to more clearly define key cell types, gene products, and signaling pathways intimately involved in joint inflammation. In addition to inducing the production of inflammatory chemokines, TNF-
promotes the abnormal proliferation of RA synoviocytes and enhances their survival. Nakazawa et al. have studied the Notch family proteins in RA synoviocytes, due to the association of these factors with cell fate and proliferative capacity [155]. Notch-1 was found to be present in the nucleus of RA patient synoviocyte samples, whereas it was largely restricted to the cytoplasm in normal and osteoarthritis synoviocytes, suggesting disease-specific activation of the pathway. Nuclear Notch-1 was characterized as Notch-1 intracellular domain and was inducible by TNF-
treatment. Notch-1 ASO treatment reduced the basal and TNF-
-induced proliferation of RA synoviocytes, identifying the Notch signaling pathway in altered proliferation of RA synoviocytes. The protective role of TNF-
in RA synoviocytes was demonstrated to be dependent upon FLICE inhibitory protein (FLIP), as TNF-
up-regulated FLIP expression in RA fibroblast-like synoviocytes and FLIP ASO treatment sensitized these cells to Fas-mediated apoptosis [156]. Since Fas ligand is present in RA synovium, these data suggest that TNF-
-induced protection of RA synoviocytes from normal homeostatic mechanisms of apoptosis is mediated through up-regulation of FLIP. FLIP is overexpressed in inflamed colonic lesions of Crohn’s disease [157] and appears to play a role in protection of LPL from Fas-mediated apoptosis in CD [158]. Fas ligand (FasL) -induced apoptosis was lower in CD3 LPL from patients with CD than in CD3 LPL from UC patients or normal subjects, although normal expression of Fas and FasL were found in CD LPL and mucosal cells, respectively. Enhanced FLIP expression was observed in CD LPL compared to UC or normal LPL, with the FLIP S isoform that contains two death effector domains but no caspase-binding domain highly up-regulated following anti-CD3 cross-linking. A FLIP PS-ODN that inhibited both FLIP isoforms restored susceptibility of CD LPL to Fas-induced apoptosis, suggesting that chronic inflammatory conditions present in CD promote overexpression of FLIP, resulting in resistance of LPL to normal mechanisms of programmed cell death.

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24.7.2 Cell Activation T lymphocytes and monocyte/macrophages have been implicated in the pathogenesis of fibrotic diseases [159,160]. Consistent with this notion, immunosuppressive drugs such as cyclosporin and FK506 have demonstrated clinical efficacy in some systemic sclerosis (SSc) or scleroderma patients [161]. Several studies have evaluated expression of inflammatory cell activation markers in either immune cells or tissue fibroblasts using ASO, resulting in a proposed causal role for interaction of hematopoietic cells or their products and fibroblasts in at least the early stages of fibrotic disease. The chemokine (C-C motif) ligand 2 (CCL2, monocyte chemoattractant protein 1, MCP-1) interaction with CCR2 has been implicated in the fibrogenesis process via paracrine and autocrine regulation of myofibroblast differentiation. Increased CCR2 expression has been demonstrated on myofibroblasts, lymphocytes, macrophages, and endothelial cells from SSc patients [162]. Treatment of CD14 human peripheral blood macrophages with CCL2 enhanced expression of transforming growth factor (TGF)-1 and a pro-
1 chain of type 1 collagen (COL1A1) [163] while amplifying macrophage CCL2/CCR2 expression. CCL2 down-regulation with phosphorothioate ASO in these macrophages reduced autocrine stimulation of CCL2/CCR2 expression [160]. Fibroblasts from SSc patients with a profibrotic phenotype (elevated
-smooth muscle actin and connective tissue growth factor expression) but not control fibroblasts were CCR2 . It has been shown that cultured SSc fibroblasts continue to produce excessive amounts of extracellular matrix proteins [163], suggesting that once these cells have been appropriately activated, a constitutive autoregulatory activation pathway becomes engaged. SSc fibroblasts express elevated levels of TGF-R and the
v3 integrin, recently demonstrated to promote activation of the latent form of TGF-1 [164]. Treatment of normal fibroblasts overexpressing
v3 or SSc fibroblasts with TGF-1 ASO blocked
2 (I) collagen gene expression [165], suggesting that an autocrine TGF- loop exists in SSc fibroblasts that may result from increased endogenous expression of genes that mediate extracellular TGF- protein processing from the latent to the active form. 24.7.3 Cell Migration and Adhesion Expression of chemoattractant molecules by RA synovial tissue recruits mononuclear cells to the joints where they release TNF-
, IL-1, and IL-12, among other factors involved in inflammation. Ruth et al. utilized a human RA synovial tissue SCID mouse chimera to study the key signals required for homing of mononuclear cells to the RA synovium [166]. In these studies, a synovial biopsy from an RA patient was coimplanted with human cartilage just under the renal capsule in SCID mice and peripheral blood leukocytes from RA subjects were used to reconstitute the animal’s immune system. Recruitment of human mononuclear cells to the synovial tissue was induced by intragraft injection of human CXC chemokine ligand (CXCL)16 or TNF-
. Ex vivo transfection of mononuclear cells with extracellular signal-related kinase (ERK)1/2 ASO before reconstitution of the mice resulted in a 50% decrease in recruitment of mononuclear cells to the engrafted RA synovial tissue. This study suggests that the MAPK pathway regulates chemoattraction of human inflammatory leukocytes to RA synovial tissue. 24.7.4 Gene Expression and Receptor Signaling NF-B signaling has been implicated in cytokine-induced synovial fibroblast adhesion molecule and chemokine production. IL-18, a proinflammatory cytokine found in synovial fluid from RA patients, activates NF-B in human RA synovial fibroblasts and up-regulates the secretion of CXCL8 (IL-8), CXCL1 (growth-regulated oncogene, gro-alpha), and CXCL5 (epithelial-neutrophil activating protein, ENA-78) [167]. Pretreatment of RA synovial fibroblasts with p65 subunit of NF-B phosphorothioate ASO produced a 44% reduction in CXCL8 secretion, compared to control sense oligonucleotide [168]. In a separate study, ASO inhibition of IL-1 receptor associated

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kinase (IRAK)-1 also blocked IL-18-induced NF-B expression [169]. IL-18 also induces adhesion of leukocytes to synovial tissue fibroblasts via activation of ERK1/2 and Akt-1 through Srcdependent pathways that result in increased synovial fibroblast vascular cell adhesion molecule (VCAM)-1 expression. These results suggest that IL-18 utilizes IRAK-1- and Src-dependent signaling pathways to induce chemokine and VCAM-1 expression in RA synovial fibroblasts, resulting in the promotion of interactions between bone marrow–derived leukocytes and synovial fibroblasts resident in the joint that contribute to RA pathology. Identification of the effector molecules mediating  islet cell apoptosis in type 1 diabetes mellitus has lagged behind delineation of the immune cell types involved. Clearly, perforin contributes to CD8 cell-mediated  islet cell death but the roles for other implicated factors such as Fas, TNF-
, IL-1, and nitric oxide are less clear. The combination of TNF-
and IFN- treatment was shown to synergistically produce caspase-dependent apoptosis in insulinoma cells and primary  islet cells [169]. Interferon regulatory factor (IRF)-1, a key regulator of IFN- signaling, was found to mediate the cytotoxic effects of the cytokine combination, as IRF-1 ASO treatment abolished TNF-
plus IFN- induced apoptosis of insulinoma cells. These data implicate the IFN- signaling pathway as a major contributor to the development of autoimmune diabetes by acting in concert with TNF-
in a final effector pathway of  islet cell death. TNF-
plays a central role in the pathogenesis of UC. In normal human intestinal lamina propria mononuclear cells (LPMC), treatment with the immunosuppressive factor TGF-1 markedly suppresses TNF-
-induced activation of NF-B through TGF- receptor (TGF-R) -mediated phosphorylation of Smad2 and Smad3 and their subsequent association with Smad4 to form a transcriptionally active complex [170]. However, in LPMC from patients with IBD, TGF-1 treatment failed to suppress NF-B activation following TNF-
stimulation, suggesting that a defect in the TGF-1 signaling pathway may be in part responsible for sustained NF-B activation observed in these cells. Further investigation demonstrated that the antagonistic Smad7 TGF- signaling pathway family member, known to interfere with phosphorylation of Smad2/ Smad3 by preventing its association with TGF-1 R, was overexpressed in LPMC from IBD patients. Inhibition of Smad7 with a targeted ASO restored the inhibitory effect of TGF-1 treatment on TNF-
-induced NF-B activation in IBD LPMC, demonstrating that Smad7 overexpression during gut inflammation results in unchecked activation of NF-B and identifying Smad7 as a potential therapeutic target in IBD. 24.7.5 Mediator Release Th2 cytokines are believed to mediate allergic inflammation in asthma and allergic rhinitis. Although an inhaled soluble IL-4 receptor protein inhibitor and IL-5 monoclonal antibody therapeutic strategies have failed in clinical trials, a locally delivered oligonucleotide Th2 inhibitor has not yet been evaluated in man and may have different pharmaceutical properties/activities. IL-4 antisense decreased the levels of IL-4 mRNA and IL-4 protein in nasal biopsy tissues from patients with seasonal ragweed allergic rhinitis following coincubation of the tissue with ragweed antigen ex vivo [171]. IL-4 ASO treatment also suppressed ragweed-induced  germline (immunoglobulin E, IgE) and eotaxin-1 transcription, downstream IL-4-regulated genes. The epithelial side was largely unstained, indicating that diffusion of oligonucleotides is limited through the tissue, most likely due to their protein-binding characteristics. Effects of the Th2 cytokine IL-13 on pulmonary airway smooth muscle cells (ASMC) appear to promote AHR. The signaling pathway utilized by IL-13 to induce the production of eotaxin-1 in ASMC was shown to require the transcription factor signal transducer and activator of transcription 6 (STAT6), as transfection of ASMC with STAT6 PS-ODN reduced IL-13- or IL-4-induced eotaxin-1 release by 81% and 75%, respectively [172]. STAT6-mediated regulation of eotaxin-1 production was specific for Th2 cytokines, as IL-1-induced eotaxin-1 secretion was not affected by STAT6 ASO treatment.

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Mast cells are believed to play a central role in the pathology of asthma, by triggering the early phase response to allergen exposure following allergen-mediated cross-linking of their high-affinity IgE receptors, resulting in release of proinflammatory mediators and in inducing the onset of the late-phase reaction characterized by pulmonary infiltration of eosinophils. A recent report has shown that an IgE-independent mechanism of human mast cell activation occurs in vitro when lung or cord blood mast cells are incubated with eosinophil major basic protein (MBP) in the presence of human lung fibroblasts [173]. Coculture of cord blood mast cells with 3T3 fibroblasts produced more histamine and PGD2 upon stimulation with MBP than mast cells maintained without contact with fibroblasts. A 2
-O-methyl modified SCF ASO preincubated with the fibroblasts reduced MBP-mediated histamine release from cord blood mast cells by 83% and completely blocked the production of PGD2. Coincubation of mast cells with MBP and soluble SCF did not increase their histamine and PGD2 production, indicating that the membrane-bound form of SCF was required for optimal mast cell activation. These results point to an IgE-independent mast cell activation pathway in the lung interstitium that may promote prolonged lung inflammation following allergen exposure. 24.7.6 Immunomodulation and Immune Surveillance Excessive interferon- (IFN-) production is linked to the pathology of MS. Both IL-12 and IL-23 are primarily dendritic cell (DC) products known to induce IFN- expression in vitro. DC from MS patients secrete increased levels of IL-23 and EAE cannot manifest in mice lacking IL-23 [174], suggesting that IL-23 may play a critical role in MS. Mature monocyte-derived human DC transfected with morpholino ASO targeting IL-23 produced decreased amounts of TNF-
and increased levels of IL-10 [175], a known product of regulatory T cells. In addition, IL-23 ASO–treated DC displayed impaired antigen presentation activity to autologous T cells, indicating that IL-23 promotes Th1-biased autoimmunity and DC maturation. In a model of type 1 diabetes mellitus, transfer of pancreatic islet DC from nonobese diabetic (NOD) mice into prediabetic NOD mice conferred protection from induction of diabetes, presumably due to acquisition of islet antigens by the DC that allowed them to promulgate regulatory immune cells [176]. In a similar approach with potential therapeutic implications, bone marrow–derived DC were transfected ex vivo with CD40, CD80, and CD86 ASOs before injection into prediabetic NOD mice or cotransfer with splenic T cells into NOD-SCID recipients [177]. In mice receiving ASOtreated DC, the onset and incidence of diabetes were reduced, with 25% of the mice remaining disease-free up to 45 weeks following a single injection of ASO-treated DC. These animals were able to mount a normal immune response to alloantigen, indicating that they were not generally immunosuppressed. Furthermore, splenocytes from mice receiving ASO-treated DC had a higher frequency of CD4 CD25 regulatory T cells (Treg) than control animals, suggesting that phenotypically immunosuppressive DC may promote the appearance of regulatory T cells and offer a method for protecting remaining pancreatic  islet cells in type 1 diabetes from immune-mediated cell death. Interactions between immune and nonhematopoietic cells are critical for the preservation of normal tissue homeostasis. In the gastrointestinal tract, mechanisms of tolerance must be in place to prevent the inappropriate activation of mucosal T cells by the frequent presence of bacterial and dietary antigens. Intestinal fibroblasts have been shown to act as regulators of mucosal immunity, although the mechanisms by which they control lymphocyte-dependent responsiveness have not been defined. Ina and colleagues have recently demonstrated that mucosal human T-cell viability after exposure to growth factor withdrawal was maintained by IL-10 derived from human intestinal fibroblasts (HIF) [178]. Antisense inhibition of fibroblast IL-10 blocked the antiapoptotic activity of conditioned medium from cultured HIF. These results suggest that survival of regulatory or memory T cells in the gastrointestinal tract requires fibroblast IL-10 production and may provide a potential mechanistic explanation for the basis of IBD in IL-10-deficient mice.

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Recent studies have indicated that oncogenic signaling molecules can aid tumor immune evasion by production of immunosuppressive factors and inhibition of DC maturation [179,180]. A common pathway through which tumor-mediated immunosuppression occurs involves activation of signal transducer and activator of transcription STAT3. STAT3 is commonly activated in tumors and promotes expression of immunosuppressive factors such as vascular endothelial cell growth factor (VEGF) and IL-10 [181]. Genetic deletion [182,183] and antisense [181] strategies have shown that STAT3 suppression blocked tumor cell production of immunosuppressive factors and up-regulated tumor cell apoptotic cascades. Reduction of STAT3 expression also had multiple positive effects on host antitumor immunity. These data suggest that tumors evade immune surveillance by active mechanisms and that ASOs may have therapeutic activity in oncological applications by either direct tumor cell targeting or inducing antitumor immune responses in host hematopoietic cells. Control of Treg production and function is of great current interest as a therapeutic approach to chronic disease modification. There is evidence suggesting that mammalian T cell–mediated immunity depends on both innate immune signals and the antigen receptor–costimulatory molecule pathway of acquired immunity. The TLR4/MD2/CD14 receptor complex that mediates recognition of bacterial lipopolysaccharide (LPS) and is negatively regulated by RP105-MD1 represents one example of an innate immune system signaling element linked to the generation of Treg in mice [184]. Reduction of MD1 expression in splenic mouse DCs following in vivo treatment with PS-ASOs resulted in suppression of LPS-stimulated CTL induction in mixed lymphocyte cultures. Administration of MD1 ASO to mice also reduced allogeneic graft rejection responses in vivo (with or without LPS cotreatment). Higher numbers of CD4 CD25 and Foxp3 Treg cells were recovered from MD1 KO mice or mice treated with MD1 PS-ODNs used for allogeneic graft rejection experiments, suggesting that MD1 antagonizes a high level of TLR4 signaling required to generate Treg cells and that inhibition of MD1 expression may therefore be a useful approach for transplantation and autoimmunity.

24.8 FUTURE DIRECTIONS Understanding how distinct genes influence the immune and inflammatory processes that contribute to the initiation and progression of chronic diseases is at an early stage. The application of antisense technologies in basic and applied research continues to provide novel insights into the complex network of molecular structures, interactions, and processes that support physiological function and health as well as those involved in disease pathogenesis. Significant progress in the development and clinical assessment of putative antisense therapies has been recently made. Firstand second-generation ASOs have demonstrated limited utility for safely and effectively targeting sites of inflammation located outside the liver and kidney using parenteral or oral administration. Topical administration strategies including enema and aerosol formulations have produced promising early clinical results in UC, pouchitis, and asthma. Current chemistries offer the promise of once-daily or less frequent topical administration of antisense therapies with acceptable safety profiles to support chronic treatment, but substantial safety and efficacy hurdles remain for these compounds. Technical hurdles, including oligonucleotide delivery to the skin, CNS, and musculoskeletal tissues must be overcome before antisense is likely to become a mainstay technology for treatment of chronic inflammatory diseases in these tissues. Current first- and second-generation ASOs have demonstrated consistent and reproducible activities in vivo in antigen-presenting cells, including dendritic cells, macrophages, eosinophils, and certain epithelial and endothelial cell populations, and in parenchymal cells of the liver, kidney, lung, and brain. Target down-regulation and pharmacological activity in relatively resistant cell populations such as lymphocytes, fibroblasts, and smooth muscle cells may require additional formulation development to facilitate ASO targeting and uptake.

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Finally, improvements in the oral bioavailability of antisense therapies would be a welcome and significant breakthrough. ISIS 104838 was demonstrated to be orally bioavailable in humans after administration of solid dose forms. The estimated tissue bioavailability was approximately 15%. Optimized formulations would be expected to provide equivalent results for all second-generation 2
-methoxyethyl antisense drugs, and this work is currently underway.

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154. Pap, T. et al., Role of synovial fibroblasts in the pathogenesis of rheumatoid arthritis, Arthritis Res., 2, 361, 2000. 155. Nakazawa et al., Role of Notch-1 intracellular domain in activation of rheumatoid synoviocytes, Arthritis Rheum., 44, 1545, 2001. 156. Palao et al., Down-regulation of FLIP sensitizes rheumatoid synovial fibroblasts to Fas-mediated apoptosis, Arthritis Rheum., 50, 2803, 2004. 157. Hagiwara K, et al., Identification of genes upregulated in the inflamed colonic lesions of Crohn’s disease, Biochem. Biophys. Res. Commun., 283, 130, 2001. 158. Monteleone, I. et al., A functional role of FLIP in conferring resistance of Crohn’s disease lamina propria lymphocytes to FAS-mediated apoptosis. Gastroenterology, 130, 389, 2006. 159. Roumm, A.D. et al., Lymphocytes in the skin of a patient with progressive systemic sclerosis, Arthritis Rheum., 27, 645, 1984. 160. Sakai, N. et al., MCP-1/CCR2-dependent loop for fibrogenesis in human peripheral CD14-positive monocytes, J. Leukocyte Biol., 79, 555, 2006. 161. Morton, S.J. and Powell, R.J., Cyclosporin and tacrolimus: their use in a routine clinical setting for scleroderma, Rheumatology, 39, 865, 2000. 162. Carulli, M.T. et al., Chemokine receptor CCR2 expression by systemic sclerosis fibroblasts: evidence for autocrine regulation of myofibroblast differentiation, Arthritis Rheum., 52, 3772, 2005. 163. LeRoy, E.C., Increased collagen synthesis by scledroderma skin fibroblasts in vitro: a possible defect in the regulation or activation of the scleroderma fibroblast, J. Clin. Invest., 54, 880, 1974. 164. Asano, Y. et al., Increased expression of integrin
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180. Melani, C. et al., Myeloid cell expansion elicited by the progression of spontaneous mammary carcinomas in c-erbB-2 transgenic BALB/c mice suppresses immune reactivity, Blood, 102, 2138, 2003. 181. Wang, T. et al., Regulation of the innate and adaptive immune response by Stat-3 signaling in tumor cells, Nat. Med., 10, 48, 2004. 182. Kortylewski, M. et al., Inhibiting Stat3 signaling in the hematopoietic system elicits multicomponent antitumor immunity, Nat. Med., 11, 1314, 2005. 183. Niu, G. et al., Over expression of a dominant-negative signal transducer and activator of transcription 3 variant in tumor cells leads to production of soluble factors that induce apoptosis and cell cycle arrest, Cancer Res., 61, 3276, 2001. 184. Gorczynski, R.M. et al., MD1 expression regulates development of regulatory T cells, J. Immunol., 177, 1078, 2006.

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CHAPTER

25

Antisense Oligonucleotides for the Treatment of Cancer Boris A. Hadaschik and Martin E. Gleave

CONTENTS 25.1 Introduction .........................................................................................................................699 25.2 Antisense Targets in Cancer ................................................................................................700 25.2.1 BCL2 and BCL-xL................................................................................................701 25.2.2 Survivin and XIAP ................................................................................................704 25.2.3 Protein Kinase C-
and RAF1 ..............................................................................705 25.2.4 Clusterin ................................................................................................................706 25.2.5 HSP27....................................................................................................................708 25.2.6 STAT3....................................................................................................................709 25.2.7 Insulin Growth Factor Binding Proteins ...............................................................710 25.2.8 Ribonucleotide Reductase .....................................................................................710 25.2.9 Other Promising ASO ...........................................................................................711 25.3 Summary .............................................................................................................................711 References ......................................................................................................................................712 25.1 INTRODUCTION The development of therapeutic resistance is the underlying basis for most cancer deaths and therefore of great interest in medical research. Despite the introduction of a number of new agents, cure for advanced tumors remains infrequent and a distant goal. For example, prostate cancer is the most common cancer and the third most common cause of cancer related mortality in men in the United States [1]. While early detection has increased with the advent of serum prostate-specific antigen (PSA) testing, the disease is often advanced when patients present with symptoms. For those with metastatic disease, androgen withdrawal is the most effective form of systemic therapy. Unfortunately, caused by clonal selection and adaptive responses androgen-independent progression is inevitable and death occurs within a few years in the majority of cases [2]. Historically, chemotherapy was thought to have minimal clinical efficacy. However, more recently for docetaxelbased chemotherapy a 20% prolongation in survival was demonstrated [3,4]. These improvements are significant but modest, since median survival for patients with hormone-refractory prostate cancer (HRPC) is still only 18 months. 699

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Therapeutic resistance results from multiple, stepwise changes in DNA structure and gene expression—a Darwinian interplay of genetic and epigenetic factors, arising in part from selective pressures of treatment. This highly dynamic process cannot be attributed to singular genetic events, involving instead cumulative changes in gene expression that facilitate escape from normal regulatory control of cell growth and survival. In prostate cancer, for instance, changes in the hormonal environment precipitate a cascade of events in gene expression and signaling networks that provide a selective survival and growth advantage for subpopulations of tumor cells, thereby accelerating androgen-independent progression and rendering cells more resistant to chemotherapy. New therapeutic strategies designed to inhibit the emergence of this phenotype must be developed. Over the last years and fueled from the development of high-throughput genomic, transcriptomic, and proteomic technologies, increased understanding of the molecular basis for cancer progression and therapeutic resistance has identified many gene targets that regulate apoptosis, proliferation, and cell signaling. Since many of these gene products are not easily amenable to agents like small molecules or antibodies, antisense inhibition is an attractive concept. Antisense oligonucleotides (ASOs) offer a tool for the selective silencing of their targets at the gene expression level. Like antibody-based therapeutics, which evolved to become a clinically useful therapeutic class through years of optimization, ASOs are evolving through chemical modifications to prolong in vivo half-life, improve tissue distribution, increase potency and reduce off-target toxicity. ASOs promise good specificity for malignant cells and favorable side-effect profiles due to well-defined modes of action. Indeed, recent clinical trials confirm the ability of this class of drugs to significantly suppress target gene expression in cancer tissues. In this chapter, the current status and future directions of several antisense drugs that have potential clinical use in cancer are reviewed. The chemistry and clinical safety profiles are described elsewhere in this volume. Owing to the rapid progress of the antisense field it is beyond the scope of this review to cover all targets in development. Since the power of siRNA for systemic in vivo applications remains to be determined, we will focus on ASO anticancer drugs. Particular emphasis will be placed on interpreting the recent phase III trial failures of several ASO compounds, and on highlighting advances that promise to overcome the hurdles that confront nucleotide-based therapeutics on their way to become successful treatment options in oncology.

25.2 ANTISENSE TARGETS IN CANCER Advances in tumor biology have identified many attractive molecular targets for new drug discovery. The most promising candidates for antisense therapy are those targets that become upregulated during and are causally related to cancer progression and therapeutic resistance, and not otherwise amendable to inhibition with antibodies or small molecules. Moreover, the target should be selectively overexpressed in tumor cells to minimize side effects as tumor-selective uptake of ASOs cannot be ensured by standard protocols. Although potential gene target libraries developed by microarray technology are valuable, this information must be balanced by the inherent limitations of microarray analyses. These include the inability to examine translational and posttranslational regulatory mechanisms that impact the activity of various cellular proteins. In our laboratory, one method of identifying potential therapeutic targets for prostate cancer starts with use of comparative hybridization of high-density cDNA arrays to rapidly and efficiently characterize changes in gene expression after androgen withdrawal in xenograft models that mimic the human condition of castration-induced regression followed by androgen-independent progression. Computer-assisted subtractive analyses of arrays highlight increases or decreases in gene expression at various time points during progression. Northern or Western analysis is then used to confirm the array data which can be verified in human tissue microarrays of untreated and posthormone therapy-treated cancers. Figure 25.1 shows some targets validated by this strategy.

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ANTISENSE OLIGONUCLEOTIDES FOR THE TREATMENT OF CANCER Androgen-dependent growth

Androgen-independent growth

Regression Hormone withdrawal

701

Progression

Clonal selection + adaptive responses ++ PSA ++ AR - BCL2 - Clusterin - IGFBP-5 - YB1 ++ Survivin - HSP27

- PSA - AR ++ BCL2 +++ Clusterin ++++ IGFBP-5 + YB1 - Survivin + HSP27

++ PSA ++ AR ++ BCL2 +++ Clusterin ++ IGFBP-5 ++ YB1 ++ Survivin +++ HSP27

Figure 25.1 Changes in gene expression of prostate cancer after castration-induced regression and during androgen-independent progression.

ASOs have been reported to specifically silence the expression of many different genes and delay tumor progression in various preclinical and some clinical models. ASO drugs that target the apoptotic rheostat, interfere with signaling pathways involved in cell proliferation and growth, or target the tumor’s microvasculature, are particularly promising not only as single agents, but in combination with conventional anticancer treatments. A survey of a number of ASO drugs in clinical development is given in Table 25.1 and several compounds are highlighted below. 25.2.1 BCL2 and BCL-xL Apoptotic pathways, which are well modulated and strictly controlled in nonmalignant cells, are frequently disrupted in tumor cells. This dysregulation of apoptosis is mainly affected by members of two families of antiapoptotic factors: the BCL2 family and the inhibitors of apoptosis (IAP) gene family. The BCL2 gene (B-cell leukemia-lymphoma gene 2) is the prototype of a class of oncogenes that contribute to neoplastic progression by enhancing cell survival through inhibition of apoptosis. Initially identified in follicular lymphoma due to the characteristic t14;18 translocation, BCL2 is a mitochondrial membrane protein that functions to stabilize the mitochondrial membrane, thereby inhibiting the release of cytochrome c and subsequent activation of the apoptotic cascade [5,6]. BCL2 heterodimerizes with BAX and other proapoptotic regulators and the selective and competitive dimerization between pairs of these antagonists and agonists determines how a cell responds to an apoptotic signal. Several lines of evidence have implicated overexpression of BCL2 with treatment resistance [7–10], highlighting BCL2 as an attractive target to improve the efficacy of conventional treatment by enhancing chemotherapy-induced apoptosis. Several BCL2 ASOs have been reported good hormone or chemosensitization activity in many preclinical models [11–15]. G3139, also referred to as oblimersen sodium or Genasense (Genta Inc.), is a first-generation 18-mer phosphorothioate ASO complimentary to the first six codons of the initiating sequence of the human BCL2 mRNA. G3139 moved into clinical trials based on promising activity in preclinical models of many cancers [12,13]. In 1997, the first clinical study evaluating G3139 enrolled 21 patients with non-Hodgkin’s lymphoma (NHL) and treated them with subcutaneous infusion of G3139 as a single agent [16]. Local inflammation at the infusion site was the most common side effect observed, while the maximum-tolerated dose was determined to be 147.2 mg/m2/d and

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Table 25.1 Anticancer Oligonucleotides in Late-Stage Pre-Clinical or Clinical Development Company/ Investigator

Phase of Development

Target

Compound

BCL2

G3139 (Oblimersen, Genasense)

Genta

II–III

Melanoma, AML, CLL, MM, NHL, HRPC, NSCLC, SCLC

Tumor Type

PKC


ISIS 3521 (Affinitak, Aprinocarsen)

Lilly/Isis

II–III

NSCLC, colon, ovarian, NHL, prostate

CLU

OGX-011

OncoGeneX

II

HRPC, breast, NSCLC

RAF1

ISIS 5132

Isis

II

NSCLC, colon, prostate

HRAS

ISIS 2503

Isis

II

NSCLC, breast, pancreas

DNA Methyltransferase

MG98

MethylGene

II

Metastatic renal cancer, head, and neck cancer

Protein kinase A

GEM231

Hybridon/Idera

II

SCLC, colon, pancreas, prostate

RNR (R1 and R2 component) TGF2

GTI-2501, GTI-2040 AP12009

Lorus

I–II

Antisense Pharma

I–II

HRPC, renal cell, breast Glioma, pancreas, melanoma-

Survivin

LY2181308/ISIS 23722

Lilly/Isis

I

cMYB

LR3001

Genta

I

CML

XIAP

AEG35156/GEM 640

Aegera

I

Solid tumors

HSP27

OGX-427

OncoGeneX

I

HRPC

eIF4E

LY2275796

Lilly/Isis

Preclinical-I

Xenografts (broad)

STAT3

ISIS 345794

Isis

Preclinical

Xenografts (broad), MM

MDM2

GEM240

Hybridon/Idera

Preclinical

Xenografts (broad)

IGFBP-2 + IGFBP-5

OGX-225

OncoGeneX

Preclinical

HRPC, breast, glioma

MCL1

ISIS 20408

Isis

Preclinical

Xenografts (broad)

Solid tumors

dose-limiting toxicity was thrombocytopenia. One complete response and two minor responses were observed, but only half of the evaluable patients had measurable decreases in BCL2 protein levels following treatment with G3139 and there was no apparent relationship to the dose [17]. Subsequent clinical trials of G3139 employed continuous intravenous (i.v.) infusions necessitated by the short tissue half-life of first-generation phosphorothioate ASOs, most often in combination with another cytotoxic agent. In a phase I/II trial in patients with advanced melanoma, continuous i.v. infusion of G3139 in combination with full-dose dacarbazine (DTIC) reduced BCL2 protein levels in serial melanoma biopsies, and this pharmacodynamic activity was associated with significant clinical responses [18]. Transient thrombocytopenia at 12 mg/kg/d was dose limiting in patients who also received full-dose DTIC treatment. An international, phase III, randomized trial was recently completed in patients with advanced melanoma using a 5-day pretreatment regimen of 7 mg/kg/d G3139 administered by continuous i.v. infusion followed by DTIC at 1000 mg/m2 [19,20]. Analysis on an intent-to-treat (ITT) basis after a minimum follow-up of 24 months reported a median survival of 9 months in patients treated with G3139 plus dacarbazine, compared with 7.8 months for dacarbazine alone (n ⫽ 771, p ⫽ 0.077). The addition of G3139 to dacarbazine significantly improved median progression-free survival (2.6 versus 1.6 months, p ⬍ 0.001) and

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overall response rates (13.5 versus 7.5%, p ⫽ 0.007). However, this drug failed to obtain FDA approval because overall survival of the ITT population was not significantly prolonged, and the increased time to progression was not deemed clinically significant. Results of a phase III trial of G3139 in combination with fludarabine and cyclophosphamide (Flu/Cy) in patients with advanced chronic lymphocytic leukemia (CLL) were presented in 2004 [21]. In this trial, 120 patients were randomized to receive Flu/Cy with or without G3139 at 3 mg/kg/d continuous i.v. infusion (day 1-7). This dose of G3139, which is significantly lower than doses used in other trials in solid tumors with lower tumor burden, based on phase I/II studies in CLL patients that showed dose-limiting development of a cytokine release syndrome and modest single-agent activity [22,23]. Patients in the phase III trial had failed prior therapy and the primary endpoint of this study was complete response plus nodular partial response. Nineteen patients (16%) treated with G3139 plus Flu/Cy achieved the primary endpoint, compared with eight patients (7%) treated with Flu/Cy alone ( p ⫽ 0.039). However, the overall response rate was similar between the treatment groups when partial responders were included. G3139 was generally well tolerated, with specific adverse events associated with G3139 treatment being nausea, fever, fatigue, and back pain. According to the manufacturer, the FDA will review G3139 as treatment for CLL by the end of October 2006 (www.genta.com). At time of submission of this chapter however, the Oncologic Drugs Advisory Committee voted not to recommend approval of Genasense. The results of a phase III trial of G3139 in combination with high-dose dexamethasone in patients with refractory multiple myeloma (MM) has also been disclosed [24]. In this trial, 224 patients were randomized to receive standard therapy using high-dose dexamethasone with or without 7 mg/kg/d G3139. The primary objective of the study was to evaluate whether addition of G3139 would significantly increase the time to progression, with monitoring of secondary endpoints that included objective response and overall clinical benefit. Prior to entering the study, patients in both groups had received extensive treatment with corticosteroids. No clinical benefit was observed in this trial for patients receiving G3139 plus dexamethasone versus dexamethasone alone. The median time to progression was similar for both treatments and no difference was observed in response rates or toxicity between the groups. Several trials in hormone refractory prostate cancer demonstrated that standard doses of docetaxel or mitoxantrone could be delivered with G3139 without apparent increased toxicity [25,26]. A lately reported phase II trial in men with metastatic hormone refractory prostate cancer combined G3139 (7 mg/kg/d for 7 days) with docetaxel (75 mg/m2 on day 6), repeated every 21 days until progression or toxicity. Partial responses were noted in 4 of 12 patients with measurable disease and a ⬎50% reduction in PSA was measured in 15 of 27 patients [26]. On the basis of this phase II data, a randomized phase III trial was scheduled, but with the negative results from trials in melanoma and myeloma, and the subsequent breakup of the Aventis/Genta partnership, this trial has been put on hold. Issues persist about the regimen of G3139, and whether treatment at the doses and schedules tested is enough to suppress target gene expression sufficiently. Moreover, Anderson et al. [27] demonstrated recently using microarray studies that the mechanism by which G3139 produces cytostatic effects might not only be related to BCL2. While both the first-generation ASO and BCL2 targeting siRNA strongly downregulated BCL2 expression in vitro, the effects of these two classes of molecules on cell proliferation and apoptosis were distinct, suggesting that the mechanism of action of G3139 was not exclusively the result of its target-specific action on BCL2. Application of their approach that combines ASO and siRNA with gene expression profiling may be used to assess validity of new drug candidates in the future. BCL-xL is another antiapoptotic BCL2 family member. In tumors where BCL2 and BCL-xL are coexpressed, it is difficult to predict which of the two proteins is more critical for survival and some tumor cells have been reported to switch expression from BCL2 to BCL-xL [28,29]. ASOs against BCL-xL have been reported to induce apoptosis in various tumor cells and sensitize tumor cells to chemotherapy [30–35]. While BCL-xL ASO marginally enhanced chemosensitivity and

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delayed androgen-independent progression of prostate cancer xenografts, combined BCL-xL plus BCL2 ASOs acted synergistically to improve efficacy of chemotherapy beyond that of either agent alone [36]. Simultaneous downregulation of both BCL2 and BCL-xL protein expression by a single bispecific ASO has been accomplished by taking advantage of the similarity of specific regions of BCL2 and BCL-xL mRNA. This bispecific second-generation 2⬘-O-methoxyethyl (2⬘-MOE) modified ASO with complete sequence identity to BCL2 and three mismatches to BCL-xL inhibits expression of both BCL2 and BCL-xL in tumor cells and is a potent inducer of apoptosis in several tumor cell types [37–39]. Future development plans for this particular ASO are yet unknown, but the reported findings illustrate that combination regimen that inhibit two or more specific gene targets can produce additive effects. Recently, small molecule inhibitors against antiapoptotic BCL2 family members have been developed [40] and several are in early phase clinical trials. This further raises the possibility of strategies, in which a small molecule inhibitor is combined with antisense gene suppression of BCL2 family members. 25.2.2 Survivin and XIAP The inhibitors of apoptosis (IAP) gene family encodes proteins that protect cells from undergoing apoptosis through, at least in part, inhibition of caspases which are key effector proteins of apoptotic cell death [41–44]. In addition, IAPs have roles in seemingly caspase-unrelated functions including cell division and signaling [45–47]. IAPs have been found to be expressed in multiple malignancies including human prostate cancer [48] and limited expression in normal tissues. Second-generation ASOs have been designed against two IAP family members: Survivin (LY2181308/ISIS 23722, Eli Lilly and Co. in collaboration with Isis Pharmaceuticals) and X-linked IAP (AEG35156/GEM640, Aegera Therapeutics Inc.). Survivin plays an important role in both cell growth and apoptosis inhibition [49,50]. Survivin is highly expressed in a wide variety of human cancer types, including lung, colon, pancreas, prostate, breast, and gastric tumors [49,51]. However, Survivin is generally not expressed in differentiated normal tissue, with expression limited to a few cell types including angiogenic endothelium, thymus, testis, activated T cells and intestinal epithelium crypts. Survivin levels correlate with lower apoptotic index in tumor cells and poor prognosis in cancer patients [52,53], and gene expression studies have indicated that Survivin is one of the top genes uniformly expressed in cancer cells but not in normal tissues [54]. Furthermore, overexpression of Survivin in tumor cells inhibits chemotherapy-induced, BAX-induced, and FAS-induced apoptosis, and expression of dominant negative mutants of Survivin induces apoptosis in many tumor cell lines [55,56]. Taken together, these observations make Survivin an obvious target for novel cancer therapy. LY2181308/ISIS 23722 is a second-generation 2⬘-MOE ASO that potently and specifically downregulates Survivin expression in a broad range of human cancer cells including lung, colon, pancreas, breast, and prostate [57,58]. Survivin inhibition in tumor cells by LY2181308 results in caspase-3-dependent apoptosis, cell cycle arrest in the G2/M phase, and in sensitization of tumor cells to chemotherapy-induced apoptosis [57–60]. Moreover, LY2181308 has been reported to produce potent antitumor activity against a broad range of tumor types in human tumor xenograft models (www.isispharm.com). Anticancer activity displayed by LY2181308 in these models is sequence-specific and associated with reduced Survivin levels. On the basis of these preclinical results, LY2181308 has been selected for clinical development and phase I studies have been initiated against a broad range of human cancers. The X-linked mammalian inhibitor of apoptosis protein (XIAP) was the first IAP identified and has been shown to bind several partners. By inhibiting caspase-3, -7 and -9 activity, XIAP suppresses apoptosis triggered by multiple stimuli: intrinsic—mitochondrial-mediated—as well as extrinsic—death receptor-mediated. Overexpression of XIAP reduces apoptosis arising from chemotherapy, radiation, and growth factor deprivation [43,46,61]. XIAP antisense knockdown in

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cancer cells under stress and primed for apoptosis (e.g., when challenged by chemotherapeutic agents), enhances proapoptotic signals and tips the apoptotic rheostat towards death. XIAP is highly expressed in acute myeloid leukemia (AML), glioblastoma, prostate, pancreatic, gastric, and colorectal tumors [47]. In AML, this overexpression has been associated with poor clinical outcome. AEG35156/GEM640 (Aegera Therapeutics Inc.) is a 19-mer ASO targeted to human XIAP mRNA that incorporates 2⬘-O-methyl chemistry with a phosphorothioate backbone. In vitro and in vivo preclinical proof-of-concept studies have demonstrated that inhibition of XIAP protein expression by AEG35156/GEM640 enhances the antitumor activity of chemotherapy in several xenograft models [62,63]. A phase I dose-escalation tolerability study of AEG35156 as a single agent is currently underway in the United Kingdom as a 7-day continuous i.v. infusion in patients with advanced tumors and phase I trials evaluating shorter infusion schedules in combination with docetaxel or reinduction chemotherapy for AML are also recruiting patients. In addition to standard safety and efficacy endpoints, these trials will also measure pharmacodynamic endpoints [64].
and RAF1 25.2.3 Protein Kinase C-
ISIS 3521 and ISIS 5132 (Isis Pharmaceuticals) are first-generation phosphorothioate ASOs directed against the mRNA of Protein kinase C-
[PKC
] and RAF1, respectively. Protein kinase C (PKC) belongs to a class of serine threonine kinases that adjust numerous intracellular responses arising from G-protein-coupled receptors, receptors with tyrosine kinase activity and nonreceptor tyrosine kinases [65]. Increased PKC expression has been implicated in both oncogenesis and tumor progression [66,67]. PKC
inhibitors affect growth and survival of tumors, promote apoptosis, and sensitize tumor cells to chemotherapeutic agents [68,69]. ISIS 3521 (also referred to as LY900003 or Affinitak or Aprinocarsen) potently inhibits PKC
expression in a wide range of human tumor types in an isoform-specific manner [70,71]. Systemic administration of ISIS 3521 to nude mice bearing subcutaneously implanted human tumors suppresses PKC
levels in tumor tissue and inhibits tumor growth of numerous human tumor cell lines, including glioblastoma, breast, pancreatic and lung carcinoma [72–74]. On the basis of the biological evidence implicating PKCs in human tumorigenesis, and the preclinical activity of ISIS 3521 clinical trials were initiated for the treatment of cancer. In phase I single-agent studies of ISIS 3521 [75,76], responses were noted in two patients with low-grade non-NHL and three patients with ovarian cancer. Dose-limiting toxicities were thrombocytopenia and fatigue at a dose of 3.0 mg/kg/d, and the recommended phase II dose was 2 mg/kg/d via continuous i.v. infusion. Toxicity at this dose appeared mild, with one of six patients developing grade 3 thrombocytopenia. A similar phase I clinical study using ISIS 5132, a first-generation ASO targeted to the MAP kinase signaling pathway protein RAF1 [77], escalated doses up to 5 mg/kg/d without reaching a maximum tolerated dose [78]. The difference in apparent tolerability of ISIS 3521 and ISIS 5132 is not clear, and may not even be real, since it may reflect nondrug related adverse events, or differences in patient population. Nevertheless, based in part on these observations and on preclinical models demonstrating activity at equivalent dosing, the phase II dose for ISIS 3521 as well as ISIS 5132 was set to 2 mg/kg/d. Both agents were compared in a National Cancer Institute of Canada randomized phase II trial in patients with hormone-refractory prostate cancer [79]. Scheduling for both ASOs was 2 mg/kg/d for 21 days by continuous infusion followed by a 7-day rest period. Overall, treatment was well tolerated, with fatigue and mild thrombocytopenia as the main treatment-related adverse events. However, although some patients had stable disease, no biochemical or objective responses were observed. A following phase I/II combination trial tested ISIS 3521 at 2 mg/kg/d by continuous infusion on days 0–14 with cisplatin at 80 mg/m2 (day 1) and gemcitabine at 1000 mg/m2 (day 1 and 8) [80] in 55 chemotherapy-naïve patients with advanced nonsmall cell lung cancer (NSCLC). Sixteen of 48 evaluable patients had a response (1 complete response and 15 partial responses). The median survival time for the entire group of 55 patients was 8.9 months and 10.5 months for the 45 patients receiving ⱖ 2 full cycles

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of treatment. Toxicity was moderate but included thrombocytopenia, neutropenia, anemia, fatigue, dehydration, sepsis, and neutropenic fever. On the basis of this phase II data and another combination study by Yuen et al. [81] with carboplatin and paclitaxel that had a reported 42% response, two large randomized phase III trials were conducted as first line treatment in patients with NSCLC. The first enrolled 600 patients using ISIS 3521 in combination with carboplatin and paclitaxel in patients with Stage IV NSCLC, and the results were disappointing [82]. No difference was observed in time to progression or overall survival between groups. There were, however, indications of antitumor activity, as patients who completed the prescribed course of therapy (6 cycles) receiving ISIS 3521 had a median survival of 17.4 months compared to 14.3 months in patients who did not ( p ⫽ 0.048). Negative results were also obtained in the other phase III NSCLC trial testing ISIS 3521 in combination with gemcitabine and cisplatin. Therapy was fairly well tolerated, but median survival was 10 months in both groups [83]. Several factors may have accounted for the lack of clinical efficacy for ISIS 3521. First, because measuring target levels in tumors is difficult in this patient population, patients were not screened and it is unknown whether the target was actually expressed in the majority of patients. Therefore the target itself may not have been relevant in the studied cohort. Moreover, recent preclinical data suggests that in NSCLC other PKC isoforms than PKC
may be a driving force for cancer cell survival [84]. Secondly, inhibition of PKC
may not produce a large enough effect on tumor growth and only result in a cytostatic effect, which the study design would not detect. Third, inhibiting a single molecular target may be insufficient to exert a clinically detectable effect beyond what can be achieved with combination chemotherapy alone. Finally, since target knockdown was not assessed, the dose of ASO used may not have been the optimal biological dose and hence not potent enough to inhibit PKC
expression sufficiently. Longer-lived second-generation chemistry or higher doses may have been required to produce anticancer activity and efficacy. 25.2.4 Clusterin The Clusterin gene encodes a cytoprotective chaperone protein also known as testosteronerepressed prostate message-2, apolipoprotein J, or sulphated glycoprotein-2. It is a secretory heterodimeric disulphide-linked glycoprotein that is expressed in virtually all tissues, and found in all human fluids at relatively high concentrations [85–87]. Clusterin (CLU) was first described for its ability to cause clustering of a variety of cell types. Since then it has been revealed to be involved in a variety of physiological processes relevant for carcinogenesis including apoptotic cell death, cell cycle regulation, DNA repair, cell adhesion, tissue remodeling, lipid transportation, membrane recycling, as well as immune system and complement regulation. Because CLU binds to numerous biological ligands, and is regulated by heat shock transcription factor 1, an emerging view suggests that Clusterin functions similarly to the small heat shock proteins and stabilizes conformations of proteins at times of cellular stress [88]. Indeed, CLU is substantially more potent than other heat shock proteins at inhibiting stress-induced protein precipitation [89]. Significant differences exist, however, in amino acid sequence, which suggests that Clusterin is a unique protein without any closely related family members yet identified. Increased CLU mRNA and protein levels have been consistently detected in various tissues undergoing stress, including heart, brain, liver, kidney, breast, and retinal tissues [90]. Several observations have indicated an association of CLU expression with contradictory functions, either cell survival, tumor progression, treatment resistance or apoptosis [85,91–95]. These opposing functions are likely attributed to two functionally divergent CLU protein isoforms, a secreted glycosylated form (sCLU), and a nonglycosylated nuclear form (nCLU). sCLU is a highly conserved 80 kDa heterodimer comprised of 40 and 60 kDa subunits derived from the first AUG codon of the full-length Clusterin mRNA, while the other isoform starts from the second AUG codon and therefore omits the endoplasmic reticulum-targeting signal. nCLU is a 55-kDa protein, which translocates from the cytoplasm to the nucleus following several cytotoxic stimuli. It has been suggested that tumor cell survival is connected with overexpression of the

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prosurvival form (sCLU) and loss of the proapoptotic form (nCLU) [86,90]. Clusterin upregulation has been reported in many varied human malignancies including lymphoma [96], prostate [97], breast [98], bladder [99], kidney [100], and colon cancers [86]. Overexpression of sCLU protects cells from a variety of agents that otherwise induce apoptosis. For example, Clusterin levels increase dramatically in response to castration-induced apoptosis in rat prostate epithelial cells [101], androgen-dependent mouse Shionogi tumors [95,102], and human prostate cancer xenografts [103]. In patients, Clusterin levels are low in hormone-naïve tissue, but increase significantly within weeks after neoadjuvant hormone therapy [104]. CLU has also been shown to suppress apoptotic cell death from cytotoxic chemotherapy [105,106], radiation [93,107], and oxidative stress [92]. Consequently, CLU gene silencing is an attractive anticancer therapeutic. OGX-011 (OncoGeneX Technologies Inc.) is a second-generation ASO complementary to the translation-initiation site of human Clusterin mRNA. OGX-011 incorporates a phosphorothioate backbone with 2⬘-MOE modifications to the four bases on either end of the 21-mer molecule [108]. Such gapmer modifications maintain the improved tissue pharmacokinetic profile of the secondgeneration chemistry but preserve the high affinity for target mRNA and the recruitment of RNase H necessary for activity. In primates, tissue half-life of OGX-011 was in the order of 7 days, and intermittent schedules of OGX-011 were therapeutically equivalent to continuous dosing of unmodified phosphorothioate CLU ASO. Therefore, more relaxed dosing schedules are possible while maintaining biologic efficacy of target inhibition. In prostate cancer models, OGX-011 improved the efficiency of androgen withdrawal, chemotherapy, and radiation by silencing of Clusterin and enhancing the apoptotic response [93,95,106]. Additional preclinical activity was reported in lung [109], renal cell [110], urothelial [111], osteosarcoma [85], and breast [112] cancers. In a phase I clinical trial, OGX-011 was recently reported to potently suppress CLU expression in prostate cancer tissues in combination with androgen deprivation therapy [113]. This trial had a unique design in that patients with localized prostate cancer were administered OGX-011 prior to radical prostatectomy, allowing for dose-dependent correlations between Clusterin expression and tissue concentrations. Surrogate tissues for markers of biological effect (CLU expression in peripheral blood mononuclear cells and serum CLU) were also assessed and could be associated with those effects found in target tissue. Thus, the presurgery study design allowed for the determination of an optimal biologically effective dose and tissue drug levels in addition to the usual parameters of toxicity. Patients having localized prostate cancer with high-risk features and candidates for prostatectomy were enrolled to this dose-escalation trial. OGX-011 was given by i.v. infusion over 2 h at a starting dose of 40 mg on days 1, 3, 5, 8, 15, 22, and 29. Androgen deprivation therapy was started on day 1 and prostatectomy was performed on days 30–36. Twenty-five patients were enrolled to six cohorts with doses of OGX-011 up to 640 mg delivered. Toxicity was mild or moderate only and adverse events included fevers, rigors, fatigue, and transient liver function elevations. Plasma pharmacokinetic analysis showed linear increases in OGX-011 with a half-life of 2 h and mean peak concentrations of 80 µM at 640 mg dose. More importantly, prostate tissue concentrations of OGX-011 increased with dose, and tissue concentrations associated with preclinical effect could be achieved and observed even 7 days after dosing. Dose-dependent decreases in prostate cancer cell CLU expression were also observed. At 640 mg dosing, CLU mRNA levels were decreased by ⬃92% compared with lower dose levels and historical controls as assessed by a quantitative RT-PCR assay of microdissected cancer cells. By immunohistochemistry, mean percentage of cancer cells staining negative for CLU at 640 mg dosing was 54% compared with 2–15% for lower dose levels and historical controls. As shown in Figure 25.2, Clusterin levels were also suppressed significantly in regional lymph tissues. This phase I trial demonstrated that OGX-011 is well tolerated and potently inhibits Clusterin expression in prostate cancers. The phase II dose for OGX-011 is now set to 640 mg based on pharmacokinetic and pharmacodynamic parameters. This is significantly higher than doses selected for G3139 and the Isis compounds reviewed above, especially when tissue exposure is considered given the prolonged tissue half-life of OGX-011, highlighting a potential explanation for the lack of demonstrated efficacy with the first-generation ASO agents. Two other phase I trials combined increasing

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(a)

(b)

(c)

Figure 25.2 (See color insert following page 270.) Immunostaining of OGX-011 drug distribution (a) and Clusterin expression (b, c) in human lymph tissue. (a) An antibody raised against the 2⬘-MOE backbone of OGX-011 enabled the immunohistochemical staining (brown) of resected human lymph tissue to verify that the drug had reached its target. (b, c) Clusterin protein expression (brown) in lymph node samples. Figure 25.2b shows an untreated control specimen while Figure 25.2c demonstrates downregulation of Clusterin in lymph tissue from a trial subject treated with OGX-011 at 640 mg dosing.

doses of OGX-011 with docetaxel in patients with metastatic breast, nonsmall cell lung, and hormone-refractory prostate cancers and with cisplatin and gemcitabine in patients with advanced NSCLC. Both confirmed the phase II dose for OGX-011 of 640 mg also in combination regimen [114,115]. Four phase II trials of OGX-011 in combination with chemotherapy are now underway in patients with prostate, breast, and lung cancers. 25.2.5 HSP27 Heat shock proteins (HSPs) were first discovered in 1962 as a set of highly conserved proteins that were induced by hyperthermia [116] and other kinds of cellular insults such as oxidative stress, activation of the FAS death receptor, and cytotoxic drugs that were subsequently reported [117–119]. They are ubiquitous proteins and have been characterized as cytoprotective molecular chaperones. The typical function of a chaperone is to assist proteins to attain their functional conformation, to mediate interaction with other proteins, and to prevent nonfunctional side reactions such as precipitation of misfolded proteins [120–122]. Mammalian HSPs have been classified into groups according to their electrophoretic characteristics. The four principal HSP families are HSP90, HSP70, HSP60, and the small HSPs including HSP27. High-molecular-weight HSPs are ATP-dependent chaperones while small HSPs act ATP independently. HSPs are important for signaling and protein traffic even in the absence of stress and regulated by specific heat shock transcription factors [123].

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However, the need of HSPs increases markedly after environmental assaults as a defense mechanism to allow cells to survive otherwise lethal conditions. HSPs have attracted attention as new targets for cancer therapy, especially since the discovery and characterization of geldanamycin as an inhibitor of HSP90 [124] and the targeting of the Clusterin gene as discussed above, whose product has small heat shock protein-like function. High levels of HSP27 are commonly detected in many cancers including prostate [125–128], breast [129,130], ovarian [131,132], glial [133], and gastric tumors [134]. HSPs have been associated with multidrug resistance and are functionally linked to increased tumorigenicity in breast [135,136] and colon cancers [137]. Recent evidence reveals that HSP27 provides cytoprotection to cells via many complex cell survival pathways, including interference with caspase activation, modulation of oxidative stress and stabilization of the cytoskeleton [138–144]. HSP27 appears to be crucial in maintaining the balance between cell death and cell survival. Accordingly, overexpression of HSP27 contributes to disequilibrium in this balance that leads to suppression of apoptosis and resistance to treatment. Consequently, HSP27 is an important therapeutic target. To specifically silence HSP27 gene expression the use of ASO and siRNA against the human translation-initiation site is a rational approach. Rocchi et al. [145,146] showed recently using prostate cancer cell lines, that HSP27 ASOs potently reduced HSP27 levels and significantly decreased cell growth in vitro. Pretreatment of PC-3 cells with HSP27 ASO enhanced apoptosis via caspase-3 activation, supporting recent data showing that HSP27 functions as a negative regulator of cytochrome c-dependent activation of procaspase-3. Concannon et al. [140] also reported that HSP27 inhibits caspase activation by sequestering both procaspase-3 and cytochrome c. Consistent with these in vitro data, systemic administration of HSP27 ASO monotherapy suppressed PC-3 tumor growth in vivo and considerably enhanced paclitaxel efficacy. Similarly, HSP27 overexpression was reported to confer resistance to doxorubicin in breast cancer cells [147]. Overexpression of HSP27 in human prostate LNCaP cells caused these normally androgen-dependent cells to become androgen-independent and more resistant to cytotoxic chemotherapy [146]. These findings suggest that increased levels of HSP27 after androgen withdrawal provide a cytoprotective role during development of androgen independence and that ASO-induced silencing can enhance apoptosis and delay tumor progression. A second-generation MOE gapmer ASO targeting HSP27 (OGX-427, OncoGeneX Technologies Inc.) is planned to enter phase I/II clinical trials in solid cancers and multiple myeloma in 2007. 25.2.6 STAT3 The signal transducer and activator of transcription (STAT) factors function as downstream effectors of many cytokine and growth factor receptors. Upon specific receptor stimulation and dimerization, activation of the Janus tyrosine kinases or SRC family members results in the phosphorylation and activation of STAT family members [148]. Once activated, STATs dimerize and translocate to the nucleus where they bind to specific DNA regulatory elements. A critical role for STAT3 in malignant transformation was first proposed in studies demonstrating constitutive activation of STAT3 in oncogene-transformed cells [149]. Since then, an abundance of studies have presented strong evidence that persistent STAT3 signaling activity participates in malignant transformation. This consequently leads to increased expression of genes associated with proliferation, cell survival and angiogenesis [150–152], and inhibition of inflammatory signals, thereby facilitating evasion of the immune system by tumor cells [153]. On the basis of this evidence, as well as the difficulties associated with discovering small molecule inhibitors against transcription factors, antisense strategies are underway to target STAT3 as a novel approach to treat human cancers. Screening of second-generation 2⬘-MOE ASOs against human STAT3 identified a highly potent and selective ASO that inhibits STAT3 expression in vitro and in vivo (ISIS 345794, Isis Pharmaceuticals). Reduced STAT3 level promote tumor cell death and increase sensitivity to chemotherapeutic agents in a variety of tumor types in vitro and human xenograft models in vivo,

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including multiple myeloma, melanoma, lymphoma, and prostate cancer [154,155]. ISIS 345794 has now been selected for clinical development and initiation of phase I studies is expected in the near future for the treatment of multiple myeloma, lymphoma, and other forms of cancer. 25.2.7 Insulin Growth Factor Binding Proteins Among the more promising ASO agents for prostate cancer in preclinical development are those that target the insulin-like growth factor binding proteins (IGFBPs). Insulin-like growth factor 1 (IGF1) plays an important role in the pathophysiology of prostatic disease and its activity is regulated by various factors in the microenvironment, including the IGFBPs [156,157]. In different physiological contexts, IGFBPs can either increase or decrease IGF1 signaling. After castration, the expression levels of certain IGFBPs change rapidly in the rat ventral prostate [158] and Shionogi tumors [159]. Differences in the expression of various IGFBPs in benign and malignant prostatic epithelial cells have been reported, with increases in IGFBP-2 and IGFBP-5, and decreases in IGFBP-3 in malignant versus benign cells [160]. After castration, higher levels of IGFBP-5 have been shown to be an adaptive cell survival response that helps potentiate the antiapoptotic and mitogenic effects of IGF1, thereby accelerating androgen-independent progression [161,162]. Furthermore, IGFBP-5 is present in high concentrations in bone, the most frequent site of metastases from prostate cancer. Systemic administration of IGFBP-5 ASO in mice bearing Shionogi tumors after castration attenuated castration-induced increases in IGFBP-5 and significantly delayed time to progression. IGFBP-2 expression also increases in human prostate tumors after castration and during androgen-independent progression and like IGFBP-5, appears to accelerate time to progression by enhancing IGF1 responsiveness [163]. IGFBP-2 levels have been shown to be increased in hormone-refractory clinical tumors [125] and forced overexpression of IGFBP-2 in LNCaP tumors produced an androgen-independent phenotype with a growth advantage compared to parental cells only in the absence of androgens. Moreover, IGFBP-2 ASOs decreased IGFBP-2 levels and reduced LNCaP cell growth rates in vitro and in vivo. Increased IGFBP-5 and IGFBP-2 levels after androgen ablation therefore represent adaptive mechanisms to potentiate IGF1-mediated survival and mitogenesis. The use of ASOs to target IGFBP-modulation of IGF1 signaling is undergoing further study, and a bispecific ASO that can simultaneously suppress both IGFBP-2 and IGFBP-5 is under development for clinical applications (OGX-225, OncoGeneX Technologies Inc.). 25.2.8 Ribonucleotide Reductase Ribonucleotide reductase (RNR) is an important enzyme for cell division and tumor growth that is required for the reductive conversion of ribonucleotides into deoxyribonucleotides, which is a crucial step in the synthesis and repair of DNA [164,165]. Mammalian RNR has a dimeric structure composed of two dissimilar subunits, R1 and R2, encoded on different chromosomes and each inactive on its own [166]. Both subunits consist of a nucleotide binding site (M1) and a metal binding site (M2). M1-affecting RNR inhibitors are nucleoside analogs, for example, gemcitabine. M2 contains nonheme iron and a tyrosine-free radical, which are required for the enzymatic reduction of ribonucleotides. Inhibitors of M2 act by destroying the free radical. Hydroxyurea is a clinically approved RNR inhibitor acting at the iron/free radical site, but the inhibition is reversible due to the ease in regenerating the tyrosine-free radical by mammalian cells [167]. The R1 subunit protein levels are constant during cell cycle, however, the expression of the R2 subunit increases in late G1/early S phase of the cell cycle when DNA replication occurs. The R2 subunit was also shown to be overexpressed in tumor tissues and appears to influence transformation and malignant potential of some oncogenes [165]. GTI-2501 and GTI-2040 (Lorus Therapeutics Inc.) are first-generation phosphorothioate antisense molecules that target and inhibit expression of the R1 and R2 subunit of RNR, respectively [168]. A phase I trial of GTI-2040 has been reported, and dose-limiting toxicity of hepatic enzyme elevation

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was observed [169]. The recommended phase II dose was determined to be 185 mg/m2/d given as a 21-day continuous i.v. infusion. Phase I trials of GTI-2501 and GTI-2040 in combination with docetaxel have been completed and phase II trials of these combination regimens are underway in patients with chemotherapy-naïve HRPC. The phase II trial of GTI-2040 and docetaxel has been reported in abstract form by Sridhar et al. [170] in 2006, with 9 patients out of 22 being described as having a PSA response. GTI-2040 is also currently in a phase II clinical trial for patients with advanced or metastatic renal cell carcinoma. The study investigates the effectiveness of the combined use of GTI-2040 and capecitabine, an oral chemotherapeutic cancer treatment. Data presented from the ongoing clinical study reported that more than 50 percent of patients showed disease stabilization (www.lorusthera.com). Recently, a RT-PCR method to quantify ribonucleotide reductase M2 mRNA in tumor samples and peripheral white blood cells (WBC) from breast cancer patients treated with GTI-2040 in another phase II trial was described [171]. By providing quantitative measurement of changes in target gene expression, this method offers an opportunity to determine correlation between target response and clinical response. 25.2.9 Other Promising ASO There have been many recently published in vitro studies applying ASO technology to various targets. One of those targets, MCL1, is a member of the BCL2 protein family, and is strongly associated with suppression of tumor apoptosis and promotion of malignancy [172,173]. MCL1 is overexpressed in many human tumor specimens, and confers resistance to chemotherapy-induced apoptosis [174–176]. A second-generation 2⬘-MOE ASO (ISIS 20408) that potently inhibits MCL1 expression in a variety of human tumor cell types has been identified [177–182]. Treatment of tumor cells in vitro promotes apoptosis and sensitizes tumor cells to chemotherapy-induced apoptosis, while in vivo administration delays growth of a variety of human xenograft models. Preclinical evaluation of ISIS 20408 continues with the goal of initiating clinical trials in the near future. Members of the protooncogenic RAS family are G-proteins involved in receptor signaling pathways controlling cell growth and division. Point mutations can convert RAS proteins into constitutively activated oncogenes mediating unrestricted cell proliferation and transformation [183]. Activated HRAS is supposed to contribute to therapeutic resistance against chemotherapy and radiation [184,185]. ISIS 2503 is a first-generation ASO that binds to the translation-initiation region of the human mRNA for HRAS and has shown promising results in a couple of single agent or combination phase I/II trials [186–188]. Additional genes currently validated as targets for antisense therapy include the p53 regulator MDM2 (GEM240, Idera Pharmaceuticals Inc., formerly Hybridon Inc.) [189–191], DNA methyltransferase (MG98, MethylGene Inc.) [192,193], Protein kinase A (GEM231) [194,195], TGF2 (AP12009, Antisense-Pharma) [196], eIF4E (LY2275796, www.isispharm.com) [197], BCR-ABL [198]. HSP70 [199], cMYB [200,201], hTERT [202], VEGF [203,204], and Lipoxygenase [205].

25.3 SUMMARY Antisense inhibition of relevant genes involved in cancer progression remains an area of hope for therapeutic development. ASO technology has quickly moved from preclinical models to testing in the clinic. Challenges remain to optimize tissue exposure, cellular uptake and demonstration of mechanism and antitumor activity in the clinic. The lack of success in the recent randomized phase III trials in lung cancer, CLL, myeloma, and melanoma has dampened enthusiasm for ASO therapeutics. However, next generation ASO chemistry, such as second-generation MOE gapmers, holds significant potential advantages for patient friendly dosing and routes of administration, enhanced activity and improved toxicity profile. Similar to the difficulties in developing any of the targeted therapies, there are several issues that need to be addressed in the early phase clinical trials

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of antisense therapeutics, and the failure of the first-generation ASOs in randomized trials only emphasizes this more. These issues include determination of biologically effective dose, ensuring the target is relevant in the patient population being studied, study designs that can detect meaningful cytostatic activity if appropriate, and rational use of combination strategies with study designs that will yield unambiguous endpoints. Addressing these issues early on will allow optimal use of these agents clinically and best ensure success in upcoming phase III trials. The clinical experience to date should still be considered part of the beginning of the era of antisense treatment for cancer.

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174. Zhou, P. et al., Mcl-1, a Bcl-2 family member, delays the death of hematopoietic cells under a variety of apoptosis-inducing conditions, Blood, 89, 630–643, 1997. 175. Miyamoto, Y. et al., Immunohistochemical analysis of Bcl-2, Bax, Bcl-X, and Mcl-1 expression in pancreatic cancers, Oncology, 56, 73–82, 1999. 176. Chung, T.K. et al., Expression of apoptotic regulators and their significance in cervical cancer, Cancer Lett, 180, 63–68, 2002. 177. Derenne, S. et al., Antisense strategy shows that Mcl-1 rather than Bcl-2 or Bcl-x(L) is an essential survival protein of human myeloma cells, Blood, 100, 194–199, 2002. 178. Thallinger, C. et al., Mcl-1 antisense therapy chemosensitizes human melanoma in a SCID mouse xenotransplantation model, J Invest Dermatol, 120, 1081–1086, 2003. 179. Aichberger, K.J. et al., Identification of mcl-1 as a BCR/ABL-dependent target in chronic myeloid leukemia (CML): Evidence for cooperative antileukemic effects of imatinib and mcl-1 antisense oligonucleotides, Blood, 105, 3303–3311, 2005. 180. Skvara, H. et al., Mcl-1 blocks radiation-induced apoptosis and inhibits clonogenic cell death, Anticancer Res, 25, 2697–2703, 2005. 181. Sieghart, W. et al., Mcl-1 overexpression in hepatocellular carcinoma: A potential target for antisense therapy, J Hepatol, 44, 151–157, 2006. 182. Wacheck, V. et al., Mcl-1 is a relevant molecular target for antisense oligonucleotide strategies in gastric cancer cells, Cancer Biol Ther, 5, 2006. 183. Adjei, A.A., Blocking oncogenic Ras signaling for cancer therapy, J Natl Cancer Inst, 93, 1062–1074, 2001. 184. Kim, I.A. et al., The influence of Ras pathway signaling on tumor radiosensitivity, Cancer Metastasis Rev, 23, 227–236, 2004. 185. Li, C. et al., H-Ras oncogene counteracts the growth-inhibitory effect of genistein in T24 bladder carcinoma cells, Br J Cancer, 92, 80–88, 2005. 186. Cunningham, C.C. et al., A phase I trial of H-ras antisense oligonucleotide ISIS 2503 administered as a continuous intravenous infusion in patients with advanced carcinoma, Cancer, 92, 1265–1271, 2001. 187. Adjei, A.A. et al., A phase I trial of ISIS 2503, an antisense inhibitor of H-ras, in combination with gemcitabine in patients with advanced cancer, Clin Cancer Res, 9, 115–123, 2003. 188. Alberts, S.R. et al., Gemcitabine and ISIS-2503 for patients with locally advanced or metastatic pancreatic adenocarcinoma: A North Central Cancer Treatment Group phase II trial, J Clin Oncol, 22, 4944–4950, 2004. 189. Sigalas, I. et al., Alternatively spliced mdm2 transcripts with loss of p53 binding domain sequences: Transforming ability and frequent detection in human cancer, Nat Med, 2, 912–917, 1996. 190. Wang, H. et al., Experimental therapy of human prostate cancer by inhibiting MDM2 expression with novel mixed-backbone antisense oligonucleotides: In vitro and in vivo activities and mechanisms, Prostate, 54, 194–205, 2003. 191. Zhang, Z. et al., Antisense therapy targeting MDM2 oncogene in prostate cancer: Effects on proliferation, apoptosis, multiple gene expression, and chemotherapy, Proc Natl Acad Sci U S A, 100, 11636–11641, 2003. 192. Davis, A.J. et al., Phase I and pharmacologic study of the human DNA methyltransferase antisense oligodeoxynucleotide MG98 given as a 21-day continuous infusion every 4 weeks, Invest New Drugs, 21, 85–97, 2003. 193. Winquist, E. et al., Phase II trial of DNA methyltransferase 1 inhibition with the antisense oligonucleotide MG98 in patients with metastatic renal carcinoma: A National Cancer Institute of Canada Clinical Trials Group investigational new drug study, Invest New Drugs, 24, 159–167, 2006. 194. Chen, H.X. et al., A safety and pharmacokinetic study of a mixed-backbone oligonucleotide (GEM231) targeting the type I protein kinase A by two-hour infusions in patients with refractory solid tumors, Clin Cancer Res, 6, 1259–1266, 2000. 195. Goel, S. et al., A phase I safety and dose escalation trial of docetaxel combined with GEM231, a second generation antisense oligonucleotide targeting protein kinase A R1alpha in patients with advanced solid cancers, Invest New Drugs, 24, 125–134, 2006. 196. Schlingensiepen, K.H. et al., Targeted tumor therapy with the TGF-beta2 antisense compound AP 12009, Cytokine Growth Factor Rev, 17, 129–139, 2006.

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197. De Benedetti, A., Graff, J.R., eIF-4E expression and its role in malignancies and metastases, Oncogene, 23, 3189–3199, 2004. 198. Rapozzi, V. et al., Antisense locked nucleic acids efficiently suppress BCR/ABL and induce cell growth decline and apoptosis in leukemic cells, Mol Cancer Ther, 5, 1683–1692, 2006. 199. Nylandsted, J. et al., Selective depletion of heat shock protein 70 (Hsp70) activates a tumor-specific death program that is independent of caspases and bypasses Bcl-2, Proc Natl Acad Sci U S A, 97, 7871–7876, 2000. 200. Ratajczak, M.Z. et al., Acute- and chronic-phase chronic myelogenous leukemia colony-forming units are highly sensitive to the growth inhibitory effects of c-myb antisense oligodeoxynucleotides, Blood, 79, 1956–1961, 1992. 201. Luger, S.M. et al., Oligodeoxynucleotide-mediated inhibition of c-myb gene expression in autografted bone marrow: A pilot study, Blood, 99, 1150–1158, 2002. 202. Kraemer, K. et al., Chemosensitization of bladder cancer cell lines by human telomerase reverse transcriptase antisense treatment, J Urol, 172, 2023–2028, 2004. 203. Forster, Y. et al., Antisense-mediated VEGF suppression in bladder and breast cancer cells, Cancer Lett, 212, 95–103, 2004. 204. Krause, S. et al., Vascular endothelial growth factor antisense pretreatment of bladder cancer cells significantly enhances the cytotoxicity of mitomycin C, gemcitabine and Cisplatin, J Urol, 174, 328–331, 2005. 205. Ikemoto, S. et al., Antitumor effects of lipoxygenase inhibitors on murine bladder cancer cell line (MBT-2), Anticancer Res, 24, 733–736, 2004.

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26

Targeting Neurological Disorders with Antisense Oligonucleotides Richard A. Smith and Timothy M. Miller

CONTENTS 26.1 26.2 26.3

Introduction ......................................................................................................................721 Distribution ......................................................................................................................723 Nucleic Acid-Based, Nonantisense Gene Silencing ........................................................726 26.3.1 DNAzymes and Ribozymes ..............................................................................726 26.3.2 RNAi ..................................................................................................................727 26.4 Safety and Toxicity ..........................................................................................................728 26.5 Amyotrophic Lateral Sclerosis ........................................................................................730 26.6 Huntington’s Disease .......................................................................................................734 26.7 Pain ..................................................................................................................................735 26.8 Glioblastoma ....................................................................................................................735 26.9 Prion Disorders ................................................................................................................736 26.10 Dementias ........................................................................................................................737 26.11 Neuropathy ......................................................................................................................738 26.12 Spinal Muscular Atrophy .................................................................................................739 26.13 Muscular Dystrophy ........................................................................................................739 26.14 Conclusions ......................................................................................................................740 References .....................................................................................................................................740

26.1 INTRODUCTION Neurological disorders represent a major health burden that is increasing in magnitude in parallel with the demographic shift in the age of the population of advanced societies. In the United States alone it is estimated that 3.5 million people are afflicted with Alzheimer’s disease, which typically runs its fatal course over 3–5 years [1]. While advances in medicine have been one of the defining achievements of modern times it is fair to say that these have had little impact on the fortunes of persons afflicted with Alzheimer’s disease, multiple system atrophy, frontotemporal dementia, and the like. Modest success has been achieved with symptomatic treatment; the best example being the treatment of Parkinson’s disease with L-dopa and dopamine agonists.

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The ability to regulate gene expression in the nervous system has broad scientific and clinical implications. From the basic science perspective turning a gene off offers the promise of understanding complicated systems, particularly when the effects of multiple receptor subtypes cannot be distinguished due to the lack of ligands with the required specificity. In this regard, antisense oligonucleotides (ASOs) have been used successfully to target a variety of receptors (e.g., dopamine) and neuropeptides in the central nervous system (CNS) such as corticotrophinreleasing factor and cholecystokinin [2–6]. And, of course, the medical uses for such a technology would be immediately applicable to diseases whose pathogenesis involves the undesirable activation of a receptor or the accumulation of a toxic protein. A number of strategies have been devised to regulate gene expression, albeit it recently has become evident that nature has long developed the means to accomplish such a feat and, in fact, does so throughout the plant and animal world. It is all but certain that more such processes remain to be discovered and that human inventiveness will result in others. Considering the variety of neurological disorders and their complexity it may seem naïve to suggest that a particular treatment strategy could be applicable to diseases ranging from prion disease to amyotrophic lateral sclerosis. But for obvious reasons the use of a technology as adaptable as antisense therapy offers unlimited therapeutic utility in instances for which an appropriate target has been identified. In the case of neurodegenerative disease, one overarching principle may underlie these conditions, namely, that collectively they are examples of proteinopathies that result from aging, genetic, or environmental causes that lead to the misfolding of proteins [7]. Accordingly, the development of appropriate antisense therapeutics would seem to be a reasoned means of treating conditions for which there is currently no meaningful treatment. The evidence that perturbation of proteins is a leading cause of neurodegeneration comes primarily from the discovery that genetic disorders such as Huntington’s disease, familial amyotrophic lateral sclerosis, and Parkinson’s disease are linked to mutations of proteins [7]. In the instance of Huntington’s disease, which is a dominantly inherited disorder leading to cognitive decline and severe choreaform movements, it has been discovered that the mutant gene is expanded due to polyglutamine repeats. The size of the repeat varies. Individuals with larger repeats experience earlier onset of their disease, which typically commences in the 40s, but the size of the repeat does not affect survival [8]. Huntington’s is just one of several examples of such a mutation. In contrast to mutations involving large segments of a gene, single-nucleotide mutations have also been etiologically linked to brain disorders including familial amyotrophic lateral sclerosis and familial Alzheimer’s disease. In spite of knowing the exact cause of Huntington’s and other genetic disorders the mechanism(s) by which a specific mutation leads to nerve cell degeneration remains mostly conjectural. Much of what has been learned has been extrapolated from the study of animal models. This has the effect of increasing the opportunity to select appropriate treatment targets. The discovery in 1993 that a proportion of ALS is caused by mutation in the ubiquitously expressed enzyme superoxide dismutase 1 (SOD1) represents one such example [9]. Experiments in mice and rats have demonstrated that a number of human mutants cause motor neuron death in rodents. While the basis for their toxic property(ies) remains unknown it is evident from rodent models that the timing of disease onset is specified by the level of expression of the mutant protein [10]. Transgenic mice that express higher levels of mutant protein have earlier onset; lines that express lower levels have later onset or do not develop disease at all [11] and animals that spontaneously lose copy number enjoy increased survival [12]. From this it is reasonable to assume, whether toxicity is achieved within motor neurons or transferred indirectly from mutant expression in astrocytes or microglia; toxicity should be ameliorated if mutant SOD1 expression is reduced. On this basis one can predict that a treatment strategy that targets SOD in the instance of familial amyotrophic lateral sclerosis (FALS) could be successful. By analogy, targeting amyloid and tau in the instance of Alzheimer’s disease might similarly be of clinical use.

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While wild-type SOD1 and other proteins implicated in neurodegenerative disease are biologically important it is likely that a modest reduction of these will be well tolerated. For example, even though SOD1 provides protection from oxidative damage, especially during recovery from ischemia, mice with only 50% of the normal amount of enzyme do not generate any reported phenotype [13]. Even complete loss of SOD1 does not produce disease or compromise life span in mice [14]. In the instance of disease caused by SOD1 mutation the evidence leads to the conclusion that toxicity will be alleviated, possibly eliminated, by as little as 50% reduction in mutant SOD1 expression. In contrast, simultaneous reduction in wild-type SOD1 would be predicted to have little consequence and might be of benefit.

26.2 DISTRIBUTION One of the limiting factors in the use of ASOs for the treatment of brain disorders is the obligation to introduce them directly into the CNS compartment or modify them in an effort to circumvent the blood–brain barrier (BBB). As a general rule, molecules of the size of ASOs would not be expected to breach the BBB and this has been demonstrated repeatedly [15]. Following systemic administration by intraperitoneal injection, ASOs are taken up by numerous organs including the liver and kidneys but the amount detected in brain, muscle, and nerve is negligible and insufficient to the task of modifying the production of a mutant protein (Figure 26.1). On this background Banks et al. [16] report that a phosphorothioate-modified ASO directed against amyloid precursor protein (APP) crosses the BBB following intravenous administration to SANP8 mice, a strain that carries a natural mutation affecting APP. Steady-state tissue levels were reached within 30–60 min. The spleen had the highest level and the brain the least. Brain and cerebrospinal fluid (CSF) levels were in equilibrium. Since uptake could be saturated by coadministration of unlabeled oligo the evidence was interpreted to favor active transport into the CNS, which the authors suggest might be coupled to a protein transporter. Comparing tissue concentrations achieved with intravenous and intraventricular (ICV) administration, a 100-fold difference in favor of ICV delivery was found. Extrapolating this result to the treatment of humans would, by our calculations, require the systemic administration of ASOs that vastly exceed tolerated doses. A seemingly ideal solution to the problem of delivering an antisense therapeutic to the nervous system is the use of peptide nucleic acids (AS-PNAs). The purported advantages of these DNA analogs include their resistance to nucleases and proteases, and their high affinity for nucleic acids. In contrast to phosphorothioate ASOs (P⫽S ASOs), AS-PNA molecules more readily penetrate the BBB and can be detected in both the brain and CSF after systemic administration [17]. As opposed to conventional antisense therpeutics, AS-PNAs do not appear to act via an RNAase H mechanism since mRNA levels remain normal in spite of a reduction in protein synthesis. This has suggested that the mechanism of action involves a translation block, a potentially advantageous property in the instance of a CNS therapeutic since neurons, nondividing cells, have less RNAase H levels than their counterparts [15]. In a study designed to test the therapeutic effect of downregulating a neurotrophin in an ALS animal model, peritoneal administration of an AS-PNA targeting p75ntr was shown to delay disease onset and increase survival [18]. Further, this result was accompanied by reduction in spinal cord levels of p75ntr and diminished caspase activation, which is under p75npr regulation. It has been suggested that conjugation of AS-PNAs to biotin or another carrier might further facilitate delivery of these molecules to the CNS. But thus far no commercial sponsor has chosen to develop this class of therapeutics. Further, it has been assumed that by transiently interrupting the BBB with administration of a highly osmotic agent it might be possible to circumvent the BBB but such treatment would, in most therapeutic instances, require repeated administration, be impractical, and likely to be dangerous. Some investigators have employed positive pressure infusion to facilitate delivery of ASOs to the CNS, an idea promulgated by the notion that ASOs do not permeate the nervous system after

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Figure 26.1 (See color insert following page 270.) Distribution of antisense oligonucleotides after infusion into the right lateral ventricle in rat and Rhesus monkey. (A, B) Antisense oligonucleotides were continuously infused at 100
g/day (A) or 1 mg/day (B) for two weeks via infusion pump into the right lateral ventricle of (A) normal rats or (B) Rhesus monkey. Tissues were collected and extracts of them analyzed for oligonucleotide content by capillary gel electrophoresis. Mean values ± standard deviations are shown (A) n ⫽ 6; (B) n ⫽ 2. (C–G) A 24-mer modified oligonucleotide Isis13920 was infused for 2 weeks into the right lateral ventricle at 100
g/day in (C–E) rats or 1 mg/day in (F–M) Rhesus monkey. After perfusion, distribution of the oligonucleotide was determined by immunohistochemistry using a monoclonal antibody that recognizes the oligonucleotide (C–E, F, H) or astrocytes (GFAP; G, I). No oligonucleotide staining was seen in animals (D, H) infused with saline only or (E) an oligonucleotide infused animal but using secondary antibody only. Bar, 50
m. (Copyright 2006 by American Society for Clinical Investigation. Reproduced with permission of American Society for Clinical Investigation in the format Textbook via Copyright Clearance Center.)

intrathecal or intraventricular administration [19]. A variant of this technique has been utilized as a means of enhancing the treatment of malignant brain tumors [20]. As a result of limitations to the delivery of ASOs, direct administration to the nervous system appears to offer promise although this route of administration has not yet been widely adopted for

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clinical use. In contrast to blood, spinal fluid exhibits minimal nuclease activity [21]. But this has little practical importance since brain nucleases rapidly degrade unmodified ASOs, primarily due to the activity of exonucleases acting at the 3⬘ end of the molecule. This problem has, more or less, been obviated by the use of phosphorothioate-modified oligonucleotides. In a seminal study designed to evaluate the clearance and disposition oligonucleotides, Whitesell et al. [21] administered labeled 15-mer oligonucleotides intraventricularly in adult rats. Since bolus infusions were thought to be unreliable, the clearance of an ASO and inulin were determined after continuous CSF infusion for 1 week. CSF samples were removed serially from the cisterna magma. At steady-state clearance t½ was determined to be 17.2 ⫾ 4.7 min for the ASO and 23 ⫾ 7.5 min for inulin. Because steady-state levels of ASO were comparable to inulin the authors concluded that tissue uptake was minimal although this conclusion is seemingly undercut by their microscopic observations. Using double staining, prominent uptake was noted in the soma of many cells, especially those of presumed glial origin as judged by florescent microscopy. Using in situ hybridization, Yaida and Nowak [22] compared the distribution of phospodiester and phosphorothioate ASOs after intraventricular and intraparenchymal injection. The distribution of phosphodiester ASOs was limited in both circumstances. Predominately, periventricular staining was seen following ICV administration, whereas phosphorothioate ASOs were detected in the ipsilateral striatum and hippocampus. The extent of brain penetration did not increase with repeated injections leading to the conclusion that ASOs were being rapidly cleared from the spinal fluid as a result of bulk flow and that drug delivery to the brain via this route depends strongly on proximity to the ventricular space. This, along with similar reports cast doubt on the general utility of antisense as a viable therapy in the instance of brain disorders such as Alzheimer’s disease, which are associated with widespread pathology [23]. More recent evidence challenges these assumptions. Chauhan studied the tissue distribution of a florescein-labeled 2⬘ methoxyethyl ASO (MOE ASO) directed against the  secretase cleavage site of APP. This was injected intraventricularly into mice as a bolus and animals were sacrificed at four time points. In the first 15 min, intense staining was noted within the ependymal lining and adjacent tissue. Subsequently, staining spread. By 3 h there was overall diffusion and at 8 h postinjection, clearance was complete. Both neuronal and nonneuronal stainings were noted. Nuclear and cytoplasmic uptake was observed in some neurons, in others only perikaryl localization was observed. While experiments demonstrating widespread uptake of ASOs in the brain following ICV administration are relevant to the treatment of Alzheimer’s disease and the like, they do not shed light on the treatment of diseases such as ALS that predominately affect the lower brain and spinal cord. Further, the question arises as to whether the results seen in rodents can be extrapolated to primates whose nervous systems are exponentially larger. With this in mind the authors have studied the distribution of ASOs within the entire CNS following ICV administration of 5-10-5 MOE gapmers in both rats and subhuman primates [24]. These modified oligonucleotides retain RNAase activity and are characterized by long half-lives due to their stability [25]. Using an antibody that recognizes motifs in a tracking oligo robust uptake was demonstrated in motor neurons in the lumbar spinal cord in both species after continuous ICV infusion of oligonucleotides for 1 month (Figure 26.1). Additionally, uptake was seen in the striatum, thalamus, cerebellum, and pons following chronic ICV administration. Using capillary gel electrophoresis capable of measuring as little as 0.35
g/g of tissue, it was further demonstrated that tissue concentrations in all brain and spinal cord tissues could be sustained over long intervals and that they were of sufficient concentration to be biologically active (Figure 26.1). A parallel study was conducted in subhuman primates using the intrathecal (IT) route of administration. While the expected findings were noted: greater spinal cord ASO levels after IT administration and greater hemispheric concentrations after ICV delivery, the finding that substantial levels of ASO could be detected within the brain after IT administration was unexpected (Figure 26.2). Since spinal fluid is produced within the choroid plexus and flows out of the ventricles, ultimately ending up in the subarachnoid space surrounding the hemispheres where it is readsorbed,

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Figure 26.2 Comparison of antisense oligonucleotide concentrations in Rhesus monkey with intraventricular or intrathecal infusion. Two Rhesus monkeys in each group were infused with antisense oligonucleotide (Isis13920) for 14 days at 1 mg/day. Animals were sacrificed and oligonucleotide concentrations were measured by capillary gel electrophoresis.

one would not have anticipated that a molecule introduced in the lumbar space would enjoy widespread access to deep brain structures, such as the striatum. If this is the case in humans, IT delivery would obviously be the preferred route of administration in most clinical circumstances since this would obviate the need to place an intraventricular catheter with its small but attendant risks. 26.3 NUCLEIC ACID-BASED, NONANTISENSE GENE SILENCING As might be expected, a number of competing strategies to the use of ASOs have emerged, several of which are nucleic acid based. Thus far, none of the demonstrated strategies have advanced, beyond proof of principle, to a clinical application. It is too early to know which of these approaches will be the most adaptable to clinical use. A number of factors will determine this, including stability, ease of manufacture, and tolerability. And a key determinant will be the ability to moderate the dose, including the ability to terminate treatment. 26.3.1 DNAzymes and Ribozymes Nucleic acids were long considered to be passive molecules whose function was restricted to the passage of information embodied in the genetic code. More recently, nucleic acids have been shown to have catalytic properties. Naturally occurring ribozymes have been demonstrated to inactivate viral and messenger RNA and DNAs with similar properties have been synthesized. The 10–23 deoxyribozyme is a catalytic DNA species discovered by Gerald Joyce at Scripps Research Institute using directed evolution [26]. It has two user-friendly features: sequence specificity and catalytic efficiency. The 10–23 DNA consists of a catalytic core of 15 nucleotides with side arms. The substrate requirements are also simple. The target RNA must be amenable to Watson–Crick pairing, must contain a purine–pyrimidine junction, and the target purine must be unpaired. Further design features include an analysis of the binding characteristics of the side arms. If they bind too tightly, turnover is hindered, lowering the catalytic efficiency. Finally, the secondary structure is analyzed. A molecule that folds upon itself is unlikely to work.

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Fraction SOD1 mRNA cleaved

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In collaboration with Dr. Joyce we have synthesized 10–23 DNAzymes that cleave SOD mRNA with the intent of developing a therapeutic that might have application to the treatment of familial ALS. Putative cleavage sites were selected based on screening a panel of antisense oligos, reasoning that a site accessible to an antisense molecule should be accessible to a 10–23 DNA. To test this in vitro, a plasmid containing full-length SOD was linearized with BAM I and transcribed using T3 polymerase in the presence of the 10–23 DNA and a control. Cleavage was demonstrated by gel electrophoresis (Figure 26.3). Subsequently, two candidate DNAzymes were injected intraventricularly into rats for 1 month using an Alzet pump connected to an indwelling cannula. Animals were then sacrificed and SOD protein was quantified using Western blots. No effect on protein was apparent, but given the small number of DNAzyme tested this is not a surprising result. In the instance of identifying an antisense therapeutic it is often necessary to screen 100 oligonucleotides to find one that works in the brain as well as it does in vitro. 26.3.2 RNAi The discovery, first in plants and then in animals, that small RNAs are involved in gene regulation and can silence viral gene products has vitalized interest in gene therapy [27]. In the initial processing steps, specialized nucleases, RNAase 111-like enzymes that are part of the Dicer complex, cleave double-stranded stretches of RNA that have been exported to the cytoplasm [28]. Subsequently, the double-stranded (ds) RNAs are modified in the RISC complex, which brings together the target mRNA, an RNAase H-like nuclease (recently identified as an Argonaute protein) and the 22 nucleotide ds RNA processed by the Dicer complex. In this configuration either the ds RNA or a single derivative antisense strand of RNA act as a guide for the nuclease that cleaves the target mRNA in a sequence-specific fashion or sets in motion a process leading to a translation block. This latter event could result from a steric effect or through the recruitment of proteins that bind to the target mRNA, interfering with its translation [29]. RNA interfering (RNAi) molecules synthesized in vitro require modification to maintain their stability and enhance their resistance to degradation, both serious issues in a clinical settting [30]. Overcoming these limitations, Thakker and his colleagues [31] were able to achieve approximately 50% knockdown of a target mRNA following instillation of stabilized RNAi molecules into the lateral ventricle, although distribution within the CNS was limited [31].

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As an alternative, a gene therapy approach employing a viral vector allows for the continuous production of RNAi molecules. Such viral vectors can be injected directly into the CNS or injected into muscle, provided the viral coat proteins mediate synaptic uptake and subsequent retrograde delivery to nuclei of motor or sensory neurons. Using these techniques, an effect on disease in an animal model has been demonstrated for spinal cerebellar ataxia type 1 targeting ataxin [32], Huntington’s disease targeting huntingtin [33], ALS targeting SOD1 [34–36], and Alzheimer’s disease targeting BACE1 [37]. A potential concern of RNAi-based therapy is that the level of gene silencing from the current generation of viruses cannot be regulated. Further, there is the possibility of saturating the endogenous capacity for processing small RNAs, resulting in fatal dysregulation of endogenous genes [38].

26.4 SAFETY AND TOXICITY Phosphorothioate-modified antisense molecules (PS⫽ASOs) have been in development for a number of years but the use of 2⬘ MOE gapmers dates to the late 1990s. These compounds have been administered to hundreds of patients, primarily cancer patients via systemic administration. In general, treatment is well tolerated but with prolonged treatment constitutional effects including malaise, fever, anorexia, etc. have been observed. When side effects occur they have been attributed to the chemistry of the molecule rather than to sequence-specific effects of the drug. Only recently has an antisense therapeutic been administered directly to the brain of a patient. Details surrounding this treatment trial have not been published but it has been reported that patients with malignant glioma do tolerate intraparenchymal injection of an antisense molecule that targets TGF- [6]. With systemic administration drug is concentrated preferentially in the liver and kidneys. Both these organs show minor histological evidence of injury and in clinical studies mild elevations of liver enzymes are seen. Surprisingly, our own studies demonstrate considerable uptake of oligos in both these organs after intraventricular administration (Figure 26.4C). Enlargement of the spleen is also observed. Effects on coagulation are the most common side effects observed thus far in clinical trials. This is manifested by prolongation of the partial thromboplastin time. The effect is dose and schedule dependent and has resolved within hours of the cessation of therapy. Mild activation of complement has been observed in both preclinical and clinical studies at a threshold concentration of approximately 50
g/mL. To prevent cardiovascular collapse, administration is designed to avoid these peak concentrations. Prolonged administration of P⫽S ASOs has been associated with thrombocytopenia in approximately one-third of treated patients. This has generally not required dose adjustment and in some cases platelet counts have been noted to increase with continued treatment. The basis for this appears to be different among species. In mice, thrombocytopenia has been thought to be due to sequestration of platelets in enlarged organs, such as the spleen. Phosphorothioate oligodeoxynucleotides (PS ODNs) are well recognized to activate cells of the immune system predominantly through interaction with Toll-like receptor 9 (TLR-9), although there are TLR-9-independent pathways as well [39]. Rodents are known to be much more sensitive to the immune stimulatory effects of PS ODNs than primates, including humans. Although sequences with appropriate CpG motifs are very potent activators of immune cells, many sequences are capable of activating TLR9 receptors at higher concentrations. In 1998, Peng Ho and his colleagues [15] synthesized a number of oligonucleotide analogs to identify chemistries that maximized potency and minimized the occurrence of side effects that were associated with first-generation phosphorothioatemodified oligonucleotides. These most notably have been associated with febrile responses and weight loss after intraventricular administration [40]. In a trade-off between potency and side effects a chimeric oligonucleotide that replaced the central phoshodiester linkages with phosphorothioate

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Figure 26.4 Identifying antisense oligonucleotides that reduce rat SOD1 in vitro and in vivo. (A) Seventy-eight 24-mer-modified oligonucleotides complementary to rat SOD1 mRNA were synthesized and then transfected (at 150 nM) into primary rat A10 cells, RNA prepared 24 h posttransfection and SOD1 mRNA levels were measured by quantitative RT-PCR. Mean values⫾standard deviations are shown (n ⫽ 4). Oligonucleotides are displayed relative to their positions on the 462 nucleotide SOD1 coding sequence. (B) Oligonucleotides identified by the in vitro screen in (A) were evaluated in a similar transfection paradigm again using rat A10 cells and transfection of increasing concentrations of oligonucleotide to produce a dose response curve. (C) Oligonucleotides SODr/h146144, SODr/h146145, SODr146192, and SODscrambled (a control oligo) were injected (with 37.5 mg/kg) three times per week intraperitoneally into adult rats for 3 weeks after which time mRNA levels were measured in the liver, kidney, and brain. Mean values ± standard deviations are shown (n ⫽ 6). (D) SOD1 protein levels in liver extracts from animals treated with oligonucleotides SODr/h146144, SODr/h146145, SODr146192, and SODscrambled were measured by immunoblotting with an antibody to SOD1. Bottom: Immunoblot for tubulin to verify protein loading. (Copyright 2006 by American Society for Clinical Investigation. Reproduced with permission of American Society for Clinical Investigation in the format Textbook via Copyright Clearance Center.)

ones proved to be the best choice. An iteration of this molecule (2⬘ MOE phosphorothioate oligonucleotide) is now in clinical development for the treatment of FALS although more effort might be profitably expended to identify oligonucleotides that are tailored for use in the CNS [25]. To our knowledge formal toxicology studies have not been undertaken in the instance of intraventricular or intrathecal administration of these molecules. Following intraventricular administration of 2⬘ MOE

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gapmers targeting SOD we noted inconsistent inflammatory changes at the site of administration or in a subependymal distribution. These were usually manifested as macrophage infiltration. In separate experiments, at high doses (300–400
g/day), we noted hind limb paralysis in some animals suggesting that oligos were pooling in the lumbar space with the result that nerve roots, the spinal cord, or possibly its coverings are adversely affected. Studies that do not optimize the oligonucleotide for the target may be at risk for increasing the incidence of adverse events. Numerous studies have demonstrated marked differences in the effectiveness of oligonucleotides designed to bind to different regions of the mRNA. Thus, studies requiring use of high doses of nonoptimized oligonucleotides may have pushed the therapeutic index. In this regard second-generation 2⬘-O-methoxyethyl-modified oligonucleotides have significantly greater affinity for RNA than PS ODNs. This translates to a 10- to 15-fold increase in potency and these molecules exhibit a longer duration of action in tissues due to increased metabolic stability. They also have decreased propensity for immune stimulation in rodents, primates, and man compared to PS ODNs. Thus, 25- to 50-fold lesser drug is needed to produce the same effect. Over 500 subjects have been exposed to these second-generation oligonucleotides.

26.5 AMYOTROPHIC LATERAL SCLEROSIS The majority (⬎ 90%) of cases of amyotrophic lateral sclerosis (ALS) are sporadic. Typically, ALS causes progressive severe weakness resulting in loss of use of the limbs, inability to swallow or speak, and ultimately death from respiratory failure 3–5 years after the onset of the disease. There are approximately 9000 new cases of ALS every year in the United States. There are no treatments that substantially slow the disease. The best therapy, Rilutek, is only marginally effective [41]. While the cause(s) of sporadic ALS is/are currently unknown a general understanding of the events leading to neurodegeneration has evolved [10]. Attractive targets for slowing disease progression in sporadic ALS have arisen from recognition that an intraneuronal cell death pathway represents the final step in the demise of motor neurons in both inherited and sporadic ALS. One such potential target is Bax, a member of the BCL-2 family of proteins that triggers cell death, including cell death in neurons [42]. Accordingly, targeting Bax with an ASO is an example of a speculative target for treating a fatal disorder whose cause(s) remain unknown. Less speculative are targets known to cause FALS. Approximately 20% of FALS cases are due to a mutation of the superoxide dismutase gene. Over a hundred such mutations have been described, but in the United States half of these cases are the so-called A4V variant [10]. This is the most virulent mutation and survival after disease onset is 1 year or less in almost all instances. While the number of A4V patients in the United States is small, probably 150 or so at any one time, the development of a treatment for this group of ALS patients would be a medical milestone since the only effective therapy for any form of ALS is palliative. Along with the gravity of the disease, there are other reasons to believe that A4V patients are an ideal population to test a novel therapy. First, ALS patients, inspite of the seriousness of their underlying disease, are otherwise in good health. As a group, patients are usually in middle life. Their vital organs are not involved in the disease process and they generally retain their cognitive abilities. Accordingly, patients are able to provide informed consent and they are hearty enough to withstand whatever medical challenges they might encounter as part of the treatment regimen. With the initial goal of treating familial ALS due to mutation in the SOD1 gene, ASOs that target rat SOD1 were selected from a panel of 80 ASOs (Figure 26.4) and lead oligos were screened further for ability to decrease endogenous SOD1 in the liver. After 1 month of intraventricular administration of lead rat oligos, SOD1 mRNA and protein were decreased by about 50% (Figure 26.5).

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g/day. (A) Endogenous SOD1 mRNA levels from brain and spinal cord regions were measured by quantitative real time PCR. Mean values ⫾ standard deviations are shown (n ⫽ 6). (B) SOD1 and -tubulin protein levels analyzed by immunoblotting following infusion. Top panel: Coomassie stained gel demonstrates equal loading. (C, D) Protein levels for tubulin and SOD1 quantified for right cortex, cervical cord, and lumbar cord after infusion as in (B). Mean values ⫾ standard deviations are shown (n ⫽ 6). (E) Antisense oligonucleotides against presenilin1 or GSK3 were infused for 2 weeks into the right lateral ventricle of nontransgenic mice and mRNA levels were measured by quantitative RT-PCR in the right frontal temporal cortex (n ⫽ 6). (Copyright 2006 by American Society for Clinical Investigation. Reproduced with permission of American Society for Clinical Investigation in the format Textbook via Copyright Clearance Center.)

Subsequently, a lead oligo, Isis oligo 333611, effective against human SOD1 was identified. This oligo has been shown to downregulate both the expression of SOD1 mRNA and protein throughout the brain and spinal cord of transgenic animals carrying the mutant human gene (Figure 26.6).

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Figure 26.6 Antisense oligonucleotides complementary to human SOD1 mRNA decrease SOD1 protein levels in SOD1G93A rat liver and spinal cord. (A) An oligonucleotide active against human SOD1 mRNA as well as a rat mRNA specific oligonucleotide (SODr146192) was injected intraperitoneally three times per week (37.5 mg/kg at a concentration of 3 M) into adult rats expressing a low copy number human SOD1G93A transgene (line L26L). After 3 weeks, liver extracts were prepared and analyzed by (A) immunoblotting using an antibody that recognizes rat and human SOD1 with equal affinity. (B–D) Antisense oligonucleotides complementary to human SOD1 mRNA were infused into the right lateral ventricle of 65-day-old SOD1G93A rats at 100
g/day for 28 days. (B) RNA was prepared from tissue extracts and SOD1 RNA levels were measured by real-time PCR. (C–D) Protein levels for SOD1 and -tubulin were analyzed in parallel extracts (C) by immunoblotting with an antibody recognizing human and rat SOD1 with equal affinity and quantified (D) for cervical cord. * indicates p ⬍ 0.05 for students t-test compared with SODscrambled. Mean values ⫾ standard deviations are shown (SOD1scrambled, n ⫽ 4; SODr/h333611, n ⫽ 8). (Copyright 2006 by American Society for Clinical Investigation. Reproduced with permission of American Society for Clinical Investigation in the format Textbook via Copyright Clearance Center.)

Finally, treatment of these animals with Isis oligo 3336111, delivered intraventricularly using an Alzet pump, resulted in slowed disease progression after onset and prolonged survival of affected animals in comparison to animals treated with saline or a scrambled ASO (Figure 26.7). As a further test, fibroblasts were cultured from a patient meeting clinical criteria for ALS and carrying an SOD1A4V mutation (Figure 26.8). Fibroblasts were treated with 300 nM of Isis oligo 333611 for 72 h and then analyzed for SOD1 mRNA levels. As demonstrated, treatment with oligo 333611 and another SOD acting oligo had a marked effect in vitro.

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g/day for 28 days (A) Disease onset defined as the peak animal weight and (B) early disease defined as the point where the animals had lost 10% of their peak weight, and (C) survival defined as the inability of the animal to right itself after 30 s after being placed on its side. Saline infused, n ⫽ 11; SODr/h333611, n ⫽ 12. (Copyright 2006 by American Society for Clinical Investigation. Reproduced with permission of American Society for Clinical Investigation in the format Textbook via Copyright Clearance Center.)

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On the basis on these data, formal toxicity studies with Isis333611 are underway and a clinical trial for familial SOD1 ALS has been planned. 26.6 HUNTINGTON’s DISEASE Huntington’s disease is a progressive inherited disorder characterized by disabling, uncontrollable movements, a change in personality, and a loss of cognitive abilities [43]. With progression, patient’s speech and swallowing are affected, contributing further to eventual incapacitation and death. The disease affects approximately 15,000 patients in the United States. All instances of Huntington’s disease are caused by a dominant mutation in the huntingtin gene. Disease typically begins between the ages of 35 and 40 and is fatal within 15 years. There are no therapies that slow disease onset or progression. Each disease-causing mutation results in the expansion of the huntingtin protein as a result of the incorporation of an excess stretch of polyglutamine repeats [44]. There is an inverse relation between the size of the expansion and the onset of the disease, with longer expansions resulting in early onset. But the size of the expansion does not influence the severity of the disease. Expression of mutant huntingtin in mice causes dysfunction of the nervous system [45]. Decreasing mutant huntingtin in adult mice not only slows the progressive deterioration of the nervous system, but, in fact, reverses some of the symptoms. Thus, it is very likely that decreasing huntingtin in humans would provide a therapeutic benefit, even in adult patients. ASOs that decrease huntingtin protein when infused into a normal mouse have already been identified, as have ASOs targeting the human protein. Although complete deletion of huntingtin, using genetic strategies, is incompatible with normal development of the mouse; this is not anticipated to represent a significant impediment to the use of ASOs to lower mutant Huntington synthesis for the following reasons: a. ASOs typically reduce protein by 50% rather than completely and the degree of target knockdown can be regulated by the amount of ASO delivered. b. Initial experiments with ASOs to decrease endogenous mouse huntingtin protein by 50% have not resulted in any untoward side effects.

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26.7 PAIN Intractable pain represents a major health burden that has multiple causations. Common causes related to the nervous system include peripheral neuropathy, post herpetic neuralgia, thalamic syndrome, multiple sclerosis, and the like. The magnitude of the problem is illustrated by diabetic neuropathy that affects approximately 350,000 patients in the United States [46]. A large percentage of these patients experience pain at some point in their disease. Neuropathic pain, usually results from both peripheral and central mechanisms [47]. In the periphery it is thought that spontaneous discharges of nociceptive fibers result from dysregulation of sodium channels [48]. Clinically, sodium channel antagonists, such as carbamazipine, have long been used to treat neuropathic pain [49]. Central sensitization may involve a cascade of events, starting with repetitive firing of C fibers that ultimately leads to activation of protein kinase C and phosphorylation of NMDA receptors decorating neurons in the dorsal horn [50]. This results in increased central sensitization that may be further enhanced by the release of ATP, which facilitates glutamate release by activating purine receptors (P2X) in sensory afferents in the dorsal horn. This, along with phosphorylation of the NMDA receptor cumulatively leads to increased calcium influx. Similar, perhaps identical events, are associated with the development of tolerance, which is a major obstacle to the continued use of opioids such as morphine. On this basis Hua et al. [51] targeted spinal cord PKC using intrathecal delivery of an ASO in a rat model of opioid tolerance. After treatment for 5 days, using a 2⬘ MOE PKC ASO, spinal cord PKC protein was diminished approximately 50% and treatment prevented the development of tolerance resulting from chronic administration of morphine. Employing an injury model of pain, Honore et al. demonstrated a reduction in mechanical allodynia in rats treated intrathecally for 7 days with an ASO that targeted the expression of P2X receptors in the spinal cord. In this model the L5-6 nerve roots are traumatized, leading to an exaggerated response to touch that is manifest 1 week postoperatively [52].

26.8 GLIOBLASTOMA Glioblastoma is the most common form of primary brain tumor with glioblastoma multiforme being the most common and malignant of the glial tumors. Few patients diagnosed with glioblastoma multiforme survive longer than 1 year from the time of diagnosis [53]. Over the past 20 years, survival has not improved, thus, current therapies are inadequate. Recent scientific studies have provided valuable insights into the genetic and biological changes that occur in glioblastoma. These studies have identified several potential molecular targets for therapeutic intervention. Unfortunately, other than epidermal growth factor, most targets are not amenable to traditional drug discovery programs. In that ASOs are capable of inhibiting virtually any RNA in the cell, antisensebased therapeutics may be ideally suited for treatment of this disease. Some investigators have placed emphasis on local, direct administration into CNS tissue, presuming that systemic side effects can be avoided. The rationale for local therapy, in our opinion, is problematic since the combination of surgery and radiotherapy are effective in reducing tumor burden and it is the local spread of tumor that ultimately is fatal. Based on current evidence it is presumed that intraventricular administration of an ASO that prevented local, initially microscopic spread of malignant cells, could curtail the inexorable progression of tumor. A number of glioma targets have been studied with the intent of demonstrating their therapeutic relevance. Typically, these have been assessed in vitro in a glioblastoma cell line which has been evaluated either by transfecting cells with vectors that code for an antisense cDNA or by directly treating cells with antisense molecules. Extending this strategy to animal models it is possible to determine the effect of similar therapeutic manipulations following subcutaneous or intracranial injection of treated and control cell lines into nude mice. For example, ASOs that downregulate the

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expression of several growth factors, such as insulin-like growth factor (IGF-I) have moderated tumor growth in vivo [54]. Dysregulation of the epidermal growth factor receptor (EGFR) is noted in about half of the gliomas studied [55]. The receptor is a member of a family of transmembrane glycoproteins that may be overexpressed in some tumors. This has the effect of stimulating tumor growth and invasiveness. Further, the EGFR may be mutated in some tumors leading to a truncated extracellular domain, again enhancing tumorigenicity. Considering its pivotal role in the biology of gliomas, the EGFR receptor has been thought to be a suitable treatment target. Several small molecules and monoclonal antibodies have been developed with the idea of inhibiting phosphorylation of the receptor and the resulting cascade of cellular effects implicated in the malignant process. Several of these therapies are being tested in clinical trials. Relevant to the use of an antisense therapeutic, an RNAi-based therapy targeting the EGFR achieved a marked extension of survival in a murine model of brain tumor [56]. A marked reduction in mortality was observed in mice bearing an experimental glioma following daily intraperitoneal administration of aprinocarsen, an oligo that inhibits PKC. Based on these results a clinical trial was conducted to test the efficacy of the drug in patients with recurrent high-grade astrocytoma [57]. Patients received a continuous intravenous infusion for 21 days (2 mg per kg/day) in repeated cycles. While the treatment was well tolerated, no beneficial effect was seen and, in fact, there was a suggestion that treatment may have accelerated tumor progression, and possibly disrupted the BBB. A phase I/II trial with an antisense molecule directed against TGF-2 has been undertaken in patients with high-grade glioma [20]. Malignancies, including glioma, are known to overexpress TGF-, which may facilitate tumor progression as a result of its effect on metastasis, cell proliferation, and angiogenesis. The drug was administered directly into the tumor and adjacent brain by high-flow micro perfusion. The results have not been published but preclinical studies involving rabbits and subhuman primates have been reported. In normal rabbits, continuous intraparenchymal infusion of AP 12009 at a rate of 1
L/h was tolerated for 7 days without ill effects. On postmortem examination, both leptomeningeal and parenchymal inflammations were noted microscopically. Both lymphocytes and macrophages were observed and phagocytosed material, presumably oligonucleotides, were seen in the latter. In spite of these findings the investigators were reassured about the safety of their method since there were no macroscopically visible changes, concluding that there are no reservations against the local administration of AP120019 in patients with malignant glioma.

26.9 PRION DISORDERS Prion diseases, once thought to be a biological and medical curiosity, have become a serious health concern based on the occurrence of variant Creutzfeldt Jakob disease (CJD) in Britain and elsewhere that has been linked to the consumption of tainted meat and meat products. Further is the concern that the disease may be transmissible via the medical use of blood products [58]. CJD has long been known to be capable of transmission through the transplantation of corneal tissue from affected donors, inadequate sterilization of surgical instruments, etc. Affected persons suffer from an inexorable neurological disorder characterized by mental deterioration, and characteristic encephalographic and pathological (spongiform degeneration) findings. Death ensues rapidly, usually within 1 year. Following infection, as currently understood, a protease resistant form of prion protein (PrPres) recruits the normal cellular protein (PrPsens); the result being that normal protein is converted into the protease-resistant form [59]. Evidence suggests that several sugar polymers can interfere with this process, presumably by binding to PrPsens and thereby interfering with the conversion to PrPres. Reasoning that the efficacy of polyanionic glycans is related to their sulfate moieties, Kocisko et al. synthesized a random assortment of phosphorothioate oligonucleotides and found that they similarly exhibited activity against scrapie. The

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optimum length of the oligomers was found to be 20–40-mer and there was no obvious sequence requirement [60]. Although untested it is assumed that targeting the expression of PrPsens protein would itself be therapeutic since this would reduce the amount of native protein that could be recruited to the disease process. This result is inferred from the fact that knockout animals lacking PrPsen are resistant to experimental infection [61]. Accordingly, several potent PrPsens oligos have been synthesized and are scheduled to be tested for efficacy in a transgenic mouse overexpressing PrPres. These will be administered intraventricularly, initially before experimental inoculation with scrapie and subsequently after inoculation so as to determine whether treatment confers prophylactic protection in the first instance and confers a treatment effect in the second instance. Should this be the case, further studies will be warranted to determine whether the benefit is due to a singular effect, for example, lowering of PrPsens or a combination of effects, including interference due to binding of the oligo to PrPsens. However, this strategy may need revision if a human homologue for Doppel, a second murine prion-like protein is found to be etiologically linked to human prion disease [62,63].

26.10 DEMENTIAS In a clinical context, the term dementia has descriptive value, connoting a general deterioration of mental processes including impairment of memory and reason but the term lacks specificity unless elaborated upon. It would be fair to say that much of the progress made in the nineteenth and twentieth centuries toward understanding the causes of dementia were made through clinical and pathological description. While the classic findings in Alzheimer’s disease of senile plaques and neurofibrillary tangles have been recognized for over 100 years, the molecular makeup of these and other changes in the brain (Lewy bodies) have only recently been characterized. With this advance it has been possible to further refine the classification of the dementias. On this basis, for example, frontotemporal dementia is considered a tauopathy whereas Lewy body dementia is considered a synucleinopathy [64]. The recognition that amyloid plaques are composed of A peptides and that the longest of these are the most likely to aggregate and accumulate in the extracellular space led to the formulation of the amyloid hypothesis [65]. Support for this has come from the finding that mutations in APP, or the enzymes that process APP, are responsible for familial variants of Alzheimer’s disease. Patients with Down’s syndrome who carry an extra copy of the APP gene may develop Alzheimer’s disease at an early age. However, the amyloid hypothesis, as originally conceived, has undergone revision since the presence of neurofibrillary tangles correlates better with the clinical and pathological features of Alzheimer’s disease than the presence of amyloid plaques [66] and amyloid plaques may be seen in nondemented individuals. In its current incarnation the amyloid hypothesis places more emphasis on the toxicity of soluble forms of A, which accumulate early, and maximally, prior to the appearance of clinical features of the disease. By as yet unknown means a cascade of events subsequently ensues with the result that there is inexorable loss of neurons, and synaptic loss accompanied by gliosis and the formation of neurofibrillary tangles, which are characterized by the presence of hyperphosphorylated tau [65]. On this continuum, patients exhibit memory loss, disorientation, and ultimately enfeeblement until they die, usually within 3–6 years from onset. The mainstay of dementia treatment has been the use of drugs that inhibit acetylcholinesterase based on the findings that cholinergic neurons in the basal forebrain are preferentially affected in Alzheimer’s disease [67]. This therapeutic approach, pioneered by William Summers in 1986, has shown modest symptomatic benefit in numerous treatment trials [68,69]. Both cognitive and behavioral performances are ameliorated with treatment and this is associated with lessening of caretaker burden and a delay in nursing home placement.

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Interestingly, a subset of cholinergic neurons appears to preferentially accumulate A 42, leading ultimately to the formation of dense-core amyloid plaques that are most likely due to cell lysis. This may account for the selective vulnerability of cholinergic neurons observed in Alzheimer brains [70]. On this basis it has been suggested that a drug that targeted the 7nAChR might diminish the tendency of cholinergic neurons to take up A42 [70]. With the goal of reducing A, research has focused on identifying drugs that inhibit  or  secretase [71]. Several inhibitors that reduce spinal fluid and brain levels of A have been reported but enthusiasm for this has been diminished by the realization that a number of substrates, including Notch, are processed by  secretase. Unfortunately, efforts to develop a  secretase inhibitor have not been productive, in part because of the difficulty in designing a drug that distinguishes between BACE 1 and other aspartyl proteases and BACE may also process multiple substrates [72]. Also, an immunologic therapy to promote clearance of A 42, remarkably effective in animal studies, was accompanied by the unacceptable occurrence of encephalomyelitis in a small percentage of patients [73]. Potential dementia disease targets that could be amenable to treatment with ASOs or similar strategies are not abundantly expressed in the brain compared to superoxide dismutase. These include APP (0.05% [74]), presenilin 1 (0.0003% [75]), tau (0.01-0.1% [76]), and alpha-synuclein (0.5% [77]), all of which have been implicated in the pathogenesis of one or more of the dementias. Such a treatment strategy is especially appealing for targets that are nonessential genes whose absence does not compromise life span in mice, including APP [78], the BACE protease whose action is required to generate the A peptide in Alzheimer’s disease [79] and tau [80]. In mice, in whom a tau transgene was under control of a doxycycline promoter, improvement in memory was demonstrated when the transgene was suppressed and neuronal numbers and brain weights were similarly increased compared to untreated animals. But interestingly, there was no effect on neurofibrillary tangles [81]. We have identified ASOs that direct degradation of mRNAs encoding target proteins involved in Alzheimer’s disease, including presenilin 1, part of the -secretase complex that processes APP to produce the A peptide [75], and GSK3, a kinase thought to be responsible for the aberrant phosphorylation of tau in intraneuronal tangles [82,83]. Oligonucleotides effective in targeting presenilin 1 or GSK3 mRNAs were identified by screening a series of oligonucleotides in cell culture for inhibition of their respective targets. Intraventricular administration of the most effective of these for 14 days into normal mice substantially reduced the corresponding mRNAs in regions primarily affected in Alzheimer’s disease, including the frontal and temporal cortices (Figure 26.5E).

26.11 NEUROPATHY Neuropathies represent a vast and varied group of disorders ranging from hereditable ones such as Charcot Marie Tooth disease and familial amyloidosis to metabolic ones including diabetic peripheral neuropathy. In the instance of familial amyloidosis, more than 85 mutations of the transthyretin (TTR) gene have been identified. The most prevalent mutation, Val30Met, is found in approximately 5% of the Portuguese population. The familial disease has protean manifestations leading to cardiomyopathy, nephropathy, and neuropathy. Because most of the serum transthyretin produced is of hepatic origin, liver transplantation has been a mainstay of therapy. But disease progression, while slowed, has continued, most likely due to the deposition of wildtype TTR. On this basis an antisense treatment strategy offers the likelihood of being less invasive and more efficacious, in part because systemically administered ASOs are avidly taken up by the liver where they are biologically active. Using transgenic mice containing the entire TTR ILe84Ser coding region and the upstream human promoter, Benson et al. [84] demonstrated a marked reduction in serum TTR over a 6-week course of treatment. Following treatment twice a week with

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subcutaneous injections serum TTR levels dropped over 70% with the most effective ASO. This was dose dependent: each mg increase in dose was associated with a 1–2% reduction in the serum level. Unfortunately, this animal model fails to replicate the human disorder in that characteristic amyloid deposits are not seen despite the fact that serum levels of TTR are double the normal human serum concentration. But considering the results in the animal model and the apparent safety of the therapy one would anticipate that an antisense therapeutic targeting TTR offers the most immediate and promising strategy for treating familial amyloidosis.

26.12 SPINAL MUSCULAR ATROPHY Spinal muscular atrophy (SMA) is the leading hereditable cause of infant mortality, with an incidence of 1 in 10,000 births. In the most severe of the three forms, motor milestones are never reached and children usually die within the first year of life. Type 111 SMA patients are able to walk and may enjoy a normal life span. The gene coding for the motor neuron survival gene (SMN) is located on chromosome 5 and consists of an inverted repeat with a telomeric (SMN1) and a centromeric (SMN2) copy [85]. While both genes code for the identical product, mutations of the SMN1 gene alone lead to SMA. But the severity of phenotype is moderated by the character of the SMN2 transcript, which is subject to differential RNA splicing, leading in most cases to an isoform lacking exon 7. Unfortunately, this isoform cannot adequately compensate for a mutation of the SMN1 gene. The recognition that a single nucleotide substitution accounts for the splicing event that excludes exon 7 has led to the realization that SMA might be favorably treated by a therapeutic strategy that targets the splicing machinery that edits SMN2 expression. Ultimately the editing process is determined by the interplay between the splicing machinery and exonic splicing enhancers (ESEs). In an effort to bias splicing in favor of the inclusion of exon 7, several strategies employing oligonucleotides have been demonstrated in vitro. The most promising of these have incorporated a noncomplementary tail that contains sequences that have the effect of mimicking the function of ESEs [86]. The body of these 2⬘-O-methylphosphorothioate oligos is complementary to the 5⬘ end of SMN exon 7 and the tail component contains GGA repeats. These sequences are known to exhibit enhancer effects, most likely by the recruitment of splicing proteins such as SF2/ASF that are known to bind ESEs.

26.13 MUSCULAR DYSTROPHY Muscular dystrophy, a childhood muscular disorder resulting from mutations of the dystrophin gene, affects 1 in 3500 males [87]. In its severest form muscle weakness and wasting become apparent in infancy, usually about 3 years of age, and by adolescence, affected individuals develop contractures and become wheelchair bound. As the disease progresses inexorably, respiratory and cardiac functions are compromised, leading to death. Mutations of the dystrophin gene in these cases results in the production of a biologically defective protein as a result of nonsense or frameshift mutations [88]. A variant of the disease, the Becker variant, is associated with a milder phenotype because the gene product, while truncated, is still biologically active. Even in its severest form it is apparent that splice variants occur spontaneously in scattered muscle fibers [89]. This is demonstrated in muscle biopsies from affected patients in which rare fibers containing dystrophin can be visualized by histochemistry. In short, nature has provided therapeutic guidance, leading to the notion that a treatment that could bias splicing might have the effect of moderating the severity of the disease process. Considering the size and complexity of the dystrophin gene—greater than 2.3 million base pairs—a conventional gene therapy approach appears to be a daunting challenge. But fortuitously,

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the majority of the mutations in the dystrophin gene occur in the rod domain, which itself is not critical to the function of the protein. Exploiting this a number of researchers have demonstrated that ASOs that lead to exon skipping can enhance synthesis of a functional, albeit truncated dystrophin transcript following direct injection into the muscle of mdx mice, a murine animal model of muscular dystrophy, or following systemic delivery [89,90]. Thus far the effect within muscles and between muscles is variable and the heart has been refractory to treatment, a serious limitation to a promising therapy. Preliminary results in humans confirm the findings in animals, namely, that dystrophin can be upregulated in the muscle of an affected patient but formal clinical trials have not been undertaken [91].

26.14 CONCLUSIONS There is an obvious need for therapies that offer the opportunity to selectively target the expression of any protein in the nervous system. This is particularly compelling since it has become increasingly apparent that a large number of neurodegenerative disorders can be considered proteinopathies. It is paradoxical that we know the exact cause of many of these diseases, most notably those that are caused by mutation but thus far we do not know the cellular basis for any of these conditions. In this context, a therapeutic strategy based on the use of ASOs makes sense because there is good reason to believe that reducing the amount of an offending protein should be of therapeutic benefit. While this treatment strategy has been considered for sometime it has long been believed that it was difficult to target the CNS, first, because the BBB excludes molecules the size of oligonucleotides and second because a number of investigators have reported that oligonucleotides were not well distributed throughout the nervous system after direct instillation into the neural parenchyma or to the spinal fluid bathing the brain. Whatever the basis for these conclusions there is overwhelming evidence that second- and thirdgeneration ASOs readily penetrate the brain substance after intrathecal and intraventricular administrations. What remains to be demonstrated is that such therapies are safe. Such studies are currently being undertaken but irrespective of the outcome it is likely that the ideal chemistry for a molecule intended for neurological use has yet to be identified, most certainly because emphasis has been placed on the development of a universal therapeutic. As the field matures one might speculate that some chemistries might be better suited to one application than another and it will become clear which of the many strategies available to regulate genes will be most suited for clinical use.

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89. Lu, Q. L., Morris, G. E., Wilton, S. D., Ly, T., Artem’yeva, O. V., Strong, P., and Partridge, T. A. (2000). Massive idiosyncratic exon skipping corrects the nonsense mutation in dystrophic mouse muscle and produces functional revertant fibers by clonal expansion. J Cell Biol 148, 985–996. 90. Alter, J., Lou, F., Rabinowitz, A., Yin, H., Rosenfeld, J., Wilton, S. D., Partridge, T. A., and Lu, Q. L. (2006). Systemic delivery of morpholino oligonucleotide restores dystrophin expression bodywide and improves dystrophic pathology. Nat Med 12, 175–177. 91. Takeshima, Y., Yagi, M., Wada, H., Ishibashi, K., Nishiyama, A., Kakumoto, M., Sakaeda, T., Saura, R., Okumura, K., and Matsuo, M. (2006). Intravenous infusion of an antisense oligonucleotide results in exon skipping in muscle dystrophin mRNA of Duchenne muscular dystrophy. Pediatr Res 59, 690–694.

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Mechanisms and Therapeutic Applications of Immune Modulatory Oligodeoxynucleotide and Oligoribonucleotide Ligands for Toll-Like Receptors Jörg Vollmer and Arthur M. Krieg

CONTENTS 27.1 27.2 27.3 27.4 27.5

Introduction .........................................................................................................................747 History of Immune Activation by Synthetic ODN and Identification of the CpG Motif ..748 MOA of CpG ODN and Role of TLR9 ..............................................................................748 Classes of Immune Modulatory ODN and Their Immune Stimulatory Effects.................751 Structure–Activity Relationship of CpG ODN...................................................................753 27.5.1 Characteristics of the CpG B-Class ......................................................................753 27.5.2 Characteristics of the CpG A-Class ......................................................................754 27.5.3 Characteristics of the CpG C-Class ......................................................................754 27.5.4 ODN Lacking CpG Motifs and Their TLR-Dependent Effects ...........................754 27.5.5 Characteristics of the S-Class ...............................................................................755 27.6 Therapeutic Applications of CpG ODN .............................................................................755 27.6.1 Infectious Disease Monotherapy...........................................................................755 27.6.2 Infectious Disease Vaccines ..................................................................................757 27.6.3 Cancer ...................................................................................................................758 27.6.4 Asthma/Allergy .....................................................................................................759 27.6.5 Autoimmunity .......................................................................................................759 27.6.6 Safety of CpG ODN..............................................................................................759 27.7 Identification and Immune Stimulatory Effects of Oligoribonucleotide Ligands for TLR7 and TLR8 ............................................................................................................760 27.8 Conclusion ..........................................................................................................................762 References ......................................................................................................................................762

27.1 INTRODUCTION Most of the therapeutic applications for synthetic oligodeoxynucleotides (ODN) and oligoribonucleotides (ORN) relate to the role of DNA as the genetic blueprint, and to mechanisms of manipulating gene expression based on Watson–Crick base pairing to endogenous nucleic acids. 747

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However, in recent years it has become clear that the immune system has evolved defense mechanisms against infections that are based on the detection of infecting viral/bacterial nucleic acids. In some cases, synthetic ODN and ORN can trigger these defenses. Although this is generally considered to be an unwanted toxicity when the goal is developing a drug based on antisense or siRNA mechanisms, immune stimulatory ODN have recently proven to be promising drug candidates in their own right, and are currently in phase III human trials for several indications, while immune stimulatory ORN are at a much earlier stage of clinical development. The purpose of this chapter is to review this field of therapeutic immune activation by ODN and ORN.

27.2 HISTORY OF IMMUNE ACTIVATION BY SYNTHETIC ODN AND IDENTIFICATION OF THE CpG MOTIF Early during the development of antisense ODN, reports began to emerge on the appearance of strong and unexpected immune stimulatory effects of certain phosphorothioate (PS) ODN that had been designed to be antisense, and also for some control ODN [1–4]. At first, the immune stimulation seemed to be rather unpredictable, and there was no apparent common sequence motif to unify these different observations. While pursuing our own antisense studies, we (AMK) became intrigued by these immune stimulatory effects, and performed a series of experiments to define their structure–activity relationship. Eventually, we discovered that the immune stimulation in each case resulted from the presence of a CpG dinucleotide, in the presence of certain flanking bases on the 5⬘ and 3⬘ sides, which have become known as “CpG motifs” ([5] reviewed in [6]). For example, if a CpG is preceded by a C and followed by a G, the immune stimulation is generally reduced, compared to other CpG motifs. As will be described in greater detail below, the most important base positions for determining the immune stimulatory effect of a CpG dinucleotide are the two bases on its 5⬘ and 3⬘ sides, which comprise the CpG motif. While the CpG motif explained the previously observed immune stimulatory effects of antisense ODN, it raised a new question of whether these effects served any physiological purpose, and if so, what? It is now accepted that immune recognition of CpG motifs allows the immune system to distinguish self DNA from invading viral or bacterial DNAs, which differ markedly from vertebrate DNA in their CpG content and methylation [7]. Bacterial and many viral DNAs generally contain the expected frequency of about one CpG dinucleotide per 16 bases, but CpG dinucleotides are markedly suppressed in vertebrate genomes to about 1/4 of the expected frequency if base utilization was random. Furthermore, the bases flanking CpGs in vertebrate genomes are not random: the most common base preceding a CpG is a C and the most common base following a CpG is a G [8]. As noted, these types of CpG motifs have reduced immune stimulation, so their predominance in our genomes may contribute to the usual lack of immune activation from self DNA. In addition, CpG dinucleotides are not methylated in viral or bacterial DNAs, but in vertebrate genomes, the C of the CpG is usually methylated at the 5 position [7]. The functional effect of DNA methylation is clear—CpG ODN in which the C is replaced with a 5-methyl C have greatly decreased immune stimulatory effects, especially if the ODN backbone is phosphodiester (PO) [5,38]. In the case of ODN with PS backbones the reduction tends to be less complete. Thus, immune recognition of unmethylated CpG motifs functions as a defense mechanism for the detection of invading viruses and bacteria.

27.3 MOA OF CpG ODN AND ROLE OF TLR9 The most important single advance in understanding the mechanism of action of the CpG motif was probably the identification of its receptor, Toll-like receptor 9 (TLR9) [9]. Including TLR9, 10 human TLRs have been identified to date, and function as one family of what have been termed

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“pattern recognition receptors” (PRRs) (reviewed in [10]). PRRs have a general ability to detect certain molecular structures that are conserved in certain pathogens, but are not present or are not accessible to the appropriate PRRs in self tissues. Besides the CpG motif as a ligand for TLR9, examples of such TLR ligands include certain lipopeptides (detected by TLR2), double-stranded RNA (TLR3), endotoxins (TLR4), and flagellin (TLR5). The immune system appears to use the presence of any of these molecular structures as a danger signal that indicates the presence of one general type of infection and activates appropriate defense pathways. Vertebrates have two complementary immune systems, the innate and the adaptive. The primary function of the innate immune system is to detect infection and to initiate an appropriate response. TLRs are thought to play a very important role in this earliest phase of the immune response. The innate immune activation results in the induction of appropriate parts of the adaptive immune system, which is a highly sophisticated and far more specific set of defenses including most notably B and T cells, that specifically target the invader, and provide a memory response to prevent a repeat of the infection. The appropriate immune defense pathways differ for different types of pathogens. At the risk of oversimplifying, if a pathogen replicates extracellularly, then the correct type of immune response to contain the infection is a Th2-like response, which is characterized by the predominant production of cytokines such as IL-4 and IL-5, and by production of antipathogen antibodies. Conversely, if a pathogen replicates intracellularly, the innate immune system should induce a Th1-like immune response, which is characterized by the predominant production of interferons (IFNs) and IL-12, and by cellular responses such as natural killer cells (NK) and cytolytic T cells, to kill infected cells. The family of TLRs is thought to play an important role in the detection and initial classification of infectious agents by detecting these PRRs. The classification of the pathogens as intracellular or extracellular may be assisted by the fact that the innate immune system has compartmentalized TLR expression: TLR2, 4, 5, and 6 are expressed on the cell surface of certain immune cells, where they detect components of extracellular pathogens, while TLR3, 7, 8, and 9 are expressed within the endosomal compartments of some immune cells, where they appear to be looking inward to detect nucleic acid components of intracellular pathogens [11]. The endosomal localization of TLR9 allows efficient detection of invading viral nucleic acids, while preventing accidental stimulation by CpG motifs within self DNA [12]. TLR9 is absolutely required for all known CpG-specific responses to synthetic PS ODN [9,13,14]. Although beyond the scope of this review, it should be noted that one or more TLR9-independent cytosolic pathways of DNA detection has recently been demonstrated, but this pathway appears to be specific for PO DNA, and requires that the DNA is transfected into the cells, and so may not be relevant to investigations using modified ODN [15–18]. TLR9-independent pathways have also been reported for PO DNA vaccines, though they are not yet characterized [19–21]. High concentrations of non-CpG PS ODN also have certain immune stimulatory effects, but these are almost completely TLR9-dependent, and are qualitatively different from the immune effects of a CpG ODN [14]. Each of the TLRs has a unique pattern of cellular expression, which likely enables the immune system to tailor its responses against different pathogen classes [10]. Among resting human immune cells, TLR9 is expressed primarily or exclusively in B cells and in plasmacytoid dendritic cells (pDC), which produce most of the type I IFN that is made in response to viral infection [22] (reviewed in [10]). Some studies have also reported functional TLR9 expression in activated but not in resting human neutrophils [23], monocytes and monocyte-derived cells [24,25], activated CD4 T cells [26], pulmonary epithelial cells [27,28], and intestinal epithelium [29,30]. In some studies, natural killer cells (NK) cells have been reported to express TLR9 and respond directly to CpG ODN [31–33], but in other studies this has not been observed, or they showed no direct response to CpG ODN [34–39]. However, the culture conditions used may not recreate the in vivo setting, in many cases the TLR9 expression was assessed using antibodies of uncertain specificity, and the functional significance of this TLR9 expression has not always been rigorously established. In some cases, the purity of the cells used may have been insufficient to completely exclude effects due to

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contaminating pDC, which are activated even at concentrations of less than 0.1%. For example, we and others [40,41] have found that CpG-induced activation of human monocytes was indirect and dependent on the presence of contaminating pDC and IFN-
. Eosinophils, neutrophils (polymorphonuclear leukocytes [PMN]), and basophils were reported to express TLR9 mRNA, but CpG ODNs in contrast to other TLR ligands such as lipopolysaccharide (LPS) did not induce direct immune modulatory effects in isolated human eosinophils or basophils [42]. To date, the only human cell types that universally and constitutively have been found to express TLR9 and respond to ODN stimulation in a CpG-specific manner are B cells and pDC. The tissue distribution of TLR expression in normal human tissues has been examined using PCR by the codiscoverers of TLR7, 8, and 9. Studies by these and other investigators have concluded that human hTLR9 mRNA has a more limited tissue expression profile than any of the other TLRs: “In contrast to all the other hTLRs, the hTLR9 is preferentially expressed in immune-cell-rich tissue including spleen, lymph node, bone marrow, and peripheral blood leukocytes” [43]. Generally, similar findings have been reported by the other groups that have performed quantitative PCR tissue distribution analyses of the hTLRs, with agreement that TLR9 mRNA is absent or only weakly detectable in adrenal gland, CNS, heart, kidney, liver, lung, pancreas, placenta, prostate, salivary gland, small intestine, spinal cord, testis, thyroid gland, trachea, and uterus [43–47]. Unfortunately, the cellular patterns of TLR expression vary between different species, so the results of TLR stimulation in one species may not be predictive of what will occur in another. For example, mice differ from primates in that they express TLR9 not only in pDC and B cells, but also in monocytes and myeloid DC (reviewed in [10]). This makes it difficult at best to use observations with CpG ODN in murine studies to predict accurately the effects of TLR9 activation in humans. In contrast to hTLR9, hTLR7 and hTLR8 appear to have a broader cell-type-specific expression. TLR7 RNA expression seems to be strongest in human pDC and B cells [48,49], and at least low hTLR7 RNA expression was reported in, for example, monocytes, monocyte-derived DC, mDC, and macrophages [47,48,50–54], although other reports showed lack of TLR7 expression from all or some of these cell types [41,55]. In contrast to hTLR7, hTLR8 RNA was readily observed in monocyte-derived DC, mDC, macrophages, Langerhans cells, or regulatory T cells [48,49,52,54–56], and was maximal in CD14+ mononuclear cells [47,48,57], but could not be detected in pDC and B cells [41,48,49,58]. Therefore, hTLR7 and hTLR9 colocalize in pDC and B cells, whereas hTLR8 and hTLR7 seem to be expressed in myeloid cells. Both receptors appear not be expressed in other cell types such as human NK cells and T cells, although some conflicting reports describe their RNA expression or lack of expression in these cell types [32,37,48,59]. Nevertheless, owing to the lack of appropriate antibodies it is difficult to judge functional TLR7/8 protein expression in the tested cells. In addition to the reported unfunctionality of murine mTLR8 [60], mTLR7 appears to be expressed in a wide variety of cells including murine pDC, CD8
⫺ DC, B cells, regulatory T cells and macrophages [61–63]. TLR7 expression was also observed in CD8
⫹ DC or T cells, although these cells did not respond to TLR7 activation [61,62,64]. In both rodents and humans, administration of a CpG ODN activates pDC to secrete IFN-
, promoting Th1 adaptive immune responses [65]. TLR9-stimulated B cells and pDC show increased expression of costimulatory molecules, resistance to apoptosis, upregulation of the chemokine receptor CCR7, and secretion of Th1-promoting chemokines and cytokines such as MIP-1, IP-10, and other IFN-inducible genes [6]. These effects drive the migration and clustering of pDC in the T cell regions of lymph nodes and other lymphoid tissues. Coactivation of naïve, germinal center, or memory B cells through the B cell antigen receptor and TLR9 can be strong enough to drive their differentiation into antibody-secreting plasma cells [66]. In the case of memory B cells, which have been stimulated previously, activation through TLR9 alone is sufficient to drive differentiation to plasma cells [58,67]. We are unaware of any other single B cell mitogen that is as strong as an optimal B-Class CpG ODN, which has provided applications for CpG in promoting the production of antigen-specific human antibodies. The efficiency of hybridoma generation from purified primary

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human memory B cells is improved from 1–2% without a CpG ODN to 30–100% with the addition of PF-3512676 (formerly known as CPG 7909 or ODN 2006) [68]. CpG-induced plasma cell differentiation does not require T cell help, but its efficiency is enhanced further by interactions with pDC and by B cell receptor (BCR) crosslinking [69]. The net effect of TLR9 activation is to induce Th1-biased cellular and humoral effector functions of innate and adaptive immunity. Since we now know that TLR9 is an intracellular protein, it is not surprising that immune cell stimulation by CpG ODN requires internalization [5]. ODN internalization occurs spontaneously in culture without the need for uptake enhancers or transfection, is temperature- and energy-dependent, and appears to be relatively sequence-independent. Recent studies have shown that transfection of CpG ODN into cells can dramatically enhance certain of their immune stimulatory effects, especially the induction of IFN-
secretion [70]. The mechanism for this effect is not yet clear. Once internalized, CpG motifs appear to induce the relocalization of TLR9 from the endoplasmic reticulum into the endosomal vesicle containing the ODN, leading to the direct binding and recognition of the CpG by TLR9, possibly in a pH-dependent fashion [71,72]. The earliest described step in the CpG-induced signal transduction pathways is the generation of reactive oxygen species, which can be detected within a few minutes [73,74]. These steps lead to the rapid recruitment and activation of the adaptor molecules MyD88, IL-1 receptor-associated kinase (IRAK)-1, IRF-7, and TNF-
receptor activated factor 6 (TRAF6) [72,75–79]. This results in the rapid activation of several mitogen-activated protein kinases (MAPKs) including extracellular receptor kinase (ERK), p38, and Jun N-terminal kinase as well as the IB complex, which pathways converge on the nucleus to alter gene transcription [80–86]. All of these steps can be blocked by inhibitors of endosomal acidification/maturation [73,75,87,88], the mechanism of which are incompletely understood, or by inhibitors of phosphatidylinositol 3 kinase (PI3-kinase), which appears to have a role in the ODN internalization [89]. To date, there has been a paucity of studies examining these questions for TLR7/8, and it is unclear whether the signaling pathways triggered by TLR7 and TLR8 differ in any significant way from those induced by TLR9.

27.4 CLASSES OF IMMUNE MODULATORY ODN AND THEIR IMMUNE STIMULATORY EFFECTS Three different major immune stimulatory classes of CpG ODN were identified that induce diverse immune modulatory profiles: the A-, B- and C-Classes. The earliest identified CpG ODN class, the B-Class, are linear molecules that strongly activate B cells, stimulate the release of Th1like cytokines and chemokines including moderate levels of IFN-
and upregulate costimulatory and MHC molecules on the cell surface of professional antigen presenting cell (APC) [5,90]. In contrast, another class of CpG ODN, the A-Class, forms higher ordered structures that appear to be responsible for the induction of high IFN-
production from pDC [91,92]. Nevertheless, A-Class ODN are surprisingly weak in mediating TLR9-dependent NFB signaling or other TLR9-dependent effects such as pDC maturation and B cell stimulation [91,93]. The third class of CpG ODN, the C-Class, combines the characteristics of the A- and B-Classes and stimulates strong IFN-
production and B cell stimulation [93,94]. The composition of linear CpG motifs (as in B-Class ODN) with sequences forming secondary and tertiary structures (as in A-Class ODN) in the C-Class CpG ODN seems to be critical for the combined activities [93]. CpG A-Class ODN are retained for longer periods in endosomes together with the MyD88-IRF-7 complex [70], and the A- and C-Classes localize to different endolysosomal compartments than the B-Class CpG ODN [90,95]. The formation of secondary and tertiary structures appears to control compartmental retention and intracellular distribution, and results in the triggering of IRF-7-mediated intracellular signaling pathways from early endosomes by the A- and C-Classes leading to their strong IFN-
induction. Human TLR9 triggering induces particularly the release of antiviral and antitumoral Th1 and Th1-like cytokines and chemokines. All type I IFN subtypes, as well as the type I IFN proteins

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IFN- and IFN- are produced upon CpG stimulation [91,96,97]. In addition, the recently described type III IFNs, IL-28A/B and IL-29, that exhibit IFN-like antiviral activity, induce typical IFN-inducible genes and share homology with IFN-
, are stimulated by all three classes of CpG ODN, although to different degrees as observed for type I and II IFNs [97]. A- and C-Class ODN induce highest upregulation of IL-28, IL-29, and IFN-
mRNA, as well as strongest production of type I IFNs [91,93,94,98], whereas the B-Class induces moderate amounts of these IFNs. In contrast to type I and III IFNs, the production of IFN- from human immune cells is weaker, and most probably results from an indirect activation of the IFN- producers NK, NKT and T cells [36,37,99–103]. Additional cytokines exhibiting antiviral and antitumor effects induced by CpGdependent TLR9 stimulation include IL-18 and TNF-related apoptosis-inducing ligand (TRAIL). TRAIL enables monocytes to kill tumor cells and IL-18 augments the cytolytic activity of NK cells [104–106]. In contrast, IL-12 production from human immune cells appears to be weaker compared to murine cells, but nevertheless contributes along with IFN-
and TNF-
to the CpG-mediated cytolytic activities of human NK cells [33,36,92]. CpG stimulation also results in the secretion of IFN-inducible proteins, including OAS, Mx1, MCP-1, or IP-10 [40,98,107]. Although the A- and C-Classes produce highest IFN-
levels, and IP-10 production strongly depends on IFN-mediated monocyte activation, B-Class ODN stimulate levels of IP-10 secretion that are higher than expected from their modest IFN-
induction [108]. This appears to be due to direct stimulation of IP-10 production in TLR9-expressing B cells and pDC, that is synergistically enhanced by IFN-
[40,108]. In contrast to the Th1 and Th1-like cytokines, CpG ODN induce relatively few proinflammatory cytokines such as TNF-
from human immune cells, only consistent IL-6 production from B cells that is lower than LPS-mediated IL-6 is observed upon in vitro CpG stimulation [93,109,110]. When administered to normal humans by subcutaneous injection B-Class CpG ODN induced an increase in serum levels of IFN-
as well as of multiple IFN-inducible genes, whereas serum IL-6 and IL-12p40 were still lower in magnitude compared to increases usually observed in mice upon CpG injection. No statistically significant change for other cytokines such as TNF-
was observed, despite the previously reported strong induction of serum TNF-
and IL-6 in various rodent studies [6,111,112]. The immune effects reported in humans appear to correlate with the observed in vitro effects, and suggest that the cytokine-mediated toxicities observed in rodents are unlikely to occur in humans, presumably due to the species- and cell-specific differences in TLR9 expression. CpG stimulation also induces the production of negative regulators of the CpG response dependent on the ODN class [113]. For example, IL-10 is secreted by murine macrophages and murine and human B cells upon CpG B- and C-Class stimulation [93,114]. The CpG-induced IL-10 negatively affects pDC IFN-
secretion perhaps functioning to limit an otherwise potentially dangerous IFN-mediated immune response [115–117]. Besides the CpG-dependent stimulation of cytokines and chemokines, CpG ODN also induce an enhanced expression of activation markers, cytokine and chemokine receptors, costimulatory and MHC molecules on several immune cell types including antigen-presenting cells that are important for triggering adaptive immune responses. Such molecules include the early activation marker CD69 on NK, NKT, and T cells, the costimulatory molecules CD80 and CD86 (B7-1 and B7-2) on B cells, monocytes, mDC or pDC that provide an important costimulatory signal to T cells, MHC I and II molecules on DC, monocytes or B cells leading to enhanced antigen-presentation and antigen-specific T cell responses [32,93,98,99,103,109,118,119]. In contrast to the CpG A-, B-, and C-Classes that efficiently trigger diverse TLR9-mediated stimulatory effects, inhibitory ODN sequences were identified that block TLR9-dependent activation. The mode of action of these suppressive ODN (S-Class) is incompletely understood, but appears not to be due to competition for uptake of immune stimulatory DNA [120–122]. S-Class ODN may directly interfere with binding of the CpG ligand to TLR9 and can contain defined sequence motifs selectively acting not only on TLR9 but also on TLR7 and TLR8 [98,123,124]. The identification of ODN selectively suppressing TLR activation results in a tool to specifically inhibit, modulate, or tailor TLR-mediated immune responses. S-Class ODN not only block

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pathogen DNA- or RNA-mediated stimulation, but also suppress immune stimulatory effects mediated by self DNA and RNA [123–127]. This is especially of interest as TLR signaling appears to be involved in autoimmunity. In autoimmune responses to DNA-containing chromatin–IgG or RNA-containing snRNP–IgG complexes TLR9 or TLR7 act in concert with Ig receptor engagement to promote autoreactive B cell or pDC activation that can be blocked by S-Class ODN [123,126,127]. Moreover, S-Class ODN were demonstrated in vivo to be effective as a treatment for rheumatoid arthritis or lupus in animal models [128,129]. These data link TLR activation to disease development, and, therefore, S-Class ODN may represent promising candidates for suppressing or limiting inflammatory responses in therapeutic indications such as systemic lupus erythematosus (SLE), where TLRs may drive inappropriate and pathogenic immune responses to self nucleic acids and their associated proteins.

27.5 STRUCTURE–ACTIVITY RELATIONSHIP OF CpG ODN 27.5.1 Characteristics of the CpG B-Class The activity of CpG ODN is determined by their sequence composition including the CpG motif(s), the number of these motifs, their spacing, position, and the surrounding bases, as well as the ODN length and the secondary and tertiary structure. B-Class CpG ODN are linear molecules containing 6mer CpG motifs with 5⬘-GTCGTT-3⬘ representing the human CpG motif, and 5⬘-GACGTT-3⬘ the murine CpG motif, reflecting the species-specific differences in CpG recognition [5,80,90,110]. The most potent B-Class ODN for activating human immune cells usually have two or three CpG motifs preceded by a 5⬘-TC and are between 20 and 26 nucleotides in length [6,14,110,130]. A 5⬘-TCG induces strongest immune effects, and the more 3⬘ the first CpG dinucleotide is positioned, the less stimulatory the B-Class ODN is: ideally the first CpG should be within two or three bases of the 5⬘ end that is critical for immune stimulation [110,130,131]. The CpG motifs are preferably spaced with at least two intervening Ts, and the overall T content of an ODN has a strong positive impact on immune modulation [14,110,132,133]. Chemical modifications positively or negatively affect the activity of B-Class CpG ODN. A PS backbone modification stabilizes CpG ODN against nuclease degradation and enhances their B cell stimulatory activity by about 10- to 100-fold compared to PO ODN [134], although this may be associated with some relative decrease in induction of IFN-
secretion. In principle, the most stimulatory CpG sequence in a PO backbone is also most stimulatory with a PS backbone [121]. [Rp] diastereoisomer PS CpG ODN even evoke higher cell activation than the corresponding [Sp] ODN, suggesting that the P-chirality impacts the CpG-mediated activity [135,136]. In addition to backbone modifications, nucleobase modifications of the CpG dinucleotide(s) strongly affect the outcome of the TLR9dependent response [137–139]. Most cytosine modifications in CpG dinucleotides do cause a strong decrease to loss of immune stimulatory effects. It appears as if both the primary exocyclic amino group as well as the spatial requirements at C5 of cytosine are very important for the immune modulatory effects stimulated by CpG ODN via TLR9. In contrast, TLR9 appears to be more forgiving to modifications at the guanosine position. The recognition of the guanine base in the CpG motif appears to be primarily determined by the N7 and exocyclic O6 functions, meaning that guanine is recognized from the Hoogsteen base pairing site by TLR9. In addition, receptor recognition of CpG ODN appears to require a DNA-like rather than a RNA-type sugar conformation at the CpG motif: 2⬘ modifications or locked nucleic acids (LNA) at the CpG dinucleotides reduce or eliminate the immune stimulatory effects of CpG ODN [139–142]. Nevertheless, certain 2⬘ substitutions at positions distal from the CpG motif increase or decrease the immune modulatory response, although depending on the position, the kind, and number of modifications [143,144]. In addition to the species-specific differences observed for TLR9 CpG motif recognition, additional data generated by comparing human and mouse TLR9 signaling stimulated by ODN with chemical

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modifications at the CpG indicate that the spatial requirements at the mouse receptor differ substantially from those of the human receptor [138]. Some CpG modifications that were reported not to alter immune effects observed in murine in vitro and in vivo models appeared to affect signaling induced via human TLR9. Therefore, the use of mouse models for ranking CpG ODN for human use appears to be of limited value. 27.5.2 Characteristics of the CpG A-Class In contrast to B-Class ODN, A-Class ODN form higher ordered structures and contain PS linked G runs at the 5⬘ and 3⬘ ends surrounding a PO palindromic CpG sequence [90–92]. These G residues and the palindromic core sequence are responsible for the formation of intermolecular G tetrads and highmolecular-weight aggregates. Preventing the formation of higher ordered structures by introducing 7-deaza-guanosine nucleotides or destroying the palindrome prevents IFN-
production indicating that the G tetrads and the palindrome both are essential for the A-Class activity [91,145]. A-Class CpG ODN with palindromic 5⬘-purine-purine-C-G-purine-pyrimidine-C-G-pyrimidine-pyrimidine-3⬘ or 5⬘-purine-pyrimidine-C-G-purine-pyrimidine-3⬘ sequences are remarkably strong inducers of type I IFN secretion from human pDCs [91,145]. As observed for the B-Class, the immune modulatory effects of A-Class ODNs are influenced by their sequence, length, and chemical modifications. Nucleobase modification of the guanosine is relatively well tolerated, but cytosine modifications usually result in a strong decrease and loss of type I IFN induction [91,146]. In contrast to the B-Class, the A-Class strongly depends on the presence of a chimeric PO/PS backbone. Complete PS A-Class ODN in principle lack the capability to induce strong IFN-
production from pDCs [91,92]. 27.5.3 Characteristics of the CpG C-Class C-Class CpG ODN combine the A- and B-Class characteristics and stimulate strong B cell and pDC type I IFN responses [93,94]. Similar to the B-Class, typical C-Class ODN preferably contain a linear stimulatory hexameric CpG motif 5⬘-GTCGTT-3⬘ positioned near the 5⬘ end, preferably adjacent to a 5⬘-TC and linked by a T spacer to a GC-rich palindromic sequence [93,98]. A wide range of modifications that maintain the GC-rich palindrome are well tolerated, but full immune activity requires physical linkage between the palindrome and the 5⬘ stimulatory sequence. The palindromic sequence similar to the A-Class appears to be involved in the formation of higher ordered structures that affect stability, uptake characteristics, and intracellular localization [147]. Indeed, C-Class ODN were demonstrated to be taken up in early as well as late endosomes, whereas B-Class ODN mainly reside in the late, and A-Class ODN in the early endosomes, suggesting that different signaling pathways are stimulated from these compartments leading to the differential CpG class effects [95]. The stimulatory capacities of C-Class CpG ODN are dependent on the length and the base content and are influenced by chemical modifications. C-Class ODN similar to the B-Class require unmodified 5⬘ CpG dinucleotides, modifications such as 5-methylation of the cytosine result in a strong reduction of the immune modulatory activity [93,94,98]. In addition, the C-Class requires a DNA-type sugar conformation for the CpG dinucleotides in the 5⬘ stimulatory sequence. In contrast, 2⬘ modifications in the 3⬘ palindromic sequence are allowed and do not strongly affect immune stimulatory activity [98]. 27.5.4 ODN Lacking CpG Motifs and Their TLR-Dependent Effects CpG-mediated B cell stimulation appears to be accomplished not only by CpG ODN but even appears to be achieved by PS thymidine-rich non-CpG ODN, although such ODN fail to induce detectable type I IFN production in pDC, despite promoting pDC maturation, as assessed by expression of costimulatory markers [14,132,133]. Thymidine-containing non-CpG ODN, and even more

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so non-CpG ODN containing a 5⬘-TC motif stimulate TLR9-dependent effects, although with lower efficiency compared to unmodified CpG ODN [14,148]. They not only lack stimulation of Th1 and Th1-like cytokines and chemokines, but induce Th2-biased responses in vivo [14,149]. In addition, Th2-dominated effects are predominantly observed upon mucosal ODN application, and appears to be associated with the PS backbone, but is shifted to Th1 by CpG motifs [149]. Therefore, it appears as if TLR9 can mediate either Th1- or Th2-dominated responses with varying efficacy depending on whether it is stimulated by CpG or non-CpG ODN, and the route of administration. An additional important factor determining the activity of non-CpG ODN appears to be the intracellular localization and the amount of ODN delivered to the endosomal compartments. Enforced uptake by using a cationic lipid results in a several fold higher accumulation of ODN in LAMP-1-positive late endosomes [15], the same intracellular vesicles found to be the primary site of CpG A-Class localization [95]. Localization to LAMP-1-positive endosomes appears to be a prerequisite for IFN-
induction, and by introducing high amounts of TLR9 ligands with low affinity, e.g., certain non-CpG ODN, a threshold ligand concentration in late endosomes may be reached that allows for some degree of IFN-
induction even by non-CpG ODN. 27.5.5 Characteristics of the S-Class Certain sequences in PS ODN efficiently block TLR9-dependent effects. Inhibitory sequences in S-Class ODN do not contain classical CpG motifs, but sometimes differ only slightly from stimulatory motifs (e.g., 5⬘-GCGGG-3⬘ instead of 5⬘-GCGTT-3⬘) [120,121,150]. The identified murine suppressive motif is 5⬘-CCNnotCnotCNNGGGN-3⬘, where N is any base, and 5⬘-NCCNGGGN-3⬘ represents the human suppressive motif [98,120], and the activity of S-Class ODN depends on the presence of the consecutive 3⬘ Gs [98,121,124,151]. In addition, PS S-Class ODN exist that specifically interfere with the activation of either one of TLRs 7,8,9. PS ODN inhibiting TLR8-mediated signaling by the synthetic TLR7/8 ligand R-848 require a TLR8 suppressive motif comprising a 3⬘ GT dinucleotide [98], and ODN with a TLR7 suppressive 5⬘ GC motif inhibit R-848 mediated and TLR7-dependent IFN-
production [124], although the PS backbone itself appears to interfere with TLR7-mediated immune modulation [98,138]. In summary, fine-tuning of the immune modulatory profiles of A-, B-, C-, or S-Class ODN is possible by introducing appropriate modifications including sequence mutations, alteration of the number and position of CpG motifs, chemical modifications inside and outside the CpG dinucleotides, or introduction of secondary and tertiary structures, allowing targeted generation of ODN for different therapeutic indications such as cancer, infectious disease, allergy, and autoimmune disease.

27.6 THERAPEUTIC APPLICATIONS OF CpG ODN 27.6.1 Infectious Disease Monotherapy Since the biologic function of TLR9 appears to be to stimulate protective immunity in response to infection by intracellular pathogens, we and others [152–157] have hypothesized that prophylactic or therapeutic treatment with a CpG ODN would protect against an intracellular infectious challenge and eliminate a chronic infection. Indeed, studies in mice have demonstrated that the innate immune defenses activated by B-Class CpG ODN (almost no studies have been reported with A- or C-Class) given by injection, inhalation, or even by oral administration can protect against a wide range of viral, bacterial, and even some parasitic pathogens, including lethal challenge with Category A agents or surrogates such as B. anthracis, vaccinia virus, F. tularensis, and Ebola, as well as more common pathogens such as L. monocytogenes, M. tuberculosis and influenza virus [152–169]. The mechanisms of protection have only been partially investigated. Protection in an L. monocytogenes model has been linked to CpG-activated DC, which protects

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naïve mice upon adoptive transfer [170–172]. Additional cell types may also be able to provide some protection, since naïve mice that received CpG-pretreated spleen cells depleted of CD11c+ DC still had a partial survival benefit. In a herpes simplex virus challenge model mice depleted of pDC no longer were protected by CpG pretreatment and IFN-
was also needed, since mice genetically deficient in the type I IFNR were no longer fully protected [173]. However, in a L. monocytogenes challenge model type I IFN were not required for CpG-induced protection, even though the protection was abolished when pDC were depleted [174]. In this and many other animal models IFN- was found to be critically required for the CpG-induced protection. Postexposure therapy with TLR9 activation is generally ineffective against rapidly progressive acute infectious agents. However, there may be a role for TLR9 activation in the therapy of chronic viral infections, since HBV transgenic mice treated with a CpG ODN showed a significant decrease in viral expression [175]. As hepatocytes normally do not express TLR9, the antiviral effect in this model is presumably indirect. HBV expression was not suppressed in mice genetically deficient in the type I IFN receptor, suggesting that the antiviral effect of CpG therapy in this model results from the CpG-induced IFN-
secretion, presumably by pDC. Hepatitis C virus is an important human pathogen that chronically infects approximately 170 million people worldwide. Infection can lead to liver cirrhosis and death, and is currently the major cause of liver failure requiring transplantation in North America. Fewer than half of North American patients respond to the current standard of care treatment for HCV, which consists of 48 weeks of a combination therapy with IFN-
and ribavirin. In up to 20% of acutely infected HCV patients, the immune system is able to clear the infection without specific therapy. This spontaneous viral clearance is associated with early and strong innate immune activation leading to the development of a strong and diverse adaptive immune response with anti-HCV Th1 and CD8 cytolytic T cells [176]. Since TLR9 activation can drive a similar pattern of innate and adaptive immune responses to that seen in the spontaneous resolvers, we investigated whether a C-Class CpG ODN, CPG 10101, may have activity against HCV. In a 4 week phase Ib blinded randomized controlled trial involving 60 HCV-infected subjects, monotherapy with once or twice weekly subcutaneous injection of CPG 10101 caused a dose-dependent decrease in blood viral RNA levels [177]. At the highest dose level of 0.75 mg/kg weekly, there was up to a 1.6 mean log reduction in viral RNA, which was associated with biomarkers for TLR9 activation, including NK cell activation and serum IFN-
and IFN-inducible chemokines. Treatment was generally well tolerated, with the most common side effects being mild to moderate flu-like symptoms and injection site reactions, and the maximal tolerated dose was not reached. This trial showed encouraging anti-HCV activity for CpG 10101 as a monotherapy. Based on in vitro studies showing that exogenous IFN-
primes human PBMC for stronger responses to CpG [178] and other studies suggesting synergy between TLR9 activation and ribavirin, we decided to perform a clinical trial using the CPG 10101 in a combination regimen, together with the conventional, partially effective therapy of pegylated IFN and ribavirin. The randomized phase Ib clinical study enrolled 74 evaluable genotype 1 patients chronically infected with Hepatitis C virus. All subjects had previously received at least 24 weeks of treatment with the standard of care (pegylated IFN and ribavirin), and achieved viral negativity, but had subsequently relapsed within 6 months of treatment. Patients in the study were randomly assigned to one of five groups, receiving 12 weekly doses of: CPG 10101 alone, CPG 10101 in combination with pegylated IFN, CPG 10101 with ribavirin, CPG 10101 with pegylated IFN and ribavirin, or pegylated IFN and ribavirin. CPG 10101 was administered by subcutaneous injection at a dose of 0.2 mg/kg once weekly. Patients who achieved a greater than 2 log (⬎99%) reduction in HCV RNA were eligible to continue on therapy for a total of 48 weeks and be followed for an additional 24 weeks to monitor for sustained virological responses. At 12 weeks, 50% (7 of 14) of treatment-refractory patients in the CPG 10101–pegylated IFN–ribavirin arm of the study achieved HCV RNA of undetectable levels, or viral negativity, versus only 2 of 13 of those patients who received pegylated IFN and ribavirin alone ( p ⫽ 0.050). The triplet combination of CPG 10101 with pegylated IFN and ribavirin resulted in a 3.3 mean log reduction in HCV RNA levels, versus a 2.3 mean log reduction

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( p ⬍ 0.050) among patients receiving the control combination. The CPG 10101 combinations were generally well tolerated. Adverse events were similar to pegylated IFN and ribavirin treatment and were predominantly mild to moderate in intensity and consisted of flu-like symptoms, headache, and injection site reactions. CPG 10101 recently received fast track status from the FDA for the therapy of these treatment refractory patients, and is currently being evaluated in a phase II trial. 27.6.2 Infectious Disease Vaccines CpG ODN have become well established as a gold standard vaccine adjuvant, capable of inducing powerful antigen-specific antibody and Th1 cellular immune responses in many vertebrate species, including humans. The range of vaccines in which this has been demonstrated include peptide or protein antigens, live or killed viruses, dendritic cell vaccines, autologous cellular vaccines, and polysaccharide conjugates. The strong vaccine adjuvant activity of CpG ODN probably results from the following factors: (i) synergy between TLR9 and BCR preferentially stimulates antigen-specific B cells [5]; (ii) inhibition of B cell apoptosis [179]; (iii) enhanced IgG class switch DNA recombination [180–182]; and (iv) DC maturation and differentiation, resulting in enhanced activation of Th1 cells and strong CTL generation, even in the absence of CD4 T cell help [183,184]. CpG ODN show even greater adjuvant activity when formulated or coadministered with other adjuvants or in formulations such as microparticles, nanoparticles, lipid emulsions, or similar formulations [185]. In humans, CpG ODN have been used as adjuvants for hepatitis B vaccination either in combination with alum [186] or alone [187]. In a randomized, double-blind controlled phase I/II dose escalation study, healthy individuals received three intramuscular (IM) injections (using the FDA-approved vaccination regimen of 0, 4, and 24 weeks) of an alum-absorbed HBV vaccine either in saline or mixed with a B-Class ODN, CPG 7909, at doses of 0.125, 0.5, or 1.0 mg [186]. HBsAg-specific antibody responses (anti-HBs) appeared earlier and had higher titers at all time points from 2 weeks after the initial prime up to 48 weeks in CPG 7909 recipients compared to those individuals who received vaccine alone. Moreover, most of the subjects who received CPG 7909 as adjuvant developed protective levels of anti-HBs IgG within just 2 weeks of the priming vaccine dose, compared to none of the subjects receiving the commercial vaccine alone [186]. The addition of the CpG ODN also improved the quality of the antigen-specific antibody response, with an increased proportion of high-avidity antibodies [188]. The ability of CPG 7909 to accelerate seroconversion has also been demonstrated when used as an adjuvant to the approved anthrax vaccine in a randomized controlled trial in healthy volunteers. Control subjects reached their peak titer of toxin-neutralizing antibody at day 46, but this titer was achieved in the subjects receiving CPG 7909 already at day 22, more than 3 weeks earlier [189]. More rapid seroconversion to the anthrax toxin could be of great importance in the setting of a bioterror attack. Furthermore, the addition of CPG 7909 induced a statistically significant 8.8-fold increase in the peak titer of toxin-neutralizing antibody, and increased the proportion of subjects who achieved a strong IgG response to the anthraxprotective antigen from 61 to 100% [189]. These results indicate great potential for TLR9 agonists as vaccine adjuvants. Certain populations are hypo-responsive to vaccination, especially immune-suppressed individuals such as those infected with HIV. A randomized double-blind controlled trial in HIV-infected humans who previously had failed to respond to Engerix-B® alone demonstrated that addition of CPG 7909 to the vaccine significantly enhanced both the mean titers of anti-HBs and the antigenspecific T cell proliferative response [190]. The proportion of HIV patients who had seroprotective levels at 12 months following vaccination was increased from 63% in the controls to 100% in the group receiving CPG 7909 [190]. One of the limitations in vaccine development is the cost of antigen production, especially for vaccines such as the flu vaccine that have to be produced in large quantity in a short time frame. The use of a CpG ODN as vaccine adjuvant in mice enables the antigen doses to be reduced by approximately two orders of magnitude, with comparable antibody responses to the full-dose vaccine without CpG [191]. In a phase Ib randomized, double-blind

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controlled clinical trial, subjects vaccinated with a one-tenth dose of a commercial trivalent killed split influenza vaccine (Fluarix®) had reduced levels of antigen-specific IFN- secretion from restimulated PBMC compared to those measured in PBMC from subjects vaccinated with the full-dose vaccine alone [192]. However, the coadministration of CPG 7909 with the one-tenth dose of Fluarix restored the antigen-specific IFN- secretion to the level seen with full-dose vaccine [192]. This suggests that addition of a CpG ODN to a flu vaccine could enable the effective use of the vaccine with lower antigen doses. 27.6.3 Cancer When used as a monotherapy, CpG ODN have antitumor activity in many mouse models (reviewed in [193]). In relatively small tumors, CpG monotherapy can be sufficient to induce a T cell-mediated rejection of established tumors, but to induce rejection of larger tumors the CpG ODN often needs to be combined with other effective antitumor strategies, such as monoclonal Ab, radiation therapy, surgery, and chemotherapy. Monotherapy with the human-optimized CpG ODN PF-3512676 (formerly known as CPG 7909) has induced objective tumor regressions in patients with advanced renal cell carcinoma, melanoma, cutaneous T cell lymphoma, and late regressions in non-Hodgkin’s lymphoma. PF-3512676 and a different B-Class ODN have been shown to induce a Th1-like cytokine response in lymphoma patients treated with the CpG ODN alone or in combination with an antitumor antibody [112,194]. For improving the response rates to TLR9 activation therapy we have been investigating various combination therapy approaches that may show synergy. Surprisingly, chemotherapy combinations with CpG ODN have shown substantial survival improvements in mouse tumor models using chemotherapy regimens ranging from the topoisomerase I inhibitor, topotecan, to the alkylating agent cyclophosphamide and the antimetabolite 5-fluorouracil [195–197]. These combination approaches appear to promote the development of an antitumor T cell response capable of controlling the tumor and improving survival. Therefore, we investigated the effect of adding the PF-3512676 to standard taxane/platinum chemotherapy for first-line treatment of stage IIIb/IV nonsmall cell lung cancer in a phase II randomized controlled human clinical trial. 112 chemotherapy-naïve patients were randomized to receive four to six 3-week cycles of standard chemotherapy alone or in combination with 0.2 mg/kg subcutaneous PF-3512676 on weeks 2 and 3 of each cycle. The primary end point for the trial, response rate (by response evaluation criteria in solid tumors [RECIST], intention-to-treat), was significantly improved ( p ⬍ 0.05) from 19% in the patients randomized to standard chemotherapy to 37% in the patients who also received PF-3512676 [198]. The secondary end point of this trial, survival, shows a trend to improvement from a median survival of 6.8 months in the chemotherapy arm, versus 12.3 months in the combination arm and an improvement in the 1 year survival from 33 to 50% [198]. As in the other clinical trials with TLR9 agonists, the most common side effects were mild to moderate injection site reactions and transient flu-like symptoms. Grade 3 or 4 neutropenia was more common in the combination arm, and is thought to reflect neutrophil redistribution, but febrile neutropenia and grade 3/4 infections were actually slightly less common in the combination arm than in the chemotherapy alone arm. Thrombocytopenia, a PS-backbone effect that has occurred in many trials of antisense ODN, was seen more commonly in the combination arm, but there was no apparent increase in bleeding events. Based on these encouraging results, two phase III human clinical trials of this regimen were initiated by Pfizer in late 2005. CpG ODN have also found application as adjuvant to human cancer vaccines. In a small phase I tumor vaccine trial using a 1 mg dose of CPG 7909 as adjuvant to recombinant MAGE-3 tumor antigen for triweekly vaccination in six patients with metastatic melanoma, there were two stable disease and two partial responses beginning after 7–10 vaccinations, and lasting at least 1 year by RECIST [199]. In eight melanoma patients CPG 7909 at a dose of 0.5 mg stimulated strong and rapid CD8 T cell responses to a Melan-A tumor peptide antigen when used with Montanide as a

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cancer vaccine adjuvant [200]. GlaxoSmithKline is currently performing several human clinical trials using CPG 7909 as one component in a breast cancer vaccine (phase II) and a prostate cancer vaccine (phase I). 27.6.4 Asthma/Allergy Allergic diseases such as asthma result from an inappropriate Th2 immune response against harmless environmental antigens. Because the Th1 type of immune response triggered through TLR9 tends to oppose Th2 immunity, there has been much interest in exploring the application of CpG ODN for allergy/asthma immunotherapy. The Th1-biased immune effect of CpG ODN have dramatically improved the efficacy of allergy vaccines in mouse models, even in mice with established allergic disease [201,202]. A conjugate of a CpG ODN to a portion of the ragweed allergen has been evaluated in human clinical trials as an allergy vaccine, with encouraging evidence for a selective and specific redirection of the allergic Th2 response toward a nonallergic and noninflammatory Th1 response, and showing significant clinical benefit with reduced allergic symptoms [203,204]. Mouse and primate models also support the development of CpG ODN as a monotherapy for allergic diseases. This may seem counterintuitive, since these diseases are inflammatory, and TLR9 activation can induce powerful inflammatory effects. However, there are many types of inflammatory responses, and the Th1 type of inflammation that is induced through TLR9 antagonizes the Th2 type of inflammation that prevails in the allergic subject. In addition, TLR9 activation triggers counterregulatory pathways that feedback to reduce local and systemic inflammation, through mechanisms such as the systemic expression of IL-10 or TGF-, and pulmonary expression of indoleamine (2,3)-dioxygenase IDO [205,206]. Inhaled CpG ODN monotherapy given repeatedly can prevent or treat allergic airway responses not only in mouse models [207] but also in primates [208]. One CpG ODN given by inhalation to allergic subjects induced a Th1 immune activation pattern in the airways, but without any obvious clinical benefit, suggesting a need for further optimization of the ODN design (the ODN was not optimized for activating human TLR9), or dose and schedule [209]. The approach of a CpG ODN as monotherapy for asthma/allergy is undergoing further clinical development by sanofi-aventis. 27.6.5 Autoimmunity Recent studies have implicated inappropriate activation of TLR9 by endogenous DNA and DNA–protein complexes in the generation of autoimmune anti-DNA antibodies, and in the pathogenesis of systemic lupus erythematosus and rheumatoid arthritis [126,210–213]. More recently, we and other groups [123,124,127,214] have reported similar findings for TLR7/8: certain endogenous RNAs and RNA protein complexes can stimulate TLR7/8, leading to the development of autoimmune responses against RNA containing self antigens. The results of these studies offer a new direction in targeting TLR7/8/9; they suggest that TLR7/8/9 antagonists may be useful in the treatment of these autoimmune diseases, by blocking this inappropriate activation of B cells and pDC. Indeed, in mouse models, suppressive ODN (S-Class) designed to block TLR9 have shown substantial benefit in preventing or reversing both systemic lupus erythematosus and rheumatoid arthritis [124,128,129]. 27.6.6 Safety of CpG ODN Even if they do not contain a CpG motif, all PS ODN can have a variety of sequenceindependent backbone-related effects that have been characterized in detailed studies of antisense ODN [215–217]. These effects are most prominent in rodents, which show on chronic dosing of ODN dose-dependent mononuclear cell infiltration in the organs of ODN deposition [215,218], and largely depend on TLR9 [108,215]. Such changes have not been described in monkeys or humans.

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Presumably these species-specific findings are a consequence of the cellular pattern of TLR9 expression, which determines the cytokines that will be produced in response to administration of a CpG ODN, and thus the safety profile of the drug. Since TLR9 is expressed in a broader range of immune cells in rodents compared to primates, the rodent tends to overpredict toxicities that will occur in primates. For example, rodents respond to CpG ODN administration with high serum concentrations of proinflammatory cytokines such as TNF-
, which can result in a lethal cytokine storm [219] but in humans and primates there is no change in serum TNF-
following CpG injection, which is generally well tolerated [111]. In terms of mechanism of action-related effects, CpG ODN treatment clearly can exacerbate autoimmunity in mouse models of lupus [220], multiple sclerosis [221], colitis [222], and arthritis [223]. However, CpG ODN protect against autoimmunity and inflammatory diseases in other murine experimental systems, through mechanisms ranging from induction of IFN- secretion to expression of IDO [206,224–226]. The safety profile of several TLR9 agonists in man has been observed in the clinical trials described above over a more than 1000-fold dose range from 0.0025 to 0.81 mg/kg. A maximal tolerated dose in humans has not been reported to date. The primary adverse events are dosedependent local injection reactions (e.g., erythema, pain, swelling, induration, pruritus, or warmth at the site of injection) or systemic flu-like reactions (e.g., headache, rigors, myalgia, pyrexia, nausea, and vomiting), and are consistent with the known TLR9 agonist mechanism of action. Depending on the dose, systemic symptoms typically appear within 12–24 hours of dosing and persist for 1–2 days. At the low doses used in vaccine trials there appears to be a slight increase in the frequency of injection site reactions, which are generally mild, above the frequency observed with the vaccine alone. So far there are no subjects who have been reported to develop an autoimmune disease following CpG therapy, but the duration of therapy has usually been less than 6 months; only a few patients have received chronic therapy with CpG ODN for longer than 3 years. Definite conclusions on the safety of chronic TLR9 activation with CpG ODN await the completion of clinical trials involving larger numbers of patients followed for longer periods of time.

27.7 IDENTIFICATION AND IMMUNE STIMULATORY EFFECTS OF OLIGORIBONUCLEOTIDE LIGANDS FOR TLR7 AND TLR8 Vertebrate TLRs are currently assigned to six major TLR subfamilies that are dominated by their evolutionary pressure to maintain the recognition of a specific class of pathogen-expressed molecules [227]. Examples are the TLR family recognizing pathogen-specific lipopeptides consisting of TLR1, TLR6, and TLR10, the family sensing double-stranded RNA currently encompassing TLR3, and the family responding to stimulation with single-stranded pathogen DNA and RNA with TLR7, TLR8, and TLR9 [227,228]. The members of the TLR9 family share high sequence homologies, sense pathogen-derived nucleic acids that are generated during intracellular infections and virus replication, and trigger signaling cascades involving similar signal transduction molecules dependent on MyD88. In contrast to TLR9 that recognizes bacterial and viral single-stranded DNA, the single-stranded genomes of RNA viruses such as influenza, vesicular stomatitis virus, Sendai virus, and HIV trigger endosomal recognition and production of type I IFN dependent on TLR7 [229–232]. The first ligands identified to induce TLR7-dependent immune modulatory effects included synthetic antiviral guanosine derivatives, as well as imidazoquinolines that stimulate TLR7- and TLR8-dependent signaling [60,233–235]. Although TLR7 is activated selectively by the guanosine analogue loxoribine, and the imidazoquinoline derivative Resiquimod (R-848) activates both TLR7 and TLR8, the combination of loxoribine or R-848 with a thymidine-rich PS ODN completely abolished TLR7-dependent signaling, but redirected activity to TLR8-mediated effects [138]. The unexpected effect of combining a single-stranded DNA with a TLR7 ligand to induce TLR8

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signaling reveals a plasticity in the ligand specificities of TLR7 and TLR8, probably explained by the homologies between the TLR9 family members, and suggests a sequence-selective interaction between these receptors and synthetic PS ODN. The identification of nucleic acid-like structures such as loxoribine triggered the search for natural ligands of TLR7 and resulted in the observation that only the mixture of the unmodified RNA nucleosides uridine and guanosine resulted in the stimulation of human peripheral blood cells [236]. Furthermore, the immune modulatory effects of single-stranded viral RNA was mimicked by synthetic ORN containing uracil and guanosine demonstrating that single-stranded RNA motifs rich in these nucleotides function as physiological pathogen-derived ligands for TLR7 and TLR8 [231,236]. Nevertheless, TLR7 reportedly is also stimulated by small interfering RNAs lacking high GU content besides the reported GU-containing siRNAs [57,237], suggesting that other RNA sequences are also capable of triggering immune stimulation via this receptor [57,238,239]. A prerequisite for the potent ORN-mediated TLR7/8 activation appears to be its formulation with cationic lipids [231,236,239] that may be explained by the need for their protection from rapid degradation by RNAses [240] and the requirement for their delivery into intracellular compartments due to the endosomal localization of their receptors [239,241,242]. Similar to CpG-mediated immune responses chemical modifications in the GU-rich sequences can result in an abrogation of TLR7/8-dependent signaling. For example, replacement of the 2⬘-hydroxyl positions with 2⬘-O-methyl or incorporation of modified nucleotides such as 5-methylcytidine or pseudouridine inhibits the immune effects observed with unmodified ORNs [57,243]. Most host-derived single-stranded RNA molecules contain a high frequency of such modified nucleotides, and it is tempting to speculate that specific posttranscriptional modifications of host-derived RNA interferes with TLR-mediated effects and acts as a potential mechanism to prevent immune stimulation by self RNA. Another interesting aspect of TLR7/8-dependent recognition is the reported species-specific differences. Mice deficient in TLR7 fail to respond to ORN or imidazoquinoline stimulation, although genetic complementation of nonresponder cells with either human TLR7 or TLR8 restores responsiveness to imidazoquinolines [60,233]. Therefore, murine TLR8 appears to be defective in recognizing nucleic acid-like structures, may be incapable of triggering intracellular signaling, or possibly senses a different yet unidentified physiological ligand. Stimulation with single-stranded ORN results similar to small molecule synthetic TLR7/8 ligands in the production of Th1, Th1-like, and proinflammatory cytokines including IFN-
from pDCs, TNF-
and IL-12p70 from monocytes and mDCs, as well as IL-12p40, IL-6, and IFN- [57,123,231,235,236,244–248]. The induction of early innate immune effects including the production of cytokines such as IL-12 or type I IFNs is essential for the stimulation of adaptive immune responses by TLRs. In addition, adaptive immunity depends on the expression of costimulatory molecules by professional APCs. Single-stranded ORN stimulate enhanced expression of such molecules on murine and human APC, mediate the expression of the early-activation marker CD69 on T cells, NK cells, or NKT cells, and enhance the proliferation of alloreactive T cells [236,238,244,245]. These hallmarks of adjuvant effects suggest that ORN can facilitate and enhance the priming of antigen-specific adaptive immune responses. Indeed, injection of lipidencapsulated ORN in mice results in the production of Th1 and proinflammatory cytokines, and addition of antigens such as Ova or Hepatitis B antigens induces enhanced levels of antigen-specific antibodies, as well as increased numbers of antigen-specific IFN- producing T cells and stronger antigen-specific CTL responses compared to mice immunized with lipid-encapsulated antigen alone [244,247,249,250]. In contrast to single-stranded RNA, double-stranded RNA is normally absent in mammalian cells and only occurs as a replication intermediate of RNA viruses in cells [251]. In vitro-generated viral double-stranded RNA appears not to be recognized by TLR7 [252], but TLR3 is activated by extracellular double-stranded RNA after entering the endosomal pathway [253]. Nevertheless, DCs that lack TLR3 are still stimulated to secrete type I IFN after intracellular delivery of doublestranded RNA, and the cytoplasmic helicase domain of the helicase protein retinoic acid-induced

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gene 1 (RIG-1) was demonstrated to be the mediator of the antiviral immune responses [252,254]. In addition, the double-stranded RNA-activated protein kinase (PKR) was identified to induce inflammatory signals upon binding double-stranded RNA and, therefore, represents together with the cytoplasmic melanoma differentiation-associated gene 5 (Mda5) another candidate sensing pathogen double-stranded RNA [255–257]. TLRs, RIG-1, Mda5, and PKR sense different pathogen RNAs in a cell-type and compartment-specific manner and, therefore, contribute each to the innate and adaptive antiviral responses that are triggered upon virus infection.

27.8 CONCLUSION Synthetic ODN ligands for TLR9, and more recently, ORN ligands for TLR7 and TLR8, contain immune stimulatory sequence motifs that can mimic molecules of infectious agents and activate therapeutic immune responses. The specificity of these immune responses can be directed against cancer, infectious diseases, or can redirect allergic immune responses, resulting in a more normal immune balance. In one class of therapeutic application low doses of CpG ODN have been used as vaccine adjuvants, in which case the specificity of the immune response is determined by the vaccine antigen. In a second type of clinical application, higher doses of CpG ODN, alone or preferably in combinations with other nonspecific therapeutic agents, such as chemotherapy, can induce therapeutic antigen-specific immune responses. The encouraging clinical results in human phase I and phase II trials and the relative lack of serious toxicities observed to date demonstrate the potential of this class of innate immune activators for improving human and animal health.

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28

Aptamer Opportunities and Challenges Charles Wilson

CONTENTS 28.1 28.2 28.3

Introduction ........................................................................................................................773 The History of Aptamers and SELEX ...............................................................................774 Applying SELEX to Generate Therapeutic Aptamers .......................................................776 28.3.1 Pool Design ..........................................................................................................776 28.3.2 Pool Composition ................................................................................................776 28.3.3 Positive and Negative Selection Pressures ...........................................................778 28.4 Post-SELEX Optimization .................................................................................................779 28.4.1 Minimization ........................................................................................................780 28.4.2 Affinity Optimization ...........................................................................................780 28.4.3 Nuclease Resistance .............................................................................................781 28.4.4 PEGylation ...........................................................................................................783 28.5 Binding and Functional Properties of Aptamers ...............................................................784 28.6 In Vivo Properties ...............................................................................................................786 28.7 Representative Therapeutic Aptamers ...............................................................................788 28.7.1 Macugen (Pegaptanib) .........................................................................................788 28.7.2 AS1411 ................................................................................................................790 28.7.3 ARC183 ...............................................................................................................791 28.7.4 REG1 ....................................................................................................................792 28.7.5 Preclinical Programs ............................................................................................793 28.8 Conclusions ........................................................................................................................794 28.8.1 Strengths and Opportunities .................................................................................794 28.8.2 Weaknesses and Challenges .................................................................................795 References .....................................................................................................................................795

28.1 INTRODUCTION Aptamers can be considered as the oligonucleotide analogs of antibodies, functioning to bind specific molecular targets with high affinity and high specificity. Since the invention of the SELEX process used to generate aptamers over 15 years ago, significant advances have been made in both our understanding of how aptamers fold and function and in our ability to generate aptamers with 773

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properties suitable for practical therapeutic applications. As with the development of monoclonal antibody therapeutics, a number of challenges have been addressed in the move from the lab into the clinic, including dramatic improvements in the pharmacokinetic properties of aptamers, the ability to cost-effectively synthesize large quantities of clinical-grade aptamers, and the ability to tune the specificity and affinity of aptamers for appropriate therapeutic targets. This chapter reviews the process by which these molecules are discovered and summarizes the properties of those that have been developed for therapeutic applications. A handful of example therapeutic programs are described, focusing in particular upon Macugen (pegaptanib), the first aptamer to be approved and marketed for therapeutic use. Remaining challenges in broadening the scope of aptamer therapeutic applications are presented, together with initial efforts at addressing the limitations of the current generation of molecules.

28.2 THE HISTORY OF APTAMERS AND SELEX In contrast to most other therapeutic modalities considered in this compendium, aptamers function by directly interacting with their targets at the protein level rather than at the gene or transcript level. Thus, whereas simple Watson-Crick base pairing defines the basic structural rules that govern the design of antisense and siRNA molecules, it is the ability of aptamers to fold into unpredictable, noncanonical structures that enables them to function. That it is, in fact, possible to create specific binding molecules solely from nucleotide building blocks was not at all obvious when the first aptamers targeting proteins and small molecules were discovered and reported in 1990 [1]. Prior to that time, characterization of a handful of biological RNAs (perhaps the transfer RNAs and the autocatalytic ribozymes serving as the best examples) had shown that certain nucleic acids could adopt discrete three-dimensional conformations to directly confer molecular recognition of proteins and small molecules—aminoacyl synthetases by the tRNAs, nucleotide cofactors by the self-splicing introns. The extent to which such properties could be generalized to other targets was considered limited. It was assumed, for example, that tRNA synthetases had evolved to specifically complement the tRNAs (rather than vice versa) and that recognition of nucleotides by ribozymes such as the group I intron was probably a highly specialized form of base pairing. Early work by Larry Gold on the mechanisms of translational regulation in bacterial and bacteriophage mRNAs provided some key insights that suggested to him that opportunities for structured nucleic acids to recognize protein targets could be larger than generally appreciated [2]. These observations led directly to the first systematic evolution of ligands by exponential enrichment (SELEX) experiment, carried out in his lab by Craig Tuerk and aimed at generating RNA molecules that could recognize bacteriophage T4 DNA polymerase (Figure 28.1) [1]. In this experiment, the translational operator sequence of the polymerase transcript (previously shown to interact with the polymerase itself [3]) was randomized at all eight positions within a loop domain. To facilitate downstream steps in the experiment, the operator sequence was flanked with constant sequences that could serve as binding sites for RT-PCR primers and which would enable individual molecules to be efficiently amplified. The corresponding library of 48 molecules was prepared by transcription with T7 RNA polymerase and purified. The pool was combined with the protein target (T4 DNA polymerase) and captured on a nitrocellulose filter to separate functional (protein-binding) from nonfunctional sequences. The isolated sequences were reverse transcribed, PCR-amplified, and then retranscribed with T7 RNA polymerase to yield an enriched pool of molecules with improved binding activity. This process of selection for binding followed by enzymatic amplification lies at the heart of the SELEX process and survives in every subsequent variant that has been developed in the past 15 years. In this first example, four cycles of selection and amplification yielded an enriched pool with binding activity comparable to the naturally occurring operator sequence. Once the desired level of functional activity had been reached and could not be improved with further

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Template construction (a)

1 5′ 3′

T7 PRO

3′

3

2

5′

nnnnnnnn

3′

4

Ligation

5′ 5

Ligation

In vitro transcription (b)

n n n n n n n n

(c)

Variable sequence

gp43 3′ 5′ gp43 recognition sequence

5′

3′ primer annealing site Selection by gp43 3′ 5′

RT 5

(d) cDNA synthesis of selected RNAs eluted from filters

(e) Second strand synthesis and PCR

T7 PRO 5′ DNA Pol 5′

1

DNA Pol

DNA Pol

5

5′ 3′ 5′

(f) In vitro transcription to begin the next round of SELEX

Figure 28.1 SELEX process. The original scheme for performing SELEX to isolate aptamers as described by Tuerk and Gold (reproduced from [1]).

selection, individual molecules in the pool were cloned and sequenced. Intriguingly, of the approximately 65,000 possible sequences in the starting library, only two—the natural sequence and a four-nucleotide variant—appear in the enriched library. This result is striking as it speaks both to the complexity of molecular recognition by nucleic acids (functional sequences do not occur frequently) and to the power of the SELEX method (despite their rarity, functional sequences can be rapidly isolated). The results of the first SELEX experiment clearly do not speak to the generality of the aptamer phenomenon (i.e., the extent to which aptamers might exist for any arbitrary target). The initial SELEX target was, after all, a protein known to bind nucleic acids and at least one functional aptamer (the natural operator sequence) was known to exist within the starting library. Despite these limitations, Tuerk and Gold immediately grasped the significance of their results and in the first publication of the SELEX method went on to propose several significant variant methods by which aptamers can be identified and the range of targets to which aptamers might be generated. They predict, for example, that small molecules (including transition-state analogs) immobilized

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on solid supports could be used as substrates for carrying out SELEX (and that aptamers isolated against such molecules might include catalysts). These predictions were borne out by later publications from the Szostak [4] and Schultz [5] labs showing that small-molecule aptamers did exist and that aptamers targeting transition-state analogs could have catalytic activity. Ellington and Szostak coined the term “aptamer” (obtained from the Latin aptus, “to fit”) and the term is now generally used to encompass nonnatural nucleic acids that bind to molecular targets through means other than Watson-Crick base pairing [2]. As described in the following sections, work by many other academic and industrial groups has gone on to demonstrate the extent to which the SELEX method can be broadly applied to generate aptamers against a wide range of targets, including proteins with no intrinsic nucleic acid–binding propensity [6,7].

28.3 APPLYING SELEX TO GENERATE THERAPEUTIC APTAMERS While the basic SELEX method developed by Tuerk and Gold and described above remains at the core of the process for generating aptamers, it is worth highlighting important refinements that have been developed over the past 15 years that are particularly important in generating these molecules for therapeutic applications. 28.3.1 Pool Design The first SELEX library consisted of RNA molecules with eight random positions [1]. The complexity of this library is low and, in the general case, insufficient to yield aptamers against an arbitrary target. Today’s libraries are typically designed with 30–40 random positions (e.g., [8]). A library prepared at the micromole scale using standard solid-phase methods generally contains approximately 1014–1015 different molecules and correspondingly spans the space of all 28-mers (i.e., an arbitrary 28-mer sequence will on average appear once within the pool). While the first SELEX experiment was carried out using a pool designed to fold as a stem-loop, most current SELEX experiments do not have defined secondary structures built into the library design. Instead, they rely upon fortuitous base pairing within the random region of the library or between the random region and either of the flanking constant regions to drive aptamer folding [9]. 28.3.2 Pool Composition The first SELEX experiments in both the Gold and Szostak labs were performed using pools of RNA molecules. While RNA clearly has the ability to fold into well-defined tertiary structures, RNA as a composition presents a number of challenges for therapeutic use. RNA is marginally stable in vivo as endonucleases and exonucleases act rapidly to cleave both single-stranded and duplex regions. Additionally, the monomers used to chemically synthesize RNA are expensive in comparison to other compositions such as DNA and the efficiency of monomer couplings (as defined by the number of equivalents required to drive coupling and the overall yield) is relatively low. It is difficult to identify workup conditions following solid-phase synthesis that fully deprotect the molecule while avoiding the introduction of other lesions. Together, these properties make RNA aptamers difficult and expensive to prepare and short-lived in the body. Alternative compositions with improved properties—cheaper, more efficient synthesis, and longer in vivo half-life—are universally sought for today’s therapeutic applications. Shortly after the first RNA SELEX experiments (and indeed envisioned in the initial Tuerk and Gold paper [1]), SELEX was carried out using DNA pools [10]. As described in the following sections, the ability to perform SELEX with a variety of other pool compositions has now been demonstrated and these compositions form the basis for most therapeutic aptamer SELEX experiments performed today.

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Serum nucleases responsible for turnover of RNAs are largely directed toward pyrimidine nucleotides and, as such, modifications to cytosine and uridine have the most beneficial effects on serum stability. Replacement of the ribopyrimidine 2⬘-hydroxyl group with 2⬘-amino, 2⬘-fluoro, or 2⬘-O-methyl substituents yields modified transcripts with significantly improved serum stability. In principle, these modifications can be introduced pre-SELEX, into the initial pools that are subjected to repeated cycles of selection and amplification. To be used in this manner, the polymerases responsible for transcribing the pool or for reverse-transcribing the selected molecules must accommodate the modified nucleotide as either a substrate or template. Before some of the earliest SELEX experiments directed at therapeutic protein targets, it was shown that T7 RNA polymerase could efficiently generate transcripts when the ribonucleotides CTP and UTP were substituted with the corresponding 2⬘-amino variants [11]. Pools of mixed composition—2⬘-ribopurines, 2⬘-aminopyrimidines—could readily yield specific aptamers to a variety of tested targets [12,13]. Whereas natural RNA aptamers exhibit a very short serum half-life (estimated at 10 s), 2⬘-aminopyrimidine-containing transcripts have a serum half-life of 170 h. Despite these properties, the 2⬘-amino modification has several shortcomings and the modification is not widely used in current SELEX approaches. While 2⬘-amino substitution increases nuclease stability, it decreases base pairing thermodynamic stability, potentially limiting the affinity of resulting aptamers [14]. Furthermore, difficulties with synthesis, deprotection, and subsequent preparation of site-specific conjugates using standard nucleophile-targeted strategies (e.g., PEGylation with NHS-activated esters) disfavor the use of 2⬘-amino-modified nucleotides [15]. As a successor to 2⬘-amino modifications, libraries containing 2⬘-fluoro modifications at uridine and cytosine have been prepared and subjected to SELEX to successfully yield functional aptamers against a number of targets. Transcripts containing 2⬘-fluoropyrimidines are somewhat less nuclease resistant than the 2⬘-amino variants (t½ ⬃ 90 h) but are still dramatically improved relative to RNA. Wild-type T7 RNA polymerase will catalyze the synthesis of 2⬘-ribopurine, 2⬘-fluoropyrimidine transcripts but the efficiency is low compared to native RNA transcription. Yields, however, are significantly improved by using the Y639F active site mutant discovered by Sousa and coworkers [16]. In contrast to the 2⬘-amino modification, introduction of the 2⬘-fluoro modification increases relative base-pair stability [17] and, in at least three head-to-head comparisons, aptamers generated using 2⬘-fluoro nucleotides show better affinity than 2⬘-amino aptamers generated to the same target [15,18,19]. Transcription of 2⬘-ribopurine, 2⬘-fluoropyrimidine pools has yielded some of the best characterized aptamers, including pegaptanib, the only aptamer currently approved and marketed as a therapeutic. Despite its many favorable properties, the 2⬘-fluoro modification has its own set of issues. 2⬘-fluoropyrimidine phosphoramidites are expensive in comparison to more commonly used chemistries and commercial supplies are limited. Deprotection of mixed ribo/fluoro oligonucleotides presents its own set of issues that seems to vary from one sequence to another. While there is no evidence for specific toxicities associated with 2⬘-fluoropyrimidines, in vitro biochemical studies have shown that these nucleotides can be used as substrates by mitochondrial DNA polymerases [20] and that, indeed, animals dosed with high concentrations incorporate these fluoropyrimidines into mitochondrial DNA [21]. The 2⬘-O-methyl modification combines many of the favorable properties of the 2⬘-fluoro modification (nuclease resistance, thermodynamic stability) without the apparent liabilities (high cost, complicated deprotection, potential for incorporation into cellular DNA). A key limitation preventing widespread use of 2⬘-O-methyl nucleotides in SELEX has been the inability to generate 2⬘-O-methyl-containing transcripts. Recent work from several groups has addressed this limitation and ultimately yielded the first 2⬘-O-methyl-containing aptamers where such modifications have been introduced pre- rather than post-SELEX. In one approach, Chelliserrykattil and Ellington reported the application of an activity-based protein selection for T7 RNA polymerase mutants with the ability to generate transcripts using 2⬘-O-methyl-containing NTPs as substrates [22]. Through this means, they successfully identify variant polymerases that accept 2⬘-OMe A, C, and U (but not G).

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In principle, such mutants could be used to generate largely 2⬘-O-methylated aptamers by transcription. Burmeister et al. have achieved this end result using a fundamentally different approach [8]. Relying initially upon the well-characterized Y639F and Y639F/H784A T7 RNAP mutants [16,23], transcription conditions were exhaustively screened to identify combinations that allowed all four 2⬘-O-methyl nucleotides to be simultaneously incorporated into optimized pool sequences. Using these modified conditions, it became possible to generate full-length 2⬘-O-methyl pool transcripts and to turn repeated rounds of SELEX. Using otherwise conventional SELEX methods, Burmeister et al. succeeded in isolating a fully 2⬘-O-methyl aptamer to vascular endothelial growth factor (VEGF) that compares favorably with Macugen in several respects (e.g., ease of synthesis, serum stability, in vivo stability). While much work has focused on enabling SELEX with pools bearing different 2⬘ modifications, it is worth noting in passing that a wider set of modifications spanning other sites within the backbone or nucleobases has been conceived and reduced to practice in the context of SELEX. Phosphorothioate modifications can be readily introduced via polymerization using
-thio-NTPs as substrates. King et al. have used this approach to generate aptamers targeting NF-B [24] while Tam et al. have generated phosphorothioate-containing aptamers to CD28 [25]. Modifications to the nucleobases, most commonly at the 5 position of pyrimidines, are often tolerated by the polymerases used for SELEX and raise the possibility of introducing entirely new functional groups with altered recognition properties into aptamers. One of the earliest examples of such a modification was the replacement of thymidine by 5-(1-pentynyl)-2⬘-deoxyuridine in a DNA library, which was subsequently used to isolate thrombin aptamers [26]. More recently, several groups have shown that amino acid-like functional groups can be synthetically appended in the same way and that these modified nucleotides can be incorporated by T7 RNA polymerase or KOD DNA polymerase to yield pools suitable for SELEX [27,28]. 28.3.3 Positive and Negative Selection Pressures The SELEX process depicted in Figure 28.1 shows a single selective pressure for function: positive selection for binding to the target of interest. In virtually all SELEX experiments performed today, negative selection is also used as a means for preventing the enrichment of nonspecific binding molecules. In the general case, the pool is initially contacted with the partitioning matrix (e.g., the nitrocellulose filter) and nonspecific matrix-binding molecules are discarded. To optimize aptamer specificity for therapeutic applications, cross-binding to undesired targets can be minimized by also including such targets in the negative selection step. An interesting example of the utility of negative selection is afforded by the related heterodimeric cytokines IL-23 and IL-12. Both molecules play important roles in the activation of T cells with IL-12 exerting its effects preferentially upon naïve TH0 cells and IL-23 acting upon memory TH1 cells [29]. The cytokines share a common p40 subunit and most monoclonal antibodies reported to date bind this shared subunit and knockout the activities of both cytokines. On the basis of both theoretical understandings of the roles of the cytokines and on knockout experiments in animal models for disease, there are good reasons to believe that specifically targeting IL-23 is likely to have beneficial effects in a range of autoimmune indications (e.g., multiple sclerosis, rheumatoid arthritis, psoriasis, and Crohn⬘s disease) while targeting IL-12 may increase the risk for infection through general immunosuppression [30]. To isolate aptamers with specificity favoring IL-23 over IL-12, SELEX was carried out using IL-12 in the negative selection step and IL-23 in the positive selection step [31]. The resulting molecules exhibit greater than 100-fold discrimination in binding. Whereas the aptamers fully inhibit IL-23-triggered release of interferon- at nanomolar concentrations, IL-12 release cannot be fully blocked at any concentration. There are some situations in which cross-specificity to multiple targets may be desirable, and in these instances successive positive selection steps can be used (either with or without an intervening amplification step) to favor the isolation of aptamers with broadened specificity. For example, it can

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be useful to generate aptamers that recognize both the human and rodent forms of a target protein such that the molecule that will be developed as a human-specific therapeutic can also be tested in preclinical disease models to demonstrate efficacy and to assess potential toxicities. Aptamers with such properties can in principle be generated by performing rounds of SELEX with the human form of the target and interspersing or following with rounds of SELEX against the rodent form. This concept has been successfully applied to generate aptamers that recognize a VEGF receptor from human (KDR) and mouse (flk-1) [32]. Five rounds of SELEX were initially performed using a soluble form of the human protein. A pool of protein-binding aptamers was identified but it failed to show significant cross-binding to the soluble mouse protein. Using the human-specific round 5 pool as a starting point, five additional rounds of SELEX were subsequently performed using the mouse protein as a target. The pool resulting from a total of 10 rounds of selection shows good cross-reactivity, hitting both human and mouse soluble receptors with comparable affinities. It is worth noting that this strategy presents some risks in the general case. If the two positive selection targets are not closely related, it is likely that the best binding molecules to either target will be lost through the combined selection process. In the worst case, SELEX will fail outright and no target-specific aptamers to either protein will be enriched.

28.4 POST-SELEX OPTIMIZATION Molecules isolated by the SELEX process typically exhibit good affinity and specificity for the targets against which they have been selected to bind. They suffer, nonetheless, from several shortcomings that may significantly limit their potential as therapeutics. Functional activity: Unless specifically designed otherwise, SELEX isolates aptamers simply on the basis of their ability to bind to a target of interest. High-affinity binding does not necessarily correlate with functional activity to the extent that aptamers may localize to nonneutralizing epitopes on their targets. As with other strategies based on enrichment for binding (e.g., antibodies, phage display peptides), aptamer clones must be screened in biochemical and cellular assays to identify those that not only bind a target but which modulate an interaction between the target and its downstream effectors to alter its biological function (e.g., block receptor-ligand binding, prevent enzyme-substrate turnover). Synthesis length: The initial aptamer sequences isolated by SELEX are typically 70–80 nucleotides long. This constraint is imposed by the initial pool design since they must generally include a 30–40 nucleotide long random region and ⬃20 nucleotide long constant sequences at both 5⬘ and 3⬘ ends. While it may be possible to chemically synthesize such full-length molecules using current solid-phase synthesis methods, the cost for synthesis is very high and yields are low, making commercialization of such molecules difficult or impossible. In general, aptamer sequences must be truncated and minimized to enable efficient large-scale synthesis. Affinity: While varying significantly from target to target, aptamers initially isolated from random pools often exhibit KDs in the 1–10 nanomolar range. Depending upon other factors (e.g., target concentration, the affinity of the target for downstream effectors, etc.), affinities in this range may or may not be sufficient for therapeutic applications. Metabolic stability: Serum nucleases have a profound impact on the metabolic stability of aptamers. Depending upon the initial pool composition used to perform the SELEX experiment, resulting molecules may show sensitivity to serum nucleases that is incompatible with the desired therapeutic application. While there are some exceptions, it is often desirable to increase the nuclease resistance of the initial aptamer clones to extend their in vivo half-life. Renal filtration/biodistribution: While aptamers are large molecules from a synthetic perspective, they are small compared to most proteins and, unless appropriately modified, are subject to rapid elimination from the body via renal filtration. In those indications where long duration of action is a desired attribute, blocking renal filtration is essential.

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Minimization

3′-cap

Optimization

PEGylation

Figure 28.2 Stepwise process for optimizing aptamers for therapeutic applications. Aptamer clones obtained by SELEX are (a) initially minimized to remove excess nucleotides not required for function, (b) capped to prevent 3⬘→ 5⬘ exonuclease degradation (typically using an inverted nucleotide to create a 3⬘-3⬘ linkage), (c) optimized by replacement of starting nucleotides (open circles) with base and backbone modifications (shaded and filled circles) to improve endonuclease resistance and target affinity, and (d) PEGylated to prevent rapid renal filtration.

Despite the limitations listed above, there are relatively straightforward means to address each through post-SELEX optimization (Figure 28.2). The following sections outline the available strategies, supported by specific examples. 28.4.1 Minimization While aptamer sequences isolated from random sequence pools are relatively long, it is almost invariably possible to remove a significant fraction (typically half to two-thirds) of the total oligonucleotide without compromising and in some cases improving binding activity. Most high-affinity aptamers generated against protein targets can be shortened to approximately 25–35 nucleotides although there are occasional examples of significantly smaller (e.g., ARC183, a 15-nucleotide aptamer targeting thrombin [10]) and larger aptamers [33]. It is generally not the case that only the random region within the starting library contains the functional aptamer core. Often, a portion of the constant sequence will base-pair with a complementary region within the random sequence to define the secondary structure required for folding and function. There are many ways in which the minimal core within an aptamer sequence may be defined. In the optimal case, visual inspection of independent clone sequences may reveal a conserved sequence or structural motif present in each (e.g., [34]). Constructs corresponding to the conserved motif can be chemically synthesized and tested for binding to confirm the prediction. Algorithms for predicting RNA secondary structure (e.g., MFOLD [35]) may assist this process. Alternatively, biochemical experiments or brute-force, systematic synthesis of truncations and internal deletions can be used to map the binding domain directly [36]. 28.4.2 Affinity Optimization A combination of both sequence and chemical modifications can be used to improve aptamer affinities for their targets, often converting low nanomolar binders into low- to mid-picomolar binders. One approach to optimization parallels the biological affinity maturation process of antibodies [37,38]. In a typical experiment, a parent aptamer sequence to be optimized is used to direct synthesis of a degenerate library of molecules with the parent sequence nucleotide dominating at each position but with each of the non-wild-type possibilities doped into the pool at low frequency (typically 5–10%). Most molecules in this library incorporate mutations by chance that prevent proper functioning of the aptamer and thus the starting pool typically exhibits minimal binding activity. By subjecting the pool

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(b)

KD= 3 nM Figure 28.3

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KD= 35 pM

KD= 2 nM

KD= 20 pM

Examples of aptamer optimization. Aptamers directed toward therapeutic targets with applications in inflammation (a) and oncology (b) were optimized through an iterative process of testing individual backbone modifications (2⬘-deoxy → 2⬘-OMe, 2⬘-OMe → 2⬘-deoxy, phosphodiester → phosphorothioate) and base modifications (guanosine → inosine) to identify those that could improve aptamer affinity and nuclease stability. Beneficial modifications were combined to yield the composite molecules shown. In the first example, modifications at roughly half the nucleotides in the aptamer yield approximately 100-fold improvement in affinity. In the second example, a single modification accomplishes comparable effects. Open circles: 2⬘-deoxy nucleotides; closed circles: 2⬘-OMe nucleotides; and shaded circles: phosphorothioate, inosine, and 3⬘-idT nucleotides.

to a few cycles of SELEX, reselecting for the ability to bind to the target, functional activity may be rapidly recovered. By progressively increasing selective pressure (e.g., using lower concentrations of target or increasing the concentration of nonspecific competitors during the partitioning step), it is possible to drive enrichment of those sequence variants with the strongest affinity. Sequencing individual clones within the reselected library typically reveals a handful of preferred sequence changes that can often be combined in composite variants to yield the highest-affinity sequences. Through this process, five- to tenfold improvements in KD are often observed. An alternative means for improving affinity involves the introduction of site-specific chemical modifications that cannot be accessed through simple changes in nucleotide sequence. 2⬘ modifications (e.g., 2⬘-deoxy → 2⬘-O-methyl, 2⬘-hydroxy → 2⬘-O-methyl), phosphate modifications (e.g., phosphodiester → phosphorothioate), and nucleobase modifications (e.g., uridine → thymidine, guanosine → inosine) have all been shown to have beneficial effects on aptamer affinity when introduced in the appropriate context [15,39]. Some of these modifications likely function by improving the folding of the aptamer, favoring either a more rigid structure or preventing its misfolding into inactive conformations. Other modifications likely function to optimize the overall steric fit between the aptamer and the target or introduce additional stabilizing pairwise interactions to drive binding. Sites where modifications are beneficial (or even tolerated) cannot generally be predicted, even when the secondary structure of the aptamer is clearly defined. As such, a typical optimization approach involves systematically synthesizing and testing a series of single-site variants in which a given type of substitution is individually introduced at each successive position within an aptamer. The effect of substitutions is assessed by comparing the variant target-binding affinity to the parental affinity—all modifications that individually improve target binding may be simultaneously combined to create a single composite molecule. Effects are quite often additive with multiple incremental improvements in binding combining to yield substantial net gains in affinity [39]. In the course of a typical optimization process, approximately 100 different variants may be tested and affinity improved by 100- to 1000-fold (Figure 28.3). 28.4.3 Nuclease Resistance Depending upon the composition of the starting SELEX pool and the desired therapeutic application, it may be necessary to engineer increased nuclease resistance into an aptamer to ultimately achieve a desired in vivo half-life. Both exonucleases and endonucleases play a role in defining the rate and pathway by which aptamers are degraded in serum. As described in the following sections, several different types of modifications can be introduced to limit the activity of these enzymes.

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3⬘→ 5⬘-directed exonucleases are responsible for the much of the cleavage of unstabilized nucleic acids [40]. Nucleotide or nonnucleotide caps attached to the 3⬘ end of an aptamer can effectively block such exonucleases, making endonucleolytic cleavage the preferred pathway for degradation. For historical reasons, the inverted deoxythymidine (idT) cap has been used in many therapeutic aptamers although other approaches (e.g., PEGylation, biotinylation) appear to be equally effective. By simply conjugating a deoxythymidine nucleotide via a 3⬘-3⬘ linkage to an otherwise unstabilized DNA aptamer, serum half-life is increased from minutes to approximately 1 h [41]. 5⬘→ 3⬘-directed exonucleases play a less prominent role in defining the stability of aptamers but can similarly be blocked using nonnucleotide caps. The most commonly used cap with therapeutic aptamers has been an alkylamine, typically introduced in the last step in solid-phase synthesis to create a reactive nucleophile for subsequent conjugation reactions. Even without subsequent conjugation, the alkylamine itself is sufficient to stabilize aptamers to subsequent exonuclease-mediated degradation. Di Giusto and King recently described a novel strategy for blocking exonucleases by circularizing aptamers via intra- or intermolecular ligation [42]. Joining the ends of the aptamer eliminates the termini altogether and thus obviates the requirement for stabilizing cap structures. The aptamer constructs they generate are remarkably stable, exhibiting serum half-lives exceeding 10 h. Modifications to the aptamer backbone can hinder both rate-limiting cleavages at internal sites by endonucleases and processive degradation by exonucleases. As noted previously, the replacement of the 2⬘-hydroxyl of ribopyrimidines with 2⬘-fluoro, 2⬘-amino, or 2⬘-O-methyl modifications can dramatically improve the serum stability of aptamers. While 2⬘ substitutions to pyrimidines have the predominant effect on stability, 2⬘ modifications to purines further extend aptamer half-lives (Figure 28.4). In contrast to cap modifications, which are invariably well tolerated, 2⬘-modifications introduced post-SELEX must be evaluated for their effect on aptamer activity since they have the potential to interfere with either folding or target binding [15].

Aptamer (ng/mL)

10000.0

1000.0

MNA mRfY

100.0 rRfY

10.0

0

1000

r/mRfY

2000 3000 Time (min)

4000

Figure 28.4 Effects of 2⬘-modifications on aptamer in vivo stability. A series of related aptamers were prepared in which a common base sequence was synthesized with a variety of different backbone substitutions including 2⬘-ribopurine, 2⬘-fluoropyrimidine (rRfY), partial replacement of some 2⬘-ribopurines with 2⬘-OMe nucleotides (r/mR,fY), replacement of all 2⬘-ribopurines with 2⬘-OMe nucleotides (mRfY), and fully 2⬘-OMe substitution (MNA). The oligonucleotides were conjugated with a 40 kDA PEG and administered to Sprague-Dawley rats as a 1 mg/kg bolus intravenous injection. Pharmacokinetic profiles were determined by measuring aptamer in serum at the indicated times following injection.

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28.4.4 PEGylation With appropriate modifications to control nuclease-mediated degradation, elimination via renal filtration becomes the major limitation to aptamer half-life. A typical minimized and stabilized aptamer has a molecular weight of 5–15 kDa, well below the effective cut-off size of the glomerular filter responsible for sieving macromolecules in the bloodstream (which generally excludes species greater than 30–50 kDa). As such, even highly nuclease-resistant aptamers exhibit relatively short functional half-lives (i.e., less than 10 min) unless additional modifications are introduced to increase their apparent size. Among the different strategies that have been explored, conjugation with high-molecular-weight poly(ethylene glycols) (PEGs) has had the most practical success. Alternative methods yielding some reduction in renal clearance have included conjugation to carrier proteins (e.g., streptavidin via biotinylated aptamer [43]) and association with liposomes through conjugation with lipid tags [44]. A handful of additional small molecules (including Tat, Ant, or poly-arginine peptides and cholesterol derivatives) have been linked to aptamers with the objective of modifying aptamer clearance and biodistribution [45]. For the most part, however, the pharmacokinetic properties of these conjugates remain largely unchanged relative to the unmodified aptamers. PEGylation is a widely used method for controlling the pharmacokinetic properties of therapeutics [46]. PEGs used for this purpose can range in size from 5 kDa up to 60 kDa. PEGs are linear molecules, generally synthesized by the catalytic polymerization of ethylene oxide. One end of the PEG polymer is usually derivatized with a cross-linking group to facilitate subsequent conjugation to a therapeutic. The preparation of high-molecular-weight molecules suitable for incorporation into therapeutics (i.e., consistent batch-to-batch mean molecular weight, low polydispersity, and high conjugation efficiency) is technically challenging. To facilitate preparation of larger PEGs (e.g., 40 kDa and 60 kDa), two or more shorter PEGs may be covalently joined to yield branched polymers. Depending upon the chemistry of the PEG activating group and the therapeutic to be conjugated, one or more PEGs may be linked to a single molecule. Very often, PEG is provided as an activated ester (e.g., N-hydroxy-succinimide) to react with available primary amines on the target (e.g., the multiple lysine side chains of a protein therapeutic) to form stable amide linkages. The basic chemical structure of aptamers (including the standard modifications previously described) generally excludes reactive nucleophiles. As such, the sites of PEG conjugation can be well defined by introducing reactive groups at specified sites into the aptamer during synthesis. Most often, a 5⬘-alkylamine phosphoramidite modifier is provided as the last solid-phase coupling and a single PEG subsequently conjugated in a solution reaction to the 5⬘ end of the aptamer. Recent work by Kurz and colleagues has relied upon both 5⬘- and 3⬘-amine modifications to aptamers to prepare diPEGylated conjugates [47]. While synthesis of these molecules imposes some additional challenges relative to monoderivatized conjugates, it avoids the requirement to use costly branched PEGs to generate molecules with the highest PEG-loading/largest apparent size. In contrast to the general case with PEGylation of biologics, PEGylation of aptamers generally does not alter their functional activity although effects must be experimentally evaluated on a case-by-case basis. This observation can be directly tied to the site-specific manner in which PEGs are attached at the termini of the molecule, typically well removed from the structural core responsible for target binding. If, in contrast, reactive amines are introduced via nucleobase modifications into the functional core of an aptamer and subsequently PEGylated, loss of functional activity is frequently observed. As shown in Figure 28.5, modification with PEGs of progressively increasing molecular weight yields aptamer conjugates with progressively longer half-lives. This simple relationship can be exploited to engineer an aptamer with a prespecified duration of action to optimize its use for a particular therapeutic indication.

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Aptamer (nM)

1,000 20 kDa PEG 100

no PEG

10

1 0

5

10

15

20

25

Time (h) Figure 28.5

Effects of PEGylation on aptamer in vivo half-life. Pharmacokinetic profiles of a 39-nucleotide dRmY composition aptamer bearing stabilizing 2⬘-OMe chemical substitutions and either no PEG, a 20-kDa linear PEG, or a 40-kDa branched PEG following intravenous administration of a 10-mg/kg bolus to CD-1 mice (n = 3 per time point).

28.5 BINDING AND FUNCTIONAL PROPERTIES OF APTAMERS Aptamers have been reported targeting over 100 different proteins [6,7]. Examining the range of targets that have yielded high-affinity binders suggests that aptamers as a class are capable of recognizing a very broad range of protein types. Successful SELEX targets include intracellular and extracellular proteins, soluble and membrane-associated proteins, and acidic and basic proteins. With respect to the biological function of targets, aptamers have been successfully generated against cytokines, hormones, growth factors, cell signaling receptors, coagulation and complement factors, enzymes, immunoglobulins, structural proteins, intracellular signaling molecules, splicing factors, transcription factors, translation factors, and toxins. There are no systematic differences in the properties of aptamers across the different functional classes that would suggest any particular protein family is particular well or poorly suited for generating high-affinity binders (Figure 28.6). Along these lines, it is interesting also to note that while some proteins clearly exhibit nonspecific low-affinity nucleic acid binding, such binding is not a pre-requisite for the generation of highaffinity aptamers—subnanomolar binders have been generated to targets that show no detectable binding to the naïve libraries used to perform SELEX. Similar to antibodies, aptamers are generally highly specific for the targets against which they have been elicited. In general, they do not cross-react to orthologs within a protein family unless a high level of sequence homology exists between the targets (⬎70%). As a specific example [48], aptamers generated to reverse transcriptase from HIV-1 bind with a KD in the low picomolar range. The reverse transcriptase of HIV-2 is recognized by the same aptamer with ⬎3000-fold weaker affinity. Along the same lines, an aptamer elicited to basic fibroblast growth factor (bFGF) binds its target with 350 pM affinity [49]. As shown in Table 28.1, other proteins within the FGF protein family are recognized with approximately 1000- to 10,000-fold weaker affinity. Finally, aptamers generated to L-selectin bind its target with 8000- to 15,000-fold and 200- and 500-fold specificity versus P-selectin and E-selectin, respectively [50]. In each of these cases, no active measures (e.g., negative selection) were taken to ensure lack of cross-reactivity. Conceptually, nonspecific protein binding could have a number of effects on the in vivo properties of aptamers. Binding to serum proteins might reduce the fraction of aptamer available to interact with a specific target and limit aptamer potency (presuming serum protein binding competes for specific binding). At the same time, association with serum proteins might help

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(a) 45 Other (toxin, structural protein, etc.)

40

Transcription/splicing/translation factor

# Targets

35

Intracellular signal transduction

30

Immunoglobulin

25

Enzyme

20

Cytokine; chemokine; hormone; growth factor

15

Coagulation/complement

10

Cell surface receptor; adhesion molecule; viral coat protein

5 0

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Affinity (nM)

00

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00 10

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00 1 0. 01

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35 # Targets

30 25 20 15 10 5 00 00 0 10

0 10

10

10

1

1 0.

00 1 0. 01

0.

0

0

Affinity (nM) Figure 28.6 Reported affinities for aptamers. Aptamers reported in publications and patents were tabulated with respect to (a) target protein class, and (b) stage of aptamer development. Overall, most reported aptamers have affinities in the 1–10 nM range. Aptamer affinities are relatively uniform across protein classes with no particularly favored or disfavored target types. Aptamer affinities uniformly improve following the development process outlined in Figure 28.2, which pool affinities at the low end of the spectrum and aptamers having undergone medicinal chemistry optimization showing the best affinities.

Table 28.1 Example of Aptamer Specificity: Basic Fibroblast Growth Factor (bFGF) Protein BFGF (FGF-2) Denatured FGF-1 FGF-4 FGF-5 FGF-6 FGF-7 VEGF PDGF AB AT III Thrombin

KdbFGF/Kdprotein 1.0 0.0008 0.0003 0.0006 0.041 0.0005 0.0007 0.0008 0.002 0.000008 0.00003

Note: An aptamer specifically selected for binding to bFGF was assessed for binding to related protein targets. Results are expressed as the ratio between the KD for bFGF (350 pM) and that for the nonspecific target.

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drive distribution of aptamers out of the bloodstream into tissues and also limit clearance by renal filtration. Certain chemical modifications to oligonucleotides, most notably phosphorothioates, are known to confer both serum protein binding and the predicted pharmacokinetic effects [51]. With a handful of exceptions, aptamers lack the extensive phosphorothioate modifications routinely used for antisense therapeutics, and the rapid renal clearance of (unPEGylated) aptamers suggests minimal influence by serum protein binding. The vast majority of therapeutic aptamers that have been generated are designed to function as antagonists by competitively blocking protein-protein interactions between an aptamer target and a biological effector of the target. In general, aptamers generated to proteins that exist as single folded domains (e.g., cytokines) have a high likelihood of functioning as antagonists with a potency directly related to the balance between aptamer-target affinity and target-effector affinity. Aptamers generated against multidomain protein targets (e.g., cell-surface receptors) may or may not function as antagonists, depending largely upon where they bind relative to the biological effector. Using appropriate experimental strategies, e.g., SELEX against a defined ligand binding domain rather than the complete protein, it is possible to direct the binding specificity of resulting aptamers in ways that maximize the likelihood of obtaining functional inhibitors. A handful of potential therapeutic applications have been described in which an aptamer functions as a targeting vehicle for delivery of an active agent. One of the best developed examples of this strategy is provided by aptamers selected to bind PSMA, a cell-surface protein preferentially expressed on the surface of prostate tumour cells. Aptamers generated to PSMA bind with affinities in the range of 1–10 nM and specifically label PSMA(⫹) LNCaP cells [33]. Aptamers conjugated to the protein toxin gelonin [52] or to cytotoxin-loaded nanoparticles [53] have demonstrated efficient in vitro killing of PSMA-expressing cells and improved efficacy in mouse xenograft models [54].

28.6 IN VIVO PROPERTIES The preceding sections have highlighted the range of chemical modifications that can be introduced into an aptamer in the course of optimization. Through the judicious choice of backbone modifications and conjugation chemistries, it is possible to generate aptamers with widely divergent pharmacokinetic properties (Figure 28.7). As such, the in vivo properties of an aptamer can be tailored to optimize its utility for a particular indication. As an example at one end of the spectrum, ARC183 is a thrombin-specific aptamer developed for use as an anticoagulant in CABG and PCI surgeries where rapid pharmacological off-set is highly desired (discussed in more detail in Section 28.7.3). Because this molecule lacks backbone modifications that would prevent nuclease attack and PEGylation that would limit renal filtration, it exhibits a very short in vivo half-life, making it possible to predictably and rapidly dial in an appropriate level of anticoagulant activity by simply adjusting the rate at which drug is infused. At the other extreme, pegaptanib (marketed as Macugen) is an anti-VEGF aptamer developed for the treatment of age-related macular degeneration (AMD). Because administration of the drug requires direct injection into the eye, very long half-life is desired to enable as infrequent dosing as possible. As discussed in Section 28.7.1, pegaptanib incorporates extensive backbone modifications (introduced both pre- and post-SELEX) to block nucleases and conjugation to a high-molecular-weight branched PEG to slow absorption from the eye into the systemic circulation (the rate-limiting step in the disposition of the aptamer [55]). Intravitreally administered pegaptanib exhibits an average apparent half-life of 10 days [56], sufficient to enable dosing in patients once every 6 weeks (to be contrasted with dosing once every 4 weeks for Lucentis, the anti-VEGF antibody fragment). With these and other examples spanning a range of aptamer compositions, it is possible to draw some general conclusions regarding the ADME properties of aptamers. With appropriate stabilizing

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modifications using the range of previously discussed chemistries, serum nuclease-mediated metabolism is not a limitation for many therapeutic applications. The terminal half-life of a typical stabilized and PEGylated aptamer following intravenous bolus administration ranges from 3 to 22 h in rodents and 30–60 h in primates [57]. The observed volume of distribution (Vss) is typically two- to fivefold larger than the plasma volume (⬃40 mL/kg) in rodents and primates with 40-kDa PEG conjugates falling at the low end of the range and unconjugated and 20-kDa PEG conjugates reaching the upper end. PEGylated aptamers composed fully by 2⬘-O-methyl modified nucleotides (enabled via recent modifications to the SELEX process) exhibit the longest in vivo half-lives studied to date [8]. Figure 28.7 shows the profile of a fully 2⬘-O-Me aptamer administered to CD-1 mice at 10 mg/kg. Aptamer concentrations in these plasma samples were assessed by fluorescence using the intercalating dye Oligreen™ (Molecular Probes, Eugene, OR) by comparison with standard curves. The aptamer shows monophasic pharmacokinetics with an elimination half-life of 22 h and a volume of distribution of 87 mL/kg. The ability to penetrate and persist in target tissues is a requisite property for therapeutics targeting factors that are expressed or exert their function in tissue. Aptamer tissue distribution has been studied as a function of composition using tritium exchange as means for labeling aptamer and with either traditional tissue oxidation followed by liquid scintillation counting (LSC) [45] or quantitative whole-body autoradiography (QWBA) [58] to quantify aptamer levels. Using these methods, both PEGylated and unPEGylated aptamers are shown to access all tissues with the exception of the central nervous system and the testis (similar low penetration across the blood-brain barrier has been previously observed for antisense oligonucleotides). There are, however, significant differences across tissue types with the proportions influenced by composition and PEGylation. For all compositions examined, the highest concentrations of radioactivity are observed in the kidney and bladder, consistent with urinary elimination as a primary route of clearance. An unPEGylated, fully 2⬘-O-Me aptamer is more rapidly cleared from the circulation than other compositions and appears to be eliminated through the kidney without metabolism. PEGylation

(a) 1000

(b) primate rodent

100

anti-C5 (rat)

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pegaptanib(rat) anti-PDGF (mouse)

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0.01 DNa

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Figure 28.7 Tailored pharmacokinetics. (a) The range of aptamer half-lives accessible via backbone modifications and PEGylation. (b) Pharmacokinetic profile of a 23-nucleotide-long fully 2⬘-OMe aptamer following intravenous administration of a 10-mg/kg bolus to female CD-1 mice (n = 3 per time point).

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significantly increases the total level of radioactivity in well-perfused organs and tissues, including liver, lungs, spleen, bone marrow, and myocardium. The ability of aptamers to penetrate into inflamed tissue has been directly studied using the carrageenan mouse paw model [58]. Boomer et al. treated animals initially with direct carrageenan injection into the right hind paw, followed 10 min later with injection of tritiated aptamer. After 3 h, animals were sacrificed and analyzed by QWBA. Inflamed tissues contained significantly more aptamer than normal tissue, suggesting that inflammation increases permeability to aptamer uptake. While all aptamers could be detected at significant concentrations in the inflamed paw tissue, a PEG-conjugated aptamer accumulated to higher levels than did unconjugated aptamers. This observation parallels those that have been made regarding the distribution of PEGylated biologics into tumors where a phenomenon of enhanced permeability and retention (EPR) similarly leads to elevated drug levels [59]. It has been postulated that the leaky vasculature of tumors (and inflamed tissue) facilitates influx into tissue but that PEGylation slows efflux via the lymphatics responsible for draining the tissue. Traditional “cut and burn” and QWBA studies described above suffer two key limitations. First, while they inform on the quantity of drug-derived material in tissues, it is impossible to discriminate between parent drug and metabolites. It remains possible that tissue-associated radioactive label reflects the distribution of metabolites as much as it does active aptamer. Second, they do not inform on the subcellular distribution of aptamer and thus the actual availability of aptamer for binding to its target. Microautoradiography has been used to examine the localization of labeled aptamer (and metabolites) in extracted tissue sections and it is clear that some fraction of labeled material resides within cells, most notably the sinusoidal lining cells in the liver and the proximal and distal tubular cells in the kidney [58]. At the same time, it is clear from in vivo pharmacology studies using aptamers directed against tissue-based targets (e.g., PDGF-BB, complement C5, IL-23) that they are able to penetrate into the extravascular space with sufficient efficiency to achieve efficacy. Future studies will be required to characterize the proportion of intra- and extracellular aptamer in tissues and understand the effects of composition (oligonucleotide and PEG) on the relative balance.

28.7 REPRESENTATIVE THERAPEUTIC APTAMERS Aptamers directed against specific protein targets have progressed by varying degrees toward development and approval as therapeutics. The following sections highlight those for which significant public data are available. 28.7.1 Macugen (Pegaptanib) Macugen, an anti-VEGF165 aptamer co-developed by OSI Eyetech and Pfizer, is approved and marketed for the treatment of AMD. As the first aptamer to run the full course from discovery to clinical development to regulatory approval, it provides a comprehensive example of some of the principles outlined previously in the chapter. Work initially pioneered by the laboratory of Judah Folkman helped establish the role of VEGF as a central regulator of angiogenesis. By the mid-1990s, the role of angiogenesis in pathology was strongly indicated by preclinical studies in both oncology (where inhibitors of VEGF signaling were shown to limit tumor growth [60]) and conditions of ocular neovascularization (diabetic retinopathy [61], retinopathy of prematurity [62], and macular degeneration [63]). With this impetus, Gold, Janjic, and coworkers at NeXstar began efforts to generate therapeutic aptamers that could bind and block VEGF. Following initial efforts with suboptimal aptamer compositions [12,64], Ruckman et al. succeeded in generating high-affinity VEGF aptamers using conventional SELEX partitioning from a starting library of 2⬘-fluoropyrimidine, 2⬘-ribopurine oligonucleotides [15].

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A large number of independent clone sequences were initially identified but many could be assigned to one of three dominant sequence families on the basis of a conserved sequence motif in each. Representatives from each of the major sequence families were minimized using a combination of biochemical fragmentation experiments and synthetic truncations, yielding aptamers ranging from 23 to 29 nucleotides long for the three different families. The tolerance of each 2⬘-ribopurine to substitution by 2⬘-OMe was biochemically mapped in an effort to remove potential sites of nuclease attack. For each of the three different minimized aptamers, a handful of sites were identified where a significant loss of affinity accompanied introduction of the 2⬘-OMe group. Simultaneously introducing 2⬘-OMe modifications at all sites that were well tolerated yielded stabilized variants with affinities for VEGF ranging from 40 to 130 pM. All three molecules were shown to block binding of VEGF to its receptors, Flt-1 and KDR, in in vitro assays with potencies correlated to their intrinsic ligand affinity. The optimized leads were evaluated in an in vivo test known as the Miles assay to identify a single candidate for further development. In this experiment, VEGF is injected into the dermis to induce changes in cutaneous vascular permeability, causing intravenously administered Evans blue dye to escape out of the blood and color surrounding tissue. Despite the relatively similar in vitro properties of the three aptamers, only one, t44-OMe, showed significant in vivo activity. Following capping of the 3⬘ end with an inverted deoxythymidine to block exonuclease activity and modification with a branched 40-kDa PEG, treatment with this aptamer (known subsequently as NX1838, EYE001, Macugen, or pegaptanib) was able to almost completely prevent dye leakage into the VEGF injection site, suggesting complete blockade of VEGF function [65]. In a second in vivo model, polymer pellets containing VEGF were implanted in the corneal stroma of rats to induce the growth of blood vessels into the normally avascular cornea [65]. Pegaptanib-treated animals showed up to 65% reduction in angiogenic index (a quantitative scoring of blood vessel density and length) relative to controls. Additional animal studies included a retinopathy of prematurity (ROP) model in which 7-day old mice were exposed to hyperoxic conditions for an extended period to induce the growth of blood vessels through the limiting membrane of the retina into the vitreous [65]. Histological scoring showed that pegaptanib treatment caused an 80% reduction in retinal neovascularization. While not directly relevant to ophthalmic applications, pegaptanib was also tested in the A673 rhabdomyosarcoma xenograft model [65]. Aptamer-treated animals showed tumor growth inhibition of ⬃70–75% relative to vehicle-treated animals, results comparable to those achieved with Avastin (bevacizumab), an anti-VEGF monoclonal antibody [60]. The pharmacokinetic properties of pegaptanib have been explored following intravenous or intravitreal administration [66,67]. When provided as an intravenous bolus injection of 1 mg/kg to cynomolgus macaques, pegaptanib exhibits a half-life of approximately 9.3 h. Its intravitreal half-life, defined by the rate of absorption, is roughly tenfold higher than the systemic half-life (94 h in monkeys and 83 h in rabbits). Intravitreal bioavailability varies slightly across species but is uniformly high (70–100%). Limited pharmacokinetic data on pegaptanib exist in humans. Following intravitreal administration at 10 times the recommended dose, pegaptanib reaches maximal plasma concentrations after 1–4 days and displays a terminal half-life of 10 days [56]. In its initial phase I clinical trial, 0.25–3.0 mg pegaptanib was administered by intravitreal injection to patients diagnosed with exudative AMD (wet AMD) [65]. Dose-limiting toxicity was not achieved. 80% of patients receiving the aptamer had improved or stabilized vision after 3 months and 27% had a ⱖ 3 line improvement on the Early Treatment for Diabetic Retinopathy Study Chart (ETDRS). With repeat dosing in a phase II study, 88 % of AMD patients demonstrated stabilized or improved vision after 3 months and 25% of eyes showed ⱖ 3 line improvement in the ETDRS [68]. Two identically designed multicenter phase III clinical trials including a total of 1186 patients were used to generate the data that supported regulatory approval for pegaptanib use in AMD [69]. Patients were randomized to receive 0, 0.3, or 3.0 mg pegaptanib every 6 weeks over a course of 54 weeks. All patients receiving pegaptanib had significantly less disease progression than untreated patients; 70% of patients receiving the approved dose of pegaptanib (0.3 mg) were

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classified as responders (losing less than 15 letters of visual acuity after 54 weeks) as compared to 55% of patients receiving sham injections ( p ⬍ 0.001). Focusing upon those patients with the best response during the treatment period, 6% of aptamer-treated patients showed a ⱖ 3 ETDRS line improvement compared with only 2% of the control group. Conversely, a significantly ( p ⬍ 0.001) smaller proportion of aptamer-treated patients experienced severe vision loss than did patients receiving the sham injection (10% versus 22%). Pegaptanib was generally well tolerated and most adverse ocular events were mild, transient, and unrelated to the drug. With safety and efficacy established for AMD through the clinical trial experience described above, pegaptanib was approved by the FDA in December 2004 for marketing in the United States under the trade name Macugen. By 2006, approval from regulatory agencies had been granted allowing its use in Canada and Europe as well. Potential applications in the treatment of diabetic macular edema (DME) and retinal vein occlusion (RVO) are currently being explored through ongoing clinical trials [70]. 28.7.2 AS1411 AS1411 is an aptamer currently being developed by Antisoma (UK) as a potential cancer treatment. The G-rich aptamer (also known as GRO29A and AGRO100) was discovered fortuitously as a designed control molecule through in vitro experiments exploring the cytostatic activity of oligonucleotides against a number of different tumor cell lines [71]. Subsequent characterization established that the oligonucleotide associated as a dimer, stabilized by the formation of intermolecular stacked G-quartets [72]. The folded dimer was shown to bind nucleolin, a large, highly expressed protein that has been implicated in a number of cellular functions that include ribosome biogenesis. While normally found in the nucleolus (hence its name), a proportion of the expressed protein is localized to the plasma membrane in transformed cells. This extracellular localization may explain both the ability of the aptamer to be efficiently taken up into cells and the observation that activity is limited to transformed and not normal cells. Recent data suggest that the aptamer additionally has the capacity to interact with NF-
B essential modulator (NEMO), a regulatory subunit of the inhibitor of
B kinase (IKK) complex, and that this additional activity may partially account for the aptamer’s biological activity [73]. The IKK complex is normally responsible for the phosphorylation of I
B
and ultimately drives the transcriptional activation activity of NF-
B. Treatment of transformed cells with AS1411 was shown to inhibit IKK activity and reduce I
B
phosphorylation observed following induction with TNF-
. As expected from these results, AS1411 was shown to block both induced and constitutive NF
B activity in human cancer cell lines derived from a variety of tumor types (including cervical, prostate, breast, and lung). The initial in vitro studies used to identify AS1411 have been followed by a number of in vivo xenograft studies. In a pancreatic cancer model, a combination of AS1411 with gemcitabine was shown to be significantly more effective in slowing tumor growth than gemcitabine alone [74]. AS1411 accumulates rapidly in lung cancer xenografts, reaching levels at least 9 times those seen in any normal tissue after 1 h. An initial phase I trial with AS1411 was carried out to define potential limiting toxicities, to explore the pharmacokinetic properties in humans, and, by exploring responses wide range of cancer types, to identify target indications for further development [75]. Patients with documented progressive metastatic disease, who had failed multiple previous therapies, received aptamer administered as a continuous intravenous infusion over 96 h at 1 mg/kg/day. A follow-up treatment at 10 mg/kg/day over 7 days was provided if no toxicity had been observed following the initial treatment. A total of 17 patients received 22 cycles of AS1411 treatment. The aptamer appears to be well tolerated as no drug-related toxicities were observed in any patients. Detailed pharmacokinetic data have not been reported from the trial but aptamer was detected in the serum throughout the infusion period and serum levels correlated with the administered dose. Two months after treatment, eight patients (50%) showed stable disease. Disease remained stable in these patients for 2–9 months

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before progression. At the time the trial results were initially presented, one patient was maintaining a nearly complete response more than 6 months after treatment. On the basis of the profile of responses in the initial trial, a second phase I trial was designed targeting patients with advanced renal cell carcinoma (RCC) or non-small cell lung cancer (NSCLC) [76]. The aptamer was delivered as a single continuous 7-day intravenous infusion with an optional second treatment cycle. At the time interim trial results were presented (June 2006), no serious adverse events had been observed. Doses of 22 mg/kg/day yielded peak plasma levels of 1.5 M, a concentration shown to be effective at killing cancer cells in in vitro studies. One of five RCC patients receiving AS1411 therapy maintained a near-complete response 18 months after treatment. Among the four other patients, three had stable disease while a fourth had progressed. Among three NSCLC patients receiving therapy, one had stable disease and two had progressed. As the first aptamer tested for the treatment of cancer in humans, AS1411 continues to show promising results. 28.7.3 ARC183 Originally discovered by Bock and colleagues at Gilead in the early 1990s [10], ARC183 (previously known as GS522) is an extensively characterized direct thrombin inhibitor that has been explored as a potential short-acting anticoagulant for use in cardiac surgeries (e.g., coronary artery bypass graft [CABG] procedures). Heparin, the current standard of care in this setting, has a number of practical advantages including a long history of physician experience with the drug, low cost, straightforward monitoring, and the ability to reverse activity with protamine. Despite these advantages, heparin has a number of widely recognized limitations. The long half-life of heparin necessitates its reversal with an antidote and, while protamine is effective in most patients in this capacity, it can trigger adverse immune reactions and its short half-life (⬃5 min) may result in postoperative unopposed (rebound) heparin effects [77]. Repeated exposure to heparin can result in the generation of antibodies directed against complexes of heparin with Platelet Factor 4 and these antibodies can induce thrombocytopenia upon subsequent heparin treatment [78]. Current estimates suggest that up to 5% of patients receiving heparin treatment will experience heparin-induced thrombocytopenia (HIT), a condition characterized by thromboembolic events in 30–50 % of patients and mortality in 10–20%. The inability of heparin to inhibit clot-bound thrombin and its tendency to induce platelet aggregation and dysfunction represent further limitations that may impact heparin’s effectiveness and which have driven the development of alternative therapies [79,80]. ARC183 is a 15-nucleotide-long all-DNA aptamer composed entirely of guanosine and thymidine residues. Structural studies indicate that the aptamer adopts a stable chair-like structure [81] in which guanosines self-associate to form two stacked tiers of G-quartets. Two dithymidine loops at the bottom of the chair structure appear to be critical for the high-affinity interaction with thrombin, directing binding to exosite I on the surface of the coagulation cascade protease. The measured affinity of the aptamer for thrombin is dependent upon both environmental conditions and assay method. KDs ranging from 1.4 nM to 100 nM have been reported [82,83]. Affinity for prothrombin is approximately an order of magnitude weaker than that for thrombin. Binding to other serum proteins or proteolytic enzymes is essentially undetectable [10]. In vitro studies using whole human blood show that ARC183 is an effective anticoagulant, inhibiting both thrombin-catalyzed generation of fibrin from fibrinogen and thrombin-induced platelet aggregation [10]. In contrast to heparin, ARC183 is effective at inhibiting clot-bound thrombin, potentially limiting the propagation of venous thrombi or rethrombosis. In vivo studies have explored the anticoagulant properties of ARC183 using dog and monkey models of cardiopulmonary bypass [84,85]. Because it lacks stabilizing backbone modifications to limit nuclease attack and PEGylation to limit renal filtration, ARC183 exhibits a very short in vivo half-life (⬃2 min). Upon infusion into cynomolgus monkeys, anticoagulation (measured as prothrombin times or ACTs) is rapidly induced and plateaus at a constant level within 10 min of beginning the treatment. Because the aptamer is rapidly cleared, drug levels sustained in the blood

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are directly proportional to the infusion rate and the anticoagulation level can be rapidly adjusted by increasing or decreasing the infusion rate. Ten minutes after stopping the infusion, measures of coagulation status return to normal, coincident with clearance of the aptamer from the blood. The aptamer has been tested in a series of phase I clinical trials in normal human volunteers and patients with coronary artery disease. Results from these experiments show that infusion of ARC183 results in a rapid onset of anticoagulation and that stable, dose-related anticoagulation can be maintained with continuous infusion. Rapid reversal and return to normal hemostasis was observed after drug infusion stopped. While the pharmacokinetic profile observed in humans was well predicted by the preclinical studies, the limited potency of the aptamer meant that the amount of drug needed to achieve the desired anticoagulation required for CABG surgery resulted in a suboptimal dosing profile. Efforts correspondingly have now focused on a second-generation aptamer, designated NU172 (ARC2172), which possesses a pharmacokinetic profile similar to that for ARC183 but with significantly higher potency as an anticoagulant. 28.7.4 REG1 REG1 is an anti-Factor IXa aptamer that is currently being explored in phase I studies for development as an anticoagulant for use in coronary procedures, driven by many of the same factors that motivated the development of ARC183 (Section 28.7.3). A unique feature of REG1 is its codevelopment with a targeted antidote that is designed to rapidly reverse its pharmacological activity. Coagulation Factor IXa (FIXa) is a component of the intrinsic tenase complex responsible for generating Factor Xa, the enzyme that converts prothrombin to thrombin. Inihibitors of Factor IXa can have potent anticoagulant effects and may offer advantages with respect to bleeding complications relative to alternative anticoagulant targets [86]. Aptamers to Factor IXa were initially isolated using standard SELEX methods from a pool of 2⬘-fluoropyrimidine, 2⬘-ribopurine oligonucleotides [34]. All aptamer sequences that bound to the target contained a conserved structural motif consisting of a hairpin containing a common asymmetric bulge. A truncated version of one of the aptamers, designated 9.3t, bound Factor IXa with 580 pM affinity and suppressed Factor X activation in vitro. As expected, the aptamer had significant activity as an anticoagulant when measured using assays that measure the intrinsic pathway (e.g., activated partial thromboplastin time (aPTT)) but minimal effects in assays based on extrinsic pathway activity (e.g., prothrombin time [PT]). REG1 appears to be a modified version of the 9.3t aptamer incorporating additional stabilizing backbone modifications and a high-molecular-weight PEG [87]. As discussed for ARC183, the ability to rapidly control and reverse anticoagulant activity is essential for safe therapeutic use. In contrast to ARC183, which achieves rapid reversibility as a result of its short in vivo half-life, REG1 has an intrinsically long half-life. Reversal of anticoagulant effects has been achieved in this case using a complementary antidote oligonucleotide that, via base-pairing to the active aptamer, is able to block proper aptamer folding and thus interfere with its interaction with the target [34]. Initial in vitro studies have been recently extended with testing of an anti-Factor IXa aptamer in a porcine model of cardiopulmonary bypass (CPB) [88]. In these experiments, aptamer was initially administered as a bolus injection to achieve a relatively constant level of anticoagulation that persisted throughout 60 min of the CPB procedure. At the end of the procedure, a specific complementary antidote oligonucleotide was administered by bolus injection and it affected an immediate return to normal coagulation status. In these experiments, the aptamer/antidote achieved a pharmacokinetic profile very similiar to that observed with heparin/protamine (i.e., rapid onset, constant activity throughout the procedure, followed by rapid offset). In addition, a number of ancillary observations point toward potential competitive advantages for the aptamer relative to heparin. For example, inflammatory responses triggered by thrombin generation (including IL-1 and IL-6 release) may contribute to the morbidity and mortality observed in patients undergoing open-heart procedures. By blocking the generation

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of thrombin upstream in the coagulation cascade rather than inhibiting thrombin itself, the aptamer has the potential to minimize these effects. In the porcine CPB model, heparin/protamine-treated animals showed 15 times higher levels of IL-1 and 2 times higher levels of IL-6 than aptamer/ antidote-treated animals. Protamine treatment itself has a number of potentially adverse effects in addition to its reversal of heparin, including increases in pulmonary artery pressue, decreases in systolic and diastolic blood pressure, impairment of myocardial oxygen consumption, and decreased cardiac output. To define whether aptamer/antidote treatment would be expected to have the same effects (and correspondingly adverse patient outcomes), cardiac physiology was assessed by measuring mean arterial pressure (MAP) during and after the CPB procedure. While aptamer- and heparin-treated animals showed no significant differences during CPB, significant differences were observed postreversal. Protamine caused a consistent and dramatic drop in MAP in heparin-treated animals in the period up to 30 min following administration. The antidote oligonucleotide in contrast had inconsistent and comparatively minor effects on MAP. To date, data on Factor IXa aptamers and the potential to reverse their activity with complementary oligonucleotides appear very encouraging. REG1 is currently undergoing testing in normal human volunteers in a phase I trial and results will be presented before the end of 2006. It remains to be seen what other types of aptamer therapeutic applications might similarly benefit from rapid reversal using an antidote approach. 28.7.5 Preclinical Programs Aptamers targeting a number of additional targets have been isolated, optimized for in vivo use, and tested in animal disease models for efficacy. Many of these are currently being assessed in IND-enabling studies with expections to enter formal clinical development in the near future. The following represents a partial listing. ●

●

ARC1779 is a PEGylated, stabilized aptamer targeting the A1 domain of von Willebrand Factor (vWF). It inhibits arterial thrombus formation by blocking platelet recruitment to collagen exposed at sites of vascular injury. In primate electrical injury studies, the aptamer has provided antiplatelet activity comparable to that of the most potent available therapies (e.g., GPIIb/IIIa inhibitors) and thus may be useful as a treatment for acute coronary syndromes (ACS), percutaneous coronary intervention (PCI), and possibly thrombotic thrombocytopenic purpura (TTP). The limited role of vWF in thrombus formation under conditions of low shear flow is expected to translate into reduced bleeding risk and thus a wider therapeutic index. The half-life of the aptamer has been optimized to allow for relatively rapid pharmacological off-set, thereby enabling its use in patients who may need to undergo subsequent surgical intervention. Clinical testing of ARC1779 is expected to start in early 2007. Anti-PDGF-B aptamers have been tested in a variety of animal models where the effects of PDGF-B in driving proliferation, differentiation, and migration of mesenchymal cells have been shown to play a role in pathology. In a model for restenosis, aptamer-treated animals exhibited significantly reduced intimal hyperplasia in response to balloon injury relative to vehicle-treated animals [89]. In a rat model of mesangioproliferative glomerulonephritis, aptamer treatment significantly reduced the number of glomerular mitoses [90]. With treatment limited to 4 days following disease induction, long-term benefit in kidney function (including reduced proteinuria, tubulointerstitial damage, and renal extracellular matrix accumulation) extended for 100 days. Anti-PDGF aptamers have shown benefit in tumor models [91,92] where they reduce interstitial fluid pressure (IFP) presumably through effects on both proliferation of fibroblasts within the tumour stroma and on recruitment of pericytes to endothelial cells in the neovasculature. Aptamer-induced reduction in IFP allows higher levels of cytotoxics to penetrate into the tumor and thus correspondingly potentiates cytotoxic effects in reducing tumor growth. The angiogenic effects of anti-PDGF agents in combination with anti-VEGF agents have been explored in the context of ocular models for neovascularization [93]. It is in this context that E10030, an anti-PDGF aptamer under development by OSI Eyetech, is likely to enter clinical testing in the near future.

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ARC1905 is a PEGylated, stabilized aptamer targeting complement factor C5 that effectively inhibits activation of the downstream proinflammatory complement cascade (including generation of C5a and the membrane attack complex). Complement activation has been implicated in a number of acute and chronic conditions. Some of the strongest clinical data suggesting a role for complement inhibitors come from pexelizumab, an anti-C5 antibody fragment that has been clinically tested for use in CABG surgeries [94]. Along similar lines, the aptamer has been tested in the isolated perfused mouse heart model. In this setting, ARC1905 was shown to block complement activation occurring both in the bloodstream and within cardiac tissue to indefinitely preserve heart function.

28.8 CONCLUSIONS The preceding sections have provided a sense of how aptamers are developed for specific molecular targets and tailored for in vivo use as therapeutic agents. The approval of Macugen (pegaptanib) as a therapy for AMD validates the aptamer approach and provides a measure of both the opportunities and the challenges associated with aptamer development. In conclusion, I briefly review the general features currently defining aptamers as a therapeutic platform. 28.8.1 Strengths and Opportunities ●

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Rapid discovery: Using standard SELEX methods, aptamers can be rapidly discovered and optimized once a particular molecular target has been specified. Arguably, the discovery timelines from target to IND are faster for aptamers than they are for both small molecules (which are typically slowed by the challenges of lead optimization) and antibodies (which are slowed by the requirement to optimize GMP manufacture). The ability to rapidly generate new aptamers makes it possible to integrate results from clinical trials with other compounds to generate second-generation therapeutics with optimal properties. Target specificity: Similar to antibodies, aptamers isolated via the SELEX process demonstrate high specificity for their targets. This specificity minimizes the potential for off-target toxicities and should ultimately translate into large therapeutic indices. (It is worth noting that this specificity comes with potential costs. Aptamers may not cross react with unidentified isoforms that may be important for efficacy or with orthologs from other species that could facilitate preclinical testing). Stability: Modern aptamer compositions are typically resistant to degradation under a relatively wide range of environmental conditions (e.g., extremes of pH or temperature). In contrast to the monoclonal antibodies that they functionally resemble, aptamers can be readily denatured and will spontaneously refold into their active conformation. This combination of properties renders them appropriate for settings that cannot be readily developed with biologics (e.g., field use in the absence of a refrigeration chain, formulation with novel controlled-release technologies that involve denaturing solvents, etc.). Blocking protein-protein interactions: Aptamers, similar to antibodies, can robustly prevent interactions between ligand-receptor and substrate-enzyme pairs, interactions that often have proven refractory to inhibition using small-molecule approaches. Tailored pharmacokinetics: The range of backbone modification and conjugation chemistries allows aptamer half-life and biodistribution to be predictably adjusted over a very wide range— enabling extremely rapid off-set for acute indications and relatively infrequent dosing for chronic indications. Lack of immunogenicity: Similar to other nucleic acid–based modalities and in contrast to proteinbased biologics, aptamers have minimal propensity to elicit an antibody response. The standard chemistries used by aptamers (including 2⬘-OMe backbone modifications and PEGylation) have also been shown to prevent Toll-like receptor–mediated immunostimulatory responses that may be undesirable for many therapeutic applications. Manufacturing: The basic process of milligram-scale aptamer synthesis that is used during discovery and optimization can be used to readily scale up to gram and kilogram scale for clinical and commercial

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manufacture. Both the relative investment required to build aptamer GMP manufacturing capacity and the timeframe for aptamer GMP process development provide advantages over competing monoclonal antibodies.

28.8.2 Weaknesses and Challenges ●

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ADMET unknowns: Our current understanding of aptamers as therapeutics is limited by the body of data defining their in vivo properties. Recent studies have continued to suggest that aptamer disposition can be understood in terms that generalize to the entire class but exceptions remain likely. The fate of aptamers and aptamer metabolites in tissues remains broadly undefined. Half-life limits: Using current compositions, the longest-lived aptamers appear to have systemic half-lives approaching 1 week in primates. In contrast, half-lives of 3 weeks and longer have been demonstrated for monoclonal antibodies in humans. Unless more stable compositions or new controlled-release technologies are developed, aptamers may not be able to achieve the multiweekly dosing intervals that contribute to competitive advantage in the monoclonal antibody space. Parenteral administration: While the feasibility of delivery via nonparenteral routes (e.g., inhalation, topical, oral, enema) has been explored for other types of oligonucleotides, no such studies have been described for aptamers. For applications where small-molecule drugs make oral delivery a possibility, aptamers may not be competitive if parenteral administration is the only delivery option. Cost of goods: Aptamers are complicated molecules to manufacture and, despite continued progress in cost-effective manufacture, are unlikely to be routinely competitive with traditional small-molecule drug manufacture. Requirements for tertiary structure: In contrast to small-molecule and antisense therapeutics, aptamers require a specific three-dimensional folded structure to function. This need imparts unique requirements for analytical and bioanalytical characterization. Effector functions: Aptamers are pure binding molecules, selected solely for the ability to bind to a therapeutic target with high affinity and specificity. In contrast to antibodies, aptamers do not intrinsically allow for target-directed immune effector functions such as complement-dependent cytotoxicity (CDC) or antibody-dependent cellular cytotoxicity (ADCC). For some specific applications (e.g., certain oncology targets), these effector functions may be important contributors to efficacy and their absence with aptamers represents a limitation. Intracellular targets: Aptamers can be readily generated to intracellular targets and can, if transcribed intracellularly via transfected expression vectors, block intracellular targets. No studies to date, however, have shown that an exogenously provided aptamer can functionally block an intracellular target.

In conclusion, aptamers as a therapeutic platform offer a number of advantages that complement existing small-molecule and biologic approaches to drug discovery. Continued efforts to better understand the properties of aptamers and to refine the technology through changes in chemical composition will broaden the opportunities for aptamers as a class of therapeutics.

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Index A acetylcholinesterase (AChE) antisense therapies, 737–738 multiple sclerosis therapy, 677–678 splice switching oligonucleotide targeting, 99–100 acetyl-coA carboxylases (ACCs), nonalcoholic steatohepatitis, antisense reduction, 656 activated partial thromboplastin time (APTT) ISIS 113715 compound, clinical safety profiles, 657 locked nucleic acid, 553–554 systemic ASO therapy, clinical safety experiments, 374–375 toxicology of ASOs and, 337–339 activator solution reagents, 2⬘-methoxyethyl (MOE)modified oligonucleotide manufacture, 409 active targeting mechanisms, liposomal drug delivery systems, nucleic acids, 242 activity assays, antisense oligonucleotide design, 119–123 primary assays, 128–134 acyl-coenzyme A: cholesterol acyltransferases (ACATs), dyslipidemias, ASO liver-targeted inhibition, 608–610 acyl-coenzyme A: diacylglycerol acyltransferase 2 (DGAT2), nonalcoholic steatohepatitis, antisense therapy, 655–656 adenosine 1 receptor delivery system, toxicologic effects of ASOs, 334 adipose tissue 2⬘-methoxyethyl oligonucleotide pharmacology in, 287 type 2 diabetes, 648-651 AEG35156/GEM640 antisense, mechanisms, 705 aerosol delivery systems, toxicologic effects of ASOs, 333–334 affinity mechanisms antisense drugs, 12–13 aptamer optimization, 780–781 oligonucleotide medicinal chemistry, 145–146 age-related macular degeneration, antisense oligonucleotide therapies, 592–593 alicaforsen profiles clinical safety experiments, 378–385 Crohn’s disease therapy, 667–668 ulcerative colitis, 668–669 allergies, CpG oligodeoxynucleotide therapy, 759 allogeneic graft rejection, inflammatory disease therapy, 684 α-L-locked nucleic acid modification oligonucleotide medicinal chemistry, 160 RNase H recruitment, 540 alternative splicing current technologies and applications, 90 global strategies, 102–103 hybridization strategies, 100–101 levels and ratios in, 92–93

mechanisms of, 20–25 modification as therapeutic tool, 98–99 RNA intermediary metabolism, 8–9 small interfering RNA, 103 small molecules, 103–104 small nuclear RNA, 102–103 trans-splicing mechanisms, 101–102 Alzheimer’s disease, splice switching oligonucleotide targeting, 96 amides, furanose nonphosphate backbones, oligonucleotide medicinal chemistry, 151–152 amidites, locked nucleic acid synthesis, 522–524, 528–529 amino- and thio-LNA amidites, 524–525 base modifications, 528 diastereoisomer amidites, 526–527 amino-LNA amidites heteroduplex thermal denaturation, 533–534 RNase H recruitment, 540 synthesis, 524–526 AMP-activated protein kinase-α, ApoB-100 inhibition therapies, 614–616 amyloid precursor protein (APP), dementia antisense therapies, 737–738 amyotrophic lateral sclerosis, antisense oligonucleotide therapy, 730–734 analytical testing, 2⬘-methoxyethyl (MOE)-modified oligonucleotides, 419–430 drug substance intermediates, 421–422 identity testing, 422 impurity tests and assay, 422–430 reagents and solvents, 421 starting materials, 419–421 angiogenesis, ophthalmology therapy and, 592–593 animal studies ApoB-100 inhibition therapies hamster combination studies, 616 monkey studies, 613–616 murine pharmacology, 612–616 siRNA inhibition, 625–626 species-specific in vivo pharmacology, 612–613 clinical safety experiments with gen-1/gen-2 ASOs and, kidney effects, 386–389 dyslipidemias, ASO liver-targeted therapies, 605-607 locked nucleic acid pharmacology, 546–547 morpholinos basic properties, 565–566 RNA splicing alteration, 577 neurological disorders, 722–723 pharmacokinetics/pharmacodynamics of ASOs, 308–316 second-generation antisense oligonucleotide optimization, gap size effects, 492–500 toxicological effects of ASOs in kidney, 343–345

801

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Page 802

802 animal studies (contd.) proinflammatory effects, 348–351 species specificity, 350–351, 356–357 anion exchange (AX) HPLC, 2⬘-methoxyethyl (MOE)modified oligonucleotide manufacture, purification process, 415–416 antagomirs, micro-RNA silencing pathways, 456–458 antibacterial agents morpholinos, 572 peptide nucleic acids, 512–513 antibodies, RNA interference drug development, 478 anti-PDGF-B aptamer, 793 antisense oligonucleotides (ASOs). See also specific ASOs, e.g. morpholinos; specific compounds, e.g. phosphorothioate oligonucleotides (PS-ODNs) antisense therapeutics and, 70–71 cancer therapy, 699–701, 711–712 cardiovascular therapy, 602–630 chimeric ASOs, human RNase H1 and, 65–70 clinical trials future research issues, 394–395 kidney effects, 386–389 liver effects, 391–392 local adminstration protocols, 371, 393–394 proinflammatory effects, 377–386 selectivity and reduction research overview, 365–367 subject characteristics and disease conditions, 367–369 systemic administration trials aPTT prolongation, 374–375 complement activation, 375–377 duration of treatment, 370–371 results, 371–392 schedules and dosages, 369–370 thrombocytopenia, 389–391 cross-species, 124–127 current technologies and applications, 90 delivery routes and formulations, 217–218, 233–234 design principles, 118–128 DNA-like structure, human RNases H and, 63–65 drug action human RNases and, 70–71 occupancy-activated destabilization, 26–39 occupancy-only mechanisms, 19–26 phases of, 7 selectivity mechanisms, 12–19 exclusion criteria for, 123 follow-up assays, 134–135 human RNase H2 biochemistry, 57–61 inflammatory disease, 666 local administration, 221–225 multiple mechanisms, 277–279 neurological disorders, 721–741 ophthalmology therapies, 585–596 oral and gastrointestinal delivery, 227–233 pharmacokinetics, 184–211 polymorphism sequence variants, 123 primary screening assays, 128–134 propyne modifications, 165

INDEX research background, 118 screening and identification, 16–19 systemic administration, 218–221 targeted oligonucleotide silencers of splicing (TOSS), 101 terminating mechanisms, 47–48 in vitro vs. in vivo activity, 135–138 antisense theory pharmacology, 5–7 second-generation antisense oligonucleotide optimization, 487–488 antiviral therapies morpholinos, 570–572 ophthalmology therapies, 591–592 ApoB-100 antisense compound, 316–320 dyslipidemias, ASO liver-targeted therapies, 611–626 apolipoprotein C-III, dyslipidemias, ASO liver-targeted inhibition, 606–608 apoptotic pathways, antisense oligonucleotide development, cancer therapy, 701–704 aprinocarsen, glioblastoma antisense therapy, 736 aptamer structures ARC183, 791–792 AS1411, 790–791 binding and functional properties, 784–786 development of, 773–774 future research issues and applications, 794–795 history of, 774–776 macugen (pegaptanib), 788–790 2⬘-methoxyethyl (MOE)-modified oligonucleotides, manufacturing and analysis of, 402–403 oligonucleotide medicinal chemistry, 171 ophthalmology therapies, 586–587 optimization strategies, 779–784 affinity optimization, 780–781 minimization technology, 780 nuclease resistance, 781–782 PEGylation, 783–784 preclinical programs for, 793–794 REG1, 792–793 representative structures, 788–792 RNA interference drug development, 475 therapeutic applications, 776–779 thrombin-binding aptamers, toxicological effects, 339 toxicologic effects, 328–331 in vivo properties, 786–788 ARC183 aptamer, 791–792 ARC1779 aptamer, 793 ARC1905 aptamer, 794 Argonaute proteins, RNAi enzymes basic properties, 80–81 pathways, 439 AS1441 aptamer, 790–791 asthma antisense oligonucleotide therapy, 678–682 CpG oligodeoxynucleotide therapy, 759 pathology and current therapy, 678 “asymmetry rule,” short interfering RNA specificity, 445–446 ATL1102, multiple sclerosis therapy, 676 autoimmune disease, CpG oligodeoxynucleotide therapy, 759

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Page 803

INDEX

803

average mass spectrum measurements, 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 426–430 AVI-4020, antiviral therapy, clinical trials, 571–572 AVI-4065, antiviral therapies, clinical trials, 571–572 AVI-4126 animal studies, 566–567 cancer therapy, 574–575 cardiovascular therapy, 573–574 restenosis inhibition, 629 AVI-4457, animal studies, 566–567

B BACE protease, dementia antisense therapies, 738 backbone modifications antisense oligonucleotides gastrointestinal drug delivery systems, 225–233 system administration systems, 218–219 oligonucleotide medicinal chemistry, 148–154 furanose-nonphosphate backbones, 151–153 furanose-phosphate backbones, 148–151 sugar and backbone replacements, 153–154 peptide nucleic acid, 508 short interfering RNAs, 440 base modifications locked nucleic acids chemistry, 528 thermal denaturation, 535 short interfering RNAs, 442–443 basic fibroblast growth factor (bFGF), aptamer development, 784–786 basophilic granules, toxicological effects of ASOs in kidney and, 341–345 Bax gene family, amyotrophic lateral sclerosis antisense therapy, 730–734 BCL2 (B-cell leukemia-lymphoma gene 2) gene antisense oligonucleotide development, cancer therapy, 701–704 2⬘-methoxyethyl oligonucleotide pharmacology, in liver, 285–286 bcl-x gene, splice switching oligonucleotide targeting, 98 BCL-xL gene, cancer therapy, 703–704 Bc1-x gene, splicing alteration, 20 Becker muscular dystrophy (BMD), splice switching oligonucleotide targeting, 95 β-globin gene, splice switching oligonucleotide targeting of, 94 β-globin RNA, splicing modulation, 20–21 bicyclic sugars locked nucleic acid, 522 oligonucleotide medicinal chemistry, 158–161 bioavailability parameters antisense oligonucleotides gastrointestinal drug delivery systems, 228–231 subcutaneous injections, 220–221 liposomal nucleic acid delivery formulations, 254–256 locked nucleic acid, 548–551 peptide nucleic acids, 514–515

Bligh-Dyer monophase, reverse-phase evaporation nucleic acid encapsulation, 248–249 blood-brain barrier (BBB), antisense oligonucleotide delivery, 221 neurological disorders, 723–726 pharmacokinetics and metabolism, 307 blood-urea-nitrogen (BUN) levels, clinical safety experiments with gen-1/gen-2 ASOs and, kidney effects, 387–389 blunt-ended perfect duplex design, short interfering RNAs (siRNAs), 443–445 bone tissue, 2⬘-methoxyethyl oligonucleotide pharmacology in, 287–288 boranophosphate backbone, oligonucleotide medicinal chemistry, 149 brain-targeted drug delivery antisense oligonucleotide delivery, 221 pharmacokinetics and metabolism, 307 toxicology of ASOs and, 334

C C5 alkyl/halogen substitution, pyrimidine 5⬘-position modifications, oligonucleotide medicinal chemistry, 163–164 cancer therapy antisense oligonucleotides, 699–712 clinical safety experiments with gen-1/gen-2 ASOs, 367–369, 378–381 CpG oligodeoxynucleotides, 758–759 immunomodulation and immune surveillance, 688–689 morpholino compounds, 574–575 synthetic short interfering RNA, systemic drug administration, 450–453 5⬘ capping mechanism, occupancy-activated destabilization, 27 capping reagents, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 409–410 cardiovascular disease antisense oligonucleotide therapies advantages of, 603–604 C-reactive protein inhibition, 626–628 current treatment paradigm, 602 dyslipidemias, liver targets in, 605–626 evolution of, 601–602 future research and applications, 629–630 hypertension inhibitors, 628 restenosis affects, 628–629 unmet therapeutic needs and, 602–603 morpholino therapies, 572–574 catalytic domain, human RNase H1, 52–54 cationic lipids, liposomal drug delivery systems, nucleic acids, 239–240 cationic 2⬘-O-alkyl modifications, oligonucleotide medicinal chemistry, 157 CD40 cell membrane protein, splice switching oligonucleotide targeting, 99 C/D RNAs, intermediary metabolism, 10–12

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Page 804

804 cell activation, inflammatory disease antisense therapy, 686 cell-free assays, antisense oligonucleotide design, activity correlation with, 122 cell migration and adhesion, inflammatory disease antisense therapy, 686 cell proliferation, maturation, and survival, inflammatory disease antisense therapy, 685 cellular delivery systems, peptide nucleic acids, 513–514 cell uptake locked nucleic acid, 551–552 ophthalmology therapies, 587 central nervous system administration neurological diseases, ASO distribution, 723–726 RNA interference drug development, “naked” siRNA, 472–474 toxicologic effects of ASOs, 333–334 chemical modifications antisense drug activity and, 25 small interfering RNAs, 439–443 translation arrest and, 26 chemical penetration enhancers, topical drug delivery systems, 222–223 chemokine release asthma therapy, 679 inflammatory disease antisense therapy, cell activation, 686 toll-like receptors, immunomodulation and immune stimulation effects, 751–753 toxicological properties of chimeric antisense oligonucleotides, proinflammatory effects, 348–351 chimeric antisense oligonucleotides human RNase H1 and, 65–71 metabolic diseases drug discovery schematic, 642–644 evaluation rationale for, 642 evolution of, 642 future research issues, 659 nonalcoholic steatohepatitis, 655–656 obesity drug discovery, 652–655 phase 1/2 clinical program overview, 657–659 type 2 diabetes, 644–652 hepatic glucose output inhibition, 648–649 kidney targeting, 651–652 protein phosphatase targeting, 644–647 tissue selectivity and pharmacokinetic properties, 649–651 transcription factor targeting, 647 second-generation antisense oligonucleotide optimization, gapmer structures, 490 toxicologic properties chronic administration, 351 clotting inhibition, 338–339 complement activation, 339–340 genetic effects, 353–354 hematopoietic effects, 345 kidney effects, 340–345 liver effects, 345–346 mechanisms and clinical correlates, 336–337 oral administration, 334–336

INDEX phosphorothioate comparisons, 328–329 plasma protein binding effects, 337–340 proinflammatory effects, 346–351 reproductive toxicology, 352–353 safety assessment strategies, 330–331 safety pharmacology, 354–356 sequence motifs, 329–330 single-strand ASO, siRNA, and aptamers, 328 species-specific effects, 356–357 systemic vs. local administration, 333–334 target organ accumulation and effect, 340–346 toxicokinetics, 332–333 chitosan nanoparticles, RNA interference drug development, 477–478 cholesterol conjugates cardiovascular therapies, 601–602 ISIS 301012 (ApoB-100), 316–320 oligonucleotide medicinal chemistry, 167–168 RNA interference drug development, 474–475 synthetic short interfering RNA, systemic drug administration, 449–451 cholesteryl esters, dyslipidemias, ASO liver-targeted therapies, acyl-coenzyme A;cholesterol acyltransferases (ACAT) inhibition, 608–610 cholesteryl ester transfer protein (CETP), dyslipidemias, ASO liver-targeted therapies, 605 chromatin, human RNase H2 biochemistry, 62–63 chronic administration systems, toxicological effects of ASOs, 351 c-Jun N-terminal kinases, obesity therapy, antisense targeting, 654–655 class-related toxicologic properties antisense oligonucleotides, 336–338 safety assessment strategies for, 331 cleavage mechanisms chimeric antisense oligonucleotides, human RNase H1 and, 69–70 human RNase H1 catalytic domain, 52–54 wild-type and mutant proteins, 50–52 human RNases and, DNA-like antisense oligonucleotides, 63–65 locked nucleic acid synthesis, amidites, 522–524 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 406–407 clinical trials antisense oligonucleotides (ASOs) future research issues, 394–395 kidney effects, 386–389 liver effects, 391–392 local adminstration protocols, 371, 393–394 metabolic diseases, 657–659 proinflammatory effects, 377–386 selectivity and reduction research overview, 365–367 subject characteristics and disease conditions, 367–369 systemic administration trials aPTT prolongation, 374–375 complement activation, 375–377 duration of treatment, 370–371 results, 371–392

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Page 805

INDEX schedules and dosages, 369–370 thrombocytopenia, 389–391 aptamer therapeutic agents, pegaptanib, 789–790 ARC183 aptamer, 791–792 AS1441 aptamer, 790–791 asthma therapy, 678–679 cancer therapy, G3139 antisense, 702–704 cardiovascular therapy current treatment paradigm, 602 ISIS 301012 ApoB inhibitor, 619–626 inflammatory bowel disease therapy, alicaforsen therapy, 667–671 locked nucleic acids, 557–558 morpholinos, 567–572 REG1 aptamer, 792–793 RNA interference drug development, 478–480 clotting time inhibition locked nucleic acid, 553–554 toxicology of ASOs and, 338 clusterin antisense compound cancer therapy, 706–708 clinical safety experiments, two-hour infusion profile, 378–381 pharmacokinetics and pharmacodynamics, 321–322 c-myc proto-oncogene, antisense inhibitors of, 629 coated cationic liposomes (CCL), reverse-phase evaporation nucleic acid encapsulation, 248–249 complement activation systemic ASO therapy, clinical safety experiments, 375–377 toxicology of ASOs and, 339–340 complementary DNAs (cDNAs) antisense oligonucleotide design, 119 locked nucleic acid hybridization, 531–532 complexation techniques, synthetic short interfering RNA, systemic drug administration, 451–453 conjugation strategies morpholinos, antibacterial agents, 572 oligonucleotide medicinal chemistry, 167–168 RNA interference drug development, 474–475 synthetic short interfering RNA, systemic drug administration, 449–451 controlled pore glass (CPG) supports, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 410–411 coronary heart disease. See cardiovascular disease corticosteroids asthma therapy, 678 inflammatory bowel disease therapy, 667 cost-competitive manufacturing, oligonucleotide medicinal chemistry, 147 covalent modification, target nucleic acids, covalent modifications, 38 CpG oligodeoxynucleotides (ODNs) A-class structure-activity relationships, 754 antisense oligonucleotide toxicology, 329–330 B-class structure-activity relationships, 753–754 C-class structure-activity relationships, 754 immune activation, 748 immunomodulation and immune stimulation effects, 751–753

805 rheumatoid arthritis antisense therapy, 674–675 structure-activity relationships, 753–755 therapeutic applications, 755–760 TLR9 and mechanism of action, 748–749 toxicological properties of chimeric antisense oligonucleotides, proinflammatory effects, 347–351 C-reactive protein, antisense oligonucleotide targeting of, 626–628 Creutzfeldt Jakob disease, antisense therapies, 736–737 Crohn’s disease clinical safety experiments with gen-1/gen-2 ASOs, 367–369 complement activation, 376–377 duration studies, 367–370 hypersensitivity reactions, 385–386 subcutaneous injection responses, 383–385 two-hour infusion profile, 378–381 FLICE inhibitory protein expression, 685 intercellular adhesion molecule (ICAM)-1, 667–668 intracellular adhesion molecule, 667–668 pathology and current therapy, 667 cross reactor identification antisense oligonucleotide design, 119 2⬘-methoxyethyl oligonucleotide pharmacology, in vitro conditions, 280–282 cross-species antisense oligonucleotide design, 124–127 2-cyanoethoxymethyl (CEM), short interfering RNA chemical synthesis, 447–448 (2-ctabietgtk)-N 3-thymine (CNET) residue, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, deprotection process, 415 cyclohexenyl HNA analogs (CeNA), oligonucleotide medicinal chemistry, 163 CYP3A family, morpholino redirection of, 575–576 cystic fibrosis transmembrane conductance regulator (CFTCR), splice switching oligonucleotide targeting, 95–96 cytochrome P450 enzymes, morpholino redirection of, 575–576 cytokines asthma antisense therapy and, 678–682 clinical safety experiments with ASOs, intravenous infusion profiles, 381–383 CpG dinucleotide motifs, toll-like receptors and mechanism of action, 749–751 CpG oligodeoxynucleotide therapy, asthma/allergy, 759 inflammatory disease antisense therapy, 686–687 intraocular inflammation, ophthalmology therapy, 593–594 liposomal nucleic acid delivery encapsulation, immune stimulation, 258–259 SELEX process and, 778–779 toll-like receptors, immunomodulation and immune stimulation effects, 751–753 toxicological properties of chimeric antisense oligonucleotides, proinflammatory effects, 348–351 cytoplasmic vacuolation, toxicological effects of ASOs in kidney and, 341–345

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Page 806

806

INDEX

cytosine analogs, oligonucleotide medicinal chemistry, 165–166

D dacarbazine (DTIC), cancer therapy, antisense G3139 combined with, 702–704 deazapurines, oligonucleotide medicinal chemistry, 167 dementia disorders antisense oligonucleotide therapy, 737–738 splice switching oligonucleotide targeting, 96 deposition mechanisms, pulmonary drug delivery systems, antisense oligonucleotides, 223–225 deprotection, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 415 detritylation reagent, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 408–409 drug intermediate analysis, 422 solid-phase synthesis, 417–418 diabetes therapy antisense oligonucleotides, ophthalmology therapies, 592–593 immunomodulation and immune surveillance, 688–689 second-generation antisense oligonucleotide optimization, gap size effects, rodent studies, 497–500 type 2 diabetes, drug discovery, 644–652 diaminopurine modification, oligonucleotide medicinal chemistry, 166 diastereoisomer LNA amidites heteroduplex thermal denaturation, 534–535 LNA synthesis, 526–527 2⬘-O-[2-[N,N-(dimethyl)aminoethoxyl]ethyl] (DMAEOE), oligonucleotide medicinal chemistry, 157 2⬘-O-2,4-dinitrophenyl substitution, oligonucleotide medicinal chemistry, 157 distribution kinetics, antisense oligonucleotides, pharmacokinetics, animal studies, 311–312 DNA-like antisense oligonucleotides (ASOs), human RNases and, 63–65 DNA synthesis, human RNase H1, 55 DNAzymes, neurological disorders, nonantisense gene silencing, 726–727 dosage studies, antisense oligonucleotides, clinical safety experiments, 369–370 “double overhang” design, short interfering RNAs (siRNAs), 443–445 double-stranded DNA (dsDNA) locked nucleic acid structure, 530–532 peptide nucleic acid targeting, 510–512 double-stranded RNAs (ds-RNAs) antisense transcripts, 9 oligoribonucleotide ligand identification, 760–762 RNAi pathways, 438–439 small interfering RNA silencing, 76–77 double-stranded RNase immune stimulation, 38 manufacturing, 37

occupancy-activated destabilization, 31–38 physical chemical properties, 37 sense strand device, 37 structural features and medicinal chemistry, 36–37 Down’s syndrome, splice switching oligonucleotide targeting, 96 Drosha enzyme, micro-RNA biogenesis, 11–12, 78–79 Drosophila, RISC assembly in, 81–82 drug delivery systems antisense oligonucleotides future research issues, 233–234 local administration, 221–225 oral and gastrointestinal delivery, 225–233 research background, 217–218 systemic administration, 218–221 2⬘-methoxyethyl oligonucleotide pharmacology, in vitro conditions, 279–282 morpholino compounds, cardiovascular therapies, 574 neurological disorders, 723–726 nucleic acids, liposomal delivery formulations active targeting strategies, 242 analytical methods, 251–254 encapsulation, 253–254 particle size measurement, 251–253 zeta potential, 253 basic properties, 237–239 cationic lipids, 239–240 efficacy evaluation, 261–262 encapsulation technologies, 242–251 analytical methods, 253–254 ethanol-destabilized liposomes, 247–249 ethanol drop (SALP) method, 247 passive encapsulation, 243–246 reverse-phase evaporation, 249–250 spontaneous vesicle formation by ethanol dilution method, 250–252 immune stimulation, 258–259 immunogenicity, 259–261 intracellular delivery, helper lipids and, 240 liposome constituents, 239–242 pharmacology, 254–262 polyethylene glycol lipids, 241–242 systemic administration, 254–256 toxicity issues, 256–258 ophthalmology therapies, 588–589, 595 RNA interference delivery systems, 471–478 drug discovery and development, metabolic diseases drug discovery schematic, 642–644 evaluation rationale for, 642 evolution of, 642 future research issues, 659 nonalcoholic steatohepatitis, 655–656 obesity drug discovery, 652–655 phase 1/2 clinical program overview, 657–659 type 2 diabetes, 644–652 hepatic glucose output inhibition, 648–649 kidney targeting, 651–652 protein phosphatase targeting, 644–647 tissue selectivity and pharmacokinetic properties, 649–651 transcription factor targeting, 647

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Page 807

INDEX

807

drug intermediates, 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 421–422 Duchenne muscular dystrophy (DMD) morpholino-base RNA splicing alteration in, 576–577 splice switching oligonucleotide targeting, 95 duration of action ISIS 301012 (ApoB-100), 317 phosphorothioate oligonucleotides, pharmacokinetics, animal studies, 312–314 dyslipidemias, antisense oligonucleotide therapies, liver targets for, 605–626 acyl-coenzyme A; cholesterol acyltransferase 2 inhibition, 608–610 ApoB-100 inhibition, 611–626 apolipoprotein C-III inhibition, 606–608 cholesterylester transfer protein inhibition, 605 Lp(a) inhibition, 605–606 dystrophin gene morpholino-base RNA splicing alteration in, 576–577 muscular dystrophy antisense therapies and, 739–740 splice switching oligonucleotide targeting, 95, 103 splicing alteration in, 20

E efficacy analysis cardiovascular therapy, ISIS 301012 ApoB inhibitor, 619–626 liposomal nucleic drug delivery systems, 260–262 encapsulation technologies, liposomal delivery formulations nucleic acids, 242–251 ethanol-destabilized liposomes, 247–248 ethanol drop (SALP) method, 247 passive encapsulation, 243–246 reverse-phase evaporation, 248–249 spontaneous vesicle formation by ethanol dilution method, 250–252 pharmacology analysis, 253–254 enema formulations, clinical safety experiments with ASOs, 394 enhanced green fluorescence protein (EGFP), splice switching oligonucleotide assay, 104–105 enzymology. See also specific human RNases antisense oligonucleotides, gastrointestinal drug delivery systems, 225–233 RNase H mechanism, 30 EPI-2010, asthma therapy, 679 epidermal growth factor receptor (EGFR), glioblastoma antisense therapy, 736 ethanol-destabilized liposomes, nucleic acid encapsulation, 247–248 ethanol drop (SALP) method, nucleic acid encapsulation, liposomal drug delivery systems, 247 eukaryotic initiation factor binding protein 2 (eIF4E-BP2), type 2 diabetes, antisense targeting of, 647 exaggerated pharmacology strategy, toxicology of ASOs, 330–331 excretion mechanisms, locked nucleic acid, 552–553

exon characteristics, antisense drug activity and, 24 exonic splicing enhancers (ESEs) antisense drug activity and, 24–25 pre-mRNA splicing, 90–92 spinal muscular atrophy antisense therapy, 739 exonic splicing silencers (ESSs) antisense drug activity and, 25 pre-mRNA splicing, 91–92 exon-specific splicing enhancement by small chimeric effectors (ESSENCE) technique, RNA splicing hybridization, 100–101 experimental autoimmune encephalomyelitis, multiple sclerosis therapy, 676–677 exploration studies, morpholinos, antiviral therapies, 570–572

F facilitated hybridization, RNA targeting, 14–15 Factor IXa, REG1 aptamer inhibition, 792–793 FALS mutation, amyotrophic lateral sclerosis antisense therapy, 730–734 familial amyloidosis, antisense therapies, 738–739 familial hypobetalipoproteinemia (FHBL), ApoB-100 inhibition therapies, 612 Fas genes antisense oligonucleotides, pharmacokinetics, animal studies, 314 2⬘-methoxyethyl oligonucleotide pharmacology, in liver, 285–286 fatty acid conjugates, oligonucleotide medicinal chemistry, 168 five-membered ring structures, oligonucleotide medicinal chemistry, sugar analogs, 162 FLICE inhibitory protein (FLIP) Crohn’s disease, 685 rheumatoid arthritis antisense therapy, cell proliferation, maturation, and survival, 685 flow-through supports, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 410–411 2⬘-deoxy-2⬘-fluoro-D-arabinonucleic (FANA), oligonucleotide medicinal chemistry, 156 2⬘-fluoro modifications, oligonucleotide medicinal chemistry, 154–155 folate hydrolase (FOLH1) gene, splice switching oligonucleotide targeting, 98 Fomivirsen ocular delivery system, 221–222 ophthalmology therapy, 586–587 antiviral compounds, 590–592 tolerability parameters, 588–589 forkhead transcription factor (FKHR), type 2 diabetes, antisense targeting of, 647 formacetal backbone, oligonucleotide medicinal chemistry, 152–153 free energy binding, antisense oligonucleotide design and, 120–122 freeze-drying, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 419

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Page 808

808

INDEX

fructose 1,6 bisphosphatase (FBP-1), type 2 diabetes, antisense inhibition, 648–649 furanose nonphosphate backbones locked nucleic acid structure, 530–532 oligonucleotide medicinal chemistry, 151–153, 158 furanose phosphate backbones, oligonucleotide medicinal chemistry, 148–151, 158 fusogenic liposomes, intracellular delivery systems, 240

G G3139 antisense compound, cancer therapy, 701–704 gapmer ASO structures cardiovascular therapies, 603–604 gastrointestinal drug delivery systems, 229–231 medicinal chemistry design strategies, 169–170 methylene(methylimino) backbone, 152 metabolic diseases drug discovery schematic, 642–644 evaluation rationale for, 642 evolution of, 642 future research issues, 659 nonalcoholic steatohepatitis, 655–656 obesity drug discovery, 652–655 peripheral targets for, 654–655 PTP-1B antisense inhibitor, 652–653 phase 1/2 clinical program overview, 657–659 type 2 diabetes, 644–652 hepatic glucose output inhibition, 648–649 kidney targeting, 651–652 protein phosphatase targeting, 644–647 tissue selectivity and pharmacokinetic properties, 649–651 transcription factor targeting, 647 2⬘-methoxyethyl (MOE)-modified oligonucleotides administration systems, 289–290 first-generation PS-ODN comparisons, 275–277 manufacturing and analysis of, 402–403 oncology models, 289–290 in vitro conditions, 279–282 in vivo conditions, 282–290, 284 liver, 285–286 lymphoid tissue and inflammatory cells, 288–289 second-generation antisense oligonucleotide optimization, 488–490 oligonucleotide length, 501–503 “1-10-1” gapmer structures gap size effects, animal studies, 498–501 second-generation antisense oligonucleotide optimization, oligonucleotide length, 502–504 “5-10-5” gapmer structures gap size effects, animal studies, 598–501 second-generation antisense oligonucleotide optimization, oligonucleotide length, 502–504 gap size, second-generation antisense oligonucleotide optimization, 490–501 limitations of, 500–501 monkey studies, 500

RNase H1, 490–492 rodent studies, 492–500 gastrointestinal delivery systems, antisense oligonucleotides local GI uptake, 231–233 permeability, 227–228 presystemic metabolism, 225–226 systemic bioavailability, 228–231 G-clamp substitution, oligonucleotide medicinal chemistry, 166 gene expression antisense oligonucleotides cancer therapy, 700–701 design-activity correlation, 119–120 inflammatory disease antisense therapy, 686–687 peptide nucleic acid modulation anti-infective agents, 512–514 basic properties, 507–508 chemistry, 508 dsDNA targeting, 510–512 future applications, 515 mRNA targeting, 508–510 in vivo bioavailability, 514–515 splice switching oligonucleotide restoration of, 94–98 toxicologic effects of ASOs and, 353–354 gene regulation human RNase H1, 55 human RNase H2, 63 gene sequence alignment, antisense oligonucleotide design, 118–119, 123 gene target inhibition, antisense oligonucleotides, pharmacokinetics, animal studies, 308–311 genomics human RNase H2, 63 human RNase H1 and, 55 GE OligoProcess synthesizer, 2⬘-methoxyethyl (MOE)modified oligonucleotide manufacture, 411–412 glaucoma management, oligonucleotide ophthalmology therapy, 594–595 glioblastoma, antisense oligonucleotide therapy, 735–736 global splicing strategies, therapeutic targeting, 103–104 glucocorticoids, type 2 diabetes, antisense reduction of, 649–650 glucose-6-phosphatase (G6P), type 2 diabetes, antisense inhibition, 648–649 golden retriever muscular dystrophy (GRMD) animal model, morpholino-base RNA splicing alteration in, 577 G-quartets, antisense oligonucleotide toxicology, 329–330 green fluorescent protein (GFP), small interfering RNA silencing, 76–77 GSK3β kinase, dementia antisense therapies, 738 guanine analogs, oligonucleotide medicinal chemistry, 167

H half-life concentrations, locked nucleic acid, 548–551 heat shock proteins, cancer therapy, 708–709 helper lipids, liposomal drug delivery systems, 240

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Page 809

INDEX

809

hematopoiesis, toxicological properties of ASOs and, 346 hepatic steatosis ApoB-100 inhibition therapies, animal studies, 614–616 nonalcoholic, antisense drug discovery, 655–656 hepatic tissue, type 2 diabetes, 648–651 hepatitis C virus (HCV), morpholino antiviral therapy, clinical trials, 571–572 hepatotoxicity, clinical safety experiments with gen-1/ gen-2 ASOs and, 391–392 heterocyclic modifications, oligonucleotides, 163–167 heteroduplex locked nucleic acid, thermal denaturation, 532–535 heteroduplex substrates, human RNase H1, catalytic domain, 53–54 heterogeneous nuclear ribonucleoprotein complexes (hnRNP), facilitated hybridization, 14–15 hexitol-derived nucleic acids (HNA), oligonucleotide medicinal chemistry, 163 high-density lipoprotein cholesterols, dyslipidemias, ASO liver-targeted therapies, cholesteryl ester transfer protein inhibition of, 605 Hoogsteen base-pairing, peptide nucleic acids, 510–512 human immunodeficiency virus patients, clinical safety experiments with gen-1/gen-2 ASOs, 367–369 human tumor xenografts, antisense oligonucleotides, pharmacokinetics, animal studies, 310–311 Huntington’s disease, antisense oligonucleotide therapy, 734 Hutchinson-Gilford progeria syndrome, splice switching oligonucleotide targeting, 97 hybridization antisense drug action, 7 activity correlation with secondary structure, 120–122 facilitated hybridization, RNA targeting, 14–15 locked nucleic acids, 530–532, 536–537 occupancy-activated destabilization, off-target effects, 34–35 hydroxyproline backbone, oligonucleotide medicinal chemistry, 153 hyperalgesia, inflammatory disease therapy, 684–685 hypercholesterolemia, ISIS 301012 ApoB inhibitor, 623–626 hypersensitivity reactions, clinical safety experiments with gen-1/gen-2 ASOs, 385–386 hypertension, antisense oligonucleotide inhibitors, 628

I identity testing, 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 422 IL-1 receptor associated kinase (IRAK)-1, inflammatory disease antisense therapy, 686–687 immune response, short interfering RNA reduction of, 446–447 immune stimulation liposomal nucleic acid delivery systems, 258–259 oligodeoxynucleotides, 751–753 oligoribonucleotide ligand identification, 760–762

toxicological properties of chimeric antisense oligonucleotides, proinflammatory effects, 347–351 immune surveillance, inflammatory disease antisense therapy, 688–689 immunofluorescence staining, human RNase H2 biochemistry, 61–63 immunogenicity, liposomal nucleic acid drug delivery systems, 259–260 immunomodulation inflammatory disease antisense therapy, 682–683 cellular and molecular pharmacology, 688–689 oligodeoxynucleotides, 751–753 immunoprecipitation assay, human RNase H2 biochemistry, 57–61 impurity tests and assays, 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 422–430 indole compounds, global splicing strategies, 104 infectious disease CpG oligodeoxynucleotides, 755–758 morpholino rapid response, 571–572 peptide nucleic acid anti-infective agents, 512–513 inflammatory bowel disease (IBD) antisense therapies, preclinical applications, 671 intracellular adhesion molecule antisense therapy, 667–668 pathology and current therapy, 667 inflammatory disease antisense therapy development, 666 (See also specific diseases) cellular and molecular pharmacology, 684–689 defined, 666 future research issues, 689–690 intraocular inflammation, ophthalmology therapy, 593–594 2⬘-methoxyethyl oligonucleotide pharmacology in, 288–289 preclinical in vivo pharmacology models, 681–685 inhibitors of apoptosis (IAP) gene, cancer therapy BCL2/BCL-xL development, 701–704 survivin and XIAP development, 704–705 in situ hybridization, neurological diseases, ASO distribution, 725–726 insulin growth factor binding proteins (IGFBPs) cancer therapy, 710 glioblastoma antisense therapy, 736 intercellular adhesion molecule (ICAM)-1 antisense Crohn’s disease therapy, 667–668 immunomodulation and transplantation, 682–683 preclinical applications, 671 rheumatoid arthritis therapy, 672–674 interferons CpG dinucleotide motifs, toll-like receptors and mechanism of action, 749–751 immunomodulation and immune stimulation effects, 751–753 infectious disease monotherapies, 755–757 liposomal nucleic acid delivery encapsulation, immune stimulation, 258–259 multiple sclerosis therapy, 675 interleukins, asthma antisense therapy and, 680–682

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Page 810

810 internal ribosome entry (IRE), translation arrest and, 25–26 intracellular delivery systems, liposomal drug delivery, helper lipids, 240 intraocular inflammation, ophthalmology therapy, 593–594 intratracheal administration, pulmonary drug delivery systems, 225 intravenous infusion antisense oligonucleotide delivery, 219 clinical safety experiments complement activation, 375–377 constitutional symptoms, 377–383 continuous infusion (14- and 21-day), 381 dosage and scheduling studies, 369–370 twenty-four-hour infusions, 381 two-hour infusions, 378–381 toxicologic effects of ASOs, 334 intravitreal injection, antisense oligonucleotide delivery, 221–222 clinical safety experiments, 372, 393–394 intron characteristics, antisense drug activity and, 24 intronic splicing enhancers (ISEs), pre-mRNA splicing, 91–92 intronic splicing silencers (ISSs), pre-mRNA splicing, 91–92 in vitro characterization antisense oligonucleotide screening, 131–132 follow-up assays, 134–138 chemical class effects on, 25 ISIS 301012 ApoB inhibitor, 616–618 locked nucleic acids, 540–545 2⬘-methoxyethyl oligonucleotide pharmacology, 279–282 micro-RNA pathways, 456 peptide nucleic acids, 514–515 RISC assembly, 81–82 RNA interference drug development, 466–470 splicing mechanisms in, 20–21 toxicologic effects of ASOs, hERG assay, 354–356 in vivo characterization antisense oligonucleotide screening, follow-up assays, 135–138 ApoB-100 inhibition therapies, species-specific pharmacology, 612–613 aptamer structures, 786–788 chemical class effects on, 25 coronary stent restenosis insufficiency, morpholinos and, 573–574 inflammatory disease antisense therapy, 682–685 locked nucleic acid pharmacology, animal studies, 546–547 2⬘-methoxyethyl (MOE)-modified oligonucleotide pharmacology, 282–290 adipose tissue, 287 bone tissue, 287–288 gapmer administration, 289–290 kidney, 286–287 liver, 285–286 lymphoid tissues and inflammatory cells, 288–289 oncology models, 289 RNA interference delivery systems, 471–478

INDEX antibodies, 478 conjugated compounds, 474–475 liposomes and lipoplexes, 475–477 naked siRNA, 471–474 peptides and polymers, 477–478 splice switching oligonucleotide targeting, β-thalassemia, 94 splicing mechanisms in, 20–21 ion chromatography, 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 424–430 iontophoresis, antisense oligonucleotide delivery, 222 IP-HPLC-ES-MS chromatography, 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 429–430 IP-HPLC-UV chromatography, 2⬘-methoxyethyl (MOE)modified oligonucleotide analysis, 424–430 ISIS 3521, cancer therapy, 705–706 ISIS 5132, cancer therapy, 705–706 ISIS 36945, asthma therapy, 679 ISIS 301012 cardiovascular therapies ApoB-100 inhibition, 611–626 combined lipid-lowering agent regimens, 621–626 hypercholesterolemia treatment, 623–626 duration of action, 317 onset of action, 316–317 plasma trough concentration-effect determination, 317–318 subchronic dosing, 318–320 toxicology of, 335 ISIS 13312 antiviral, ophthalmology therapy, 591–592 ISIS 104838 compound pharmacokinetics and pharmacodynamics, 321 rheumatoid arthritis therapy, 672–674 ISIS 113715 compound clinical trials, 657–659 metabolic disease and, drug discovery protocol, 642–644 obesity effects of, 652–653 pharmacokinetics, 657–658 pharmacology, 658–659 second-generation antisense oligonucleotide optimization, gap size effects, rodent studies, 497–500 type 2 diabetes, 645–646 ISIS 333611 compound, amyotrophic lateral sclerosis antisense therapy, 731–734

K (KFF)3K peptide, anti-infective agents, 512–513 kidney cardiovascular ASO therapies and, 604 clinical safety experiments with gen-1/gen-2 ASOs and, 386–389 2⬘-methoxyethyl oligonucleotide pharmacology in, 286–287 toxicological effects of ASOs in, 340–345 type 2 diabetes, antisense therapeutic targeting of, 651–652

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Page 811

INDEX

811

kinetic RT-PCR assay, antisense oligonucleotide design, 129–130 Kupffer cell hypertrophy, toxicological effects of ASOs and, 345–346

L lamin A protein, splice switching oligonucleotide targeting, 96–97 large unilamellar vesicles (LUVs), liposomal drug delivery systems nucleic acids, 238–239 passive nucleic acid encapsulation, 244–246 Lewy body dementia, antisense therapies, 737–738 LIC-101 complex, synthetic short interfering RNA, systemic drug administration, 452–453 linker materials 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 406–407 peptide nucleic acid, 508 “Lipinski Rules,” small molecule pharmaceuticals, 437–438 lipophilic conjugates, oligonucleotide medicinal chemistry, 167–168 lipophilic 2⬘-O-alkyl modifications, oligonucleotide medicinal chemistry, 156–157 lipoplex systems nucleic acid drug delivery, encapsulation technologies, 242–251 RNA interference drug development, 475–477 liposomal delivery formulations nucleic acids active targeting strategies, 242 analytical methods, 251–254 basic properties, 237–239 cationic lipids, 239–240 efficacy evaluation, 261–262 encapsulation technologies, 242–251 analytical methods, 253–254 ethanol-destabilized liposomes, 247–248 ethanol drop (SALP) method, 247 passive encapsulation, 243–246 reverse-phase evaporation, 248–249 spontaneous vesicle formation by ethanol dilution method, 249–251 immune stimulation, 258–259 immunogenicity, 259–261 intracellular delivery, helper lipids and, 240 liposome constituents, 239–242 pharmacology, 254–262 polyethylene glycol lipids, 241–242 systemic administration, 254–256 toxicity issues, 256–258 ophthalmology therapies, 595 RNA interference drug development, 475–477 liver, drug targeting of. See also hepatic tissue cardiovascular ASO therapies, 604 ApoB-100 inhibition therapies, hepatic steatosis effects, 614–616 ISIS 301012 ApoB inhibitor, 624–626

clinical safety experiments with gen-1/gen-2 ASOs and, 391–393 dyslipidemias, antisense oligonucleotide therapies, 605–626 acyl-coenzyme A;cholesterol acyltransferase 2 inhibition, 608–610 ApoB-100 inhibition, 611–626 apolipoprotein C-III inhibition, 606–608 cholesterylester transfer protein inhibition, 605 Lp(a) inhibition, 605–606 2⬘-methoxyethyl oligonucleotide pharmacology, 285–286 nonalcoholic steatohepatitis, 655–656 toxicological effects of ASOs and, 345–346 type 2 diabetes, 648–651 local administration systems antisense oligonucleotides, 221–225 pharmacokinetics, animal studies, 310 clinical safety experiments with gen-1/gen-2 ASOs, 372, 393–394 gastrointestinal uptake mechanisms, 231–233 toxicologic effects of ASOs, 333–334 locked nucleic acid (LNA) biochemical properties, 537–540 biophysical properties, 532–537 chemistry, 521–528 chimeric antisense oligonucleotides, human RNase H1 and, 69–70 drug development, 557 evolution of, 520–521 future research issues, 557–558 liposomal drug delivery systems, immune stimulation, 258–259 oligonucleotide medicinal chemistry, 158–159 pharmacokinetics, 548–553 pharmacology, animal studies, 546–547 structure, 530–532 synthesis, 528–529 toxicology, 553–557 in vitro inhibition, 540–544 Loquacious (Loqs) protein, micro-RNA biogenesis, 79 low-density lipoprotein cholesterol (LDL-C) antisense oligonucleotide therapies, 601–602 current therapeutic limitations, 602–603 ISIS 301012 (ApoB-100), 316–320 oligonucleotide medicinal chemistry, 167–168 RNA interference drug development, 474–475 synthetic short interfering RNA, systemic drug administration, 449–451 Lp(a) lipoprotein particles, dyslipidemias, ASO liver-targeted therapies, 605–607 LY2181308/ISIS 23722, cancer therapy, 704–705 lymphoid tissue, 2⬘-methoxyethyl oligonucleotide pharmacology in, 288–289, 288(c)

M Macugen aptamer therapeutic agents, 788–790 in vivo characterization, 786–788

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Page 812

812 magnesium ions, human RNase H1 biochemistry, 49 manganese human RNase H1 biochemistry, 49 human RNase H2 biochemistry, 56–61 manufacturing process, 2⬘-methoxyethyl (MOE)-modified oligonucleotides, 403–419 deprotection, 415 detritylation, 417–418 freeze-drying, 419 future issues in, 430 oligonucleotide synthesis, 413–415 precipitation, 418 purification, 415–416 reagents, 408–410 solid-phase synthesis, purification, and isolation, 413–419 solid support, 410–411 solution preparation, 413 starting materials, 404–408 synthesizers, 411–413 yield and purity, 419 MAP kinase cascade, ophthalmology therapy, angiogenesis mechanisms, 593 mass spectrometry, 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 424–430 mast cells, asthma antisense therapy, 687–688 matrix metalloproteinases, morpholino-targeted cancer therapy, 574–575 MCL1 molecular target, antisense cancer therapy, 711 mediator release mechanisms, inflammatory disease antisense therapy, 687–688 messenger ribonucleoprotein particles (mRNP), facilitated hybridization, 14–15 messenger RNA (mRNA) locked nucleic acid inhibition, 540–544 2⬘-methoxyethyl oligonucleotides, multiple antisense mechanisms, 278–279 ophthalmology therapies angiogenesis mechanisms, 592–593 pharmacodynamics, 589–590 peptide nucleic acid targeting, 508–510 phosphorothioate oligonucleotides, pharmacokinetics, 308 animal studies, 309–316 short RNA-directed destabilization and translational repression, 82–83 short RNA silencing pathways, 458–459 toxicology of ASOs, off-target antisense effects, 331 metabolic diseases, antisense oligonucleotide drug development drug discovery schematic, 642–644 evaluation rationale for, 642 evolution of, 642 future research issues, 659 nonalcoholic steatohepatitis, 655–656 obesity drug discovery, 652–655 phase 1/2 clinical program overview, 657–659 type 2 diabetes, 644–652 hepatic glucose output inhibition, 648–649 kidney targeting, 651–652 protein phosphatase targeting, 644–647

INDEX tissue selectivity and pharmacokinetic properties, 649–651 transcription factor targeting, 647 metabolism antisense oligonucleotide toxicology, 330 gastrointestinal drug delivery systems, 225–226 morpholino redirection of, 575–576 2⬘-methoxyethyl (MOE)-modified oligonucleotides analytical testing, 419–430 drug substance intermediates, 421–422 identity testing, 422 impurity tests and assay, 422–430 reagents and solvents, 421 starting materials, 419–421 aptamer development, SELEX process, 776–778 asthma therapy, 679–682 cancer therapy, 704–705 STAT3, 709–710 cardiovascular therapies ApoB-100 inhibition therapies, species-specific pharmacology, 612–613 current protocols, 603–604 ISIS 301012 human ApoB inhibitotr, 616 cardiovascular therapies and, 603–604 chimeric antisense oligonucleotides human RNase H1 and, 65–70 toxicologic properties chronic administration, 351 clotting inhibition, 338–339 complement activation, 339–340 genetic effects, 353–354 hematopoietic effects, 345 kidney effects, 340–345 liver effects, 345–346 mechanisms and clinical correlates, 336–337 oral administration, 334–336 phosphorothioate comparisons, 328–329 plasma protein binding effects, 337–340 proinflammatory effects, 346–351 reproductive toxicology, 352–353 safety assessment strategies, 330–331 safety pharmacology, 354–356 sequence motifs, 329–330 single-strand ASO, siRNA, and aptamers, 328 species-specific effects, 356–357 systemic vs. local administration, 333–334 target organ accumulation and effect, 340–346 toxicokinetics, 332–333 clinical safety experiments future research issues, 394–395 kidney effects, 386–389 liver effects, 391–392 local adminstration protocols, 371, 393–394 proinflammatory effects, 377–386 hypersensitivity reactions, 385–386 infusion-associated symptoms, 377–383 subcutaneous injection site responses, 383–385 selectivity and reduction research overview, 365–367 subject characteristics and disease conditions, 367–369

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Page 813

INDEX systemic administration trials aPTT prolongation, 374–375 complement activation, 375–377 duration of treatment, 370–371 results, 371–392 schedules and dosages, 369–370 thrombocytopenia, 389–391 drug delivery systems gastrointestinal systems, 225–233 systemic administration systems, 218–219 evolution of, 402–403 human RNase H1, 51–52 inflammatory disease therapy, 666 hyperalgesia, 684–685 immunomodulation and transplantation, 682–683 manufacturing process, 403–419 deprotection, 415 detritylation, 417–418 freeze-drying, 419 future issues in, 430 oligonucleotide synthesis, 413–415 precipitation, 418 purification, 415–416 reagents, 408–410 solid-phase synthesis, purification, and isolation, 413–419 solid support, 410–411 solution preparation, 413 starting materials, 404–408 synthesizers, 411–413 yield and purity, 419 medicinal chemistry cationic 2⬘-O-alkyl modifications, 157 lipophilic 2⬘-O-alkyl modifications, 156–157 methylene(methylimino) backbone, 152 optimization strategies, 170–171 propyne modifications, 165 sugar modification, 156 metabolic diseases drug discovery schematic, 642–644 evaluation rationale for, 642 evolution of, 642 future research issues, 659 nonalcoholic steatohepatitis, 655–656 obesity drug discovery, 652–655 phase 1/2 clinical program overview, 657–659 type 2 diabetes, 644–652 hepatic glucose output inhibition, 648–649 kidney targeting, 651–652 protein phosphatase targeting, 644–647 tissue selectivity and pharmacokinetic properties, 649–651 transcription factor targeting, 647 metabolism, 307 neurological disorders antisense pain therapy, 735 delivery and distribution, 725–726 ophthalmology therapy, 586–587 antiviral compounds, 591–592 formulations, 595 pharmacodynamics, 589–590

813 pharmacokinetics, 587–588 tolerability parameters, 589 pharmacokinetic/pharmacodynamic properties animal studies, 308–316 duration of action, 312–314 multiple-dose regimens, 314 onset of action, 311–312 plasma-tissue linkage, accumulation and clearance, 314–316 target tissues, 308–311 basic principles, 307–308 in humans, 316–321 ISI 104838, tumor necrosis factor-a, 321 ISIS 301012, ApoB-100 compound, 316–320 OGX-011, clusterin antisense, 321–322 pharmacological properties evolution and development, 273–275 future research issues, 291–292 human pharmacology, 290–291 multiple antisense mechanisms, 277–279 phosphorothioate oligonucleotide comparisons, 276–277 structural properties, 275–276 in vitro properties, 279–282 in vivo concentrations, 282–290 adipose tissue, 287 bone tissue, 287–288 gapmer administration, 289–290 kidney, 286–287 liver, 285–286 lymphoid tissues and inflammatory cells, 288–289 oncology models, 289 pulmonary drug delivery systems, 223–225 second-generation antisense oligonucleotide optimization gapmer structures, 489–490 gap size effects, animal studies, 492–500 oligonucleotide length, 501–503 splice switching oligonucleotide assay, 104–105 5-methyl cytosine (5-MeC), antisense oligonucleotide toxicology, 329–330 3⬘-methylene phosphonate, oligonucleotide medicinal chemistry, 150 methylene(methylimino) (MMI) backbone, oligonucleotide medicinal chemistry, 152 2⬘-O- methyl and methoxyethyl modifications, oligonucleotide medicinal chemistry, 156 methyl phosphates, discovery of, 5–7 methylphosphonate, oligonucleotide medicinal chemistry, 151 micro-RNAs (miRNAs) antagomir silencing, 453–458 antisense therapeutics and, 10–12 locked nucleic acid inhibition, 544–545 mechanisms of, 39 2⬘-methoxyethyl oligonucleotides, multiple antisense mechanisms, 279 mRNA destabilization and translation repression and, 82–83 oligonucleotide medicinal chemistry, optimization strategies, 169–171

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Page 814

814 micro-RNAs (contd.) pathways for, 453–456 precursor biogenesis, 78–79 precursor maturation process, 79 silencing pathways, 77–79 microsomal triglyceride transfer protein (MTP), cardiovascular therapies ApoB-100 inhibition, 611 hepatic steatosis and, 614–616 midazolam metabolism, morpholino redirection of, 575–576 Millipore 8800 synthesizer, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 411–412 minimization algorithms, aptamer optimization, 780 mitochondrial localization signal (MLS), human RNase H1 and, 55 modification sites, oligonucleotide medicinal chemistry, 147 molecular mechanisms inflammatory disease antisense therapy, 685–689 locked nucleic acid, 521–522 RNA interference, 438–439 monkey studies, ApoB-100 inhibition therapies, 613–616 morpholinos alterned RNA splicing, Duchenne muscular dystrophy, 576–577 antibacterial applications, 572 antiviral applications, 570–572 basic properties, 565–566 cancer applications, 574–575 cardiovascular applications, 572–574 formulations, 577 future research issues, 577 metabolic redirection, 575–576 pharmacokinetic profile, 569 phosphorodiaminidate backbone, oligonucleotide medicinal chemistry, 153 safety profile, 567–569 mucosal delivery systems, RNA interference drug development, 476–477 multilamellar vesicles (MLVs), liposomal drug delivery systems nucleic acid delivery, 238–239 passive nucleic acid encapsulation, 243–246 multiple-dose regimens, antisense oligonucleotides, pharmacokinetics, animal studies, 314 multiple sclerosis (MS) pathology and current therapy, 675 preclinical models of antisense therapy, 676–678 very late activation antigen (VLA)-4 antisense therapy, 674–676 murine pharmacology studies, ApoB-100 inhibition therapies, 612–616 muscular dystrophies antisense oligonucleotide therapy, 739–740 splice switching oligonucleotide targeting, 95 MyD88 protein splice switching oligonucleotide targeting, 99 splicing modulation, 21

INDEX

N nanosized materials, ophthalmology therapies, 595 natural ligands, RNA interference drug development, 475 nebulization formulas pulmonary drug delivery systems, antisense oligonucleotides, 223–225 toxicologic effects of ASOs, 333–334 N-ethylmaleimide (NEM), human RNase H1 biochemistry, 49 neurological disorders. See also specific diseases antisense oligonucleotide therapies current therapies, 721–723 distribution barriers, 723–726 future research issues, 740–741 safety and toxicity, 728–730 mechanisms of, 722–724 nucleic acid-based, nonantisense gene silencing, 726–728 neuropathies, antisense oligonucleotide therapy, 738–739 NF-ΚB signaling, inflammatory disease antisense therapy, 686–687 NittoPhase supports, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 411 nonalcoholic steatohepatitis, antisense drug discovery, 655–656 non-antisense activities, antisense oligonucleotide design and, 123 noncoding RNAs, intermediary metabolism, 9–12 nontarget genes, antisense oligonucleotide design and reduction of, 123 nonviral targeting strategies, synthetic short interfering RNA, 453 Northern blot assays, antisense oligonucleotide design, 130–131 nose-only administration, pulmonary drug delivery systems, 225 nuclear localization peptides, peptide nucleic acid conjugation, 512 nuclease resistance aptamer optimization, 781–782 locked nucleic acids, 537–539 nucleic acids covalent modifications, occupancy-activated destabilization, 38 liposomal delivery formulations active targeting strategies, 242 analytical methods, 251–254 basic properties, 237–239 cationic lipids, 239–240 efficacy evaluation, 261–262 encapsulation technologies, 242–251 analytical methods, 253–254 ethanol-destabilized liposomes, 247–248 ethanol drop (SALP) method, 247 passive encapsulation, 243–246 reverse-phase evaporation, 248–249 spontaneous vesicle formation by ethanol dilution method, 249–251 immune stimulation, 258–259 immunogenecity, 259–261

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Page 815

INDEX

815

intracellular delivery, helper lipids and, 240 liposome constituents, 239–242 pharmacology, 254–262 polyethylene glycol lipids, 241–242 systemic administration, 254–256 toxicity issues, 256–258 nonantisense gene silencing, neurological disorders, 726–728 sequences, specificity mechanisms, 13–14 nucleoside-loaded solid support, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 406–407 nucleoside phosphoramidites and precursors, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 404–405 impurities in, 405–406 number-surface mean diameter, particle size measurement, liposomal delivery systems, 251–253

O obesity, antisense drug discovery, 652–655 oblimersen clinical safety experiments, subcutaneous injection, 383–385 G3139 antisense compound, cancer therapy, 701–704 occupancy-activated destabilization, antisense drug action, 26–39 5⬘ capping, 27 double-strand RNase (siRNA), 31–38 oligonucleotide-induced RNA cleavage, 38 3⬘-polyadenylation inhibition, 27 RNase H mechanism, 27–31 RNase L-mediated cleavage, 38–39 target nucleic acids, covalent modifications, 38 “occupancy-mediated antisense interference,” secondgeneration antisense oligonucleotide optimization, 487–488 occupancy-only mechanisms antisense drugs, 19–26 oligonucleotide medicinal chemistry, design strategies, 170–171 ocular albinism type 1 gene, splice switching oligonucleotide targeting, 97–98 ocular drug delivery. See also ophthalmology therapies antisense oligonucleotides, 221–222 clinical safety experiments with ASOs, 372, 393–394 RNA interference drug development clinical trials, 478–479 “naked” siRNA, 471–472 toxicologic effects of ASOs, 333–334 ocular pressure, ASO compounds for, 594–595 off-target silencing RNA interference drug development, specificity criteria, 468 short interfering RNA specificity, 445–446 toxicology of ASOs, 331

OGX-011 compound cancer therapy, 707–708 clinical safety experiments, two-hour infusion profile, 378–381 pharmacokinetics and pharmacodynamics, 321–322 Okazaki fragments, human RNase H1 and, 55 oligodeoxynucleotides (ODNs) CpG structure-activity relationships, 753–755 CpG therapeutic applications, 755–760 asthma/allergy, 759 cancer therapies, 758–759 infectious disease monotherapy, 755–757 infectious disease vaccines, 757–758 safety profiles, 759–760 future research issues, 762 immune activation, 748–751 immune modulatory classifications, 751–753 S-class characteristics, 755 oligonucleotides affinity mechanisms, 12–13 antisense, terminating mechanisms, 47–48 antisense theory and, 5 backbone modifications, 148–154 basic properties, 145–147 conjugates, 167–168 heterocyclic modifications, 163–167 length parameters, second-generation antisense oligonucleotide optimization, 501–502 medicinal chemistry, 5–7, 144 affinity limitations, 145–146 cost-competitive manufacture, 147 drug optimization, 169–171 aptamer designs, 171 gapmer designs, 169 occupancy only designs, 170–171 siRNA designs, 170 future research and applications, 171–172 pharmacokinetics, 144–146 receptor specificity, 146 stability limitations, 144 therapeutic index criteria, 147 modification sites, 147 specificity, 13–14 sugar modifications, 154–163 bicyclic sugars, 158–161 furanose substitution positions, 158 ribofuranose sugar, 161–163 2⬘-modifications, 154–157 target RNA cleavage, 38 therapeutic specificity (therapeutic index), 19 oligoplex systems, nucleic acid drug delivery, encapsulation technologies, 242–251 oligoribonucleotide (ORN) ligands future research issues, 762 immune stimulatory effects, TLR7 and TLR8, 760–761 oncology models, 2⬘-methoxyethyl oligonucleotide pharmacology, 289–290

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Page 816

816

INDEX

onset of action mechanisms antisense oligonucleotide pharmacokinetics, animal studies, 311–312 ISIS 301012 (ApoB-100), 316–317 ophthalmology therapies, antisense oligonucleotides angiogenesis, 592–593 antiviral therapies, 591–592 classification, 586–587 drug delivery options, 595 formulations, 595 future research and applications, 595–596 glaucoma, 594–595 intraocular inflammation, 593–594 pharmacodynamics, 589–590 pharmacokinetics, 587–588 research background, 585–586 tolerability, 588–589 optimal RNA target sites, translation arrest and, 25–26 optimization strategies aptamer development, 779–784 locked nucleic acid synthesis, 528–529 oligonucleotide medicinal chemistry, 169–171 second-generation antisense oligonucleotides basic principles, 487–488 future research issues, 503–504 gapmer designs, 488–490 gap size limitations of, 500–501 monkey studies, 500 RNase H1, 490–492 rodent studies, 492–500 oligonucleotide length, 501–503 oral delivery systems antisense oligonucleotides local GI uptake, 231–233 permeability, 227–228 presystemic metabolism, 225–226 systemic bioavailability, 228–231 toxicology of ASOs and, 334–336 organ accumulation effects second-generation antisense oligonucleotide optimization, gap size effects, rodent studies, 494–500 toxicology of ASOs and, 340–346 osmotic nephrosis, toxicological effects of ASOs in kidney and, 342–345 overexpression mechanisms, human RNase H2 biochemistry, 56–61 OX26 monoclonal antibody, brain drug delivery system, 221

P packed-bed supports, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 410–411 pain management antisense oligonucleotide therapy, 735 inflammatory disease therapy, 684–685 par-1 locus, RNA silencing studies, 76

particle size measurement, liposomal delivery systems, nucleic acid analysis, 251–253 Pasha enzyme, micro-RNA biogenesis, 11–12, 78–79 passive nucleic acid encapsulation, liposomal drug delivery systems, 243–246 pathogen-associated molecular pattern (PAMP) receptors, toxicological properties of chimeric antisense oligonucleotides, proinflammatory effects, 347–351 “pattern recognition receptors” (PRRs), CpG dinucleotide motifs, toll-like receptors and mechanism of action, 749–751 PAZ domain, Argonaute protein structure, 80–81 pegaptanib aptamer therapeutic agents, 788–790 ophthalmology therapy, 586–587 tolerability parameters, 588–589 in vivo characterization, 786–788 PEGylation technology, aptamer optimization, 783–784 in vivo characterization, 786–788 PEPCK enzyme, type 2 diabetes, antisense inhibition, 648–649 peptide nucleic acid (PNA) anti-infective agents, 512–513 cellular delivery, 513–514 basic properties, 507–508 chemistry, 508 dsDNA targeting, 510–512 future applications, 515 mRNA targeting, 508–510 neurological diseases, ASO distribution, 723–726 oligonucleotide medicinal chemistry, backbone substitution, 154 topical drug delivery systems, 222–223 in vivo bioavailability, 514–515 peptide structures, RNA interference drug development, 477–478 percutaneous coronary intervention (PCI), restenosis, antisense inhibitors of, 628–629 percutaneous transluminal coronary angioplasty (PTCA), morpholino therapies, 572–574 peripheral blood mononuclear cells (PBMCs) immune response, short interfering RNA reduction of, 446–447 inflammatory disease therapy, immunomodulation and transplantation, 684 permeability, gastrointestinal drug delivery systems, 227–228 permeation enhancers gastrointestinal drug delivery systems, 227–231 topical drug delivery systems, 222–223 pharmacodynamics antisense oligonucleotides animal studies, 308–316 duration of action, 312–314 multiple-dose regimens, 314 onset of action, 311–312 plasma-tissue linkage, accumulation and clearance, 314–316 target tissues, 308–311 in humans, 316–321

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Page 817

INDEX ISI 104838, tumor necrosis factor-a, 321 ISIS 301012, ApoB-100 compound, 316–320 OGX-011, clusterin antisense, 321–322 ophthalmology therapies, 589–590 pharmacokinetics antisense oligonucleotides absorption, 191–192 animal studies, 308–316 distribution, 192–198 excretion, 202–206 metabolism, 198–202, 307 methoxyethyl modifications, 188–190 ophthalmology therapies, 587–588 phosphorothioate backbone, 185–188 research background, 184 treatment regimen applications, 206–211 type 2 diabetes therapy, 649–651 gastrointestinal drug delivery systems, 228–231 ISIS 113715 compound, 657–658 liposomal nucleic acid delivery formulations, 254–256 locked nucleic acid biodistribution and tissue half-life, 548–551 cell uptake, 551–552 excretion, 552–553 plasma, 548 morpholinos, 569 oligonucleotide medicinal chemistry, 144–146 toxicology of ASOs and, 332–333 pharmacology inflammatory disease antisense therapy, 682–689 liposomal delivery formulations, nucleic acids, 254–262 locked nucleic acids, animal studies, 546–547 oligonucleotide medicinal chemistry, 146 pulmonary drug delivery systems, 224–225 toxicology of ASOs exaggerated pharmacology strategy for, 330–331 safety pharmacology, 354–356 pharmacophore, development of, 5–7 phenoxazine modification, oligonucleotide medicinal chemistry, 165–166 phenylacetyl disulfide (PADS) reagent, 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 420–421 manufacturing, 407–408 pH levels, liposomal drug delivery systems, nucleic acids, 239–240 phosphatases, type 2 diabetes, novel antidiabetic target therapies, 646–647 phosphodiester oligonucleotides dyslipidemias, ASO liver-targeted therapies, cholesteryl ester transfer protein ihibition of, 605 local administration, in brain, 221 phosphonoacetate, oligonucleotide medicinal chemistry, 150 phosphonoformate, oligonucleotide medicinal chemistry, 150 phosphoramidate backbone, oligonucleotide medicinal chemistry, 150

817 phosphoramidites locked nucleic acid synthesis, 528–529 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 419–420 short interfering RNA chemical synthesis, 446–448 phosphorodiaminidate-linked morpholino oligomers (PMOs) alterned RNA splicing, Duchenne muscular dystrophy, 576–577 antibacterial applications, 572 antiviral applications, 570–572 basic properties, 565–566 cancer applications, 574–575 cardiovascular applications, 572–574 formulations, 577 future research issues, 577 metabolic redirection, 575–576 pharmacokinetic profile, 569 safety profile, 567–569 phosphorodithioate backbone, oligonucleotide medicinal chemistry, 149 phosphorothioate cyclooxygenase, rheumatoid arthritis antisense therapy, 674–675 phosphorothioate oligodeoxynucleotides (PS-ODNs). See also oligodeoxynucleotides (ODNs) asthma therapy, 679–682 cancer therapy, 701–704 cardiovascular therapies, 603–604 chemical structure, 306–307 chimeric antisense oligonucleotides, human RNase H1 and, 65–70 clinical safety experiments, antisense oligonucleotides (ASOs) future research issues, 394–395 kidney effects, 386–389 liver effects, 391–392 local adminstration protocols, 371, 393–394 proinflammatory effects, 377–386 hypersensitivity reactions, 385–386 infusion-associated symptoms, 377–383 subcutaneous injection site responses, 383–385 selectivity and reduction research overview, 365–367 subject characteristics and disease conditions, 367–369 systemic administration trials aPTT prolongation, 374–375 complement activation, 375–377 duration of treatment, 370–371 results, 371–392 schedules and dosages, 369–370 thrombocytopenia, 389–391 gastrointestinal drug delivery systems, 228–231 impurity tests and assays, 423–430 inflammatory disease therapy, 666 hyperalgesia, 684–685 local administration, in brain, 221 locked nucleic acids, 537–539 medicinal chemistry, 5–7 modification, 148–149

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Page 818

818 phosphorothioate oligodeoxynucleotides (contd.) metabolism, 307 ophthalmology therapies, 589–595 pharmacokinetic/pharmacodynamic properties animal studies, 308–316 in humans, 316–321 physical and chemical properties, system administration systems, 218–219 pulmonary drug delivery systems, 223–225 restenosis inhibition, 629 rheumatoid arthritis antisense therapy, 674–675 toxicologic effects class-related toxicologic properties, 336–338 2-O-methoxyethyl chimeric ASO vs., 328–329 organ accumulations, 340–346 proinflammatory effects, 347–351 reproductive systems, 352–353 safety pharmacology, 354–356 toxicokinetic properties, 332–333 toxicology of ASOs and, oral administration of, 334–336 Pick’s disease, splice switching oligonucleotide targeting, 96 PIWI domain Argonaute protein structure, 80–81 RNAi pathways, 439 plasma binding proteins, toxicology of ASOs, acute and transient changes, 337–340 plasma disposition, antisense oligonucleotides, pharmacokinetics, animal studies, 314–316 plasma pharmacokinetics, locked nucleic acid, 548 plasma trough concentration-effect determination, ISIS 301012 (ApoB-100), 317–318 platelet function clinical safety experiments with gen-1/gen-2 ASOs and, 389–391 toxicological properties of ASOs and, 346 p75 low-affinity neurotrophin receptor multiple sclerosis antisense therapy, 676–677 neurological diseases, ASO distribution, 723–726 PNA oligomers, 99, 104–105 polyadenylation regions, splice switching oligonucleotide targeting, 100 3⬘-Polyadenylation inhibition, occupancy-activated destabilization, 27 polyethylene glycol (PEG) lipids aptamer optimization, 783–784 liposomal drug delivery systems immunogenicity, 259–260 nucleic acids, 241–242 polyethyleneimine (PEI) formations RNA interference drug development, 477–478 synthetic short interfering RNA, systemic drug administration, 451–453 polymer systems, RNA interference drug development, 477–478 polymorphism, antisense oligonucleotide design, gene sequence variants, 123 posthybridization, antisense drug action, 7 posttranscriptional gene silencing (PTGS), small interfering RNAs, 76–77 posttranscriptional modification, RNA, 15–16

INDEX potency studies morpholinos, 569 formulation and enhancement technologies, 577 RNA interference drug development, 467–468 second-generation antisense oligonucleotide optimization, 500–501 pouchitis disease activity index (PDAI), alicaforsen therapy, 670–671 P50 protein, facilitated hybridization, 14–15 precipitation, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 418 preclinical applications aptamer therapeutics, 793–794 asthma antisense therapy, 679–682 inflammatory bowel disease antisense therapy, 671 inflammatory disease antisense therapy, in vivo characterization, 682–685 multiple sclerosis antisense therapy, 676–677 rheumatoid arthritis antisense therapy, 674–675 pre-mir transcript, micro-RNA biogenesis, 11–12 pre-mRNA RNA intermediary metabolism, 7–12 splicing mechanisms, 90–92 pre-/non-hybridization, antisense drug action, 7 presenilin 1 complex, dementia antisense therapies, 738 presystemic metabolism, gastrointestinal drug delivery systems, 225–226 primary cell assays, antisense oligonucleotide design, 131 primary miRNA (pri-miRNA), biogenesis, 78–79 prion diseases antisense oligonucleotide therapy, 736–737 splice switching oligonucleotide targeting, 96 progressive supranuclear palsy (PSP), splice switching oligonucleotide targeting, 96 proinflammatory effects clinical safety experiments with gen-1/gen-2 ASOs, 377–386 hypersensitivity reactions, 385–386 infusion-associated symptoms, 377–383 subcutaneous injection site responses, 383–385 ophthalmology therapy, tolerability parameters, 589 toxicological properties, chimeric antisense oligonucleotides, 346–351 propyne modifications, oligonucleotide medicinal chemistry, 164–165 prostaglandins compounds, inflammatory disease therapy, hyperalgesia, 684–685 prostate-specific membrane antigen (PSMA) splice switching oligonucleotide targeting, 98 synthetic short interfering RNA, systemic drug administration, 450–451 protamine-antibody fusion protein, synthetic short interfering RNA, systemic drug administration, 452–453 protein binding, 14–15 protein kinase C-α antisense cancer therapy, 705–706 pain therapy, 735 protein kinase R (PKR) activating protein (PACT), micro-RNA biogenesis, 79

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Page 819

INDEX

819

protein phosphatases, type 2 diabetes, 644–647 protein tyrosine phosphatase 1B (PTP-1B) metabolic disease, drug discovery protocol, 642–644 obesity, ISIS 113715 compound effects, 652–653 type 2 diabetes, 644–647 prothrombin time (PT) systemic ASO therapy, clinical safety experiments, 374–375 toxicology of ASOs and, 338–339 proximal tubule morphology, toxicological effects of ASOs in, 341–345 PrPres protein, prion disease antisense therapy, 736–737 PSMA protetin, aptamer binding, 786 PTEN cell lines antisense oligonucleotides pharmacokinetics, animal studies, 309–311 screening, 131–134 oligonucleotide medicinal chemistry peptide nucleic acid backbone substitution, 154 propyne modifications, 165 second-generation antisense oligonucleotide optimization, gap size effects, rodent studies, 492–500 type 2 diabetes, 644 antisense targeting of, 644–647 PTM RNA, trans-splicing mechanisms, 102 pulmonary drug delivery systems antisense oligonucleotides, 223–225 RNA interference drug development clinical trials, 480 “naked” siRNA, 472–474 toxicologic effects of ASOs, 333–334 purification, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 415–416 yield and, 419 purine modifications antisense pain therapy, 735 locked nucleic acid synthesis, amidites, 524 oligonucleotide medicinal chemistry, 166–167 peptide nucleic acids, 510–512 pyrimidine modifications locked nucleic acid synthesis, amidites, 524 oligonucleotide medicinal chemistry, 163–166 peptide nucleic acids, 510–512

Q quantitative reverse transcriptase polymerase chain reaction (qRT-PCR), antisense oligonucleotide design, 130–131 quantitative whole-body autoradiography (QWBA), aptamer in vivo characterization, 787–788 “quelling” silencing phenomenon, small RNA systems, 75–76

R RAF1 antisense, cancer therapy, 705–706 ranibizumab, ophthalmology therapy, tolerability parameters, 589

RANK protein expression, 2⬘-methoxyethyl oligonucleotide pharmacology, bone tissue, 286–287 RAS ongene family, antisense cancer therapy, 711 reagents, 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 421 manufacturing, 407–410 receptor signaling, inflammatory disease antisense therapy, 686–687 REG1 aptamer, 792–793 relative standard deviation (RSD), 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, 429–430 renal function clinical safety experiments with gen-1/gen-2 ASOs and, 386–389 toxicological effects of ASOs in kidney and, 341–345 renin-angiotensin system (RAS), antisense inhibitors and, 628 reproductive systems, toxicological effects of ASOs on, 352–353 respiratory syncytial virus (RSV), RNA interference drug development, clinical trials, 480 restenosis antisense inhibitors of, 628–629 morpholinos and, 573–575 retention enema formula, antisense oligonucleotide delivery and, 232–233 reverse-phase evaporation, nucleic acid encapsulation, liposomal delivery formulations, 248–249 reverse-phase HPLC, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, purification process, 416–417 reverse transcriptase polymerase chain reaction (RT-PCR) primers, antisense oligonucleotide design, 119 kinetic RT-PCR, 129–130 rheumatoid arthritis antisense therapies cell migration and adhesion, 686 cell proliferation, maturation, and survival, 685 clinical evaluation, 672–674 gene expression and receptor signaling, 686–687 preclinical applications, 674–675 pathology and current therapy, 672 Ribofuranose sugars, oligonucleotide medicinal chemistry, 161–163 ribonuclease protection assays, antisense oligonucleotide design, 130–131 ribonucleic acid (RNA) antisense drugs, 12–13 antisense theory and, 5 chemical synthesis, 446–448 intermediary metabolism, 7–12 locked nucleic acid inhibition, 540–544 posttranscriptional modifications, 15–16 structural disruption, 26 targeting of, 14–15 ribonucleotide reductase (RNR), cancer therapy, 710–711

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Page 820

820 ribozymes, neurological disorders, nonantisense gene silencing, 726–727 ring structures, oligonucleotide medicinal chemistry, 162–163 RNA-binding domain (RNA-BD), human RNase H1 structure and enzymology, 49–52 RNA-induced silencing complex (RISC). See also Silencing pathways assembly mechanisms, 81–82 facilitated hybridization, 14–15 2⬘-methoxyethyl oligonucleotides, multiple antisense mechanisms, 278–279 micro-RNA biogenesis, 11–12 occupancy-activated destabilization, 33–35 ophthalmology therapies, 586–587 RNAi enzymes and, 79–80 RNA interference drug development, potency criteria, 467–468 RNAi pathways, 438–439 small interfering RNA strand selection, 77 RNA-induced transcriptional silencing (RITS), micro-RNA biogenesis, 11–12 RNA interference (RNAi) chemical modifications, 439–443 clinical trials, 478–480 discovery and development evolution of, 465–466 in vitro selection of lead candidates, 466–470 duplex oligoribonucleotides, 12 future research and applications, 480–481 liposomal drug delivery systems, immune stimulation, 258–259 2⬘-methoxyethyl oligonucleotides, multiple antisense mechanisms, 278–279 molecular mechanism, 438–439 neurological disorders, nonantisense gene silencing, 727–728 oligonucleotide medicinal chemistry, 156 therapeutic applications, 437–438 immune response reduction, 446 in vivo delivery systems, 471–478 antibodies, 478 conjugated compounds, 474–475 liposomes and lipoplexes, 475–477 naked siRNA, 471–474 peptides and polymers, 477–478 RNA primers, human RNase H1 and, 55 RNase H enzymes antisense therapeutics and, 70–71, 586–587 characeristics, 48 human RNase H1 biochemical properties, 49 biological roles, 55 catalytic domain, 52–54 chimeric antisense oligonucleotide activity, 65–70 genomics and regulation, 56 RNA-binding domain, 50–52 second-generation antisense oligonucleotide optimization, gap size effects, 490–492 structure and enzymology, 49–54

INDEX human RNase H2 biological roles, 61–63 genomics and regulation, 63 structure and enzymology, 56–61 human RNases H, 48–49 DNA-like antisense oligonucleotides, 63–65 locked nucleic acid recruitment, 539–540 2⬘-methoxyethyl oligonucleotides first-generation pharmacological comparisons, 273–277 multiple antisense mechanisms, 277–279 occupancy-activated destabilization, 27–31 double-stranded RNase vs., 32–33, 36 enzymology of, 30 robustness of, 30–31 target site accessibility, 28–29 oligonucleotide medicinal chemistry, 146 furanose substitution positions, 158 peptide nucleic acid, mRNA targeting, 509–510 phosphorothioate oligonucleotides, pharmacokinetics, 308 second-generation antisense oligonucleotide optimization, 488 future research issues, 503–504 gapmer structures, 488–490 oligonucleotide length, 501–503 RNase III enzymes, second-generation antisense oligonucleotide optimization, 488 RNase L cleavage mechanism, occupancy-activated destabilization, 38–39 RNA splicing, morpholino alteration of, 576–577 robustness RNase H mechanism, 30–31 translation arrest, 26 RP-HPLC UV chromatography, 2⬘-methoxyethyl (MOE)-modified oligonucleotide analysis, phosphoramidites, 419–420

S safety profiles antisense oligonucleotides metabolic diseases, 657–658 neurological diseases, 728–730 cardiovascular therapy, ISIS 301012 ApoB inhibitor, 619–626 clinical safety experiments future research issues, 394–395 kidney effects, 386–389 liver effects, 391–392 local adminstration protocols, 371 results, 393–394 proinflammatory effects, 377–386 hypersensitivity reactions, 385–386 infusion-associated symptoms, 377–383 subcutaneous injection site responses, 383–385

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Page 821

INDEX selectivity and reduction research overview, 365–367 subject characteristics and disease conditions, 367–369 systemic administration trials aPTT prolongation, 374–375 complement activation, 375–377 duration of treatment, 370–371 results, 371–392 schedules and dosages, 369–370 thrombocytopenia, 389–391 CpG oligodeoxynucleotides, 759–760 morpholinos, 567–569 toxicology of ASOs, 330–331 pharmacological aspects, 354–356 SARS virus, morpholino rapid response to, 571 S-class oligodeoxynucleotides (ODNs), 755 screening processes antisense inhibitor identification, 16–19 antisense oligonucleotides (ASOs), primary assays, 128–134 activity assay, 128–134 kinetic RT-PCR, 129–130 positive and negative controls, 134 “selective” benzylation, locked nucleic acid synthesis, amidites, 522–524 selectivity mechanisms, antisense drugs, 12–19 affinity, 12–13 facilitated hybridization, 14–15 inhibitor screening and identification, 16–19 nucleic acid sequence specificity, 13–14 posttranscriptional RNA modifications, 15–16 protein binding and RNA targeting, 14 target RNA levels, 15 terminating mechanism, 15 therapeutic specificity (index), 19 type 2 diabetes, 649–651 SELEX process aptamer development binding and functional properties, 784–786 historical background, 774–776 optimization strategies, 779–784 therapeutic applications, 776–779 in vivo characterization, 786–788 pool composition, 776–778 pool design criteria, 776 positive and negative selection pressures, 778–779 sequence motifs antisense oligonucleotide toxicology, 329–330 morpholinos, 569 toxicological effects of ASOs, proinflammatory mechanisms, 349–351 sequence-specific hybridization-independent effects, toxicology of ASOs and, 331 serum creatinine levels, clinical safety experiments with gen-1/gen-2 ASOs and, kidney effects, 386–389 serum transaminases, clinical safety experiments with gen-1/gen-2 ASOs, liver function and, 391–392

821 SH2-domain containing inositol 5-phosphatase 2 (SHIP2), type 2 diabetes, 644 signaling cascade inflammatory disease antisense therapy, 686–687 ophthalmology therapy, angiogenesis mechanisms, 593 signal tranducer and activator of transcription (STAT3) cancer therapy, 709–710 immunomodulation and immune surveillance, 689 silencing pathways argonaute protein structure, 80–81 micro-RNAs, 77–79, 453–457 RISC system, 79–82 RNAi enzyme complex, 79–81 small interfering RNAs, 76–77 small molecule systems mRNA destabilization and translational repression, 82–83 overview, 75–76 “single overhang” design, short interfering RNAs (siRNAs), 443–445 single-strand antisense oligonucleotides ophthalmology therapies, 586–587 toxicologic effects, 328 single-strand RNA, oligoribonucleotide ligand identification, 760–762 site accessibility, RNase H mechanism and, 28–29 six-membered ring structures, oligonucleotide medicinal chemistry, 162–163 skin barriers, topical delivery systems, 222–223 small interfering RNAs (siRNAs) antisense therapeutics and, 10–12 ApoB-100 inhibition therapies, animal studies, 625–626 argonaute protein structure, 80–81 backbone modifications, 440 base modifications, 442–443 cancer therapy, HSP27 gene silencing, 709 chemical synthesis, 446–448 design, 443–445 enzyme complex, 79–81 global splicing strategies, 103 human RNase H2 biochemistry, 57–61 immune response reduction, 446 liposomal drug delivery systems efficacy analysis, 260–262 immune stimulation, 258–259 nucleic acids, 238–239 pharmacokinetics and biodistribution, 254–256 2⬘-methoxyethyl oligonucleotides, multiple antisense mechanisms, 278–279 neurological disorders, nonantisense gene silencing, 727–728 oligonucleotide medicinal chemistry cholesterol conjugates, 168 design strategies, 170 2⬘-fluoro modifications, 156 2⬘-O- methyl and methoxyethyl modifications, 156 ophthalmology therapies angiogenesis mechanisms, 592–593 classification, 587

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Page 822

822 small interfering RNAs (contd.) pharmacokinetics, 587–588 research background, 586 tolerability parameters, 589 RISC system, 79–80 RNA interference drug development antibodies, 478 clinical trials, 478–480 conjugation strategies, 474–475 liposomes and lipoplexes, 475–477 “naked” siRNA, 471–474 peptides and polymers, 477–478 potency criteria, 467–468 specificity criteria, 468 stability properties, 468–469 therapeutic applications, 468–470 RNAi pathways, 439 sliencing pathways, 76–77 specificity improvements, 445–446 RNA interference drug development, 468 sugar modifications, 440–442 systemic delivery complexation techniques, 451–453 conjugation techniques, 448–451 toxicologic effects, 328 small molecules. See also short interfering RNAs (siRNAs) global splicing strategies, 103–104 “Lipinski Rules” for, 437–438 RNA interference drug development, 475 small noncoding RNAs, intermediary metabolism, 10–12 small nuclear ribonucleoproteins (snRNPs), pre-mRNA splicing and, 90–92 small nuclear RNAs (snRNAs) hybridization of splicing with, 102–103 pre-mRNA splicing and, 90–92 small RNA systems, silencing pathways, overview, 75–76 SMN gene, spinal muscular atrophy antisense therapy, 739 SNAIL transcription repressor, morpholino-targeted cancer therapy, 575 sodium glucose transport protein 2 (SGLT2) second-generation antisense oligonucleotide optimization, 502–504 type 2 diabetes, antisense therapeutic targeting of, 651–652 solid-phase synthesis, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 413–419 deprotection, 415 detritylation, 417–418 freeze-drying, 419 precipitation, 418 purification, 415–417 solution preparation, 413–415 yield and purity, 419 solid support materials, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 410–411 solution preparation, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, solid-phase synthesis, 413–415 sparged reactor vessels, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 410–411

INDEX species sensitivity ApoB-100 inhibition therapies, 612–613 toxicological effects of ASOs, 350–351, 356–357 specificity aptamer development, 784–786 nucleic acid sequences, 13–14 oligonucleotide medicinal chemistry, 146 impurity tests and assays, 424–430 RNA interference drug development, 468 short interfering RNAs, improvements in, 445–446 therapeutic specificity (therapeutic index), 19 spinal muscular atrophy (SMA) antisense oligonucleotide therapy, 739 splice switching oligonucleotide targeting, 97 spliceosome, structure and function, 90–92 splice switching oligonucleotides (SSOs) basic properties, 93–94 defective gene function restoration, 94–98 natural alternative splicing modification, 98–100 therapeutic targets, 94–100 acetylcholinesterase, 99–100 bcl-x gene, 98 β-globin, 94 CD40 cell membrane protein, 99 cystic fibrosis transmembrane conductance regulator, 95–96 dystrophin, 95 lamin A protein, 96–97 MyD88 protein, 99 ocular albinism type 1 gene, 97–98 polyadenylation regions, 100 prostate-specific membrane antigen, 98 survival motor neurons, 97 tau protein, 96 Wilms’ tumor suppressor (WT1) gene, 99 splicing mechanisms. See also alternative splicing global strategies, 103–104 hybridization strategies, 100–103 modulation of, 19–25 antisense drug position, 24 B-cell lymphoma/leukemia cell x (Bc1-x), 20 β-globin RNA splicing, 20–21 chemical class influences, 25 dystrophin splicing alteration, 20 exonic enhancer/silencer binding, 25 intron/exon characteristics, 24 MyD88 protein, 21 signal strength, 21–24 pre-mRNA, 90–92 RNA intermediary metabolism, 8–12 splice-site position, 24 splice-site strength, 21–24 splice switching oligonucleotides assay, 104–105 basic properties, 93–94 defective gene function restoration, 94–98 natural alternative splicing modification, 98–100 spontaneous vesicle formation by ethanol dilution (SNALP), nucleic acid encapsulation, 249–251

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Page 823

INDEX SRB-1 gene expression, 2⬘-methoxyethyl oligonucleotide pharmacology, liver concentrations, 285–286 stability properties locked nucleic acids, 539 oligonucleotide medicinal chemistry, 144 RNA interference drug development, 468–469 stabilized antisense-lipid particles (SALPs), ethanol drop nucleic acid encapsulation, liposomal drug delivery systems, 247 stabilized plasmid lipid particles (SPLPs), liposomal nucleic drug delivery systems immunogenicity, 259–260 spontaneous vesicle formation by ethanol dilution, 249–251 stable nucleic acid lipid particle (SNALP) ApoB-100 inhibition therapies, combined regimen, animal studies, 625–626 liposomal nucleic acid delivery encapsulation efficacy analysis, 260–262 pharmacokinetics and biodistribution, 254–256 toxicity measurement, 256–258 liposomal nucleic delivery encapsulation, spontaneous vesicle formation by ethanol dilution, 249–251 RNA interference drug development, liposomes and lipoplexes, 475–477 synthetic short interfering RNA, systemic drug administration, 451–453 statin therapies, cardiovascular disease, antisense oligonucleotides combined with current treatment paradigm, 602 hamster combination studies, 616 ISIS 301012 combined regimen, 621–626 limitations of, 602–603 stearoyl-CoA desaturase (SCD), nonalcoholic steatohepatitis, antisense reduction, 656 stent devices, morpholino therapies and, 572–574 streptavidin, brain drug delivery system, 221 “structural saturation,” locked nucleic acid structure, 530–532 structure-activity relationships (SARs) CpG-oligodeoxynucleotides, 753–755 human RNase H1, 51–54 oligonucleotide medicinal chemistry, 146 subcellular localization, human RNase H2 biochemistry, 61–63 subchronic dosing regimen, ISIS 301012 (ApoB-100), 318–320 subcutaneous injection antisense oligonucleotide drugs, 219–220 clinical safety experiments aPTT prolongation, 374–375 ASO responses, 383–385 dosage and scheduling studies, 369–370 local administration results, 372, 393–394 ISIS 301012 ApoB inhibitor, 623–626 toxicologic effects of ASOs, 334

823 sugar modifications locked nucleic acid structure, 530–532 oligonucleotide medicinal chemistry, 154–163 backbone replacements, 152–154 bicyclic sugars, 158–161 furanose substitution positions, 158 ribofuranose sugar, 161–163 2⬘-modifications, 154–157 second-generation antisense oligonucleotide optimization, gapmer structures, 489–490 short interfering RNAs, 440–442 sulfur transfer reagent, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 407–408 superoxide dismutase 1 (SOD1) amyotrophic lateral sclerosis antisense therapy, 730–734 neurological disorders, 722–723 survival motor neurons, splice switching oligonucleotide targeting, 97 survivin antisense, cancer therapy, 704–705 synthesizers locked nucleic acid synthesis, 528–529 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 411–413 synthons, locked nucleic acid synthesis, base modifications, 528 systemic drug administration antisense oligonucleotides clinical safety experiments aPTT prolongation, 374–375 complement activation, 375–377 dosage studies, 369–370 duration of treatment, 370–371 results, 371–392 intravenous infusion, 219 pharmacokinetics, animal studies, 308–309 physical and chemical properties, 218–219 subcutaneous injection, 219–221 liposomal nucleic acid delivery formulations, pharmacokinetics and biodistribution, 254–256 synthetic short interfering RNA, 448–453 complexation techniques, 451–453 conjugation techniques, 448–451 toxicologic effects of ASOs, 333–334

T targeted oligonucleotide silencers of splicing (TOSS), RNA splicing hybridization, 101 target sites accessibility in RNAs, RNase H mechanism and, 28–29 antisense oligonucleotide design and selection, 119–123 antisense oligonucleotides, pharmacokinetics, animal studies, 308–311 toxicology of ASOs and, 340–346

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Page 824

824 tau protein-related diseases, splice switching oligonucleotide targeting, 96 TBDMS chemistry, short interfering RNA chemical synthesis, 446–448 T-bet transcription factor, multiple sclerosis antisense therapy, 676–677 temperature effects, oligonucleotide concentrations, subcutaneous injections and, 219–220 terminal plasma concentrations, antisense oligonucleotides, pharmacokinetics, animal studies, 315–316 terminating mechanism in antisense drugs, 15 antisense oligonucleotides, 47–48 tetrabutylammonium fluoride (TBAF), short interfering RNA chemical synthesis, 447–448 β-thalassemia, splice switching oligonucleotide targeting of, 94 small nuclear RNA hybridization, 103 therapeutic index, oligonucleotide medicinal chemistry, 147 therapeutic specificity (therapeutic index), antisense drugs, 19 thermal denaturation, locked nucleic acid, 532–537 thermodynamics, locked nucleic acids, 536 thiazole modification, oligonucleotide medicinal chemistry, 165 thioformacetal, oligonucleotide medicinal chemistry, 152–153 thio-LNA amidites heteroduplex thermal denaturation, 533–534 RNase H recruitment, 540 synthesis, 524–526 thiophosphonoacetate, oligonucleotide medicinal chemistry, 150 thiophosphoroamidates, oligonucleotide medicinal chemistry, 150 4⬘-thioribose, oligonucleotide medicinal chemistry, 161–162 threofuranosyl-(3→2⬘)-linked nucleic acid analog (TNA), oligonucleotide medicinal chemistry, 163 thrombin-binding aptamers, toxicological effects, 339 thrombocytopenia, clinical safety experiments with gen-1/gen-2 ASOs and, 389–391 tissue concentrations antisense oligonucleotides, pharmacokinetics accumulation and clearance, 314–316 animal studies, 308–311 gastrointestinal drug delivery systems and, 231–233 locked nucleic acid, 548–551 TNF receptor-associated adaptor protein (TRADD), second-generation antisense oligonucleotide optimization, gap size effects, rodent studies, 495–500 tolerability parameters ISIS 301012 ApoB inhibitor, 623–626 ophthalmology therapies, 588–589 toll-like receptors (TLRs) autoimmune disease therapy, 759 cancer therapies, 758–759

INDEX CpG dinucleotide motifs, TLR9 and mechanism of action, 748–751 immunomodulation and immune stimulation effects, 751–753 infectious disease monotherapies, 755–757 inflammatory disease therapy, 666 oligodeoxynucleotides with CpG, 754–755 oligoribonucleotide ligand identification and immune stimulation, 760–762 RNA interference drug development, specificity improvements, 468 safety profiles, 759–760 toxicological effects of ASOs, proinflammatory mechanisms, 349–350 topical delivery systems antisense oligonucleotides, 222–223 pharmacokinetics, animal studies, 310 clinical safety experiments, 372, 394 toxicity effects cardiovascular ASO therapies, ISIS 301012 ApoB inhibitor, 617–618 chimeric antisense oligonucleotides chronic administration, 351 clotting inhibition, 338–339 complement activation, 339–340 genetic effects, 353–354 hematopoietic effects, 345 kidney effects, 340–345 liver effects, 345–346 mechanisms and clinical correlates, 336–337 oral administration, 334–336 phosphorothioate comparisons, 328–329 plasma protein binding effects, 337–340 proinflammatory effects, 346–351 reproductive toxicology, 352–353 safety assessment strategies, 330–331 safety pharmacology, 354–356 sequence motifs, 329–330 single-strand ASO, siRNA, and aptamers, 328 species-specific effects, 356–357 systemic vs. local administration, 333–334 target organ accumulation and effect, 340–346 toxicokinetics, 332–333 liposomal nucleic acid delivery encapsulation, 256–258 locked nucleic acid, 553–557 2⬘-methoxyethyl oligonucleotide pharmacology, in vitro conditions, 280–282 morpholinos, safety profiles, 567–569 neurological disease antisense therapies, 728–730 TPI-ASM8, asthma therapy, 679 transactivating region (TAR) element, RNA disruption, 26 transactivating reseponse (tar) RNA-binding protein (TRBP), micro-RNA biogenesis, 79 transcription factors antisense oligonucleotides, 9 asthma pathology and, 678 multiple sclerosis antisense therapy, 676–677 occupancy-activated destabilization, siRNA repression, 35–36 peptide nucleic acids, 512

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Page 825

INDEX

825

RNA intermediary metabolism, 7–12 type 2 diabetes, antisense targeting of, 647 transfected cell lines, antisense oligonucleotide screening, 131 transforming growth factor, ophthalmology therapies and, 594–595 transforming growth factor-β, glioblastoma antisense therapy, 736 translational repression occupancy-only antisense drug mechanisms, 25–26 small RNA-directed mechanisms in mRNA, 82–83 transplantation, inflammatory disease antisense threapy, 682–683 trans-splicing mechanisms, technology for, 101–102 transthyretin (TTR) gene, neuropathies, 738–739 Treg production and function, inflammatory disease antisense therapy, 689 tricyclic cytosine analogs, oligonucleotide medicinal chemistry, 165–166 triplex structures, locked nucleic acids, thermal denaturation, 537 tumor necrosis factor-α inflammatory bowel disease antisense therapy, 671 pharmacokinetics and pharmacodynamics, 321 rheumatoid arthritis therapy, 672–674 cell proliferation, maturation, and survival, 685 RNA interference drug development, 477 ulcerative colitis therapy, 687 tumor necrosis factor receptor family, 2⬘-methoxyethyl oligonucleotide pharmacology in, bone tissue, 287–288 2⬘-sugar modifications, oligonucleotide medicinal chemistry, 154–157 type 2 diabetes, drug discovery for, 644–652 hepatic glucose output inhibition, 648–649 ISIS 113715 compound, 658–659 kidney targeting, 651–652 protein phosphatase targeting, 644–647 tissue selectivity and pharmacokinetic properties, 649–651 transcription factor targeting, 647

U ulcerative colitis, 667–671 Unylinker molecule, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 406–407 uptake mechanisms locked nucleic acid, 551–552 ophthalmology therapies, 587 pulmonary drug delivery systems, 223–225

V vaccines, CpG oligodeoxynucleotides, 757–758 vascular endothelial growth factor (VEGF) aptamer therapeutic agents, pegaptanib, 788–790 ophthalmology therapy angiogenesis and, 592–593 pegaptanib, 586–587 RNA interference drug development clinical trials, 480 “naked” siRNA, 471–472 SELEX process and, 777–779 toxicologic effects, reproductive systems, 352–353 vascular smooth muscle cell (VSMC) inhibition, morpholino therapies and, 573–574 very late activation antigen (VLA)-4 antisense, multiple sclerosis therapy, 675–676 viral miRNAs, antagomir silencing, 457–458 viral suppression, miRNA silencing, 458 viscosity, oligonucleotide concentrations, subcutaneous injections and, 219–220

W Watson-Crick base-pairing antisense oligonucleotide pharmacokinetics, 306–307 antisense theory and, 5 locked nucleic acid heteroduplexes, 532–534 oligonucleotide medicinal chemistry, 145 peptide nucleic acids, 510–512 West Nile virus (WNV), morpholino rapid response to, 571 Wilms’ tumor suppressor (WT1) gene, splice switching oligonucleotide targeting, 99

X X-linked mammalian inhibitor of apoptosis protein (XIAP), cancer therapy mechanisms, 704–705 morpholino-targeted drug development, 575

Y yield statistics, 2⬘-methoxyethyl (MOE)-modified oligonucleotide manufacture, 419

Z zeta potential, liposomal drug delivery systems, nucleic acids, 253

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(A)

Page 1

(B) NR IgG

Anti-H1 Ab

(C) Anti-H1 Ab

Infected with virus

us vir 1 H1 rol Size _H 6(−) ont L standard C 2 F

50 kDa

Uninfected Hela cell

Anti-H2 Ab

Uninfected Hela cell

Anti-H1 Ab

FL_H1 virus infected Hela cell

RNase H1

36 kDa

infected Hela cell

30 kDa

Mitotracker red Purified H1 Ab

Uninfected Hela cell

26(−)H1 virus infected Hela cell

Anti-H2 Ab

Anti-H1 Ab

FL_H2 virus infected Hela cell

FL_H1 virus infected Hela cell

Figure 2.10

FL_H1 virus infected Hela cell

26(−)H1 virus infected Hela cell

Anti-H1 Ab mitotracker red merge

FL_H1 virus infected Hela cell

26(−)H1 virus infected Hela cell

Immunofluoresence staining of human RNases H with purified anti-H1 or H2 antibody. Normal rabbit IgG (NR IgG) was used as control. (A) Human RNase H1 and H2 immunostaining of normal (uninfected) or virus infected Hela cells. (B) RNase H1 staining of Hela cells infected with H1 virus and costaining with mitochondrial-specific stain. (C) Expression of human full length (FL) and N-terminal 26 amino acid (⫺26) minus RNase H1 in Hela cells. Hela cells were infected with FL or ⫺26 minus RNase H1 virus or control virus (LoxP) for 24 h. The cell lysates were prepared and subjected to immunoprecipitation with RNase H1 Ab. The immunoprecipitated samples were then used to the Western blot with same H1 Ab.

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Liver

Kidney

Adipose

Bone

Spleen

Human tumor xenograft

Figure 10.4 Immunolocalization of 2⬘-MOE gapmer in different mouse tissues following systemic treatment. Mice were dosed with ISIS 13920, a 2⬘-MOE gapmer recognized by the 2E1 monoclonal antibody. Tissues were collected and stained with the 2E1 antibody for presence of oligonucleotide as described by Butler et al. [133]. Antibody bound to the oligonucleotide appears as the brown stain in the histological sections. The blue stained structures are the result of staining with hematoxylin, used as a counterstain. G—glomerulus, PT—proximal tubules, O—osteoclast, E—endosteum, T— tumor xenograft, S—stromal cells.

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Granular cells Purkinje cell

Motor neuron Molecular layer

Lumbar cord, ICV

Cerebellum, IT

R/C

Lung, inhalation

IN

Eye, intravitreal

Figure 10.5 Immunolocalization of 2⬘-MOE gapmer in different tissues following local administration. Animals were dosed with the 2⬘-MOE gapmer ISIS 13920 and oligonucleotide localized using immunocytochemistry as previously described [133]. Lumbar cord, ICV: 13920 was infused into the lateral ventricle for 14 days and ISIS 13920 localized (brown stain) in the lumbar cord. Cerebellum, IT: Rhesus monkeys were infused with ISIS 13920 into the intrathecal space for 14 days and ISIS 13920 localized in the cerebellum. Lung, inhalation: Mice were treated with ISIS 13920 by aerosol administration and localization of ISIS 13920 determined in lung tissue. Eye, intravitreal: ISIS 13920 was injected intravitreally into a mouse eye and localized in different cell population by immunohistochemical staining (brown stain). R/C—cell bodies of the rods and cones in the retina, IN—inner nuclear layer of the retina.

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Figure 10.6 Immunolocalization of 2⬘-MOE gapmer in normal and inflamed tissue. Inflammation was induced in mouse ears by either treating topically with DNFB [200] or in intestinal tissue by treating with dextran sulfate [163]. Once the inflammation was established, mice were treated with ISIS 13920 and tissue collected approximately 24 h after dosing. The 2⬘-MOE gapmer was localized in normal and inflamed tissue by staining with 2E1 antibody [133]. Tissues were counterstained with hematoxylin (blue stain). Brown staining represents localization of ISIS 13920.

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(a)

(c)

2

1.5

1

75% MM inhibition p = 0.001 vsMM

* Scheffer’s post hoc analysis

49% inhibition vs MM p = 0.18MM

Neovascularization (mm2)

2.5

Irrelevant siRNA

0.5

0 No Inj

PBS

siMM 60 µg

siVEGF 3 µg

siVEGF Pegaptanib VEGF Rc Ig 60 µg 16 µg 1 µg

(d)

(b) 100

Normal vascular area (%)

90 80 70 60

ALN-VEG01

50 40 30 20 10 0 No Inj

Figure 16.4

BS

siMM 60 µg

siVEGF 3 µg

siVEGF Pegaptanib VEGF Rc Ig 60 µg 16 µg 1 µg

ALN-VEG01 specifically inhibits retinal neovascularization in a rat oxygen-induced retinopathy model. Newborn rats were exposed to alternating high oxygen concentrations from days 0–14 as outlined previously [101]. On day 14, therapeutic agents were given once intravitreally (5
l volume) at the amounts indicated and rats placed in room air for the following 6 days (days 14–20). On day 20, rats were sacrificed and flat mount retinal preparations stained with ADPase was used to quantitate (a) pathologic neovascularization and (b) normal vascular development; representative ADPase flat mount preparations are shown following administration of (c) irrelevant control siRNA or (d) ALN-VEG01. Experimental groups: no injection (No Inj), saline (PBS), highand low-dose ALN-VEG01 siRNA (siVEGF), high-dose ALN-VEG01 mismatch siRNA (siMM), clinical-grade VEGF aptamer (Pegaptanib), and research-grade VEGF receptor immunoglobulin fusion protein from R&D Systems (VEGF Rc Ig). All groups were scored blinded; N ⫽ 10 per group. Neovascularization data are expressed as mean neovascular area (mm2) ⫾ SE (A) and normal retinal vasculature data are expressed as percentage vascular area (⫾ SE). Scheffe’s post-hoc analysis was employed to identify significant differences in both neovascular area and normal vascular area. One of three representative experiments.

(a)

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Light field

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900

Dark field

Concentration (µg / gram tissue)

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Retina

RPE Choroid/ Sclera

800 700

Equivalent to 45,000 nM in vitro IC50: < 1 nM

600 500 400 300 200 100 0

Vitreous fluid Retina Ocular compartment

2 days post Intravitreal Injection Magnification 400x

(b)

0.5 h

Unmodified siRNA

6h

24 h

72 h

168 h

10000 Unmodified siRNA Chemically modified siRNA

modified siRNA

1000 100 10

IC50for VEGF silencing

1 0.5 0.1 0.5 h

3 days

7 days

Vitreous fluid

0.5 h

3 days

7 days

Choroid/sclera

Figure 16.5 Beneficial effect of nuclease stabilization on intact ocular drug levels and anatomical distribution of siRNA following intravitreal injection. A 7-day rabbit study was performed to determine the pharmacokinetics of a chemically modified siRNA (P ⫽ S) and its unmodified counterpart. (a) Rapid uptake and distribution of siRNA within ocular tissues. 33P-radiolabeled unmodified and modified compounds (0.4 mg) were injected intravitreally and ocular tissues analyzed after 0.5, 6, 24, 72, and 168 h. Counts per minute (CPM) were measured for different eye compartments (aqueous humor, vitreous, retina, iris, sclera/choroid). Negligible counts were detected in aqueous humor and iris. Analysis of total CPM in retina for the modified siRNA is shown; unmodified siRNA exhibited a similar profile. Concentration of siRNA/gram of tissue was calculated based on CPM assuming 100% intact duplex. Microautoradiography (as visualized under light and dark field microscopy) shows distribution of radiolabeled modified siRNA throughout retina and sclera two days following intravitreal injection. Radiolabel is detected as dark spots under light field and bright spots under dark field. (b) In vivo benefit of exonuclease protection. Ocular tissues (vitreous and choroid/sclera) were subjected to polyacrylamide gel electrophoresis and percentage of intact 33P-labeled duplex was determined by autoradioluminogram. Exonuclease protection results in greater stability within the vitreous and ocular tissue than the same duplex in its unmodified form. Estimated ocular levels of intact nucleases protected siRNA (based on CPM ⫻ % intact duplex) are approximately 50-fold above the in vitro IC50 7 days after a single intravitreal injection.

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H&E

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ASO

Lumen Muscle

Foamy macrophages in aortic plaque

Trichrome staining

Macrophages

Figure 22.1 Localization of a representative 20-mer phosphorothioate within an atherosclerotic lesion of a Watanabe Heritable Hyperlipidemic rabbit. Animals were administered 25 mg/kg ASO twice weekly for 3 weeks. Top left panel: H&E staining of the aortic plaque indicating the presence of foamy macrophages in the lesion. Bottom left panel: Masson’s Trichrome staining of the lesion to highlight excess collagen deposition. Top right panel: Localization of the ASO using immunostaining. Bottom right panel: Localization of macrophages using a monoclonal mouse anti-macrophage antibody (RAM 11).

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(b)

(a)

160 140

100

Serum triglycerides (mg/dL)

ApoC-III mRNA (% saline control)

120

80 60 40 20

120 100 80 60 40 20 0

0 Saline Treated

Saline

ISIS 167878 ISIS 167880 50 mg/kg/week 50 mg/kg/week Treatment

(c)

ISIS 167878 50 mg/kg/week Treatment

ISIS 167880 50 mg/kg/week

(d)

Saline

167878

Liver triglycerides (mg/g)

300 250 200 150 100 50 0 167880

Saline

ISIS 167878 50 mg/kg/week Treatment

ISIS 167880 50 mg/kg/week

Figure 22.3 Pharmacological effect of two ASOs targeting murine apoC-III in high fat–fed C57BL/6 mice. In this representative experiment, mice were administered 50 mg/kg/week ISIS 167878 and ISIS 167880 for 6 weeks. (a) Reduction in hepatic apoC-III mRNA analyzed by qRT-PCR. Data are expressed as the mean percentage of mRNA levels in saline treated animals. (b) Reduction in serum triglyceride levels of treated animals. (c) ApoC-III ASOs reduced hepatic steatosis as assessed by Oil Red O staining of livers. (d) Quantitation of liver triglyceride levels.

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Saline

SMI

ISIS 147764

ISIS 158661

ISIS 144477

Figure 22.5 Microsomal triglyceride transfer protein (MTP) inhibitors exacerbate hepatic steatosis in high fat–fed C57BL/6 mice. In this representative experiment, mice were administered 50 mg/kg/week ASOs for 6 weeks and 1 mg/kg daily of a small-molecule MTP inhibitor. Oil Red O–stained liver sections of high fat–fed C67BL/6 mice administered either saline (top left panel), ISIS 147764, the apoB inhibitor (top right panel), small-molecule MTP inhibitor (bottom left panel), ISIS 158661 and ISIS 144477, antisense inhibitors to MTP (bottom middle and right panel, respectively).

P 0.6 0.5

P P Saline

0.4 0.3 0.2

ISIS 301012 50 mg/kg/week

301012 20 mg/kg/week

Volume mm3

Normal intima

Saline

0

301012 50 mg/kg/week

*

0.1

*P = 0.033 (one-tailed t test) Figure 22.7 ISIS 301012, the human apoB antisense inhibitor, reduces aortic sinus plaque volume in human apoB transgenic/Ldlr–deficient mice. Transgenic mice were administered 20 or 50 mg/kg/week ASO for 14 weeks. Top left panel: Aortic sinus region of saline treated mice. P indicates the plaque that is characterized by neointimal hyperplasia, macrophage foam cells, intracellular lipid, and fibrous caps. Lower left panel: Aortic sinus region of ISIS 301012–treated animals indicating the decrease in plaque volume. Right panel: Quantitative imaging analysis of total plaque volume within the aortic sinus. Administration of ISIS 301012 reduced total aortic sinus plaque volume in a dose-dependent fashion, with the highest dose group reducing plaque by approximately 60%.

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Saline

VLA-4 ASO

(a)

(b)

(c)

(d)

(e)

(f)

VLA4+ cells

CD4+ T cells

BM8+ Mφs

Figure 24.6 VLA-4, T cell, and macrophage immunostaining on spinal cords from EAE mice. Saline-treated mice (a, c, e) had a Grade 2 paralysis but were symptom free while receiving treatment with a VLA4 antisense inhibitor (b, d, f). Antibodies were used to detect VLA-4 (a, b), CD4⫹ T cells (c, d), or BM8⫹ macrophages (e, f). Magnification: 250X. (From Myers, K.J. et al., J. Neuroimmunol., 160, 12, 2005. With permission.)

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(b)

(c)

Figure 25.2 Immunostaining of OGX-011 drug distribution (a) and Clusterin expression (b, c) in human lymph tissue. (a) An antibody raised against the 2⬘-MOE backbone of OGX-011 enabled the immunohistochemical staining (brown) of resected human lymph tissue to verify that the drug had reached its target. (b, c) Clusterin protein expression (brown) in lymph node samples. Figure 25.2(b) shows an untreated control specimen while Figure 25.2(c) demonstrates downregulation of Clusterin in lymph tissue from a trial subject treated with OGX-011 at 640 mg dosing.

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Rat lumbar ventral horn

(A) Oligonucleotide (µM)

Rat

(C)

(E)

9 6 3

(B)

Lumbar cord

Thoracic cord

Cerivical cord

Left cortex

Brain stem

Right cortex

0

Rhesus monkey

Isis13920treated

50 µm

Oligo treated secondary antibody only

Salinetreated

Rhesus monkey ventral horn (F)

Anti-oligo astrocyte

Anti-GFAP astrocyte

(G)

Oligo-treated

Oligonucleotide (µM)

(D)

12

12 9 6 50 µm

50 µm

3

Lumbar cord

Thoracic cord

Cerivical cord

Brain stem

Left cortex

Right cortex

(I)

Saline-treated

(H)

0

50 µm

50 µm

Rhesus monkey brain (J) Hippocampus Pyramidal neuron

(K)

Substantia nigra Dendritic neuron

(I)

Pons

(M)

Cerebellum

Pontine nucleus Granular neuron

Dentate granular neuron

60 µm

50 µm

50 µm

Purkinje cell

50 µm

Figure 26.1 Distribution of antisense oligonucleotides after infusion into the right lateral ventricle in rat and Rhesus monkey. (A, B) Antisense oligonucleotides were continuously infused at 100
g/day (A) or 1 mg/day (B) for 2 weeks via infusion pump into the right lateral ventricle of (A) normal rats or (B) Rhesus monkey. Tissues were collected and extracts of them analyzed for oligonucleotide content by capillary gel electrophoresis. Mean values ± standard deviations are shown (A) n ⫽ 6; (B) n ⫽ 2. (C–G) A 24-mer-modified oligonucleotide Isis13920 was infused for two weeks into the right lateral ventricle at 100
g/day in (C–E) rats or 1 mg/day in (F–M) Rhesus monkey. After perfusion, distribution of the oligonucleotide was determined by immunohistochemistry using a monoclonal antibody that recognizes the oligonucleotide (C–E, F, H) or astrocytes (GFAP; G, I). No oligonucleotide staining was seen in animals (D, H) infused with saline only or (E) an oligonucleotide infused animal but using secondary antibody only. Bar, 50 nm. (Copyright 2006 by American Society for Clinical Investigation. Reproduced with permission of American Society for Clinical Investigation in the format Textbook via Copyright Clearance Center.)