Pharmaceutical Aspects of Oligonucleotides (Taylor & Francis Series in Pharmaceutical Sciences)

Pharmaceutical Aspects of Oligonucleotides

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Pharmaceutical Aspects of Oligonucleotides Edited by

PATRICK COUVREUR University of Paris-Sud CNRS Unit ‘Physico-chimie, Pharmacotechnie, Biopharmacie’, Chatenay-Malabry, France

CLAUDE MALVY CNRS, Institut G.Roussy, Villejuif, France

First published 2000 by Taylor & Francis 11 New Fetter Lane, London EC4P 4EE Simultaneously published in the USA and Canada by Taylor & Francis Inc., 325 Chestnut Street, 8th Floor, Philadelphia, PA 19106 Taylor & Francis is an imprint of the Taylor & Francis Group This edition published in the Taylor & Francis e-Library, 2005. “To purchase your own copy of this or any of Taylor & Francis or Routledge’s collection of thousands of eBooks please go to www.eBookstore.tandf.co.uk.” © 2000 Taylor & Francis Limited All rights reserved. No part of this book may be reprinted or reproduced or utilised in any form or by any electronic, mechanical, or other means, now known or hereafter invented, including photocopying and recording, or in any information storage or retrieval system, without permission in writing from the publishers. Every effort has been made to ensure that the advice and information in this book is true and accurate at the time of going to press. However, neither the publisher nor the authors can accept any legal responsibility or liability for any errors or omissions that may be made. In the case of drug administration, any medical procedure or the use of technical equipment mentioned within this book, you are strongly advised to consult the manufacturer’s guidelines. British Library Cataloguing in Publication Data A catalogue record for this book is available from the British Library Library of Congress Cataloging in Publication Data Pharmaceutical aspects of oligonucleotides/edited by Patrick Couvreur, Claude Malvy. p. cm.—(Taylor & Francis series in pharmaceutical sciences) Includes bibliographical references and index. 1. Oligonucleotides—Therapeutic use. I. Couvreur, Patrick. II. Malvy, Claude. III. Series. [DNLM: 1. Oligonucleotides—therapeutic use. 2. Oligonucleotides— adverse effects. QU 57 P536 2000] RM666.N87P47 2000 615′.31—dc21 99–34210 ISBN 0-203-30566-3 Master e-book ISBN

ISBN 0-203-34211-9 (Adobe eReader Format) ISBN 0-748-40841-X (Print Edition)

Contents

PART ONE

Contributors

xii

Preface

xiv

General Features 1

1

Mechanisms of Action of Antisense Oligonucleotides D.M.Tidd and R.V.Giles

2

1.1

Introduction

2

1.2

Cell Proliferation Arrest through Release of Deoxynucleosides

3

1.3

Extracellular Aptameric Effects of Phosphorothioate Oligodeoxynucleotide Analogues

5

1.4

Aptameric Effects of G-quartet Oligonucleotides and Analogues

6

1.5

Immune Stimulation by Oligodeoxynucleotides Containing CpG Motifs

10

1.6

Other Activities of Oligodeoxynucleotides Containing CpG Motifs

11

1.7

Antisense Inhibition of Gene Expression by Steric Block

11

1.8

Antisense Inhibition of Gene Expression through Ribonuclease H-mediated Destruction of Target mRNA

13

1.9

Oligonucleotides in Vivo

20

Conclusions

21

Acknowledgements

21

References

22

1.10

vii

PART TWO

Chemical Aspects 2

32

Chemistry of Oligonucleotides J.W.Engels and E.Uhlmann

33

2.1

Introduction

33

2.2

Design of Oligonucleotides

37

2.3

Oligonucleotide Modifications: Synthesis and Properties

40

2.4

Analysis of Oligonucleotides

59

2.5

Conclusion

68

Acknowledgement

68

References

68

3

The Oligonucleotide Prodrug Approach: The 80 Pro-oligonucleotides F.Morvan, J.-J.Vasseur, E.Vivès, B.Rayner and J.-L.Imbach

3.1

Introduction

80

3.2

What Kind of Enzymolabile Group?

81

3.3

First Pro-oligonucleotide Models

84

3.4

Pro-oligonucleotides of the Second Generation

87

3.5

Conclusion

94

Acknowledgements

96

References

96

4

Peptide Nucleic Acids P.E.Nielsen

100

4.1

Introduction

100

4.2

Antisense

101

4.3

Antimicrobials

103

4.4

Anti-telomerase

103

4.5

Antigene

103

4.6

Anti-HIV

105

4.7

Pharmacology

105

4.8

Further Developments

106

viii

Acknowledgement

106

References

107

PART THREE Delivery 5

110

Peptide-mediated Delivery of Oligonucleotides E.Vivès and B.Lebleu

111

5.1

Delivery Vehicles for the Improved Uptake of Nucleic Acids: a Survey

111

5.2

The Potential of Peptides for Nucleic Acids Delivery

113

5.3

Strategies for the Coupling of Peptides to Oligonucleotides

114

5.4

Poly (L-lysine)-based Delivery Systems

116

5.5

Conjugation to Fusogenic Peptides Allowing Membrane Fusion or Membrane Translocation

117

5.6

Conclusions

122

Acknowledgements

124

References

124

Polymeric Nanoparticles and Microparticles as Carriers for Antisense Oligonucleotides E.Fattal and P.Couvreur

129

6.1

Introduction

129

6.2

Nanoparticles

130

6.3

In Vitro Stability of ONs Adsorbed onto Nanoparticles

138

6.4

Cell Interactions with ON Loaded Nanoparticles

139

6.5

In Vitro Pharmacological Activity of Oligonucleotideloaded Nanoparticles

140

6.6

In Vivo Studies with Oligonucleotide Nanoparticles

140

6.7

Microparticles

141

6.8

Conclusion

142

References

142

Liposomes for the Delivery of Oligonucleotides P.Couvreur, C.Malvy, C.Dubernet and E.Fattal

148

Introduction

148

6

7 7.1

ix

7.2

Anionic Liposomes

149

7.3

Cationic Liposomes

152

7.4

pH-sensitive Liposomes

159

7.5

Immunoliposomes and Other Molecularly Targeted Liposomes

163

7.6

Fusogenic Liposomes and Proteoliposomes

165

7.7

Conclusions

167

References

168

Comb-type Polycation Copolymer for Antigene Strategy and DNA Carrier A.Maruyama

176

8.1

Introduction

176

8.2

Comb-type Polycations as a Stabilizer for DNA Duplex and Triplex

176

8.3

Comb-type Polycation Copolymers with Cell-specific Polysaccharide Side-chains as cell-specific DNA Carrier

189

Acknowledgements

196

References

196

8

PART FOUR Biopharmaceutics 9

203

Delivery of Antisense Oligonucleotides in Vitro: Experimental Points G.B.Takle and C.A.Stein

204

9.1

Introduction

204

9.2

Oligonucleotide-Binding Proteins on the Cell Surface

206

9.3

Intracellular Compartmentalization

208

9.4

Oligonucleotide Delivery Reagents—Practical Considerations

210

9.5

Experimental Antisense: Points to Consider

211

References

214

Mechanisms of Transmembrane Transport of Oligonucleotides

217

10

x

R.L.Juliano 10.1

Overview of Cellular Uptake of Antisense Oligonucleotides

217

10.2

Permeation of Oligonucleotides across Membranes

219

10.3

Mechanisms of Enhancement of Oligonucleotide Permeation across Membranes

222

10.4

Summary

225

References

225

Pharmacokinetics of Oligodeoxynucleotides A.Gouyette

230

11.1

Introduction

230

11.2

Pharmacokinetics

231

11.3

Chemistry of Oligonucleotides and Formulations

233

11.4

Cellular Pharmacokinetics

233

11.5

Preclinical Pharmacokinetics

235

11.6

Clinical Pharmacokinetics

239

11.7

Conclusions

240

References

241

11

PART FIVE

Pharmacological Activity 12

246

Antisense as a Novel Therapy for Cancer B.P.Monia

247

12.1

Novel Approaches for Anticancer Therapy

247

12.2

Antisense Approaches for Cancer

249

12.3

Antisense as a Novel Anticancer Approach against ras

255

12.4

Conclusions and Future Prospects

263

Acknowledgements

264

References

264

Modulation of Inflammatory Processes with Antisense Oligonucleotides C.F.Bennett

268

Introduction

268

13

13.1

xi

13.2

ICAM-1, a Case Study

269

13.3

Other Examples

281

13.4

Regulation of Immune Response by Non-antisense Mechanisms

285

13.5

Conclusions

286

References

286

Oligonucleotides as Antiparasite Compounds J.-J.Toulmé

293

14.1

Introduction

293

14.2

Design of Antisense Oligonucleotides for Antiparasite Use

294

14.3

Antiparasite Effects of Antisense Oligonucleotides

297

14.4

RNA Structures are Valid Targets for Regulatory Oligonucleotides

305

14.5

Conclusion

310

Acknowledgements

311

References

311

Index

318

14

Contributors

C.F.BENNETT, ISIS Pharmaceutical, 2292 Faraday Avenue, Carlsbad, CA 92008, USA. P.COUVREUR, Laboratoire de Physico-Chimie-PharmacotechnieBiopharmacie, UMR CNRS 8612, Université Paris-Sud, Faculté de Pharmacie, 92296 Châtenay-Malabry Cedex, France. C.DUBERNET, Laboratoire de Physico-Chimie-PharmacotechnieBiopharmacie, UMR CNRS 8612, Université Paris-Sud, Faculté de Pharmacie, 92296 Châtenay-Malabry Cedex, France. J.W.ENGELS, University of Frankfurt, Institut for Organische Chemir, Marie Curie Str 11, 60439 Frankfurt am Main, Germany. R.V.GILES, School of Biological Sciences, The University of Liverpool, Life Sciences Building, Crown Street, Liverpool L69 7ZB, UK. E.FATTAL, Laboratoire de Physico-chimie-Pharmacotechnie-Biopharmacie, UMR CNRS 8612, Université Paris-Sud, Faculté de Pharmacie, 92296 Châtenay-Malabry Cedex, France. A.GOUYETTE, Institut Gustave Roussy, CNRS, 140 rue Camille Desmoulins, 94805 Villejuif Cedex, France. J.-L.IMBACH, Laboratoire de Chimie Bio-Organique, UMR CNRS-UMII 5625, Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France. R.L.JULIANO, Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, NC 27599, USA. B.LEBLEU, Institut de Génétique Moléculaire, CNRS-UMR 5535, 1919, Route de Mende, 34293 Montpellier Cedex 5, France. C.MALVY, Laboratoire de Régulation Artificielle de l’Expression Génétique, UMR CNRS 8532, Institut Gustave Roussy, rue Camille Desmoulins, 94805 Villejuif, France. A.MARUYAMA, Department of Biomolecular Engineering, Faculty of Bioscience and Biotechnology, Tokyo Institute of Technology, 4259 Nagatsuda-Cho, Midori-Ku, Yokohama 226–8501, Japan.

xiii

B.P.MONIA, Department of Molecular Pharmacology, ISIS Pharmaceuticals, 2280 Faraday Avenue, Carlsbad, CA 92008, USA. F.MORVAN, Laboratoire de Chimie Bio-Organique, UMR CNRS 5625, Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France. P.E.NIELSEN, Center for Biomolecular Recognition, Department of Medical Biochemistry and Genetics, Biochemical Laboratory B, The Panum Institute, Blegdamsvej 3c, 2200 Copenhagen N, Denmark. B.RAYNER, Laboratoire de Chimie Bio-Organique, UMR CNRS 5625, Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France. C.A.STEIN, Departments of Medicine and Pharmacology, Columbia University, 630 W 168th Street, New York, NY 10032, USA. G.B.TAKLE, Innovir Labs Inc., 510 E 73rd Street, New York, NY 10021, USA. D.M.TIDD, School of Biological Sciences, The University of Liverpool, Life Sciences Building, Crown Street, Liverpool L69 7ZB, UK. J.-J.TOULMÉ, INSERM U 386, IFR Pathologies Infectieuses, Université Victor Segalen, 146 rue Léo-Saignat, 33076 Bordeaux Cedex, France. E.UHLMANN, Hoehst Marion Rounel Deutschland GmbH, Chemical Research G838 D-65 926, Frankfurt am Main, Germany. J.-J.VASSEUR, Laboratoire de Chimie Bio-Organique, UMR CNRS 5625, Université de Montpellier II, Place Eugène Bataillon, 34095 Montpellier Cedex 5, France. E.VIVÈS, Institut de Génétique Moléculaire, CNRS-UMR 5535, 1919, Route de Mende, 34293 Montpellier Cedex 5, France.

Preface

The dream of modern drug research is to discover a biologically active molecule or a class of biologically active molecules, 100% specific, able to act efficiently only on the function responsible for the disease. If this dream became a reality, treatment of fatal illnesses (AIDS, cancer, chronic immune disorders, etc.) would be possible without the severe side-effects and toxicities that are the main limitations of these treatments. Many drugs work by interfering with critical proteins which have been identified as responsible for dysfunction of cells or tissues. However, conventional therapeutic agents now on the market which tend to act on proteins in the body also often bind to non-target proteins, or exert an effect through unknown interactions. Fortunately, progress in genetics and genomics has enabled definitive studies on some of the fundamental molecular mechanisms that regulate the expression of genes, as well as the dysfunction of these mechanisms. From this mass of knowledge, the idea has emerged of drugs that may turn off genes by targeting the RNA that codes for the protein instead of the protein product. This strategy displays several advantages: (i) an active antisense oligonucleotide can be identified in a short time, (ii) the subcellular location of the protein is not important, (iii) first results do not show any antigenicity of oligonucleotides. One antisense oligonucleotide was approved by the FDA as a drug in 1998. The ‘Antisense’ concept is based on the ability of cellular RNA macromolecules to bind to complementary (or antisense) sequences of oligonucleotides. Since the antisense oligonucleotides interact with their targets by Watson-Crick base pairing, this should lead, in theory, to the perfect specificity and affinity as dreamed of by the drug researcher. However, despite their exciting potential for selectively modulating the expression of an individual gene, oligonucleotides are still far from becoming drugs. They suffer from numerous limitations: they are rapidly degraded in vivo by nucleases, they diffuse poorly through the biological barriers (including cell membranes), and they fail to reach the right intracellular compartments. There are also questions about reproducibility of biological efficacy of oligonucleotides, and about whether the pharmacological effects are definitely the result of an antisense activity.

xv

This book is intended as a condensed work on oligonucleotides from the perspective of their pharmaceutical application, trying to answer the following questions: what technological bolts are hindering the development of oligonucleotides as pharmaceuticals, and how can these be overcome? Contributors were asked to emphasize their own experience with oligonucleotides, and to deal with aspects not normally covered in specialized reviews. The objective was to provide a book which, although written by specialists, could be easily understood by people in different fields of life science, and especially scientists interested in drug discovery and pharmaceutical development. The book starts with a general account of how oligonucleotides work at the molecular level, and goes on to discuss the challenge of improving the administration of these compounds. Therefore, particular emphasis is placed on the development of the chemistry (peptidic nucleic acids) as well as of the formulation and the controlled delivery of oligonucleotides with the aid of molecular (peptides, polymers) or particular/supramolecular systems (liposomes, nanoparticles). Background information concerning intracellular trafficking and tissue distribution/ pharmacokinetics is reviewed and discussed, because these areas should also give new ideas for improving oligonucleotide delivery. The last part of the book is devoted to therapeutical aspects (treatment of cancer and of inflammatory and parasitic diseases). Clearly, the potential provided by oligonucleotides as shown in this book will be realized only through a multidisciplinary and innovative research effort. Patrick Couvreur and Claude Malvy (Eds) Université Paris-Sud. CNRS. France

PART ONE General Features

1 Mechanisms of Action of Antisense Oligonucleotides D.M.TIDD and R.V.GILES

1.1 Introduction As well as inhibiting gene expression through steric blockade of pre-mRNA splicing or initiation of translation, or through ribonuclease H-mediated ablation of mRNA, antisense oligonucleotides have been found to induce a variety of biological effects by mechanisms other than, or in addition to, those which might result from hybridization to the targeted mRNA. Such additional mechanisms of action include: 1 release of pharmacologically active concentrations of deoxyribonucleosides, through nucleolytic degradation 2 aptameric binding to proteins 3 immune stimulation by oligodeoxynucleotides containing CpG motifs 4 other activities of oligodeoxynucleotides containing CpG motifs. The interpretation of results of experiments with antisense oligonucleotides requires that account be taken of all potential mechanisms that might have been entrained, as well as the possibility that the expression of non-targeted genes may have been suppressed by antisense mechanisms, through substantial partial complementarity between their mRNAs and the antisense effector. Antisense oligonucleotides were initially hailed as agents for achieving highly specific inhibition of pathologic gene expression, whether that be of a virus (Cohen, 199la; Degols et al., 1992), a cancer cell (Calabretta, 1991; Dolnick, 1991; Carter and Lemoine, 1993), a neuropathology (Eng, 1993; Wahlestedt, 1994), or indeed of almost any disease of humans imaginable (Miller and Ts’o, 1987; Cohen, 1991b; Crooke, 1992; Gura, 1995). In reality, these molecules have turned out to induce a variety of biological effects, many of which may be ascribed to mechanisms other than Watson-Crick hybridization to the miscreant mRNA (Stein, 1995; Neckers and Iyer, 1998). Furthermore, the recognition that oligonucleotides may bind to proteins and subvert their function has led to a

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 3

growing following for the pursuit of such aptamers, through their evolution in repetitive selection—amplification procedures from libraries of random base sequence (Bock et al., 1992). Apart from such non-antisense mechanisms, there is increasing awareness also that a specific base sequence in an oligonucleotide might exert its antisense effects on several of the mRNAs from the as yet mostly undiscovered 60 000 to 80 000 genes thought to be contained in the human genome. Despite a lack of complete complementarity between the oligonucleotide and the non-targeted mRNAs, the activity at these sites could be as potent as, or more potent than, that achieved against the intended target, if the former and not the latter were devoid of secondary and tertiary structure, and therefore more readily accessible (Giles et al., 1998). In this chapter we will discuss some of the mechanisms by which antisense oligonucleotides may achieve their biological effects. 1.2 Cell Proliferation Arrest through Release of Deoxynucleosides Consideration of the intracellular trafficking (see also Chapters 9 and 10) of any drug is fundamental to an understanding of its mechanism of action upon the cell. Antisense oligonucleotides simply added to the bathing medium do not gain access to the cytoplasm and hence their target mRNAs in cells in culture. Small amounts of oligonucleotide are taken up through processes of endocytosis and/or fluid phase pinocytosis, but they remain in endosomes or vacuoles, excluded from the nucleus and still topologically exterior to the cell, being separated by a membrane from the cytoplasm (Spiller and Tidd, 1992; Giles et al., 1993; Crooke and Lebleu, 1993; Wagner et al., 1993). Experiments in which the media were replaced by oligonucleotide-free solution have demonstrated that the bulk of such molecules are subsequently quite rapidly expelled from cells by exocytosis (Tonkinson and Stein, 1994). There is no evidence to support the suggestion that oligonucleotides are even delivered to lysosomes to any significant extent, although Tonkinson and Stein (1994) have demonstrated that phosphorothioate oligodeoxynucleotide analogues penetrate into a deeper, acidic vesicular compartment within cells, as well as the shallow compartment attained predominantly by normal phosphodiester oligodeoxynucleotides. In contrast, when fluorescently tagged oligonucleotides are specifically delivered into the cytoplasm by direct microinjection, they have been observed to accumulate rapidly in the nuclei, presumably as a result of their affinity for binding to nuclear structures (Chin et al., 1990; Leonetti et al., 1991; Fisher et al., 1993; Wagner et al., 1993). In fact, nuclear accumulation of oligonucleotides is indicative that intracytoplasmic delivery has been achieved by other means, such as electroporation (Bergan et al., 1996; Spiller et al., 1998a), reversible plasma membrane permeabilization with streptolysin O (Spiller and Tidd, 1995; Giles et al., 1997), or lipofection with cationic liposomes (see also Chapter 7) (Bennett et

4 MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES

al., 1992; Gewirtz et al., 1996; Lewis et al., 1996; Marcusson et al., 1998). However, such observations require the concomitant exclusion of propidium iodide by the cells in order to establish that the cells are still living, since dead cells readily accumulate oligonucleotide in their nuclei by passive diffusion of the molecules through the holes in their ruptured plasma membranes. It was originally reported that primary cultures of keratinocytes did not require any special manipulation for intracytoplasmic delivery of oligonucleotides from the media (Nestle et al., 1994), but it has now been shown that all such cells exhibiting nuclear localization of oligonucleotide were also stained by propidium iodide or merocyanine 540, indicating that they were either already dead or undergoing apoptosis, respectively (Wingens et al., 1998; Giachetti and Chin, 1996). Also, it is important that observations of the intracellular localization of oligonucleotides be made on living and not fixed cells, since vesicular-contained oligonucleotide in living cells has been seen to translocate instantaneously to their nuclei upon fixation with ethanol (Tidd, 1998). It is evident from the foregoing considerations that early reports of inhibition of cell proliferation by a variety of oncogene antisense oligodeoxynucleotides added to the culture media were unlikely to have been achieved by an antisense mechanism against the targeted mRNA. In addition, such unprotected normal phosphodiester oligodeoxynucleotides are rapidly degraded to mononucleotides by endonuclease and 3′-phosphodiesterase activities present in the serum component of the culture medium, where the latter is the most important activity (Tidd and Warenius, 1989). Although monodeoxynucleotides also do not gain access to the cytoplasm of cells when presented exogenously, phosphatases present on the cell surface and possibly released into the media readily catalyse their hydrolysis to deoxynucleosides (unpublished observations), which are then rapidly transported into the cells and rephosphorylated by intracellular deoxynucleoside kinases (Henderson and Paterson, 1973). It is likely that at least some of the reported inhibitions of cell proliferation observed with oncogene antisense oligodeoxynucleotides were the result of cell uptake of their deoxynucleoside breakdown products. The consequent induction of gross imbalances in intracellular deoxynucleoside triphosphate pools would have prevented passage of the cells through the S-phase of the cell cycle. Limited exposure to excess thymidine is a long-established method for synchronizing cells for their entry into S-phase (Doida and Okada, 1967). Progressive replacement of internucleoside phosphodiester linkages by nuclease-resistant methylphosphonates (Miller and Ts’o, 1987; Miller, 1998) from each end of a cmyc antisense oligodeoxynucleotide resulted in proportionately less inhibition of proliferation of the cells by the same base sequence (Tidd, 1993). The terminal methylphosphonates protected the oligodeoxynucleotide from exonuclease attack and reduced its susceptibility to endonuclease-mediated hydrolysis. The predominant 3′-exonucleolytic degradation of oligodeoxynucleotides also permits an apparent sequence specificity for inhibition of cell proliferation. Vaerman et al. (1997) have demonstrated that oligodeoxynucleotides terminating

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 5

at the 3′-end in the bases adenine, guanine or thymine were inhibitory, while those ending in cytosine at either of the two most terminal 3′-positions were not. They determined the toxicity of deoxynucleoside 5′-monophosphates to haematologic cells and demonstrated that only deoxycytidine 5′-monophosphate was non-toxic, and that this mononucleotide neutralized the cytotoxicity of deoxyadenosine, deoxyguanosine and thymidine monophosphates. Added to this, there is the possibility of observing spurious, apparent inhibition of proliferation when 3H-thymidine or 5-bromodeoxyuridine incorporation assays are used (Matson and Krieg, 1992). Thymidine released from degraded oligodeoxynucleotides competes with these labelled deoxynucleosides, reducing their incorporation into DNA. This effect is greatest when the thymidine nucleotides are present at the 3′-end of an oligodeoxynucleotide. 1.3 Extracellular Aptameric Effects of Phosphorothioate Oligodeoxynucleotide Analogues Recognition of the poor biological stability of phosphodiester oligodeoxynucleotides led to a search for analogues of the structure which would be resistant to nucleases while retaining the hybridization properties of the natural molecule. A variety of structures have been developed, but phosphorothioate oligodeoxynucleotides, in which a non-bridging oxygen of the internucleoside linkage is replaced by sulphur, have become the most popular analogues. These are comparatively, though not completely, nuclease-resistant, while their hybridization potential with complementary nucleic acids is only moderately reduced relative to the corresponding phosphodiester molecules (Stein et al., 1988; Hoke et al., 1991). There has been a plethora of reports of putative antisense effects on gene expression induced through application of phosphorothioate oligodeoxynucleotides, where no special measures were taken to ensure intracytoplasmic delivery of the molecules. However, more careful examination of some of these claims has demonstrated that the biological effects observed were unrelated to inhibition of expression of the target gene. Barton and Lemoine (1995, 1997) have demonstrated that the established antiproliferative effects of phosphorothioate p53 antisense oligodeoxynucleotides applied exogenously to cells were unrelated to any effects on p53 expression. Growth of breast and lung cancer cell lines was inhibited by the c-myc (see also Chapter 12) antisense, phosphorothioate oligodeoxynucleotide via a sequencespecific, non-antisense mechanism which was correlated with inhibition of cell adhesion (Watson et al., 1992; Saijo et al., 1997). Similarly, growth inhibition of chronic myelogenous leukaemia cells, induced by phosphorothioate bcr-abl antisense oligodeoxynucleotides added to the culture medium, represented non-antisense, base sequence-dependent but not base sequence-specific toxicity of the oligomers, where the lower toxicity of the sense sequence controls was fortuitous (O’Brien et al., 1994; Smetsers et al., 1995).

6 MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES

Coulson et al. (1996) have also reported that a phosphorothioate oligodeoxynucleotide antisense to the epidermal growth factor receptor inhibited the proliferation and altered the morphology of A431 cells by a sequenceselective, but non-antisense, mechanism apparently affecting receptor tyrosine kinase activity. Phosphorothioate oligodeoxynucleotide analogues bind promiscuously to proteins (Stein and Cheng, 1993; Stein and Krieg, 1994; Stein, 1995) such as protein kinase C isoforms (Khaled et al., 1995), HIV gp120 (Stein et al., 1993) and heparin binding proteins such as members of the fibroblast growth factor family (Guvakova et al., 1995), integrin and fibronectin (Khaled et al., 1996) and soluble recombinant CD4 (Yakubov et al., 1993). Furthermore, since intracytoplasmic access would not have been achieved by the oligomers in the experiments described in the preceding paragraph, it is likely that the observed biological activities were the result of aptameric effects of their competitively binding essential growth factors (Fennewald and Rando, 1995; Guvakova et al., 1995), proteins in the plasma membrane or membrane proteins within endosomes. Antiviral effects of phosphorothioate antisense oligodeoxynucleotides may for the most part be accounted for by their binding to viral envelope proteins and/or cell surface receptors and thereby inhibiting cell entry, rather than through antisense inhibition of targeted viral gene expression (Stein, 1995). Likewise, sequence-specific inhibition of cell adhesion may be the result of direct interactions with proteins at the cell surface and substratum, rather than the consequence of altered gene expression (Neckers and Iyer, 1998). 1.4 Aptameric Effects of G-quartet Oligonucleotides and Analogues Guanine-rich oligonucleotides and polynucleotides can form intermolecular or intramolecular structures whereby four guanines are arranged in a planar array, known as a G-tetrad or G-quartet. In general, intermolecular G-quartets require four, or more, consecutive guanine residues and present structures in which all four sugar-phosphate backbones are in a parallel arrangement. Intramolecular Gquartets fold into a structure with a minimum of two adjacent G-tetrads and with the backbone oriented so as to present two parallel and two antiparallel strands. These arrays are stabilized by hydrogen bonding in which each of the guanines acts as an acceptor and a donor for two bonds (discussed in Williamson, 1994). ) In solution, G-quartets are stabilized by alkali metal ions ( which occupy a position in the core between two square arrays of guanine, and it would appear that potassium ions are preferred due to their possessing an optimal radius (Williamson, 1994). Residues outside the G-quartet region can stabilize the structure, particularly with intramolecular G-tetrads, by T–T or T–A hydrogen bonding (Schultze et al., 1994; Murchie and Lilley, 1994; Keniry et al., 1997). Intermolecular and intramolecular G-quartets may be identified and

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 7

distinguished by X-ray crystallography, circular dichroism (CD) spectroscopy (Williamson, 1994), nuclear magnetic resonance (Feigon et al., 1995) and thermal denaturation with UV spectroscopy (Mergny et al., 1998). Inhibition of smooth muscle cell hyperplasia by phosphorothioate oligodeoxynucleotides antisense to c-myb in vitro (see also Chapter 12) (Simons and Rosenburg, 1992) and in vivo (Simons et al., 1992) and to c-myc in vitro and in vivo (Shi et al., 1994; M.R.Bennett et al., 1994), but not by control oligonucleotide analogues, was presented as a potential antisense approach to treatment of restenosis following balloon angioplasty. However, these effects were subsequently shown to occur by a non-antisense mechanism (Villa et al., 1995; Burgess et al., 1995; Castier et al., 1998). The presence of four contiguous guanines in the c-myb (Villa et al., 1995; Burgess et al., 1995; Castier et al., 1998) and the c-myc (Burgess et al., 1995; Saijo et al., 1997) antisense oligodeoxynucleotides, and in otherwise random oligodeoxynucleotides (Castier et al., 1998; Saijo et al., 1997) was responsible for their antiproliferative activity. Such runs of G residues permit the formation of intermolecular G-quartets which stabilize oligodeoxynucleotide quadruplexes, and it was intimated that these phosphorothioate quadruplex structures were responsible for at least some of the aptameric activities observed. On the other hand, it has been suggested that the c-myc and c-myb antisense phosphorothioate oligodeoxynucleotides are incapable of forming intermolecular G-tetrad stabilized quadruplexes under physiological ionic and temperature conditions (Basu and Wickstrom, 1997). However, other studies have indicated that pre-formed phosphorothioate intermolecular quadruplexes are quite stable under such conditions (50–80% quadruplex remaining at six days; Wyatt et al., 1994). To complicate the picture further, it appears that non-G-quartet forming phosphorothioate oligonucleotides (e.g. S-dC28) may also potently inhibit smooth muscle cell proliferation in vitro and in vivo (Wang et al., 1996). The mechanism responsible for the biological effects of a number of ‘antisense’ phosphodiester and phosphorothioate oligomers, which contain motifs that admit the potential to form intermolecular quadruplexes, has been investigated. Phosphodiester oligodeoxynucleotides antisense to the vpr gene of HIV1 were shown to be potent inhibitors of HIV replication and presented CD spectra consistent with the formation of intermolecular quadruplexes. Heat denaturation prior to use or mutation of the G4 run, so as to inhibit G-quartet formation, was found to abolish activity (Tondelli et al., 1996). Antisense NB1 phosphorothioate oligodeoxynucleotides which contained G4 runs, but not sense or antisense analogues lacking G4 tracts, were found to alter the morphology and inhibit proliferation of normal breast epithelial cells without concom itant reduction in NB1 mRNA or protein expression. Further control compounds were synthesized and activity was found to correlate directly with the presence of four contiguous G residues. Examination of phosphodiester or alternating phosphodiester/phosphorothioate congeners revealed that activity also correlated with the degree of phosphorothioate substitution (Yaswen et al., 1993). It is not

8 MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES

clear whether these results reflected the relative nuclease stability of the phosphorothioate internucleoside linkage or indicated a requirement for the phosphorothioate backbone, i.e. a ‘phosphorothioate’ effect overlaying the Gquartet effect, in this system. Specific inhibition of human type II phospholipase A2 was shown to require both the phosphorothioate backbone and the G-quartet forming sequence. Nuclease-resistant, 2′-modified congeners with a phosphodiester backbone were inactive and heat denaturation of the phosphorothioate oligonucleotide stock, prior to use, abolished activity (C.F.Bennett et al., 1994). Phosphorothioate oligomers antisense to the mRNA of the RelA subunit of NF-κB have been shown to inhibit adhesion of a range of tumor cell lines to substrata, at the same time as down-regulating relA mRNA, and NF-κB and Sp 1 DNA binding activities by non-antisense mechanisms. For the most part, single base mismatches which destroyed the G4 tract in the antisense effector abolished both inhibition of adhesion and down-regulation of expression, whereas sequential 3′-end deletions, which retained the G4 run, were found to inhibit adhesion but not to reduce NF-κB DNA binding activity (Maltese et al., 1995). Further investigations were carried out using oligonucleotides with long GN tracts (N = 7 or 9), or mutations which interrupted the G4 run (Khaled et al., 1996), or 7-deazaG substitutions (which inhibit G quadruplex formation but not normal base-pairing; Benimetskaya et al., 1997). These confirmed that a significant proportion of the biological activity of the relA antisense effector resulted from its potential to form G-quartet structures that interacted with cell surface or extracellular proteins. An in vitro selected phosphorothioate octamer, S-T2G4T2, has been isolated by its ability to inhibit HIV1 replication efficiently (Wyatt et al., 1994). This compound was shown to form stable intermolecular quadruplex structures under physiological conditions. Nuclear magnetic resonance (NMR) of a similar compound (TG4T) demonstrated that the structure in solution comprised an allparallel arrangement of the phosphodiester backbones (Aboul-ela et al., 1994). Heat denaturation of S-T2G4T2 prior to use abolished anti-HIV l activity, confirming that the active species was a quadruplex. In addition, replacement of the phosphorothioate backbone with phosphodiester internucleoside linkages also abolished activity. Clearly, as for the phospholipase A2 system described above, the phosphorothioate internucleoside structure was required for activity since nuclease-resistant, quadruplex forming, α-phosphodiester congeners were equally inactive (Wyatt et al., 1994). It was shown that the mode of action of ST2G4T2 involved binding to the v3 loop of HIV1 gp120, thereby inhibiting adsorption of the virus onto CD4+ cells. Another oligonucleotide containing only T and G residues, but with the sequence GTG2TG3TG3TG3T and a phosphodiester backbone, has been described as a potent inhibitor of HIV1 integrase but an inefficient inhibitor of the gp120-CD4 interaction (Ojwang et al., 1994, 1995). The folded intramolecular quadruplex structure adopted by this molecule has been assessed by nondenaturing gel electrophoresis and NMR, and shown to possess two adjacent G-

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 9

quartets with the backbones aligned two parallel and two antiparallel (Rando et al., 1995). The partially phosphorothioate-protected analogue of this structure (a single phosphorothioate residue between the terminal and penultimate base at both the 5′ and the 3′ end) has been most extensively investigated. Comparison to a 10–15 fold less active compound, G2T2G2TGTG2T2G2 (thrombin binding aptamer; Bock et al., 1992), and intermediates between that and the parent oligodeoxynucleotide, which adopt similar three-dimensional structures (Macaya et al., 1993; Wang et al., 1993; Schultze et al., 1994), revealed that the precise sequence in the loops surrounding the G-quartets was important for interaction with HIV1 integrase (Mazumder et al., 1996). Detailed analysis of the mode of action indicated that the GTG2TG3TG3TG3T oligonucleotide bound directly to a zinc finger domain of the integrase (Mazumder et al., 1996) and that this inhibited oligomerization of the enzyme into the active complex (Cherepanov et al., 1997). However, it was also noted that a HIV1 isolate which was insensitive to this aptamer, in cell culture replication assays, did not possess a mutated integrase gene. In fact, the gp120 gene was mutant (Cherepanov et al., 1997), and thus it would appear that the major effect of this compound in cell culture is inhibition of viral adsorption to the cell surface. In addition to the oligodeoxynucleotide G-quartet aptamers (see also Chapter 14) that have been evolved using in vitro selection protocols (Bock et al., 1992; Wyatt et al., 1994), a number of RNA (or 2′-NH2 modified RNA) intramolecular quadruplex compounds have been selected which interact with specific proteins. Hamm (1996) demonstrated that a G-quartet RNA bound at, or very close to, the active site of an anti-ferritin antibody (H107) in vitro. Normal prion protein (PrPc) from hamster, mouse and cow, and recombinant prion protein (rPrP), but not PrPSc from scrapie-infected mice, was recognized by an RNA aptamer. The aptamer was shown to interact specifically with the N terminus of rPrP and the intramolecular quadruplex (composed of three adjacent G-quartets) was shown to be essential for the interaction (Weiss et al., 1997). A 2′-NH2 nuclease protected RNA aptamer was evolved (Wiegand et al., 1996) which bound to human immunoglobulin E (IgE) with a Kd of ~30 nM. The dissociation constants for mouse and rat IgE or human IgG were found to be at least 300 and 600 fold higher, respectively. Binding of the RNA aptamer to human IgE inhibited the interaction between the antibody and its receptor, FcεRI on RBL SX-38 cells and inhibition of the antibody-receptor binding on sensitized RBL SX-38 cells inhibited serotonin release in cell culture. G-quartet forming oligodeoxynucleotides and oligoribonucleotides are capable of forming intermolecular and intramolecular quadruplexes. The quadruplexes facilitate binding to proteins, and protein binding may result in substantial biological effects. The specific protein bound by a given quadruplex structure appears to depend in part on the backbone structure of the oligonucleotide and in part on the sequence context of the quadruplex. It may be that the quadruplex defines a rigid structure that permits reproducible presentation of the aptamer to its protein binding site and that the specificity of interaction depends on other

10 MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES

aspects of the chemistry of the compound. However, there appears to be no way to predict, a priori, what the protein binding specificity of a given quadruplex structure will be. 1.5 Immune Stimulation by Oligodeoxynucleotides Containing CpG Motifs Krieg (1998) has reviewed the original reports of unexpected, base sequencespecific, immune stimulation, B-cell proliferation, and immunoglobulin secretion induced by antisense and control sense phosphorothioate oligodeoxynucleotides. Intrigued by the profound magnitude of the immune stimulation, he synthesized and tested several hundred oligodeoxynucleotides to identify the base sequences responsible. These results established that all the previously observed effects could be accounted for by the presence in the oligodeoxynucleotides of a simple motif based on a cytosine phosphate guanine (CpG) dinucleotide in the optimal consensus sequence context, ‘R1R2CGY1Y2, where R1 is a purine (mild preference for G), R2 is a purine or T (preference for A; T can be substituted with minimal loss of activity if the rest of the motif is intact), and Y1 and Y2 are pyrimidines (preference for T)’ (Krieg et al., 1995; Krieg, 1998). The effects of the motif were enhanced when preceded on the 5′side by T, whereas a C on the 5′-side of the CpG greatly reduced activity. The optimal minimal sequence that would stimulate B-cells was TCAACGTT. Krieg (1998) has re-evaluated his own previous, apparently successful antisense experiments, where immune stimulation was the expected result, as well as those of others, in terms of the possible non-antisense, involvement of active CpG motifs. In addition, although phosphorothioate oligodeoxynucleotides containing CpG motifs were reported to exhibit immune stimulatory activity while the corresponding phosphodiester oligomers were inactive, the differences may have been more apparent than real, and related to the greater biological stability of the former. Phosphodiester oligodeoxynucleotides with CpG motifs mediated B-cell activation in media containing foetal calf serum which had been heat inactivated at 65°C for 30 minutes to reduce nucleases. Oligodeoxynucleotide-induced B-cell activation was achieved without any specific intervention to secure intracytoplasmic delivery of the oligomers, and consequently it is almost certainly the result of interactions at the cell surface or within endosomes. However, Krieg (1998) has reported that active oligodeoxynucleotides failed to trigger B-cell proliferation when immobilized on surfaces or on beads. Apart from the significance of the above findings in terms of their impact on antisense strategies, they have broader biological implications. CpG dinucleotides are far less prevalent in vertebrate genomes as compared to bacteria, and where they occur they are often methylated on the 5 position of the cytosine. Krieg et al. (1995) have advanced the hypothesis that the established immunostimulatory activity of bacterial DNA is mediated through detection of

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 11

unmethylated CpG motifs by B lymphocytes, and that the lymphoproliferative response may represent a first line, innate immune defence against infection when such DNA is detected in host tissues. In support of their hypothesis they demonstrated that methylation of bacterial DNA abolished its mitogenicity. Likewise, replacement of cytosine by 5-methylcytosine in CpG motifs of B-cell mitogenic oligodeoxynucleotides led to loss of their activity, whereas molecules in which other cytosines were methylated retained their stimulatory properties. 1.6 Other Activities of Oligodeoxynucleotides Containing CpG Motifs In addition to the immune stimulatory properties of CpG containing oligodeoxynucleotides, Krieg et al. (1997) have identified a sequence motif (CGT [C]GA) in phosphorothioate modified oligodeoxynucleotides that specifically inhibited the enzymatic activity of several recombinant or immunoprecipitated protein tyrosine kinases in vitro, without affecting the enzyme activity of a serine/threonine protein kinase. It is conceivable that antisense oligodeoxynucleotides containing these sequence motifs might have profound effects when introduced into cells, which would be unrelated to any suppression of the targeted gene. In our own work, we have observed that suitably endprotected phosphodiester oligodeoxynucleotides containing CpG motifs, rather than stimulating proliferation, rapidly induced fulminating apoptosis, when delivered into the cytoplasm of human leukaemia cells by reversible plasma membrane permeabilization with streptolysin O (unpublished observations). The minimum length of an oligodeoxynucleotide required to elicit this effect was just five bases, and inversion of the sequence from 5′–3′ to 3′–5′ completely abolished activity, as did mutation of the CpG dinucleotide. However, oligodeoxynucleotides in which the cytosine of the CpG dinucleotide was replaced by 5-methylcytosine retained the ability to induce apoptosis. 1.7 Antisense Inhibition of Gene Expression by Steric Block Early investigations of the potential for inhibiting gene expression with antisense oligonucleotides were founded on the premise that their hybridization to the complementary site in the target mRNA might present a block to the passage of the translating ribosome complex. Such a mechanism, which was referred to as hybrid arrest of translation, was invoked to account for apparent antisense effects of oligonucleotides and cDNA in early work on cell-free systems and microinjected Xenopus oocytes (Paterson et al., 1977; Kawasaki, 1985). However, other experimental results demonstrated that the fully assembled ribosomal complex is able to locally destabilize secondary structures as it moves along the message such that DNA antisense to the translated region of the mRNA was

12 MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES

without effect, and that hybridization to part of the 5′-untranslated region extending from the cap site to the initiation codon was required for inhibition of translation (Liebhaber et al., 1984; Shakin and Liebhaber, 1986). These conflicting results in cell-free protein synthesizing systems were resolved by the observations that where inhibition of translation was achieved with antisense DNA targeting the coding region of mRNA, the latter was found to have been degraded during the course of the incubations (Haeuptle et al., 1986; Minshull and Hunt, 1986). The enzyme present in the cell extracts responsible for elimination of intact mRNA was shown to be ribonuclease H (RNase H) (Haeuptle et al., 1986; Minshull and Hunt, 1986; Cazenave et al., 1987; Walder and Walder, 1988), a ubiquitous activity that catalyses the hydrolysis of RNA in RNA—DNA heteroduplexes (Hausen and Stein, 1970). Degradation of mRNA by RNase H was also shown to contribute to inhibition of cell-free protein synthesis achieved by antisense oligodeoxynucleotides targeting 5′-untranslated and translation initiation codon regions (Walder and Walder, 1988; Cazenave et al., 1987). However, oligonucleotide analogues that do not form substrate hybrids for RNase H, such as a-anomeric (Bertrand et al., 1989; Boiziau et al., 1991) or morpholino (Partridge et al., 1996; Summerton et al., 1997) structures, may block translation in cell-free and cellular systems through hybridization to 5′-untranslated and initiation codon sites. Such activity apparently results from their preventing formation of the translation initiation complex, and they are generally inactive when targeted further downstream. The only exception to the rule would appear to be for RNase H-inactive, peptide nucleic acid oligomers (see also Chapter 4), oligonucleotide analogues in which a polyamide structure replaces the phosphodiester backbone. When these are synthesized as triplex forming homopyrimidine base sequences, they have sufficient affinity for RNA that they are able to withstand the unwinding activity of the fully assembled ribosomal complex, and are thus able to inhibit peptide chain elongation through a non-RNase H mechanism (Hanvey et al., 1992; Bonham et al., 1995; Knudsen and Nielsen, 1996). Another approach to inhibition of gene expression with antisense oligonucleotides which may exploit a steric blocking mechanism is through targeting splice sites in pre-mRNA. There have been several reports of the application of splice site antisense oligonudeotides, but in the absence of definitive evidence, it is not possible to conclude that their observed biological effects on living cells were necessarily induced through inhibition of maturation of the targeted pre-mRNA. However, Kole (1998) has provided convincing evidence for inhibition of splicing by RNase H-inactive 2′-O-methyloligoribonucleotides in nuclear splicing extracts from HeLa cells, and by phosphorothioate 2′-O-methyl-oligoribonucleotides delivered into HeLa cells by lipofection with Lipofectamine. In this case the strategy was somewhat different from that of the usual antisense approach, since the oligonudeotides were targeted at cryptic splice sites activated by mutation in introns of the β-globin gene, which are responsible for a majority of β-thalassemia cases worldwide.

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 13

Oligonucleotide-induced restitution of normal pre-mRNA splicing and normal βglobin synthesis in HeLa cells transfected with mutant constructs confirmed that steric blocks to splicing at the cryptic sites were established within living cells. We have observed strong evidence for the inhibition of gene expression with a splice site targeted antisense oligonucleotide in living cells. A 28-mer RNase Hinactive, morpholino oligonucleotide analogue, antisense to human c-myc premRNA across the intron 1/exon 2 boundary and initiation codon, induced the accumulation of high molecular weight, myc probe hybridizing RNA species, when introduced into a number of human leukaemia cell lines by reversible plasma membrane permeabilization with streptolysin O (Giles et al., 1999). The p64 Myc protein had completely disappeared from the cells by four hours and a Myc epitope reactive protein of approximate molecular weight 47 kDa was readily apparent on Western blots by 24 hours. However, mature c-myc mRNA seen on Northern blots was apparently unaffected throughout, if not somewhat increased in amount. Kole (1998) has suggested that accumulation of unspliced RNA is unlikely to occur even if splice sites are blocked by oligonudeotides that do not support RNase H, since the likely outcome of the oligonucleotide’s action would be skipping of the exon or removal of a portion of the exon due to activation of a cryptic splice site (Mayeda et al., 1990). Subsequent reverse transcription—polymerase chain reaction (RT-PCR) amplification of c-myc mRNA followed by DNA sequencing demonstrated that the morpholino oligonucleotide had indeed forced the splicing machinery to skip the normal acceptor site at exon 2 in favour of a cryptic site downstream of the initiation codon within the exon. The aberrant, c-myc epitope bearing, 47 kDa protein was probably the product of a fortuitously in-frame initiation of translation at an AUG codon within exon 2 of the truncated c-myc mRNA. However, the inhibition of splicing at the normal site was not complete, and therefore, the total loss of p64 Myc protein was probably the result of the combined effects of steric blocks to both splicing of pre-mRNA and initiation of translation from normally spliced mRNA. 1.8 Antisense Inhibition of Gene Expression through Ribonuclease H-mediated Destruction of Target mRNA Most antisense oligonucleotide analogues are unable to direct RNase H, and have generally proved less effective inhibitors of translation in cell-free systems and microinjected cells than RNase H-active oligomers that induce irreversible destruction of the message, even when targeting the cap, 5′-untranslated, and initiation codon sites on the mRNA (Bonham et al., 1995). At low concentrations, phosphorothioate oligodeoxynucleotide analogues do form hybrids with RNA which are still recognized as substrates by RNase H, but at higher concentrations the analogue oligomers apparently bind non-specifically to the enzyme and inhibit its activity (Gao et al., 1992). The protein-binding avidity

14 MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES

of phosphorothioate oligodeoxynucleotides seemed once again to counter their ability to induce sequence-specific antisense suppression of gene expression, by obstructing the very enzyme that would mediate it. Following the discovery of the involvement of RNase H in the antisense oligodeoxynucleotide-induced inhibition of protein synthesis in cell-free systems, it became generally assumed that the enzyme activity was also responsible for at least some of the biological effects of oligodeoxynucleotides on living cells. However, tell-tale fragments of the targeted mRNA which should have been produced by RNase H-mediated cleavage could not be detected in RNA isolates from treated cells. This led to a general consensus that such fragments would be extremely unstable in the biological milieu and would be degraded by ribonucleases as rapidly as they were produced. In fact, failure to detect mRNA cleavage products probably indicated that the observed biological effects of the antisense oligodeoxynucleotides were not the result of their hybridization to the complementary regions in mRNAs within the cells. RNase H-generated fragments of mRNA are actually readily detectable on Northern blots of RNA isolated at early times following the introduction of antisense oligodeoxynucleotides into cells by lipofection with cationic lipids, electroporation or reversible plasma membrane permeabilization with streptolysin O (Dean et al., 1998; Marcusson et al., 1998; Giles et al., 1995a, 1995c; Spiller et al., 1998b). RNase H cleaves RNA to produce 3′-fragments bearing phosphate groups at their 5′-ends (Eder et al., 1993). The identity of such fragments may be positively confirmed by DNA sequencing of their reverse ligation-mediated polymerase chain reaction (RLPCR) products, derived without an intermediate polynucleotide kinase phosphorylation step prior to ligation to the RNA linker (Bertrand et al., 1993). RNA was isolated from human leukaemia cells previously permeabilized with streptolysin O in the presence of antisense oligodeoxynucleotides, and samples were subjected to ligation reactions with an oligoribonucleotide of known sequence, such that any RNase Hgenerated 3′-fragments present would be tagged with the linker at their 5′-ends. The RNA was then reverse transcribed from primers specific for the antisense targeted genes and the cDNA products amplified by polymerase chain reactions using nested, gene-specific 3′-primers and linker sequence 5′-primer. Subsequent DNA sequencing of the amplified products confirmed not only that endogenous RNase H of the leukaemia cells had cleaved the targeted mRNAs, but also that cleavage had occurred in vivo at the site of complementarity to each of the antisense oligodeoxynucleotides (Giles et al., 1995a, 1995c). It has generally been the case that early RNase H-mediated antisense effects of phosphorothioate oligodeoxynucleotides, delivered into cells by streptolysin O permeabilization, have been less dramatic, in terms of the extent of ablation of the target mRNA, than those of the corresponding molecules with normal phosphodiester internucleoside linkages (Giles et al., 1995a, 1995b, 1995c; Spiller et al., 1998a). Indeed, in a comparison of mixed structure, antisense oligonucleotides, the extent of loss of efficacy correlated with the degree of

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 15

phosphorothioate substitution in the molecules (Giles et al., 1998). This is more likely due to sequestration of phosphorothioates through binding to cellular proteins, with less being freely available to interact with target mRNA, than to non-specific inhibition of RNase H. Of course, it is not only the immediate extent to which the target mRNA is ablated that is important if phenotypic effects of antisense inhibition of gene expression are to ensue. It is also essential that antisense activity be maintained for a sufficient duration to enable the pre-existing protein product of the gene to decay by natural turnover (Spiller et al., 1998a). Normal phosphodiester oligodeoxynucleotides, even when 3′-end protected against exonuclease with a 3hydroxypropyl phosphate group, are rapidly degraded by nucleases in living cells and their potent, RNase H-mediated antisense effects on mRNA are short-lived. At the same time, nuclease-resistant oligodeoxynucleotide analogues other than phosphorothioates are unable to recruit RNase H activity to cleave the target mRNA. Our solution to this problem has been to replace several internucleoside linkages at both ends of a phosphodiester oligodeoxynucleotide by nucleaseresistant, non-ionic methylphosphonate groups, while retaining a central, RNase H-active, phosphodiester section (Tidd and Warenius, 1989). The methylphosphonate modification (Miller and Ts’o, 1987; Miller, 1998) served to protect the oligodeoxynucleotides against exonuclease attack when incorporated into such chimeric molecules, while at the same time reducing their susceptibility to endonuclease-initiated degradation (Giles et al., 1995b). The methylphosphonate sections of the oligodeoxynucleotides are unable to direct RNase H activity against the target mRNA (Maher and Dolnick, 1987; Furdon et al., 1989; Quartin et al., 1989), and therefore serve to focus enzyme activity to the central section which may contribute towards achieving single base mismatch specificity against point mutations (Giles et al., 1995a). The concept of using antisense chimeric structures of non-activating analogue and enzyme directing phosphodiester oligodeoxynucleotide to achieve site-directed RNase H cleavage of RNA was originally developed by Ohtsuka’s group (Inoue et al., 1987b; Shibahara et al., 1987). They utilized the 2′-O-methyl analogue of RNA (Inoue et al., 1987a) in chimeras with normal phosphodiester oligodeoxynucleotide and demonstrated RNase H-mediated cleavage in vitro at a single internucleoside linkage in target RNA when the phosphodiester section was sufficiently short. However, the 2′-O-methyl-oligoribonucleotide structure was apparently of insufficient resistance to nucleases for effective application against living cells (Shibahara et al., 1989). In a recent comparison of the activities of c-myc antisense, methylphosphonodiester/phosphodiester oligodeoxynucleotides and chimeric oligonucleotides with the more nuclease-resistant 2′-Oalkyloligoribonucleotide wings, having 2′-methoxyethoxy or 2′methoxytriethoxy substituents, and either phosphodiester or phosphorothioate internucleoside linkages, the former proved to be greatly superior in promoting RNase H-mediated ablation of c-myc mRNA in living human leukaemia cells (Giles et al., 1998). It may well be that the 2′-O-alkyl substituents distort the

16 MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES

helix such that hybrids between RNA and chimeric oligonucleotides with these structures are less efficient substrates for RNase H (Crooke et al., 1995), than duplexes formed between RNA and methylphosphonodiester/phosphodiester oligodeoxynucleotides. In addition, the 2′-O-alkyl modifications are helixstabilizing (Cummins et al., 1995; Monia et al., 1996), which tends to counter the activity of RNase H. Measurements of the hybridization potential of methylphosphonate oligodeoxynucleotide analogues indicated that, while replacement of phosphodiester internucleoside linkages by methylphosphonate was moderately helix-destabilizing for complexes with a complementary phosphodiester oligodeoxynucleotide as the second strand, the modification induced profound reductions in stability of hybrids with RNA oligomers (Tidd, 1990; Giles and Tidd, 1992a). Hybrids of methylphosphonodiester/phosphodiester chimeric antisense oligodeoxynucleotides with sense oligoribonucleotides exhibited melting temperatures (Tm) for half dissociation of the hybrids that were intermediate between those of their allphosphodiester and all-methylphosphonate counterparts, and were directly related to the degree of methylphosphonate substitution. However, the reduced hybridization potential of such chimeric oligodeoxynucleotides correlated inversely with increases rather than decreases in the initial rates of hydrolysis of the complementary oligoribonucleotide in the presence of Escherichia coli RNase H (Tidd, 1990; Giles and Tidd, 1992a). Possibly their lower affinity enhanced turnover rates for the enzyme by facilitating dissociation of enzyme—product complexes. Whatever the mechanism, it was evident that avid binding to target mRNA need not necessarily be of prime concern for optimizing antisense oligonucleotide activity, when this is mediated by RNase H. The last point was brought home by experiments in cell-free systems with in vitro transcribed, high molecular weight RNAs. Phosphodiester and phosphorothioate antisense oligodeoxynucleotides induced fragmentation of the RNAs by E.coli RNase H and RNase H activity in human leukaemia crude cell extracts, not only at the target site of full base complementarity, but also at nontargeted sites of partial complementarity within the same and different RNA molecules (Giles and Tidd, 1992b; Giles et al., 1993, 1995a, 1995b). Similar observations by others caused general concern about the ability to achieve specific antisense effects on gene expression with oligodeoxynucleotides under constant physiological conditions in living cells. Herschlag (1991) discussed helix destabilization as a means to enhance the specificity of ribozymes and antisense oligonucleotides, and Woolf et al. (1992) suggested the need ‘to devise chemically modified oligomers that hybridize less strongly’ on the basis of their demonstration of RNase H cleavage of RNAs at imperfectly matched target sites in Xenopus oocytes microinjected with antisense oligodeoxynucleotides. In fact, the helix-destabilizing property of the methylphosphonate substitution in chimeric, methylphosphonodiester/ phosphodiester, antisense oligodeoxynucleotides goes some way towards achieving this function, and

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 17

improved specificity for cleavage of RNA at the target site was observed with such antisense effectors in assays with E.coli, human and wheat germ RNase H (Giles and Tidd, 1992b; Giles et al., 1993, 1995a, 1995b; Larrouy et al., 1992). That the gain in specificity was due to increased stringency of hybridization was demonstrated by the observation that corresponding 2′-O-alkyloligoribonucleotide/phosphodiester oligodeoxynucleotide chimeras antisense to α-globin, with enhanced rather than reduced hybridization potential, directed non-targeted cleavage of β-globin mRNA by RNase H, and did not elicit similar selective inhibition of α-globin synthesis in the same in vitro protein synthesizing system (Larrouy et al., 1995). However, in using chimeric methylphosphonodiester/phosphodiester oligodeoxynucleotides to achieve single base mismatch discrimination in targeting point mutations in p53 mRNA (Giles et al., 1995a; Ruddell et al., 1996), as well as stringency of hybridization achieved through the methylphosphonate modifications, there would appear to be a contribution from the mismatch at the central phosphodiester section in reducing RNase H activity. Corresponding mutant and wild type, chimeric, antisense oligomers with 2′-Omethyl oligoribonucleotide modifications exhibited a degree of discrimination between perfect and partial complementarity in the extent of RNase H-mediated ablation of the p53 mRNAs. Essentially, complete discrimination between wildtype and Harlow point mutant sequences for cleavage of p53 mRNA was achieved in living MOLT-4, human leukaemia cells following streptolysin Omediated delivery of 15-mer chimeric oligodeoxynucleotides with three methylphosphonate linkages at each end and antisense base sequences centred on codon 273. However, it would seem that secondary and tertiary structure in this region of the mRNA contributed to the achievement of this degree of specificity by effectively competing out binding by the mismatched oligomer. In targeting a point mutation in an apparently more readily accessible region of p53 mRNA at codon 248, in KYO1 leukaemia cells, further methylphosphonate substitutions were required in order to destabilize the interaction between the chimeric antisense oligodeoxynucleotides and the mRNA, and thereby to maximize the difference in RNase H cleavage activity at this site induced by mutant and wild type sequences. The degree of accessibility of the chosen target site within an mRNA can have a profound effect on the level of activity of an antisense oligodeoxynucleotide. The b3a2 junction in bcr-abl mRNA of human chronic myeloid leukaemia would appear to be less readily accessible than the b2a2 junction, and antisense oligodeoxynucleotides of equivalent structure consistently produced greater reductions in b2a2 mRNA than b3a2 mRNA, when introduced into leukaemic cells (Giles et al., 1995b). Also, the popular translation initiation region for targeting c-myc mRNA is in fact a poor antisense target site. Profound reductions in intracellular levels of c-myc mRNA and protein were achieved shortly after reversible plasma membrane permeabilization of human leukaemia cells with streptolysin O in the presence of comparatively high concentrations of a chimeric

18 MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES

15-mer oligodeoxynucleotide with three methylphosphonate internucleoside linkages at each end and base sequence complementary to codons 1–5 (Giles et al., 1997; Spiller et al., 1998a). However, the effects were short-lived, the mRNA and protein began to recover by four hours as degradation of the antisense effector proceeded by endonucleolytic attack, and the proliferation of the cells was essentially unaffected. In addition, replacement of just one more phosphodiester linkage by a helix-destabilizing methylphosphonate effectively abolished antisense activity of the chimeric oligodeoxynucleotide, suggesting that the target was involved in secondary and tertiary structure, which the oligomer no longer had the hybridization potential to invade (Spiller et al., 1998a). In contrast, a methylphosphonodiester/phosphodiester chimeric oligodeoxynucleotide with just six phosphodiester linkages exhibited high activity against the accessible p53 codon 248 site. However, it was evident that a site in the middle of c-myc mRNA (bases 1147–1166, ‘HSMYC1’, GenBank accession number V00568) that had previously shown exquisite sensitivity to non-targeted, RNase H cleavage, through partial complementarity to the initiation codon antisense sequence (Giles and Tidd, 1992b; Giles et al., 1993), must necessarily be quite accessible. Chimeric methylphosphonodiester/ phosphodiester, 20-mer oligodeoxynucleotides complementary to this site were shown to suppress c-myc mRNA and protein expression in human leukaemia cells for more than 24 hours, during which time cell proliferation ceased and cells started to die (Giles et al., 1998; Spiller et al., 1998a). Even a chimeric effector with just four phosphodiester linkages induced essentially undiminished RNase H cleavage activity (unpublished observations). Dose—response curves showed substantial antisense effects on c-myc mRNA and protein persisting for more than 24 hours when cells were initially permeabilized in the presence of concentrations of oligodeoxynucleotide as low as 0.2 µM (Giles et al., 1998). Evidently, very low concentrations of an antisense oligodeoxynucleotide are able to sustain high rates of RNase H-mediated cleavage of the target mRNA if the complementary region is located in an open loop which is freely available for hybridization. Therefore, sustained inhibition of gene expression may be achieved by targeting an accessible site, since the threshold level of intact chimeric oligodeoxynucleotide required for effective ablation of the mRNA will be low and will be exceeded for longer in the cell as the oligomer is degraded. These experiments also highlighted another reason why it is essential to locate open loops in mRNA if sequence-specific antisense effects resulting from inhibition of expression of the selected gene alone are to be achieved. The oligodeoxynucleotides targeted to c-myc, bases 1147–1166, were partially complementary to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA (bases 965–975, 10 base pairs in total and including nine contiguous base pairs, ‘HSGAPDR’, GenBank accession number X01677), and permeabilization of human leukaemia cells with streptolysin O in the presence of 20 µM chimeric cmyc antisense effector resulted in total ablation, not only of c-myc mRNA, but also of GAPDH mRNA (Giles et al., 1998), and probably the mRNAs of several

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 19

other genes which were not assayed. However, the non-targeted cleavage of GAPDH mRNA was reduced to more acceptable levels by lowering the concentration of the oligodeoxynucleotide, without seriously affecting activity against c-myc. Therefore, the identification of good antisense sites in the mRNA of the gene of interest may permit a reduction in the concentration of antisense oligodeoxynucleotide required to induce the desired effect, which may at the same time minimize the extent of non-targeted, RNase H-mediated cleavage of other mRNAs through their partial complementarity to the antisense effector. Undesired side-effects may also be countered by reducing the central phosphodiester section of chimeric oligodeoxynucleotides to the minimum compatible with RNase H activity, although this did not appear to ameliorate the situation with regard to the c-myc antisense sequence and GAPDH mRNA (unpublished observations). Attempts to predict open loop regions in mRNA secondary structure using computer RNA folding routines have not been particularly successful (Giles et al., 1998; Spiller et al., 1998a). Stull et al. (1996) have described a gel shift assay for determining the efficiency of binding of a series of complementary radiolabelled oligodeoxynucleotides to in vitro transcribed RNA in order to identify accessible sites. A similar type of approach has been to measure binding of RNA to large oligodeoxynucleotide arrays as an empirical method for selecting effective antisense oligodeoxynucleotides (Milner et al., 1997; Southern et al., 1997). Accessible sites on in vitro transcribed RNA have also been identified in cell-free assays with RNase H and oligonucleotide libraries (Ho et al., 1996, 1998). However, there are doubts about the validity of using naked RNAs in in vitro assays as surrogates for intracellular RNAs, which exist in interaction with proteins (Branch, 1998). In the absence of a rational strategy for determining good antisense sites in mRNAs, an empirical approach using living cells would appear to be the optimum. Dean et al. (1998) have adopted this method by simply synthesizing a series of phosphorothioate oligodeoxynucleotide sequences designed to hybridize to multiple sites throughout the target mRNA, and evaluating them for their ability to reduce its expression. However, this approach should be treated with caution as it retains the potential to select sequences which induce potent down-regulation of the mRNA and protein studied by non-antisense aptameric effects, such as those obtained with RelA/NF-κB antisense oligonucleotides (see above). In our own work, we have synthesized selected sequences as normal phosphodiester oligodeoxynucleotides, end-protected against exonuclease degradation by 3′hydroxypropyl phosphate and 5′-fluorescein groups, where the latter also served as a reporter to monitor cell delivery. Sequences inducing substantial RNase Hmediated cleavage of the target mRNA, within 30 minutes after reversible membrane permeabilization of the cells with streptolysin O, were resynthesized for further evaluation as the more nuclease-resistant, methylphosphonodiester/ phosphodiester, chimeric antisense effectors.

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Ablation of target mRNA occurs rapidly after introduction of antisense oligodeoxynucleotides into cells (Giles et al., 1997; Spiller et al., 1998a), but development of the phenotypic effects of blocking expression of a gene requires that its pre-existing protein product be degraded by the normal mechanisms of protein turnover within the cell. In the case of Myc protein, which has a very short half-life of around 10 minutes, the antisense oligodeoxynucleotide-induced reduction in its intracellular concentration closely followed the reduction in the level of c-myc mRNA (Giles et al., 1997; Spiller et al., 1998a). However, drastic reduction in mutant p53 mRNA in human chronic myeloid leukaemia KYO1 cells produced a much delayed and more modest decrease in p53 protein, as a result of the comparatively long half-life of the mutant protein of approximately eight hours (Spiller et al., 1998a). Endonuclease-mediated degradation of the chimeric, methylphosphonodiester/phosphodiester, p53 antisense oligodeoxynucleotide was such that levels of p53 mRNA were well on the way to recovering before all pre-existing p53 protein had time to decay. Methylphosphonodiester/ phosphorothiodiester, chimeric oligodeoxynucleotides exhibit greater nuclease resistance than their chimeric phosphodiester counterparts, but bcr-abl antisense oligomers with this structure had previously shown no activity against the relatively inaccessible b3a2 junction in mRNA in chronic myeloid leukaemia cells (Giles et al., 1995b). This was probably the result of partial sequestration of the molecules through protein binding via the phosphorothioate section, coupled with the combined helix-destabilizing effects of methylphosphonate and phosphorothioate substitutions reducing the hybridization potential of the oligomers below that required to invade the secondary structure at the b3a2 site. However, the mutant codon 248 site in p53 mRNA was apparently more readily accessible, and in this case a p53 antisense methylphosphonodiester/phosphorothiodiester oligodeoxynucleotide targeted to this site was able to maintain reduced p53 mRNA concentrations in KYO1 cells for sufficiently long that p53 protein decayed to low levels in the cells (Spiller et al., 1998a). On the other hand, experiments in which the general protein synthesis inhibitor cycloheximide was applied to KYO1 cells indicated that p210 Bcr-Abl protein had an apparent half-life in excess of 48 hours, and no effects whatsoever on the intracellular concentration of this protein were observed with any antisense oligodeoxynucleotide structure targeting bcrabl mRNA. 1.9 Oligonucleotides in Vivo There appears to be an increasing move towards using antisense oligonucleotides in animal models of disease, relegating cell culture work to the role of identifying compounds optimally active at suppressing the expression of the gene of interest. One of the ideas underlying this trend is that oligonucleotide uptake into cells in vivo may be completely different from that observed with cultured cells. Furthermore, targets may be identified where antisense suppression

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 21

actually alters the progression of a given disease in the organism, rather than targets which may be predicted to do so from in vitro work. Clearly, any compound found to produce a therapeutic advantage in such systems is of great interest, whatever the actual mechanism of action. However, it is vital to differentiate between real antisense effects and aptameric, and other nonantisense mechanisms so that rational progress may be maintained in targeting other genes in other disease states. One way that an antisense mechanism may be demonstrated to be at least part of the overall activity of an oligonucleotide in vivo is by RLPCR detection of mRNA 3′-fragments resulting from RNase H cleavage at the targeted site of RNA-oligomer hybridization, as described above for cell culture models. However, certain caveats need to be borne in mind. It is important that RLPCR is performed on RNA extracted from viable cells, as oligodeoxynucleotides rapidly redistribute to intracellular locations when the integrity of the cytoplasmic membrane is violated (Tidd, 1998), and RNase H activity is readily observed when cells are Iysed at room temperature under non-denaturing conditions in the presence of antisense compounds (Giles et al., 1995c). Therefore, care should be taken to address these concerns when samples are taken from a biopsy, and RNA should be extracted using a protocol which efficiently denatures proteins. DNA sequencing provides the means to identify positively any amplified fragments produced when the extracted RNA samples are subjected to the RLPCR procedure. 1.10 Conclusions The variety of potential mechanisms of action of antisense oligonucleotides makes it difficult to assign their biological effects to inhibition of expression of the targeted gene with any degree of certainty, even when the protein product of that gene is shown to have been suppressed. The question of the appropriate controls for antisense experiments is also still a matter of some debate (see Chapter 9 this volume) (Stein and Krieg, 1994; Wagner, 1995; Tidd, 1996, 1998), with certain participants arguing that each and every oligonucleotide is unique, and no ideal control exists (Neckers and Iyer, 1998). However, it would seem that inhibition of expression of a particular gene would be more strongly implicated in a biological response if the same phenotypic effects were induced by targeting different sites in its pre-mRNA/mRNA with both RNase H inactive, steric blocking, and RNase H active, antisense oligonucleotides. Acknowledgements The authors’ research is supported by the Leukaemia Research Fund of the UK and The Liposome Company, Inc., Princeton, NJ, USA.

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GILES, R.V., SPILLER, D.G., CLARK, R.E. and TIDD, D.M., 1999, Antisense morpholino oligonucleotide analogue induces missplicing of c-myc mRNA, Antisense Nucl. Acid Drug Dev., 9, 213–220. GILES, R.V., SPILLER, D.G., GREEN, J.A., CLARK, R.E. and TIDD, D.M., 1995b, Optimization of antisense oligodeoxynucleotide structure for targeting bcr-abl mRNA, Blood. 86. 744–754. GILES, R.V., SPILLER, D.G., GRZYBOWSKI, J., CLARK, R.E., NICKLIN, P. and TIDD, D.M., 1998, Selecting optimal oligonucleotide composition for maximal antisense effect following streptolysin O-mediated delivery into human leukaemia cells, Nucl. Acids Res., 26, 1567–1575. GILES, R.V., SPILLER, D.G. and TIDD, D.M., 1993, Chimeric oligodeoxynucleotide analogues: enhanced cell uptake of structures which direct ribonuclease H with high specificity, Anti-Cancer Drug Des., 8, 33–51. GILES, R.V., SPILLER, D.G. and TIDD, D.M., 1995c, Detection of ribonuclease H generated mRNA fragments in human leukaemia cells following reversible membrane permeabilisation in the presence of antisense oligodeoxynucleotides, Antisense Res. Dev., 5, 23–31. GILES, R.V. and TIDD, D.M., 1992a, Enhanced RNase H activity with methylphosphonodiester/ phosphodiester chimeric antisense oligodeoxynucleotides, Anti-Cancer Drug Des., 7, 37–48. GILES, R.V. and TIDD, D.M., 1992b, Increased specificity for antisense oligodeoxynucleotide targeting of RNA cleavage by RNase H using chimeric methylphosphonodiester/ phosphodiester structures, Nucl. Acids Res., 20, 763–770. GURA, T., 1995, Antisense has growing pains, Science, 270, 575–577. GUVAKOVA, M.A., YAKUBOV, L.A., VLODAVSKY, L., TONKINSON, J.L. and STEIN, C.A., 1995, Phosphorothioate oligodeoxynucleotides 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, 2620–2627. HAEUPTLE, M.T., FRANK, R. and DOBBERSTEIN, B., 1986, Translation arrest by oligodeoxynucleotides complementary to mRNA coding sequences yields polypeptides of predetermined length, Nucl. Acids Res., 14, 1427–1445. HAMM, J., 1996, Characterisation of antibody-binding RNAs selected from structurally constrained libraries, Nucl. Acids Res., 24, 2220–2227. HANVEY, J.C., PEFFER, N.J., BISI, J.E., THOMSON, S.A., CADILLA, R., JOSEY, J.A., RICCA, D.J., HASSMAN, C.F., BONHAM, M.A., AU, K.G., CARTER, S.G., BRUCKENSTEIN, D.A., BOYD, A.L., NOBLE, S.A. and BABISS, L.E., 1992, Antisense and antigene properties of peptide nucleic acids, Science, 258, 1481–1485. HAUSEN, P. and STEIN, H., 1970, Ribonuclease H. An enzyme degrading the RNA moiety of DNA—RNA hybrids, Eur. J. Biochem., 14, 278–283. HENDERSON, J.F. and PATERSON, A.R.P., 1973, Nucleotide Metabolism, New York: Academic Press. HERSCHLAG, D., 1991, Implications of ribozyme kinetics for targeting the cleavage of specific RNA molecules in vivo: more isn’t always better, Proc. Natl Acad. Sci. USA, 88, 6921–6925. HO, S.P., BAO, Y., LESHER, T., MALHOTRA, R., MA, L.Y., FLUHARTY, S.J. and SAKAI, R.R., 1998, Mapping of RNA accessible sites for antisense experiments with oligonucleotide libraries, Nat. Biotech., 16, 59–63.

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HO, S.P., BRITTON, D.H.O., STONE, B.A., BEHRENS, D.L., LEFFET, L.M., HOBBS, F.W., MILLER, J.A. and TRAINOR, G.L., 1996, Potent antisense oligonucleotides to the human multidrug resistance-1 mRNA are rationally selected by mapping RNAaccessible sites with oligonucleotide libraries, Nucl. Acids Res., 24, 1901–1907. HOKE, G.D., DRAPER, K., FREIER, S.M., GONZALEZ, C., DRIVER, V.B., ZOUNES, M.C. and ECKER, D.J., 1991, Effects of phosphorothioate capping on antisense oligonucleotide stability, hybridization and antiviral efficacy versus herpes simplex virus infection, Nucl. Acids Res., 19, 5743–5748. INOUE, H., HAYASE, Y., IMURA, A., IWAI, S., MIURA, K. and OHTSUKA, E., 1987a, Synthesis and hybridization studies on two complementary nona(2′-Omethyl)-ribonucleotides, Nucl. Acids Res., 15, 6131–6148. INOUE, H., HAYASE, Y., IWAI, S. and OHTSUKA, E., 1987b, Sequence-dependent hydrolysis of RNA using modified oligonucleotide splints and RNase H, FEBS Lett., 215, 327–330. KAWASAKI, E.S., 1985, Quantitative hybridization-arrest of mRNA in Xenopus oocytes using single-stranded complementary DNA or oligonucleotide probes, Nucl. Acids Res., 13, 4991–5004. KENIRY, M.A., OWEN, E.A. and SHAFER, R.H., 1997, The contribution of thymine— thymine interactions to the stability of folded dimeric quadruplexes, Nucl. Acids Res., 25, 4389–4392. KHALED, Z., BENIMETSKAYA, L., ZELTSER, R., KHAN, T., SHAMARA, H.W., NARAYANAN, R. and STEIN, C.A., 1996, Multiple mechanisms may contribute to the cellular anti-adhesive effects of phosphorothioate oligodeoxynucleotides, Nucl. Acids Res., 24, 737–745. KHALED, Z., RIDEOUT, D., ODRISCOLL, K.R., PETRYLAK, D., CACACE, A., PATEL, R., CHIANG, L.C., ROTENBURG, S. and STEIN, C.A., 1995, Effects of suramin-related and other clinically therapeutic polyanions on protein-kinase-C activity, Clin. Cancer Res., 1, 113–122. KNUDSEN, H. and NIELSEN, P.E., 1996, Antisense properties of duplex- and triplexforming PNAs., Nucl. Acids Res., 24, 494–500. KOLE, R., 1998, Modification of alternative splicing of pre-mRNA by antisense oligonucleotides, in Stein, C.A. and Krieg, A.M. (eds), Applied Antisense Oligonucleotide Technology, pp. 451–469, New York: Wiley-Liss. KRIEG, A.M., 1998, Leukocyte stimulation by oligodeoxynucleotides, in Stein, C.A. and Krieg, A.M. (eds), Applied Antisense Oligonucleotide Technology, pp. 431–448, New York: Wiley-Liss. KRIEG, A.M., MATSON, S., CHENG, K., FISHER, E., KORETZKY, G.A. and KOLAND, J.G., 1997, Identification of an oligodeoxynucleotide sequence motif that specifically inhibits phosphorylation by protein tyrosine kinases, Antisense Nucl. Acid Drug Dev., 7, 115–123. KRIEG, A.M., YI, A.-K., MATSON, S., WALDSCHMIDT, T.J., BISHOP, G.A., TEASDALE, R., KORETZKY, G. and KLINMAN, D., 1995, CpG motifs in bacterial DNA trigger direct B-cell activation, Nature, 374, 546–549. LARROUY, B., BLONSKY, C., BOIZIAU, C., STUER, M., MOREAU, S., SHIRE, D. and TOULMÉ, J.-J., 1992, RNase H-mediated inhibition of translation by antisense oligodeoxyribonucleotides: use of backbone modification to improve specificity, Gene, 43, 189–194.

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LARROUY, B., BOIZIAU, C., SPROAT, B. and TOULMÉ, J.-J., 1995, RNase H is responsible for the non-specific inhibition of in vitro translation by 2′-O-alkyl chimeric oligonucleotides: high affinity or selectivity, a dilemma to design antisense oligomers, Nucl. Acids Res., 17, 3434–3440. LEONETTI, J.P., MECHTI, N., DEGOLS, G., GAGNOR, C. and LEBLEU, B., 1991, Intracellular distribution of microinjected antisense oligonucleotides, Proc. Natl Acad. Sci. USA, 88. 2702–2706. LEWIS, J.G., LIN, K.Y., KOTHAVALE, A., FLANAGAN, W.M., MATTEUCCI, M.D., DEPRINCE, R.B., MOOK, R.A., JR, HENDREN, R.W. and WAGNER, R.W., 1996, A serum-resistant cytofectin for cellular delivery of antisense oligodeoxynucleotides and plasmid DNA, Proc. Natl Acad. Sci. USA, 93, 3176–3181. LIEBHABER, S.A., CASH, F.E. and SHAKIN, S.H., 1984, Translationally associated helix-destabilizing activity in rabbit reticulocyte lysate, J. Biol. Chem., 259, 15597–15602. MACAYA, R.F., SCHULTZE, P., SMITH, F.W., ROE, J.A. and FEIGON, J., 1993, Thrombinbinding DNA aptamer forms a unimolecular quadruplex structure in solution, Proc. Natl Acad. Sci. USA, 90, 3745–3749. MAKER, L.J. and DOLNICK, B., 1987, Comparative hybrid arrest by tandem antisense oligodeoxyribonucleotides or oligodeoxyribonucleoside methylphosphonates in a cellfree system, Nucl. Acids Res., 16, 3341–3358. MALTESE, J.-Y., SHARMA, H.W., VASSILEV, L. and NAYARANAN, R., 1995, Sequence context of antisense RelA/NF-κB phosphorothioates determines specificity, Nucl. Acids Res., 23, 1146–1151. MARCUSSON, F.G., BHAT, B., MANOHARAN, M., BENNETT, C.F. and DEAN, N.M., 1998, Phosphorothioate oligodeoxyribonucleotides dissociate from cationic lipids before entering the nucleus, Nucl. Acids Res., 26, 2016–2023. MATSON, S. and KRIEG, A.M., 1992, Nonspecific suppression of 3H-thymidine incorporation by control oligonucleotides, Antisense Res. Dev., 2, 325–330. MAYEDA, A., HAYASE, Y., INOUE, H., OHTSUKA, E. and OSHIMA, Y., 1990, Surveying cisacting sequences of pre-mRNA by adding antisense 2′-Omethyloligoribonucleotides to a splicing reaction, J. Biochem. (Tokyo), 108, 399–405. MAZUMDER, A., NEAMATI, N., OJWANG, J.O., SUNDER, S., RANDO, R.F. and POMMIER, Y., 1996, Inhibition of the human immunodeficiency virus type 1 integrase by guanosine quartet structures, Biochemestry, 35, 13762–13771. MERGNY, J.-L., PHAN, A.-T. and LACROIX, L., 1998, Following G-quartet formation by UV spectroscopy, FEBS Lett., 435, 74–78. MILLER, P.S., 1998, Oligonucleoside methylphosphonates: synthesis and properties, in Stein C.A. and Krieg, A.M. (eds), Applied Antisense Oligonucleotide Technology, pp. 3–22, New York: Wiley-Liss. MILLER, P.S. and TS’O, P.O.P., 1987, A new approach to chemotherapy based on molecular biology and nucleic acid chemistry: Matagen (masking tape for gene expression), Anti-Cancer Drug Des., 2, 117–128. MILNER, N., MIR, K.U. and SOUTHERN, E.M., 1997, Selecting effective antisense reagents on combinatorial oligonucleotide arrays, Natl Biotechnol., 15, 537–541. MINSHULL, J. and HUNT, T., 1986, The use of single-stranded DNA and RNase H to promote quantitative ‘hybrid arrest of translation’ of mRNA—DNA hybrids in reticulocyte lysate cell-free translations, Nucl. Acids Res., 14, 6433–6451.

28 MECHANISMS OF ACTION OF ANTISENSE OLIGONUCLEOTIDES

MONIA, B.P., JOHNSTON, J.F., SASMOR, H. and CUMMINS, L., 1996, Nuclease resistance and antisense activity of modified oligonucleotides targeted to Ha-ras, J. Biol. Chem., 271, 14533–14540. MURCHIE, A.I.H. and LILLEY, D.M.J., 1994, Tetraplex folding of telomere sequences and the inclusion of adenine bases, EMBO J., 13, 993–1001. NECKERS, L.M. and IYER, K., 1998, Nonantisense effects of antisense oligonucleotides, in Stein, C.A. and Krieg, A.M. (eds), Applied Antisense Oligonucleotide Technology, pp. 147–159, New York: Wiley-Liss. NESTLE, F.O., MITRA, R.J., BENNETT, C.F., CHAN, H. and NICKOLOFF, B.J., 1994, Cationic lipid is not required for uptake and selective inhibitory activity of ICAM–1 phosphorothioate antisense oligonucleotides in keratinocytes, J. Invest. Dermatol., 103, 569–575. O’BRIEN, S.G., KIRKLAND, M.A., MELO, J.V., RAO, M.H., DAVIDSON, R.J., MCDONALD, C. and GOLDMAN, J.M., 1994, Antisense bcr-abl oligomers cause non-specific inhibition of chronic myeloid leukaemia cell lines, Leukaemia, 8, 2156–2162. OJWANG, J., BUCKHEIT, R.W., POMMIER, Y., MAZUMDER, A., DE VREESE, K., ESTE, J.A., REYMEN, D., PALLANSCH, L.A., LACKMAN-SMITH, C., WALLACE, T.L., DE CLERCQ, E., MCGRATH, M.S. and RANDO, R.F., 1995, T30177, an oligonucleotide stabilised by an intramolecular guanosine octet, is a potent inhibitor of laboratory strains and clinical isolates of human immunodeficiency virus type 1, Antimicrob. Agents Chemother., 39, 2426–2435. OJWANG, J., ELBAGGARI, A., MARSHALL, H.B., JAYARAMAN, K., MCGRATH, M.S. and RANDO, R.F., 1994, Inhibition of human-immunodeficiency-virus type-1 activity in vitro by oligonucleotides composed entirely of guanosine and thymidine, J. Acquired Immune Defic. Syndr., 7, 560–570. PARTRIDGE, M., VINCENT, A., MATTHEWS, P., PUMA, J., STEIN, D. and SUMMERTON, J., 1996, A simple method for delivering morpholino antisense oligos into the cytoplasm of cells, Antisense Nucl. Acid Drug Devel., 6, 169–175. PATERSON, B.M., ROBERTS, B.E. and KUFF, E.L., 1977, Structural gene identification and mapping by DNA.mRNA hybrid-arrested translation, Proc. Natl Acad. Sci. USA, 74, 4370–4374. QUARTIN, R., BRAKEL, C. and WETMUR, J., 1989, Number and distribution of methylphosphonate linkages in oligodeoxynucleotides affect exo- and endonuclease sensitivity and ability to form RNase H substrates, Nucl. Acids Res., 17, 7253–7263. RANDO, R.F., OJWANG, J., ELBAGGARI, A., REYES, G.R., TINDER, R., MCGRATH, M.S. and HOGAN, M.E., 1995, Suppression of human immunodeficiency virus type 1 activity in vitro by oligonucleotides which form intramolecular tetrads, J. Biol. Chem., 270, 1754–1760. RUDDELL, C.J., GREEN, J.A. and TIDD, D.M., 1996, Antisense oligonucleotidemediated inhibition of mutant p53 expression, Biochem. Soc. Trans., 24, 410S. SAIJO, Y., UCHIYAMA, B., ABE, T., SATOH, K. and NIKIWA, T., 1997, Contiguous fourguanosine sequence in c-myc antisense phosphorothioate oligonucleotide inhibits cell growth on human lung cancer cells: possible involvement of cell adhesion inhibition, Jpn J. Cancer Res, 88, 26–33. SCHULTZE, P., MACAYA, R.F. and FEIGON, J., 1994, Three-dimensional solution structure of the thrombin-binding DNA aptamer d(GGTTGGTGTGGTTGG), J. Mol. Biol., 235, 1532–1547.

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SHAKIN, S.H. and LIEBHABER, S.A., 1986, Destabilization of messenger RNA/ complementary DNA duplexes by the elongating 80S ribosome, J. Biol. Chem., 261, 16018–16025. SHI, Y., FARD, A., GALEO, A., HUTCHINSON, H.G., VERMANI, P., DODGE, G.R., HALL, D.J., SHAHEEN, F. and ZALEWSKI, A., 1994, Transcatheter delivery of cmyc antisense oligomers reduces neointimal formation in a porcine model of coronary artery balloon injury, Circulation, 90, 944–951. SHIBAHARA, S., MUKAI, S., MORISAWA, H., NAKASHIMA, H., KOBAYASHI, S. and YAMAMOTO, N., 1989, Inhibition of human immunodeficiency virus (HIV-1) replication by synthetic oligo-RNA derivatives, Nucl. Acids Res., 17, 239–252. SHIBAHARA, S., MUKAI, S., NISHIHARA, T., INOUE, H., OHTSUKA, E. and MORISAWA, H., 1987, Site directed cleavage of RNA, Nucl. Acids Res., 15, 4403–4415. SIMONS, M., EDELMAN, E.R., DEKEYSER, J.-L., LANGER, R. and ROSENBURG, R.D., 1992, Antisense c-myb oligonucleotides inhibit intimal arterial smooth muscle cell accumulation in vivo, Nature, 359, 67–70. SIMONS, M. and ROSENBURG, R.D., 1992, Antisense nonmuscle myosin heavy chain and c-myb oligonucleotides suppress smooth muscle cell proliferation in vitro, Circ. Res., 70, 835–843. SMETSERS, T.F.C.M., VAN DE LOCHT, L.T.F., PENNINGS, A.H.M., WESSELS, H.M.C., DE WITTE, T.M. and MENSINK, E.J.B.M., 1995, Phosphorothioate BCR —ABL antisense oligonucleotides induce cell death, but fail to reduce cellular Bcr— Abl protein levels, Leukaemia, 9, 118–130. SOUTHERN, E.M., MILNER, N. and MIR, K.U., 1997, Discovering antisense reagents by hybridization of RNA to oligonucleotide arrays, Ciba Found. Symp., 209, 38–44. SPILLER, D.G., GILES, R.V., BROUGHTON, C.M., GRZYBOWSKI, J., RUDDELL, C.J., TIDD, D.M. and CLARK, R.E., 1998a, The influence of target protein half-life on the effectiveness of antisense oligonucleotide analog-mediated biologic responses, Antisense Nucl. Acid Drug Dev., 8, 281–293. SPILLER, D.G., GILES, R.V., GRZYBOWSKI, J., TIDD, D.M. and CLARK, R.E., 1998b, Improving the intracellular delivery and molecular efficacy of antisense oligonucleotides in chronic myeloid leukemia cells: a comparison of streptolysin O permeabilization, electroporation, and lipophilic conjugation, Blood, 91, 4738–4746. SPILLER, D.G. and TIDD, D.M., 1992, The uptake kinetics of chimeric oligodeoxynucleotide analogues in human leukaemia MOLT-4 cells, Anti-Cancer Drug Design, 7, 115–119. SPILLER, D.G. and TIDD, D.M., 1995, Nuclear delivery of antisense oligodeoxynucleotides through reversible permeabilization of human leukemia cells with streptolysin O, Antisense Res. Dev., 5, 13–21. STEIN, C.A., 1995, Does antisense exist? Nat. Med., 1, 1119–1121. STEIN, C. and CHENG, Y., 1993, Antisense oligonucleotides as therapeutic agents—is the bullet really magical? Science, 261, 1004–1012. STEIN, C.A., CLEARY, A.M., YAKUBOV, L. and LEDERMAN, S., 1993, Phosphorothioate oligodeoxynucleotides bind to the third variable loop domain (v3) of human immunodeficiency virus type 1 gp120, Antisense Res. Dev., 3, 19–31. STEIN, C.A. and KRIEG, A.M., 1994, Problems in interpretation of data derived from in vitro and in vivo use of antisense oligodeoxynucleotides, Antisense Res. Dev., 4, 67–69.

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PART TWO Chemical Aspects

2 Chemistry of Oligonucleotides J.W.ENGELS AND E.UHLMANN

2.1 Introduction Oligonucleotides as drugs have attracted substantial interest in recent years. Sequencespecific interference with RNA function by complementary Oligonucleotides, as proposed by Belikowa et al. (1973) and experimentally investigated by Stephenson and Zamecnik (1978) and Zamecnik and Stephenson (1978), resulted in the ‘antisense approach’. Here the expression of a specific gene is blocked on the mRNA level by a complementary oligonucleotide called antisense oligonucleotide (Figure 2.1). Hybridization, according to Watson-Crick base pairing, should in theory provide high specificity and affinity. Thus Zamecnik and Stephenson (1978) could inhibit Rous sarcoma replication in a cellular assay. In these early days, the available chemistry and the inaccessibility of a large pool of synthetic Oligonucleotides, respectively, limited progress in this field. Some years later, the discovery of catalytically active RNA—so-called ribozymes—that can cleave in either cis or trans has created much interest. Their development as therapeutics has so far been limited to hammerhead and hairpin ribozymes. For practical purposes we shall limit our discussion to synthetic hammerhead ribozymes. The hammerhead ribozyme, originally found in the avocado sunblotch virus (Hutchins et al., 1986), owes its name to the two-dimensional description (Figure 2.2). Now, based on two X-ray structures (Pley et al., 1994; Scott et al., 1995), the two colinear helices, helices II and III, mean that it would be better described as ‘paperclip’. The hammerhead ribozyme catalyses a phosphodiester transesterification reaction at a specific NUH triplex (shown in italics in Figure 2.2) within a given RNA sequence. The final product is a 2′-3′-cyclic phosphate and a fragment with a 5′-hydroxyl group. Magnesium ions are essential for that cleavage (Uhlenbeck, 1987). Although conceptually appealing, antisense or ribozyme action does not play a major role in natural gene regulation. Here the regulation at the level

34 CHEMISTRY OF OLIGONUCLEOTIDES

Figure 2.1 Schematic presentation of a cell with the antigene, antisense ribozyme and sense/aptamer approach

Figure 2.2 Hammerhead consensus sequence, conserved nucleotides in bold: N=A, C, G, U; R=A, G; H=A, C, U; numbering according to Hertel et al. (1992)

of transcription initiation is clearly favoured. A complex interaction of various transcription factors and the DNA itself guarantees the sequence-specific recognition. To date no simple polypeptide can recognize a particular DNA in a practical fashion. As early as 1957 (Felsenfeld and Miles, 1957) a triple-stranded nucleic acid structure was first reported.

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 35

Figure 2.3 Triple helices, Hoogsteen base pairing: above, pyrimidine motif (parallel); below, purine motif (antiparallel)

The basic recognition principle of the triplex is the Hoogsteen base pairing scheme (Figure 2.3). Here the major groove purine site is additionally involved in base pairing. Since there are several motifs, the TAT and C+GC as well as the GGC and AAT, the third strand can orient in either a parallel or an antiparallel fashion. With this concept in mind, it is obvious that so far only purine strands can be targeted. Furthermore, the thermodynamic stability of Hoogsteen base pairs is poorer than that of Watson-Crick base pairs. Therefore, triplex binders for physiological studies are either longer or have additional stabilizing features as compared to antisense agents. Sequence-specific recognition of DNA based on the pyrimidine motif was successfully shown by Moser and Dervan (1987). The recognition was detected by tethering an Fe(II)-EDTA chelate to the oligodeoxynucleotide (ODN) and observation of strand cleavage by footprinting techniques. Le Doan et al. (1987) shortly thereafter observed triplex formation using photocrosslinking techniques with psoralene. For the purine recognition motif, Cooney et al. (1988) demonstrated binding of a guanosine-rich ODN to a guanosine-rich promoter element of the human c-myc gene. An ODN with superior affinity to a complementary DNA strand can in theory replace one strand in a DNA duplex. Homopyrimidine peptide nucleic acid (PNA) (see also Chapter 4) binds under low salt condition to one strand of an AT

36 CHEMISTRY OF OLIGONUCLEOTIDES

DNA-duplex forming a triple helix, thus leaving the second strand looped (Ploop). Table 2.1 Degree and type of modification: (A) 3′-end-capped; (B) 3′5′-end-capped; (C) minimally modified, which is 3′5′-end-capping plus protection at internal pyrimidines; (D) all-phosphorothioate; (E) all-phosphorothioate ‘gap-mer’ with 2′-Omethylribonucleoside wings and a seven deoxynucleotide window for activation of RNase H; (F) ‘mixed backbone’ with methylphosphonate wings and a phosphorothioate window A B

5′-N N N R Y R R Y R Y Y Y R YsNsNsN-3′ 5′-NsNsN R Y R R Y R Y Y Y R YsNsNsN-3′ 5′-NsNsN R YsR R YsR YsYsY R YsNsNsN-3′ 5′NsNsNsRsYsRsRsYsRsYsYsYsRsYsNsNsN3′ 5′NsNsNsRsYsRsRsYsRsYsYsYsRsYsNsNsN3′ 5′NmNmNmRmYsRsRsYsRsYsYsYmRmYmNm NmN-3′

C D

E

F

Abbreviations: N, any nucleotide; Y, pyrimidine; R, purine; s, phosphorothioate; m, methylphosphonate; italics, 2′-O-methylribonucleotide. Table 2.2 Clinical studies with antisense oligonucleotides Phase

Indication

Target protein

Name

Company

FDA approval August 1998

CMV-retinitis/ AIDS

CMV-protein

ISIS/Novartis

Phase II

Morbus Crohn, colitis ulcerosa, rheumatoid arthritis, psoriasis, kidney transplantation Solid tumors Solid tumors CMV-infection Non-Hodgkin lymphoma Solid tumors HIV-infection HIV-infection

ICAM-1

ISIS 2922 (Fomivirsen) Vitravene ISIS 2302

PKC α c-raf kinase CMV-protein bcl-2

ISIS 3521 ISIS 5132 GEM 132 G3139

ISIS/Novartis ISIS/Novartis Hybridon Genta

Ha-ras HIV-protein HIV-protein

ISIS 2503 ISIS 5320 Gps0193

ISIS ISIS/NCI Chugai

Phase I

ISIS/ Boehringer Ingelheim

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 37

Phase

Indication

Target protein

Name

Company

HIV-infection HIV-infection CML AML

HIV-protein HIV-protein − −

Arl77 Gem 92 − –

Aronex Hybridon Lynx Lynx

Abbrevations: CMV, cytomegalie virus; ICAM-1, intercellular adhesion molecule 1; PKC α, protein-kinase C α; c-raf kinase mitogen activated protein kinase; bcl-2, B cell leukemia/lymphoma 2; Ha-ras, Harvey-ras oncogene; HIV, human immuno deficiency virus; NCI, National Cancer Institute; CML, chronic myeloic leukaemia; AML, acute myeloic leukemia.

This is topologically equivalent to the D-loop structure formed under Rec A mediated recombination (Cherny et al., 1993). In gene transfer experiments a synthetic gene, a double-stranded piece of DNA, coding for a polypeptide, is introduced into a cell and expressed within it. This so-called gene therapy also relies in part on synthetic oligonucleotides, mostly unmodified ones so far. Synthetic oligonucleotides are defined as repetitive nucleotide units where the individual bases are properly spaced to interact specifically with the target nucleic acid. Originally, the oligonucleotide was derived from either DNA or RNA repetitive units. More recently, modifications have been undertaken in all the relevant subunits, i.e. sugar, base and phosphate backbone (Table 2.1 and Figure 2.4). Thus we shall discuss backbone-modified ODNs, notably phosphorothioates. These derivatives have so far met the criteria of antisense ODNs as the best drugs (Table 2.2). Repetitive nucleotides or analogues thereof can also be used as, for example, PNA units. Since Chapter 4 deals with PNA, only the analogues will be considered here when discussing chimeras, i.e. mixed backbone ODNs. A different way of dividing ODNs is the concept of their way of action. They can target either DNA or RNA. In the former case we deal with the so-called triplex or antigene approach, or even with strand invasion; in the latter with the antisense or ribozyme approach. 2.2 Design of Oligonucleotides Optimal design of ODNs for biological application demands good knowledge of the target to be addressed. The first decision is whether to target DNA or RNA. What is known about their accessibility, stability, and availability? Though several of the criteria for design are in parallel, we shall discuss the abovementioned approaches separately.

38 CHEMISTRY OF OLIGONUCLEOTIDES

2.2.1 Design Criteria for Antisense, Ribozymes, Triplex Synthetic oligonucleotides designed to interact with specific stretches of RNA or DNA have to meet several criteria. For design purposes, three points are worth mentioning: the length, the type of modification and the mode of action. These in turn determine the affinity and specificity of the oligonucleotide utilized. In order to bind in a Watson-Crick fashion a given RNA in a human cell specifically, a minimum length of a 12-mer has been calculated; for triplex this may be closer to a 20-mer. Though it is difficult to define an upper limit in length, depending on the chemistry and base composition, 12–28mer oligonucleotides are most often used. By optimizing oligonucleotide length and concentration, high specificity of binding can be achieved in a cell. Second, the stability of synthetic oligonucleotides has to be ascertained. Natural oligonucleotides are susceptible to exonuclease and endonuclease degradation. Here a large variety of modified oligonucleotides will be discussed which circumvent this problem. Third, target selection plays an important role in finding the optimal binding site. Very often the region around the translation initiation site is targeted. It is well accepted now that the structure of RNA is of great importance for its accessibility and only a limited part of it can be reached. RNA folds into a complex secondary structure consisting of double-and singlestranded regions. This stem loop geometry is further folded into a tertiary structure of spatially arranged helices and single strands. It has been shown that therapeutic oligonucleotides bind well to only a limited number of sites. Site selection could in principle be based on secondary structure predictions (Zuker and Stiegler, 1981). Since our knowledge about folding and stability of RNA is rather limited, experimental approaches are advisable. These are based either on hybridization techniques (Milner et al., 1997) or on RNase H activity (DorisKeller, 1979). Though oligonucleotides directed to the translational initiation site often decrease the level of a particular mRNA, ODNs targeted to alternative regions frequently show superior downregulation of mRNA. Therefore screening of a set of overlapping ODN is necessary to find potent candidates. Recently a statistical analysis was presented of the correlation between in vitro accessibility and ODN intracellular activity (Matveeva et al., 1998). 2.2.2 Degree of Modification Uniformly phosphorothioate-modified oligonucleotides are still the best investigated and most broadly used type of antisense agents. Although numerous compounds of this first generation antisense oligonucleotide are in clinical trials, great effort is devoted to the development of second generation antisense

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 39

compounds in which the beneficial properties needed for antisense therapeutics are combined. Two basic aspects have to be discussed with respect to side-effects caused by phosphorothioates. First, uniformly phosphorothioate-modified oligonucleotides have a tendency for non-antisense effects which are mainly due to undesired binding to proteins (see also Chapter 9) (Milligan et al., 1993; Stein, 1995; Stein and Cheng, 1993; Uhlmann and Peyman, 1990). Second, the activation of RNase H by partial oligonucleotide/RNA duplices of only five to seven nucleotides in length can cause, under certain conditions, non-specific degradation of mRNA (Woolf et al., 1992). Both limitations may in principle be overcome by reducing the number of phosphorothioate linkages in oligonucleotides by modifications which do not lead to increased binding to proteins or which do not activate RNase H. This enzyme recognizes a duplex between DNA and RNA resulting in the cleavage of the RNA strand of this duplex. However, in view of the lability of unmodified oligonucleotides against nucleases and the weak antisense effects observed for most derivatives which do not activate RNase H, the development of mixed-backbone oligonucleotides or chimeric oligomers, in which the advantages of the individual structural elements are combined, appears to be the approach of choice at present. Although the metabolism of ODN involves cleavage by exonucleases and endonucleases, the major degrading activity in serum and cells is a 3′exonuclease (Shaw et al., 1991; Uhlmann and Peyman, 1990). Capping of the 3′end, or both the 3′- and 5′-ends, by phosphorothioate linkages was reported to protect the oligonucleotide significantly against 3′-exonuclease degradation (Daum et al., 1992; Gillardon et al., 1994; Gillardon et al., 1995; Hoke et al., 1991; Shaw et al., 1991; Stein et al., 1988). However, in in vivo experiments this protection strategy has proved to be only of limited success, since end-capped oligonucleotides are still subject to endonuclease degradation. Recently, a ‘minimal protection’ strategy has been suggested which is a combination of the end-capping technique and the protection at internal pyrimidine residues which are the major sites of endonuclease degradation (Peyman and Uhlmann, 1996; Uhlmann and Peyman, 1998). The endonuclease cleavage becomes especially serious if two or more pyrimidines are adjacent (Uhlmann et al., 1997). Minimally phosphorothioate modified oligonucleotides turned out to be particularly useful, since they are sufficiently stable to exonucleases and endonucleases, while at the same time undesirable non-antisense effects are strongly reduced (Peyman et al., 1997; Tanaka et al., 1996; Uhlmann and Peyman, 1998). Furthermore, the minimal protection scheme can be combined with secondary modifications, such as 2′-O-alkyl modification of ribose (Inoue et al., 1987; Sproat et al., 1991) or C5-alkynyl modification of the pyrimidine bases (Froehler et al., 1992; Uhlmann et al., 1997), in order to improve binding to the target mRNA. The activation of RNase H is not compromised by the C5alkynyl modification, whereas 2′-modified nucleotides do not stimulate RNase H. One possibility to limit the activation of RNase H to a certain region of the oligonucleotide is realized in the so-called gap-mers (see also Chapter 1)

40 CHEMISTRY OF OLIGONUCLEOTIDES

(Crooke et al., 1995; Inoue et al., 1987), in which 2′-O-methylribonucleotide wings are introduced to enhance binding to mRNA, while a window of six to eight deoxynucleotides limits the stimulation of RNase H cleavage to this part of the duplex. Similarly, mixed-backbone oligonucleotides (Agrawal et al., 1997) consisting for example of methylphosphonate and phosphorothioate linkages, show improved properties: the methylphosphonate provides excellent nuclease resistance and non-ionic nature, while the phosphorothioate linkage is also sufficiently stable to nucleases and in addition allows activation of RNase H. Peptide nucleic acids (PNAs) are extremely stable during incubation in serum, but show poor solubility in aqueous medium and do not stimulate RNase H in a duplex with RNA (Uhlmann et al., 1998b). In contrast, 5′-DNA-PNA(pseudo-3′) chimeras (Uhlmann et al., 1996) with more than four deoxynucleotides at the 5′-part of the oligomer can induce RNase H and are completely resistant to 3′-exonucleases (Uhlmann, 1998). Additionally, these chimeras show improved binding affinity as compared to the natural counterparts. It has been found that phosphorothioate oligonucleotides with a CG sequence motif have immunostimulatory effects (Krieg et al., 1995). Substitution of 2′-Omethylribonucleosides for CG deoxynucleotides could minimize the undesired immunostimulatory effect in the 18-mer oligonucleotide 5′CCGCTCTTCCTCACTGGT-3′ (Agrawal and Zhao, 1998). Since the CG motif in this oligonucleotide was at the 5′-end, substitution of four deoxynucleotides at both the 5′- and the 3′-end resulted not only in reduced toxicity, but also in increased biological activity due to improved binding and enhanced stability against nuclease degradation. 2.3 Oligonucleotide Modifications: Synthesis and Properties In the following we will outline the structural chemistry and properties of antisense oligonucleotide derivatives, and discuss the different routes for synthesis of unmodified oligonucleotides and selected modified derivatives. Chemical variation of the natural oligonucleotide structure is necessary to render these compounds useful in biological systems, as follows. 1 They must be sufficiently stable against nucleases in serum and within cells. 2 They should enter the various organs of the body. After distribution to the desired tissue, they must be able to penetrate cellular membranes to reach their site of action. 3 They must form stable Watson-Crick or Hoogsteen complexes with complementary target sequences under physiological conditions. In recent years, a plethora of different oligonucleotide analogues have been described (Figure 2.4), in which the natural phosphodiester linkage, the sugar

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 41

part or the heterocyclic bases of the oligonucleotides were modified aiming at the improvement of properties (Beaucage, 1993; Beaucage and Iyer, 1993; Milligan et al., 1993; Uhlmann and Peyman, 1990). 2.3.1 Unmodified Oligonucleotides having 3′5′-Phosphodiester Linkages Although unmodified oligonucleotides are widely used as tools in molecular biology, they have serious limitations with respect to their use as gene expressioninhibiting therapeutics. In cellular and animal experiments one has to take account of the fact that oligonucleotides with a natural phosphodiester internucleoside linkage are degraded in serum within a few hours, mainly by the action of fast cleavaging 3′-exonucleases which are accompanied by slower cleavaging endonucleases. In certain tissues a significant 5′-exonuclease activity has been observed. In the following sections chemically modified oligonucleotides will be described. However, it should be noted that oligonucleotides can also be protected against nucleolytic cleavage to a certain extent by packaging into liposomes or nanoparticles which at the same time serve as carriers for in vivo application of these compounds (see Chapters 6 and 7). Unmodified oligonucleotides are taken up to a substantial extent by cultured living cells in a time- and energy-dependent endocytodic-like process (see also Chapters 9 and 10). However, a punctate intracellular distribution of oligonucleotides is observed using fluorescence microscopy. As the oligonucleotides are obviously trapped in endosomes or lysosomes, only a comparatively low concentration of ‘free’ hybridizable oligonucleotide in the cytoplasm or in the nucleus is obtained. This problem can be overcome by microinjection (Clarenc et al., 1993) of the antisense oligonucleotides into the cytosol from which they rapidly penetrate into the nucleus, or by administration of the oligonucleotides with cationic lipids, such as lipofectin™ (Bennett et al., 1993). The observed efficacy of unmodified oligonucleotides as antisense inhibitors can be explained by an antisense oligonucleotide-stimulated degradation of the target mRNA which is brought about by the cellular enzyme RNase H. In the following two sections, the basic possibilities for the synthesis of natural phosphodiester oligonucleotides are discussed briefly. We will see that both methods frequently also form the basis for the synthesis of modified oligonucleotides.

42 CHEMISTRY OF OLIGONUCLEOTIDES

Figure 2.4 Different possibilities for the modification of oligonucleotides

2.3.1.1 Chemical synthesis The synthesis of unmodified oligonucleotides can be performed either in solution or on solid supports. At low or medium scale, oligonucleotide synthesis using polymeric supports and step-wise addition of monomeric building blocks is preferred. Although different chemistries, such as the phosphodiester, the phosphotriester and H-phosphonate methods have been used successfully in the past, the phosphoramidite chemistry clearly dominates oligonucleotide synthesis up to the multikilogram scale at present. The phosphoramidite method according to Matteucci and Caruthers (1981) involves the reaction of the 5′-hydroxy group of a nucleos(t)ide, which is bound to the solid-support via its 3′-hydroxy group, with a nucleoside-3′-β-cyanoethyl N, N-diisopropylphosphoramidite under catalysis using a weak acid (Figure 2.5). Although catalysis by 1H-tetrazole has been the method of choice for many years, more efficient catalysts, such as dicyanoimidazoles (Schell and Engels, 1998; Vargeese et al., 1998) are becoming of increasing interest. The phosphite triester resulting from the coupling is oxidized immediately with iodine/water to

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 43

Figure 2.5 Synthesis cycle for the preparation of oligonucleotides on a solid support according to the phosphoramidite chemistry

Figure 2.6 Synthesis of oligonudeotides by the H-phosphonate method

the corresponding phosphotriester. The coupling yields of the phosphoramidite method are usually >99%. As phosphate blocking group the β-cyanoethyl group (Sinha et al., 1983) is most popular; it can be cleaved with ammonia in a βelimination reaction, thus avoiding unfavourable cleavage of internucleotide linkages. For chain elongation the 5′-hydroxy group of the growing DNA chain is protected with 4, 4′-dimethoxytrityl (Dmt) group. It has the advantage that it can be introduced into the monomers quite regioselectively and renders the monomers easily soluble in organic solvents, such as methylenechloride and acetonitril. On deblocking with 3% trichloroacetic acid the resulting orange-red

44 CHEMISTRY OF OLIGONUCLEOTIDES

Figure 2.7 Oligonucleotides with 2′5′-, 3′3′- and 5′5′-phosphodiester linkages

coloured Dmt cation allows easy monitoring (498 nm) of the efficiency of coupling steps. The exocylic amino functions of the bases are protected by acyl protecting groups, which are cleaved off by ammonia treatment at the end of the synthesis. Recently, the facile synthesis of oligonudeotides without the need for nucleobase protection has been reported (Hayakawa and Kataoka, 1998). The H-phosphonate method has the advantage that no phosphate protecting group is employed. This method involves the reaction of the 5′-hydroxy group of the growing DNA chain with a nucleoside-3′-H-phosphonate (Figure 2.6). Sterically hindered carbonyl chlorides, such as pivaloyl or adamantoyl chloride (Sinha and Cook, 1988), are used as coupling reagents. Only after the construction of the whole oligonucleotide chain has been completed, the Hphosphonate internucleoside linkages are oxidized in one step to the phosphotriester linkages. This is in contrast to the phosphoramidite method, in which the phosphite linkages are oxidized after each coupling step (Froehler et al., 1986). 2.3.1.2 Enzymatic synthesis and ligation The enzymatic synthesis of oligonudeotides, especially longer ones, containing modified building units is a valuable alternative to chemical synthesis. Enzymatic incorporation of natural or suitably modified nucleoside triphosphates by phage RNA polymerases (e.g. T7) has been successfully used. This approach is limited by the acceptance of the polymerase for sugar, backbone or base modification. For the backbone linkage, phosphorothioates are the most common derivatives. All four nucleoside thiotriphosphates are good substrates for RNA as well as DNA polymerases. Only the Sp-Isomer is actually accepted. Since the incorporation proceeds with inversion of configuration by an in-line mechanism, only the Rpphosphorothioate linkage is obtained (Eckstein, 1985). For the sugar modification, 2′-deoxynucleoside triphosphates (Milligan and Uhlenbeck, 1989), especially in the presence of Mn2+ instead of Mg2+ (Conrad et al., 1995), have

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 45

been successfully incorporated. In this case even 2′-O-methyl nucleoside triphosphates were incorporated. 2′-Aminonucleoside triphosphates are good substrates (Aurup et al., 1992) whereas 2′-fluoronucleoside triphosphates are less readily incorporated. Here Huang et al. (1997) introduced mutant T7 polymerase with improved efficiency. In a post-synthetic modification step, kinase easily introduces a 5′thiophosphoryl group (Igloi, 1988). Ligation of short oligonucleotides to synthesize long oligonucleotides is very attractive. This can be accomplished either by DNA, RNA-ligases or by chemical ligation. When DNA ligases are used, a 5′-phosphoryl donor group in a preformed duplex is ligated to a free 3′hydroxyl group acceptor (Maunders, 1993). In contrast, RNA ligases, particularly T4 RNA ligase, catalyse the formation of phosphodiester bonds between two single-stranded oligomers, whereby a 5′-phosphoryl donor and a 3′hydroxylacceptor are coupled. This enzyme accepts a variety of nucleoside analogues even at the ligation junction. By this method, longer RNA-sequences, which can be fluorescence labelled (Cosstick et al., 1984), in particular can be synthesized. Alternatively, T4 DNA ligase has been used to ligate two oligoribonucleotides with the aid of a short complementary oligodeoxynucleotide at the ligation junction (Moore and Sharp, 1992). 2.3.2 Oligonucleotides with 2′5'-Linkages and 3'3'-/ 5'5'Inversions A special type of minimal modification is oligonucleotide analogues which differ from their naturally occuring 3′, 5′-phosphodiester compounds only by changes in the linkage, but do not include new chemical entities. Oligonucleotides with 3′, 3′- or 5', 5'-end inversions (Seliger et al., 1991) (Figure 2.7) are very stable against exonucleases, and their half-life in human serum is several hours, as compared to minutes for completely 3′5′-linked oligonucleotides. The WatsonCrick base-pairing of the antiparallel portion of the oligonucleotide appears not to be disturbed by the inverted termini. In order to extend base recognition and to improve flexibility in triplex formation, the 3′3′- and 5′5′-inversions were also exploited. The 3′3′-inversions, also called 3′, 3′-switches, in which the polarity of the strand changes via a 3′, 3′linkage within the sequence, allow jumping from one purine tract of one strand to another purine tract on the complementary strand (McCurdy et al., 1991). Synthesis of these analogues is straightforward by using the corresponding ‘inverted’ nucleoside-5′-O-phosphoramidites or nucleoside-5′-O-succinylsupports combined with standard phosphoramidite chemistry (Seliger et al., 1991). The naturally occurring 2′, 5′-oligoadenylates (Figure 2.7) are involved in the stimulation of cellular RNase L as part of the cell’s defence system, e.g. after viral infection. RNase L cleaves single-stranded RNA adjacent to double-

46 CHEMISTRY OF OLIGONUCLEOTIDES

stranded RNA in response to interferon treatment or viral infection. Interestingly, chimeric molecules having 5′-phosphorylated 2′, 5′-tri-adenylate conjugated to a normal 3′, 5′-linked antisense oligonucleotide have been successfully used to cleave a target RNA by activation of 2′, 5′-adenylatedependent RNase L (Torrence et al., 1993). Because 2′, 5′-adenylate-dependent RNase L is present in most mammalian cells, this strategy is of potential use when applied in combination with antisense oligonucleotide derivatives which do not stimulate RNase H cleavage, e.g. in combination with 2-Oalkylribonucleotide analogues or PNA. Synthesis of 2′5′-oligoribonucleotides can be achieved using protected nucleoside-2′-phosphoramidite building blocks, in which the 3′-hydroxy group is protected by the fluoride cleavable tert.-butyl dimethylsilyl protecting group (Ogilvie and Iwacha, 1973). 2.3.3 Modification on Phosphorus The nucleases achieve oligonucleotide degradation by nucleophilic attack at the phosphodiester linkage. Therefore, replacement of one non-bonding phosphate oxygen by other atoms is an easy way to make oligonucleotides resistant against nucleases. It also allows the alteration of other properties of oligonucleotides, such as binding affinity and cellular permeation. The most common types of modification are depicted in Figure 2.8. 2.3.3.1 Phosphorothioates Phosphorothioates, in which one non-bridging oxygen is replaced by a sulphur, are among the most obvious analogues of naturally occurring phosphodiesters (Eckstein, 1983). The phosphorothioate modification provides significant stabilization against degradation by nucleases. It must be mentioned, however, that nuclease resistance depends strongly on the configuration on the phosphorus. The Sp diastereomers are substrates of nucleases S1 or P1, while the Rp diastereomers are cleaved by snake venom phosphodiesterase. Cellular uptake of phosphorothioate oligonucleotides is similar to phosphodiester oligonucleotides. An important property of phosphorothioates is the ability to mediate RNase H degradation of the RNA after hybridization. One drawback of uniformly phosphorothioate-modified oligonucleotides is their propensity for non-specific effects, which result mainly from the interaction with cellular proteins (Stein, 1996). Phosphorothioate-containing oligonucleotides are easy to synthesize, as the same synthons as for phosphodiester oligonucleotide synthesis (phosphoramidite or H-phosphonate chemistry) can be used. The only difference is in the oxidation step, where sulphur is introduced instead of oxygen. The most widely used

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 47

Figure 2.8 Examples of the modification on phosphorus

sulphurization agents are Beaucage’s reagent (Iyer et al., 1990) and tetraethylthiuram disulphide (Vu and Hirschbein, 1991). The phosphorothioate bridge also represents a centre of chirality. The hybridization properties of diastereoisomeric phosphorothioates are sufficiently good to use them under in vivo conditions, although there is an average loss of 0. 5 K/ phosphorothioate linkage in the melting temperature for the racemic mixture. Heterodimers formed between oligoribonucleotides and all-RPphosphorothioates showed a higher Tm as compared with the less stable heterodimers formed with all-Sp-phosphorothioates or the random mixture of diastereomers (Koziolkiewicz et al., 1995). The DNA—RNA complex containing the phosphorothioate of all-Rp configuration was found to be more susceptible to RNase H-dependent degradation than hybrids having either all-Sp counterparts or the random mixture of diastereomers. Interestingly, 3′exonucleases present in human plasma appear to degrade phosphorothioates of Rp configuration, but not of those of Sp configuration (Koziolkiewicz et al., 1997). 2.3.3.2 Alkylphosphonates and arylphosphonates After the phosphorothioates, the methylphosphonates are probably the second best investigated class of oligonucleotide derivatives with a modification on the phosphorus, and they were used very early for specific antisense inhibition of gene expression (Miller et al., 1985). In methylphosphonates the negatively charged phosphate oxygen is replaced by a neutral methyl group. The methylphosphonate linkage is highly stable against degradation by nucleases. However, a major problem is posed by the chirality of the methylphosphonate

48 CHEMISTRY OF OLIGONUCLEOTIDES

bridge, which can have either the Rp or the Sp configuration. Therefore, methylphosphonate oligonucleotides as phosphorothioates resulting from standard synthesis usually consist of a mixture of 2n diastereoisomers, where n is the number of such linkages. It has been shown for short oligonucleotides with a methylphosphonate backbone that the all-Rp backbone shows a significantly higher melting point (Tm) than a mixture of diastereomers or even the corresponding phosphodiester oligonucleotide when hybridized to complementary nucleic acids. Uniformly methylphosphonate-modified oligonucleotides do not form duplexes with RNA that induce RNase H cleavage. This may be a limitation to their use as antisense oligonucleotides and could explain the relatively high concentration required for effective translation arrest in antisense experiments. As discussed in more detail in section 2.2, chimeric oligonucleotides containing both methylphosphonate and a window of at least five to seven phosphodiester linkages retain RNase H activity, and the poor solubility of uniformly modified compounds in biological systems can be overcome. Alkylphosphonates are available through automated oligonucleotide synthesis using methylphosphonamidites as synthons (Engels and Jaeger, 1982). Since the alkylphosphonate bridge is more base-labile than the natural phosphodiester linkage, much milder conditions are necessary for cleavage from the support and for deprotection. Similarly, phenylphosphonate—and phenylphosphonothioatecontaining oligonucleotides can be prepared from the corresponding nucleoside-3′-phenylphosphonamidites (Mag et al., 1997). Binding affinity of the arylphosphonate-containing oligomers depends strongly on the sequence, so that some duplexes are destabilized by −0.3 to −1.3 K, whereas others are stabilized by +0.2 to +0.5 K relative to their natural congeners. The synthesis of oligodeoxynucleotide pentadecamers containing two octylphosphonate linkages with stereoregular or stereorandom chirality has also been described (Mag et al., 1996). The difference in Tm was −3.4 K per modification for the stereorandom and about −2.3 to −4.0 K per modification for the stereoregular configurated oligonucleotides. 2.3.3.3 Other modifications on phosphorus The phosphoramidate linkage is also attractive, as introduction can easily be achieved using H-phosphonate chemistry with a final CCl4/amine oxidation step (Froehler, 1986). Alternatively, the oxidation of phosphite triesters by I2/amine allows the specific introduction of phosphoramidates in specific positions with moderate yields. However, the P–N bond is relatively easily cleaved by acid, resulting in phosphodiesters. Phosphoramidate oligonucleotides have a decreased binding affinity to complementary DNA (−1.5 K/modification). Phosphotriesters (Marcus-Sekura et al., 1987), like the alkylphosphonates, have been used as non-ionic analogues of oligonucleotides. However, because of

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 49

the lability of the phosphotriester function towards basic deblocking conditions (P-OCH3>P-OC2H5>P-OiC3H7) and their potential as alkylating reagents, they are only infrequently used as antisense agents at present. An interesting class of analogues are those with a ‘boronated’ internucleotide linkage (Sood et al., 1990), since they appear also to induce RNase H in a duplex with RNA. As phosphorothioates and methylphosphonates, they are also very resistant to degradation by nucleases. 2.3.4 Oligonucleotides Containing Dephospho Linkages Due to the stereochemical problems associated with methylphosphonates and phosphorothioates, there is a growing interest in oligonucleotide analogues containing achiral, uncharged and phosphate-free linkages (Uhlmann and Peyman, 1993). All investigated oligonucleotide analogues of this type are completely stable to nuclease degradation. Furthermore, many oligonucleotide analogues of this type form more stable complexes with complementary nucleic acids. Problems with these analogues include poor water-solubility and poor hydrolytic stability. Most of the methods reported so far for the synthesis of dephospho oligonucleotide analogues are difficult and not easily amenable to automated solid-phase synthesis. Oligonucleotide analogues have been described containing formacetal, 5′thioformacetal and 3′-thioformacetal linkages (Figure 2.8), which are uncharged, achiral isosteres of the normal phosphodiester linkage (Jones et al., 1993; Matteucci, 1991). In all cases dimers or trimers containing the modified linkages were synthesized in solution. After incorporation of these building blocks into oligonucleotides at appropriate sites, chimeric formacetal-phosphodiester oligonucleotides were obtained. Oligonucleotides containing formacetal linkages have favourable hybridization properties when hybridized to RNA. Thus, a tetradecamer containing four formacetal linkages showed a Tm value of 59°C when hybridized to single-stranded RNA, as compared to 60°C for the allphosphodiester-linked tetradecamer. In contrast, the binding affinity of formacetal analogues to single-stranded DNA turned out to be considerably lower (Tm=39°C) than that of the all-phosphodiester-linked oligomer (Tm=50°C). Oligonucleotides containing alternating 3′-thioformacetal-phosphodiester linkages showed improved binding affinity on hybridization with single-stranded RNA. In a DNase I footprinting assay, triple-helix formation equivalent to that of the control oligonucleotide was observed. Oligonucleotide analogues with dialkylsilyl internucleoside linkages were prepared by one-pot reactions starting from dialkyldichloro or chloro (dialkylamino)silane derivatives (Seliger and Feger, 1987). Phosphodiesterlinked oligonucleotides containing one or two diisopropylsiloxane linkages were synthesized using phosphoramidite derivatives of siloxane-linked dimers and trimers. The Tm values for undecamers containing one or two diisopropylsiloxane

50 CHEMISTRY OF OLIGONUCLEOTIDES

Figure 2.9 Examples of the modification of the sugar moiety

links were 2–5 K less than that of the control of all-phosphodiester oligonucleotides. The diisopropylsiloxane link is again stable to 3′-exonuclease digestion (Cormier and Ogilvie, 1988). A plethora of other dephospho oligonucleotide analogues have been reported in the literature (Uhlmann and Peyman, 1993), including carbonate-, carboxymethyl-, acetamidate-, carbamate-, thioether-, sulphonate-, sulphonamide-, oxime, methyleneimino-, methylene methylimino (MMI)-, methylene dimethylhydrazo (MDH)-, methyleneoxy methylimino-, urea-, guanidine-, all-carbon-, riboacetal-, and amide-linked nucleosides. Among the most promising derivatives are the MMI-linked derivatives (Debart et al., 1992). 2.3.5 Modification of the Sugar Moiety The 2′-deoxy-β-D-ribofuranose unit of the DNA backbone is another suitable site for oligonucleotide modification (Figure 2.9). There are quite a number of sugar modifications that have been synthesized and tested for improvement of antisense oligonucleotide properties, such as improved binding affinity and enhanced stability against nucleases.

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 51

2.3.5.1 α-Anomeric oligonucleotides α-Anomeric oligonucleotides (Bloch et al., 1988; Morvan et al., 1991) show excellent binding properties. In some cases the melting temperature of a duplex with RNA is almost doubled compared to β-anomeric oligonucleotides. Furthermore, α-anomeric oligonucleotides are highly nuclease-resistant. Despite their excellent binding properties, they are not generally efficient in antisense experiments, since they do not activate RNase H, although good inhibition was obtained when the α-anomeric oligonucleotide was directed against the 5′-nontranslated mRNA region (Bertrand et al., 1989). The synthesis of α-anomeric oligonucleotides is very similar to the one used in β-deoxyribonucleotide synthesis, whereby α-anomeric monomeric phosphoramidite building blocks are employed (Debart et al., 1992). Similarly to their β-anomeric analogues, the α-oligodeoxynucleoside phosphorothioates can be obtained easily by oxidation with elemental sulphur or Beaucage’s reagent following the standard conditions. 2.3.5.2 2′-Modified oligonucleotides 2′-Modified oligonucleotides can be considered as analogues of oligoribonucleotides. They can be subdivided into 2′-O-alkyl RNA and other modifications at the 2′-position such as 2′-fluoro or 2′-amino-substituted oligonucleotides. The 2′-O-methyl ether of RNA is a naturally occurring modification which is found, for example, at certain positions in tRNAs, rRNAs and snRNAs. The stability of (RNA)•(2′-O-alkyl-RNA) heteroduplexes (Freier and Altmann, 1997) clearly depends on the nature of the 2′-O-alkyl group and decreases in the order 2′-methoxyethoxy>2′-O-methyl>2′-O-allyl>2′-OH>2′-O-butyl>2′-Odimethylallyl. As early as 1987 it was reported that 2′-O-methyl RNA forms a more stable duplex with a complementary RNA strand than unmodified DNA or even RNA (Inoue et al., 1987). The 2′-O-alkylribonucleotides prefer the C(3′)endo conformation in a duplex with RNA. This sugar pucker has been found as a key structural element in RNA•RNA duplexes which are generally more stable than DNA•DNA duplexes of the same sequence. It is worth mentioning that the 2′-O-methoxyethoxy modification not only results in enhanced binding affinity to RNA, but at the same time renders the oligomer more stable against nuclease degradation. Synthesis starts from the corresponding nucleosides containing 2′-O-alkyl groups combined with standard 2-cyanoethyl phosphoramidite chemistry (Shibahara et al., 1987). Replacement of the 2′-hydroxy groups in RNA by fluorine or the amino group can also be employed to obtain analogues with improved binding affinity or

52 CHEMISTRY OF OLIGONUCLEOTIDES

enhanced stability towards nucleases, respectively. Unfortunately, the corresponding monomeric nucleosides are difficult to prepare and they also tend to be toxic. A uniformly modified 2′-deoxy-2′-fluoro oligonucleotide (Kawasaki et al., 1993) exhibits a considerably increased binding affinity for RNA as compared to the DNA oligonucleotide without compromising base pair specificity, whereas the 2′-amino modification (Aurup et al., 1994) destabilizes the duplex with RNA. However, additional modifications, such as phosphorothioate bridges, are necessary to render these oligonucleotide derivatives sufficiently stable to nucleases. As with 2′-O-alkyl RNA, introduction of uniform 2′-deoxy-2′-fluoro sugars lead to the loss of RNase H activation. 2.3.5.3 Sterically locked nucleic acid analogues Nucleic acid analogues with conformationally restricted sugar phosphate backbones based on (3′S, 5′R)-2′-deoxy-3′5′-ethano-β-D-ribofuranosyladenine and-thymine (bicyclo-DNA) were reported to form more stable duplexes than the corresponding natural congeners (Tarkov et al., 1993). The bicyclo DNA (A10) formed more stable triplexes with d(T10) of the pyrimidine-purine-pyrimidine motif than natural d(A10). Recently, a new DNA analogue, ‘bicyclo-[3.2.1]-DNA’, has been described (Epple and Leumann, 1998) which has a rigid phosphodiester backbone, emulating a B-DNA-type conformation, and to which the nucleobases are attached via a flexible open-chain linker. Although bicyclo-[3.2.1]-DNA forms less stable duplexes with complementary DNA than natural DNA, basemismatch discrimination is slightly enhanced compared to pure DNA duplexes. Importantly, bicyclo-[3.2.1]-DNA oligomers are resistant to 3′-exonuclease degradation. A novel modification with unusually high binding affinity is LNA (Locked Nucleic Acid), in which the 2′ oxygen is linked by a methylene bridge to the 4′ carbon forming a methylene-linked bicyclic ribofuranosyl nucleoside. The sugar conformation of this derivative is locked in the N-type (3′-endo) conformation (Koshkin et al., 1998). An unprecedented increase of 3 to 8 K per modification in the thermal stability of duplexes towards both DNA and RNA was reported when evaluating mixed sequences of partly or fully modified LNA. 2.3.5.4 Other sugar-modified analogues Oligonucleotide analogues consisting of 1′, 5′-anhydrohexitol nucleoside building blocks (hexitol nucleic acids, HNAs) (Hendrix et al., 1997) were shown to form stable duplexes with natural DNA and RNA. The stabilizing effect amounts to +1.3 K/base pair with DNA as complement and +3.0 K/base pair with RNA as complement. HNA is completely stable towards 3′-exonucleases.

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 53

Figure 2.10 Structure of peptide nucleic acids (PNAs) and DNA—PNA chimeras

A great number of other sugar modifications have been incorporated in antisense oligonucleotides, e.g. L-2′-deoxyribose (Garbesi et al., 1993; Urata et al., 1992), 2′-deoxy-β-D-xylofuranose (Rosemeyer et al., 1991), l-(Darabinofuranose) (Resmini and Pfleiderer, 1993), 4′-thio-β-D-ribose (Bellon et al., 1993), and carbocyclic (Szemzo et al., 1990) sugar analogues. 2.3.6 Peptide Nucleic Acids Peptide nucleic acids (PNAs) (see also Chapter 4), in which the entire sugarphosphate backbone is replaced by an N-aminoethylglycine-based polyamide structure (Nielsen et al., 1991), bind with higher affinity to complementary nucleic acids than their natural counterparts following the Watson-Crick base-pairing rules (Egholm et al., 1993; Hyrup and Nielsen, 1996; Uhlmann et al., 1998b). The Nterminus of PNA corresponds to the 5′-end and the C-terminus to the 3′-end of DNA (Figure 2.10). In the case of the antiparallel PNA•DNA or PNA•RNA duplex, the melting temperature is increased by approximately 1 K/base or 1.5 K/base, respectively, as compared to the corresponding DNA•DNA or DNA•RNA duplex. A further advantage of PNA is that base-mismatches often give rise to a significantly larger reduction in the Tm value as compared to DNA. PNA is extremely stable to nucleases and peptidases. One limitation of PNA, however, is that it cannot stimulate RNase H cleavage. Depending on the targeted mRNA sequence, antisense oligonucleotides having the ability to stimulate RNase H (e.g. phosphodiester and phosphorothioate oligonucleotides) are often more effective antisense inhibitors than those without this ability. Consequently, it has been found that

54 CHEMISTRY OF OLIGONUCLEOTIDES

Figure 2.11 Examples of the modification of the heterocyclic bases

PNAs are less effective antisense agents than phosphodiester (van der Laan et al., 1998) and phosphorothioate (Bonham et al., 1995) oligonucleotides. In contrast, DNA•PNA chimeras (Uhlmann, 1998) (Figure 2.10) with more than four nucleotides are able to stimulate the cleavage of RNA by RNase H on formation of a chimera•RNA duplex. RNA cleavage occurs at the ribonucleotides which base-pair with the DNA part of the chimera. PNA—DNA chimeras also obey the Watson-Crick rules on binding to complementary DNA and RNA (Uhlmann et al., 1996; van der Laan et al., 1997) whereby the Tm value strongly depends on the PNA/DNA ratio in the chimeras. The Tm value of 5′DNA–3′-PNA chimeras, in which the PNA and DNA parts are of equal length, lies roughly between those observed for the corresponding pure PNA and DNA. In contrast to pure PNA, 5′-DNA—PNA chimeras bind exclusively in the antiparallel orientation to DNA and RNA under physiological conditions. The chimeras show also much better cellular uptake than pure PNA. PHONAs are analogs of PNA in which the peptide bond has been replaced by a phosphonic acid ester bridge, rendering this negatively charged analogue more soluble in aqueous medium than PNA (Peyman et al., 1996, 1998). PNA can be synthesized by the Boc strategy (Egholm et al., 1992) or the Fmoc strategy (Breipohl et al., 1996; Thomson et al., 1995) in analogy to the established peptide solid-phase synthesis methods. PNA—DNA chimeras containing purine nucleotides cannot be synthesized by standard PNA synthesis methods, since strong acid is used for deprotection or cleavage of the PNA from the solid support. Therefore, a mild method for PNA synthesis has been introduced which is fully compatible with DNA and RNA synthesis (van der Laan et al., 1995; Will et al., 1995). This strategy makes use of the monomethoxytrityl group for temporary protection of the N-terminus and acyl groups for protection of the exocyclic amino groups of the nucleobases (Uhlmann et al., 1997). The monomethoxytrityl group can be cleaved under very

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 55

mild conditions (3% trichloroacetic acid in dichloromethane), thus avoiding depurination in the DNA part. The DNA part is synthesized according to standard methods. 2.3.7 Modification on Heterocyclic Bases Modified nucleobases (Figure 2.11) are mainly introduced into oligonucleotides to improve binding affinity and specificity, though they always influence other parameters, such as nuclease stability and induction of RNase H, as well. Thus, enhanced binding affinity is observed by incorporation of C5 propynyl (Froehler et al., 1992) and C5 hexynyl (Ojwang et al., 1997) pyrimidines into oligonucleotides which at the same time leads to enhanced specificity. In case of C5 substituted pyrimidines, significant stabilization to nucleolytic digestion was observed as compared to their unmodified controls (Uhlmann et al., 1997). Very recently, phenoxazine-substituted oligonucleotides were described as selfpermeable antisense therapeutics (Flanagan et al., 1999). Phenoxazine, a planar tricyclic cytosine analogue, was introduced to improve stacking interactions between the heterocycles of oligonucleotide•RNA hybrids while retaining the ability to activate RNase H. The substitution of phenoxazine for C5 propynynl cytosine in a heptamer antisense oligonucleotide resulted in a five-fold increase in the relative binding affinity. The ability to induce RNase H cleavage was retained, although the pattern of cleavage was altered relative to the propynyl analogue. However, the most unique feature of the phenoxazine analogue was its ability to confer cellular permeation to a short heptameric oligonucleotide. N4, N4-ethano-5-methyl-cytosine (Shaw et al., 1991) has been used for irreversible modification of target sequences in vitro. For triplex formation, cytosine must be protonated (C+) to allow binding to a G-C base-pair. Therefore, under physiological conditions cytosine is replaced by 5-methylcytosine to effect triplex formation. In addition, incorporation of pseudoisocytidine, e.g. as its 2′-Omethyl derivative, has been used successfully for triplex formation at neutral pH (Ono et al., 1992). To allow triplex formation on a homopurine stretch containing a pyrimidine inversion, N4-(3-acetamidopropyl)-cytosine was introduced opposite the inversion site (Huang et al., 1993). Substitution of guanine by 7-deazaguanine in GGGG-motifs was used to suppress non-antisense effects caused by these G-tetrads (Benimetskaya et al., 1997). This substitution retains Watson-Crick base pair hybridization but prevents Hoogsteen base-pair interactions. 2.3.7.1 Universal bases and abasic sites A universal base is defined as a base analogue that can substitute for all four natural bases without significant loss of duplex stability. This is mainly

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accomplished by base stacking energy rather than hydrogen bond interaction, though the latter has been postulated for the imidazole-4-carboxamide (Sala et al., 1996). 3-Nitropyrrole (Nichols et al., 1994) and 5-nitroindole (Loakes and Brown, 1994) are among the best accepted base substitutions used for PCR so far. Deletion of a nucleoside base from the sugar unit gives rise to an abasic site. Any effect observed by this change is directly correlated to the loss of the heterocycle. Substitution of abasic ribonucleosides in the hammerhead ribozyme significantly impairs its catalytic activity (Peracchi et al., 1996). 2.3.8 Oligonucleotide conjugates By covalent attachment of non-nucleosidic molecules to either the 3′- or the 5′end of oligonucleotides, the properties of antisense oligonucleotides can also be modulated. This type of derivatization is chemically relatively simple and allows modulation of nuclease stability, cellular uptake and organ distribution of oligonucleotides. Besides the conjugation to the 5′- or the 3′-end of the oligonucleotide, the internucleoside linkages, the C5-position of pyrimidine bases and 2′-position of ribose are favoured. Conjugation to the 3′-end of oligonucleotides usually results in strong stabilization against 3′-exonucleases, which are the predominant nucleases in human serum. 2.3.8.1 5′-end conjugates Conjugation of molecules to the 5′-end of oligonucleotides is straightforward by coupling a phosphoramidite or H-phosphonate derivative of the desired molecule to the 5′-hydroxy group of the oligomer following chain elongation by solidphase synthesis. A broad range of phosphoramidite derivatives of ligands, such as fluorescein, biotin, cholesterol, dinitrophenyl, acridine and psoralen derivatives, are commercially available. Alternatively, the 5′-terminal hydroxy group of the oligonucleotide is reacted with an aminoalkyl linker phosphoramidite, which after deprotection results in a free aminoalkyl function. The amino function of the oligonucleotide can then be reacted post-synthesis in solution with suitably activated conjugate molecule derivatives, such as active esters, isothiocyanates, or iodo-acetamides. 2.3.8.2 3′-end conjugates The conjugation of molecules to the 3′-end of oligonucleotides is conveniently achieved by using correspondingly functionalized solid supports which in addition harbour a hydroxyl function from which the oligonucleotide chain is

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 57

extended during solid-phase synthesis. After oligonucleotide synthesis is complete, the oligonucleotide conjugate is cleaved from the solid support and deprotected by ammonia treatment. Using this method, relatively exotic derivatives, such as the anionophoric moiety of pamamycin (Uhlmann et al., 1998a) can be easily introduced. Similarly to the 5′-conjugation, appropriate 3′amino-modifier solid supports are commercially available which allow coupling of suitably activated derivatives of the molecule to be conjugated with the 3′amino alkyl group in a post-synthetic solution-phase step. 2.3.8.3 Conjugation at internucleoside linkages, nucleobases and ribose The attachment of conjugate molecules to the internucleoside linkages of oligonucleotides is possible via oxidative amination. On one hand, this can be accomplished by replacement of the normal iodine/water oxidation of the phosphite triester intermediate in phosphoramidite oligonucleotide synthesis by treatment with a solution of iodine and a derivative of the conjugate molecule bearing an amino function. On the other hand, replacement of the normal iodine/ water oxidation of the H-phosphonate intermediate in H-phosphonate oligonucleotide synthesis by treatment with a carbon tetrachloride solution of a derivative of the conjugate molecule bearing an amino function also leads to a modified phosphoramidate linkage. Although the oxidative amination method allows the conjugation of suitable molecules at any internucleoside linkage in an oligonucleotide, it has mainly been used for the synthesis of oligonucleotides with conjugate molecules attached to the 5′-and/or 3′-terminal internucleoside linkages. For conjugation of molecules to the heterocyclic bases, C5 trifluoroacetylprotected aminoalkenyl pyrimidine and C7-aminoalkynyl-7deazapurine nucleoside phosphoramidites can be used, which after deprotection can be further coupled with active esters and the like as described above. Similarly, suitably protected 2′-O-aminoalkylribonucleoside phosphoroamidites are useful for site-specific introduction of conjugate groups into the 2′-position of ribose of oligonucleotides. 2.3.8.4 Types of conjugate Many different oligonucleotide conjugates have been described in recent years aiming at the improvement of cellular uptake, exonuclease stability, binding affinity, or enhancement of biological activity due to cross-linking reactions or artificial nuclease cleavage. The relatively poor cellular uptake and unfavourable intracellular distribution of antisense oligonucleotides is still one of the major obstacles to their use as therapeutic agents. Much work has been devoted to the

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conjugation of oligonucleotides with lipophilic molecules or with positively charged polypeptides (see also Chapter 5). Cholesterol groups (Krieg et al., 1993; Letsinger et al., 1989) were introduced by oxidative animation of H-phosphonate internucleoside linkages. Alternatively, H-phosphonate and phosphoramidite derivatives allowed the synthesis of 5′-cholesteryl oligonucleotides, whereas cholesterol derivatized controlled-pore glass solid supports are available for the synthesis of 3′-cholesteryl oligonucleotides. Interestingly, studies using fluorescently labelled or radiolabelled cholesteryl oligonucleotides showed that attachment of cholesterol to the oligonucleotide increases its cell association and uptake. In addition, cholesterol conjugation appears to influence the intracellular localization of the oligonucleotide in that the nuclear fraction is increased. However, the interaction of the cholesterol moiety with cellular membranes can also lead to complications in the interpretation of biological activity experiments. Vitamin E (Will and Brown, 1992) and polyalkyl chains (MacKellar et al., 1992) of various lengths have also been attached to oligonucleotides at both the 3′-and 5′-ends. In many cases higher cellular association and uptake are reported for these conjugates. Furthermore, 3′-and 5′-polylysine-modified oligonucleotides (Lemaitre et al., 1987) (see also Chapter 8 this volume) have revealed remarkable antisense activity in antiviral assay systems. However, the potential cytotoxicity of polylysine and some reports of sequence non-specific effects may limit the applicability of this approach. In order to enhance the duplex stability of oligonucleotides with its target sequence, intercalating agents were conjugated to both the 3′-and the 5′-end. Similarly, the stability of triple helices could be enhanced by attachment of intercalators to triple-helix-forming oligonucleotides. Acridine-derived moieties (Thuong et al., 1987) are commonly used as intercalating conjugates and a phosphoramidite of 9-amino-6-chloro-2-methoxy-acridine is commercially available for coupling to the 5′-end of oligonucleotides. Cross-linking agents were used for permanent inactivation of target nucleic acids (Knorre et al., 1985, 1989), which were based for example on aromatic (2-chloroethyl) amino (Vlasov et al., 1988) groups. Ideally, the alkylation reaction at the purine bases on the complementary strand should only occur on hybridization of the oligonucleotide to its target sequence. Chlorambucil, proflavine, azidoproflavine and pazidophenacyl cross-linkers have also been used for attachment to oligonucleotides. Conjugation of psoralen is frequently used, which reacts with thymine bases on exposure to UV light. As artificial endonucleases, which induce non-specific DNA cleavage, oligonucleotide conjugates with metal chelators were mainly employed, such as EDTAFe(II), o-phenanthroline-Cu(II), Bleomycin-Fe(II), bipyridinyl-Cu(II) and porphyrin chelators. Although oligonucleotide conjugates of this type have proved to be useful tools for probing oligonucleotide interactions in vitro, this approach cannot be applied to the in vivo situation. Of potential use for therapy are oligonucleotides with texaphyrins (ring expanded porphyrin analogues) (Magda et al., 1996, 1997) attached at the ends, which, depending on the

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 59

chelated lanthanide metal cation, can be used to cleave either single-stranded RNA (with europium) or DNA (with lutetium). 2.4 Analysis of Oligonucleotides The analysis of oligonucleotides (see also Chapter 11) and the analytical techniques are refined in the same way as synthesis methods are improved. Today a great diversity of analytical tools is available, ranging from classical electrophoresis via chromatographic techniques to nuclear magnetic resonance (NMR) and mass spectrometry (Schweitzer and Engels, 1997). For pharmaceutical application the purity and identity (base composition and sequence) of the oligonucleotides has to be proven. An incomplete coupling results in a contamination of the full length oligonucleotide with n–1 failure sequences. These can be monitored by chromatography or electrophoresis. Furthermore, the protecting groups applied in the synthesis have to be separated from the oligonucleotide product after cleavage. HPLC is a good method to verify the purity of the product. For physiological applications modification of the oligonucleotides is essential, since unmodified oligonucleotides are readily degraded in biological systems. The introduction of all kinds of modifications, such as phosphorothioates, methylphosphonates, O-methyl oligonucleotides or amino groups for the attachment of reporter groups has to be monitored. Mass spectrometry is a powerful tool for this purpose, and NMR allows the determination of the average amount of sulphurization. Radioactive or fluorescent-labelled molecules are used to monitor the course of the oligonucleotide in vivo and to determine organ distribution, half-life and degradation products. The stability of hybridization to the target sequence is an important criterion for antisense application. Here recording of UV melting curves allows the determination of the Tm value and the thermodynamic parameters of the transition. 2.4.1 UV Spectroscopy of Oligonucleotides UV Spectroscopy is an easy and sensitive technique, and among the first methods employed in the analysis of nucleic acids. A typical UV spectrum of an oligonucleotide shows the absorbance maximum at around 260 nm and the minimum at approximately 230 nm. Because of its sensitivity and nondestructive nature, UV spectroscopy is frequently utilized as a detection method for analytical procedures such as HPLC or CGE. UV spectra of nucleic acids show pronounced hypochromicity. The absorbance of a native DNA duplex is 20–30% lower than the absorbance of the

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single strands in random coil conformation. This phenomenon is employed to measure the melting behaviour (Tm) of oligonucleotides. 2.4.1.1 Quantification of oligonucleotides UV-spectrophotometry is the main technique for quantification of nucleic acids, since the ionic character of these molecules results in an extensive hydration in the solid state. This and the possibility of salt contamination complicates the direct weighing of dry DNA samples. The determination of the absorbance or optical density (OD) is a good way to measure oligonucleotide amounts when the extinction coefficient (ε) of the molecule is known. The concentration (c) of the oligonucleotide solution is correlated with the absorbance reading (OD) according to Beer’s law: OD=εcl (l is the path length of the cuvette). The concentration of an oligonucleotide solution can be determined by hydrolysis of the oligonucleotide into mononucleotides, so minimizing stacking interactions between the bases. Provided that its base composition is known, this is the most accurate way to obtain the concentration but, especially for modified oligonucleotides, hydrolysis is not always possible. A method for the calculation of the extinction coefficient considering the nearest neighbour interactions between the bases is presented by Gray et al. (1989). An easier method that does not respect the sequence of the oligonucleotide is given by Brown and Brown (1991). In this formula the stacking interaction is estimated and considered by multiplying the sum of the individual extinction coefficients by a factor of 0.9. Both methods are valid only for single-stranded oligonucleotides. The extinction coefficient is almost independent of the nature of the phosphate group, so these calculations are applicable to backbone-modified oligonucleotides without corrections as well. Measurements are routinely performed in 1 cm cuvettes with a volume of 1 ml. The absorbance at 260 nm is then referred to as OD260. Calculate the extinction coefficient at 260 nm as follows:

a, t, g, c correspond to the number of the respective bases in the oligonucleotide sequence. 2.41.2 Melting curves of DNA In order to evaluate these effects and to determine the hybridization characteristics of an oligonucleotide, a melting curve has to be recorded (Figure 2.12). In UV melting experiments the change of absorbance with increasing temperature is measured (Puglisi and Tinoco, 1989). The measured hypochromicity is due to the stacking interaction between the chromophores of the nucleobases (sugar and phosphate backbones do not contribute to the UVabsorbance), resulting in a lower absorbance than expected for the sum of the

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 61

Figure 2.12 UV melting curve of a 20-mer ODN (Tm=62°C) and its derivative d (absorbance)/dT

extinction coefficients of the free nucleotides. The UV-absorbance increases as a duplex (with stacked bases) melts into two single strands with random coil conformation. The extent of hypochromicity is wavelength-dependent. For AT base pairs, maximum hypochromicity is observed at 260 nm while for CG base pairs the maximum is 280 nm. The optimum wavelength is between these two values, and depends on the base composition of the oligonucleotide investigated. The melting temperature Tm is commonly used as a measure for the binding affinity. It is the temperature at which 50% of the double strand has dissociated into its single strands. Once a curve is recorded, it has to be analysed to extract the Tm value and, if desired, the thermodynamic parameters of the transition. Several procedures for data analysis have been reported (Albergo et al., 1981; Marky and Breslauer, 1987; Puglisi and Tinoco, 1989): these are outside the scope of this chapter. A good way to obtain thermodynamic parameters of the transition is to plot 1/Tm versus In c. The resulting plot should be linear, the slope corresponds to R/∆H° and the intercept is ∆S°/∆H° (where H°, S° and R are enthalpy, entropy and the gas constant, respectively). Furthermore, the duplex to random coil transition is dependent on the ionic strength and the pH of the buffer. 2.4.2 Analysis of Oligonucleotides by High-Performance Liquid Chromatography High-performance liquid chromatography (HPLC) is the most widespread tool in the analysis of oligonucleotides (Oliver, 1989). Chromatography is the separation

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technique mostly applied to modified antisense oligonucleotides with neutral backbone such as methylphosphonates and PNA. Among HPLC techniques, two main forms are distinguished according to their principle of separation: reversed-phase (RP) and anion-exchange chromatography. In RP-HPLC the oligonucleotide is bound by hydrophobic interaction to a nonpolar matrix and eluted with a gradient of increasing amount of organic solvent. For anion-exchange HPLC a positively charged ion-exchange material serves as stationary phase. The oligonucleotide binds to this material with the negatively charged phosphorodiester backbone, and elution is accomplished by a gradient of increasing ionic strength. 2.4.2.1 Reversed-phase HPLC Reversed-phase HPLC is most frequently used for the analysis of crude oligonucleotide synthesis mixtures. Since the separation is based on the hydrophobic interactions with the column material, it may be advisable to leave the terminal dimethoxytrityl (DMTr) protection group on the oligonucleotide. This unpolar group retards the migration and allows a good separation of the fulllength product from truncated sequences without a DMTr group. An important application of RP-HPLC is the analysis of backbone-modified oligonucleotides which cannot be accomplished by other techniques. Neutral oligonucleotide analogues do not bind to ion-exchange resins and do not migrate in an electric field, so that RP-HPLC is the only method for their separation at physiological pH. The modification of the backbone can introduce chirality at the phosphorus atom. Methylphosphonates or phosphorothioates consist of 2n diastereomers (n=number of modifications in the oligonucleotide) if no attempts at a diastereoselective synthesis were undertaken. This large amount of diastereoisomers can lead to a broadening of the peaks. A wide variety of RP columns for oligonucleotide analysis, differing in the type of packing material and particle diameter, is commercially available. For each of these columns a slightly different gradient is recommended. Furthermore, the choice of gradient depends on the nature and length of the oligonucleotide to be analysed. Triethylammonium acetate is most commonly used as elution buffer. This volatile compound can be removed in vacuo from the purified oligonucleotide upon preparative separations. 2.4.2.2 Anion-exchange HPLC Anion-exchange HPLC is a very effective analysis for nucleic acids (Drager and Regnier, 1985; Metelev and Agrawal, 1992). The resolution of n from n–1 chain length can be achieved for up to 30-mer oligonucleotides, and even attempts to increase the resolution up to 50-mer samples have been successfully undertaken.

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Figure 2.13 Strong anion exchange HPLC of a crude oligonucleotide A-16-mer and a 15mer phosphorothioate

The individual resolution depends significantly on the column type, gradient and pH chosen. Strong anion-exchange material remain ionized at pH values up to 12, whereas weak exchangers possess lower pK values and are operated at neutral pH values. The elution of the sample is accomplished with a gradient of increasing salt concentration; sometimes organic solvent is added to the mobile phase which gives a better separation of certain analytes by minimizing hydrophobic interactions between the sample and the column packing material. Metal ions may poison the exchange resin of certain columns or hydrolyse RNA, so these should be used with metal-free HPLC systems. Short failure sequences with fewer negative charges elute before the fulllength product. The retention time is almost independent of the base composition of the oligonucleotide (Figure 2.13). Phosphorothioates bind strongly to anion-exchange columns. An elution can be obtained by increasing the ionic strength (e.g. 2.0 M NaCl) and by prolonged chromatography time. For these compounds weak anion-exchange columns have been employed successfully. The broadening of the peaks due to the diastereomeric nature of the phosphorothioates is not so pronounced as by RPHPLC. 2.4.3 Electrophoretic Techniques The electrophoretic analysis of oligonucleotides (Rickwood and Hames, 1990) is based on the migration of charged molecules under the influence of an electric field. During electrophoresis the molecules are separated due to their ratio of mass to charge and to interaction with a supporting medium. For DNA fragments

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of the size of antisense oligonucleotides, cross-linked polyacrylamide is commonly used as supporting gel; for larger molecules agarose gels are suitable. Only molecules that carry a charge under electrophoresis conditions can be analysed by this technique. There have been few changes in the basic protocols for gel electrophoresis since the introduction of the method in the middle of the 20th century. In recent years capillary gel electrophoresis (CGE) arose as a new and potent alternative to the traditional electrophoretic techniques. 2.4.3.1 Polyacrylamide Gel Electrophoresis (PAGE) on slab gels Although the electrophoresis of nucleic acids on slab gels has been practised for several decades, it remains a powerful tool for the analysis of oligonucleotides. The main advantage of this method is a good resolution of the full-length oligonucleotide from truncated sequences. Furthermore, it combines the possibility of running several samples on one gel with a high sensitivity, especially in combination with radioactively labelled oligonucleotides. The interaction with the stationary polyacrylamide (PAA) matrix retards this migration, depending on size and shape of the moving species. For short oligonucleotides high concentrations of PAA (20%) are necessary, while lower concentrations allow the separation of oligonucleotides up to 1000 base pairs (3%). The electrophoresis is usually carried out in the presence of urea as denaturing agent. Under these conditions the oligonucleotides are separated by their length, and sequence-specific effects are minimized. Analysis at low temperature in the absence of denaturing agents may provide further information, such as secondary structure or duplex formation. Depending on the type and amount of oligonucleotide used, different techniques for the visualization of the molecules may be performed. A practical method for the detection of oligonucleotides is to stain the gel. Stains all (Green and Pastewka, 1974) is a suitable dye for the visualization of oligonucleotide samples in PAA gels. The most sensitive method for the detection is the autoradiography of radioactively labelled oligonucleotides (Sambrook et al., 1989). The irradiation of 32P is detected with a phosphorus imager or by covering the gel with an X-ray film which allows a quantification of the oligonucleotide. 2.4.3.2 Capillary Gel Electrophoresis (CGE) Capillary electrophoresis (CE) is a special form of electrophoresis where the charged molecules are separated in a capillary with 25 to 100 µm diameter (Cohen et al., 1988; Engelhardt et al., 1993; Khur and Moning, 1992). For the separation of oligonucleotides capillaries filled with a sieving gel (CGE) are used almost exclusively, although some publications have described the separation of

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DNA in open capillaries. Several types of polymer have been described, such as the traditional cross-linked PAA, linear PAA without cross-linking agent, cellulose, agarose and hydroxyethylcellulose. The sample is injected electrokinetically by short immersion of one end of the capillary in the oligonucleotide solution and application of an electric field. The molecules migrate into the capillary, which is then shifted to a buffer reservoir, and electrophoresis is performed typically at 300 V/cm, corresponding to 12 kV, on a commercial 40 cm long PAA capillary. The sample molecules are resolved on their ratio of mass to charge by the same principles valid for PAGE on slab gels. At the end of the capillary, a window in the coating material allows direct detection of the oligonucleotides by UV-or laser-induced fluorescence (LIF). Coupling with an electrospray ionization (ESI) mass spectrometer is possible and can provide valuable information about the chemical composition of the detected peaks. CGE is a good method for the analysis of all phosphorothioate oligonucleotides, since these molecules bind strongly to ion-exchange HPLC columns. The main advantage of this method is the high resolution (single base resolution up to 300 nucleotides), and chemically modified oligonucleotides such as phosphorothioates, 5′-aminohexyl or biotinylated oligonucleotides are well resolved from unmodified molecules. The analysis is rapid and automation is possible, but the method is limited by salt or buffer ions present in the sample. 2.4.4 Mass Spectrometry of Oligonucleotides Mass Spectrometry (MS) is a valuable analytical technique that can provide additional information to chromatographic and electrophoretic data. The determination of the molecular mass allows the verification of length and base composition of the molecule. Fragmentation of the sample and analysis of the resulting pattern permit direct sequencing of shorter (<50 nucleotides) oligonucleotides. Furthermore, the introduction of modifications, particularly those introduced directly during the synthesis process, can be monitored by MS. Today the analysis of oligonucleotides is mainly performed by ESI and matrixassisted laser desorption ionization (MALDI) in combination with various mass separation and detection techniques. 2.4.4.1 Electrospray Ionization Mass Spectrometry (ESI-MS) In electrospray (Little et al., 1995; Potier et al., 1994), a special form of atmospheric pressure ionization, the sample is delivered in solution through an HPLC flow system to a stainless steel capillary set on a high negative or positive voltage. The strong electric field on the point of this capillary induces an aerosol composed of small solvent droplets carrying an excess of negative or positive

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charge on their surface. The formation of this spray is assisted by a stream of gas (nitrogen) that passes the capillary concentrically. This special form of electrospray is sometimes referred to as ion spray. The charged particles are introduced through a small hole into the high-vacuum area of the source, where remaining solvent molecules are completely stripped off the ions. At the end of this process single desolvated ions carrying several charges remain; they are accelerated and focused in the source, conducted to the spectrometer, and analysed by their mass to charge ratio. The formation of multiple charged ions is a characteristic of ESI and allows the analysis of high molecular mass compounds on quadrupole mass filters whose upper mass limit is typically some thousands of m/z units (Da). The charge distribution pattern depends on the characteristics of the sample molecule. The molecular mass of the oligonucleotide can be determined at each of these peaks, provided that the charge state of the series is known. Commercial instruments offer analysis software that allows the determination of the molecular mass from at least two ion series. The spectrum can then be transformed into a virtual spectrum of single charged ions by a mathematical algorithm. For oligonucleotides, which carry negative charges on the phosphate backbone, the negative ionization mode is chosen. Problems arise when some of the negative charges are neutralized due to adduct formation with alkali metal cations such as sodium (Stults and Marsters, 1991). This will result in the detection of a series of peaks representing the molecular ion, the molecular ion plus one sodium atom, the molecular ion plus two sodium atoms, and so on ( ; ;…). Although these adducts allow the determination of the charge state of the series (which corresponds to 22 ( ) divided through the measured distance of two peaks of one series), sodium is not acceptable in oligonucleotide samples because of the loss of sensitivity when the signal spreads over several peaks. This problem may be reduced when the cations in the oligonucleotide sample are exchanged to ammonium ions which dissociate into ammonia and H+ in the vacuum of the spectrometer. The ammonia form of oligonucleotides may be obtained by ammonium acetate precipitation, treatment with ammonium-loaded ionexchange resin, dialysis towards 50 mM ammonium acetate solution, or gel permeation chromatography. 2.4.4.2 Matrix-Assisted Laser Desorption Ionization (MALDI) MALDI, a new mass spectrometric technique, is frequently used in combination with time-of-flight (TOF) mass detection (Kirpekar et al., 1994; Pieles et al., 1994; Wu et al., 1994). For a MALDI-TOF analysis the sample solution is mixed with a concentrated solution of a matrix compound and spotted on a target, usually a stainless steel disc. Upon evaporation of the solvent the matrix

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 67

crystallizes on the surface, thereby including sample molecules. The target is then inserted into the high vacuum of a mass spectrometer and a laser pulse is fired on the matrix crystals leading to an evaporation and ionization of matrix and sample molecules. The ions are accelerated in an electric field and move towards a detector placed at the end of a flight tube. The time from the laser pulse until the impact of the ions at the detector is used to determine their mass to charge ratio. The raw spectrum is a plot of the measured ion intensity versus time after the laser pulse. The measurement of compounds with known molecular mass allows the transformation into an intensity versus mass spectrum. The choice of the matrix substance depends on the wavelength of the applied laser, and a great diversity of compounds has been described. For UV laser instruments (337 nm N2 laser), 2, 4, 6-trihydroxyacetophenone (Pieles et al., 1993) or 3-hydroxypicolinic acid is a good matrix. A variation of this TOF technique is the electrostatic reflection of the ions at the end of the flight tube. The advantage of the reflection mode is an enhanced resolution and a suppression of undesired matrix peaks in the spectrum. Furthermore, this technique offers the possibility of investigating fragments of the primary ions which have formed during the first flight period (post-source decay). MALDI-TOF is a valuable technique due to the small amount of sample required, the ease of sample preparation and the high mass range covered by the TOF mass detector. The analysis of enzymatically synthesized RNA up to 150 kDa (461 nts) has been published (Kirpekar et al., 1994). As for ESI, the sodium adduct formation is critical for MALDI, but considerable advantages have been achieved by the use of ammonium citrate or ammonium loaded anion-exchange resin to convert the oligonucleotide into the ammonium form. Further improvement of mass resolution and sensitivity is expected from new forms of MALDITOF such as delayed ion extraction MALDI (Vestal et al., 1995). Sequencing of smaller RNA has recently been accomplished (Faulstich et al., 1997). 2.4.5 NMR of Oligonucleotides Nuclear magnetic resonance is not routinely employed in the purity analysis of oligonucleotides. Nevertheless, 31P-NMR spectra can provide valuable information on the extent of modification of antisense oligonucleotides (Stein et al., 1995). The introduction of phosphorothioate moieties during the synthesis cycle is particularly conducive to incomplete sulphurization. The resulting oligonucleotide is a mixture of phosphorothioate and phosphodiester. Electrophoretic techniques usually fail in resolving these species, while separation by HPLC can frequently be achieved. Mass spectrometry allows detection of incomplete sulphurization, but the resulting isotope pattern can get quite complicated.

68 CHEMISTRY OF OLIGONUCLEOTIDES 31P-NMR

spectroscopy allows the differentiation of the two species by observation of the chemical shift. The shift of each phosphate fragment is approximately independent from the other groups present in the molecule. Two sets of signal appear in a typical 31P-NMR of a phosphorothioate oligonucleotide, representing the phosphodiesters at ~0 ppm and the phosphorothioates at ~55 ppm (~110 ppm for phosphorodithioates). The integration of these peaks gives the average rate of sulphurization of the oligonucleotide. It should be mentioned here that the chirality of the phosphorothioate moieties leads to the formation of 2n (n=number of thioates in the molecule) diastereomers that give signals at different chemical shift. The signals of this large amount of diastereomers (e.g. 524 288 diastereomers for a 20-mer phosphorothioate oligonucleotide) are generally not resolved in the spectrum but result in a broadening of the peaks. 2.5 Conclusion Modified oligonucleotides have been immensely helpful in understanding and utilizing these molecules in a physiological environment. For the future of oligonucleotide therapeutics, the easy availability of tailor-made derivatives is of prime importance. Thus, to address a biological question specifically, the derivative of choice has to be incorporated into nucleic acids at a given position, be it chemically or enzymatically. Acknowledgement We thank Markus Schweitzer for supporting analytical material, Uwe Parsch and Thorsten Strube for help with the manuscript, and Valentin Wittmann for a literature search. References AGRAWAL, S., JIANG, Z., ZHAO, Q., SHAW, D., CAI, Q., ROSKEY, A., CHANNAVAJJALA, L., SAXINGER, C. and ZHANG, R., 1997, Mixed-backbone oligonucleotides as second generation antisense oligonucleotides: in vitro and in vivo studies, Proc. Natl Acad. Sci. USA, 94, 2620–2625. AGRAWAL, S. and ZHAO, Q., 1998, Mixed backbone oligonucleotides: improvement in oligonucleotide-induced toxicity in vivo, Antisense Nucl. Acid Drug Dev., 8, 135–139. ALBERGO, D.D., MARKY, L.A., BRESLAUER, K.J. and TURNER, D.H., 1981, Thermodynamics of (dG-dC)3 double-helix formation in water and deuterium oxide, Biochemistry, 20, 1409–1413.

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3 The Oligonucleoticle Prodrug Approach The Pro-oligonucleotides

F.MORVAN, J.-J.VASSEUR, E.VIVÈS, B.RAYNER AND J.L.IMBACH

3.1 Introduction Oligonucleotide (ON)-based therapy promises to be a highly specific tool for the treatment of numerous human diseases. However, to date the effectiveness of ONs has been limited by several problems such as instability in serum, inability to reach their target site because of non-specific disposition, poor cell penetration and adverse pharmacokinetics. These hurdles have stimulated efforts to prepare neutral or charged backbone-modified oligonucleotides, but the limitations have been only partly solved (Agrawal and Iyer, 1997). The reason the ONs are not delivered efficiently is probably that the internucleosidic linkages are anionic at physiological pH; these linkages derivatized with enzymolabile protecting groups provide neutral phosphotriester oligonucleotides (the pro-oligonucleotides or pro-oligos) (Figure 3.1). In cells, enzymes cleave the protecting groups to release the parent drug (the ON). Application of this prodrug concept to various phosphorylated drugs is well established (Krise and Stella, 1996), but has never been envisaged for ONs before our preliminary presentation at the Cambridge Meeting on Synthetic Oligonucleotides and Analogues in 1993. Since then, other groups of researchers have being developing this approach (lyer et al., 1995a; Mauritz et al., 1997). But before releasing the ON, the lipophilic pro-oligonucleotides may present a completely different behaviour ranging from increased nuclease stability and low non-specific proteins interactions to more favourable pharmacokinetic/ pharmacodynamic properties. Therefore, to evaluate this concept we first had to select a suitable enzymolabile phosphodiester protecting group and to set the corresponding chemical approach in order to obtain some pro-oligos models and then to gain information on their behaviour. The enzymolabile pivaloyloxymethyl (POM) (Sastry et al., 1992) and Sacylthioethyls (SATEs) (Périgaud et al., 1993) phosphate-protecting group were previously introduced on mononucleotide, and this approach was taken to highly potent in vitro antiviral agents for a review see Périgaud et al.. (1997). It was further shown that the SATE group is selectively removed in vitro (Lefebvre

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 81

Figure 3.1 The pro-oligonucleotide approach

Figure 3.2 Mechanism of hydrolysis

et al., 1995) as well as in vivo (Martin et al., 1998) upon carboxyesterase activation according to the mechanism shown in Figure 3.2 (way a). This chapter is therefore devoted to the validation of the pro-oligo concept and to the preliminary evaluation of some model SATE prododecathymidylates. We will show that this approach may represent an interesting alternative in order to overcome some of the abovementioned shortcomings. 3.2 What Kind of Enzymolabile Group? 3.2.1 Structure Three kinds of enzymolabile group were evaluated: lipophilic, hydrophilic and cationic (Figure 3.3). The lipophilic ones are pivaloyloxymethyl (POM), Sacetylthiomethyl (SATM), and the methyl, tert-butyl or phenyl-Sacylthioethyls (Me-, tBu- or Phe-SATE); the hydrophilic ones are derivatives of

82 THE OLIGONUCLEOTIDE PRODRUG APPROACH

Figure 3.3 Structure of the enzymolabile groups

glucuronic acid with glucuronoyloxyethyl (GOE) and glucuronoyl-Sacylthioethyl (G-SATE) (Mignet et al., 1997a); and the cationic ones are derivatives of γ-aminobutyric acid with γ-aminobutyroyloxyethyl (GABOE) and γ-trimethylaminobutyroyloxyethyl (Me3-GABOE) (Figure 3.2). 3.2.2 Mechanism of Hydrolysis The carboxyesterases attack the carbonyl of the ester or thioester function to yield an unstable intermediate which decomposes to phosphodiester linkage with a release of formaldehyde or thioformaldehyde when POM or Me-SATM respectively are used, and a release of episulphide for the other enzymolabile groups. An attack by nucleophiles present in medium (e.g. water) on the phosphorus atom (way b, Figure 3.2) may occur when pro-oligos are incubated in culture medium, but only a selective attack on carbonyl (way a) occurs in the presence of carboxyesterases (Figure 3.2). 3.2.3 Dimers′ Stability in Biological Media To determine which enzymolabile group will possess the required characteristics for the pro-oligo approach, we synthesized, as models, dithymidine

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 83

Table 3.1 Stability of pro-dithymidine phosphorothiolates bearing an enzymolabile group in culture medium, in total cells extract and in the presence of RLE (8 units/ml) Half-life of pro-dithymidine phosphorothiolate

R POM SATM Me-SATE GOE G-SATE GABOE Me3GABOE

Culture medium 6.0 h l.0h 7.0h 2.1h ND* 17h 26 h

Total cells extract 40min <5 min <5 min 4.7 h ND* 15h 22 h

PLE ND ND ND 2.2 h ND* l0h 26 h

ND, not determined. * Unstable in water.

phosphorothiolates masked with the corresponding enzymolabile group. They were obtained by alkylation of dithymidine phosphorothioate or dithioate with the iodo derivative of the enzymolabile groups. The dimers were incubated in culture medium (CM; RPMI 1640 with 10% of inactivated foetal calf serum) to mimic the extracellular fluids and in total CEM cells extract (TCE) to mimic the cellular compartment. A good candidate for the pro-oligo approach should exhibit a high stability in CM and a rapid hydrolysis in TCE. Among the enzymolabile groups tested, the hydrophilic and the cationic ones displayed a poor substrate capacity for the carboxyesterases with high halflife in TCE or in the presence of pig liver esterase (PLE) (Tables 3.1 and 3.2), and hence they were not investigated further. Among the lipophilic ones the MeSATM is too unstable in CM and was not used further. In contrast, POM and SATE are appropriate enzymolabile groups since they exhibit a good to high stability in CM and a rapid hydrolysis in TCE or with PLE. In TCE, the hydrolysis proceeds with a diastereo-differentiation since each Sp and Rp diastereoisomer exhibits a different substrate capacity for carboxyesterases (lyer et al., 1994, 1995a, 1996; Tosquellas et al., 1996; Mignet et al., 1997b, 1997c). Furthermore, the SATE-protected dimers are stable in gastric juice (Table 3.2), which is compatible with the use of pro-oligos with an oral administration. It was also shown that lipophilic groups such as acyloxyalkyl (lyer et al., 1994, 1995a) or acyloxyaryl (lyer et al., 1996b, 1997) may also be used for prodrugs of oligonucleotides. A new protecting group (a-hydroxy-p-nitrophenyl-phosphonate linkage) for pro-oligo was introduced, but in this case removal proceeds through an enzyme-independent mechanism (Meier and Mauritz, 1995).

84 THE OLIGONUCLEOTIDE PRODRUG APPROACH

Table 3.2 Stability of pro-dithymidine thiono-phosphorothiolates bearing an enzymolabile group in culture medium, in total cells extract, in the presence of RLE (8 units/ml) and in gastric juice Half-life of pro-dithymidine thiono-phosphorothiolate

R Me-SATE tBu-SATE Phe-SATE GOE

CM 6.0/9.8 h 42.7 h 46.5 h 3.0 h

TCE <5 min 0.9/1.5 h 0.6/1.3 h 5.0 h

PLE <2min <4min 5 min l.0h

Gastric juice 7 days Stable* Stable* ND

* No degradation observed up to 30 days.

3.3 First Pro-oligonucleotide Models 3.3.1 Post-synthesis Alkylation The first pro-oligo models (Figure 3.4) were synthesized by alkylation of mono or dithioate phosphodiester linkages with the iodide derivative of the enzymolabile group. Thus 12-mers with a central gap of three (1 in Figure 3.4) or six (2 and 3) phosphorothioate linkages were alkylated with the iodide POM. To study the influence of the nature of the flanks, pro-oligos with a gap of six POMs with either neutral (methyl-phosphonate 2) or charged (phosphodiester 3) flanks were synthesized (Morvan et al., 1997; Tosquellas et al., 1997). Finally, to determine the half-life value for the last POM hydrolysis, we synthesized a 12mer bearing only one central phosphorothiolate POM with charged phosphodiester flanks (4). This will give us information on the possibility that all the POMs could be removed from a pro-oligo. The synthesis of SATE pro-oligos was less efficient, because SATE iodides are less reactive than POM iodide. For this reason we used a 12-mer with a central gap of three phosphorodithioates and neutral methyl-phosphonate flanks (5) (Tosquellas et al., 1996), because phosphorodithioates are more nucleophilic than phosphoromonothioates. 3.3.2 Pro-oligonucleotides′ Stability in Biological Media We determined the half-lives in CM of pro-oligos 1 and 5 and in TCE of prooligos 1 to 5 for the first hydrolysis, as well as half-times of appearance of

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 85

Figure 3.4 Structure of pro-oligos synthesized by alkylation

fully unmasked oligos (Tosquellas et al., 1996, 1997; Morvan et al., 1997), using an Table 3.3 Stability of first pro-oligos in CM and TCE Pro-oligos

RT (HPLC C18) (min)

First*

Total†

1 2 3 4 5

51.0 59.0 51.0 31.5 51.8

t½ in CM (h)

t½ in TCE (h)

4.5 ND ND ND 32

0.1 1.4 0.1 22 0.25

9.6 31 30 NA 1.3

ND, not determined; NA, not applicable. * First hydrolysis. † Appearance of fully unmasked oligo.

on-line cleaning HPLC method (Pompon et al., 1992) (Table 3.3). As expected, 1 is stable in CM (t½ 4.5 h for 1) and 5 displays a higher half-life due to the lack of nucleophilic attack on the phosphorus atom (avoiding way b, Figure 3.2). In TCE, we observed that half-lives for the first hydrolysis varied strongly from one to

86 THE OLIGONUCLEOTIDE PRODRUG APPROACH

another pro-oligo (t½ from 0.1 h to 22 h). In addition, for the same pro-oligo each successive hydrolysis proceeded at a different rate. These data could be explained by the decrease of lipophilicity of the substrate compound due to the progressive removal of POM or SATE groups. Lipophilicity of each pro-oligo was evaluated by its retention time (RT) on reverse-phase HPLC. We showed that the rate of hydrolysis is highest when optimal lipophilicity is achieved. The low hydrolysis of 4 (t½ 22 h) results from carboxyesterase preference for neutral substrates (Satoh, 1987; Satoh and Hosokawa, 1998). Moreover, it appears that excessively lipophilic substrates are also poorly hydrolysed. Indeed, 2 displays the highest lipophilicity and the highest half-life for the first hydrolysis. The halftimes of appearance of fully unmasked 2 and 3 are similar (about 30 h). This slow demasking certainly results from the low lipophilicity of the pro-oligos with only one POM, which are a poor substrate for carboxyesterases. Comparison of pro-oligos 1 and 5 showed that SATE groups are more efficiently hydrolysed than POM groups, and also display a better stability in culture medium. These data prompted us to consider the SATE group as a good candidate for the pro-oligo approach. Furthermore, the possibility to use either methyl or tert-butyl-SATE will allow the lipophilicity of the pro-oligos to be adjusted. 3.3.3 Limitations Alkylation of oligonucleotides to obtain pro-oligos proceeds slowly and only allows the synthesis of phosphorothiolate or thionophosphorothiolate pro-oligos, excluding the phosphotriester ones. In addition, a concomitant alkylation of the nucleobases is likely (lyer et al., 1995a). Finally, the finding that a desulphurization side-reaction (Tosquellas et al., 1997) occurs during the alkylation of phosphorothioate oligos represents a hurdle for the further development of the pro-oligo approach. 3.3.4 Conclusion We have shown that chimeric dodecamers bearing a central gap of six enzymolabile POM groups and neutral or charged flanks, and a chimeric dodecamer bearing a central gap of three enzymolabile Me-SATE groups with neutral flanks, could be fully unmasked in TCE by carboxyesterase activity with a half-life of about 30 h and 10 h respectively. We have to keep in mind that a total CEM cell extract is a rough mimic of a cell content, and we can expect enzyme activities to be much more potent in intact cells, hence the rate of removal should be higher. Therefore our data suggest that a dodecamer chimeric prooligonucleotide bearing a 6 POM or a 3 SATE gap is compatible with the prooligonucleotide approach. We observed that the rate of hydrolysis of the

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 87

Figure 3.5 Solid support with photolabile linker and phosphoramidites building blocks

enzymolabile groups is connected with the global lipophilicity of the pro-oligo with an optimal lipophilicity. Because of the limitations of the synthesis of prooligos by post-synthesis alkylation, it was necessary to develop their automated synthesis on solid support. 3.4 Pro-oligonucleotides of the Second Generation 3.4.1 Phosphorous Environment We determined that the two best phosphorous environments for the pro-oligos approach are oxo-and thiono-SATE-phosphotriesters. Indeed, both structures displayed a high stability in MC and an effective release in TCE of the phosphodiester and the phosphorothioate linkage respectively (Mignet et al., 1997b, 1997c). Furthermore, the thiono-phosphotriesters are less prone to a nucleophilic attack on the phosphorus atom, and release phosphorothioate linkages that are resistant to nuclease degradation. It should be noted that these structures could not be obtained by alkylation. 3.4.2 Automated Synthesis on Solid Support The pro-oligos, being phosphotriester (or thiono-phosphotriester) derivatives with a thioester function, are sensitive to base or nucleophile treatments, therefore a completely new strategy was set for their synthesis on solid support (Tosquellas et al., 1998a). We designed (i) a new solid support (Dell’Aquila et al., 1997) which is cleaved under non-basic and non-nucleophilic conditions, and (ii) phosphoramidite building blocks bearing the SATE group. First pro-oligo models were constituted only with thymidine; later we developed nucleobase

88 THE OLIGONUCLEOTIDE PRODRUG APPROACH

Figure 3.6 Structures of the protecting groups on the nucleobases

protecting groups which could be removed readily under non-basic and nonnucleophilic conditions. 3.4.2.7 Solid Support For our purposes we conceived a new photolabile solid support (Figure 3.5) which releases the pro-oligo after a 20 min irradiation with a high-pressure Hg lamp (Dell’Aquila et al., 1997). This solid support yields pro-oligos with only one charge at their 3′-end. 3.4.2.2 Phosphoramidite building blocks The suitable structures for the pro-oligos (oxo-and thiono-SATEphosphotriester) are reached using the phosphoramidite building block bearing a Me-or tBu-SATE group instead of a regular cyanoethyl group (Figure 3.5) (Tosquellas et al., 1998a). During the synthesis of the pro-oligo the oxophosphotriester linkage is obtained by oxidation with tert-butyl hydroperoxide

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 89

(Hayakawa et al., 1986) and the thiono-phosphotriester one by treatment with Beaucage reagent (lyer et al., 1990). 3.4.2.3 Protecting groups on nucleobases In the current synthesis of regular oligonucleotide heteropolymers as well as their analogues, the exocyclic function of the nucleobases C, G and A is generally protected by base-labile protecting groups (Beaucage and lyer, 1992). Pro-oligo phosphotriesters as well as thionophosphotriesters are sensitive to bases and nucleophiles and decompose under the standard basic conditions required for removal of the common heterocyclic N-acyl protecting groups. The synthesis of these base-sensitive oligonucleotides requires new nucleobase amino protecting groups which could be removed under non-basic and non-nucleophilic conditions. We have evaluated several protecting groups (Figure 3.6), such as the sulphenyl protections 2-nitrophenylsulphenyl (NPS) (Heikkila et al., 1983; Alvarez et al., 1998) and tritylsulphenyl (TrS) (Sekine and Seio, 1993). The removal of these protecting groups could be achieved under neutral reductive conditions (tributylphosphine/water) for NPS and mild oxidative conditions (I2/ water) for TrS. Such redox conditions are compatible with the stability of SATEphosphotriester oligonucleotides. Unfortunatly, these sulphenyl protecting groups cannot act as efficient protecting groups in an oligonucleotide synthesis on solid support using the phosphoramidite approach. Alternatively, the use of the pent-4-enoyl (PNT) group (Figure 3.6) removable by treatment with iodine was proposed as a nucleobase protecting groups, and allowed the synthesis of various base-sensitive dimer and trimer analogues (lyer et al., 1995b, 1996a). But no evidence was provided that iodine treatment alone was able to remove quantitatively every PNT group present on oligonucleotides (Devlin et al., 1996). Our effort is now focused on photocleavable protecting groups as nucleobase protections during the synthesis of SATE pro-oligos. The efficiency of 2nitoveratryloxycarbonyl (NVOC) (Fodor et al., 1991) and 2, 2′-di-(2nitrophenyl)-ethoxycarbonyl (diNPEOC) (Hasan et al., 1997) groups was demonstrated during the synthesis of short SATE heteropolymers (pentamers; unpublished data). The removal of the protecting groups and the cleavage of the pro-oligos from the solid support are accomplished in a single photolysis procedure upon UV irradiation at wavelengths > 300 nm. Synthesis of longer heteropolymers is under way. 3.4.3 Fully SATE Pro-oligonucleotides Four pro-dodecathymidines (6–9) were synthesized which correspond to two different SATE masking groups, i.e. Me-SATE or tBu-SATE and phosphate or

90 THE OLIGONUCLEOTIDE PRODRUG APPROACH

Figure 3.7 Structure of pro-oligos of second generation

thionophosphate triester internucleosidic linkages (Figure 3.7). It is noteworthy that 6–9 contained a thionophosphotriester internucleoside link at their 5′ end in order to be eventually labelled with 35S. These pro-oligos were synthesized applying our chemistry using a highly loaded photolabile CPG solid support (Dell’Aquila et al., 1997). A modified cycle was utilized which included an

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 91

extended coupling time (180 s) for the first four coupling steps to ensure high coupling yields. tert-Butyl hydroperoxide (Hayakawa et al., 1986) and Beaucage reagent (lyer et al., 1990) were used as oxidizers for the formation of phosphate and thionophosphate triester internucleoside linkages, respectively. After photolysis, reverse-phase HPLC analysis of the crude materials indicated satisfactorily homogeneous products. Among the four pro-oligos 6–9, the least lipophilic one is the dodecanucleotide 9 containing Me-SATE phosphate triesters, and the most lipophilic one is 6 made of tBu-SATE thionophosphate linkages (Table 3.4). In each case a broad peak was obtained which reflects the presence of a diastereoisomeric mixture due to Rp and Sp isomers at each internucleoside linkage. 31P-NMR spectra of 6 and 7 exhibit two sets of peaks corresponding to internucleoside thionophosphate triesters (δ≈68 ppm) and 3′terminal thiophosphate diester (δ≈57 ppm) in the expected ratio whereas spectra of 8 and 9 present three sets of peaks corresponding to 5′-thionophosphate triester (δ≈68 ppm), internal phosphate triesters (δ≈−1 ppm) and 3′-terminal phosphodiester (δ≈0ppm). MALDI-TOF mass spectra of crude 6–9 are in agreement with the expected structure. Unfortunately, pro-oligos 6–9 are poorly soluble in aqueous media (especially 6 and 7). This low hydrosolubility may be a hurdle for the development of this approach. Table 3.4 Stability of second-generation pro-oligos in TCE and in the presence of RLE (8 units/ml) Pro-oligo

RT (HPLC C18) (min)

TCE

PLE

6 7 8 9 10 11 12 13 14 15

>80 76.6 70.2 57.6 58.4 51.9 51.7 48.8 60.5 54.7

Half-life (h) Stable Stable 9.7 (20) 0.35 (22) 17 3 2.5 1 1.2 (33) 0.25 (38)

Stable Stable Stable 4.6 Stable Stable Stable Stable 0.6 0.1

Values in parentheses give the incubation time necessary for the formation of 50% corresponding fully-unmasked dodecathymidylate

92 THE OLIGONUCLEOTIDE PRODRUG APPROACH

3.4.4 Mixed Phosphodiester and SATE Phosphotriester Prooligonucleotides To modulate the lipophilicity of the pro-oligos and also to increase their hydrosolubility, it is necessary to introduce some charges in them. The synthesis of mixed tBu-SATE pro-oligos was designed using the phosphoramidite approach with the regular cyanoethyl (CNE) group and tBu-SATE phosphoramidite building block. A post-synthesis treatment using DBU (as a strong non-nucleophilic base in anhydrous media) eliminates, by a β-elimination mechanism, the CNE groups without affecting either the tBu-SATE phosphotriester linkage or the solid support anchoring (Tosquellas et al., 1998b). The pro-oligos 10 to 15 were synthesized by this method using a 60 or 20 min treatment with a solution of 90 mM DBU in dry THF for 10–11 and 12–15, respectively, according to the thiono- or oxo-CNE-triester. These pro-oligos display different charge arrangements that lead to alternating (10–13) and gap (14–15) pro-oligos with either neutral or charged flanks. These pro-oligos were characterized by mass spectrometry (ESI) and their substrate capacity in TCE and in the presence of PLE was evaluated. 3.4.5 Stability of Pro-oligonucleotides of Second Generation in Biological Media We therefore synthesized ten different SATE pro-oligo models, 6–15 (Figure 3.7). Compounds 8 and 9 were masked with Me-SATE groups and the others with tBu-SATE ones. Me-SATE are more easily removed by esterases than the bulkier tBu-SATE one (Lefebvre et al., 1995; Tosquellas et al., 1996). Compounds 6–9 and 14 have only one negative charge at their 3′-end; the others, 10–13 and 15, have seven negative charges. According to their structure the prooligos release after hydrolysis oligos with phosphodiester, phosphorothioate or mixed phosphodiester and phosphorothioate linkages. In addition, the lipophilicity order of the ten pro-oligos is expected to be 6>7>8>14>10>9>15>11>12>13, as reflected by their HPLC retention time under the same conditions (Crooke et al., 1996) (Table 3.4). Stability of 6 to 15 was evaluated in TCE and in the presence of PLE using an HPLC on-line cleaning method (Pompon et al., 1992) (Table 3.4). Only 9, 14 and 15 were substrates for PLE. These three compounds have in common a structure with several successive SATE in a row and a lipophilicity in the same range. In contrast, 10 possesses a lipophilicity in the same range but alternating SATE groups, and was not a substrate. Our results suggest that the esterase activities of PLE are very sensitive to the presence of negative charges adjacent to the SATE groups, since all alternating pro-oligos (10 to 13) were not substrates while gap pro-oligos (14 and 15) and fully Me-SATE (8 and 9) were

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 93

hydrolysed. This discrepancy could be explained by the fact that only in gap structures can carboxyesterases hydrolyse a SATE group surrounded by neutral phosphotriesters. Hence, the difference of structure appears to be determinant for a pro-oligo to be a PLE substrate. This is not surprising, since it was reported that most carboxyesterases have higher affinity for lipophilic esters than for polar or charged substrates (Satoh, 1987; Satoh and Hosokawa, 1998). In addition, it appears that PLE does not accept substrates with too high a lipophilicity, since 6 and 7 are not substrates. In TCE only 6 and 7 were not substrates for esterases, probably because of their excessively high lipophilicity and their poor hydrosolubility. The other prooligos may be classed in two series: the fully SATE (8–9) and gap (14–15) on one hand, and the alternating ones (10–13) on the other. The half-life values for the first hydrolysis, reported in Table 3.4, brought out the difference of substrate capacity between these two series of pro-oligos. Thus, for each series, the higher the lipophilicity of pro-oligo, the lower is the rate of hydrolysis for the first SATE group. Our results suggest that when a pro-oligo possesses too high a lipophilicity, its substrate capacity for carboxyesterases decreases. We reported similar results with pro-oligos bearing pivaloyloxymethyl (POM) enzymolabile groups with either neutral or charged wings (see section 3.3.2) (Morvan et al., 1997; Tosquellas et al., 1997). It is noteworthy that compounds 8 and 9 with Me-SATE are fully deprotected to the parent oligo with a half-life of 20 h and 22 h respectively, and that compounds 14 and 15 with tBu-SATE are fully deprotected with a half-life of 33 h and 38 h respectively. This difference is probably due to the nature of the enzymolabile group (Me- versus tBu-SATE). In addition, the ex vivo stability studies herewith presented should not fully reflect the in vivo behaviour of the prooligos. We have further observed that the esterase activity in total cell extracts decreases after 3–4 h at 37°C (data not shown). Thus, demasking of prooligos in intact cells should proceed more rapidly. In addition, 9 was not degraded by snake venom and calf spleen phosphodiesterases (Tosquellas et al., 1998a). The behaviour of compounds 8 and 9 was also evaluated in human serum and in human gastric juice. No degradation was observed upon seven-day incubation in gastric juice, which is in agreement with the expected stability of phosphotriesters in acidic media. In human serum, no degradation was observed for 8 and the half-life of 9 was 3 h. 3.4.6 Preliminary Data of Pro-oligos′ Cell Uptake Four pro-oligos with different lipophilicity (Figure 3.8) were labelled with fluorescein and their uptake was studied on HeLa cells (unpublished data). Results showed (Figure 3.9) that whereas the control T12 phosphorothioate 16 was not taken up, the other pro-oligos were easily internalized in the HeLa cells, and that the higher the lipophilicity of pro-oligos, the higher was the uptake. It is

94 THE OLIGONUCLEOTIDE PRODRUG APPROACH

Figure 3.8 Structure of the fluorescein labelled pro-oligos

noteworthy that compounds 16–19 contain respectively 12, 6, 6 and 4 negative charges. 3.5 Conclusion In conclusion, we have shown that SATE-prododecathymidylates (fully SATE or mixed SATE and phosphodiester) can be efficiently synthesized on solid support. As expected, the fully SATE pro-oligos are not degraded by nucleases (Tosquellas et al., 1998a) and present lower affinity to proteins as compared to the parent oligonucleotides (Vlassov et al., unpublished results). In addition, the pro-oligos are selectively hydrolysed to the parent oligonucleoside phosphodiester or phosphorothioate in total cell extract, but are much more stable in human serum. It is noteworthy that substrate capacity of each pro-oligo was dependent on its global lipophilicity, but also on the surroundings of the thioester to hydrolyse. The fact that some pro-oligos are substrates for esterases present in TCE and not for PLE is not surprising, since a large variety of carboxyesterases from various origins but with different substrate specificity have been isolated and characterized (Heymann, 1980; Mentlein and Heymann, 1984; Satoh, 1987; Aldridge, 1993; Satoh and Hosokawa, 1998). One can hypothesize that the bioavailability of such neutral or partially charged but still lipophilic pro-oligos will be different from that of the phosphodiester oligos. Furthermore, their pharmacokinetics may be modulated according to the nature of the enzymolabile protecting group, i.e. Me-SATE versus tBu-SATE, and the number and the disposition of the charges. It appeared that lipophilicity and hydrosolubility are two major components to be optimized.

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 95

Figure 3.9 Microscopy fluorescence of labelled pro-oligos incubated for 1 h at 10 µm with HeLa cells and retention time on reverse phase HPLC C18

The validation of the pro-oligo approach must be envisaged in vivo and experiments to this end are in progress. It is now possible to synthesize pro-oligos partially substituted with tBu-SATE protecting groups in any given positions. After labelling, such compounds, of various lipophilicities, are under evaluation for their ability to be taken up into intact cells. Preliminary data showed that a very important uptake was obtained when the lipophilicity of the pro-oligos was increased. Much more work will have to be done in this area before any definitive conclusion is reached, but we hope that the pro-oligo approach herewith presented could be the basis for the

96 THE OLIGONUCLEOTIDE PRODRUG APPROACH

development of a new generation of antisense oligonucleotides that could easily enter into intact cells. Acknowledgements We thank Dr I.Barber, Dr N.Mignet and Dr G.Tosquellas, who have worked on this approach, and K.Alvarez, C.Dell’Aquila, J.-C.Bologna and A.Meyer, who still are involved in this project. We thanks T.Beltran for mass spectrometry analysis. This project was supported by grants from the Agence Nationale de Recherche sur le SIDA (ANRS), the Association pour la Recherche sur le Cancer (ARC), and the CNRS. We thank ISIS Pharmaceuticals for financial support. References AGRAWAL, S. and IYER, R.P., 1997, Perspectives in antisense therapeutics, Pharmacol. Ther.,76, 151–160. ALDRIDGE, W.N., 1993, The esterases: perspectives and problems, Chem.-Biol. Interactions, 87, 5–13. ALVAREZ, K., TWORKOWSKI, I., VASSEUR, J.J., IMBACH, J.L. and RAYNER, B., 1998, A reinvestigation of sulfenyl groups as amino protecting groups for the synthesis of oligonucleotides on solid support by phosphoramidite chemistry, Nucleosides Nucleotides, 17, 365–378. BEAUCAGE, S.L. and IYER, R.P., 1992, Advances in the synthesis of oligonucleotides by the phosphoramidite approach, Tetrahedron, 48, 2223–2311. 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., 1996, Pharmacokinetic properties of several novel oligonucleotide analogs in mice, J. Pharmacol. Exp. Ther., 277, 923–937. DELL’AQUILA, C., IMBACH, J.L. and RAYNER, B., 1997, Photolabile linker for the solidphase synthesis of base-sensitive oligonucleotides, Tetrahedron Lett., 38, 5289–5292. DEVLIN, T., IYER, R.P., JOHNSON, S. and AGRAWAL, S., 1996, Mixed backbone oligonucleotides containing internucleotidic primary phosphoramidate linkages, Bioorg. Med. Chem. Lett., 6, 2663–2668. FODOR, P.A., LEIGTHON-READ, J., PIRRUNG, M.C., STRYER, L., LU, A.T. and SOLAS, D., 1991, Light-directed spacially addressable parallel chemical synthesis, Science, 251, 767–773. HASAN, A., STENGELE, K.P., GIEGRICH, H., CORNWELL, P., ISHAM, K.R., SACHLEBEN, R.A., PFLEIDERER, W. and FOOTE, R.S., 1997, Photolabile protecting groups for nucleosides: synthesis and photodeprotection rates, Tetrahedron, 53, 4247–4264. HAYAKAWA, Y., UCHIYAMA, M. and NOYORI, R., 1986, Nonaqueous oxidation of nucleoside phosphite to the phosphates, Tetrahedron Lett., 27, 4191–4194.

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HEIKKILA, J., BALGOBIN, N. and CHATTOPADHYAYA, J., 1983, The 2nitrophenylsulfenyl (NPS) group for the protection of amino functions of cytidine, adenosine, guanosine and their 2′-deoxysugar derivatives, Acta Chem. Scand. Ser. B, 37, 857–864. HEYMANN, E., 1980, Carboxyesterases and amidases, Enzymatic Basis of Detoxification, pp. 291–323, New York: Academic Press. IYER, R.P., DEVLIN, T., HABUS, I., YU, D., JOHNSON, S., and AGRAWAL, S., 1996a, Oligonucleoside phosphoramidates from N-pent-4-enoyl nucleoside Hphosphonates, Tetrahedron Lett., 37, 1543–1546. IYER, R.P., EGAN, W., REGAN, J.B. and BEAUCAGE, S.L., 1990, 3H-1, 2Benzodithiole-3-one 1, 1-dioxide as an improved sulfurizing reagent in the solidphase synthesis of oligodeoxyribonucleoside phosphorothioates, J. Am. Chem. Soc., 112, 1253–1254. IYER, R.P., HO, N.H., YU, D. and AGRAWAL, S., 1997, Bioreversible oligonucleotide conjugates by site-specific derivatization, Bioorg. Med. Chem. Lett., 7, 871–876. IYER, R.P., YU, D. and AGRAWAL, S., 1994, Stereospecific bio-reversibility of dinucleoside s-alkyl phosphorothiolates to dinucleoside phosphorothioates, Bioorg. Med. Chem. Lett., 4, 2471–2476. IYER, R.P., YU, D. and AGRAWAL, S., 1995a, Prodrugs of oligonucleotides: the acyloxyalkyl esters of oligodeoxyribonucleoside phosphorothioates, Bioorg. Chem., 23, 1–21. IYER, R.P., YU, D., DEVLIN, T., HO, N.H. and AGRAWAL, S., 1996b, Acyloxyaryl prodrugs of oligonucleoside phosphorothioates, Bioorg. Med. Chem. Lett., 6, 1917–1922. IYER, R.P., YU, D., HO, N.-H., DEVLIN, T. and AGRAWAL, S., 1995b, Methyl phosphotriester oligonucleotides: facile synthesis using N-pent-4-enoyl nucleoside phosphoramidites, J. Org. Chem., 60, 8132–8133. KRISE, J.P. and STELLA, V.J., 1996, Prodrugs of phosphates, phosphonates, and phosphinates, Adv. Drug Delivery Rev., 19, 287–310. LEFEBVRE, I., PÉRIGAUD, C., POMPON, A., AUBERTIN, A.-M., GIRARDET, J.-L., KIRN, A., GOSSELIN, G. and IMBACH, J.-L., 1995, Mononucleoside phosphotriester derivatives with S-acyl-2-thioethyl bioreversible phosphateprotecting group: intracellular delivery of 3′-azido-2′, 3′-dideoxythymidine 5′monophosphate, J. Med. Chem., 38, 3941–3950. MARTIN, L.T., FARAJ, A., SCHINAZI, R.F., IMBACH, J.L., GOSSELIN, G., MCCLURE, H.M. and SOMMADOSSI, J.P., 1998, Pre-clinical pharmacology of βL-2′, 3′-dideoxy-5-fluorocytidine and its prodrug bis-[(t-butyl)SATE]-(β-L-2′, 3′dideoxy-5-fluorocytidine monophosphate, Proc. 11th International Conference on Antiviral Research, San Diego, CA. MAURITZ, R.P., MEIER, C. and UHLMANN, E., 1997, Synthesis of 3′, 5′-dithymidylylα-hydroxyphosphonate dimer building blocks for oligonucleotide synthesis—A new pro-oligonucleotide approach, Nucleosides Nucleotides, 16, 1209–1212. MEIER, C. and MAURITZ, R., 1995, Synthesis of protected 3′, 5′-di-2′-deoxythymidine (alpha-hydroxy-2-nitrobenzyl)-phosphonate diesters as dimer building blocks for oligonucleotides, Nucleosides Nucleotides, 14, 803–804. MENTLEIN, R. and HEYMANN, E., 1984, Hydrolysis of ester-and amide-type drugs by the purified isoenzymes of nonspecific carboxylesterase from rat liver, Biochem. Pharmacol., 33, 1243–1248.

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MIGNET, N., CHAIX, C., RAYNER, B. and IMBACH, J.L., 1997a, Synthesis and evaluation of glucuronic acid derivatives as alkylating agents for the reversible masking of internucleoside groups of antisense oligonucleotides, Carbohyd. Res., 303, 17–24. MIGNET, N., MORVAN, F., RAYNER, B. and IMBACH, J.L., 1997b, The prooligonucleotide approach. 5. Influence of the phosphorus atom environment on the hydrolysis of enzymolabile dinucleoside phosphotriesters, Bioorg. Med. Chem. Lett., 7, 851–854. MIGNET, N., TOSQUELLAS, G., BARBER, I., MORVAN, F., RAYNER, B. and IMBACH, J.L., 1997c, The pro-oligonucleotide approach—synthesis and stability of chimeric pro-oligonucleotides in culture medium and in total cell extract, New J. Chem., 21, 73–79. MORVAN, F., TOSQUELLAS, G., MIGNET, N., BARBER, I., RAYNER, B. and IMBACH, J.L., 1997, The pro-oligonucleotide approach—chimeric dodecamers bearing six bioreversible protecting groups, Nucleosides Nucleotides, 16, 1213–1214. PÉRIGAUD, C., GOSSELIN, G. and IMBACH, J.-L., 1997, Minireview: from the pronucleotide concept to the SATE phosphate protecting groups, Curr. Topics Med. Chem., 2, 15–29. PÉRIGAUD, C., GOSSELIN, G., LEFEBVRE, I., GIRARDET, J.L., BENZARIA, S., BARBER, I. and IMBACH, J.L., 1993, Rational design for cytosolic delivery of nucleoside monophosphates—SATE and DTE as enzyme-labile transient phosphate protecting groups , Bioorg. Med. Chem. Lett., 3, 2521–2526. POMPON, A., LEFEBVRE, I. and IMBACH, J.L., 1992, On-line internal surface reversedphase cleaning—the direct HPLC analysis of crude biological samples— application to the kinetics of degradation of oligonucleotides in cell culture medium. Biochem. Pharmacol., 43, 1769–1775. SASTRY, J.K., NEHETE, P.N., KHAN, S., NOWAK, B.J., PLUNKETT, W., ARLINGHAUS, R.B. and FARQUHAR, D., 1992, Membrane-permeable dideoxyuridine 5′-monophosphate analogue inhibits human immunodeficiency virus infection, Mol. Pharmacol., 41, 441–445. SATOH, T., 1987, Role of carboxyesterases in xenobiotic metabolism, Rev. Biochem. Toxicol., 8, 155–181. SATOH, T. and HOSOKAWA, M., 1998, The mammalian carboxylesterases: from molecules to functions, Annu. Rev. Pharmacol. Toxicol., 38, 257–288. SEKINE, M. and SEIO, K., 1993, Synthesis and properties of N-tritylthio nucleoside derivatives and reductive removal of the tritylthio group by use of tributyltin hydride and tris(trimethylsilyl)silane, J. Chem. Soc. Perkin Trans., 1, 3087–3093. TOSQUELLAS, G., ALVAREZ, K., DELL’AQUILA, C., MORVAN, F., VASSEUR, J.J., IMBACH, J.L. and RAYNER, B., 1998a, The pro-oligonucleotide approach—solid phase synthesis and preliminary evaluation of model pro-dodecathymidylates, Nucl. Acids Res., 26, 2069–2074. TOSQUELLAS, G., BARBER, I., MORVAN, F., RAYNER, B. and IMBACH, J.L., 1996, The prooligonucleotide approach. 3. Synthesis and bioreversibility of a chimeric phosphorodithioate prooligonucleotide, Bioorg. Med. Chem. Lett., 6, 457–462.

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TOSQUELLAS, G., BOLOGNA, J.C., MORVAN, F., RAYNER, B. and IMBACH, J.L., 1998b, First synthesis of alternating SATE phosphotriester/phosphodiester prooligonucleotides on solid support, Bioorg. Med. Chem. Lett., 8, 2913–2918. TOSQUELLAS, G., MORVAN, F., RAYNER, B. and IMBACH, J.L., 1997, The prooligonucleotide approach. 4. Synthesis of chimeric prooligonucleotides with 6 enzymolabile masking groups and unexpected desulfurization side reaction, Bioorg. Med. Chem. Lett., 7. 263–268.

4 Peptide Nucleic Acids P.E.NIELSEN

4.1 Introduction

Figure 4.1 Chemical structures of DNA and PNA: B is a nucleobase

Peptide nucleic acids (PNAs) are not oligonucleotides and not even DNA analogues, but rather DNA mimics. Chemically PNA is a pseudopeptide in which the individual nucleobase units are amino acids joined together by amide bonds (Figure 4.1) (Nielsen et al., 1991; Egholm et al., 1992). Therefore, although PNA is a very good structural mimic of DNA in terms of its ability to form stable and sequence-selective complexes with complementary DNA or RNA oligonucleotides, it has unique chemical and physicochemical properties (Hyrup and Nielsen, 1996; Good and Nielsen, 1997). These can be exploited advantageously for development of pharmaceuticals, but also limit the degree to

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which results, such as bioavailability and pharmacokinetics, obtained with oligonucleotides or their close chemical analogues can be used in developing PNA. It is therefore not surprising that ‘PNA drug development’ is lagging behind the much larger field of oligonucleotide (phosphorothioate) drug development. Nonetheless, many exciting results have been reported within the past five years, and some of these are highly relevant for discussing pharmaceutical aspects of PNA and also hold promise that highly efficient PNA-based gene therapeutic drugs may not be too far away. 4.2 Antisense Antisense inhibition of in vitro translation by PNA has been demonstrated in a variety of systems (Hanvey et al., 1992; Bonham et al., 1995; Knudsen and Nielsen, 1996; Gambacorti-Passerini et al., 1996; Good and Nielsen, 1998a). Not surprisingly, PNA—RNA duplexes are not substrates for RNase H, and thus antisense inhibition must take place by an RNase H-independent mechanism (Hanvey et al., 1992; Bonham et al., 1995; Knudsen and Nielsen, 1996). Therefore, sensitive sequence targets on specific mRNAs identified by phosphorothioates are not necessarily good targets with PNA. Indeed, it has been found that sensitive PNA targets are found at the AUG initiation codon (Knudsen and Nielsen, 1996; Gambacorti-Passerini et al., 1996) or upstream from this (GambacortiPasserini et al., 1996), but no thorough ‘gene-walks’ (Monia et al., 1996) have so far been reported for PNA. It is also worth noting that triplexforming PNA decamers are able to cause translation elongation arrest, and homopurine targets, even if these are situated within the coding region, should be prime targets for PNA antisense (Hanvey et al., 1992; Bonham et al., 1995; Knudsen and Nielsen, 1996). Recently a very exciting study reported antisense downregulation of the galanin receptor activity in cells in culture using PNAs conjugated to membranepenetrating small peptides (e.g. the 16 amino acid homeodomain of the antennapedia protein from Drosophila) (Pooga et al., 1998), and these authors were even able to demonstrate downregulation of the galanin receptor activity in live rats upon intrathecal injection of the PNA. In another, less thorough, study, ‘naked’ PNAs targeted to either the mRNA of the opioid(mu)- or the neurotensin-receptor were injected into the brain of rats, and decreased ligand-binding activity of the respective receptor was reported (Tyler et al., 1998). The latter results are surprising in view of the fact that it is generally observed that PNAs are taken up very poorly by eukaryotic cells in culture (Bonham et al., 1995). However, as with oligonucleotides, cells in tissues and in particular neurons may well behave differently from cells in culture. These reports demonstrate that it is possible to find ways and/or systems in which PNAs exhibit antisense compatible effects in vivo. Formally, these two

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Figure 4.2 Secondary structure of E.coli 23S RNA indicating the positions of the peptidyl transferase centre and the α-sarcin loop. The targets for the PNAs, H-Lys-TTJTJJJTTTJT(eg1)3-TCTTTCCGTCTT-LysNH2 and H-Lys-JTJTJJT-(eg1)3-TCCTCTC-LysNH2 are shown in bold below (positions 2051–2062 and 2659–26659). J is pseudoisocytosine (cf. Figure 4.4)

studies have not shown that the biological effects on the rats are bona fide antisense. and therefore more studies are warranted.

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4.3 Antimicrobials Quite surprisingly, PNAs are to a limited extent taken up by E.coli bacterial cells, and by designing triplex forming bis-PNAs targeted to essential loop regions of 23S ribosomal RNA (Figure 4.2), PNAs with antibiotic-like properties were obtained (Good and Nielsen, 1998b). Specifically, it was found that two such PNAs (Figure 4.2) inhibited translation in vitro at nanomolar concentrations (Good and Nielsen, 1998b) and were able to inhibit the growth of both wild type E.coli K12, and, especially, a permeable mutant (AS 19) at micromolar concentrations. Provided the bacterial uptake of such PNAs can be improved by chemical modification and/or formulation and that similar or improved effects can be demonstrated with pathogenic strains of bacteria, these results open the way for the development of novel PNA-based antibacterial drugs. Along similar lines, it was demonstrated that antisense downregulation of gene expression in E.coli is possible with PNAs (Good and Nielsen, 1998a). Specifically, PNAs targeted to the AUG-region of the mRNA of the β-lactamase gene were found to resensitize formerly penicillin-resistant (due to β-lactamase production) bacteria to penicillin by six to seven orders of magnitude using micromolar concentrations of the PNAs. Thus the antisense principle could also be a means for development of ‘antibiotics’ by targeting essential genes in pathogenic bacteria. 4.4 Anti-telomerase The enzyme, telomerase, which is responsible for maintaining telomere ends on chromosomes during mitosis, and which appears to be overexpressed in at least some cancer cells, is an RNA—protein complex in which the RNA is essential for actitivity. The RNA actually provides the template for the new telomere. PNAs targeted to this RNA are very potent inhibitors of the enzyme in vitro (C50 in the sub-nanomolar range) (Norton et al., 1996), and, provided telomerase activity is indeed required for malignant growth, such PNAs could be developed into anticancer drugs. 4.5 Antigene Homopyrimidine PNAs bind to sequence complementary homopurine targets in double-stranded DNA by invasion into the DNA duplex by forming an internal PNA2—DNA triplex in a strand displacement complex (Figure 4.3) (Nielsen et al., 1994a). Such PNA strand displacement complexes are extremely stable and inhibit protein (transcription factor) binding to an overlapping (or even adjacent) binding site (Nielsen et al., 1993; Vickers et al., 1995), and can even arrest

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Figure 4.3 Schematic drawing of a PNA—dsDNA strand displacement complex (right) consisting of two PNA strands (composed of thymines (T) and cytosines (C)) that invade the DNA duplex and bind to the complementary DNA strand (composed of adenines (A) and guanines (G)) by Watson-Crick and Hoogsteen base pairing (left). The noncomplementary DNA strand is extruded as a singlestranded loop

Figure 4.4 Hoogsteen recognition of guanine by pseudoisocytosine, a ‘permanently protonated’ cytosine mimic

transcription elongation (Hanvey et al., 1992; Nielsen et al., 1994b; Vickers et al., 1995; Praseuth et al., 1996). The most stable complexes form with two antiparallel PNAs, of which one binds antiparallel by Watson-Crick base pairing to the DNA target and the other binds antiparallel by Hoogsteen base pairing. Thus even more potent PNAs are obtained by chemically joining such two PNAs into a bis-PNA (Egholm et al., 1995). Furthermore, pH independent binding can

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be accomplished by exchanging cytosines in the Hoogsteen PNA strand with pseudoisocytosines (Figure 4.4) (Egholm et al., 1995). Once formed, the PNA2—DNA strand invasion complexes are exceedingly stable and are formed with exquisite, kinetically controlled sequence selectivity (Demidov et al., 1995; Kuhn et al., 1997). However, because the DNA double helix must be opened (denatured) in the binding process, the binding rate is very sensitive to elevated ionic strength, and binding is in fact very slow at physiological ionic strength (140 mM K+). Thus, most in vitro experiments demonstrating antigene effects were done using PNA—dsDNA complexes that were preformed at low ionic strength and then transferred to higher ionic strength buffers. It might therefore be feared that biological antigene effects of PNA would be very weak. None the less, a recent study reported mutagenic effects of a bis-PNA target to a gene in mouse cells (Faruqi et al., 1998). Although it is not possible from these data to determine the degree of PNA binding to the dsDNA target, the result clearly indicates that some level of binding was achieved. Indeed, two observations from in vitro experiments could provide an explanation for facilitated binding in vivo. It has been found that the transcription process itself, presumably via the single-stranded transcription bubble, catalyses PNA binding (Larsen and Nielsen, 1996), and transcription may also indirectly facilitate PNA binding via induction of negative supercoiling upstream from the transcription complex, because it has been found that negative supercoiling can accelerate PNA binding up to 200-fold at 140 mM K+ (Bentin and Nielsen, 1996). 4.6 Anti-HIV In vitro studies have shown that purine/pyrimidine mixed sequence, duplexforming PNAs as well as homopyrimidine triplex-forming PNAs targeted to, for example, gag-RNA are very potent inhibitors of HIV reverse transcriptase by blocking the elongation by the enzyme (Koppelhus et al., 1997; Lee et al., 1998). Thus further developments in this area, especially regarding cellular uptake and bioavailability, may lead to new anti-HIV drugs. 4.7 Pharmacology Very little is known about the phamacological behaviour of PNA. It has been established that PNAs are inherently very stable in biological fluids such as serum and cellular extracts (Demidov et al., 1994), but thorough studies on bioavailability, pharmacokinetics and pharmacodynamics are still very much needed. As already indicated (Norton et al., 1995; Pardridge et al., 1995; Pooga et al., 1998; Aldrian-Herrada et al., 1998), PNA peptide conjugates could be of high interest in this connection.

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Figure 4.5 Comparison of a diaminopurine-thymine and a guanine-cytosine base pair. The ‘extra’ 2-amino group of diaminopurine is shown in bold

Figure 4.6 Examples of a backbone functionalized PNA using the amino acid lysine, leucine, aspartic acid or asparagine instead of glycine in the PNA backbone

4.8 Further Developments Besides favourable hybridization (Egholm et al., 1993) and stability (Demidov et al., 1994) properties, PNA distinguishes itself from other gene therapeutic drug leads by what may be termed ‘chemical flexibility’: it is fairly straightforward and easy to synthesize PNA oligomers with modified backbones and/or nucleobases. For instance, substituting adenine with diaminopurine (Figure 4.5) increases the thermal stability of PNA—DNA duplexes by 2–4 K per substitution (Haaima et al., 1997). Furthermore, chemical functionality can easily be introduced in the PNA backbone by using α-amino acids other than glycine (Figure 4.6) (Haaima et al., 1996; Püschl et al., 1998) without seriously affecting the hybridization efficiency. Such modifications could be extremely valuable in tuning the pharmacokinetic and pharmacodynamic properties of PNA drug leads, and they might also improve PNA bioavailability. Acknowledgement This work was supported by the Danish National Research Foundation.

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nucleic acids as triplexing agents: binding and stoichiometry, J. Am. Chem. Soc., 117, 831–832. HAAIMA, G., HANSEN, H.F., CHRISTENSEN, L., DAHL, O. and NIELSEN, P.E., 1997, Increased DNA binding and sequence discrimination of PNA upon incorporation of diaminopurine, Nucl. Acids Res., 25, 4639–4643. HAAIMA, G., LOHSE, A., BUCHARDT, O. and NIELSEN, P.E., 1996, Peptide nucleic acids (PNA) containing thymine monomers derived from chiral amino acids: hybridization and solubility properties of D-lysine PNA, Angew. Chem., 35, 1939–1941. HANVEY, J.C., PEFFER, N.C., BISI, J.E., THOMSON, S.A., CADILLA, R., JOSEY, J.A., RICCA, D.J., HASSMAN, C.F., BONHAM, M.A., AU, K.G., CARTER, S.G., BRUCKENSTEIN D.A., BOYD, A.L., NOBLE, S.A. and BABISS, L.E., 1992, Antisense and antigene properties of peptide nucleic acids, Science, 258, 1481–1485. HYRUP, B. and NIELSEN, P.E., 1996, Peptide nucleic acids (PNA). Synthesis, properties and potential applications (review), Bioorg. Biomed. Chem., 4, 5–23. KNUDSEN, H. and NIELSEN, P.E. (1996) Antisense properties of duplex and triplex forming PNA, Nucl. Acids Res., 24, 494–500. KOPPELHUS, U., ZACHAR, V., NIELSEN, P.E., LIU, X., EUGEN-OLSEN, J. and EBBESEN, P., 1997, Efficient in vitro inhibition of HIV-1 gag reverse transcription by peptide nucleic acid (PNA) at minimal ratios of PNA/RNA, Nucl. Acids Res., 25, 2167–2173. KUHN, H., DEMIDOV, V., FRANK-KAMENETSKII, M.D. and NIELSEN, P.E., 1997, Kinetic sequence discrimination of bis-PNAs upon targeting of double stranded DNA, Nucl. Acids Res., 26, 582–587. LARSEN, H.J. and NIELSEN, P.E., 1996, Transcription-mediated binding of peptide nucleic acid (PNA) to double stranded DNA: sequence specific suicide transcription, Nucl. Acids Res., 24, 458–463. LEE, R., KAUSHIK, N., MODAK, M.J., VINAYAK, R. and PANDEY, V.N., 1998, Polyamide nucleic acid targeted to the primer binding site of the HIV-1 RNA genome blocks in vitro HIV-1 reverse transcription, Biochemistry, 37, 900–910. MONIA, B.P., JOHNSTON, J.F., GEIGER, T., MULLER, M. and FABBRO, D., 1996, Antitumor activity of a phosphorothioate antisense oligodeoxynucleotide targeted against C-raf kinase, Nat. Med., 2, 668–675. NIELSEN, P.E., EGHOLM, M., BERG, R.H. and BUCHARDT, O., 1991, Sequence selective recognition of DNA by strand displacement with a thymine-substituted polyamide, Science, 254, 1497–1500. NIELSEN, P.E., EGHOLM, M., BERG, R.H. and BUCHARDT, O., 1993, Sequence specific inhibition of restriction enzyme cleavage by PNA, Nucl. Acids Res., 21, 197–200. NIELSEN, P.E., EGHOLM, M. and BUCHARDT, O., 1994a, Evidence for (PNA)2/DNA triplex structure upon binding of PNA to dsDNA by strand displacement, J. Mol. Recognition, 7, 165–170. NIELSEN, P.E., EGHOLM, M. and BUCHARDT, O., 1994b, Sequence specific transcription arrest by PNA bound to the template strand, Gene, 149, 139–145. NORTON, J.C., PIATYCZEK, M.A., WRIGHT, W.E., SHAY, J.W. and COREY, D.R., 1996, Inhibition of human telomerase activity by peptide nucleic acid, Nat. Biotechnol., 14, 615–619.

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NORTON, J.C., WAGGENSPACK, J.H., VARNUM, E. and COREY, D.R., 1995, Synthesis and membrane permeability of PNA—peptide conjugates, Bioorg. Med. Chem., 3, 437. PARDRIDGE, W.M., BOADO, R.J. and KANG, Y.-S., 1995, Vector-mediated delivery of a polyamide (‘peptide’) nucleic acid analog through the blood—brain barrier in vivo, Proc. Natl Acad. Sci. USA, 92, 5592–5596. POOGA, H., SOOMETS, U., HÄLLBRINK, M., VALKNA, A., SAAR, K., REZAEI, K., KAHL, U., HAO, J.-X., XU, X.-J., WIESENFELD-HALLIN, Z., HÖKFELT, T., BARTFAI, T. and LANGEL, Ü., 1998, Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo, Nat. Biotechnol., 16, 857. PRASEUTH, D., GRIGORIEV, M., GUIEYSSE, A.L., PRITCHARD, L.L., HARELBELLAN, A., NIELSEN, P.E. and HELENE, C., 1996, Peptide nucleic acids directed to the promoter of the α-chain of the interleukin-2 receptor, Biochim. Biophys. Acta, 1309, 226–238. PÜSCHL, A., SFORZA, S., HAAIMA, G., DAHL, O. and NIELSEN, P.E., 1998, Peptide nucleic acids (PNAs) with a functional backbone, Tetrahedron Lett., 39, 4707–4710. TYLER, B.M., MCCORMICK, D.J., HOSHALL, C.V., DOUGLAS, C.L., JANSEN, K., LACY, B.W., CUSACK, B. and RICHELSON, E., 1998, Specific gene blockade shows that peptide nucleic acids readily enter neuronal cells in vivo, FEBS Lett., 421, 280–284. VICKERS, T.A., GRIFFITH, M.C., RAMASAMY, K., RISEN, L.M. and FREIER, S.M., 1995, Inhibition of NF-kappa B specific transcriptional activation by PNA strand invasion. Nucl. Acids Res., 23, 3003–3008.

PART THREE Delivery

5 Peptide-mediated Delivery of Oligonucleotides E.VIVÈS AND B.LEBLEU

5.1 Delivery Vehicles for the Improved Uptake of Nucleic Acids: a Survey Synthetic oligonucleotides offer interesting prospects for the control of gene expression through specific interactions with RNA (antisense oligonucleotides, ribozymes), DNA (triple helix-forming oligonucleotides) or even proteins (decoy oligonucleotides, aptamers). These strategies, and in particular ribozymes and antisense ON, have been the object of numerous studies in various in vitro and in vivo (including several human clinical trials) biological models. Whatever the successes encountered with a first generation of phosphorothioate antisense ON and synthetic ribozymes, it is generally admitted that greatly improved pharmacological properties will have to be provided. The most frequently encountered problems are degradation by nucleases, insufficient affinity for the target, lack of specificity and, more importantly, poor bioavailability (for a review see Gewirtz et al. (1998)). Synthetic oligonucleotides are negatively charged and therefore do not freely cross biological membranes, a problem also encountered with mononucleotides in antiviral chemotherapy or with plasmid DNA in gene delivery. Mere neutralization of the internucleotidic linkages does not lead to improved cellular uptake as experienced with methylphosphonates (Giles et al., 1993) or with peptide nucleic acids (Wittung et al., 1995). The synthesis of prooligonucleotides (see Chapter 3) might, however, lead to a breakthrough in this field. Somewhat unexpectedly antisense ONs are taken up by various cell types through as yet poorly explored mechanisms involving fluid phase and receptormediated endocytosis (Yakubov et al., 1989; Geselowitz and Neckers, 1992; Beltinger et al., 1995). The efficiency of these endocytotic processes for oligonucleotides is generally poor, and material remains for a large part segregated in endocytotic vesicles. Some notable exceptions have been reported, for instance primary keratinocytes or keratinocyte cell lines which efficiently

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internalize antisense ONs and concentrate them in the nuclei (for a review see Lebleu et al., 1997). Successes of in vivo studies with synthetic ribozymes or antisense phosphorothioate analogues have been assigned partly to efficient uptake of nucleic acids in primary cells, but the underlying mechanisms have not been explored to our knowledge. Although much attention has been paid to the cellular uptake of synthetic ON, intracellular trafficking or crossing through biological barriers such as the bloodbrain barrier will be of the utmost importance for the future of nucleic acid-based drugs. Many strategies have been proposed to improve cellular delivery of synthetic oligonucleotides. Direct microinjection (Leonetti et al., 1991) and permeabilization of the plasma membrane by physical or enzymatic agents, such as electroporation (Bergan et al., 1996) or streptolysin (Giles et al., 1993), have been successfully adapted to antisense ON but cannot be applicable for in vivo experimentation. Association or encapsidation of nucleic acids to particulate delivery vehicles such as liposomes or nanoparticles (reviewed in Lebleu et al., 1996) has been the object of many developments and is reviewed in Chapters 6 and 7. Chemical conjugation or physical association to cationic lipids or cationic peptides has been explored for the delivery of synthetic ONs and plasmid DNA. The conjugation of plasmid DNA or antisense ONs to various cationic lipid formulations (Zelphati and Szoka, 1996) or to polyethyleneimine (Boussif et al., 1995) allows their efficient delivery in many cell types, at least in in vitro experiments, and is the most widely used strategy so far. Studies in our group have established the potential of cationic polyaminoacids for the delivery of nucleic acids (see below). The chemical conjugation of nucleic acids to cell ligands, such as transferrin (Gotten et al., 1992) or neoglycoproteins (Midoux et al., 1993; Hangeland et al., 1995) represents an interesting strategy allowing specific recognition by cognate receptors at the cell surface and subsequent internalization. None of these strategies is devoid of problems, and most studies in this emerging field are still in an early phase. Which strategy would be preferable in a particular therapeutic application cannot be decided at the present time. Among the most commonly encountered problems in drug delivery are capture of particulate material by cells of the reticulo-endothelial system, immunogenicity or toxicity of the delivery vehicle for certain cell types, and segregation in the endocytic vesicles. Remarkably, however, cationic lipids (Zelphati and Szoka, 1996) and polyethyleneimine (Boussif et al., 1995) are taken up by endocytosis but efficient release from the endosomes is allowed through lipid fusion or endosome destabilization.

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5.2 The Potential of Peptides for Nucleic Acids Delivery So far, none of the DNA transfection protocols have approached the efficiency with which viruses internalize (and express) their nucleic acids payload in an infected cell. Many efforts have therefore been devoted to engineering virus mimics for the delivery of plasmid DNA, antisense ONs or ribozymes. Two main strategies have been evolved by viruses to bypass cellular membranes: fusion of their envelope at neutral pH with the cell plasma membrane, and endocytosis (mainly receptor-mediated) followed by escape from the endocytotic compartments. In both cases the efficiency of nucleic acids release largely relies on fusogenic and/or membrane-destabilizing events driven by fusogenic domains in viral proteins. Interestingly, several cellular or viral proteins can pass cellular membranes efficiently, as demonstrated for the Antennapaedia protein (Derossi et al., 1994) and for the HIV-1 Tat protein (Vivès et al., 1997a). Whatever the physiological relevance of these properties, they might be exploited for the delivery of nucleic acids, as will be discussed in section 5.5. Similar goals are sought for gene and synthetic oligonucleotides transfer, e.g. nucleic acids protection from nucleases in biological fluids, selective recognition by targeted cells or tissues when possible, efficient cellular uptake, and delivery and accumulation in the appropriate intracellular compartment. However, significant differences can also be found. For example, a single expressed gene is in principle sufficient for cell transformation, while many copies of a synthetic antisense ON are required in order to exert a significant biological activity. On the other hand, the large size of plasmid DNA requires compaction prior to delivery, which is not the case for antisense ONs. Finally, short ONs released in the cytoplasm will diffuse to the nucleus through the nuclear pores (Leonetti et al., 1991), while nuclear uptake is a major problem for plasmid DNA in resting cells in particular. ON analogues with improved pharmacological properties can be engineered, as discussed in several other chapters. We will restrict ourselves to a review of the literature dealing with peptide (or polypeptide)-mediated delivery of antisense ON, with emphasis on our own work dealing with poly (L-lysine)mediated delivery (reviewed in Lebleu et al., 1996), and on attempts to exploit the fusogenic properties of influenza haemagglutinin (Bongartz et al., 1994) or the membrane translocating activity of the HIV-1 Tat protein (Vivès et al., 1997a). Although most recent efforts have been focused on attempts to harness the potential of peptides for increased cellular uptake and release from (or avoidance of) the endocytotic compartment, other strategies might be beneficial. Short cellular recognition motifs could potentially be introduced to restrict uptake by certain cell types, as already demonstrated in same studies (Leonetti et al., 1990b; Hangeland et al., 1995).

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Once delivered in the cytoplasm, antisense ONs are rapidly translocated to the nuclei and bind tightly to nuclear proteins (Clarenc et al., 1993). If potential synthetic ON targets such as pre-mRNA or DNA obviously exist in the nuclei, tight binding to nuclear proteins might reduce the free intracellular ON pool. Reducing nuclear proteins trapping by appropriate chemical modification of the ON or favouring nucleo-cytoplasmic transport by conjugation to appropriate peptidic motifs might therefore be required to target cytoplasmic mRNAs. In this respect, studies with ribozyme-expressing genes have clearly demonstrated the benefits of co-localizing the effector ribozyme with the target RNA (Bertrand et al., 1997). The appending of basic peptides to oligonucleotides (or their analogues) increases affinity for their nucleic acids target, as shown in several cell-free models, and should also favour invasion of highly structured DNA or RNA targets (Iyer et al., 1995). Whether any of the strategies discussed here will be able to meet some or all of these requirements without unacceptably increasing cost and complexity is far from certain. Finally, it should be pointed out that such peptide-based delivery vectors could also be useful to improve the bioavailability of other potential drugs, and in particular of non-permeant peptides. 5.3 Strategies for the Coupling of Peptides to Oligonucleotides Several chemical routes have been proposed for the conjugation of a peptide to an ON. The most convenient method would in principle be to synthesize entirely the chimeric molecule by stepwise synthesis on a single solid support. Although feasible, this strategy is still under development, mainly to circumvent problems arising from incompatibilities of the protection schemes required for the solidphase synthesis of ONs and peptides. Several low molecular weight chimeric molecules have been synthesized by such strategies, but the final products were obtained with poor yields (Haralambidis et al., 1990; Truffert et al., 1994). It is therefore likely that the direct synthesis of larger antisense ON—peptide conjugates will lead to further synthesis and/or purification problems (Peyrottes et al., 1998). It should be recalled here that both entities require a minimal length to retain their desired activities, i.e. 10 to 15 amino acids for an efficient translocating activity of the peptide, and 12 to 15 nucleotides for maintaining a sequence-specific binding of the ON. A solid-phase strategy for the synthesis of longer ON—peptide conjugates at a scale suitable for antisense studies has, however, been described (Soukchareun et al., 1995). It concerned a fusion peptide located in the N-terminus of the HIV transmembrane gp41 glycoprotein. In this method, the peptide (11 or 16 amino acids) was assembled first by the Nfluorenylmethoxycarbonyl (Fmoc) solid-phase strategy on a derivatized controlled pore glass (CPG) resin. After derivatization of the amino terminus to a hydroxy group, the standard phosphoramidite method was used for the synthesis

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of the ON moiety (20-to 27-mer). Although this method could circumvent some peptide solubility problems sometimes encountered during a classical block conjugation scheme, the overall yield was in the range of 6–14% and will certainly have to be improved. In addition, new side-chain protection moieties will probably be required for the efficient stepwise synthesis of longer peptide— ON conjugates. It therefore appeared easier to couple separately prepared and purified entities. Peptide and nucleotide chemistries allow a large panel of derivatization schemes for both molecular synthons to allow their specific coupling. One convenient strategy for coupling appears to be disulphide bond formation between the peptide and the ON. The incorporation of a sulphhydryl group in a peptide sequence is obvious through a cystein residue available from the native sequence or appended to the N-terminal or to the C-terminal end of the peptide. Likewise, several chemical protocols allow the efficient introduction of a sulphhydryl group at either the 5′ or the 3′ end of an ON (Connolly and Rider, 1985; Li et al., 1987; Sinha and Cook, 1988; Zuckerman et al., 1987; Gupta et al., 1991). The condensation of two sulphhydryl groups containing molecules through disulphide bridge formation could lead to the loss of substantial amounts of starting material through homodimers synthesis. It is therefore worth activating first one of the thiol-containing molecules. Since it is generally easier to synthesize peptides in larger quantity (synthesis scale of several hundred micromoles) than ON (synthesis scale of some micromoles), an initial activation of the ON moiety (a reaction which is easily brought to completion) should be preferred. A two-to three-fold molar excess of the peptide over the activated ON would then lead to chimeric molecules with very good yields (Chu and Orgel, 1988; Vivès and Lebleu, 1997; Eritja et al., 1991). Several thiol-activating groups such as 2, 2-dipyridyldisulphide, 3-nitro-2pyridinsulphenyl (Npys) and 2-nitrophenyl are available, and lead to the activated molecule with very good yields (Chu and Orgel, 1988). Alternatively, it is possible to synthesize directly the peptide with an activated thiol group by incorporating a Boc-Cys(Npys) monomer (Eritja et al., 1991). The intracellular release of the transported material will be preferred in many applications, since this would prevent possible interferences with the delivery vector for target recognition. In this respect, it is generally anticipated that the intracellular reductive environment will reduce disulphide bridges and release the conjugated drug (Derossi et al., 1998; Prochiantz, 1996), although no direct evidence has been provided to our knowledge. Along these lines, a 15-mer antisense ON coupled to a KDEL peptide through a stable thioether bond was 10 times fold more efficient than the disulphide-linked chimeric molecule (Arar et al., 1995). An early reduction of the disulphide bond of the ON—peptide conjugate was proposed to explain that difference (Arar et al., 1995). Early studies on the fate of radioiodinated tyramine conjugated to peptide carriers through a disulphide bond suggested the Golgi apparatus as the most probable site of

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reduction (Feener et al., 1990). However, such mechanisms should be relevant only for conjugates which are internalized by endocytosis. In some applications, however, a stable linkage between the conjugated material and the delivery vector might be sought, for example in cases in which the conjugated peptide is also involved in intracellular trafficking or in target recognition. Several stable bonds between the peptide and the ON have been described, e.g. thioether (Arar et al., 1995) or maleimide linkages (Zhu et al., 1993; Tung et al., 1991; Eritja et al., 1991; Ede et al., 1994). For instance, a procedure for the selective coupling of an ON to a peptide was based on the reactivity of a maleimide group bound to the ON with the thiol group of a cysteine residue of the peptide (Tung et al., 1991). Following the same chemistry a series of positively charged (D-ornithine)n-cysteine peptides was coupled to an ON (Zhu et al., 1993). Binding of the nucleic acid—peptide conjugate to its complementary DNA target showed that Tm increased with the net charge on the conjugated peptide. The coupling of highly basic peptides such as Antennapaedia or Tat to an ON should improve the binding kinetics and the stability of the chimeric molecules to their target. Moreover, site-directed cleavage with RNase H demonstrated that the peptide-modified ON hybridized specifically with its RNA target sequence. 5.4 Poly (L-lysine)-based Delivery Systems The efficiency of poly (L-lysine) (PLL) conjugation to deliver ON was first established using 2–5A tetramers, a generic term for the 2′–5′ linked activators of RNase L. Activation of RNase L (see also Chapter 2) was achieved when incubating various cell lines in culture with PLL-conjugated 2–5A or 2–5A analogues at nanomolar doses, while free 2–5A oligomers were totally inactive even in the micromolar concentration range (Bayard et al., 1986). Likewise, PLL-conjugated short antisense ON were shown to be 10 to 50 times more active than unconjugated oligomers in various biological models (reviewed in Lebleu et al., 1996). For example, a PLL-conjugated 15-mer unmodified antisense ON complementary to the translation initiation site of HIV-1 tat mRNA exerted a dose-dependent antiviral activity (with an IC 50 of 150 nM) in de novo infected T cell lines, while unconjugated material was barely active (Degols et al., 1994). Moreover a sequence-specific effect could be demonstrated with PLL-conjugated material but not with unconjugated material, as often encountered when using antisense ON as antivirals (Degols et al., 1992). Likewise, short antisense ON complementary to the U5 or to the pre-PBS regions of HIV-1 genomic RNA exerted a sequence-specific reduction in viral DNA production and an antiviral activity at submicromolar concentrations (Bordier et al., 1995). The mechanism through which PLL increases the biological activity of antisense ON is not fully understood. The positive net charge of these conjugates

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should facilitate binding to the negatively charged cell membrane and subsequent internalization through adsorptive endocytosis. Proteolysis of the polyaminoacids carrier in the endocytic vesicle is probably involved in the release of the conjugated material, since poly (D-lysine)-conjugated material is inactive (Leonetti et al., 1990a). Protection against nucleases in cell culture media as well as in cells themselves should also contribute to the increased efficiency of PLL-conjugated material. Indeed, the antisense (or 2–5A) oligomers were oxydized at their 3′ end before being conjugated to ε-amino groups of the carrier polypeptide. Whatever the fate of the carrier moiety after cell uptake, such 3′ modification will confer increased resistance to phosphodiesterases which are known to play a major role in the catabolism of nucleic acids. Various modifications of these PLL carriers have been introduced in order to confer cell-targeting properties. In these studies the antisense ON was covalently linked or was complexed by electrostatic interaction with the polycationic carrier. For example, glycosylated PLL was used to allow recognition by membrane lectins. The PLL entity has also been covalently linked to polypeptidic ligands as transferrin (Citro et al., 1993) or asiologlycoproteins (Nakazono et al., 1996), thus allowing preferential capture by cells expressing transferrin or galactose receptors, respectively. Whether such PLL-based delivery vectors will be applicable for the in vivo delivery of nucleic acids remains questionable, although extensive studies have been conducted in the perspective of gene transfer. Molecular complexity of these conjugates, cytoxicity for certain cell types, immunogenicity and complement activation, as well as segregation in endocytotic compartments, will limit in vivo applications. Methods for the covalent attachment of ONs to alternative cationic polypeptides such as polyarginine (Wei et al., 1994) or polyornithine (Zhu et al., 1993) have been described, but the biological activity of these conjugates in intact cells has not been reported. More complex structures known as L-oligomers have recently been proposed as drug carriers. They consist of an oligolysine scaffold branched with basic amino acid sequences consisting of a lysine pentapeptide extended by a nuclear localization signal. In contrast to PLL, these L-oligomers were efficiently translocated to cell nuclei apparently through an active process (Sheldon et al., 1995). Their use for the transport of nucleic acids material has not been reported, however. 5.5 Conjugation to Fusogenic Peptides Allowing Membrane Fusion or Membrane Translocation As mentioned in section 5.2, most of the peptide sequences described here are short native sequences belonging to various proteins showing translocating, fusogenic or intracellular trafficking properties. Some of these sequences have

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been further studied to point out the determinants that could be involved in the observed translocating process and used for internalizing different molecular entities as reviewed below. 5.5.1 Influenza Virus Haemagglutinin Fusogenic Peptide The fusogenic properties of the influenza virus haemagglutinin protein have been extensively studied. They are currently assigned to a pH-dependent conformational change of the viral haemagglutinin occurring in the endosomal compartment leading first to the exposure of its hydrophobic N-terminal region, and second to the fusion of the viral and endosomal membranes (Plank et al., 1994). A short peptide (24 amino acids) corresponding to the fusogenic region of this protein retains the conformational change at acidic pH, and therefore its membrane-destabilizing potential. A HIV-1 Tat m-RNA specific antisense ON that has been covalently bound to this fusogenic peptide by a disulphide bridge has an increased antiviral activity in vitro, probably as a result of improved intracellular release (Bongartz et al., 1994). The increment of activity remained low, however, and the majority of the conjugated ONs remained segregated in endocytic vesicles (Bongartz, Milhaud and Lebleu, unpublished observations). The topological organization of the haemagglutinin proteins at the viral membrane surface is critical for fusion and is not maintained in these conjugates. Interestingly, such peptides increased the delivery of plasmid DNA complexed with transferrin-PLL conjugates (Wagner et al., 1992) and were found to be much more efficient when used as dimers. 5.5.2 HIV-1 gp41 Fusogenic Peptide A chimeric 27 residues peptide combining the HIV gp41 fusion sequence and the SV40 T-antigen nuclear localization sequence was used for the intracellular delivery of antisense ON and for the transfection of mRNA (Morris et al., 1997; Vidal et al., 1997; Chaloin et al., 1998). The HIV gp41 peptide was also shown to facilitate the intracellular delivery of covalently linked antisense ON (Soukchareun et al., 1995). 5.5.3 Antennapaedia Peptide Studies of the biological role of the Drosophila Antennapaedia protein led to the serendipitous observation of its capacity to be internalized in nervous cells. A 16 amino acid long peptide derived from the Antennapaedia homeodomain third helix was still able to translocate through the plasma membrane and to reach the nucleus. Extensive structure-relationship studies have been performed in order to

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define the motifs responsible for cell uptake. Such studies have excluded the involvment of an amphipathic α-helix as initially proposed by reference to the structure of several viral fusogenic peptides. The translocating activity does not involve a chiral receptor, as the uptake of a retro-inverso form of the Antennapaedia peptide was fully maintained (Derossi et al., 1996; Brugidou et al., 1995). Interestingly, the peptide was also internalized at 4°C, a temperature at which endocytosis does not occur. The formation of inverted micellar structures at the membrane level after electrostatic interactions of negatively charged phospholipids and the positively charged amino acids of the peptide has been proposed as a mechanism for internalization (Derossi et al., 1996). The tryptophan residue at position 48 might play a crucial role in the destabilization of these structures, thus leading to the release of the peptide at either side of the membrane. The intranuclear accumulation of the peptide probably involves its passive diffusion (as the phenomenon was also described for incubation at 4°C), followed by as yet unexplained concentration in this compartment. The Antennapaedia peptide has been extensively used as a vector for ONs’ and for peptides’ cellular delivery, and is commercially available under the trade name of Penetratin® (Appligen). Up to now, over 20 different ONs and peptides have been coupled and vectorized by the complete Antennapaedia homeodomain or by its shorter version corresponding to the third helix. The size of the vectorized molecule is a limiting factor for the uptake, as peptides over 100 amino acids cannot be carried across the plasma membrane by this strategy (Prochiantz, 1996). This can probably be explained by a steric hindrance which could prevent access to the plasma membrane and impair inverted micelles formation. Exogenous antigens with length in the range of 30–40 amino acids were taken up efficiently when coupled to the Antennapaedia peptide (Schutze et al., 1996). Concerning nucleic acids delivery, most of the experiments were dedicated to an antisense effect and the ON size mainly ranged between 15- and 25-mer. Plasmid DNA was not internalized by the Antennapaedia peptide (Prochiantz, 1996). The conjugate exerted an antiviral activity against vesicular stomatitis virus when added in the cell culture at doses as low as 100 nM. Likewise, an antisense ON directed against the amyloid precursor protein (APP) was coupled to the Antennapaedia peptide and inhibited neurite outgrowth at extracellular concentration ranging between 40 and 200 nM (Allinquant et al., 1995). The conjugation of an antisense PNA complementary to galanin-receptor 1 mRNA with penetratin allowed PNA internalization and reduced selectively GalRl expression in melanoma cell cultures and, excitingly, in vivo after intrathecal administration (Pooga et al., 1998) (see also Chapter 4). As mentioned above, structure-activity relationship studies of the translocating activity of the Antennapaedia peptide have led to the synthesis of several analogues. One of them contained only arginine and tryptophan residues and exhibited a stronger propensity to translocate through the plasma membrane. Tryptophan residues are known to be a key element in the transmembrane

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domain of proteins, as they are in most cases located at the interface between the lipidic and the hydrophilic medium. Additional studies based on artificial membrane models could lead to the discovery of new peptide sequences or peptide mimetics showing an increased ability to translocate as well as a higher metabolic stability. 5.5.4 Tat Basic Domain The purified HIV-1 Tat protein is able to translocate efficiently through the plasma membrane and to reach the nucleus, as attested by its ability to transactivate the viral genome (Frankel and Pabo, 1988). This translocation activity has been assigned to a region of the Tat protein centred on a cluster of basic amino acids. Indeed, the chemical coupling of a few Tat-derived peptides extending from residues 37 to 72 (Tat 37–72) to large proteins such as horseradish peroxidase allowed their functional internalization into several cell lines or tissues (Fawell et al., 1994). Tat-37–72 linked proteins were the most efficiently internalized, although all Tat peptides retaining the basic domain promoted uptake with a variable and cargo-dependent efficiency (Fawell et al., 1994). The intracellular delivery of the human papillomavirus E2 transactivator by Tat-derived peptides was also established, and biological activity was documented (Pepinsky et al., 1994). Both a genetic fusion between Tat peptide and E2 transactivator and the chemical E2 transactivator Tat-conjugate were active in inhibiting the E2 transactivation at micromolar doses. A fusion protein containing the Tat basic domain only (residues 47 to 58) was more potent than the chemically linked chimera comprising the Tat 37–72 sequence. As it is generally admitted that the ends of a protein are exposed at the protein surface, the latter results, combined with those obtained by Fawell et al. (1994), could indicate that the exposure of the basic region of the peptide at the surface of the conjugates is an important determinant for efficient delivery. A short Tat peptide (residues 49 to 57) has been used to internalize proteins into the MHC class I pathway of CD8+ T cells (Kim et al., 1997). The uptake of the proteins was observed after chemical coupling of two to three Tat peptides per molecule of protein. The expected biological response was already observed at micromolar doses. In this study, PLL-protein conjugates did not induce any biological response when used at equivalent doses. Another Tat-derived peptide sequence (residues 37 to 62) was successfully used for the intracellular delivery of anti-tumor Fab antibody fragments (Anderson et al., 1993). Taken together, these data suggested that the Tat basic cluster was responsible for its translocating activity. In order to confirm this hypothesis, we have studied the main determinants required for Tat translocation (Vivès et al., 1997a). We unambigously established that the basic cluster was both necessary and sufficient in order to cross the plasma membrane. Peptide internalization was assessed by

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direct labelling with fluorescein or by indirect immunofluorescence using a monoclonal antibody directed against the Tat basic cluster. Both approaches established that all peptides containing the basic domain were taken up by cells within less than 5 min when incubated at concentrations as low as 100 nM with various cells in culture. A peptide harbouring a deletion of three arginine residues (sequence Tat 37–53) completely lost its ability to translocate. The full translocation activity was retained in a 13 amino acids long peptide which contains a nuclear localization signal (NLS) and four additional basic amino acids (Table 5.1). We later established that activity remained significant when the three C-terminal amino acids were deleted (Vivès et al., 1997b). In addition, the cellular uptake of the Tat peptide can be assigned to the minimized sequence Tat 48–57. A sharper structure-activity relationship study has been performed to define the determinants which were important in the observed activity. One or more basic charges have been either deleted or substituted within the short basic peptide. A direct correlation between translocation efficiency and the density of positively charged amino acids was highlighted (Vivès et al., 1997b) (Table 5.1). As also observed for the peptide derived from the Antennapedia homeodomain (Derossi et al., 1994), the internalization of the Tat basic peptide does not involve an endocytic pathway. No sign of endocytic accumulation has been found even in short-term experiments, and no inhibition of the uptake was observed at 4°C. Because of that result and the similar cationic nature of Tat and Antennapaedia peptides, an internalization mechanism based on inverted micelles formation at Table 5.1 Structure-activity relationship for the translocation of Tat peptides (Vivès et al., 1997a, 1997b): greater efficacy of translocation is indicated by a greater number of V symbols. The NLS sequence [Ruben, 1989 #980] is indicated in bold face Sequence

Name

Internalization efficacy

CFITKALGISYGRKKRRQRRRPPQC C--------LGISYGRKKRRQRRRPPQC C----------------GRKKRRQRRRPPQC CFITKALGISYGRKKRR------------C GRKKRRQRR--PPQC GRKKRRQR---- PPQC GRKKRRQ-----PPQC GRKKRRQRARPPQC GRKKRRQARAPPQC GRKKRRQRRR-----C

Tat 37–60 Tat 43–60 Tat 48–60 Tat 37–53 Tat EV-1 Tat EV-2 Tat EV-3 TatRAR Tat ARA Tat EV-4

++ +++ ++++ − ++ + − ++ + ++++

the membrane level (Derossi et al., 1996) appears attractive but requires further investigation. A possible internalization of the Tat basic peptide through potocytosis was also excluded (Vivès et al., 1997a).

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A putative cytotoxicity of the Tat basic peptides was investigated for doses up to 100 µM and for incubation times up to 24 h. No significant toxicity was observed for the more potent peptide, i.e. the peptide with the full basic domain (Vivès et al., 1997a). The ability of the basic Tat peptide to promote the internalization of peptides and antisense ON was investigated in our laboratory. The efficient internalization of a non-permeant peptide was established by indirect immunofluorescence with a monoclonal antibody directed against the vectorized peptide (Vivès et al., 1997b). The cellular uptake of a rhodamin-labelled ON coupled through a disulphide bridge to the Tat carrier peptide was also demonstrated. No cellular uptake was detected when the ON or the non-permeant peptide was coupled to an inactive Tat peptide (Tat 37–53), when non-covalently bound to the translocating Tat basic peptide or when the chimeric molecule was reduced before incubation with cells (Vivès et al., 1997b and unpublished results). As shown in Figure 5.1, Tat 48–60 is able to drag antisense ON into cells. Briefly, the ON was first labelled with rhodamin on its 3′ end and then coupled to the Tat peptide through a 5′-activated sulphhydryl group as previously described (Vivès and Lebleu, 1997). When used at high concentration (50 µM), rhodamin was not taken up by cells after a 30 min incubation time at 37°C (Figure 5.1 A). At doses of 1.6 µM unconjugated rhodamin-labelled ON was very poorly taken up by cells (Figure 5.1B), but once coupled to the Tat basic peptide and incubated with cells (dose at 1 µM), the peptide—ON conjugate was rapidly detected in nuclei with a nucleolar accumulation. This translocating property was abolished when the conjugate was previously reduced by DTT treatment. Uptake of the conjugate was also efficient at 4°C (Figure 5.1E), in agreement with our previous observations showing the free peptide uptake at low temperature (Vivès et al., 1997a). Moreover, no labelling was observed after incubation of cells with 1 µM rhodamin-labelled ON coupled to the Tat 37–53 peptide, a peptide which does not translocate in cells (see Table 5.1, Figure 5.1F and Vivès et al., 1997a). In these experiments, cell uptake and intracellular distribution were monitored by fluorescence microscopy after cell fixation, but similar data were obtained in unfixed cells. 5.6 Conclusions Our increasing knowledge of the molecular determinants of polypeptides uptake and intracellular trafficking has been exploited with some success for the delivery of information-rich drugs such as antisense ON, peptides or proteins in cell culture models, as outlined in this chapter. Molecular modelling and combinatorial chemistry (see also Chapter 14) will most probably allow the definition of a new generation of shuttle peptide delivery vectors with improved pharmacological properties. Much remains to be done, however, to overcome problems which might arise in vivo. Immunogenicity could potentially cause

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 123

Figure 5.1 Tat 48–60 can internalize rhodamin-labelled antisense ON into cells when incubated for 30 min at 37°C. A: Free rhodamin (50 µM). B: Unconjugated rhodaminlabelled ON (1.6µM). C: Tat basic peptide conjugated to the rhodamin-labelled ON (1 µM). D: Same molecule as in (C) after DTT treatment. E: Uptake of the Tat-ON conjugate at 4° C. F: Incubation of cells with 1 µM rhodamin-labelled ON coupled to the Tat 37–53 peptide, a peptide which does not translocate in cells (see Table 5.1 and Vivès et al. (1997a))

problems despite the use of short peptides. The addition of targeting moieties allowing the concentration of these new drugs at the target level will certainly be preferable. The manufacturing cost of such conjugates will obviously require progress in synthetic chemistry.

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Acknowledgements Work in the authors’ laboratory has been financed by the Centre National de la Recherche sur le Cancer, the Agence Nationale pour la Recherche sur le SIDA, the Ligue Nationale Française de Lutte contre le Cancer, the Association pour la Recherche sur le Cancer, and the Fondation pour la Recherche Médicale. References ALLINQUANT, B., HANTRAYE, P., MAILLEUX, P., MOYA, K., BOUILLOT, C. and PROCHIANTZ, A., 1995, Downregulation of amyloid precursor protein inhibits neurite outgrowth in vitro, J. Cell Biol., 128, 919–927. ANDERSON, D.C., NICHOLS, E., MANGER, R., WOODLE, D., BARRY, M. and FRITZBERG, A.R., 1993, Tumor cell retention of antibody Fab fragments is enhanced by an attached HIV TAT protein-derived peptide, Biochem. Biophys. Res. Commun., 194, 876–884. ARAR, K., AUBERTIN, A.M., ROCHE, A.C., MONSIGNY, M. and MAYER, R., 1995, Synthesis and antiviral activity of peptide-oligonucleotide conjugates prepared by using N alpha-(bromoacetyl)peptides, Bioconjug. Chem., 6, 573–577. BAYARD, B., BISBAL, C. and LEBLEU, B., 1986, Activation of ribonuclease L by (2′-5′) (A)4-poly(L-lysine) conjugates in intact cells, Biochemistry, 25, 3730–3736. BELTINGER, C., SARAGOVI, H.U., SMITH, R.M., LESAUTEUR, L., SHAH, N., DEDIONISIO, L., CHRISTENSEN, L., RAIBLE, A., JARETT, L. and GEWIRTZ, A.M., 1995, Binding, uptake, and intracellular trafficking of phosphorothioatemodified oligodeoxynucleotides, J. Clin. Invest., 95, 1814–1823. BERGAN, R., HAKIM, F., SCHWARTZ, G.N., KYLE, E., CEPADA, R., SZABO, J.M., FOWLER, D., GRESS, R. and NECKERS, L., 1996, Electroporation of synthetic oligodeoxynucleotides: a novel technique for ex vivo bone marrow purging , Blood, 88, 731–741. BERTRAND, E., CASTANOTTO, D., ZHOU, C., CARBONNELLE, C., LEE, N.S., GOOD, P., CHATTERJEE, S., GRANGE, T., PICTET, R., KOHN, D., ENGELKE, D. and ROSSI, J.J., 1997, The expression cassette determines the functional activity of ribozymes in mammalian cells by controlling their intracellular localization, RNA, 3, 75–88. BONGARTZ, J.P., AUBERTIN, A.M., MILHAUD, P.G. and LEBLEU, B., 1994, Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide, Nucl. Acids Res., 22, 4681–4688. BORDIER, B., PERALA, H.M., DEGOLS, G., LEBLEU, B., LITVAK, S., SARIH, C.L. and HELENE, C., 1995, Sequence-specific inhibition of human immunodeficiency virus (HIV) reverse transcription by antisense oligonucleotides: comparative study in cellfree assays and in HIV-infected cells, Proc. Natl Acad. Sci. USA, 92, 9383–9387. BOUSSIF, O., LEZOUALC’H, F., ZANTA, M.A., MERGNY, M.D., SCHERMAN, D., DEMENEIX, B. and BEHR, J.P., 1995, A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc. Natl Acad. Sci. USA, 92, 7297–7301. BRUGIDOU, J., LEGRAND, C., MERY, J. and RABIE, A., 1995, The retro-inverso form of a homeobox-derived short peptide is rapidly internalised by cultured neurones: a

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new basis for an efficient intracellular delivery system, Biochem. Biophys. Res. Commun., 214, 685–693. CHALOIN, L., VIDAL, P., LORY, P., MERY, J., LAUTREDOU, N., DIVITA, G. and HEITZ, F., 1998, Design of carrier peptide—oligonucleotide conjugates with rapid membrane translocation and nuclear localization properties, Biochem. Biophys. Res. Commun., 243, 601–608. CHU, B.C. and ORGEL, L.E., 1988, Ligation of oligonucleotides to nucleic acids or proteins via disulfide bonds, Nucl. Acids Res., 16, 3671–3691. CITRO, G., GINOBBI, P. and SZCZYLIK, C., 1993, Receptor-mediated oligodeoxynucleotides delivery by estradiol and folic acid polylysine conjugates, Cytotechnology, S30–S34. CLARENC, J.P., LEBLEU, B. and LEONETTI, J.P., 1993, Characterization of the nuclear binding sites of oligodeoxyribonucleotides and their analogs , J. Biol. Chem., 268, 5600–5604. CONNOLLY, B.A. and RIDER, P., 1985, Chemical synthesis of oligonucleotides containing a free sulphydryl group and subsequent attachment of thiol specific probes, Nucl. Acids Res., 13, 4485–4502. COTTEN, M., WAGNER, E., ZATLOUKAL, K., PHILLIPS, S., CURIEL, D.T. and BIRNSTIEL, M.L., 1992, High-efficiency receptor-mediated delivery of small and large (48 kilobase gene constructs using the endosome-disruption activity of defective or chemically inactivated adenovirus particles, Proc. Natl Acad. Sci. USA, 89, 6094–6098. DEGOLS, G., DEVAUX, C. and LEBLEU, B., 1994, Oligonucleotide-poly(L-lysine)— heparin complexes: potent sequence-specific inhibitors of HIV-1 infection, Bioconjug. Chem., 5, 8–13. DEGOLS, G., LEONETTI, J.P., BENKIRANE, M., DEVAUX, C. and LEBLEU, B. 1992, Poly-(L-lysine)—conjugated oligonucleotides promote sequence-specific inhibition of acute HIV-1 infection, Antisense Res. Dev., 2, 293–301. DEROSSI, D., CALVET, S., TREMBLEAU, A., BRUNISSEN, A., CHASSAING, G. and PROCHIANTZ, A., 1996, Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent, J. Biol. Chem., 271, 18188–18193. DEROSSI, D., CHASSAING, G. and PROCHIANTZ, A., 1998, Trojan peptides: the penetratin system for intracellular delivery, Trends Cell Biol., 8, 84–87. DEROSSI, D., JOLIOT, A.H., CHASSAING, G. and PROCHIANTZ, A., 1994, The third helix of the Antennapedia homeodomain translocates through biological membranes, J. Biol. Chem.. 269. 10444–10450. EDE, N.J., TREGEAR, G.W. and HARALAMBIDIS, J., 1994, Routine preparation of thiol oligonucleotides: application to the synthesis of oligonucleotide-peptide hybrids, Bioconjug. Chem., 5, 373–378. ERITJA, R., PONS, A., ESCARCELLER, M., GIRALT, E. and ALBERICIO, F., 1991, Synthesis of defined peptide-oligonucleotide hybrids containing a nuclear transport signal sequence, Tetrahedron, 47, 4113–4120. FAWELL, S., SEERY, J., DAIKH, Y., MOORE, C., CHEN, L.L., PEPINSKY, B. and BARSOUM, J., 1994, Tat-mediated delivery of heterologous proteins into cells, Proc. Natl Acad. Sci. USA, 91, 664–668.

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FEENER, E.P., SHEN, W.C. and RYSER, H.J., 1990, Cleavage of disulfide bonds in endocytosed macromolecules. A processing not associated with lysosomes or endosomes, J. Biol. Chem., 265, 18780–18785. FRANKEL, A.D. and PABO, C.O., 1988, Cellular uptake of the Tat protein from human immunodeficiency virus, Cell, 55, 1189–1193. GESELOWITZ, D.A. and NECKERS, L.M., 1992, Analysis of oligonucleotide binding, internalization, and intracellular trafficking utilizing a novel radiolabeled crosslinker, Antisense Res. Dev., 2, 17–25. GEWIRTZ, A.M., SOKOL, D.L. and RATAJCZAK, M.Z., 1998, Nucleic acid therapeutics: state of the art and future prospects, Blood, 92, 712–736. GILES, R.V., SPILLER, D.G. and TIDD, D.M., 1993, Chimeric oligodeoxynucleotide analogues: enhanced cell uptake of structures which direct ribonuclease H with high specificity, Anticancer Drug Des., 8, 33–51. GUPTA, K.C., SHARMA, P., KUMAR, P. and SATHYANARAYANA, S., 1991, A general method for the synthesis of 3′-sulfhydryl and phosphate group containing oligonucleotides, Nucl. Acids Res., 19, 3019–3025. HANGELand, J.J., LEVIS, J.T., LEE, Y.C. and TS’O, P.O., 1995, Cell-type specific and ligand specific enhancement of cellular uptake of oligodeoxynucleoside methylphosphonates covalently linked with a neoglycopeptide, YEE(ah-GalNAc)3, Bioconjug. Chem., 6, 695–701. HARALAMBIDIS, J., DUNCAN, L., ANGUS, K. and TREGEAR, G.W., 1990, The synthesis of polyamide—oligonucleotide conjugate molecules, Nucl. Acids Res., 18, 493–499. IYER, M., NORTON, J.C. and COREY, D.R., 1995, Accelerated hybridization of oligonucleotides to duplex DNA, J. Biol. Chem., 270, 14712–14717. KIM, D.T., MITCHELL, D.J., BROCKSTEDT, D.G., FONG, L., NOLAN, G.P., FATHMAN, C.G., ENGLEMAN, E.G. and ROTHBARD, J.B., 1997, Introduction of soluble proteins into the MHC class I pathway by conjugation to an HIV tat peptide, J. Immunol., 159, 1666–1668. LEBLEU, B., BASTIDE, L., BISBAL, C., BONGATZ, J.-P., DEGOLS, G., LEONETTI, J.-P., MILHAUD, P., ROBBINS, I., and VIVÈS, E., 1996, Poly (L-lysine) mediated delivery of nucleic acids, Targeting of Drugs 5: Strategies for Oligonucleotide and Gene Delivery in Therapy, G.A.McCormack (ed.), pp. 115–123, New-York: Plenum Press. LEBLEU, B., ROBBINS, L, BASTIDE, L., VIVÈS, E. and GEE, J.E., 1997, Pharmacokinetics of oligonucleotides in cell culture, Ciba Found. Symp., 209, 47–54; discussion 54–59. LEONETTI, J.P., DEGOLS, G. and LEBLEU, B., 1990a, Biological activity of oligonucleotide—poly(L-lysine) conjugates: mechanism of cell uptake, Bioconjug. Chem., 1, 149–153. LEONETTI, J.P., MACHY, P., DEGOLS, G., LEBLEU, B. and LESERMAN, L., 1990b, Antibody—targeted liposomes containing oligodeoxyribonucleotides complementary to viral RNA selectively inhibit viral replication, Proc. Natl Acad. Sci. USA, 87, 2448–2451. LEONETTI, J.P., MECHTI, N., DEGOLS, G., GAGNOR, C. and LEBLEU, B., 1991, Intracellular distribution of microinjected antisense oligonucleotides, Proc. Natl Acad. Sci. USA, 88, 2702–2706.

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LI, P., MEDON, P.P., SKINGLE, B.C., LANSER, J.A. and SYMONS, R.H., 1987, Enzymelinked synthetic oligonucleotide probes: non-radioactive detection of enterotoxigenic Escherichia coli in faecal specimens, Nucl. Acids Res., 15, 5275–5287. MIDOUX, P., MENDES, C., LEGRAND, A., RAIMOND, J., MAYER, R., MONSIGNY, M. and ROCHE, A.C., 1993, Specific gene transfer mediated by lactosylated poly-Llysine into hepatoma cells, Nucl. Acids Res., 21, 871–878. MORRIS, M.C., VIDAL, P., CHALOIN, L., HEITZ, F. and DIVITA, G., 1997, A new peptide vector for efficient delivery of oligonucleotides into mammalian cells, Nucl. Acids Res., 25, 2730–2736. NAKAZONO, K., ITO, Y., WU, C.H. and WU, G.Y., 1996, Inhibition of hepatitis B virus replication by targeted pretreatment of complexed antisense DNA in vitro, Hepatology, 23, 1297–1303. PEPINSKY, R.B., ANDROPHY, E.J., CORINA, K., BROWN, R. and BARSOUM, J., 1994, Specific inhibition of a human papillomavirus E2 trans-activator by intracellular delivery of its represser, DNA Cell Biol., 13, 1011–1019. PEYROTTES, S., MESTRE, B., BURLINA, F. and GAIT, M.J., 1998, Studies towards the synthesis of peptide-oligonucleotide conjugates, Tetrahedron, 54, 12513–12522. PLANK, C., OBERHAUSER, B., MECHTLER, K., KOCH, C. and WAGNER, E., 1994, The influence of endosome-disruptive peptides on gene transfer using synthetic viruslike gene transfer systems, J. Biol. Chem., 269, 12918–12924. POOGA, M., SOOMETS, U., HALLBRINK, M., VALKNA, A., SAAR, K., REZAEI, K., KAHL, U., HAO, J.X., XU, X.J., WIESENFELD, H.Z., HOKFELT, T., BARTFAI, T. and LANGEL, U., 1998, Cell penetrating PNA constructs regulate galanin receptor levels and modify pain transmission in vivo, Nat. Biotechnol., 16, 857–861. PROCHIANTZ, A., 1996, Getting hydrophilic compounds into cells: lessons from homeopeptides, Curr. Opin. Neurobiol., 6, 629–634. RUBEN, S., PERKINS, A., PURCELL, R., JOUNG, K., SIA, R., BURGHOFF, R., HASELTINE, W.A. and ROSEN, C.A., 1989, Structural and functional characterization of human immunodeficiency virus tat protein, J. Virol., 63, 1–8. SCHUTZE, R.M., GOURNIER, H., GARCIA, P.F., MOUSSA, M., JOLIOT, A.H., VOLOVITCH, M., PROCHIANTZ, A. and LEMONNIER, F.A., 1996, Introduction of exogenous antigens into the MHC class I processing and presentation pathway by Drosophila antennapedia homeodomain primes cytotoxic T cells in vivo, J. Immunol., 157, 650–655. SHELDON, K., LIU, D., FERGUSON, J. and GARIEPY, J., 1995, Loligomers: design of de novo peptide-based intracellular vehicles, Proc. Natl Acad. Sci. USA, 92, 2056–2060. SINHA, N.D. and COOK, R.M., 1988, The preparation and application of functionalised synthetic oligonucleotides: III. Use of H-phosphonate derivatives of protected aminohexanol and mercapto-propanol or -hexanol, Nucl. Acids Res., 16, 2659–2669. SOUKCHAREUN, S., TREGEAR, G.W. and HARALAMBIDIS, J., 1995, Preparation and characterization of antisense oligonucleotide-peptide hybrids containing viral fusion peptides, Bioconjug. Chem., 6, 43–53. TRUFFERT, J.C., LORTHIOIR, O., ASSELINE, U., THUONG, N.T. and BRACK, A., 1994, Online solid phase synthesis of oligonucleotide—peptide hybrids using silica support, Tetrahedron Lett.. 35. 2353–2356.

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TUNG, C.H., RUDOLPH, M.J. and STEIN, S., 1991, Preparation of oligonucleotide— peptide conjugates, Bioconjug. Chem., 2, 464–465. VIDAL, P., MORRIS, M.C., CHALOIN, L., HEITZ, F. and DIVITA, G., 1997, New strategy for RNA vectorization in mammalian cells. Use of a peptide vector, C.R. Acad. Sci. III, 320, 279–287. VIVÈS, E., BRODIN, P. and LEBLEU, B., 1997a, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus , J. Biol Chem., 272, 16010–16017. VIVÈS, E., GRANIER, C., PREVOT, P. and LEBLEU, B., 1997b, Structure activity relationship study of the plasma membrane translocating potential of a short peptide from HIV-1 Tat protein, Lett. Peptide Sci., 4, 429–436. VIVÈS, E. and LEBLEU, B., 1997, Selective coupling of a highly basic peptide to an oligonucleotide, Tetrahedron Lett., 38, 1183–1186. WAGNER, E., PLANK, C., ZATLOUKAL, K., GOTTEN, M. and BIRNSTIEL, M.L., 1992, Influenza virus hemagglutinin HA-2 N-terminal fusogenic peptides augment gene transfer by transferrin—polylysine–DNA complexes: toward a synthetic viruslike gene-transfer vehicle, Proc. Natl Acad. Sci. USA, 89, 7934–7938. WEI, Z., TUNG, C.H., ZHU, T. and STEIN, S., 1994, Synthesis of oligoarginine— oligonucleotide conjugates and oligoarginine-bridged oligonucleotide pairs, Bioconjug. Chem., 5, 468–474. WITTUNG, P., KAJANUS, J., EDWARDS, K., HAAIMA, G., NIELSEN, P.E., NORDEN, B. and MALMSTROM, B.G., 1995, Phospholipid membrane permeability of peptide nucleic acid [corrected and republished with original paging, article originally printed in FEBS Lett. 1995 May 22; 365(1): 27–9], FEBS Lett., 375, 27–29. YAKUBOV, L.A., DEEVA, E.A., ZARYTOVA, V.F., IVANOVA, E.M., RYTE, A.S., YURCHENKO, L.V. and VLASSOV, V.V., 1989, Mechanism of oligonucleotide uptake by cells: involvement of specific receptors? Proc. Natl Acad. Sci. USA, 86, 6454–6458. ZELPHATI, O. and SZOKA, F.J., 1996, Mechanism of oligonucleotide release from cationic liposomes, Proc. Natl Acad. Sci. USA, 93, 11493–11498. ZHU, T., WEI, Z., TUNG, C.H., DICKERHOF, W.A., BRESLAUER, K.J., GEORGOPOULOS, D.E., LEIBOWITZ, M.J. and STEIN, S., 1993, Oligonucleotide-poly-L-ornithine conjugates: binding to complementary DNA and RNA, Antisense Res. Dev., 3, 265–275. ZUCKERMAN, R., COREY, D. and SCHULTZ, P., 1987, Efficient method for attachment of thiol specific probes to the 3′-ends of synthetic oligodeoxyribonucleotides, Nucl. Acids Res.. 15. 5305–5321.

6 Polymeric Nanoparticles and Microparticles as Carriers for Antisense Oligonucleotides E.FATTAL AND P.COUVREUR

6.1 Introduction Antisense oligonucleotides (ONs) displaying base sequences complementary to a specific RNA are able to modulate selectively the expression of an individual gene (Helene and Toulme, 1990; Zon, 1988). Due to this mechanism of action, ONs were proposed for the treatment of several diseases such as viral infections or cancers resulting from oncogenes’ activation (Cohen, 1991; Cooke, 1992; Milligan et al., 1993; Stein and Cheng, 1993). However, crucial problems such as the poor stability of ONs versus nuclease activity in vitro and in vivo and their low intracellular penetration have not yet been solved (Loke et al., 1989; Yakubov et al., 1989). To overcome these obstacles, different delivery approaches were proposed: electropermeabilization, microinjection, chemical modifications, and particulate delivery systems. Chemical modifications of the natural phosphodiester ONs’ backbone (see also Chapters 2 and 4) have been performed with success inducing the protection of ONs against enzymatic degradation and improving their cellular uptake (see also Chapter 9). However, in some cases, especially with the phosphorothioates’ derivatives, non-specific effects were observed (Stein, 1996). Techniques such as electropermeabilization (Mir et al., 1988) and microinjection (Dash et al., 1987) were also proposed to increase oligonucleotide penetration into cells. Both methods are supposed to introduce ONs directly into cell cytoplasm, skipping the passage across intact cell membrane. However, these methods cannot be easily applied in therapies for humans. Use of particulate carriers such as liposomes (see Chapter 7) or nanoparticles may be a more realistic approach to deliver ONs, because colloidal carriers are able to protect natural unmodified phosphodiester ONs against degradation and, since they are taken up by endocytosis, they can increase cell penetration of the ONs. Among drug carriers, nanoparticles, biodegradable or not, have shown interesting potentialities to bind and deliver ONs (Fattal et al., 1998). Indeed, nanoparticles were able to protect ONs against degradation in vitro and in vivo and also to enhance significantly their pharmacological activity in cell

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culture conditions, but also after in vivo administration (Fattal et al., 1998). More recently, slow delivery systems were proposed for ONs administration, including biodegradable microparticles (Lewis et al., 1998). These particles can be implanted very close to the target site, thus releasing the oligonucleotide in situ. This chapter will thus focus on the use of polymeric drug carriers for the delivery of ONs, the liposomal systems being reviewed in Chapter 7. 6.2 Nanoparticles 6.2.1 Rationale of Using Nanoparticles for the Delivery of ONs Nanoparticles are defined as being submicrometre (<1 µM) colloidal systems generally made of polymers (biodegradable or not). According to the process used for the preparation of nanoparticles, nanospheres or nanocapsules can be obtained. Nanocapsules are vesicular systems in which the drug is confined to a cavity (an oily or aqueous core) surrounded by a unique polymeric membrane; nanospheres are matrix systems in which the drug is dispersed throughout the particles. Nanoparticles were first developed in the mid-1970s by Birrenbach and Speiser (1976). Later, their application for the design of drug delivery systems was made available by the use of biodegradable polymers that were considered to be highly suitable for human applications (Couvreur, et al., 1979). At that time, the research on colloidal carriers was mainly focusing on liposomes, but noone was able to produce stable lipid vesicles suitable for clinical applications. In some cases, nanoparticles have been shown to be more active than liposomes due to their better stability (Fattal, et al., 1991). This is why many drugs were associated with nanoparticles in recent decades (e.g. antibiotics, antiviral and antiparasitic drugs, cytostatics, protein and peptides). The main interest of nanoparticles is their ability to achieve tissue targeting and enhance the intracellular penetration of drugs. Indeed, nanoparticles are mainly taken up by cells of the mononuclear phagocyte system (MPS) in the liver, the spleen, lungs and bone marrow (Lenaerts et al., 1984). The uptake occurs through an endocytosis process, after which the particles end up in the lysosomal compartment (Lenaerts et al., 1984) where they are degraded, producing low molecular weight soluble compounds that are eliminated from the body by renal excretion (Lenaerts et al., 1984). As a result of the MPS sitespecific targeting, avoidance of some organs was made possible, thus reducing the side-effects and toxicity of some active compounds. Due to their strong lysosomal localization, one could imagine that nanoparticles are not suitable to target ONs to the cytoplasm. To avoid their trapping within the lysosomal compartement, several compounds able to destabilize the lysosomal membrane were added to the nanoparticulate systems, e.g. cationic surfactant or cationic

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Figure 6.1 Mechanism of internalization and hypothesized intracellular release of ONs associated with biodegradable nanoparticles

hydrophobic peptides) (Chavany et al., 1992, 1994; Emile et al., 1996) (Figure 6.1) (see also Chapter 5). In one study, modified ONs with strong diffusional properties were associated to the nanoparticles (Godard et al., 1995) (Figure 6.1). Therefore, nanoparticles can deliver to the cells of the MPS ONs, which are potentially active against intramacrophagic viral infections. Recently, in order to avoid MPS uptake, several groups have developed a strategy consisting of the linkage to the nanoparticles of polyethylene glycol derivatives (Gref et al., 1994, 1997; Peracchia et al., 1997a, 1997b; Bazile et al., 1995). This results in a lower uptake of nanoparticles by the MPS and in a longer circulation time (Gref et al., 1994; Bazile et al., 1995). As a consequence, these so-called Stealth® nanoparticles would be able to extravasate across endothelium that becomes permeable due to the presence of solid tumors (Papahadjopoulos et al., 1991; Gabizon and Papahadjopoulos, 1988) or inflamed tissues (BakkerWoudenberg et al., 1993). However, this technology has not been applied so far for the delivery of ONs. Several methods have been developed for preparing nanoparticles. They can be classified into two main categories according to whether the formation of nanoparticles requires a polymerization reaction, or whether it is achieved directly from a macromolecule or a preformed polymer.

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Figure 6.2 Examples of preparation methods of nanoparticles

6.2.2 Preparation of Nanoparticles by Polymerization of a Monomer Nanoparticle preparation methods based on the polymerization of monomers generally consist of introducing a monomer into an aqueous phase or dissolving the monomer in a non-solvent of the polymer. The polymerization reactions in these systems take place in two phases: a nucleation phase followed by a growth phase. Couvreur et al. (1979) developed nanospheres consisting of poly (alkylcyanoacrylates) (PACAs). These polymers, which have been used for several years as surgical glues, are bioerodible (Lenaerts et al., 1984) which is the most significant advantage of alkylcyanoacrylates over other acrylic derivatives previously used. In contrast to other acrylic derivatives, which require an energy input for the polymerization, alkylcyanoacrylates can be polymerized easily without such a contribution, which is another advantage regarding the stability of the associated drug. These nanoparticles are prepared by emulsion polymerization of cyanoacrylic monomers dispersed in an acidic

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aqueous phase (Figure 6.2). The size of the nanoparticles obtained is approximately 200 nm, but it can be reduced to 30–40 nm using a non-ionic surfactant in the polymerization medium (Seijo et al., 1990) or by adding SO2 to the monomer (Lenaerts et al., 1989). Freeze fracture studies have revealed that the internal structure of these cyanoacrylic nanospheres consisted largely of a matrix made up of a dense polymeric network (Rollot et al., 1986). Molecular weight determinations made by gel permeation chromatography suggested that nanospheres are built up from an entanglement of numerous small oligomeric subunits rather than from the rolling-up of one or a few long polymer chains (Van Snick et al., 1985). The anionic emulsion polymerization has been turned to good account for preparing nanoparticles of poly(dialkylmethilidene malonate), a biomaterial displaying great potential as an alternative drug delivery system (De Keyser et al., 1991; Mbela et al., 1993). The method for preparing biodegradable PACA nanospheres has also provided the basis for the development of PACA nanocapsules made of an internal oily core. Thus, in 1986, Al Khouri-Fallouh et al. proposed an original method in which the monomer is solubilized in an alcohol phase containing an oil and is then dispersed in an aqueous phase containing surfactants. In contact with water, the alcohol phase is dispersed and favours the formation of a very fine oil-inwater emulsion. The monomer, insoluble in water, polymerizes at the interface of the phases to form the wall of the nanocapsules. This process, which is simple to apply, was designed to allow the encapsulation of larger quantities of lipophilic active compounds. Although non-biodegradable, cationic polystyrene nanoparticles were proposed for the adsorption of ONs. ONs that are bound to such solid support can be useful as hybridization probes and affinity matrices for binding specifically to complementary target DNA or RNA sequences. These systems might be used in clinical diagnostics for detecting pathogenic microorganisms. For this purpose, copolymerization in batch conditions of styrene in the presence of two aminocontaining monomers, aminoethyl methacrylate hydrochloride (AEMH) and vinylbenzylamine hydrochloride (VBAH), using 2, 2′-azobis(2amidino-propane) dihydrichloride as cationic initiator was achieved (Ganachaud et al., 1997). In that study, it was shown that the two monomers affected similarly the kinetics of emulsion polymerization of styrene: the overall polymerization rate and particle number increased dramatically on increasing the functional monomer concentration, and the molecular weight of polymer samples decreased with the functional monomer concentration, revealing the strong activity of VBAH and AEMH in the chain transfer. The colloidal and surface properties of both copolymers were investigated and shown to induce rather similar properties (Sauzedde et al., 1997). It was found that the final particle size decreased with increasing functional monomer concentration. In addition, on both types of latexes increasing the functional monomer concentration caused the surface amino groups’ density to increase from 0 to a plateau value at 8.2 mC/cm2 (Sauzedde et al., 1997). Furthermore, Ganachaud et al. (1995) have compared

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the synthesis of these polystyrene latex particles by different polymerization methods: either by seed particle functionalization or by the shot-growth procedure using 2,2′-azobis(2-amidinopropane) dihydrichloride as an initiator and VBAH as a cationic monomer. The seed particle functionalization is based on the functionalization of preformed particles. The seeding materials (styrene and VBAH) were added according to a batch process (one shot) or in a semicontinuous manner. The shot growth approach requires as a first step the synthesis of particles with low amounts of functional monomer in order to set the particle size. The remaining amounts of styrene and VBAH are added in a second step at about 80% conversion, which is high enough to avoid a secondary nucleation process (Ganachaud et al., 1995). It was found that functionalizing seed particles gave poor functionalization yields, whereas the shot growth method allows one to synthesize spherical monodispersion particles with differing charge densities and fairly good yields (Ganachaud et al., 1995). In this process, ‘core-shell’-like structures were formed, the shell containing most of the functional monomer added in the second process step. Most amine and amidine groups resulting from the first step were partially buried (Ganachaud et al., 1998). 6.2.3 Nanoparticles Obtained from Preformed Polymers With the exception of alkylcyanoacrylates and dialkylmethylidene malonate, most of the monomers suitable for a micellar polymerization process in an aqueous phase lead to slowly biodegradable or non-biodegradable polymers. In addition, residual molecules in the polymerization medium (monomer, oligomer, surfactant, etc.) can be more or less toxic, which necessitates tedious purification of the colloidal material. In order to avoid those limitations, methods have been proposed using preformed polymers instead of monomers. Two important methodologies have been proposed for the preparation of nanoparticles from synthetic polymers. The first is based on the emulsification of water-non-miscible organic solutions of preformed polymers in aqueous phases containing surfactants, followed by the removal of solvents under reduced pressure (Figure 6.2). This method, which relates to the methods for preparing pseudolatex or artificial latex developed by Vanderhoff et al. (1979) in polymer chemistry, has been applied to polylactic acid (Vanderhoff et al., 1979; Krause et al., 1986; Tice and Gilley, 1985). The main advantage is that polylactic polymers are generally considered as biocompatible and well tolerated. Poly(βhydroxybutyrate) is also a very promising biodegradable polymer which has been used as material for producing nanoparticles by solvent evaporation (Koosha et al., 1987, 1989). Using this polymer, it was possible by high-pressure emulsification to reduce the particle sizes to 170nm. Biodegradable nanospheres of polylactic acid were also prepared by an emulsion, microfluidization and solvent evaporation method using human serum albumin as emulsifying agent

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(Verrecchia et al., 1993). The molecular conformation of albumin at the surface of the polymer has been investigated, and the matrix structure of the system evidenced by freeze-fracture (Verrecchia et al., 1993). Another interesting technology, applicable to a wide range of polymers, is based on the selection of salts producing the salting-out of acetone from water (Ibrahim et al., 1992; Allémann et al., 1992). In fact, the saturated aqueous solution prevents acetone from mixing with water. After the preparation of an oil-in-water emulsion, water is added in a sufficient amount to allow complete diffusion of acetone into the aqueous phase, inducing the formation of nanospheres. The main advantage is that this process does not require an increase in temperature and therefore may be useful when heat-sensitive substances have to be processed (Ibrahim et al., 1992). This method has allowed polylactic, polymethylmetacrylate and ethylcellulose nanospheres to be prepared (Allémann et al., 1992). The second principle for obtaining nanospheres from synthetic polymers has been proposed by Fessi et al. (1986). It is based on the precipitation of a polymer in solution following the addition of a non-solvent of the polymer (Figure 6.2). This method thus allows the formation of nanospheres without prior emulsification. Of course, the choice of the polymer/solvent/non-solvent system is extremely important, since it governs the production of nanoparticles (Fessi et al., 1986). This is because the solvent and the non-solvent of the polymer must be mutually miscible. The progressive addition of the polymer solution to the non-solvent generally leads to the formation of nanospheres close to 200 nm in size. The nanoparticles appear to be formed, in this technique, by a mechanism comparable to the ‘diffusion and standing’ process found in spontaneous emulsification. This phenomenon, beyond the scope of this chapter, has been explained by local variations of the interfacial tension between the two nonmiscible liquids due to the reciprocal diffusion of the third liquid. This method has been successfully applied to various polymers such as polylactide and polylactide-co-glycolide, polyεcaprolactone, ethylcellulose, polyalkylcyanoacrylate and polystyrene. Nanocapsules of those polymers may also be obtained by a very similar procedure (Fessi et al., 1989). It differs from the nanosphere preparation method by the introduction of a fourth component of an oily nature miscible with the polymer solvent but non-miscible with the polymer solvent/non-solvent mixture. The polymer is deposited at the interface between the oily finely dispersed droplets and the water phase, thus forming nanoparticles with a shell-like wall and a size around 230 nm. Finally, the possibility of preparing nanocapsules with an aqueous core was recently shown by Blanco and Alonso (1998). These authors demonstrated that using a water-in-oil-in-water emulsion solvent evaporation method it was possible to obtain such products with a high entrapment efficiency for watersoluble compounds (Blanco and Alonso, 1998).

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Figure 6.3 Different possibilities of association of ONs with polymeric nanoparticles (nanospheres or nanocapsules)

6.2.4 Association of ONs to Nanoparticles Due to their hydrophilic and polyanionic character, ONs interact poorly with polymeric materials. Therefore, the association and/or encapsulation of ONs in nanoparticles is a challenge which is particularly difficult to achieve. Two main strategies can be considered for a successful entrapment: 1 ONs may be covalently linked to a hydrophobic molecule, allowing hydrophobic interaction with the polymer (Figure 6.3a) or the solubilization into the oily core of nanocapsules (Figure 6.3b)

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2 cationic polymers may be used to coat the particles (Figure 6.3c) or to confer a positive charge to the oily core of nanocapsules (Figure 6.3d). These cations are used to obtain successful interactions with the negatively charged ON molecules. Initially, ONs were associated to polyalkycyanoacrylate (PACA) (polyisobutylcyanoacrylate (PIBCA) or polyisohexylcyanoacrylate (PIHCA)). This association was found to occur only in the presence of a hydrophobic cation such as cethyltrimethylammonium bromide (CTAB) (Chavany et al., 1992). Oligonucleotide adsorption onto nanoparticles was therefore mediated by the formation of ion pairs between the negatively charged phosphate groups of the nucleic acid chain and the positively charged compound pre-adsorbed onto nanoparticles or using a hydrophobic conjugate of ONs (Chavany et al., 1992; Godard et al., 1995). The adsorption efficiency of oligonucleotide-cation complexes on PAC A nanoparticles was found to be highly dependent on several parameters: the oligonucleotide chain length, the nature of the cyanoacrylic polymer, the hydrophobicity of the cation used as ion-pairing agent, and the ionic strength of the dispersing medium (Chavany et al., 1992). More recently, Zobel et al. (1997) have replaced CTAB by DEAE—dextran that was introduced into the polymerization medium before nanoparticles’ formation. Although the amount of DEAE—dextran covalently linked to the polymer was not precisely measured, the unloaded nanoparticles displayed a positive charge, signifying that the DEAE—dextran has been associated with the nanoparticles. The highest loading of ONs (35 µmol of ONs per gram of polymer) was achieved at pH 5.5 using a 10mM phosphate buffer. Other hydrophobic compounds such as cationic lipids were also employed as adjuvants for loading ONs to nanoparticles (Balland et al., 1996). However, using compounds such as polyamines (DOGS), the adsorption was lower than with CTAB in similar conditions of adsorption (Balland et al., 1996). ONs coupled to cholesterol were also adsorbed onto polyisohexylcyanoacrylate nanoparticles (Godard et al., 1995). However, because this conjugate was negatively charged, it was partially repulsed by the negative charges displayed by the polymer surface, leading to a low loading efficiency of ONs in the absence of CTAB. ONs were also associated to poly(lactic acid)—PEG nanoparticles (Emile et al., 1996) using two types of oligopeptides with alternating hydrophobic and hydrophilic acids and containing lysines residues with a high affinity for the ONs, due to ionic interactions. ONs interacted with the lysine residues on the peptides forming non-water-soluble complexes that were able to coprecipitate with PEG—PLA copolymers (Emile et al., 1996). ONs were associated to cationic polystyrene nanoparticles (Ganachaud et al., 1997a, 1997b; Elaissari et al., 1994, 1995; Fritz et al., 1997). Owing to the presence of surface charge on the particles and the polyelectrolyte character of the oligonucleotides, it was found in a preliminary study that electrostatic forces play a major role in the adsorption process (Elaissari et al., 1995). It was also shown

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that the kinetics of adsorption of ONs to the particles was very rapid. About 70% of the final adsorption occurred within a few minutes, after which a slow second phase occurred which could be due to the reduced diffusion of ONs to the particles’ interfaces caused by the electrostatic repulsion between free and adsorbed ONs (Elaissari et al., 1995). The influence of the pH on the adsorption was found to be very important, since it was shown that raising the pH above the pKa of the amino groups decreases the adsorption (Elaissari et al., 1994). On the contrary, the lower the pH, the more ONs were adsorbed (Elaissari et al., 1995). The higher adsorption at low pH is due to an increase of the cationic surface charge at acidic pH. This confirms that electrostatic forces play a major role in the adsorption process. Ganachaud et al. (1997) have also shown that the maximum amount of ONs adsorbed was near to the theoretical value of a closepacked monolayer of flat conformation, assuming that the ON is in a cylindrical conformation. It is important to note that this conformation is not at all favourable for further hybridization with complementary sequences, as it is required in the case of bioassays. Finally, the adsorption amount of ONs at basic pH was still non-negligible (Elaissari et al., 1995). It is also worth mentioning that adsorption occurs also in the case of negatively charged particles (Elaissari et al., 1994, 1995). It is therefore likely that the adsorption is not only governed by electrostatic interactions (Ganachaud et al., 1997). Hydrophobic or hydrogen bonding may contribute to the adsorption. Such forces could be generated between the bases of ONs and the particle surface (Elaissari et al., 1995, 1998). Finally, encapsulation methods that do not involve hydrophobic or ionic interactions of the ONs with the polymer surface are also carried out. Recently, Das et al. (1998) used the multiple emulsion (water-oil-water) solvent evaporation method to entrap ONs into nanoparticles of PLGA. However, the encapsulation efficiency was very low. 6.3 In Vitro Stability of ONs Adsorbed onto Nanoparticles When adsorbed onto PIHCA nanoparticles through binding to CTAB, ONs were efficiently protected against enzymatic degradation even after 5h incubation with phosphodiesterase or in cell culture media (Chavany et al., 1992). In addition, about 90% of the oligonucleotide still remained intact after overnight incubation with the enzyme (0.1 mg/ml). Similar results were obtained by Zobel et al. (1997) with ONs adsorbed onto DEAE containing nanoparticles incubated with DNAase. ONs-DEAE-nanoparticles complexes were found to be more stable in cell culture medium containing 10% of foetal calf serum than ONs-associated nanoparticles containing CTAB. It was assumed that adsorption of plasma protein could turn to a protective coating in DEAE containing nanoparticles preventing the action of esterases (Zobel et al., 1997). On the contrary, the lower stability of the ONs in CTAB-containing nanoparticles was explained by a

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competition between CTAB and plasma protein inducing a partial desorption of the ONs from the nanoparticles (Chavany et al., 1994). In addition, PAC A nanoparticles were shown to be bioerodible, degradation by plasma esterases starting from the surface (Lenaerts et al., 1984). Therefore the difference between the nanoparticles (containing DEAE or CTAB) can be provided from the difference of stability of the coating layer, DEAE being much more strongly attached to the nanoparticles than CTAB. The displacement of CTAB—ONs complexes by plasma proteins was confirmed in the presence of more concentrated mice plasma (70%) at 37°C. The half-lives of pdT16 incubated free or bound to the nanoparticles were short: 6.0 and 12.5 min, respectively (Nakada et al., 1996). However, 30 min after incubation in plasma, the percentage of nondegraded 33P-pdT 16 was only 2.9% for free ONs, whereas 28.9% of 33P-pdT 16 associated with nanoparticles were still intact (Nakada et al., 1996). This suggests that at least some of the phosphodiester linkages were not available for nucleases degradation. 6.4 Cell Interactions with ON Loaded Nanoparticles Cell uptake studies of a 15-mer oligothymidilate adsorbed onto PIHCA nanoparticles were performed in non-toxic conditions using U937 cells. It was shown that the uptake of the ONs was dramatically increased when associated with nanoparticles. After 24 h incubation, uptake of oligonucleotide was eight times higher when adsorbed to nanoparticles than when incubated as an ON free solution (Chavany et al., 1994), and it was markedly reduced (95%) at 4°C as compared to 37°C. These results clearly show that ONs adsorbed onto nanoparticles were internalized in U937 cells by an endocytic/phagocytic process and not simply adsorbed at the membrane surface. This has been confirmed by confocal microscopy with fluorescently labelled nanoparticles: after internalization, nanoparticles accumulate into phagosomes or lysosomes. Such an intracellular distribution profile is supposed to lead to the rapid degradation of the ONs after their release from nanoparticles. This is why the intracellular stability of internally labelled 15-mer free or adsorbed onto PIHCA nanoparticles was tested (Chavany et al., 1994): no undegraded ONs were detected in cell lysate after 1.5 h incubation with free ONs, whereas the 15-mer adsorbed onto PIHCA nanoparticles remained intact even after a 6.5 h incubation period. After 24 h, some degradation products appeared, but the fraction of intact ONs remained considerable with nanoparticles. The intracellular distribution of ONs (5′ labelled oligomer) was also measured after lysis in the presence of NonidetP40, a non-ionic detergent which protects the nuclear structure. It should be stressed that the cytoplasmic fraction contains also endocellular vesicles such as lysosomes or phagosomes. When adsorbed onto nanoparticles, intact ONs were detected in both nuclear and extranuclear fraction. The increased stability of the oligonucleotide associated to the nanoparticles which is observed in the

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extranuclear fraction could be explained by the fact that nanoparticles protect them against digestion by lysosomal enzymes. How they escape from the lysosomes remains unclear, but it is supposed that CTAB, which is a quaternary ammonium, could destabilize the lysosomal membrane after a certain period of time, thus allowing the release of the oligonucleotide into the cell cytoplasm. Escape from the lysosomes was, however, not observed on Vero cells when ONs were delivered by nanoparticles containing DEAE. Indeed, this type of nanoparticle remained localized in vesicular structure in the cytoplasm, suggesting that in this case no destabilization of the lysosomal membrane occurred, which might be a strong limitation of this type of nanoparticle for ON delivery. 6.5 In Vitro Pharmacological Activity of Oligonucleotideloaded Nanoparticles The in vitro activity of oligonucleotide-loaded PIHCA nanoparticles has been demonstrated in a few studies. Schwab et al. (1994) have shown that nanoparticleadsorbed antisense ONs directed to a point mutation (G → U) in codon 12 of the Ha-ras mRNA selectively inhibited the proliferation of cells expressing the point mutated Ha-ras gene (see also Chapter 12). With nanoparticles, the effective concentration was 100 times lower than with the free ON. A sequence-specific inhibition of the profileration of T24 human bladder carcinoma cells (containing the human Ha-ras oncogene mRNA) was evidenced using the same ON conjugated to cholesterol and loaded onto PIHCA nanoparticles (Godard et al., 1995). The efficiency was comparable to that of CTAB containing nanoparticles. More recently, Lambert et al. (1998), in an attempt to inhibit PKCα expression in Hep G2 cells, observed that PIBCA in subtoxic concentration induced nanoparticles, with depletion of PKCα. The same observation was made for lipofectin, a cationic lipid transfecting agent (Filion and Phillips, 1997). The main conclusion of this study is that the commonly used strategy of ONs targeting with cationic non-viral vectors may display nonspecific effects which can lead to artefactual results. 6.6 In Vivo Studies with Oligonucleotide Nanoparticles The pharmacokinetic studies carried out with PIBCA nanoparticles (Nakada et al., 1996) have shown that although nanoparticles did not markedly increase the blood half-life of the 33P-pdT 16, its tissue distribution was significantly modified. Indeed, when transported by PIBCA nanoparticles, 33P-pdT 16 was taken up significantly by the liver whereas a subsequent reduced distribution in the other organs was observed, especially in the kidneys. These data suggest that with the aid of nanoparticles, the oligonucleotide could be delivered to the liver

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with a certain specificity and the urinary excretion decreased. To address the crucial problem of oligonucleotide degradation, the state (degraded or not) in which the oligonucleotide was in the plasma and delivered to the liver was investigated (Nakada et al., 1996). In this view, an original assay method was proposed that consists of the use of a TLC analyser allowing, in polyacrylamide gels, the amount of undegraded pdT 16 in tissues such as liver and plasma to be quantified (Aynie et al., 1996). This method was able to distinguish between ONs differing by only one nucleotide in length. Using that methodology, it was found that a significant amount of 33P-pdT 16 was kept intact in the liver and the plasma when administered in the form of nanoparticles (Nakada et al., 1996). In one study performed in vivo, nanoparticles proved to be efficient after intratumoral administration (Schwab et al., 1994). In a nude mice model HBL100ras1 cells (expressing the point mutated Ha-ras genes) were inoculated by subcutaneous administration. These clones are able to induce tumors since the mutated ras genes are directly involved in cell proliferation and tumorigenicity. Anti-ras oligonucleotide or the sense sequence adsorbed onto PIHCA nanoparticles was injected one day before and 48 h after implantation of the tumor in the same area where cells were inoculated. Twenty-three days after cell inoculation, animals were sacrificed and the tumors were excised and weighed (Schwab et al., 1994). It was shown that nanoparticles containing the antisense markedly inhibited Ha-ras-dependent tumor in a highly specific manner. Indeed, only the antisense was able to reduce significantly tumor weight and volume, as compared to the sense sequence adsorbed onto the same nanoparticles (Schwab et al., 1994). 6.7 Microparticles Microparticles made of biodegradable polymers can be obtained through a multiple emulsion solvent evaporation process. This preparation method was widely described for the preparation of peptide and protein loading (Couvreur et al., 1997) because it is the most adequate for the encapsulation of hydrophilic compounds. Using this delivery system, Akhtar and Lewis (1997) have described the encapsulation in polylactide-co-glycolide (PLGA) microparticles of several diameter ranges of a 20-mer phosphodiester and phosphorothioate ON complementary of the tat gene of HIV. Entrapment efficiencies and in vitro release rates were found to depend on microparticle size. Smaller particles (1–2 µm) display lower entrapment efficiency and released 70% of the entrapped ONs within four days, compared with 40 days for larger microparticles (10–20 µm). On the other hand, cell association of ONs entrapped within small microparticles was improved 10-fold in murine macrophages compared with free ONs. Cell uptake was enhanced when macrophages were activated with interferon-γ and/or lipopolysaccharide (LPS) treatment, but decreased significantly in the presence of metabolic and phagocytosis inhibitors (Akhtar and Lewis, 1997). Microparticles

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containing fluorescently labelled phosphorothioate ONs were found to be able to deliver the ONs to both the cytoplasm and nucleus as visualized by fluorescence microscopy. PLGA microparticles loaded with antisense ONs against rat tenascin mRNA were tested on smooth muscle cell proliferation and migration. Smooth muscle cell proliferation studies exhibited dose-dependent growth inhibition with the antisense ON-loaded microparticles. Microparticles loaded with scrambled ONs showed less growth inhibition than antisense ONs. Moreover, only the antisense ON-loaded microparticles inhibited migration (Cleek et al., 1997). Microparticles were also suggested to be useful as implants for the long-term delivery of ONs, as described more recently by Lewis et al. (1998). Although not in the form of microparticles, PLGA polymers were also used as implants for the delivery of ONs (Yamakawa et al., 1997). In our opinion, there is a lot of opportunity to develop a new generation of controlled release systems for the local delivery of ONs. 6.8 Conclusion The results presented in this review show that the association of ONs with biodegradable nanoparticles or microparticles is possible if one is using a hydrophobic counter-ion or in the case of modified more hydrophobic ONs. These systems have been proved to be efficient not only for protecting the oligonucleotide from degradation by 3′-exonucleases, but also for increasing the intracellular capture of these molecules. The tissue distribution profile of ONs could be dramatically modified after association with nanoparticles, and organs such as the liver may be targeted. Thus, new antisense treatment of liver diseases such as viral hepatitis or liver cancers or metastasis may be considered with this type of carrier. However, the encapsulation process needs to be improved in order to challenge the encapsulation and/or entrapment within the polymeric matrix, thus avoiding a single-surface adsorption of ONs which leads to unwanted effects (burst release of ONs and partial accessibility for esterases). References AKHTAR, S. and LEWIS, K.J., 1997, Antisense oligonucleotide delivery to cultured macrophages is improved by incorporation into sustained release biodegradable polymer microspheres, Int. J. Pharm., 151, 57–67. AL KHOURI-FALLOUH, N., FESSI, H., ROBLOT-TREUPEL, L., DEVISSAGUET, J.P. and PUISIEUX, F., 1986, An original procedure for preparing nanocapsules of polyalkylcyanoacrylates for interfacial polymerization, Pharm. Acta Helv., 61, 274–281. ALLÉMANN, E., GURNY, R. and DOELKER, E., 1992, Preparation of aqueous polymeric nanodispersions by a reversible salting-out process: influence of process parameters on particle size, Int. J. Pharm., 87, 247–253.

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AYNIE, I., VAUTHIER, C., FOULQUIER, M., MALVY, C., FATTAL, E. and COUVREUR, P., 1996, Development of a quantitative polyacrylamide gel electrophoresis analysis using a multichannel radioactivity counter for the evaluation of oligonucleotidebound drug carrier, Anal. Biochem., 240, 202–209. BAKKER-WOUDENBERG, I.A.J.M., LOKERSE, A.F., TENKATE, M.T., MOUTON, J.W., WOODLE, M.C. and STORM, G., 1993, Liposomes with prolonged blood circulation and selective localization in Klebsiella-pneumoniae infected lung tissue, J. Infect. Dis., 168, 164–171. BALLAND, O., SAISON-BEHMOARAS, T. and GARESTIER, T., 1996, Nanoparticles as carriers for antisense oligonucleotides, Targeting of Drugs 5: strategies for oligonucleotide and gene delivery in therapy, G.GREGORIADIS and B.MCCORMACK (eds), pp. 131–142, New York: Plenum Press. BAZILE, D., PRUD’HOMME, C., BASSOULLET, M.T., MARLARD, M., SPENLEHAUER, G. and VEILLARD, M., 1995, Stealth Me.PEG–PLA nanoparticles avoid uptake by the mononuclear phagocytes system, J. Pharm. Sci., 84, 493–498. BIRRENBACH, G. and SPEISER, P., 1976, Polymerized micelles and their use as adjuvants in immunology, J. Pharm. Sci., 65, 1763–1766. BLANCO, D. and ALONSO, M.J., 1998, Protein encapsulation and release from poly (lactideco-glycolide) microspheres: effect of the protein and polymer properties and of the co-encapsulation of surfactants, Eur. J. Pharm. Biopharm., 45, 285–294. CHAVANY, C., LE DOAN, T., COUVREUR, P., PUISIEUX, F. and HELENE, C., 1992, Polyalkylcyanoacrylate nanoparticles as polymeric carriers for antisense oligonucleotides, Pharm. Res., 9, 441–449. CHAVANY, C., SAISON-BEHMOARAS, T., LE DOAN, T., PUISIEUX, F., COUVREUR, P. and HELENE, C., 1994, Adsorption of oligonucleotides onto polyisohexylcyanoacrylate nanoparticles protects them against nucleases and increases their cellular uptake, Pharm. Res., 11, 1370–1378. CLEEK, R.L., REGE, A.A., DENNER, L.A., ESKIN, S.G. and MIKOS, A.G., 1997, Inhibition of smooth muscle cell growth in vitro by an antisense oligodeoxynucleotide released from poly(DL-lactic-co-glycolic acid) microparticles, J. Biomed. Mater. Res., 35, 525–530. COHEN, J.S., 1991, Antisense oligodeoxynucleotides as antiviral agents, Antiviral Res., 16, 121–133. COOKE, S.T., 1992, Therapeutic applications of oligonucleotides, Annu. Rev. Pharmacol. Toxicol., 32, 329–376. COUVREUR, P., BLANCO-PRIETO, M.J., PUISIEUX, F., ROQUES, B. and FATTAL, E., 1997, Multiple emulsion technology for the design of microspheres containing peptides and oligopeptides, Adv. Drug Deliv. Rev., 28, 85–96. COUVREUR, P., KANTE, B., ROLAND, M., GUIOT, P., BAUDUIN, P. and SPEISER, P., 1979, Polycyanoacrylate nanocapsules as potential lysosomotropic carriers: preparation, morphological and sorptive properties, J. Pharm. Pharmacol., 31, 331–332. DAS, S.K., MILLER, K.J. and CHATTARAJ, S.C., 1998, Facilitated delivery of oligonucleotide as inhibitor of serotonin uptake, Proc. International Symposium on Controlled Release Bioactive Materials, Las Vegas, NV, pp. 350–351.

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DASH, P., LOTAN, L., KNAPP, M., KANDEL, E.R. and GOELET, P., 1987, Selective elimination of mRNAs in vivo complementary oligonucleotide promote RNA degradation by an RnaseH like activity, Proc. Natl Acad. Sci. USA, 84, 7896–7900. DE KEYSER, J.L., POUPAERT, J.H. and DUMONT, P., 1991, Poly(diethyl methylidenemalonate) nanoparticles as a potential drug carrier: preparation, distribution and elimination after intravenous and peroral administration to mice, J. Pharm. Sci., 80, 67–70. ELAISSARI, A., CHAUVET, J.P., HALLE, M.A., DECAVALLAS, O., PICHOT, C. and CROS, P., 1998, Effect of charge nature on the adsorption of single-stranded DNA fragments onto latex particles, J. Coll. Int. Sci., 202, 251–260. ELAISSARI, A., CROS, P., PICHOT, C., LAURENT, V. and MANDRAND, B., 1994, Adsorption of oligonucleotides onto negatively and positively charged latex particles, Colloids and Surfaces A, 83, 25–31. ELAISSARI, A., PICHOT, C., DELAIR, T., CROS, P. and KURFURST, R., 1995, Adsorption and desorption studies of polyadenylic acid onto positively charged latex particles, Langmuir, 11, 1261–1267. EMILE, C., BAZILE, D., HERMAN, F., HELÈNE, C. and VEILLARD, M., 1996, Encapsulation of oligonucleotide in Stealth Me.PEG-PLA50 nanoparticles by complexation with structured oligopeptides, Drug Del., 3, 187–195. FATTAL, E., ROJAS, J., YOUSSEF, M., COUVREUR, P. and ANDREMONT, A., 1991, Liposomeentrapped ampicillin in the treatment of experimental murine listeriosis and salmonellosis, Antimicrob. Agents Chemother., 35, 770–772. FATTAL, E., VAUTHIER, C., AYNIE, I., LAMBERT, G., MALVY, C. and COUVREUR, P., 1998, Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides, J. Control. Rel., 53, 137–143. FESSI, H., DEVISSAGUET, J.P., PUISIEUX, F. and THIES, C., 1986, Procédé de préparation des systèmes colloïdaux dispersibles d’une substance sous forme de nanoparticules, French Patent Application, 8, 618, 446. FESSI, H., PUISIEUX, F., DEVISSAGUET, J.P., AMMOURY, N. and BENITA, S., 1989, Nanocapsules formation by interfacial polymer deposition following solvent displacement, Int. J. Pharm., 55, R1–R4. FILION, M.C. and PHILLIPS, N.C., 1997, Anti-inflammatory activity of cationic lipids, Br. J. Pharmacol., 122, 551–557. FRITZ, H., MAIER, M. and BAYER, E., 1997, Cationic polystyrene nanoparticles: preparation and characterization of a model drug carrier system for antisense oligonucleotides, J. Colloids Interface Sci., 195, 272–288. GABIZON, A. and PAPAHADJOPOULOS, D., 1988, Liposome formulations with prolonged circulation time in blood and enhanced uptake by tumors, Proc. Natl Acad. Sci. USA, 85, 6949–6953. GANACHAUD, F., BOUALI, B., VERON, L., LANTERI, P., ELAISSARI, A. and PICHOT, C., 1998, Surface characterization of amine-containing latexes by charge titration and contact angle measurements, Colloids Surf., 137, 141–154. GANACHAUD, F., ELAISSARI, A., PICHOT, C., LAAYOUN, A. and CROS, P., 1997a, Adsorption of single-strand DNA fragments onto cationic aminated latex particles, Langmuir, 13, 701–707. GANACHAUD, F., MOUTERDE, G., DELAIR, TH., ELAISSARI, A. and PICHOT, C., 1995, Preparation and characterization of cationic polystyrene latex particles of different aminated surface charges, Polym. Adv. Technol., 6, 480–488.

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GANACHAUD, F., SAUZEDDE, F., ELAISSARI, A. and PICHOT, C., 1997b, Emulsifier-free emulsion copolymerization of styrene with two different aminocontaining cationic monomers. I. Kinetic studies, J. Appl. Polym. Sci., 65, 2315–2330. GODARD, G., BOUTORINE, A.S., SAISON-BEHMOARAS, E. and HELENE, C., 1995, Antisense effects of cholesterol-oligodeoxynucleotide conjugates associated with poly(alkylcyanoacrylate) nanoparticles, Eur. J. Biochem., 232, 404–410. GREF, R., MINAMITAKE, Y., PERACCHIA, M.T., DOMB, A., TRUBETSKOY, V., TORCHILIN, V. and LANGER, R., 1997, Poly(ethylene glycol)-coated nanospheres: potential carriers for intravenous drug administration, Pharm. Biotechnol., 10, 167–198. GREF, R., MINAMITAKE, Y., PERACCHIA, M.T., TRUBETSKOY, V., TORCHILIN, V. and LANGER, R., 1994, Biodegradable long-circulating polymeric nanospheres, Science, 263, 1600–1603. HELENE, C. and TOULME, J.J., 1990, Specific regulation of gene expression by antisense, sense and antigene nucleic acids, Biochim. Biophys. Acta, 1049, 99–125. IBRAHIM, H., BINDSCHAEDLER, C., DOELKER, E., BURI, P. and GURNY, R., 1992, Aqueous nanodispersions prepared by a salting-out process, Int. J. Pharm., 87, 239–246. KOOSHA, F., MULLER, R.H., DAVIS, S.S. and DAVIS, M.C., 1989, The surface chemical structure of poly(b-hydroxybutyrate) microparticles produced by solvent evaporation process, J. Control Rel., 9, 149–153. KOOSHA, F., MULLER, R.H. and WASHINGTON, C., 1987, Production of polyhydroxybutyrate (PHB) nanoparticles for drug targeting, J. Pharm. Pharmacol., 39, 136P. KRAUSE, H.J., SCHWARTZ, A. and ROHDEWALD, P., 1986, Interfacial polymerization, a useful method for the preparation of polymethylcyanoacrylate nanoparticles, Drug Dev. Ind. Pharm., 12, 527–552. LAMBERT, G., FATTAL, E., BREHIER, A., FEGER, J. and COUVREUR, P., 1998, Effect of polyisobutylcyanoacrylate nanoparticles and lipofectin loaded with oligonucleotides on cell viability and PKCa neosynthesis in HepG2 cells, Biochimie, 80, 969–976, LENAERTS, V., COUVREUR, P., CHRISTIAENS-LEYH, D., JOIRIS, E., ROLand, M., ROLLMAN, B. and SPEISER, P., 1984, Degradation of poly (isobutyl cyanoacrylate) nanoparticles, Biomaterials, 5, 65–68. LENAERTS, V., NAGELKERKE, J.F., VAN BERKEL, T.J., COUVREUR, P., GRISLAIN, L., ROLAND, M. and SPEISER, P., 1984, In vivo uptake of polyisobutyl cyanoacrylate nanoparticles by rat liver Kupffer, endothelial, and parenchymal cells, J. Pharm. Sci., 73, 980–982. LENAERTS, V., RAYMOND, P., JUHASZ, J., SIMARD, M.A. and JOLICOEUR, C., 1989, New method for the preparation of cyanoacrylic nanoparticles with improved colloidal properties, J. Pharm. Sci., 78, 1051–1052. LEWIS, K.J., IRWIN, W.J. and AKHTAR, S., 1998, Development of a sustained-release biodegradable polymer delivery system for site-specific delivery of oligonucleotides: characterization of P(LA-GA) copolymer microspheres in vitro, J. Drug Target., 5, 291–302.

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LOKE, S.L., STEIN, C.A., ZHANG, X.H., MORI, K., NAKANISHI, M., SUBASINGHE, C., COHEN, J.S. and NECKERS, L.M., 1989, Characterization of oligonucleotide transport into living cells, Proc. Natl Acad. Sci. USA, 86, 3474–3478. MBELA, T.K.M., POUPAERT, J.H., DUMONT, P. and HOEMER, S.A., 1993, Development of poly(dialkylmethylidene malonate) nanoparticles as drug carriers, Int. J. Pharm., 92, 71–79. MILLIGAN J.F., MATTEUCI M.D. and MARTIN J.C., 1993, Current concepts in antisense drug design, J. Med. Chem., 36, 1923–1937. MIR, L.M., BANOU, H. and PAOLETTI, C., 1988, Introduction of definitive amounts of non permeant molecules into living cells after electropermeabilization: direct access to the cytosol, Exp. Cell. Res., 175, 15–25. NAKADA, Y., FATTAL, E., FOULQUIER, M. and COUVREUR, P., 1996, Pharmacokinetics and biodistribution of oligonucleotide adsorbed onto poly (isobutylcyanoacrylate) nanoparticles after intravenous administration in mice, Pharm. Res., 13, 38–43. PAPAHADJOPOULOS, D., ALLEN, T.M., GABIZON, A., MAYHEW, E., MATTHAY, K., HUANG, S.K., LEE, K.D., WOODLE, M.C., LASIC, D.D., REDEMANN, C. and MARTIN, F.J., 1991, Sterically stabilized liposomes— improvements in pharmacokinetics and antitumor therapeutic efficacy, Proc. Natl Acad. Sci. USA, 88, 11460–11464. PERACCHIA, M.T., DESMAELE, D., COUVREUR, P. and D’ANGELO, J., 1997a, Synthesis of a novel poly(PEG cyanoacrylate-co-alkyl cyanoacrylate) amphiphilic copolymer for nanoparticle technology, Macromolecules, 30, 846–851. PERACCHIA, M.T., VAUTHIER, C., POPA, M., PUISIEUX, F. and COUVREUR, P., 1997b, An investigation on the formation of Sterically stabilized PEG-PIBCA nanoparticles by chemical grafting of PEG during the polymerization of isobutyl cyanoacrylate, STP. Pharm. Sci., 514–521. ROLLOT, J.M., COUVREUR, P., ROBLOT-TREUPEL, L. and PUISIEUX, F., 1986, Physicochemical and morphological characterization of polyisobutyl cyanoacrylate nanocapsules, J. Pharm. Sci., 75, 361–364. SAUZEDDE, F., GANACHAUD, F., ELAISSARI, A. and PICHOT, C., 1997, Emulsifier-free emulsion copolymerization of styrene with two different aminocontaining cationic monomers. II Surface and colloidal characterization, J. Appl. Polym. Sci., 65, 2331–2342. SCHWAB, G., CHAVANY, C., DUROUX, I., GOUBIN, G., LEBEAU, J., HELENE, C. and SAISONBEHMOARAS, T., 1994, Antisense oligonucleotides adsorbed to polyalkylcyanoacrylate nanoparticles specifically inhibit mutated Ha-ras-mediated cell proliferation and tumorigenicity in nude mice, Proc. Natl Acad. Sci. USA, 91, 10460–10464. SCHWAB, G., DUROUX, I., CHAVANY, C., HELENE, C. and SAISONBEHMOARAS, E., 1994, An approach for new anticancer drugs: oncogene-targeted antisense DNA, Ann. Oncol., 5, Suppl. 4, 55–58. SEIJO, B., FATTAL, E., ROBLOT-TREUPEL, L. and COUVREUR, P., 1990, Design of nanoparticles of less than 50 nm in diameter, preparation, characterization and drug loading, Int. J. Pharm., 62, 1–7. STEIN, C.A., 1996, Phosphorothioate antisense oligodeoxynucleotides: questions of specificity, Biotechnology, 14, 147–149.

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STEIN, C.A. and CHENG, Y.C., 1993, Antisense oligonucleotides as therapeutic agents— is the bullet really magical? Science, 261, 1004–1012. TICE, T.R. and GILLEY, R.M., 1985, Preparation of injectable controlled release microcapsules by a solvent-evaporation process, J. Control. Rel., 2, 343–352. VANDERHOFF, J.W., EL AASSER, M.S. and UGELSTAD, J., Polymer emulsification process, 1979, U.S. patent 4, 177. VAN SNICK L., COUVREUR P., CHRISTIAENS-LEYH, D. and ROLAND, M., 1985, Molecular weights of free and drug-loaded nanoparticles, Pharm. Res., 1, 36–41. VERRECCHIA, T., HUVE, P., BAZILE, D., VEILLARD, M., SPENLEHAUER, G. and COUVREUR, P., 1993, Adsorption/desorption of human serum albumin at the surface of poly(lactic acid) nanoparticles prepared by a solvent evaporation process, J. Biomed. Mater. Res, 27, 1019–1028. YAKUBOV, L.A., DEEVA, E.A., ZARYTOVA, V.F., IVANOVA, E.M., RYTE, A.S., YURCHENKO, L.V. and VLASSOV, V.V., 1989, Mechanism of oligonucleotide uptake by cells: involvement of specific receptors, Proc. Natl Acad. Sci. USA, 86, 6454–6458. YAMAKAWA, I., ISHIDA, M., KATO, T., ANDO, H. and ASAKAWA, N., 1997, Release behavior of poly(lactic acid-co-glycolic acid) implants containing phosphorothioate oligodeoxynucleotide, Biol. Pharm. Bull., 20, 455–459. ZOBEL, H.P., KREUTER, J., WERNER, D., NOE, C.R., KUMEL, G. and ZIMMER, A., 1997, Cationic polyhexylcyanoacrylate nanoparticles as carriers for antisense oligonucleotides, Antisense Nucl. Acid Drug Dev., 7, 483–493. ZON, G., 1988, Oligonucleotide analogues as potential chemotherapeutic agents, Pharm. Res.. 5. 539–549.

7 Liposomes for the Delivery of Oligonucleotides

P.COUVREUR, C.MALVY, C.DUBERNET AND E.FATTAL

7.1 Introduction Clinical applications of antisense oligonucleotides depend on the development of suitable transfer vehicles. In this regard, liposomes are of potential interest because they are made of phospholipids which are natural constituents of all cell membranes; thus toxicity is not expected to be a problem. Phospholipids may organize or disorganize at a supramolecular level depending on their nature and the microenvironment. This allows the production of ‘intelligent’ vesicles which are able to fuse, aggregate, leak or turn into other supramolecular organizations at a given pH, temperature or ionic strength. In addition, liposomes are very versatile carrier systems since they can be prepared in various sizes, morphologies, compositions and surface charges, and they can be tailored to the application needed. Finally, they may be functionalized with antibodies, lectins or hydrophilic polymers such as polyethyleneglycol in order to modify their cell and/or tissue distribution. Since the effectiveness of antisense oligonucleotides is limited by a low efficiency of cellular uptake (see also Chapter 9) and the lack of stability due to degradation by nucleases, their incorporation into liposomes has been considered to improve their transfer and delivery into cells. Most liposomes used for this purpose are cationic liposomes which form stable complexes with the polyanionic oligonucleotides. However, more sophisticated liposomal systems have been considered, such as pH-sensitive liposomes which can improve the endosomal escape of oligonucleotides after endocytosis, or immunoliposomes, which can deliver these molecules intracellularly through a receptor-mediated endocytosis. Plasma or lysosome membrane fusion may also be obtained through the use of virus-derived liposomes. This chapter will focus on the advantages and disadvantages of the different strategies using liposomes for oligonucleotide delivery.

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Figure 7.1 Schematic representation of oligonucleotide encapsulation in (A) anionic liposomes, (B) cationic liposomes and (C) immunoliposomes. Note in (A) that in the case of cholesterol oligonucleotides, the cholesterol is anchored in the bilayer and the oligonucleotidic sequence is still accessible at the surface of the outer leaflet

7.2 Anionic Liposomes Liposomes with a net negative or neutral charge cannot interact ionically with oligonucleotides. However, significant amounts of oligonucleotides can be encapsulated into non-cationic vesicles. This has been shown by Akhtar et al. (1991) using liposomes composed of egg phosphatidylcholine (PC), dimyristoylphosphatidylglycerol (DMPG) and cholesterol (CH), or dipalmitoylphosphatidylcholine (DPPC), DMPG and CH. When the liposomes were subjected to five freeze and thaw cycles prior to extrusion through a polycarbonate filter, the encapsulation was still more efficient than after a single extrusion procedure (Akhtar et al., 1991). On the basis of partition coefficients data it was suggested that oligonucleotides reside in the aqueous phase, even in the case of the methylphosphonate oligomers which are more hydrophobic (Figure 7.1 A). Relatively slow efflux kinetics across liposomal membranes was observed for unmodified oligonucleotides as well as for phosphorothioates and methylphosphonates oligomers. In addition, changes in lipid composition had little influence on oligonucleotide efflux rates (Akhtar and Juliano, 1992). Thus, the vesicles were able to encapsulate the oligonucleotides in quite a stable manner. In addition to encapsulation, methylphosphonate oligonucleotides exhibited saturable binding (adsorption) to liposomal membrane. When represented as a Scatchard plot, the linear fit of the binding data suggests that a simple interaction occurs in which one molecule of oligonucleotide attaches to

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a single lipid (Akhtar et al., 1991). The interaction between oligonucleotides and phospholipidic monolayers has been investigated using a Langmuir film balance (Rhodes and LIU, 1996). It was confirmed that large polyanions such as oligonucleotides cannot cross lipid layers in meaningful quantities in the absence of specific conditions. However, in the presence of Ca++, an interaction mediated by the divalent ion between anionic lipid, DPPG, and different anionic oligonucleotides was observed. This interaction depended on the composition and/or sequence of the oligonucleotide but also on its length and backbone chemistry (Rhodes and Liu, 1996). Ca++ has been used to reduce the repulsions between negative charges of oligonucleotides and lipids in order to obtain acceptable encapsulation efficiency of anti-c-myc (see also Chapter 12) phosphorothioate oligonucleotides in anionic vesicles composed of phosphatidylserine (Loke et al., 1998). Interestingly, oligonucleotides were also synthesized with cholesteryl groups tethered near one or both termini to anchor more efficiently into the phospholipidic bilayer of liposomes (Zhang et al., 1996). Liposomes tagged with such oligonucleotides are recognized by complementary oligonucleotides, which suggests that if the cholesterol moiety is well anchored in the bilayer, the oligonucleotidic sequence is still accessible onto the surface of the vesicles (Figure 7.1 A). The method of liposome encapsulation by minimal volume entrapment (MVE) was also found to be quite efficient for oligonucleotides (Thierry and Dritschilo, 1992a). The procedure is based on thin film lipid hydration in highly concentrated oligonucleotide solution followed by sonication. This formulation composed of cardiolipin, phosphatidylcholine and cholesterol was found to have a final concentration of oligomers per liposome content of 60–70 µg oligonucleotide/mg lipid. In this case, more than 90% of encapsulated oligonucleotides are inside the anionic lipidic vesicles (Thierry et al., 1992). As demonstrated on leukemia MOLT-3 cells (Thierry and Dritschilo, 1992a) and on lung carcinoma A549 cells (Thierry et al., 1992), liposomal encapsulation made possible oligomer protection in serum-containing medium and substantially improved cellular accumulation. However, this process is saturable and may be inhibited by increased concentrations of empty liposomes (Thierry et al., 1992). The major route of cellular uptake for liposomes encapsulated oligonucleotides is endocytosis (Thierry and Dritschilo, 1992a; Thierry et al., 1992) (Figure 7.2A). This has been demonstrated using sodium azide, a known inhibitor of endocytosis (Thierry et al., 1992). Liposome uptake is accompanied by an efflux mechanism of oligonucleotide, but this efflux is rather lower than with the free oligonucleotide formulation (Thierry and Dritschilo, 1992a). Due to a higher cellular accumulation, liposomes oligonucleotide exhibited a consistently higher intracellular concentration of undegraded oligonucleotide. Using laser-assisted confocal microscopy, it was observed that liposomes oligonucleotide treatment led to cytoplasmic and nuclear localization of FITCend labelled phosphorothioate. This provided evidence of a significant release of oligonucleotide from endocytic vesicles into the cytoplasm (Thierry and

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Figure 7.2 Mechanism of intracellur penetration of (A) anionic liposomes, (B) cationic liposomes, (C) pH-sensitive liposomes, and (D) immunoliposomes

Dritschilo, 1992a). Cytoplasmic localization of fluorescent oligonucleotides encapsulated into anionic vesicles was also observed in chronic myelogenous leukemia cells (Tari et al., 1996b). Similar observations were made on K562 cells with liposomally encapsulated methylphosphonate oligodeoxynucleotides (Tari et al., 1996a). How this process can occur is still unclear with anionic vesicles. The potential of MVE liposomes loaded with 15-mer cap phosphorothioate oligonucleotides (complementary to the 5′ end of the coding region or to a loopforming site in the mdr-1 mRNA) was examined in modulating the expression of the mdr gene (Thierry et al., 1993). Multidrug resistance (mdr) is, indeed, associated with the simultaneous development of cellular resistance to

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structurally unrelated drugs. The use of antisense oligonucleotides towards mdr expression may be an interesting way to inhibit the P-glycoprotein synthesis and to restore the sensitivity of mdr tumor cells to anticancer drugs. In fact, it was observed that Pglycoprotein synthesis and doxorubicin resistance were dramatically reduced by exposure of the multidrug-resistant SKVLB cells to those liposomally encapsulated oligonucleotides (Thierry et al., 1993). Similar observations were made with MCF-7/ADR cells (Thierry and Dritschilo, 1992b). Liposome-entrapped phosphodiester and phosphorothioate oligonucleotides targeted to the breakpoint junction of the BCR-ABL mRNA can inhibit the production of the p210 bcr-Abl protein and proliferation of chronic myelogenous leukaemic cell lines in a sequence-specific manner (Tari et al., 1996b). Similar observations were made using anti-BCR-ABL methylphosphonate oligonucleotide liposomes (Tari et al., 1994). Anionic liposomes were also efficient for the delivery of anti-c-myc oligonucleotides (Loke et al., 1998). However, in this case, the use of polyethyleneglycol in serum-free medium was necessary to achieve the fusion of the liposomes with the cell membrane. This shows the limit of using anionic vesicles for the delivery of oligonucleotides to cells with a net negative charge. The pharmacokinetic and tissue distribution of these liposomes with P-ethoxy oligonucleotides (lipophilic analogues of phosphodiesters) was found to be very similar to that of other liposomal compounds with a biphasic plasma clearance rate (t½ α of 6.7 min and t½ β of 7 h) (Tari et al., 1998). The highest concentrations of liposomal P-ethoxy oligonucleotides were found in spleen and liver, with a t½ of approximately 48 h. After in vivo administration up to 180 mg of liposomal Pethoxyoligonucleotides per kg of mice’s body weight, no adverse effects were observed on either renal or hepatic functions, or on the haematological parameters (Tari et al., 1998). 7.3 Cationic Liposomes The association of oligonucleotides with cationic lipids is based on the single ionic interaction between the positive charges of the lipids and the negative charges of the oligonucleotides (Figure 7.1B). Although many papers have been published on the use of cationic liposomes for oligonucleotides delivery, little is known about the interactions between cationic lipids and oligonucleotides. It is noteworthy that any comparison with DNA is difficult because oligonucleotides are much smaller molecules and their susceptibility to tertiary structure and liposome-induced structural changes is different from DNA. Jaaskelainen et al. (1994) studied the aggregation and fusion reactions during the formation of cationic lipid/oligonucleotide complexes in solution and their interactions with lipid bilayers. Resonance energy transfer experiments showed that in addition to aggregation, oligonucleotides induced fusion of cationic liposomes, but the fusion was rate-controlled by the initial aggregation step

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which depended on lipid concentration. Increase in oligonucleotide concentration induced aggregation and fusion until a high −/+ ratio induced, again, electrostatic repulsion of negative surfaces, thus inhibiting further aggregation and fusion. The cationic liposomes used in this study were small unilamellar vesicles and consisted of 1, 2-diacyl3-trimethylammonium propane (DOTAP) or dimethyldioctadecyl ammonium bromure (DDAB)/dioleyl phosphatidyl ethanolamine (DOPE). Aggregation and fusion were also observed using freezefracture and electron microscopic observations (Jaaskelainen et al., 1998). Similar results were obtained with liposomes consisting of N-(αtrimethylammonioacetyl)didodecyl-D-glutamate chloride (TMAG), dilauroylphosphatidylcholine (DLPC) and DOPE (Hidetoshi et al., 1997). With these liposomes, aggregation and vesicle fusion occurred when the charge repulsion between the vesicles was lowered due to the addition of oligonucleotides. Recently, cationic liposomes were coated with polyethyleneglycol (Meyer et al., 1998). They were composed of positively charged lipids DOTAP, DOGS, DDAB, of the neutral lipid DOPE and of a phospholipid derivative of polyethyleneglycol (PEG—phosphatidylethanolamine (PEG-PE)). The binding of an 18-mer phosphorothioate onto these liposomes was very efficient even when containing a relatively high amount of PEG-PE: such liposomes can bind an impressive amount of oligonucleotides without a loss of structural stability in a wide range of oligonucleotide/lipid charge ratios (Meyer et al., 1998). The complexes showed intact vesicular structures similar to original liposomes, and their size was unchanged after several weeks of storage. The complexes lacking PEG-PE showed, on the contrary, progressive aggregation and/or precipitation (Meyer et al., 1998). 7.3.1 Intracellular delivery and Distribution Antisense oligonucleotides are generally more efficiently cell internalized when they are associated with cationic liposomes. This was observed, for instance, with CaSki cells for which the cell penetration of oligonucleotides without liposomes was significantly lower than for DDAB–DOPE complexed oligonucleotides (Lappalainen et al., 1994). In the absence of DDAB–DOPE, the oligonucleotides appeared to be associated with the cell membrane, whereas the oligonucleotides with DDAB–DOPE were also found in the cytoplasm. However, the cationic liposomes were toxic to the cells as demonstrated by the reduced cell number and altered cell morphology (Lappalainen et al., 1994). Also, on RAW264.7 cells, a mouse macrophage-like cell line, the cellular association of oligonucleotides was significantly enhanced with positively charged liposomes compared with neutral and negatively charged vesicles (Hidetoshi et al., 1997). Complexation of oligonucleotides with PEG-modified cationic liposomes enhanced the cellular uptake (human breast cancer cells) of

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oligonucleotides severalfold, similar to the enhancement observed with nonmodified cationic liposomes (Meyer et al., 1998). When the complex was modified with a targeting device (anti-HER2 Fab’ fragments) at the distal termini of PEG chains, further increase of the cellular uptake by a factor of 2.3–2. 6 was noted for both lipid and oligonucleotide (Meyer et al., 1998). Although cationic liposomes are efficient carriers for intracellular delivery of oligonucleotides, the mechanism of cell capture is still under discussion. It is widely accepted that cationic lipids are able to bind efficiently to the negatively charged cell membrane through ionic interactions (Figure 7.2B). The mechanism of delivery is thought by only a few authors to be based on the fusion of the cationic lipids with the negative phospholipids of the cell membrane (Bennett et al., 1992; Felgner and Ringold, 1989) (Figure 7.2B). This process of direct cell membrane destabilization allows the oligonucleotides to be delivered directly into the cell cytoplasm. Many other authors believe that the cell internalization of oligonucleotides associated with cationic liposomes occurs through a typical endocytotic pathway (Figure 7.2B), which is a rather general observation made with different cell lines using cationic liposomes of different composition (Capaccioli et al., 1993). In this view, the results of Zelphati and Szoka (1996a), who investigated the parameters influencing the intracellular trafficking of oligonucleotides delivered by DOTAP and showed that endocytosis was the main pathway for the delivery of oligonucleotides via the cationic complex with DOTAP, were very convincing. This was demonstrated using inhibitors of the endocytic pathway: nuclear accumulation of oligonucleotides could be decreased by inhibitors of actin microfilaments, energy metabolism and proteins implicated in the fusion of endosomes (Zelphati and Szoka, 1996a). This capture of cationic liposomes oligonucleotides through an endocytotic mechanism is supported by Wrobel and Collins (1995), who showed that fusion of cationic liposomes with cells occurs after endocytosis and that an early event in the endocytosis pathway probably plays a key role in liposomes destabilization. This is supported by the observation that at 15–20°C, a temperature which does not normally affect processing of materials into endosomes, no nuclear delivery occurred (Wrobel and Collins, 1995). Endocytosis was also found to be the main pathway of the cellular uptake of liposomal 2, 3 dioleyloxy-N-(sperminecarboxamino)ethyl-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) and DDAB oligonucleotides (Lappalainen et al., 1997). Then, oligonucleotides were rapidly released into the cytoplasm and transported into the perinuclear area. Using electron microscopy, this study was the first to show that the nuclear envelope can form a barrier against penetration of oligonucleotides carried by cationic liposomes into the cell nucleus (Lappalainen et al., 1997). Since microinjected oligonucleotides free in solution rapidly diffuse into the nucleus (Chin et al., 1990; Leonetti et al., 1991) and localize in association with small nuclear ribonucleoproteins, it may be hypothesized that when transported by cationic liposomes, there is a persisting association of oligonucleotides with residual cationic lipids after their release into the cytosol. This means that such

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complexes are possibly too big to enter through the pores of the nucleus. Moreover, the mystery of how the oligonucleotide is released from the cationic lipids and escapes from endosomes has been highlighted using confocal microscopy and fluorescence resonance energy transfer (FRET) techniques (Zelphati and Szoka, 1996b). Using that double-labelling technique (fluoresceinlabelled oligonucleotide and rhodamine-labelled lipid), the key observation was that the fluorescent lipids associated with the complexes were found in punctate cytoplasmic structures corresponding to the endosomes, whereas the fluorescent oligonucleotide was found alone into the nucleus. Thus, oligonucleotide separates from the lipid after internalization and enters the nucleus leaving the fluorescent lipid in cytoplasmic structures (Zelphati and Szoka, 1996b). This somewhat contradicts the poor dissociation of the complexes as suggested above, but the cells and lipid compositions are the same. Thus, the proposed mechanism is that, after internalization, the oligonucleotide/cationic lipid complex initiates a destabilization of the endosomal membrane that results in a flip-flop of anionic lipids from the cytoplasmic facing monolayer (Figure 7.2B). The anionic lipids diffuse laterally into the complex and form a charged neutralized ion-pair with cationic lipids. This displaces the oligonucleotide from the complex, and its release and free diffusion into the cytoplasm occur (Zelphati and Szoka, 1996b). Why such a fusion through a flip-flop process doesn’t occur directly via the cell membrane (which possesses exactly the same phospholipid composition as the membrane of the endosome) remains unanswered, but it may be hypothesized that DOPE is playing a key role at the acidic pH of the endosomes, helping the flip-flop process to occur. To determine what role the lipid composition of ‘target’ membranes might have in promoting fusion with cationic liposomes, biophysical investigations were also performed using artificial vesicles of different composition (Bailey and Cullis, 1997). Membrane fusion was determined by freeze-fracture electron microscopy, fluorescence microscopy and lipid mixing assay. It was found that fusion of cationic liposomes composed of DOPE/DODAC with different target membranes was dependent on the concentration of negative charges in the target. According to Bailey and Cullis (1997), the negative charges have several roles: first, promotion of a close contact between vesicles bearing an opposite charge; second, a mutual surface charge neutralization which reduces the hydrophilic nature of the opposite membranes and promotes a close contact by the loss of water previously bound to the polar heads of the lipids; and third, membrane destabilization due to the preference of the DOPE for adopting a hexagonal structure. Thus, cationic liposomes are not only able to increase significantly the cellular capture of oligonucleotides, but they also modify their intracellular distribution. When associated with cationic lipids, oligonucleotides distribute mainly into the cytoplasm and, in the case of the dissociation of the complex occurring, they efficiently diffuse into the nucleus (Bennett et al., 1992; Kanamaru, et al., 1998; Lappalainen et al., 1997; Wagner et al., 1993; Zelphati and Szoka, 1996a)

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instead of remaining in the endosomal vesicles (Deshpande et al., 1996; Jaroszewski and Cohen, 1991; Wu-Pong et al., 1992). 7.3.2 Pharmacological Efficacy in Vitro The pharmacological activity of several antisense oligonucleotides complexed with cationic liposomes has been tested in cell culture experiments. The most commonly used formulation for the evaluation of oligonucleotides in vitro is Lipofectin®, a mixture of 2, 3-bis(oleoyl)propyl trimethyl ammonium chloride (DOTMA) and DOPE. To achieve an antisense effect, it is necessary to optimize the lipid to oligonucleotide ratio in order to keep an excess of positive charges able to bind through electrostatic interactions with negative charges present on the cell surface. Thus, it is understandable why, in most experiments, serum-free medium is used, because serum proteins may interact and neutralize the net positive charge of the complex, thus decreasing its affinity for the cell membrane and consequently the efficiency of the cationic liposomes. In addition, it is supposed that serum proteins may displace the oligonucleotide molecules from their ionic interactions with the cationic lipids, thus inducing their burst release from the complex. This is why some pharmacological activity observed in vitro cannot be reproduced if the experimental conditions, especially concerning the serum concentration, are not strictly adhered to. The structural requirements for cationic lipid-mediated phosphorothioate oligonucleotides delivery to cells in culture have been investigated in detail by Bennett et al. (1997). With this aim, a series of 2, 3-dialkyloxypropyl quaternary ammonium lipids containing hydroxyalkyl chains on the quaternary amine were synthesized, formulated with dioleoylphosphatidylethanolamine and assayed for their ability to enhance the activity of the intracellular adhesion molecule ICAM-1 antisense oligonucleotide, ISIS 1570 (see also Chapter 13). The activity of cationic lipids was found to be more dramatically affected by changes in the aliphatic chains than by modifications on the quaternary amine (Bennett et al., 1997). Activity of saturated acyl groups decreased as the chain length increased. Cationic lipids synthesized with unsaturated aliphatic chains were more effective in enhancing the biological activity of the ICAM-1 antisense oligonucleotide compared to a saturated aliphatic chain of the same length (Bennett et al., 1997). One interpretation of these data by the authors is that fusion of liposomes with the cellular membrane is required for effective delivery of oligonucleotides by cationic liposomes (Bennett et al., 1997). There are some reports on the in vitro efficacy on viral infections of oligonucleotides when associated with cationic liposomes. Hatta et al. (1996) evaluated the inhibitory effect of free and liposome-encapsulated phosphodiester and phosphorothioate oligonucleotides on influenza virus RNA polymerase and nucleoprotein gene expression in response to treatment with dexamethasone. The in vitro activities of these oligonucleotides on the expression of the influenza

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virus RNA polymerase and nucleoprotein genes were assessed on the basis of their inhibition of CAT protein expression. The liposomally encapsulated oligonucleotides exhibited higher inhibitory activity than free oligonucleotides (Hatta et al., 1996). This was explained by a better protection in serumcontaining medium and an improved cellular accumulation. Inhibition of hepatitis B virus was also found to be more efficient in vitro on Hep-G2 cells when antisense phosphorothioate was used in association with Lipofectin (Yao et al., 1996). Antisense oligonudeotides were considered for the treatment of human papillomavirus (Lappalainen et al., 1994, 1996). Only a slight reduction of the cell proliferation was seen when antisense 12-mer targeted to human papillomavirus 16 E7 mRNA was protected from its 3′- and 5′-ends with thiolate and FITC, respectively. Both 12- and 23-mers with cationic liposomes inhibited cell proliferation, the inhibitory effect being longer with the 23-mer (Lappalainen et al., 1994). However, as mentioned above, the cationic lipids DDAB/DOPE used in this study proved to be toxic, as evidenced by the severe morphological changes in CaSki cells observed. The intracellular delivery of bcl-2 antisense oligonudeotides (see also Chapter 12) by means of cationic liposomes containing DDAB and DOPE has been investigated into four different myeloma cells (Ollikainen et al., 1996). Despite the enhanced cell internalization and stability of phosphodiester oligonudeotides, no effect on mRNA level was observed. One explanation is that the amount of intracellular oligonucleotide might be inadequate despite the use of cationic liposomes. It is believed that substantially higher amounts of intracellular oligonucleotide molecules are required in this model to hybridize with the target mRNA before translation is inhibited. Antisense inhibition of angiotensinogen in hepatoma cell culture has been shown to be enhanced by cationic liposome delivery; attenuation of angiotensinogen protein and decrease of mRNA were obtained in a dosedependent manner (Wielbo et al., 1997). Rat endothelial cells, when transfected in the presence of cationic liposomes with fibronectin-antisense phosphorothioate oligonudeotides, also showed significant decrease of fibronectin protein and mRNA levels relative to untransfected cells (Roy and Roth, 1997). When complexed with cationic liposomes, stem loop Syk antisense oligonucleotide was able to abrogate in the same way the Fc gamma receptor IIA-mediated phagocytic signal in monocytes (Matsuda et al., 1996). Cationic liposomes composed of N-dodecyl imidazole propionate or lipofectin were evaluated for their potential to enhance the effects of phosphorothioate oligonudeotides. The test system used a CHO cell line expressing CAT under the control of an inducible promoter. In these conditions, liposomes could also significantly enhance the effects of the oligonudeotides (Hughes et al., 1996). Finally, a recent study demonstrated that antisense phosphorothioate oligonucleotide complementary to the site of the PB2-AUG initiation codon of influenza virus RNA polymerase was able to display important inhibitory effects

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on influenza virus A replication in MDCK cells (Abe et al., 1998). Again, the liposomally encapsulated oligonudeotides exhibited higher inhibitory activity than the free oligonudeotides (Abe et al., 1998). Different oligonudeotides were screened for their antiviral effect; the antisense phosphorothioate oligomer containing the AUG initiation codon at the centre site of the oligonucleotide was found to have the highest inhibitory effects (Hatta et al., 1997). 7.3.3 Pharmacological Efficacy in Vivo There are some reports on the in vivo efficacy of cationic liposomes for the delivery of antisense molecules. Although, as explained above, the main limitation of using cationic liposomes in vivo through systemic intravascular administration is the strong interaction that occurs with the proteins of the serum, interesting results were obtained after intra-arterial administration of oligonucleotides oriented towards angiotensinogen mRNA and encapsulated into liposomes (Wielbo et al., 1996). Administration of liposome-encapsulated antisense significantly decreased hypertensive blood pressures to normotensive levels compared with scrambled control oligonucleotides, unencapsulated antisense and empty liposomes. These data were supported by biochemical changes elicited by the antisense treatment. Rats receiving liposomeencapsulated antisense had significantly lowered peripheral angiotensin and angiotensin II levels compared with control groups (Wielbo et al, 1996). Tissue distribution studies have confirmed that encapsulated antisense molecules were seen to concentrate within the liver 1 h after injection, whereas little or no uptake was observed with free oligonucleotides (Wielbo et al., 1996). When injected systemically or into the brain, antisense oligonucleotides targeted to the reninangiotensin system at the level of synthesis (angiotensin) and the receptor (AT1 receptor) were observed to reduce blood pressure in spontaneously hypertensive rats; the biological effect was prolonged using liposomes as carrier system (Phillips, 1997). An ICAM-1 antisense oligodesoxyribonucleotide associated with lipofectin has been shown to attenuate efficiently reperfusion injury and renal failure in the rat (Haller et al., 1996). An antisense oligonucleotide corresponding to the NH2terminus of the substance P(SP)/neurokinin-l receptor was constructed, encapsulated into liposomes and injected into the lateral cerebral ventricle of rats (Ogo et al., 1994); in these conditions, functional SP receptor was blocked. A 20-mer phosphorothioate oligodeoxynucleotide designed to hybridize to the AUG translation initiation codon of mRNA encoding murine protein kinase Calpha (PKC-alpha) (see also Chapter 12) was observed to reduce specifically the expression of PKC α when complexed with DOTMA/DOPE liposomes and incubated in vitro with mouse C127 mammary epithelial cells (Dean and McKay, 1994). The reduction of PKC α was both dose—and time-dependent, without any effect on the other PKC isoenzymes (δ, ε, ζ). When administered

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intraperitoneally in mice, the same oligonucleotide also caused a dosedependent, and sequencedependent reduction of PKC a mRNA in the liver with an IC50 value of 30–50 mg/kg. Again, the expression of PKC δ, PKC ε and PKC ζ were unaffected by the treatment (Dean and McKay, 1994). Very surprisingly, the oligonucleotide activity in vivo did not require the use of cationic liposomes for the inhibition of PKC α expression. This observation may be explained by the fact that oligonucleotides have a tendency to concentrate in the liver, and it is supposed that phosphorothioates are quite stable in this tissue. On the contrary, the suppression of urokinase-type plasminogen activator as a strategy to treat experimental ovarian cancer in mice was feasible only when the corresponding phosphorothioate oligonucleotide was used in concert with cationic liposomes (Wilhelm et al., 1995). Antisense suppression of protein kinase C-α and -δ was also obtained in vascular smooth muscle using phosphorothioates oligonucleotides containing C-5 propynyl modified pyrimidines and associated with DOTMA/DOPE liposomes (Lipofectin®) (Busuttil et al., 1996) (see also Chapter 12). 7.4 pH-sensitive Liposomes A lipid typically used for the preparation of pH-sensitive liposomes is dioleylphosphatidylethanolamine (DOPE) (Connor et al., 1984). However, phosphatidylethanolamine (PE) has a strong tendency to form the HII hexagonal phase under physiological conditions. Thus, the design of PE stable liposomes is not possible in conditions (pH, salts concentration, temperature, etc.) which are acceptable for biological applications. Therefore, the formation of stable PE liposomes needs the use of a second component containing titrable acidic groups able to provide electrostatic repulsions which will prevent the formation of the non-desired hexagonal phase at physiological pH. The negative charge arising from these titrable acidic functions provides electrostatic repulsion to inhibit PE intermolecular interaction, which prevents HII phase formation at neutral pH. On the contrary, protonation of the amine function of PE induced by an acidification of the medium neutralizes this negative charge which, in turn, destabilizes the vesicle as the PE component reverts to the hexagonal phase. This is the physicochemical basis for the pH sensitivity of DOPE-containing liposomes. For further information, see the excellent review by Litzinger and Huang (1992). Practically, the most common PE liposomes stabilizers are: fatty acids such as oleic acid (Duzgunes et al., 1985), cholesterol hemisuccinate (CHEMS) (Bentz et al., 1985), or palmitoylhomocystein (Connor et al., 1984). Since the negative charge of these pH-sensitive liposomes may prevent them from efficiently associating nucleic acids, new cationic pH-sensitive liposomes have been developed; they are composed of cationic lipids with an amine having a pK within the physiologic range of 4.5 to 8, which are incorporated with DOPE to form liposomes (Budker et al., 1996). The newly synthetized cationic lipids

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include 4-(2, 3-bis-palmitoyloxy-propyl)-l-methyl-lH-imidazole and 4-(2, 3-bisoleoyloxy-propyl)-l-methyl-1H-imidazole and cholesterol(3-imidazol-l-yl propyl)carbamate and 2, 3-bis-palmitoyl-propyl-pyridin-4-yl-amine (Budker et al., 1996). To avoid lysosomal degradation and in order to target the cytoplasmic compartment, the concept of pH-sensitive liposomes has been applied to the delivery of antisense oligonucleotides (Ropert et al., 1992; De Oliveira et al., 1997, 1998) because, as explained, these phospholipidic bilayered vesicles are destabilized at acidic pH, whereas in the process of endocytosis, the pH is reduced in the endosomal compartment which precedes the lysosomes. Thus, after destabilization and fusion with the endosomal membrane, the pH-sensitive liposomes may deliver their contents into the cytoplasm just like a virus (Chu et al., 1990) (Figure 7.2C). They are efficient tools able to avoid the degradation of their content by the enzymes. Such an intracellular behaviour is, of course, of great interest for the efficient delivery of genes or antisense oligonucleotides into cells. This is the rationale of using those pH-sensitive phospholipidic bilayered structures for the delivery of oligonucleotides. In this context, we have recently shown that proliferation of the Friend retro virus was specifically inhibited by the env mRNA complementary oligonucleotide encapsulated in pH-sensitive liposomes, whereas the lack of antiviral activity was noted with the same oligonucleotide but incubated free (Ropert et al., 1993). The specificity was demonstrated by the absence of activity when the random control oligonucleotide was used free or encapsulated into liposomes (Ropert et al., 1992). Interestingly, it was observed that the pHsensitive compositions were more effective than their non-pH-sensitive counterparts whatever the procedure used for the preparation of the liposomal vesicles (reverse phase evaporation or freeze-thawing). The relation between viral infection and cell uptake of pH-sensitive liposomes has been investigated (Ropert et al., 1993, 1996). The key finding of those studies was the dramatic impact of the virus on the liposomes’ penetration into the cells. For both chronic and de novo infection, the point at which liposome penetration started corresponded well to the time needed for the virus to leave the cell: approximately 6 h for chronically infected cells and 18 h for de novo infected cells (Ropert et al., 1993). In the absence of the virus, liposomes remained adsorbed at the surface of the cell membrane without any significant internalization. Further, the more virions were infecting the cells, the greater was the oligonucleotide pH-sensitive liposomes’ cell association. To investigate the relation between viral infection and cell uptake of liposomes, a defective virus was used to infect two types of cells: cells allowing virus budding (psi2neo cells) and cells bereft of a virus exit process (NIH 3T3 cells). This study (Ropert et al., 1996) revealed that cell uptake of pH-sensitive liposomes was highly dependent on the virus exit process, since it ensued only when virus budding occurred. This preferential uptake of pH-sensitive liposomes by infected cells was not carrierspecific, because similar uptake was observed with non-biodegradable

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fluorescent nanoparticles using confocal microscopy (Ropert et al., 1996). Our explanation is that the virus budding is possibly followed by the activation of the cell pinocytosis. This may result from the recapture and re-use of the vesicle membrane, so that the overall dimensions of the cells and of the vacuolar system remain constant. For these reasons, virus budding may stimulate fibroblast pinocytic activity and thus increase cell capture of particulate drug carrier. Also, inhibition of neo gene expression by oligonucleotide pH-sensitive liposomes was only observed in the cell system (psi2neo) endowed with a virus exit process. An interesting study by Jaaskelainen et al. (1998) on the release of fluorescentlylabelled oligonucleotides from DOPE/DOTAP liposomes after incubation with endosomal model membranes has established the impact of DOPE on these properties. There was a significant fluorescence dequenching, and probably release of the oligonucleotide when DOTAP/DOPE complexes of oligonucleotides were incubated with endosomal model membranes (Jaaskelainen et al., 1998). The release was highest at excess positive charge of the complexes. On the contrary, with DOTAP only, no significant dequenching was observed. This suggests that DOPE facilitates oligonucleotide release from the complex in presence of the bilayer. Freeze-fracture electron micrographs have shown that DOPE in cationic liposomes induced hexagonal tubule formation that was most pronounced in cell culture medium (Jaaskelainen et al., 1998). The in vivo efficacy of pH-sensitive liposomes remains questionable, since the intravascular administration of pH-sensitive liposomes poses at least two problems: 1 pH-sensitive liposomes are leaky in the presence of serum and display a high degree of aggregation upon incubation in mouse plasma (Lappalainen et al., 1996); thus, their stability needs to be improved without loss of pHsensitivity 2 due to the opsonization process (complement activation and interaction with other serum proteins), they are rapidly cleared from the bloodstream and accumulate in the macrophages of the MPS (liver and spleen) (Connor et al., 1986); this is an obstacle to the targeting of antisense oligonucleotides to tumors or infected organs other than the liver and the spleen. Concerning the stability, the addition of cholesterol was found efficient for preparing stable DOPE pH-sensitive immunoliposomes in presence of serum (Liu and Huang, 1989a). However, the presence of cholesterol further increased OA transfer which, in turn, decreased the acid sensitivity of the liposomes. DOPE stabilized by 1, 2 dipalmitoyl-3-succinylglycerol (1, 2-DSPG) or by 1, 2dioleoyl3-succinylglycerol (1, 2-DOSG) was claimed to be stable in human plasma at 37°C and to remain pH-sensitive (Collins et al., 1990). However, according to LIU and Huang (1989b), the pH of destabilization was shifted from 5.3 in absence of plasma to 4.2 in presence of plasma. This effect was assigned

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either to the protein insertion in the liposome bilayer or to their adsorption preventing close juxtaposition of the liposomes and content leakage (Liu and Huang, 1989b). Another very interesting strategy for preparing stable pHsensitive liposomes is to use polyethyleneglycol (PEG), which has been employed to increase the fusion ability of PC liposomes (Kono et al., 1997). Incorporation of carboxyl groups into the PEG polymer was shown to change the ability of liposomes to fuse in response to pH (Kono et al., 1994). The fusion ability of these liposomes was low at neutral pH but increased with decreasing pH. The intracellular delivery of calcein encapsulated within these succinylated poly(glycidol)-modified liposomes has been examined: the cells treated by these liposomes displayed strong and diffuse fluorescence, whereas weak and vesicular fluorescence was observed with the bare PC liposomes (Kono et al., 1997). This result suggests that calcein molecules were transferred into cytoplasm for the cells treated with the pH-sensitive polyethyleneglycol derivative, whereas calcein molecules existed in endosomes/lysosomes for the cells treated with the single PC liposomes (Kono et al., 1997). Since PEG is known to induce steric repulsions with plasma components when attached at the surface, stability of such PEG derivative-modified liposomes in the blood may be superior to conventional pH-sensitive preparations. However, this needs to be demonstrated experimentally. Thus, preparing serum-stable and still pH-sensitive vesicles remains an important challenge for in vivo administration of oligonucleotides. In this context, liposomes containing Nstearoylcysteamine were recently proposed as a pH-sensitive system for which the addition of cholesterol had a stabilizing effect but without affecting pH sensitivity (Cazzola et al., 1997). This type of liposome has never been applied to the encapsulation of oligonucleotides, but it should be interested to investigate it with this aim. The development of pH-sensitive formulations able to avoid recognition by the MPS is another very important challenge for the successful iv administration of antisense oligonucleotides. This point has been poorly addressed until now, with the exception of the inclusion of GM 1 in DOPE/1, 2 DSPG liposomes (Liu and Huang, 1990). Such liposomes revealed reduced MPS capture as well as prolonged residence time in the circulation. In addition, the use of a watersoluble marker (inulin) of the liposomes’ aqueous phase suggests that liposomes were stable, retaining the entrapped content during the circulation. However, the pH sensitivity decreased with the incorporation of GM1 in the bilayer. It was found that lipid-anchored polyethyleneglycol was able to confer steric stability and to prolong the circulation time of pH-sensitive CHEMS/DOPE liposomes (Slepushkin et al., 1997). However, this modification significantly decreased the pH-dependent release of calcein, a charged water-soluble fluorophore (Slepushkin et al., 1997). In conclusion, plasma stable liposomes which retain pH sensitivity and avoid MPS recognition need to be developed for targeted oligonucleotides delivery in vivo. For more information on the use of pH-sensitive liposomes for the delivery of oligonucleotides, see also the review by Couvreur et al. (1997).

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7.5 Immunoliposomes and Other Molecularly Targeted Liposomes A way to deliver oligonucleotides intracellularly in a targeted manner is to use immunoliposomes as a transport system (Figure 7.2D). Immunoliposomes have been demonstrated to enter cells through the endocytic pathway and to deliver the encapsulated product intracellularly (Leserman et al., 1980). However, this process is more or less efficient depending on the target antigen, the cell type expressing it and the size and nature of the liposomes used (Machy et al., 1982a, 1982b; Machy and Leserman, 1983). Thus, encapsulation of oligonucleotides in antibody-targeted liposomes could circumvent problems of extracellular degradation by nucleases and poor membrane permeability, and address these molecules more efficiently to target cells. This strategy was applied successfully by Zelphati et al. (1993) to inhibit the replication of HIV-1. Phosphodiester oligonucleotides encapsulated into immunoliposomes directed to HLA class I antigen have been observed to protect oligonucleotides from degradation, to increase intracellular delivery and to inhibit specifically the expression of the targeted HIV-1 rev and tat viral genes (Zelphati et al., 1993). The efficacy of immunoliposomes to inhibit HIV replication has been confirmed using CD4 monoclonal-antibody-targeted liposomes containing Rev antisense phosphorothioate oligonucleotides. HIV-1 infected H9 cells as well as peripheral blood lymphocytes were used as models of in vitro infection. In these conditions, HIV-1 replication was reduced by 85% with antisense immunoliposomes treatment, whereas the inhibition of HIV-1 replication was not observed either with empty immunoliposomes or with immunoliposomes containing the scrambled sequence (Selvam et al., 1996). Phosphodiester and phosphorothioate oligonucleotides in alpha and beta configurations directed against the initiation codon region of the HIV-1 rev gene were also used by Zelphati et al., (1994b) to compare oligonucleotides in solution and encapsulated into immunoliposomes in order to distinguish between oligonucleotide-mediated inhibition of viral entry and intracellular effects on viral RNA, since only the immunoliposomal formulation was able to deliver the oligonucleotides intracellularly in a significant amount. In fact, it was observed that any sequence of oligonucleotide in solution but not into liposomes interfered with virus-mediated cell fusion (Zelphati et al., 1994b). Interference with reverse transcription occurred also in a non-specific manner with phosphorothioate in alpha and beta configurations, whereas it was sequence-specific in the case of alpha and beta phosphodiester. Finally, inhibition of viral mRNA was found to be sequence-specific and RNase-H-dependent with beta-phosphorothioate oligonucleotides (Zelphati et al., 1994b) (see also Chapter 1). Because of the poor efficiency of encapsulation, Zelphati et al. (1994a) have investigated the biological activity of oligonucleotides covalently linked to cholesterol via a bioreversible disulphide bridge in order to improve the entrapment into

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liposomes. This approach was successful since 20–30% of cholesterol-coupled oligonucleotides were incorporated into liposomes instead of the 2–3% obtained with the single phosphodiester. Based on degradation experiments performed in the presence of DNAse, the authors have shown very nicely that cholesterolcoupled oligonucleotides were incorporated into the bilayer of liposomes with half of the oligonucleotides in the internal leaflet and half in the external leaflet. The oligonucleotides facing the medium were sensitive to DNAse. This formulation was tested on HIV-1 acutely infected cells and found to inhibit strongly the replication of HIV (Zelphati et al., 1994a). Since the antiviral effect was observed with the antisense but not with the scrambled anti-tat sequence, it was suggested that, in this case, the effect was due to a specific interaction with viral RNA rather than with reverse transcriptase or on the viral fusion entry process as reported previously by Stein et al. (1991) with phosphorothioate oligonucleotides coupled or not with cholesterol. Delivery of anti-myb oligonucleotides to human leukemia cells was also improved by using anti-CD32 or anti-CD2 immunoliposomes (Ma and Wei, 1996). In fact, the cellular uptake of the oligonucleotide was twice that of single liposomes or non-specific immunoliposomes (Ma and Wei, 1996). Despite these very encouraging results, it is noteworthy that even if efficiently targeted, the immunoliposomes are taken up through endocytosis and end up in the cell lysosomes, which is a clear drawback of this approach (Figure 7.2D). For that reason, other strategies have to be considered to allow the cytoplasmic release of oligonucleotides from targeted liposomal formulations. This has been done using folate, which has been observed to be an excellent ligand for delivering materials into cells expressing a folate receptor. In addition, folate endocytosis does not culminate in lysosome deposition but rather leads to nondestructive release of much of the captured material into the cytosol (Leamon and Low, 1991; Lee and Low, 1995). For that reason, liposomes conjugated to folate via polyethyleneglycol were used for the selective delivery to KB cells of antisense oligonucleotides targeted to the epidermal growth factor (EGF) receptor (Wang et al., 1995). It was found that the folate targeted liposomes were able to promote cell internalization of such oligonucleotides dramatically: nine times more than with the non-targeted counterpart liposomes and 16 times more than the free oligonucleotide (Wang et al., 1995). The important biological activity (in terms of inhibition of cell growth and suppression of EGF expression) suggested that folate-PEG-liposome antisense was delivered intracellularly in a nondegraded form. Another way to allow the cytoplasmic delivery of immunologically targeted liposomes is to combine the selective recognition due to the presence of antibodies at the surface of the liposomes with the use of pH-sensitive lipids. This was done by Collins et al., (1989), who prepared immunoliposomes covered with anti-H2K antibody and displaying various pH sensitivities due to the nature of the lipids used: DOPE/palmitoylhomocysteine (PHC), or DOPE/OA or DOPE/

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dipalmitoylsuccinylglycerol (DPSG). The following model was proposed (Collins et al., 1989). 1 Immunoliposomes of all three compositions first bind to cell surface. 2 Upon endocytosis, the immunoliposomes are taken into cells, encountering a pH of approximately 6.2 after 5 min, allowing the fusion and cytoplasmic release of DOPE/PHC immunoliposomes. 3 After 15 min, as endocytosis proceeds, pH encountered by immunoliposomes decreases to close to 6.0, allowing DOPE/OA to fuse. 4 After 25 min, the luminal pH encountered by the immunoliposomes is close to 5.0, which pH induces destabilization and cytoplasmic delivery for DOPE/ DSPG immunoliposomes. Thus, it is possible to design immunoliposomes able to fuse with the endosomal membrane at early endosomes (DOPE/PHC) or late endosomes (DOPE/DPSG) or with a kinetic intermediate between the other two immunoliposomes. Although it was not applied to oligonucleotides delivery, this type of liposome could represent, in our opinion, a promising approach for the transport and specific targeting of those molecules. Recently, PEG-coated cationic liposomes bearing oligonucleotides were conjugated to anti-HER2 Fab′ at their surface (Meyer et al., 1998). This formulation was able to deliver the oligonucleotides very efficiently into the cell cytoplasm in a diffuse manner, and to a larger extent in the nucleus of HER2overexpressing breast cancer cells (Meyer et al., 1998). Because there was no colocalization of oligonucleotides and lipids in these regions, oligonucleotide was presumably free of lipid, which is a requirement for their biological activity (Meyer et al., 1998). 7.6 Fusogenic Liposomes and Proteoliposomes Liposomal formulations containing dipalmitoyl-DL-α-phosphatidyl-L-serine (DPPS) were proposed as fusogenic preparations (Fresta et al., 1998). The fusogenic properties of DPPS vesicules were shown by evaluating the increase of fluorescence intensity at 492 and 554 nm due to the Tb(DPA)33-complex which is formed following the fusion between Tb(III)-loaded DMPC model membranes and DPA-loaded DPPS liposomes. It was observed that the formation of the highly fluorescent Tb(DPA)3 3-compound was triggered mainly by vesicle fusion but also by molecular transfer through the aqueous phase (Fresta et al., 1998). When encapsulated into those DPPS-containing vesicles, a 30-mer oligonucleotide complementary to a sequence of β-endorphin mRNA located in the 3′ region of the gene was able to inhibit the biosynthesis of this hormone in a concentration-dependent manner (Fresta et al., 1998). On the

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contrary, the free 30-mer oligonucleotide did not provide any lowering of the βendorphin production. Fusogenic proteoliposomes were also developed to avoid intracellular degradation of oligonucleotides occurring via the lysosomal pathway. The principle behind this approach is to mimic the pathway used by several viruses to infect cells: cell recognition and binding, cell membrane merging and genome release into the cytoplasm, thus bypassing endocytosis and lysosomal degradation. The typical illustration of this strategy is the incorporation of inactivated Sendai virus (haemagglutinating virus of Japan, HVJ) into liposomes (Kaneda et al., 1987; Nakanishi and Okada, 1993). Those HVJ-liposomes take advantage of the properties of HVJ, a paramyxovirus (Okada, 1969) that can fuse with cell membranes at neutral pH because it contains two envelope proteins, haemagglutininneuraminidase and fusion protein, which mediate cell attachment and membrane fusion respectively (Scheid et al., 1972). Practically, HVJliposomes are prepared by mixing UV-inactivated HVJ with neutral liposomes based on phosphatidylserine, phosphatidylcholine and cholesterol (Morishita et al., 1994). HVJ-liposomes have acquired the property of fusing with the cell membrane, thus introducing directly into the cell cytoplasm molecules which do not penetrate easily into cells, such as DNA (Kaneda et al., 1987; Morishita et al., 1993; Tomita et al., 1992). It was postulated that HVJ-liposomes should be also an effective method for the in vivo delivery of oligonucleotides (Morishita et al., 1994). In this regard, the results of Morishita et al., (1994) are very convincing, showing that the HVJliposome method enhanced the stability of fluorescently labelled FITColigonucleotides in the vessel wall of rats’ injured carotid arteries, enabling detection up to one week after in vivo transfer. Moreover, nuclear localization of the fluorescence was observed. On the contrary, naked FITC-labelled oligos were localized notably within the cell endosomes, with the fluorescence disappearing within 24 h after transfection. In another study (Yamada et al., 1996), similar results were obtained after injection into the hypothalamus: the HVJ-liposome method produced a higher concentration and more persistent fluorescence than did the oligonucleotide alone with, again, a nuclear localization. Direct injection of HVJ-liposomes containing FITC-labelled oligonucleotides in the apex of rat’s heart resulted in the fluorescent labelling of myocytes for at least one week (Aoki et al., 1997). Measurement of fluorescence also demonstrated a significantly higher level in myocardium transfected by HVJ-liposomes than using cationic liposomes (Aoki et al., 1997). Immunostaining of nuclei with propidium iodide led to a mixture of the green fluorescence of FITC and the red fluorescence of propidium iodide, indicating the migration of the oligonucleotide into the nucleus. Importantly, direct injection of HVJ-liposomes seemed not to affect cardiac function since no change in ECG was observed (Aoki et al., 1997). Given the prolongation of oligonucleotides’ half-life with HVJ-liposome delivery, the in vivo feasibility of an antisense strategy to block neointimal

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hyperplasia was investigated in an experimental model of vascular injury of the rat carotid artery (Morishita et al., 1994). Administration of either antisense CDC2 kinase or antisense cyclin B1 encapsulated into HVJ-liposomes resulted in a partial inhibition of neointima formation after balloon injury. The sense and scrambled oligonucleotides failed to show any inhibitory effect. The administration of a mixture of anti-CDC2 kinase/cyclin B1 into HVJ-liposomes led to an inhibition of neointimal formation for at least eight weeks (Morishita et al., 1994). The HVJ-liposome-antisense therapeutic approach is based on a very attractive biomimetic concept which, however, remains limited by the probable immunogenicity of this preparation. Recently, a novel lipidic system has been developed for selective delivery of oligonucleotides to target cells (Li and Huang, 1998). It involves the complexation of oligonucleotides with polylysine, followed by the addition of pH-sensitive liposomes that contain a targeting ligand, folate. These hybrid particles (polylysine/ oligonucleotides complex as a core, covered by DOPE/ CHEMS/folate-PEG-DOPE) are supposed to shield the positive charges of polylysine, thus reducing the non-desirable interactions with seric proteins. This construction was able to deliver the oligonucleotides selectively to human KB cells overexpressing the folate-binding protein (Li and Huang, 1998). The entrapment in those particles of oligonucleotides against epidermal growth factor receptors (EGFRs) resulted in dramatic downregulation of EGFR and also in cell growth inhibition, which the free oligonucleotide or the encapsulated scramble sequence did not (Li and Huang, 1998). 7.7 Conclusions Antisense technology has two important advantages over more traditional drug design: the target has a defined sequence which can be determined relatively easily, and the interaction occurs theoretically through base pairing, which leads to a very high specificity. However, these molecules have at least two drawbacks: they are rapidly degraded in the biological fluids and they pass through most of the biological barriers, including the cell membrane, with difficulty. One strategy to circumvent these problems is to associate these molecules with liposomes. These phospholipidic vesicles systems are conceived in order: • to protect the encapsulated oligonucleotides from degradation • to isolate them from the biological surrounding medium to avoid non-specific effects • to transport them to the biological cellular and/or tissular target • to allow their efficient cell internalization • to induce the cytoplasmic and/or nucleic delivery of the oligonucleotides.

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Unquestionably, none of the liposomal systems developed up to now can meet all these requirements. However, some of them can meet one or more. Thus, practically all the liposomal formulations described in this chapter are claimed to be able to protect oligonucleotides from degradation, mainly in vitro but also in vivo, and one can accept that liposomal encapsulation is, in general, an efficient technology to keep the sequences intact in a biological environment containing nucleases. To address oligonucleotides specifically to certain cells and/or tissues is a more complicated challenge, which was found to be possible in vitro using immunoliposomes. More complicated is the in vivo targeting, because of the opsonization process. The pEGgylation of the liposomes is a successful strategy for avoiding the non-specific recognition by the macrophages of the mononuclear phagocyte system, but there is no example showing whether pEGgylated liposomes covered with targeting molecules (antibodies, lectins, etc.) are really able to deliver in vivo oligonucleotides in a specific manner. In vitro, the efficient cell internalization and cytoplasmic delivery of oligonucleotides was found to be possible using either pH-sensitive liposomes or cationic liposomes. In the first case, the phase transition from a lamellar to a hexagonal phase of certain phospholipid and lipid mixtures allows fusion with the endosomal membrane and the cytoplasmic delivery of the liposomal content as soon as the pH of the endosome is decreasing. In vivo, these systems are inefficient because either those liposomes are unstable in the blood stream or they lose their pH sensitivity due to the adsorption of serum proteins. In the case of cationic liposomes, the excess of positive charges induces strong interactions with the biological environment, thus limiting the efficacy of this approach in vivo. Thus, the ideal carrier for oligonucleotides delivery in vivo is still to be discovered; however, the liposomal technologies have already solved a certain number of very challenging problems associated with the use of oligonucleotide in vitro. Future efforts should concentrate on the design of molecularly addressed cationic or pH-sensitive liposomes able to avoid opsonization and strong interactions with serum proteins. References ABE, T., HATTA, T., TAKAI, K., NAKASHIMA, H., YOKOTA, T. and TAKAKU, H., 1998, Inhibition of influenza virus replication by phosphorothioate and liposomally endocapsulated oligonucleotides, Nucleosides Nucleotides, 17, 471–478. AKHTAR, S., BASU, S., WICKSTROM, E. and JULIANO, R.L., 1991, Interactions of antisense DNA oligonucleotide analogs with phospholipid membranes (liposomes), Nucl. Acids Res., 19, 5551–5559. AKHTAR, S. and JULIANO, R.L., 1992, Liposome delivery of antisense oligonucleotides: adsorption and efflux characteristics of phosphorothioate oligodeoxynucleotides, J. Control. Rel., 22, 47–56.

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DE OLIVEIRA, M.C., FATTAL, E., COUVREUR, P., LESIEUR, P., BOURGAUX, C., OLLIVON, M. and DUBERNET, C., 1998, pH-sensitive liposomes as a carrier for oligonucleotides: a physico-chemical study of the interaction between DOPE and a 15-mer oligonucleotide in quasi-anhydrous samples, Biochim. Biophys. Acta, 1372, 301–310. DE, OLIVEIRA, M.C., FATTAL, E., ROPERT, C., MALVY, C. and COUVREUR, P., 1997, Delivery of antisense oligonucleotides by means of pH-sensitive liposomes, J. Control. Rel., 48, 179–184. DEAN, N.M. and MCKAY, R., 1994, Inhibition of protein kinase C-alpha expression in mice after systemic administration of phosphorothioate antisense oligodeoxynucleotides, Proc. Natl Acad. Sci. USA, 91, 11762–11766. DESHPANDE, D., TOLEDO-VELASQUEZ, D., THAKKAR, D., LIANG, W. and ROJANASAKUL, J., 1996, Enhanced cellular uptake of oligonucleotides by EGF receptor-mediated endocytosis in A549 cells, Pharm. Res., 13, 57–61. DUZGUNES, N., SRAUBINGER, R.M., BALDWIN, P.A., FRIEND, D.S. and PAPAHADJOPOULOS, D., 1985, Proton induced fusion of oleic acidphosphatidylethanolamine liposomes, Biochemistry, 24, 3091–3098. FELGNER, P.L. and RINGOLD, G.M., 1989, Cationic liposome-mediated transfection, Nature, 337, 387–388. FRESTA, M., CHILLEMI, R., SPAMPINATO, S., SCIUTO, S. and PUGLISI, G., 1998, Liposomal delivery of a 30-mer antisense oligodeoxynucleotide to inhibit propiomelanocortin expression, J. Pharm. Sci., 87, 616–625. HALLER, H., DRAGUN, D., MIETHKE, A., PARK, J.K., WEIS, A., LIPPOLDT, A., GROSS, V. and LUFT, F.C., 1996, Antisense oligonucleotides for ICAM-1 attenuate reperfusion injury and renal failure in the rat, Kidney Int., 50, 473–480. HATTA, T., NAKAGAWA, Y., TAKAI, K., NAKADA, S., YOKOTA, T. and TAKAKU, H., 1996, Inhibition of influenza virus RNA polymerase and nucleoprotein genes expression by unmodified, phosphorothioated, and liposomally encapsulated oligonucleotides, Biochem. Biophys. Res. Commun., 223, 341–346. HATTA, T., TAKAI, K., NAKADA, S., YOKOTA, T. and TAKAKU, H., 1997, Specific inhibition of influenza virus RNA polymerase and nucleoprotein gene expression by liposomally endocapsulated antisense phosphorothioate oligonucleotides: penetration and localization of oligonucleotide in clone 76 cells, Biochem. Biophys. Res. Commun., 232, 545–549. HIDETOSHI, A., YUKIHIKO, A. and SEISHI, T., 1997, Effects of oligodeoxynucleotides on the physicochemical characteristics and cellular uptake of liposomes, J. Pharm. Sci., 86, 438–442. HUGHES, J.A., ARONSOHN, A.I., AVRUTSKAYA, A.V. and JULIANO, R.L., 1996, Evaluation of adjuvants that enhance the effectiveness of antisense oligodeoxynucleotides, Pharm. Res., 13, 404–410. JAASKELAINEN, I., MÖNKKÖNEN, J. and URTTI, A., 1994, Oligonucleotide-cationic liposome interactions. A physicochemical study, Biochim. Biophys. Acta, 1195, 115–123. JAASKELAINEN, I., STERNBERG, B., MONKKONEN, J. and URTTI, A., 1998, Physico-chemical and morphological properties of complexes made of cationic liposomes and oligonucleotides, Int. J. Pharm., 167, 191–203. JAROSZEWSKI, J.W. and COHEN, J.S., 1991, Cellular uptake of antisense oligodeoxynucleotides, Adv. Drug Del. Rev., 6, 235–250.

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KANAMARU, T., TAKAGI, T., TAKAKURA, Y. and HASHIDA, M., 1998, Biological effects and cellular uptake of c-myc antisense oligonucleotides and their cationic liposome complexes , J. Drug Target., 5, 235–246. KANEDA, Y., UCHIDA, T., KIM, J., ISHIURA, M. and OKADA, Y., 1987, The improved efficient method for introducing macromolecules into cells using HVJ (sendai virus) liposomes with gangliosides, Exp. Cell. Res., 173, 56–69. KONO, K., IGAWA, T. and TAKAGISHI, T., 1997, Cytoplasmic delivery of calcein mediated by liposomes modified with a pH-sensitive polyethylene glycol) derivative, Biochim. Biophys. Acta, 143–154. KONO, K., ZENITANI, K. and TAKAGISHI, T., 1994, Novel pH-sensitive liposomes: liposomes bearing polyethylene glycol) derivative with carboxyl groups, Biochim. Biophys. , 1193, 1–9. LAPPALAINEN, K., URTTI, A., JÄÄSKELÄINEN, I., SYRJÄNEN, K. and SYRJÄNEN, S., 1994, Cationic liposomes mediated delivery of antisense oligonucleotides targeted to HPV 16 E7 mRNA in CaSki cells, Antiviral Res., 23, 119–130. LAPPALAINEN, K., PIRILÄ, L., JÄÄSKELÄINEN, I., SYRJÄNEN, K. and SYRJÄNEN, S., 1996, Effects of liposomal antisense oligonucleotides on mRNA and protein levels of the HPV 16 E7 oncogene , Anticancer Res., 16, 2485–2492. LAPPALAINEN, K., MIETTINEN, R., KELLOKOSKI, J., JÄÄSKELÄINEN, I. and SYRJÄNEN, S., 1997, Intracellular distribution of oligonucleotides delivered by cationic liposomes: light and electron microscopic study, J. Histochem. Cytochem., 45, 265–274. LEAMON, C.P. and LOW, P.S., 1991, Delivery of macromolecules into living cells: a method that exploits folate receptor endocytosis, Proc. Natl Acad. Sci. USA, 88, 5572–5576. LEE, R.J. and LOW, P.S., 1995, Folate-mediated tumor cell targeting of liposomeentrapped doxorubicin in vitro, Biochim. Biophys. Acta, 1233, 134–144. LEONETTI, J.P., MECHTI, N., DEGOLS, G., GAGNOR, C. and LEBLEU, B., 1991, Intracellular distribution of microinjected antisense oligonucleotides, Proc. Natl Acad. Sci. USA, 88, 2702–2706. LESERMAN, L., BARBET, J., KOURILSKY, F. and WEINSTEIN, J.N., 1980, Targeting to cells of fluorescent liposomes covalently coupled with monoclonal antibody or protein A, Nature, 288, 602–604. LI, S. and HUANG, L., 1998, Targeted delivery of antisense oligodeoxynucleotides formulated in a novel lipidic vector, J. Liposomal Res., 8, 239–250. LITZINGER, D. and HUANG, L., 1992, Phosphatidylethanolamine liposomes: drug delivery, gene transfer and immunodiagnostic applications, Biochim. Biophys. Acta, 1113, 201–227. LIU, D. and HUANG, L., 1989a, Role of cholesterol in the stability of pH-sensitive large unilamellar liposomes prepared by the detergent dialysis method, Biochim. Biophys. Acta, 981, 254–260. LIU, D. and HUANG, L., 1989b, Small, but not large unilamellar liposomes composed of dioleylphosphatidylethanolamine and oleic acid can be stabilized by human plasma, Biochemistry, 28, 7700–7707. LIU, D. and HUANG, L., 1990, pH-sensitive, plasma stable liposomes with relatively prolonged residence in circulation, Biochim. Biophys. Acta, 1022, 348–354.

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LOKE, S.L., STEIN, C.A., AVIGAN, M., COHEN, J.S. and NECKERS, L.M., 1998, Delivery of c-myc antisense phosphorothioate oligodeoxynucleotides to hematopoietic cells in culture by liposome fusion: specifie reduction in c-myc protein expression correlates with inhibition of cell growth and DNA synthesis, Curr. Top. Microbiol. Immunol., 141. 282–289. MA, D.D. and WEI, A.Q., 1996, Enhanced delivery of synthetic oligonucleotides to human leukemic cells by liposomes and immunoliposomes, Leuk. Res., 20, 925–930. MACHY, P., BARBET, J. and LESERMAN, L.D., 1982a, Differential endocytosis of T and B lymphocyte surface molecules evaluated with antibody-bearing fluorescent liposomes containing methotrexate, Proc. Natl Acad. Sci. USA, 79, 4148–4152. MACHY, P. and LESERMAN, L.D., 1983, Small liposomes are better than large liposomes for specific drug delivery in vitro, Biochim. Biophys. Acta, 730, 313–320. MACHY, P., PIERRES, M., BARBET, J. and LESERMAN, L.D., 1982b, Drug transfer into lymphoblasts mediated by liposomes bound to distinct sites on H-2 encoded IA, I-E, and K molecules, J. Immunol., 129, 2098–2102. MATSUDA, M., PARK, J.G., WANG, D.C., HUNTER, S., CHIEN, P. and SCHREIBER, A.D., 1996, Abrogation of the Fc gamma receptor IIA-mediated phagocytic signal by stem-loop Syk antisense oligonucleotides, Mol. Biol. Cell, 7, 1095–1106. MEYER, O., KIRPOTIN, D., HONG, K., STERNBERG, B., PARK, J.W., WOODLE, M.C. and PAPAHAJOPOULOS, D., 1998, Cationic liposomes coated with polyethylene glycol as carriers for oligonucleotides, J. Biol. Chem., 25, 15621–15627. MORISHITA, R., GIBBONS, G.H., KANEDA, Y., OGIHARA, T. and DZAU, V.J., 1993, Novel and effective gene transfer technique for study of vascular renin angiotensin system, J. Clin. Invest., 91, 2580–2585. MORISHITA, R., GIBBONS, G., KANEDA, Y., OGIHARA, T. and DZAU, V., 1994, Pharmacokinetics of antisense oligodeoxyribonucleotides (cyclin B1 and CDC 2 kinase) in the vessel wall in vivo: enhanced therapeutic utility for restenosis by HVJliposome delivery, Gene, 149, 13–19. NAKANISHI, M. and OKADA, Y., 1993, Liposome-mediated introduction of macromolecules into living animal cells with the aid of HVJ (sendai virus), Liposome Technology: Entrapment of drugs and other materials, Gregoriadis, G., ed., pp. 249–260, Boca Raton, FL: CRC Press. OGO, H., HIRAI, Y., MIKI, S., NISHIO, H., AKIYAMA, M. and NAKATA, Y., 1994, Modulation of substance P/neurokinin-1 receptor in human astrocytoma cells by antisense oligodeoxynucleotides, Gen. Pharmacol., 25, 1131–1135. OKADA, Y., 1969, Factors in fusion of cells by HVJ, Curr. Top. Microbiol. Immunol., 48, 102–128. OLLIKAINEN, H., LAPPALAINEN, K., JÄÄSKELÄINEN, I., SYRJÄNEN, S. and PULKKI, K., 1996, Liposomal targeting of Bc1–2 antisense oligonucleotides with enhanced stability into human myeloma cell lines, Leukemia Lymphoma, 24, 165–174. PHILLIPS, M.I., 1997, Antisense inhibition and adeno-associated viral vector delivery for reducing hypertension, Hypertension, 29, 177–187. RHODES, D.G. and LIU, J., 1996, Divalent cation mediated binding of oligonucleotides to Langmuir monolayers of charged lipids, Langmuir, 12, 1879–1883.

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ROPERT, C., LAVIGNON, M., DUBERNET, C., COUVREUR, P. and MALVY, C., 1992, Oligonucleotides encapsulated in pH-sensitive liposomes are efficient toward Friend retrovirus, Biochem. Biophys. Res. Commun., 183, 879–889. ROPERT, C., MALVY, C. and COUVREUR, P., 1993, Inhibition of the Friend retrovirus by antisense oligonucleotides encapsulated in liposomes: mechanism of action, Pharm. Res., 10, 1427–1433. ROPERT, C., MISHAL, Z., JR, RODRIGUES, J.M., MALVY, C. and COUVREUR, P., 1996, Retrovirus budding may constitute a port of entry for drug carriers, Biochim. Biophys. Acta. 1310. 53–59. ROY, S. and ROTH, T., 1997, Proliferative effect of high glucose is modulated by antisense oligonucleotides against fibronectin in rat endothelial cells, Diabetologia, 40, 1011–1017. SCHEID, A., CALIGUIRI, L.A., COMPANS, R.W. and CHOPPIN, P.W., 1972, Isolation of paramyxovirus glycoproteins, association of both hemagglutining and neuraminidase activities with larger SV 5 glycoprotein, Virology, 50, 640–652. SELVAM, M.P., BUCK, S.M., BLAY, R.A., MAYNER, R.E., MIED, P.A. and EPSTEIN, J.S., 1996, Inhibition of HIV replication by immunoliposomal antisense oligonucleotide, Antiviral Res., 33, 11–20. SLEPUSHKIN, V.A., SIMOENS, S., DAZIN, P., NEWMAN, M.S., GUO, L.S., PEDROSO DE LIMA, M.C. and DUZGUNES, N., 1997, Sterically stabilized pHsensitive liposomes. Intracellular delivery of aqueous contents and prolonged circulation in vivo, J. Biol. Chem., 272, 2382–2388. STEIN, C.A., PAL, R., DE VICO, A.L., HOKE, G., MUMBAUER, S., KINSTLER, O., SARNGADHARAN, M.G. and LETSINGER, R.L., 1991, Mode of action of 5′linked cholesteryl phosphorothioate oligodeoxynucleotides in inhibiting syncitia formation and infection by HIV-1 and HIV-2 in vitro, Biochemistry, 30, 2439–2444. TARI, A.M., TUCKER, S.D., DEISSEROTH, A. and LOPEZ-BERESTEIN, G., 1994, Liposomal delivery methylphosphonate antisense oligodeoxynucleotides in chronic myelogenous leukemia, Blood, 84, 601–607. TARI, A.M., ANDREEFF, M., KEEINE, H.D. and LOPEZ-BERESTEIN, G., 1996a, Cellular uptake and localization of liposomal-methylphosphonate oligodeoxynucleotides, J. Mol Med., 74, 623–628. TARI, A., KHODADADIAN, M., ELLERSON, D., DEISSEROTH, A. and LOPEZBERESTEIN, G., 1996b, Liposomal delivery of oligodeoxynucleotides, Leukemia Lymphoma, 21, 93–97 . TARI, A.M., STEPHENS, C., ROSENBLUM, M. and LOPEZ-BERESTEIN, G., 1998, Pharmacokinetics, tissue distribution, and safety of P-ethoxy oligonucleotides incorporated in liposomes, J. Lipos. Res., 251–264. THIERRY, A.R. and DRITSCHILO, A., 1992a, Intracellular availability of unmodified, phosphorothioated and liposomally encapsulated oligodeoxynucleotides for antisense activity, Nucl Acids Res., 20, 5691–5698. THIERRY, A.R. and DRITSCHILO, A., 1992b, Liposomal delivery of antisense oligodeoxynucleotides, Antisense Strat., 660, 300–302. THIERRY, A.R., RAHMAN, A. and DRITSCHILO, A., 1992, Liposomal delivery as a new approach to transport antisense oligonucleotides, Gene Regu. Biol. of Antisense RNA DNA, 147–160.

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THIERRY, A.R., RAHMAN, A. and DRITSCHILO, A., 1993, Overcoming multidrug resistance in human tumor cells using free and liposomally encapsulated antisense oligodeoxynucleotides, Biochem. Biophys. Res. Commun, 190, 952–960. TOMITA, N., HIGAKI, J., MORISHITA, R., KATO, K., MIKAMI, H., KANEDA, Y. and OGIHARA, T., 1992, Direct in vivo gene introduction into rat kidney, Biochem. Biophys. Res. Commun., 186, 129–134. WAGNER, R.W., MATEUCCI, M.D., LEWIS, J.G., GUTTIEREZ, A.J., MOULDS, C. and FROEHLER, B.C., 1993, Antisense gene inhibition by oligonucleotides containing C-5 propyne pyrimidines, Science, 260, 1510–1513. WANG, S., LEE, R.J., CAUCHON, G., GORENSTEIN, D. and LOW, P., 1995, Delivery of antisense oligonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via ethylene glycol, Proc. Natl Acad. Sci. USA, 92, 3318–3322. WIELBO, D., SHI, N. and SERNIA, C., 1997, Antisense inhibition of angiotensinogen in hepatoma cell culture is enchanted by cationic liposome delivery, Biochem. Biophys. Res. Commun, 232, 794–799. WIELBO, D., SIMON, A., PHILLIPS, M.I. and TOFFOLO, S., 1996, Inhibition of hypertension by peripheral administration of antisense oligodeoxynucleotides, Hypertension, 28, 147–151. WILHELM, O., SCHMITT, M., HOHL, S., SENEKOWITSCH, R. and GRAEFF, H., 1995, Antisense inhibition of urokinase reduces spread of human ovarian cancer in mice, Clin. Exp. Metastasis, 13, 296–302. WROBEL, I. and COLLINS, D., 1995, Fusion of cationic liposomes with mammalian cells occurs after endocytosis, Biochim. Biophys. Acta, 1235, 296–304. WU-PONG, S., WEISS, T.L. and HUNT, C.A., 1992, Antisense c-myc oligodeoxyribonucleotide cellular uptake, Pharm. Res., 9, 1010–1017. YAMADA, K., MORIGUCHI, A., MORISHITA, R., AOKI, M., NAKAMURA, Y., MIKAMI, H., OSHIMA, T., NINOMIYA, M., KANEDA, Y., HIGAKI, J. and OGIHARA, T., 1996, Efficient oligonucleotide delivery using the HVJ-liposome method in the central nervous system, Am. J. Physiol., 271, R1212–R1220. YAO, Z.Q., ZHOU, Y.X., GUO, J., FENG, Z.H., FENG, X.M., CHEN, C.X., JIAO, J.Z. and WANG, S.Q., 1996, Inhibition of hepatitis B virus in vitro by antisense oligonucleotides, Acta Virol., 40, 35–39. ZELPHATI, O., IMBACH, J.L., SIGNORET, N., ZON, G., RAYNER, B. and LESERMAN, L., 1994b, Antisense oligonucleotides in solution or encapsulated in immunoliposomes inhibit replication of HIV-1 by several different mechanisms, Nucl. Acids Res., 22, 4307–4314. ZELPHATI, O. and SZOKA, F.C., JR, 1996a, Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids, Pharm. Res., 13, 1367–1372. ZELPHATI, O. and SZOKA, F.C., JR, 1996b, Mechanism of oligonucleotide release from cationic liposomes, Proc. NatlAcad. Sci. USA, 93, 11493–11498. ZELPHATI, O., WAGNER, E. and LESERMAN, L., 1994a, Synthesis and anti-HIV activity of thiocholesteryl-coupled phosphodiester antisense oligonucleotides incorporated into immunoliposomes, Antiviral Res., 25, 13–25. ZELPHATI, O., ZON, G. and LESERMAN, L., 1993, Inhibition of HIV-1 replication in cultured cells with antisense oligonucleotides encapsulated in immunoliposomes, Antisense Res. Dev., 3, 323–338.

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8 Comb-type Polycation Copolymer for Antigène Strategy and DNA Carrier A. MARUYAMA

8.1 Introduction Despite a lot of efforts in the past decade, medicinal application of oligonucleotides is still hampered by insufficient delivery of oligonucleotides to the proper site of action, and a low ratio of specific to non-specific interactions. Several modifications of oligonucleotides have been adopted to overcome these issues (see also Chapters 2, 3 and 4). Though such strategies partially solved the problems, an ideal and/or general strategy that circumvents them has not yet been established. Synthetic polymers offer the opportunity for preparing tailor-made carriers, properly designed, and presenting all the desired features and moieties required for oligonuclotide and gene delivery. Block and graft copolymers, which consist of more than two polymer segments with different properties, are particularly interesting because the properties and functions of constituting polymer segments are usually preserved in the copolymers, enabling us to design welldefined DNA carriers which may fulfil required functions for controlled delivery of DNA. Further, the unique self-assembling and/or phase-separating properties of the copolymers occasionally led to unexpected findings at the interface of the biological system. This chapter focuses on unique properties of tailor-made graft (comb-type) copolymers in context of gene therapy and antigène strategy. 8.2 Comb-type Polycations as a Stabilizer for DNA Duplex and Triplex Intermolecular triplex DNA formation by sequence-specific interaction of triplexforming oligonucleotides (TFOs) to the major groove of a short homopurinehomopyrimidine stretch in native duplex DNA can be a designed strategy (i.e. an antigène strategy) for artificial gene represser through manipulating gene expression, gene-targeted mutagenesis and inhibition of viral propagation (Hélène, 1991; Grigoriev et al., 1993; Wang et al., 1996; Moser and

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Dervan, 1987). However, triplexes of either the purine motif (Beal and Dervan, 1991; Pilch et al., 1991) or the pyrimidine motif (Moser and Dervan, 1987; Rajagopal and Feigon, 1989) are unstable under physiological conditions and thus impede their therapeutic applications. For example, formation of the pyrimidine motif triplex DNA needs conditions of low pH (<6.0), because the unmodified cytosine residues, if present in the pyrimidine-rich TFOs, are to be protonated to bind with the guanine (G) of the G:C duplex (Moser and Dervan, 1987; Singleton and Dervan, 1992). In contrast, although the purine motif triplex DNA is pH-independent, triplexes involving guanine-rich (G-rich) TFOs are severely inhibited by physiological concentrations of certain monovalent cations (M+), especially K+ (Milligan et al., 1993; Cheng and Van Dyke, 1993). To date, numerous strategies, such as modification or substitution of cytosines in pyrimidine-rich TFO with non-natural bases and/or conjugation of triplexspecific or non-specific DNA intercalators to TFO, have been reported to improve triplex stability through pH-independent triplex formation to a greater extent than the regular TFO (Frank-Kamenetskii and Mirkin, 1995; Marchand et al., 1996; Silver et al., 1997). On the other hand, a chemical modification strategy with G-rich TFOs has partially overcome the inhibitory effect of K+ on purine motif triplex formation (Olivas and Maher, 1995; Gee et al., 1995; Vasquez et al., 1995; Dagle and Weeks, 1996; Faruqi et al., 1997; Joseph et al., 1997). Despite extensive efforts, significant stabilization of triplex DNA under physiological conditions is, however, yet to be achieved. A common source of the instability of triplex DNA can be ascribed to unfavourable electrostatic and entropie contribution caused by high accumulation of nucleotide phosphate anions along the triplex structure. Cationic substances such as polyamines (e.g. spermine, spermidine and putrescine) stabilized duplexes and triplexes (Hampel et al., 1991, Hanvey et al., 1991, Thomas and Thomas, 1993). Polyamines associated with DNA perturb the electrostatic potential from triplexes and duplexes. Their effect was, however, reduced considerably under physiological conditions, because association of polyamines with DNA was hampered by coexisting cations (Hanvey et al., 1991; Thomas and Thomas, 1993; Murray and Morgan, 1973). In order to enhance association of cationic substance to DNA and reduce electrostatic obstacles that accompany DNA duplex and triplex formation, macromolecular polycations such as poly(Llysine) (PLL) and poly(L-arginine) are more effective than oligovalent cations, leading to a considerable rise in the melting temperature (rj of the duplex (Olins et al., 1967; Tsuboi, 1967). Polycations, however, interact strongly with polyanions to form irreversible polyion complexes (or inter-polyelectrolyte complexes) (Tsuboi, 1967; von Hippel and McGhee, 1972). Polycations severely compacted the DNA conformation (Haynes et al., 1970; Wagner et al., 1991). Coacervation or precipitation of the complex occurred (Olins et al., 1967, von Hippel and McGhee, 1972). Single-stranded (ss) DNAs rarely form duplexes and triplexes in the presence of polycations, resulting in irreversibility of duplex and triplex transitions (melting and reassociation).

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Figure 8.1 Structural formula and schematic illustration of the PLL-g-Dex copolymer

The reversible transition of DNA might therefore be attainable by increasing the solubility of the complex and reducing the conformational changes of DNA. For this approach, the interactions of polycations with DNA have to be regulated. Modification of polycations with DNA-immiscible polymer chains such as uncharged hydrophilic polymer chains to interfere with these interactions and to improve the solubility of a DNA/polycation complex is a potential strategy for designing a polycationic stabilizer. We prepared comb-type copolymers of poly(L-lysine) with polysaccharide side chains (PLL-g-Dex, Figure 8.1) and evaluated their ability to stabilize DNA duplexes and triplexes. The comb-type copolymers were prepared by reductive amination reaction between the reducing end of dextran and e-amino groups of PLL (Maruyama et al., 1997a, 1997b, 1998). Despite the relatively high molecular weight of Dex, and hence the extremely low concentration of the reducing end of Dex in the reaction, it is possible to couple Dex to PLL with high efficacy, which allowed us to prepare various graft copolymers with well-defined graft structure including length and density of graft chains. First, we assessed the solubility of complexes between the comb-type copolymers and DNA by turbidity measurement. As shown in Figure 8.2, PLL homopolymer caused considerable turbidity when it was mixed with calf thymus DNA in phosphate buffered physiological saline (PBS). With increasing grafting

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Figure 8.2 Turbidity of electrostatically equivalent mixtures of DNA and the copolymer in phosphate-buffered saline (PBS). The copolymers were prepared with Dextran 4 (Mn = 2600, open circle) or Dextran T-10 (Mn = 5900, closed circle). Reprinted with permission from Bioconjugate Chemistry, 9, 292–299. Copyright 1998 American Chemical Society

degree (% mole fraction of dextran units coupled with polylysine) the turbidity of the mixture drastically reduced, resulting in homogeneous solution at grafting degree over 10 mol% (Maruyama et al., 1997b, 1998). Structural change of DNA was estimated by circular dichroism (CD) of the mixture. As mentioned above, PLL induced considerable change in CD spectrum. The change in CD spectrum was explained by distortion and base tilting of the B conformation or by the formation of chiral aggregates (von Hippel and McGhee, 1972; Haynes et al., 1970; Maestre and Reich, 1986; Keller and Bustamante, 1986; Kim et al., 1986). By contrast, no significant change in CD spectrum was observed for DNA/ copolymer mixtures. Similar results were obtained when the measurements were performed in a buffer with ionic strength two orders lower, where ionic interaction between DNA and the polymers should be significantly enhanced. Further, the copolymer does not alter CD spectrum of different DNAs such as poly(dA)•poly(dT) duplex. Complex formation between the copolymer and DNA was characterized by gel electrophoresis studies. The copolymer forms electrostatically stoichiometric complexes with either calf thymus DNA or 20mer oligodeoxynucleotide (ODN). This evidence revealed that the copolymer with a high degree of grafting forms a soluble and stoichiometric complex with DNA without inducing marked structural change of DNA. The ability of the copolymer to stabilize DNA duplexes and triplexes was estimated by melting temperature measurements (Maruyama et al., 1997b). Figure 8.3 shows UV-melting curves of poly(dA)•2poly(dT) triplex in the presence or absence of PLL-Dex copoloymer. The melting of DNA duplexes and triplexes is accompanied by hyperchromic effect owing to destacking of DNA bases. As shown in Figure 8.3, poly(dA)•2poly(dT) exhibits biphasic melting in PBS. The first transition at lower temperature (37°C) was the melting of the

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Figure 8.3 UV–Tm profile of poly(dA)•2poly(dT) triplex in the absence or presence of PLL-g-Dex copolymer (polymer/DNA charge ratio = 2) in 150 mM NaCI containing 10 mM sodium phophate (pH 7.2) and 0.1 mM EDTA. The UV–Tm curves were recorded at 0.2 K/min at final polynucleotide concentration of 14.5 (bp)(imol/l. Reprinted with permission from Bioconjugate Chemistry, 8, 3–6. Copyright 1998 American Chemical Society

triplex to a poly(dA)•poly(dT) duplex and a single-stranded DNA (ssDNA). The second transition at higher temperature (72°C) was that of the duplex. In the presence of small excess (based on charge ratio) of PLL-g-Dex copolymer, only one transition was observed at higher temperature (89°C). As magnitude of the hyperchromicity at Tm in the presence of the copolymer was equal to the sum of those at Tm1 and Tm2 in the absence of the copolymer, the transition is suggested to be a direct melting of the triplex to its constituting ssDNAs. The UV–Tm profile in the cooling process demonstrated reversibility of the transition even in the presence of the comb-type copolymer. We further explored the triplex reassociation in the presence of the copolymer by changing cooling rate. The triplex formation was not significantly delayed even if the cooling rate was increased from 0.2 K/min to 5 K/min (Figure 8.4). The copolymer was indicated to stabilize thermally the triplex and not to disturb triplex formation from ssDNAs. The results of circular dichromism measurements also supported the idea. As shown in Figure 8.5, whereas the CD spectrum of the poly(dA)•2poly(dT) triplex has no positive band near 220 nm, the poly(dA)•poly(dT) duplex has a positive band near this wavelength (Howard et al., 1992). The mixture of poly(dA)•2poly (dT) and the comb-type copolymers showed almost the same signals as the triplex alone, even immediately after heat treatment at 95°C. The efficacy of the comb-type copolymer for stabilizing the triplex was compared to that of spermine, a polyamine effective in triplex stabilization

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Figure 8.4 Association of poly(dA)•2poly(dT) triplex observed at various cooling rates in the absence (a) or presence (b) of PLL-g-Dex copolymer

(Thomas and Thomas, 1993), under the same conditions. An electrostatically equivalent amount ([amino group]polymer/[phosphate group]DNA) of the comb-type copolymer increased the Tm of the triplex by 50 K, while a large excess of spermine increased it by 20 K. Moreover, one-step melting of the triplex was observed in the presence of the comb-type copolymer at a stabilizer/DNA ratio over 1, whereas biphasic melting was still seen even in the presence of a large excess of spermine. The one-step melting and considerable increase in Tm observed with an electrostatically equivalent amount of the comb-type copolymer implied its stable association with DNA. as discussed below. Since the triplexes of poly(dA)•2poly(dT) contain no K+-sensitive G•G:C triplet or pH-sensitive C+•G:C triplet, it is crucial to study the stabilizing efficacy of the comb-type copolymer on triplex formation with native DNA sequences. For this purpose, we evaluated either reverse-Hoogsteen- or Hoogsteen-triplex formation in vitro using a 30bp homopurine-homopyrimidine stretch (located between −141 and −170 bp) of the rat αl (I) collagen gene promoter (Lichtler et al., 1989; Joseph et al., 1997) as a target duplex and its specific purine-(re verse Hoogsteen)and pyrimidine(Hoogsteen)-rich TFOs (Figure 8.6) by gel electrophoresis mobility shift assay (EMSA) (Ferdous et al., 1998a, 1998b). In the absence of M+ (Na+ and K+), the percentage of the reverse-Hoogsteen-triplex formation is increased in a dose-dependent manner with purine-rich TFO (Pu-20). An apparent dissociation constant (Kd) was calculated: 8×10−9 M. While the triplex formation was slightly inhibited in the presence of Na+ at three-fold higher than physiological level (33 mM), it was drastically diminished in the presence of physiologically relevant levels of K+ (150mM). Although considerable triplex formation was observed with 17 µM Pu-20 in the presence of K+, it would be quite difficult to introduce such a high concentration of TFO into the cells of a target tissue for therapeutic use. Therefore, it is of paramount importance to stabilize triplex structure while using very low concentrations of

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Figure 8.5 CD spectra at 25°C of poly(dA)•poly(dT) duplex, poly(dA)•2poly(dT) triplex and the 2:1 mixture of PLL-g-Dex copolymer and the triplex before and after heat treatment (HT) at 95°C for 5 min

Figure 8.6 ODN sequences of the target duplexes (T-1 and T-2) representing a portion of the oc1 collagen (I) gene located between −141 and −170 bp upstream of the transcription initiation site (Lichtler et al. 1989). T-2 was end-labelled and mixed with T-1 to prepare the labelled duplex

TFOs. We investigated whether the copolymer can overcome the K+-dependent inhibition of triplex formation. Figure 8.7 shows the effect of K+ on triplex formation with 0.17 µM Pu-20 in the absence or presence of the comb-type copolymer. In the absence of the copolymer, increasing concentration of K+ drastically inhibited triplex formation with an IC50 at 60 mM KC1, and triplex

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Figure 8.7 Analysis of purine motif triplex formation in the absence or presence of PLLg-Dex copolymer (polymer/DNA charge ratio = 2) with 0.17 µM Pu-20. Reprinted with permission from Nucleic Acid Research, 26, 3949–3954. Copyright 1998 Oxford University Press

formation was less than 10% at physiological concentration of K+. In the presence of the copolymer, almost no inhibitory effect of K+ was observed and we could not estimate IC50 up to 200 mM K+. It is obvious that the copolymer is capable of stabilizing Pu-motif triplex formation to overcome K+-induced destabilization of the triplex. The copolymer was compared with polyamines in their stabilizing effect under the same experimental conditions. The comb-type copolymer, but neither spermine nor spermidine, both at 0.2 mM final concentrations, was able to stabilize triplex DNA. An increase in polyamine concentraation to 1.0 mM also failed to overcome K+ inhibition (Ferdous et al., 1998b). Since pH-dependency of pyrimidine motif (Hoogsteen) triplex DNA severely limits its in vivo applications, we subsequently examined the PLL-Dex copolymer for pyrimidine motif triplex formation at physiologically relevant pH, pH 7.0 (Ferdous et al., 1998a). About 95% triplex DNA is formed when 0.5 µM of unmodified pyrimidine-rich TFO, Py-20 (Figure 8.6), is incubated with the target duplex at pH5.5 (Figure 8.8, lane 4), and the presence of the copolymer has further induced triplex formation (100%) (data not shown). No triplex formation was observed when Py-20 was incubated with the target duplex at pH 7.0 (lane 6). It is of interest that the presence of the copolymer during incubation at pH 7.0 produced 50% of the triplex DNA observed at pH 5.5 (lanes 4 and 8). It has been reported that spermine and spermidine can also stabilize pyrimidine motif triplex DNA at neutral pH (Hampel et al., 1991; Thomas and Thomas, 1993). High concentrations (1.0 mM) of spermine (lane 11) but not spermidine

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Figure 8.8 Effects of PLL-g-Dex, spermine (Spm) and spermidine (Spmd) on pyrimidine motif triplex formation at neutral pH with 1.7 mM Pu-20. The reaction mixture were incubated for 6 h at 37°C and pH 5.5 (lanes 1–4) or pH 7.0 (lanes 6–13) with (+) or without (−) PLL-g-Dex (lanes 1–9) and with 0.2 mM or 1.0 mM Spm (lanes 10 and 11 ) or Spmd (lanes 12 and 13) respectively. The samples were electrophoresed at pH 5.5 to separate the duplex (d) and Triplex (T) DNAs. Sonicated calf thymus DMA (t-DNA) was added just before the electrophoresis to dissociate the polymer from labelled DNAs. In lane 5, the reaction was processed identically as in lane 4 but without incubation, indicating that the triplex does not form during the gel electrophoresis at pH 5.5. Reprinted with permission from Nucleic Acid Research, 26, 3949–3954. Copyright 1998 Oxford University Press

(lane 13) slightly stabilize pyrimidine motif triplex DNA (−5%) at pH 7.0, but this is considerably less than what is found (50%) in the presence of the copolymer (lane 8). Since these studies demonstrated the remarkable stabilizing effect of the combtype copolymer on either pyrimidine or purine motif triplex formation, it is important to investigate the thermodynamic and kinetic effects of the copolymer. Isothermal titration calorimetry (ITC) enables us to obtain directly thermodynamical parameters such as equilibrium association constant (Ka), and free energy (∆G), entropic (∆S) and enthalpic (∆H) changes accompanied with triplex formation at a given temperature (Kamiya et al., 1996). We employed ITC measurement to obtain the inside of the stabilization effect of the copolymer on the triplex formation. For triplex formation between 15-mer-pyrimidine rich TFO and 23bp homopurine-homopyyrimidine target duplex at physiological pH and 25°C, Ka in the absence and presence of the comb-type copolymer, respectively, was estimated at 2.0 × 105 and 1.9 × 107, indicating that the copolymer increases Ka by two orders of magnitude (Torigoe et al., 1999). The result is consistent with the apparent Kd value obtained by EMSA. In contrast to the copolymer, spermine increased Ka by only 2.5-fold at the same stabilizer/

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Figure 8.9 Schematic illustration of counterion association with DNAs. Counterion condensation (ψ) increased with increasing phosphate ion density, i.e. single strand (ss) < double strand (ds) < triplex strand (ts) DNA. Duplex and triplex formation is accompanied by a counterion association process which is entropically unfavourable at low salt concentration

DNA charge ratio to that of the copolymer. It was further judged from ∆H and ∆S values obtained in the presence of the copolymer that the copolymer facilitates stoichiometric triplex formation even at physiological pH, where either DNA alone or the presence of spermine results in imperfect and nonstoichiometric triplex formation. The facilitating triplex formation in the presence of the copolymer was also demonstrated by kinetic analysis of triplex formation using a surface plasmon resonance (SPR) apparatus (Torigoe et al., 1999). The association rate constant of the TFO to the target duplex immobilized on SPR cell matrix was increased more than 45fold by the copolymer, whereas spermine at the same stabilizer/DNA charge ratio increased it only 3.6-fold. It was also estimated that the copolymer reduced the dissociation rate constant of the triplex by 0.66-fold, whereas spermine reduced it by 0.91-fold (Torigoe et al., 1999). The much larger Ka in the presence of the PLL-g-Dex copolymer resulted mainly from the increase in the association rate constant rather than the decrease in the dissociation rate constant. In other words, the copolymer is capable of promoting triplex formation. Using SPR apparatus we recently studied specificity of triplex formation using fully matched and mismatched TFOs. It was found that the copolymer stabilizes triplex DNA without changing specificity of TFOs (Torigoe et al., unpublished data). The mechanism of the triplex stabilization by the copolymer was discussed by taking the salt dependency of duplexes and triplexes transitions into account. The salt dependency of the DNA order-disorder transition has been documented by the counterion condensation and cylindrical cell Poissori-Boltzman theories, as originally proposed by Manning (1969) and elaborated by Manning (1972, 1976), Record (1975) and Record et al. (1976b). The high electrostatic potential from the negatively charged backbone of a linear nucleic acid results in the accumulation of counterions in the immediate vicinity of the nucleic acid to neutralize partially the closely spaced backbone phosphates. The extent (ψ) of

186 COMB-TYPE POLYCATION COPOLYMER

Figure 8.10 (a) Salt concentration dependency of Tm of 15-mer DMA duplex estimated in the absence (square) and presence (circle) of PLL-g-Dex copolymer. (b) Salt concentration dependencies of Tm of poly(dA)•poly(dT) duplex (open symbols) and poly (dA)•poly(dT) triplex (closed symbols) estimated in the absence (circle) and the presence of either 100 µM spermine (triangle) or PLL-g-Dex copolymer (square). The Tm of poly (dA)•poly(dT) duplex in the presence of PLL-g-Dex copolymer overlaps with that of poly (dA)•2poly(dT) triplex (Maruyama, 1999c)

the counterion condensation per phosphate, which is predicted by counterion condensation theory, is a function only of the linear charge density of the nucleic acid, the counterion charge, and dielectric constant of the solvent, and is independent of the bulk salt concentration (Manning, 1969). Since DNA in a duplex form has higher charge density than the single-stranded form (for a review, see Anderson and Record, 1982), double-strand formation is accompanied by an increase in counterion association, which is entropically unfavourable in a solution with low salt concentration. Also, triplex formation further increases counterion association (Figure 8.9). An increase in bulk salt concentration stabilizes the state with higher charge density, resulting in an increase in Tm of the helix-coil transition. At low to moderate monovalent salt (M +X−) concentrations (10−3 to 10−1 M), the T of DNA is function of the logarithm m of mean activity or concentration of M+ of the salt, as described (Manning, 1978) by (8.1) where ∆H is the helix-to-coil transition enthalpy, R is the gas constant (cal/mol per K), Nu is the number of phosphates per cooperative melting unit, and ∆ψ is the thermodynamic differential counterion association parameter per phosphate group. The 0.9 correction factor corresponds to the conversion of mean ionic activities to concentration, and 2.303 is the conversion from loge to log10 units. The ratio RTm2/∆H has been found experimentally to be constant for DNA within the small range of salt concentrations over which equation (8.1) is valid (Manning, 1978).

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We evaluated salt dependency of coil-helix transition of 15-bp duplex in the absence and presence of the comb-type copolymer (Maruyama et al., 1999e). The semilogarithmic plots of [Na+] versus Tm of the duplex are shown in Figure 8.10a. A straight relationship with a positive slope is seen for the plot obtained in the absence of the copolymer, indicating that the duplex formation is accompanied by an increase in the extent of the counterion condensation, i.e. a positive value of ∆ψ. On the other hand, log[Na+] dependency of Tm of the duplex in the presence of the copolymer was not significant, indicating that the extent of counterion condensation of Na+ is unchanged (∆ψ = 0) between the duplex form and the single-stranded form. The association of polycationic copolymer to DNA causes perturbation of the electrostatic potential surrounding the DNA and releases the condensed counterions of Na+. It could be concluded that the copolymer stabilizes the duplex by eliminating the counterion condensation effect. It was reported that the stable duplex formation between peptide nucleic acid (PNA) (see also Chapter 4) and DNA was ascribed to negligible counterion condensation effect owing to uncharged PNA backbone (Tomac et al., 1996). In contrast, the copolymer is unique because it can eliminate the counterion condensation effect on the duplex formation of unmodified DNAs. We extended this study to estimate involvement of counterion association in triplex formation of poly(dA)•2poly(dT) in the absence or presence of either the comb-type copolymer or spermine (Maruyama et al., 1999e). The plots of Tm versus log[Na+] in the absence or presence of stabilizers are shown in Figure 8.10b. A linear relationship with a positive slope between log[Na+] and Tm of poly(dA)•poly(dT) obtained in the absence of the stabilizers again demonstrates that the poly(dA)•poly(dT) duplex formation is accompanied by counterion association. Similarly, Tm of poly(dA)•2poly(dT) increases with [Na +] in the absence of stabilizers, indicating higher extent of counterion condensation at the triplex form than the single-stranded form and the duplex form. Though spermine significantly increases Tm of both the duplex and the triplex at lower [Na+], the effect of spermine is significantly reduced at an intermediate [Na+], i.e. physiological level of salts, as reported previously (Thomas and Thomas, 1993; Hampel et al., 1991). Binding of oligocationic ligands, such as polyamines, to DNA causes release of counterions into the bulk solution. The release of these counterions into a solution of low salt concentration causes a net increase in the entropy of the system, thus providing a major favourable component to the interaction free energy (Record et al., 1976a; Mascotti and Lohman, 1990). The monovalent salt dependence of the equilibrium binding constant, Kobs, for formation of the complex can be described (Record et al., 1976a) by (8.2) where z is net charge of the oligocation, indicating that zψ counterions should be thermodynamically released into bulk solution.

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As expected from equation (8.2), the binding constant of the cationic ligand decreases with increasing salt concentration. At low [Na+] the binding of spermine to the DNA is almost saturated, and shows the maximum stabilization effect. Increase in [Na+] to the physiological level, however, leads to dissociation of spermine. Indeed, at [Na+] over 160mM, spermine fails to stabilize poly(dA) •poly(dT) duplex, indicating considerable dissociation of spermine from the duplex. It is of interest that spermine still stabilizes the triplex at 150mM [Na+]. This can be explained by stronger association of spermine to the triplex than duplex owing to a larger extent of counterion condensation on the former than the latter. In the presence of the copolymer, both transitions of poly(dA)•2poly(dT) triplex and poly(dA)•poly(dT) duplex merge into one transition and their Tm values are unchanged over the [Na+] range. It is clear that PLL-g-Dex copolymer is capable of eliminating the counterion condensation effect accompanying not only duplex but also triplex transitions. As expected from equation (8.2), polyvalent cations such as PLL-g-Dex copolymer should have strong association to DNA compared with oligocations such as spermine. The association of polycations to DNA should be enhanced by a release of monovalent anions, X, that had been condensed in the vicinity of the polycationic chain. Although the binding constant of PLL-g-Dex to DNA must be a function of [NaCl] and decrease with increasing [NaCl], the binding of the copolymer to DNA may still be saturated at NaCl concentration over the physiological range, leading to the almost linear and flat relationship of Tm versus log[Na+]. This idea is supported by our previous observations that the electrostatically equivalent amount (P/D = 1) of the copolymer almost saturates the increase in Tm of poly(dA)•2poly(dT) triplex in physiological saline (Maruyama et al., 1997b) and abrogates the electrophoretic migration of 20-mer ODN (Maruyama et al., 1998). The stable association of the copolymer is further evidenced by the fact that the copolymer increases Tm of the duplex and triplex even at [Na+] = 1 M, at which spermine shows no stabilization effect on either the duplex or the triplex. In addition, we have shown that the copolymer/DNA complex is immediately dissociated by an addition of extra DNA (Maruyama et al., 1998; Ferdous et al., 1998a). Even though the copolymer has strong association to DNA, the association is seemingly highly exchangeable, allowing DNAs to recognize and associate each other. The strong but exchangeable complex formation between the copolymer and DNA is seemingly important to improve stability of DNA duplexes and triplexes without affecting reversibility of DNA transition. Recent studies indicated that the copolymer enhanced triplex-mediated protection against DNA cleavages by endonuclease (Ferdous et al., 1998e). The copolymer also promotes triplex formation to impede specific binding of transcription factor in nuclear extract that contains various components (Ferdous et al., unpublished data). From a practical point of view, polymeric stabilizer may be unfavourable for nuclear localization by simple diffusion. However, modification of ODN with

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hydrophilic polymers may result in prolonged circulation in the body or accumulation in a particular organ (Monfardini and Veronese, 1998) (see next section). Prolonged residence or accumulation of polymer/ODN conjugate in the vicinity of target cell may compensate for limitation of intracellular diffusion. Further, it is possible to modify the comb-type copolymers with other functional moieties to complement functions required for antigene strategy. On the other hand, as the comb-type copolymer significantly increases the association rate of polynucleotide, it may have wide applications in which nucleotide recognition is involved. 8.3 Comb-type Polycation Copolymers with Cell-specific Polysaccharide Side-chains as Cell-specific DNA Carrier Since the pioneering work on receptor-mediated transfection using PLL— asialoglycoprotein (ASGP) conjugates by Wu and Wu (1987, 1988) several ligands, such as transferrin (Wagner et al., 1990; Gotten et al., 1990) folate (Gottschalk et al., 1994), growth factors (Cristiano and Roth, 1996) and oligosaccharides (Plank et al., 1992), were conjugated with polycations such as PLL as non-viral vectors for a foreign gene (for review see Michael and Curiel, 1994; Guy et al., 1995; Cristiano, 1998) However, the insufficient transfection efficacy of these delivery systems limited their applications to research and medicinal tools. Lysosomal degradation and inadequate intracellular trafficking of the transgene in the target cells accounted for the insufficient expression of transgenes. In order to release DNA from lysosomal compartments and to facilitate transportation of DNA to the cytoplasm of the cells, endosomal lysis agents such as viral particles (Curiel et al., 1991; Cristiano et al., 1993) and viral peptide (Wagner et al., 1992) have been employed (see also Chapter 5). In addition to such inadequate processing at the target cells, non-specific uptake of the complexes by non-target organs and cells could be a factor which limits transfection efficacy of these delivery systems. Polycations including PLL are liable to form insoluble complex when they are mixed with DNA under physiological ionic conditions. Although soluble complex is obtainable under particular conditions such polycation excess or with particular procedures (Perales et al., 1994), colloidal stability of the resulting complexes in biological fluid should be not enough to suppress aggregation of complexes and their nonspecific interactions with serum components. Indeed, non-specific interaction of PLL/DNA or transferrin-PLL/DNA complex with cells was reported previously (Zatloukal et al., 1992; Curiel et al., 1991). Even though the soluble (dispersive) complex between PLL-ASGP and DNA resulted in higher transfection efficiency in vivo than the heterogeneous complexes, it was recently demonstrated that the efficacy of the soluble complex was considerably decreased by interactions with serum components (Lollo et al., 1997). Unsatisfactory colloidal stability and nonspecific interaction of the complex would lead to massive uptake by

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reticuloendothelial systems (RESs) and/or physical entrapment in microvascular vessels, as has been argued for liposome and nanoparticle drug carriers (see also Chapters 6 and 7) (Gregoriadis and Neerunjun, 1974; Scherphof et al., 1985; Simon et al., 1995). Thus, such complexes resulted in less accumulation in the target cells or organ, even though the complex was provided with ligand molecules which have strong affinity to the target cells. Both improvement of colloidal stability and suppression of non-specific interactions of DNA/ polycation complexes with serum components and cells are considered among the most vital issues to achieve cell-specific gene delivery. The molecular exclusion effect of water-soluble polymers, such as poly(ethylene oxide) (PEO) and polysaccharide, has been proved effective to reduce these non-specific interactions and to increase dispersion stability of colloidal drug carriers. Increasing blood circulation, i.e. stealth feature, of drug carriers including liposomes (Klibanov et al., 1990; Blume and Cevc, 1990), polymer micelles (Yokoyama et al., 1991), and nanoparticles (Illum et al., 1987) has been demonstrated when drug carriers’ surfaces were properly modified with these water-soluble polymers (for review, see Monfardini and Veronese, 1998). In order to take advantage of the molecular exclusion effect of water-soluble polymers, several researchers have synthesized series of block Table 8.1 ζ-potentials of nanoparticles treated with BSA Code

NPs alone

+BSA

PLL PLL-Amy (15–3000) PLL-Dex (7–5900)

+27.0 +3.3 +4.8

−3.3 +3.1 +4.3

Nanoparticles bearing PLL homopolymer, amylose (MW = 3000, 15 mol% grafting) grafted PLL, PLL-Amy (15-3000), or dextran (MW = 5900, 7 mol% grafting) grafted PLL were dispersed in PBS at 60 µg/ml. To 5 ml nanoparticles suspension, 1 mg BSA was added from stock solution. After 1 h incubation, ζpotential was measured. (Reprinted with permission from Bioconjugate Chem., 8, 735–742. Copyright 1997 American Chemical Society.)

copolymers consisting of PEO and cationic polymers to construct DNA carriers (Kabanov et al., 1995; Wolfert et al., 1996; Kataoka et al., 1996). To create a targeted delivery vector exhibiting the molecular exclusion effect, we have focused on polycation comb-type copolymer with polysaccharide side-chains, because various lectins that specifically recognize and bind particular saccharide chains have been identified in animal cells. As mentioned in the previous section, the polylysine comb-type copolymers with dextran side-chains form soluble complexes with DNA in physiological saline (Maruyama et al., 1997b, 1998). The polysaccharide chains of the copolymer are seemingly extruded to the complex surface to form a glycocalyxlike structure, which grants solubility and dispersive stability of the complex and reduced non-specific interaction with serum components and cells. These

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characteristics of the complexes between DNA and the comb-type copolymers were previously judged from observations employing nanoparticles whose surfaces were immobilized with the copolymers (Maruyama et al., 1997a, 1999b). The nanoparticles having the comb-type copolymer on their surfaces showed higher dispersive stability in water than bare nanoparticles or nanoparticles with PLL homopolymer. The copolymer nanoparticles are also stably dispersed in the phosphate buffer solution that caused severe aggregation of PLL nanoparticles. Surface ζ-potential measurements indicated that polysaccharide segments preferentially distributed over PLL chains in the outer surfaces of the nanoparticles. While PLL nanoparticles adsorbed a considerable amount of bovine serum albumin (BSA), the copolymer nanoparticles slightly adsorb BSA, as detemined by adsorption isotherm study. As summarized in Table 8.1, treatment of nanoparticles with BSA solution did not change surface ζpotential of the copolymer particles, whereas the same treatment drastically changed that of PLL nanoparticles owing to BSA adsorption. Irrespective of positively charged PLL chains, the interaction with negatively charged BSA is impeded by the polysaccharide graft chains, probably owing to shielding and molecular exclusion effects of polysaccharide chains. The ability of polysaccharide moieties to bind specifically to lectins was subsequently evaluated by aggregation test. The copolymer nanoparticles selectively aggregated by an addition of corresponding lectin, but did not aggregate in the presence of a competitive saccharide. These observations with copolymerimmobilized nanoparticles supported the potentiality of polysaccharide-grafted PLLs as a DNA delivery vector. The copolymer nanoparticles themselves were considered useful as carriers and/or reservoirs of oligonucleotides for sustained release (Maruyama et al., 1999b). To construct a cell-specific carrier based on the comb-type copolymer structure, we studied hyaluronic acid (HA) and its counter-receptors. HA is an unbranched high-molecular weight polysaccharide consisting of alternating Nacetyl-β-D-glucosamin and β-D-glucuronic acid residues linked at the 1–3 and 1– 4 positions, respectively (for review, see Laurent and Fraser, 1992). Liver sinusoidal endothelial cells (SECs) possess the receptors that recognize and internalize HA (Forsberg and Gustafson, 1991; Yannariello-Brown et al., 1992). More than 90% of HAs in the blood stream are known to be taken up and metabolized by SECs. Because SECs play a key role in critical responses to both physiologic and pathophysiologic stimuli, modulation of SEC functions by gene or antigene therapies could open a new therapeutic strategy for treatment of liver diseases. The comb-type copolymer (PLL-g-HA) consisting of a PLL backbone and HA graft chains was prepared by enzymic hydrolysis of high molecular weight HA by hyaluronidase (EC 3.2.1.35), followed by reductive animation reaction between the reducing end of HA and amino groups of PLL, as shown in Figure 8.11 (Asayama et al., 1998). Since HA is a polyanion, PLL-g-HA undergoes self-polyelectrolyte complex formation between the PLL backbone

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and HA sidechains, which might interfere with complex formation with DNA. We carried out a 1H-NMR study to estimate if the copolymer has an ability to form complex with DNA. The DNA was mixed with the PLL-g-HA at charge ratio ([amino group]copolymer/[phosphate group]DNA= 1), and the ionic strength of the medium was gradually decreased from 1 to 0 M NaCl by step-down dialysis. The transparent solution obtained after dialysis was then lyophilized, dissolved in D2O, and analysed by 1H-NMR. The 1H-NMR spectra of the copolymer with or without DNA are shown in Figure 8.12. It is worth noting that the signals of emethylene protons of the PLL moiety in the comb-type copolymer broadened in the presence of DNA (Figure 8.12c). This is probably caused by interpolyelectrolyte complex formation between the PLL moiety and DNA. Although the PLL signals broadened, the HA signals remained unchanged or became sharper. The sharpening signals of HA in the presence of DNA under low-ionic strength conditions (Figure 8.12c) seem to be similar to those of the PLL-g-HA copolymer alone at high ionic strengths (Figure 8.12a), where the PLL-g-HA did not form self-polyelectrolyte complexes; i.e. HA chains were free. These results suggest that the PLL backbone selectively formed the interpolyelectrolyte complex with DNA even in the presence of the HA sidechains. The preferential complex formation was further demonstrated by the acrylamide gel electrophoresis analysis of PLL-g-HA/fluorescence-labelled ODN (F-ODN) mixture. As shown in Figure 8.13, the PLL-g-HA that has excess negative charge due to high content of HA impeded the migration of 38 mer FODN at an almost electrostatically equivalent point. The preferential complex formation of DNAs to HA chains probably resulted from differences in charge density and hydrophobicity between DNA and HA. This is reasonable, because DNAs having higher charge density and hydrophobicity than HA should be favourable for interpolyelectrolyte complex formation with PLL. The PLL-g-HA/ DNA complex is expected to have the multiphase colloidal structure in which the PLL-DNA complex is surrounded by the hydrated shell of free HA. In vivo study with the PLL-g-HA/DNA complex was carried out using either 32P-labelled plasmid DNA or F-ODN (Takei et al., 1996, 1999). Radioactivity was exclusively accumulated in the liver within 1 h without remarkable uptake in other organs including spleen and lung when the complexes between 32P-labelled plasmid and PLL-g-HA were injected in the rat from the tail vein. The result was quite different from that obtained for the heterogeneous complexes between PLL and the plasmid or the ternary mixture of plasmid, PLL and HA, which resulted in massive accumulation in the lung. The intra-liver destination of DNA was evaluated by fluorescence microscopical observation of the liver sections obtained from the rat injected with the complexes between F-ODN and the PLLg-HA copolymer. The fluorescence was detected linearly and homogeneously along sinusoidal lining cells, indicating the selective uptake of the PLL-g-HA/ DNA complex in SECs. It is worth noting that the injection of reporter gene, pSV β-Gal plasmid, with PLL-g-HA resulted in PCR and RT-PCR signals from the cytosolic fraction of the liver and in expression of β-galactosidase along sinusoids,

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 193

Figure 8.11 Synthetic route and structural formula of PLL-g-HA graft copolymers. Reprinted with permission from Bioconjugate Chemistry, 9, 476–481. Copyright 1998 American Chemical Society

indicating successful gene transfer and expression in SECs. Though the injection of the 32P-labelled plasmid alone also resulted in considerable radioactivity in the liver, neither PCR nor RT-PCR studies detected the transgene from the liver. Tt can be concluded that the colloidal DNA complex bearing hyaluronanglycocalyx is a promising formulate to deliver DNA to SECs. Synergy between the steric exclusion effect and specific interaction of HA-glycocalyx with the target cells, i.e. SECs via the HA receptors, seemingly led to the observed selective delivery of DNA. It is interesting to investigate whether the glycocalyx-bearing DNA carrier is applicable for delivery to other target cells or organs. For this purpose we prepared the comb-type copolymer with arabinogalactane (AG) as side-chains. AG is a naturally occurring and highly branched polysaccharide having numerous terminal β-D-galactosyl residues (Groman et al., 1994; Prescott et al., 1995). It was reported that more than 50% of AG administered in rat was

194 COMB-TYPE POLYCATION COPOLYMER

Figure 8.12 Effect of DMA on the 1 H NMR spectra of the PLL-g-HA. PLL-g-HA alone in D20 containing 350 mM NaCI (a) and PLL-g-HA in the absence (b) or presence (c) of fragmented salmon sperm DMA (~300 bp) in D20 without NaCI. The signal of emethylene protons of PLL (3.0 ppm) and the H-2 signal of glucuronic acid of HA (3.3 ppm) are represented. Reprinted with permission from Bioconjugate Chemistry, 9, 476– 481. Copyright 1998 American Chemical Society

accumulated in the liver parenchymal cells by specific interaction with the asialoglycoprotein receptors (ASGP-Rs) (Groman et al., 1994). As ASGP-Rs has extremely high affinity to clustered β-galactosyl residues (for review, see Lee, 1991), the highly branched structure of the AG seems favourable in its interaction with ASGP-Rs. Thus, the dendritic graft copolymer (PLL-g-AG, Figure 8.14) having a PLL backbone and dendritic side-chains of AG was prepared, and its potentiality as a hepatocyte-specific DNA carrier was estimated (Park et al., 1999). The reductive amination reaction of AG with PLL proceeded only when a lower molecular weight fraction (Mn = 25 000) of AG was used. The coupling efficacy of AG to PLL was lower than that of Dex or HA. The highly branched structure and thereby bulkiness of molecular structure of AG was ascribed to the lower coupling efficacy of AG. Regardless of such bulky structure of AG graft chains, PLL-g-AG formed a complex with plasmid DNA at

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 195

Figure 8.13 PAAm gel electrophoresis of 38-mer F-ODN mixed with increasing amount of PLL-g-HA (PLL grafted with HA (Mn = 3800) at 11 mol%

Figure 8.14 Schematic illustration of dendritic graft copolymers (PLL-g-AG) prepared from PLL and AC copolymer (Park et al.,1999). The closed circles represent terminal βgalactosyl residues

almost electrostatically equivalent ratio. The turbidity measurements and microscopic observations of the complex demonstrated high dispersiveness of the PLL-g-AG/DNA complexes either in PBS or in culture medium containing serum. The specificity of the copolymer complex to target cells, i.e. hepatocyte, was assessed by in vitro study using HepG2 cells, hepatoma cells. The association of F-ODN to HepG2 was markedly increased when the oligonucleotide was added in the culture in the form of the complex with PLL-gAG copolymer. The association of the F-ODN was thoroughly inhibited in the presence of excess free AG or poly[N-p-vinylbenzyl-o-β-D-galactopyranosyl(l– 4)-D-gluconamid] (PVLA), an artificial ASGP-R ligand (Kobayashi et al., 1986, 1992; Adachi et al., 1994), indicating that the association of the PLL-g-AG/DNA complexes to HepG2 cells was mediated by ASGP-Rs. The transfection study using pGLLuc, a luciferase-coding plasmid, also supported specificity of the copolymer complexes to HepG2 cells. The copolymer exhibited 14 times and 180 times, respectively, higher luciferase activity to HepG2 culture than PLL and plasmid alone. No transfection activity of the copolymer to HepG2 cells in the

196 COMB-TYPE POLYCATION COPOLYMER

presence of PVLA, and to PANC cells that do not express ASGP-Rs in the absence of PVLA, further confirmed the specificity of the copolymer. The observations described above indicated that design of PLL-gpolysaccharide copolymers could be a promising strategy for cell-specific delivery of DNA. As described above, polycation comb-type copolymer facilitates DNA duplex and triplex formation. A comb-type copolymer which has the ability not only to deliver the ODN to desired cells but also to facilitate duplex and triplex formation on target genes might be possible, and suggests a direction for further progress in antigene medicine. On the other hand, further modification of the copolymer would be required to control intracellular sorting and to enhance transfection activity. Natural gene carriers, such as viruses, evolved various molecular mechanisms for effective entry into cells and escaping lysosomal degradation. For example, some virus particles exhibit fusogenic activity with endosomal membranes to translocate the viral gene to cytoplasmic space (Wiley and Skehel, 1987). The mechanics of the direct injection of DNA through the cellular plasma membrane were well documented for the infection of T4 bacteriophages (Simon and Anderson, 1967). The fact that these viruses display such molecular mechanisms only after their arrival at the target cells is unique. The viruses, therefore, switch on these mechanisms by sensing environmental factors such as pH and/or specific molecules at the target sites. A synthetic polymeric carrier that has such ‘intelligence’ of viral infection pathways would greatly improve the transfection efficacy of an artificial vector. Design of an ‘intelligent polymeric carrier’ is now in progress in our laboratory (Asayama et al., 1997). Acknowledgements The author sincerely acknowledges Profs. T.Akaike, S.W.Kim, Drs. H.Torigoe, A. Ferdous, and MD. Y.Takei for valuable discussion. The author also thanks all scientists who have joined in our research cited here. Part of our work was supported by a grant-inaid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan. References ADACHI, N., MARUYAMA, A., ISHIHARA, T. and AKAIKE, T., 1994, Cellular distribution of polymer particles bearing various densities of carbohydrate ligands, J. Biomater. Sci. Polym. Ed., 6, 463–479. ANDERSON, C.F. and RECORD, M.T., JR, 1982, Polyelectrolyte theories and their applications to DNA, Annu. Rev. Phys. Chem., 33, 191–222. ASAYAMA, S., MARUYAMA, A., CHO, C.-S. and AKAIKE, T., 1997, Design of combtype polyamine copolymers for novel pH-sensitive DNA carrier, Bioconjugate Chem., 8, 833–838.

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PART FOUR Biopharmaceutics

9 Delivery of Antisense Oligonucleotides in Vitro Experimental Points C.B.TAKLE and C.A.STEIN

9.1 Introduction Our intention in this chapter is to discuss antisense technology in its role as a useful tool for specific downregulation or ablation of mRNAs, while pointing out that it has a complicated methodology and a literature that contains examples of misinterpretation of true antisense effects. With this in mind, we have directed this chapter towards the practical and conclude it with a series of points to consider that will help the newcomer to the field to avoid some of the pitfalls we are aware of. Much of the detail of design and biochemistry of oligonucleotide drugs is covered in other chapters (see Chapters 1 and 2) in this book, and we have intentionally focused this chapter on delivery issues, which, to our minds, provide some of the major remaining variables in the antisense field. It seems obvious that for antisense oligos to downregulate gene expression they must penetrate the cell membrane, and very early work showed that this could occur (Koch and Bishop, 1968). The explosion of cell transfection studies using exogenously delivered genes also attests to the fact that nucleic acids can enter cells (in the case of genes, one needs a facilitator to bring this about). However, since polynucleotides are polyanions they cannot passively diffuse across cell membranes, and even neutral methylphosphonate oligos are too polar to move passively intracellularly (Shoji et al., 1991). Data from a large number of studies indicate that naked oligos are taken up by active transport, probably via the combined processes of adsorptive endocytosis and fluid phase endocytosis (pinocytosis). Several factors, such as oligomer concentration, size, chemistry, cell type and activation state, (Krieg et al., 1991) influence the relative contribution of each process. The fact that oligos are taken up by an active process was indicated by findings that internalization is slowed by the well-known metabolic poisons deoxyglucose, cytochalasin B and sodium azide, and by reduced temperature (Yakubov et al., 1989; Crooke et al., 1995; WuPong et al., 1992, 1994). The adsorptive nature of oligo uptake was suggested by the

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 205

Table 9.1 Cell surface oligonucleotide binding proteins Protein name (s)

Size (kDa)

Cell type

DNA receptor protein

30

Human haematopoiet ic

Nucleic acid binding receptor- 1 ?

75–80

HL-60, COS-1, Vero, L-671, etc.

46

?

28–34

?

28, 59,* 79

Lymphoid

CD4

59*

T-cells

PS

Mac-1 (CD11b/ CD18, CR3, aM(32) PS oligo

α 165, p 95

Neutrophils, NK cells, macrophages

PS/PO

20–22

K562

29–32 43–46 79–85 137–147 100/110

HepG2

binding proteins

Nucleolin?

Oligo

Comments

Reference

Antibodies to receptor found in patients with rheumatologi c disease

Bennett et al. (1985)

Wu-Pong et al. (1994)

Blocks binding of HIV gp120

Hawley and Gibson (1996) Goodarzi et al. (1991) Gasparro et al. (1990) Yakubov et al. (1993) Benimetskaya et al. (1997b)

Beltinger et al. (1995)

PS

Bound polyanions with low nM affinity

Yao et al. (1996)

*Possibly the same. PS, phosphorothioate; PO, phosphodiester.

fact that oligos that adsorb well to cells (the exception being haematopoietic cells, especially T-cells) are internalized, and those that do not adsorb well (uncharged oligos) are internalized very poorly. Also, if the binding of oligos to cell surface binding sites is competed with high concentrations of other polyanions, then internalization of oligos (in this case fluorescein-tagged) is inhibited (Stein et al., 1993).

206 DELIVERY OF ANTISENSE OLIGONUCLEOTIDES IN VITRO

Contributing considerably to the adsorptive process are cell surface oligonucleotidebinding proteins (see Table 9.1). A number of cell surface heparin-binding proteins have been identified, some of which have very high affinity for phosphorothioate oligomers (Guvakova et al., 1995). The best characterized of these proteins is Mac-1 (see below, and Benimetskaya et al., 1997b), which shows oligomer length dependence (highest binding at 22 nt with negligible binding <15 nt (Stein et al., 1993), but sequence and chirality independence (Benimetskaya et al., 1995). The exception to the sequenceindependent binding of Mac-1 and other heparinbinding proteins occurs in oligos containing the G-tetrad or G-quartet motif (Benimetskaya et al., 1997a), and an interesting observation is that G-tetradbearing oligos are statistically overrepresented in the published examples of oligonucleotides showing antisense activity (Smetsers et al., 1996). We feel that a reason for this may be that since the adsorptive process is very efficient at low concentration, while the converse is true for pinocytosis at concentrations of less than 0.5 mM (Krieg et al., 1991; Vlassov et al., 1994), those oligos that adsorb efficiently will be delivered well. G-tetrad-containing oligomers bind especially well to cell surface heparinbinding proteins, particularly if the G-tetrad is located close to either the 3′ or the 5′ terminus of the oligo. The presence of the G-tetrad is thought to promote the formation of higher order structures containing multiple non-covalently associated oligos, and these multimers are likely to bind to heparinbinding proteins with much greater affinity than the corresponding monomer. The behaviour of the monomeric and multimeric forms can be studied by substituting a single deazaguanosine residue for guanosine at any position in the G-tetrad, thus disrupting the formation of the higher ordered structures without diminishing the duplex Tm significantly (Benimetskaya et al., 1997a). We have focused in this introduction on the absorptive process, but need to mention the process of pinocytosis for completeness before moving on. Pinocytosis is the mechanism by which cells take up water and dissolved solute from the fluid phase. It is considered to be an inefficient process, but it may account for considerable oligomer uptake when the oligomer concentration exceeds the oligo-cell surface binding protein binding constants (Gao et al., 1993). 9.2 Oligonucleotide-Binding Proteins on the Cell Surface Heparin-binding proteins are responsible for high-affinity binding of oligonucleotides to the cell surface. Information on the identified cell-surface oligomer-binding proteins is summarized in Table 9.1. Of these proteins, three have been fairly well characterized. Several years ago, we noticed that Mac-1 (CD11b/CD 18; CR3; αMβ2) was a protein that bound to and mediated the cellular internalization of oligonucleotides (Benimetskaya et al., 1997b). This homodimer is found

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predominantly on the surface of neutrophils, natural killer (NK) cells, and macrophages, and is a heparinbinding integrin. Both phosphodiester and phosphorothioate oligomers bound to both subunits, and the binding was competed out with polyanions such as suramin, a discrete persulphated heparin analogue, and other, longer phosphorothioates. Fibrinogen, which is a natural ligand for Mac-1, was an effective competitor of oligomer binding. Treatment of human neutrophils with molecules known to upregulate cell surface Mac-1 expression, such as TNF-α, leukotriene B4, arachidonic acid, interleukin-8 and C5a, all increased oligomer binding. This binding could be blocked by mAbs directed at either chain of Mac-1. In addition, the increase in binding was correlated with a three- to four-fold increase in net oligonucleotide internalization. In Chinese hamster ovary cells which had been doubly transfected with the αM and β2 genes, increased mAb-sensitive internalization was also observed. The binding of phosphorothioate oligomers to Mac-1 has functional significance. For instance, the β2-dependent migration through Matrigel was inhibited by SdC28 (a phosphorothioate 28-mer homopolymer of cytidine), and the production of reactive oxygen species in TNF-α or formyl-met-leu-phe activated neutrophils adherent to fibrinogen was dramatically increased by SdC28, and was blocked by an anti-Mac 1 monoclonal antibody. These observations again highlight the intrinsic biological activity possessed by phosphorothioate oligonucleotides, and emphasize the care that must be taken in interpreting data from experiments in which they are employed. Mac-1 is not the only oligo-binding cell surface protein; several others have been characterized to varying degrees. Bennett et al. (1985) identified a 30 kDa DNA receptor protein in human haematopoietic cells, including neutrophils, monocytes, T- and B-lymphocytes, but not erythrocytes. Antibodies to the putative receptor were found in 60–70% of patients with various rheumatologic disorders, including lupus, mixed connective tissue disease, rheumatoid arthritis, and Sjogren's syndrome (Hefeneider et al., 1990). A family of proteins of Mr = 75–80 kDa and called nucleic acid binding receptor-1 (NABR1) was also discovered in the late 1980s (Wu-Pong et al., 1994), and is present on a variety of diverse cell types (Krieg et al., 1991). The extent to which NABR1 mediates internalization is unknown. Other nucleic acid binding proteins, of Mr 46 kDa (Hawley and Gibson, 1996), 28–34 kDa (Goodarzi et al., 1991), and 79, 59 and 28 kDa (Gasparro et al., 1990), have been found in various cell types, the last three in lymphoid cells. It is possible that the 59 kDa protein is CD4, a heparin-binding protein that binds with low nanomolar affinity to phosphorothioate oligonucleotides (Stein et al., 1989; Yakubov et al., 1993). The effect of this binding is to block, at least in part, the binding of the v3 loop of the HIV-1 envelope glycoprotein, gp120, to the cell surface, but it is not known what role this protein might play in the process of oligonucleotide internalization in T-cells.

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Beltinger et al. (1995) detected several phosphorothioate oligonucleotide binding proteins (Mr = 137–147, 79–85, 43–46, 29–32, and 20–22 kDa) in the surface membranes of K562 cells. These proteins were not further characterized, and again their role in net internalization is unclear. More recently, Yao et al. (1996) found proteins of Mr = 100 and 110 kDa on the surface of HepG2 cells, though in the absence of the protease inhibitor PMSF, 40–90 kDa digestion fragments were obtained. The proteins bound a variety of polyanions, including phosphorothioates with low nanomolar affinity. In this case, the quantity of cell surface protein seemed to correlate with the rate of cellular internalization of oligonucleotides. Sequence analysis has revealed it to be nucleolin or a nucleolin-like protein. 9.3 Intracellular Compartmentalization As seen above, the passage of naked oligonucleotide across the cell membrane is predominantly due to the process of adsorptive endocytosis that may or may not involve the binding to discrete cell surface proteins. This results in the oligonucleotide concentrating into the endosomal compartment and, consequently, the penetration of the endosomal membrane is a prerequisite for antisense activity. Oligos delivered without a carrier do not appear to escape endosomes to any great degree. However, since some naked phosphorothioate oligomers have shown antisense activity, there must be some level of endosomal release in the absence of carrier. In keeping with this assumption, it is fitting to observe that in cell culture the antisense concentrations of naked oligomers are usually 5–100 times higher than active concentrations of oligomer obtained using a delivery reagent (Ehrlich et al., 1994; Soreq et al., 1994; Anfossi et al., 1989; Gewirtz and Calabretta, 1988; Dean et al., 1994; Flanagan et al., 1996; Monia et al., 1996; Wagner et al., 1996). Confocal microscope observations of internalized naked, fluorescently labelled oligos show the oligo to be located in punctate intracytoplasmic structures (probably early endosomes with slightly acidic internal pH prior to quenching of fluorescein as the pH decreases during endosomal maturation), and the nuclei are weakly stained, if at all. The use of oligo carriers or delivery reagents alters this intracellular distribution dramatically, with clear nuclear localization under optimal conditions frequently observed. In experiments where antisense activity is obtained using oligos delivered with carrier, the same effect is rarely observed with the same oligo delivered naked, which would indicate that nuclear localization due to carrier use is necessary. There are, however, plenty of examples of naked oligos having antisense activity. To resolve this apparent paradox we present one hypothesis (among many possible hypotheses) that might explain the observations. For this hypothesis one must invoke the concept that there are two possible intracellular locations in which antisense effects may occur. One of these is nuclear, is RNAse H-dependent, and

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can occur at low extracellular oligonucleotide concentration. The other is cytoplasmic, is relatively RNAse H-independent, and requires relatively high extracellular concentrations of oligomer, perhaps to compensate for the low rate of endosomal rupture (and thus the small amount of oligonucleotide release) in the absence of a delivery vehicle. Studies utilizing the ‘molecular beacon’ approach, in which a 5′-fluorophore (whose fluorescence has been quenched by an apposed 3′-fluorophore) is activated only after Watson-Crick hybridization to its sequence-specific target, may be very useful in resolving some of these questions (Sokoletal., 1998). Endosomal Compartmentalization can be studied using the pH-dependent quenching of oligonucleotide fluorescein as a reporter for oligonucleotide localization (Tonkinson and Stein, 1994). The sodium ionophore monensin was used to break down the pH gradient between endosomal and cytoplasmic compartments. Monensin treatment resulted in a large increase in intracellular fluorescence as measured by flow cytometry for phosphorothioate, but not for phosphodiester oligos, implying that phosphorothioate resided in a low pH compartment. Using this methodology it was possible to derive a mathematical model for oligo uptake. Net uptake represents the difference between uptake and efflux. Oligonucleotides were found to undergo significant efflux from HL60 cells. The rate of efflux, for all classes of charged oligonucleotides studied, was best described by: (9.1) where Ct is the amount of oligomer remaining internalized in the cell at any time t, α and β are the rate constants of efflux, and A + B = 100%. Each exponential component of the sum in equation (9.1) is also referred to as a compartment. While this mathematical description does not assign an actual cellular structure to the compartments, it is clear on a biological basis that they are endosomes/ lysosomes. Phosphodiester oligomers predominantly enter a ‘shallow’ (relatively rapid efflux, short t½) compartment. A = 61% ± 4%; the value of a is 10 h−1. About 36% of the oligonucleotides enters the B, or deep compartment (relatively slow efflux, long t½ (β = 0.329 h−1)). Phosphorothioates, in contrast, behave differently, and the situation is reversed. For 15–28-mers, only 18% entered the shallow (A) compartment, while 80% entered the deep (B) compartment (α = 3.5 h−1; β = 0.131 h−1). Acidification of the phosphorothioate oligomer occurred in the deep compartment. Phosphodiesters, on the other hand, were not acidified. Similar efflux data were obtained with rhodamine-labelled oligomers, indicating that it was probably not the fluorescent group that was responsible for the efflux properties. Sequestration of internalized oligomers in endosomal/lysosomal compartments occurs not only in HL60 cells, but also in K562 erythroleukemia and DU145 prostate cancer cells. One method for determining the rate of loss of oligonucleotide from the vesicular compartment to the cytoplasm is a ratiometric one that takes advantage of the fact that the dye rhodamine green dextran will colocalize with fluorescently labelled oligonucleotides in the vesicular compartment.

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Because the dye is endosome-impermeable, a change in the ratio of tagged oligomer fluorescence/endosomal dye fluorescence, as determined by confocal microscopy, implies leakage of the oligomer from endosome into cytoplasm. This type of measurement has been performed in DU145 cells. Changes in the ratio even after 12 h of incubation were not observed, implying that the ‘leak rate’ is extremely low. 9.4 Oligonucleotide Delivery Reagents—Practical Considerations The reader may wish to investigate several extensive papers on this subject (Bennett, 1998; Bennett et al., 1992, 1993; Jaaskelainen et al., 1994). The described oligonucleotide carriers fall into two categories, the cationic lipids and the polyamines. The classical cationic lipids (see Chapter 7) were developed primarily as reagents for cell transfection by exogenous genes. However, their applicability to the delivery of oligonucleotides soon became obvious. Classical commercially available cationic lipids include Lipofectin™, Lipofectase™, and Lipofectamine™, and are composed of a dioleyoyl lipid containing a cationic headgroup with a neutral colipid. Some of these lipids may be covalently bound to a cell type-specific ligand for targeting (Takle et al., 1997). Oligonucleotide/ cationic lipid complexes are, like naked oligonucleotides, internalized via adsorptive and fluid-phase endocytosis (Zabner et al., 1995; Wrobel and Collins, 1995), although precisely how the oligonucleotides exit the endosome is somewhat unclear (Zelphati and Szoka, 1996). These cationic lipids can be very effective antisense delivery reagents under certain circumstances, but their cell culture and clinical use may be limited by the fact that they are serum-sensitive and relatively toxic to some cell types, particularly, in our experience, prostate cancer cells. This latter problem has been overcome by the use of cationic porphyrin delivery vehicles (Benimetskaya et al., 1998), which were initially developed for use in oligonucleotide delivery to hepatocytes (Flynn et al., 1999) and appear to be significantly less toxic in tissue culture. Several newer lipids are also available, including the serum-stable Cytofectin (Lewis et al., 1996; Gilead Sciences, Foster City, CA), and a variety of lipids made by JBL (San Luis Obispo, CA), known as Eufectins, and every week a new cationic lipid reagent seems to appear on the market with improved delivery characteristics. Also commercially available is a series of polyamines called ‘Starburst dendrimers’ (Bielinska et al., 1996). With the appropriate use of these delivery vehicles and others, it should be possible to transfect a wide variety of cell types at an oligomer concentration of 1–2 µM or less, giving antisense IC50s in the nanomolar range. It is important, however, to realize that the optimum ratio of carrier/oligomer, the transfection time, and the time of cell harvest for Western or Northern blotting will be different for each vehicle and cell line and will require optimization. Also, the physiological effects on cells of

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the carriers themselves are not well known, and it is possible that an observed antisense effect may be the result of both the sequencespecific effect of the oligonucleotide and the effect of the carrier, both being necessary but neither sufficient. This point takes on additional meaning because both lipid and porphyrin carriers dissociate intracellularly from the oligonucleotide (Benimetskaya et al., 1998; Marcusson et al., 1998) and are present in the cell in free form. It should also be mentioned that the proper control for the carrier is not an equimolar concentration of the carrier alone, but rather the carrier plus control oligonucleotides. 9.5 Experimental Antisense: Points to Consider 9.5.1 Oligonucleotides Over the past several years, the antisense biotechnology field has significantly progressed in its understanding of how to conduct meaningful experiments that do not conflate an antisense effect and non-sequence specific behaviour. An initial set of guidelines was published in 1994 (Stein and Krieg, 1994), but these have not been updated to reflect recent experience. We present here a concise series of guidelines that may help antisense researchers to avoid some pitfalls. Start with the phosphorothioate backbone. At the present time, the best and most readily commercially available oligonucleotides have the phosphorothioate backbone. Do not use unprotected phosphodiesters, as problems (e.g. inhibition of cellular growth) may be caused by the mononucleotide products of enzymatic digestion. dGMP may be the most toxic. Obtain the oligomers from a reputable manufacturer. Never store oligonucleotides in water. The pH of such a solution will fall in an acidic urban environment, leading to depurination and strand cleavage. Instead they should be stored between pH 7.5 and 8.0 in buffer, i.e. Tris-EDTA, at −20°C (although they will survive well at 4°C). Non-sequence specificity must be minimized. The lower the concentration used, the better. Phosphorothioate 3′ and 5′ end-capped oligomers (three phosphorothioates per terminus) preserve RNAse H activity and diminish sulphur content while preserving nuclease resistance. C5-propyne substitution at one or more cytidine residues in the oligomer, or 2′-O-methyl RNA substitution, may increase Tm and potentiate the antisense effect. However, use these judiciously, as irrelevant cleavage will also probably increase, and the C5propyne residues may be toxic, especially in vivo. To find an active oligomer, generate a panel of 30–40 by mRNA walking. For every 8–9 oligos tested, one will probably be active. mRNA folding programmes are not predictive, and have little or no value in selecting target sites because

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they cannot account for local, critical microenvironments at the mRNA level. The other oligomers that are not active are your backbone controls. The more control oligomers that are used, the more likely it is that an observed effect represents ‘true antisense’. Demonstrate inhibition of the target protein by Western blotting, and of the mRNA by Northern blotting or RT-PCR. 9.5.2 Delivery In general, do not treat cells with naked phosphorothioate oligonucleotides, although exceptions do exist. Phosphorothioates are very non-specifically active at the cell membrane, with concentrations as low as 2 µM causing defined effects. It is usually better to deliver oligonucleotides with a carrier. Many are commercially available, including lipofectin, lipofectase, cytofectin (serum stable), Starburst dendrimers of many generations, cationic porphyrins, and others. Remember, ‘antisense’ may be caused by the summation of effects of the carrier plus the oligomer, as the carriers dissociate from the oligomer intracellularly. You must optimize the molar ratios and concentrations for each carrier, cell line, and oligomer sequence as well. Several problems exist in the unambiguous determination of oligonucleotide internalization, and are worth discussing. First, internalized oligomer must be distinguished from cell-surface non-specifically bound material. Methods to do this have included DNase digestion of cells to remove adhered material, and washing the cells in high salt/low pH buffers (such as glycine, pH 2.0). The efficacy of either of these methods when phosphorothioate oligomers are used is open to question, and both may be toxic to the cells. A simpler approach is to add a competitor, e.g. 5 µM SdC28 for 5 min. This method is not toxic and will remove virtually all cell surface bound phosphorothioate. The non-removable fraction can be taken as being internalized, a term which will be better defined below. Although they are more easily quantifiable, the use of 32P end-labelled oligomers is currently frowned upon. This is because ubiquitous cell surface alkaline phosphatase activity will cleave the label, which may be internalized without the oligonucleotide. There are circumstances where end-labelled oligomers can be used, and this is when full-length oligo is recovered from the cells and radioactivity associated with the full-length material only is measured by phosphorimager or some other method. This would give an underestimate of full-length material inside the cell, as some full-length molecules will be undetectable, having had the 32P removed by cellular phosphatases. Also, this method relies on cell lysis or fractionation, both methods requiring a considerable number of controls.

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Optimal internalization experiments are best performed in real time using fluorescently-labelled oligomers. Real-time experiments are preferred because non-viable cells internalize significantly more oligonucleotide than viable ones (Krieg et al., 1991; Zhao et al., 1993; Juliano and Mayhew, 1972). The attachment of a fluorescently-labelled moiety to an oligonucleotide may alter its intracellular binding properties compared to the unlabelled molecule. While this is certainly true for extremely hydrophobic molecules, such as cholesterol, which will insert in the cell membrane, there is no evidence that less hydrophobic molecules such as fluorescein create similar artefacts; indeed, the available data suggests otherwise (Loke et al., 1989). 9.5.3 Controls The demonstration of true antisense activity relies on gathering as much circumstantial evidence as possible. Since target mRNA is rapidly degraded, it is almost impossible to retrieve direct evidence of antisense activity (correctly sized RNA fragments), and this has made it harder to correlate a biological effect of an oligonucleotide with antisense sequence-specific mRNA degradation. Several strategies can be used to gather evidence of specific antisense phenomena, and the more that are used, the stronger is the case for antisense. A sense oligomer is not necessary, and a total random-mer (mixture of bases at each position) is not an appropriate control. The other oligos that are active can be used to determine specificity by examining their effects on proteins that are related to the target, e.g. one with an approximately equal half-life. Don't use actin as a control protein if your target has a t½ of only a few hours. Remember, the more control oligomers tested, the more assurance there is that the result is specific (minimum of two). The use of two different oligonucleotide backbones, each producing the identical ‘antisense’ effect, is usually convincing. An additional control is to attempt to antisense-knockout your gene in an overexpressing line. If the cells are rescued from the antisense effect, your case is strengthened, but still not proven. Another useful control is to introduce the target gene with one or more mutations in the region complementary to the antisense molecule. Lack of inhibition suggests a true ‘antisense’ effect, but does not prove it if the rate of transcription and hence the copy number of the mRNA is high. An additional control is to clone your gene in the antisense orientation into an expression vector. Successful suppression of translation of the transfected gene, with the identical biological activity as seen with the antisense oligomers, is convincing. However, because of high-order mRNA structure, the antisense strand may not be able to invade the sense strand, and the method may fail.

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9.5.4 Traps It is best to avoid active oligomers with the CpG motif when possible. Depending on the sequence context, they can be highly immune-stimulating (Krieg et al., 1995). This can be a problem in xenograft models (if it is your eventual plan to use them) because of immune-mediated graft rejection. However, methylation at C5 of cytidine eliminates immune stimulation. Be very careful about G-quartet-containing oligomers. Molecules with four contiguous G-residues can form, by Hoogsteen base-pairing, tetrads which can stack to form tetraplexes. These higher order structures seem to have very high affinities for heparin-binding proteins, and may be highly non-specific. The problem is maximal if the G-quartet motif is located within 3–4 bases of either the 5′ or the 3′ terminus. Oligomers with the G-quartet in the middle of the molecule may demonstrate ‘antisense’ activity, but non-denaturing PAGE gels should be run to rule out the presence of higher order structures. Substitution of a 7-deazaG residue for G can also help resolve these questions, as the modified base cannot Hoogsteen base-pair, and Watson-Crick base pairing is left intact. The control for this type of oligo should leave the G-quartet intact and in the same position in the molecule as it was found in the antisense species. Do not treat cells with a naked G-quartet containing oligo at a concentration >5 µM. Finally, a word on interpretation. Do not attempt to correlate an observed biological effect with antisense ‘efficacy’, e.g. do not say that inhibition of proliferation is caused by an antisense effect, as the phosphorothioates are intrinsically too biologically active. Rather, demonstrate downregulation of protein by Western blotting and mRNA levels by Northern, and make no other claims. References ANFOSSI, G., GEWIRTZ, A. and CALABRETTA, B., 1989, Proc. Natl Acad. Sci. USA, 86, 3379–3383. BELTINGER, C., SARAGOVI, H.U., SMITH, R.M., LESAUTEUR, L., SHAH, N., DEDIONISIO, L., CHRISTENSEN, L., RAIBLE, A., JARETT, L. and GEWIRTZ, A.M., 1995, J. Clin. Invest., 95, 1814–1823. BENIMETSKAYA, L., BERTON, M., KOLBANOVSKY, A., BENIMETSKY, S. and STEIN, C.A., 1997a, Nucl. Acids Res., 25, 2648–2656. BENIMETSKAYA, L., LOIKE, J., KHALED, Z., LOIKE, G., SILVERSTEIN, S., CAO, L., EL-KHOURY, J., KAI, T.-Q. and STEIN, C.A., 1997b, Nat. Med., 3, 414–420. BENIMETSKAYA, L., TAKLE, G., VILENCHIK, M., LEBEDEVA, I., MILLER, P. and STEIN, C.A., 1998, Nucl. Acids Res., 26, 5310–5317. BENIMETSKAYA, L., TONKINSON, J., KOZIOLKIEWICZ, M., KARWOWSKI, B., GUGA, P., ZELTSER, R., STEC, W. and STEIN, C.A., 1995, Nucl. Acids Res., 23, 4239–4245.

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BENNETT, C.F., 1998, in STEIN, C.A. and Krieg, A. eds, Applied Antisense Oligonucleotide Technology, pp. 129–145, New York: Wiley-Liss. BENNETT, C.F., CHIANG, M., CHAN, H. and GRIMM, S., 1993, J. Liposome Res., 3, 85–102. BENNETT, C.F., CHIANG, M., CHAN, H., SHOEMAKER, J. and MIRABELLI, C., 1992, Mol. Pharmacol., 41, 1023–1033. BENNETT, R.M., GABOR, G.T. and MERRITT, M.M, 1985, J. Clin. Invest., 76, 2182–2190. BIELINSKA, A., KUKOWSKA-LATALLO, J., JOHNSON, J., TOMALIA, D. and BAKER, J., 1996, Nucl. Acids Res., 11, 2176–2182. CROOKE, R.M., GRAHAM, M.J., COOKE, M.E. and CROOKE, ST., 1995, J. Pharmacol. Exp. Ther., 275, 462–473. DEAN, N.M., MCKAY, R., CONDON, T. and BENNETT, C.F., 1994, J. Biol. Chem., 269, 16416–16424. EHRLICH, G., PATINKIN, D., GINZBERG, D., ZAKUT, H., ECKSTEIN, F. and SOREQ, H., 1994, Antisense Res. Dev., 4, 173–180. FLANAGAN, W.M., SU, L. and WAGNER, R., 1996, Nat. Biotech., 14, 1139–1145. FLYNN, S.M., GEORGE, S.T., WHITE, L., DEVONISH, W. and TAKLE, G.B., 1999, Biotechniques, 26, 736–746. GAO, W.Y., STORM, C., EGAN, W. and CHENG, Y.C., 1993, Mol. Pharmacol., 43, 45–50. GASPARRO, F.P., DALL’AMICO, R., O’MALLEY, M., HEALD, P.W. and ELSON, R., 1990, Photochem. Photobiol., 52, 315–321. GEWIRTZ, A. and CALABRETTA, B., 1988, Science, 242, 1303–1306. GOODARZI, G., WATABE, M. and WATABE, K., 1991, Biochem. Biophys. Res. Commun., 181, 1343–1351. GUVAKOVA, M.A., YAKUBOV, L.A., VLODAVSKY, L, TONKINSON, J.L. and STEIN, C.A., 1995, J. Biol. Chem., 270, 2620–2627. HAWLEY, P. and GIBSON, I., 1996, Antisense and Nucl. Acid Drug Dev., 6, 185–195. HEFENEIDER, S.H., BENNETT, R.M., PHAM, T.Q., CORNELL, K., MCCOY, S.L. and HEINRICH, M.C., 1990, J. Invest. Dermatol., 94 (Suppl. 6), 79S–84S. JAASKELAINEN, I., MONKKONEN, J. and URTTI, A., 1994, Biochim. Biophys. Acta BioMembr., 1195, 115–123. JULIANO, R. and MAYHEW, E., 1972, Exp. Cell. Res., 73, 3–12. KOCH, G. and BISHOP, J.M., 1968, Virology, 35, 9–17. KRIEG, A.M., GMELIG, M.F., GOURLEY, M.F., KISCH, W.J., CHRISEY, L.A. and STEINBERG, A.D., 1991, Antisense Res. Dev., 1, 161–171. KRIEG, A.M., TONKINSON, J., MATSON, S., ZHAO, Q., SAXON, M., ZHANG, L., BHANJA, U., YAKUBOV, L. and STEIN, C.A., 1993, Proc. Natl Acad. Sci. USA, 90, 1048–1052. KRIEG, A.M., YI, A.K., MATSON, S., WALDSCHMIDT, T.J., BISHOP, G.A., TEASDALE, R., KORETZKY, G.A. and KLINMAN. D.M.. 1995. Nature. 374. 546–549. LEWIS, J.G., LIN, K., KOTHAVALE, A., FLANAGAN, M., MATEUCCI, M., DEPRINCE, R., MOOK, R., HENDREN, R. and WAGNER, R., 1996, Proc. Natl Acad. Sci. USA, 93, 3176–3181.

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LOKE, S.L., STEIN, C.A., ZHANG, X.H., MORI, K., NAKANISHI, M., SUBASINGHE, C., COHEN, J.S. and NECKERS, L.M., 1989, Proc. Natl Acad. Sci. USA, 86, 3474–3478. MARCUSSON, E., BHAT, B., MANOHARAN, M., BENNETT, C.F. and DEAN, N., 1998, Nucl. Acids Res., 26, 2016–2023. MONIA, B., JOHNSTON, J., GEIGER, T., MULLER, M. and FABBRO, D., 1996, Nat. Med., 2, 668–675. SHOJI, Y. AKHTAR, A., PERIASAMY, A., HERMAN, B. and JULIANO, R., 1991, Nucl. Acids Res., 19, 5543–5550. SMETSERS, T.F., BOEZEMAN, J.B. and MENSINK, E.J., 1996, Antisense Nucl. Acid Drug Dev., 6, 63–67. SOKOL, D., ZHANG, X., LU, P. and GEWIRTZ, A., 1998, Proc. Natl Acad. Sci. USA, 1998, 95, 11538–11543. SOREQ, H., PATINKIN, D., LEV-LEHMAN, E., GRIFMAN, M., GINZBERG, D., ECKSTEIN, F. and ZAKUT, H., 1994, Proc. Natl Acad. Sci. USA, 91, 7907–7911. STEIN, C.A. and KRIEG, A., 1994, Antisense Res. Dev., 4, 67–69. STEIN, C.A., MATSUKURA, M., SUBASINGHE, C., BRODER, S. and COHEN, J., 1989, AIDS Res. Human Retroviruses, 5, 639–646. STEIN, C.A., TONKINSON, J.L., ZHANG, L.M., YAKUBOV, L., GERVANSONI, J., TAUB, R. and ROTENBERG, S.A., 1993, Biochemistry, 32, 4855–4861. TAKLE, G.B., THIERRY, A.R., FLYNN, S.M., PENG, B., WHITE, L., DEVONISH, W., GALBRAITH, R.A., GOLDBERG, A.R. and GEORGE, S.T., 1997, Antisense Nucl. Acid Drug Dev., 7, 177–185. TONKINSON, J.L. and STEIN, C.A., 1994, Nucl. Acids Res., 22, 4268–4275. VLASSOV, V.V., BALAKIREVA, L.A. and YAKUBOV, L.A., 1994, Biochim. Biophys. Acta., 1197, 95–108. WAGNER, R., MATTEUCCI, M., GRANT, D., HUANG, T. and FROEHLER, B., 1996, Nat. Biotech., 14, 840–844. WROBEL, I. and COLLINS, D., 1995, Biochim. Biophys. Acta., 1235, 296–304. WU-PONG, S., WEISS, T.L. and HUNT, C.A., 1992, Pharm. Res., 9, 1010–1017. WU-PONG, S., WEISS, T.L. and HUNT, C.A., 1994, Cell. Mol. Biol., 40, 843–Z850. YAKUBOV, L.A., DEEVA, E.A., ZARYTOVA, V.F., IVANOVA, E.I., RYTE, A.S., YURCHENKO, L.V. and VLASSOV, V.V., 1989, Proc. Natl Acad. Sci. USA, 86, 6454–6458. YAKUBOV, L., KHALED, Z., ZHANG, L.-M., TRUNEH, A., VLASSOV, V. and STEIN, C.A., 1993, J. Biol. Chem., 268, 18818–18823. YAO, G.Q., CORRIAS, S. and CHENG, Y.C., 1996, Biochem. Pharmacol., 51, 431–436. ZABNER, J., FASBENDER, A., MONIGER, T., POELLINGER, K. and WELSH, M., 1995, J. Biol. Chem., 270, 18997–19007. ZELPHATI, O. and SZOKA, F., 1996, Proc. Natl Acad. Sci. USA, 93, 11493–11498. ZHAO, W., MATSON, S., HERRARA, C.J., FISHER, E., YU, H., WAGGONER, A. and KRIEG. A.M.. 1993. Antisense Res. Dev.. 3. 53–66.

10 Mechanisms of Transmembrane Transport of Oligonucleotides R.L.JULIANO

10.1 Overview of Cellular Uptake of Antisense Oligonucleotides The fact that antisense Oligonucleotides can reach target mRNAs within cells is really quite remarkable from a membrane transport perspective. Cell membranes are designed by nature to be very impermeable to large, polar molecules such as Oligonucleotides, unless specific transport pathways are available. For charged Oligonucleotides, there is good evidence that initial accumulation in cells is largely due to some form of endocytosis (Akhtar and Juliano, 1992; Bennett, 1998; Stein and Cheng, 1993; Wagner, 1994). Studies using fluorescent conjugates (Beltinger et al., 1995; Shoji et al., 1991; Stein and Cheng, 1993; Wagner, 1994) have shown that Oligonucleotides taken up by intact cells have a punctate cytoplasmic distribution, and are initially localized in cytoplasmic vesicles that are probably endosomes; in most cases little fluorescence is associated with the nucleus. However, when fluorescent Oligonucleotides are microinjected directly into the cytoplasm they rapidly redistribute to the nucleus (Fisher et al., 1993; Leonetti et al., 1991). Therefore, intact Oligonucleotides leaking out of endosomes would be expected to accumulate rapidly in the nucleus, but this is not seen in most cells, indicating very limited transfer of Oligonucleotides from endosomal to cytoplasmic compartments. However, since antisense effects clearly do occur, some transfer must take place. Oligonucleotides not only accumulate in endosomes, but they can also recycle back to the cell surface and be released; this seems to be particularly true for material accumulating in so-called ‘shallow’ endosomal compartments (Stein and Cheng, 1993; see also Chapter 9). Although many cells take up Oligonucleotides via endocytosis, some interesting recent studies have suggested that the endocytotic rates for Oligonucleotides are different for the apical and basolateral regions of polarized epithelial cells (Takakura et al., 1998). Until recently there has been relatively little information on the status of oligonucleotides within the cell, once released from endosomes. However, this issue has begun tø be explored. Thus, in an elegant study, Politz et al. (1998) have used fluorescence correlation spectroscopy and fluorescence recovery after

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photobleaching to examine the intranuclear status of fluorescent oligonucleotides. These studies reveal that a fraction of injected oligo has a diffusion coefficient equivalent to that of the free molecule in aqueous solution, while another fraction has a diffusion coefficient consistent with being complexed with large RNA molecules. While this result is not necessarily surprising, it provides physical documentation of the status of oligonucleotides within the cell. In another study (Shoeman et al., 1997), a detailed microscopic examination revealed that fluorescent phosphorothioate oligonucleotides complexed strongly with intermediate filaments and with the nuclear lamina; in addition, punctate structures were observed inside the cell (this has also been reported by another group—Lorenz et al., 1998). By contrast, little organelle binding was observed for methylphosphonates or for standard phosphodiester oligonucleotides. The question of how oligonucleotides leave the endosomal compartment is an open one at this point; there is little understanding of the mechanism involved. Likewise, we are just beginning to understand the binding and compartmentation of oligonucleotides within the cytoplasm and nucleus. It seems likely that the rate and extent of endosome to cytosol transfer may be a key limiting factor in antisense pharmacology. Many strategies have been used to increase oligonucleotide uptake, promote release from endosomes, and subsequently enhance pharmacological efficacy (Juliano et al., 1999; Mahato et al., 1997). These include microinjection (Wagner, 1994), electroporation (Spiller et al., 1998), co-administration with cationic lipids (Bennett et al., 1992; see also Chapter 7) or other modified liposomes (Gokhale et al., 1997; Thierry et al., 1993; Wang et al., 1995), and complexation with polycations (Boussif et al., 1995; DeLong et al., 1997). Alternatively, the oligonucleotide itself can be chemically modified so as to retain base pairing fidelity yet exhibit enhanced membrane interactive properties; thus conjugation with cholesterol (Alahari et al., 1996; Letsinger et al., 1989), with polylysine (Lemaitre et al., 1987), or with fusogenic peptides or other delivery peptides (Bachmann et al., 1998; Basu and Wickstrom, 1997; Bongartz et al., 1994; Dokka et al., 1997; see also Chapter 5), have all been tried with moderate success. There is a paucity of information on the relative transport behaviour of various chemical types of oligonucleotides. The transport and distribution behaviour of various chemically modified oligonucleotides may be quite different from that of the parent compounds, as suggested in a recent study of the cellular uptake of various types of first and second generation oligonucleotides (Basu and Wickstrom, 1997). Although several approaches (e.g. use of cationic lipids) have markedly improved delivery of oligonucleotides to cells in culture, there is still really very little understanding of the underlying mechanisms.

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10.2 Permeation of Oligonucleotides across Membranes 10.2.1 Interactions of Oligonucleotides with the Lipid Bilayer For most drugs the basic mechanism of entry into the cell involves diffusion across the lipid bilayer of the plasma membrane. Clearly, in the case of typical polyanionic oligonucleotides, it seems far-fetched to visualize these highly polar molecules diffusing through the hydrophobic environment of the membrane bilayer. However, there are a number of chemically modified oligonucleotides that are uncharged, e.g. methylphosphonates, peptide nucleic acids (PNAs) (see also Chapter 4), and morpholino compounds (Good and Nielsen, 1998; Matteucci, 1996; Summerton and Weller, 1997). In addition, investi-gators have synthesized a variety of oligonucleotides with non-polar substituents including cholesterol and alkyl chains, these lipid modifications being intended to increase membrane binding and permeation. Thus it is conceivable that such chemically modified oligonucleotides might enter cells by passive diffusion across the membrane; however, experience thus far suggests that this is not the case. In some early work (Shoji et al., 1991), we found evidence that methylphosphonates entered cells by endocytosis rather than by diffusion. We also examined the diffusion rate of phosphodiester, phosphorothioate, and methylphosphonate oligonucleotides across lipid bilayer membranes (liposomes) (Akhtar and Juliano, 1991). Both charged Oligonucleotides and the uncharged methylphosphonate compounds exhibited extremely low diffusion rates across the lipid bilayer membrane, with T½ ranging from 4 to 10 days. In another study, we carefully measured the diffusion rates of alkyl-substituted phosphosphorothioate oligonucleotides across lipid membranes. The T½ for permeation of these oligonucleotides across the bilayer membrane was also of the order of several days (Hughes et al., 1994). Interestingly, while use of longer, more lipophilic alkyl substituents increased binding of oligonucleotides to the liposome membrane, this tactic failed to increase transmembrane permeation. Another group of investigators has used liposomes to investigate the membrane permeability of PNAs, which have an uncharged backbone. They found that the membrane permeability of PNAs was similar to that of standard oligonucleotides, with T½ of permeation in the range of 5–10 days (Wittung et al., 1995). Thus even for an uncharged oligonucleotide derivative, permeation by simple diffusion across the lipid bilayer is very slow. This is not surprising. The notion that simply reducing the charge on oligonucleotides would allow them to enter cells by passive diffusion seems to contradict decades of research on essential properties of cell membranes. Large molecules with polar residues, such as the amino, hydroxyl, or carbonyl residues found on purines and pyridines, do not easily cross the bilayer even if these molecules have no net charge; thus even phospholipid molecules ‘flip-flop’ across the bilayer at limited

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rates (Jain, 1988; Kotyk, 1985). For large molecules (such as oligonucleotides), permeation across the bilayer deviates from the classic behaviour predicted by Overton's rule, since molecular shape factors, as well as simple oil-water partition coefficients, play a role in trans-bilayer diffusion (Wolosin et al., 1978). In fact, permeation of larger molecules probably involves movement via defects in the bilayer structure rather than simple diffusion through the hydrophobic region (Lieb and Stein, 1986). Some interesting recent work on morpholino compounds, which have high Tm values, are very stable and are uncharged, suggested that some degree of antisense activity is seen with these molecules even in the absence of any delivery agent (Summerton et al., 1997). However, it is not clear that this is due to transmembrane permeation; rather, these ultrastable molecules may slowly leak from endosomes and be maintained in the cytoplasm, thus allowing an antisense effect. 10.2.2 Oligonucleotide Receptors and Transporter Proteins Since oligonucleotides are unlikely to enter the cell by simple diffusion across the lipid bilayer of the plasma membrane, an important issue in oligonucleotide transport is the possible role of membrane proteins in this process (see also Chapter 9). Cell surface proteins could act in two ways to enhance cellular uptake of antisense molecules, as receptors or as transporters. The simplest mechanism would involve a protein that acts as a receptor or binding site. This would enhance the local concentration of the oligonucleotide at the cell surface, and, if the protein were efficiently coupled to the endocytotic machinery, would directly enhance total intracellular accumulation. However, the oligonucleotides associated with the receptor would be within endocytotic vesicles and not in direct contact with the cytoplasm. Currently there is substantial evidence for various cell surface proteins playing a role in oligonucleotide binding and cell uptake. Another possibility would have the protein actually serving as a transporter that would move the antisense molecule across the membrane and into the cytoplasm. At this point there is only one report describing a transporter activity for oligonucleotides (Hanss et al., 1998). Cell surface proteins that bind radiolabelled oligonucleotides were first detected several years ago (Loke et al., 1989; Yakubov et al., 1989), with the initial candidates described as molecules in the 80 kDa range. These putative oligonucleotide receptors remain poorly characterized. More recently, Corrias and Cheng (1998) have described several cellular proteins ranging in size from about 40 to 60 kDa that bind phosphorothioate oligonucleotides, while other workers (Beltinger et al., 1995) have identified additional oligonucleotidebinding proteins. This straightforward detection of putative receptors for antisense molecules by radioligand binding has been somewhat unsatisfying,

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since it has been difficult to follow up and fully characterize the radiolabelled bands. Among the most likely candidates for cellular receptors for oligonucleotides are those proteins with a high binding affinity for polyanionic molecules (such as glycosaminoglycans). Thus, the identification as an oligonucleotide receptor (Benimetskaya et al., 1997) of MAC-1 (CD1 l b/CD18), a heterodimeric cell surface protein that is a member of the beta2 integrin sub-family, made a great deal of sense, since MAC-1 is known to bind avidly to heparin-like molecules. Another significant finding was the identification of so-called ‘scavenger receptors’ as being important oligonucleotide-binding proteins, especially in the liver and kidney (Biessen et al., 1998; Steward et al., 1998). This class of receptors is also known to bind polyanionic molecules, including denatured lipoproteins and proteoglycans. In the cases of both MAC-1 and the scavenger receptor, there is good evidence that the binding protein actually plays an important role in the intracellular accumulation of oligonucleotides. In contrast to the proteins discussed above, which are receptors for oligonucleotides, there has recently been a description of a molecule that seems to be a transporter for oligonucleotides (Hanss et al., 1998). This 45 kDa protein was purified from rat renal brush border membranes using an oligonucleotide affinity column. The oligonucleotide-binding protein was reconstituted into lipid vesicles which were then used to insert the 45 kDa protein into model planar lipid bilayers, so that electrophysiological measurements could be made. When the bilayer containing the 45 kDa protein was exposed to oligonucleotides, ion channel activity was observed, whereas no activity was seen in the absence of either the inserted protein or the oligonucleotide. A number of tests showed that the channel activity represented current carried by the oligonucleotide, indicating that the 45 kDa protein was indeed serving as an ion channel for oligonucleotides. This represents a fascinating set of observations; however, a number of issues need to be addressed. The partial purification of the 45 kDa protein needs to be followed by the cloning and full characterization of the molecule. In addition, the quantitative role of the putative oligonucleotide channel in cellular uptake of oligonucleotides needs to be defined, since most previous measurements have found no indication of direct transmembrane permeation of oligonucleotides. In addition to uptake by endogenous oligonucleotide receptors, several investigators have coupled oligonucleotides to peptides designed to bind to other types of cell surface receptors, thus promoting uptake of the oligonucleotide complex by cells. For example, peptides designed to bind to integrins (Bachmann et al., 1998) or to IGF-1 receptor (Basu and Wickstrom, 1997) have been used to enhance oligonucleotide uptake (see also Chapter 5).

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10.3 Mechanisms of Enhancement of Oligonucleotide Permeation across Membranes In most instances, some form of delivery agent has been necessary to attain clearcut antisense effects in cell culture. While a variety of strategies for oligonucleotide delivery have been employed, we will focus here on two approaches that have been widely used and that seem to hold substantial promise for further development. Thus, we will examine mechanisms involved in oligonucleotide uptake mediated by cationic lipids, and uptake mediated by several types of ‘delivery peptides’. 10.2.3.1 Mechanism of Delivery by Cationic Lipids Use of cationic lipids has become a widely accepted strategy for improving delivery of standard anionic antisense oligonucleotides to the cytoplasm and nucleus (see also Chapter 7). As originally described by Bennett et al. (1992) and widely confirmed by others (e.g. Alahari et al., 1996), most antisense compounds have virtually no effect when simply added to cells in culture, but upon complexation with various cationic lipids, the antisense molecules display potent activity. Fluorescence microscopy studies suggest an explanation for this phenomenon. When cells are incubated with ‘free’ fluorescent polyanionic oligonucleotides, most of the fluorescence is observed in vesicular structures (presumably endosomes) within the cytoplasm; there is very little fluorescent material in the nucleus. However, subsequent to incubation with oligonucleotidecationic lipid complexes, a substantial amount of fluorescence is observed in the nucleus, as well as in cytoplasmic vesicles. Thus the cationic lipids help to get oligonucleotides to key sites in the nucleus, where efficient antisense action can occur. Recent observations by two groups (Marcusson et al., 1998; Zelphati and Szoka, 1996), using fluorophores on both the lipid and the oligonucleotide, have suggested that separation of the two components occurs within endosomes, and that only the oligonucleotide passes on to the nucleus. The precise mechanism of antisense delivery via cationic lipids remains poorly defined, but presumably involves a transient destabilization of the endosomal membrane, or (much less likely) the plasma membrane. The lipid bilayer of the endosomal membrane would normally be very impermeable towards charged oligonucleotides. However, upon entering the endosome, the cationic lipid complex recruits anionic phospholipids found on the cytoplasmic face of the endosome bilayer, thus probably inducing a transition to a non-bilayer state (Zelphati and Szoka, 1996). Regions of non-bilayer lipid are known to be much ‘leakier’ to a variety of molecules than unperturbed bilayer (Jain, 1988). Given this model, a number of issues emerge regarding the use of cationic lipid complexes for antisense delivery. Since the cationic lipid destabilizes the

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endosome bilayer and permits egress of the oligonucleotide, it probably also permits egress of other molecules within the endosomes. Since at some point in the endocytotic pathway endosome to lysosome conjunction occurs, this suggests that cationic lipid might trigger the inappropriate release of lysosomal enzymes into the cytosol. While this has not, to our knowledge, been directly demonstrated, it could well be a contributor to the observed toxicity of cationic lipids. Other issues regarding cationic lipids arise when one considers them as in vivo delivery systems. The oligonucleotidecationic lipid complexes are typically quite large (micrometre) and thus would be cleared rapidly from the circulation. Further, the cationic lipid complexes are likely to have important deleterious effects on cytokine release (Scheule et al., 1997), on complement activation (Barron et al., 1998) and on the capabilities of the effector cells of the immune system (Filion and Phillips, 1997). Thus, although strong interest persists in the use of cationic lipids for gene and oligonucleotide delivery, there are clearly problems that will need to be overcome. 10.2.3.2 Mechanism of Delivery by Membrane-active Peptides Although cell membranes are generally impervious to big molecules such as proteins, there are a number of situations in nature where large proteins or even supramolecular particles move across membranes (see also Chapter 5). For example, a number of toxins (e.g. ricin, diphtheria toxin, Pseudomonas toxin), have domains that bind to and destabilize membranes, thus allowing the active domain of the toxin to enter the cytoplasm (Kaslow and Burnes, 1992; Pugsley, 1996). In addition, a number of viruses have capsid or envelop proteins that interact with membranes and permit entry of the viral nucleic acid into the cytoplasm (Greber et al., 1994). The best-studied example of this is the influenza virus haemaglutinin which undergoes a pH-sensitive conformational change within the acidic environment of endosomes, and subsequently interacts with the endosome membrane, thus permitting release of the virus. A number of investigators have tried to copy this strategy for enhancing transmembrane transport of large molecules by designing peptides that mimic the functions of toxin or viral proteins. These peptides generally are intended to assume an amphipathic helix configuration when the pH is at the level found in endosomes. Thus, these molecules should strongly associate with the membrane and induce a degree of membrane destabilization that may allow egress of material from the endosome. In the context of oligo transport, these peptides have been either chemically coupled to the oligonucleotide or used as part of a non-covalent complex. For example, a peptide sequence based on a critical region of the influenza virus haemaglutinin protein has been coupled to oligonucleotides and has demonstrated some degree of activity (Bongartz et al., 1994). A designed amphipathic helix peptide termed GALA (Parente et al., 1990) and variants thereof (Wyman et al., 1997) have also been used with some success, both for

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gene delivery (Wagner, 1998) and oligonucleotide delivery (Hughes et al., 1996; Wyman et al., 1997). The mechanism of action of amphipathic helix peptides has been carefully studied; they seem to act by forming pores in membranes and promoting phospholipid flip-flop and non-bilayer structures (Epand et al., 1995; Fattal et al., 1994). Although amphipathic helix peptides would seem a good idea for enhancing the transport of large molecules such as oligonucleotides, nature has also attained this end using other strategies. Several years ago surprising observations were made on the biological activity of two transcriptional activating factors, namely the Antennapedia protein from Drosophila and the Tat protein coded by HIV. It was observed that both of these molecules showed transcriptional activating activity when added to the outside of cells, thus indicating that these proteins were entering cells, moving across membranes, and penetrating to the nucleus (Frankel and Pabo, 1988; Joliot et al., 1991). In both cases the membrane penetrating activity was localized to short polybasic sequences; these peptides are capable of entering cells themselves and have also been shown to be able to convey other large molecules into cells (Derossi et al., 1996, 1998; Fawell et al., 1994). Additional peptide sequences have been used successfully to import other molecules into cells. This includes short hydrophobic polypeptides based on an FGF signal sequence that have been used to deliver peptides (Rojas et al., 1996), oligonucleotides (Dokka et al., 1997), and large chimeric proteins (Rojas et al., 1998) into cells. A 38 kDa basic protein from herpes simplex virus has also been shown to promote cellular uptake when linked as a chimera with other proteins (Elliott and O’Hare, 1997). In a dramatic example of the membrane penetrating power of the Tat polybasic sequence, Nagahara et al. (1998) have recently made a series of chimeric proteins containg a short Tat sequence at the N-terminal and have demonstrated delivery of proteins of up to 100 kDa into cells based on both microscopic visualization and functional criteria. An important point about the Tat peptide (and probably related polybasic peptides) is that these molecules are not predicted to form alpha-helical structures (Vives et al., 1997), and are thus likely to function quite differently from amphipathic helix peptides such as GALA. While the mechanism underlying the uptake of molecules linked to polybasic peptides such as Tat remains poorly defined, one interesting suggestion for Tatprotein chimeras is that the molecules go through the membrane in an unfolded (linear?) configuration and then are refolded, perhaps by heat-shock proteins, as they reach the cell interior (Nagahara et al., 1998). Whether this concept is relevant to transport of oligonucleotides is unclear at this point. One of the potentially exciting aspects of peptide-oligonucleotide conjugates as delivery moieties for antisense compounds is that these entities are of moderate molecular size (usually under 10 kDa). Thus their biodistribution properties in the extracellular environment may be roughly similar to those of oligonucleotides themselves, including penetration into a variety of tissues (Juliano et al., 1999). This is unlike the case for cationic lipid complexes, whose

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large size is likely to compromise uniform distribution to tissues and to promote clearance by the phagocytes of the reticuloendothelial system. Thus oligopeptide conjugates may have the potential to enhance intracellular delivery of antisense molecules without some of the problems of larger delivery complexes. 10.4 Summary The chemical nature of oligonucleotides as highly polar (usually charged) molecules of substantial molecular size precludes their ability to enter cells by simple diffusion across the lipid bilayer of the plasma membrane or endomembranes. Thus, some form of membrane disruption seems essential to enhance oligonucleotide entry into the cytoplasmic and nuclear compartments of cells. This might take the form of a major perturbation of endosomal membrane structure, with substantial regions of conversion to non-bilayer forms, as may be the case for delivery by cationic lipid particles. Alternatively, more subtle or localized perturbation resulting from the action of delivery peptides may also serve to facilitate entry of antisense molecules. The precise molecular details underlying enhanced oligonucleotide delivery by cationic liposomes, polymers or peptides remain to be elucidated. References AKHTAR, S. and JULIANO, R.L., 1991, Permeation characteristics of antisense DNA oligonucleotide analogs across model membranes (liposomes), Nucl. Acids Res., 19, 5551–5559. AKHTAR, S. and JULIANO, R.L., 1992, Cellular uptake and intracellular fate of antisense oligonucleotides, Trends Cell Biol., 2, 139–143. ALAHARI, S.K., DEAN, N.M., FISHER, M.H., BELONG, R., MANOHARAN, M, TIVEL, K.L. and JULIANO, R.L., 1996, Inhibition of expression of the multi-drug resistance associated P-glycoprotein by phosphorothioate and 5′ cholesterolphosphorothioate antisense oligonucleotides, Mol. Pharmacol., 50, 808–819. BACHMANN, A.S., SUROVOY, A., JUNG, G. and MOELLING, K., 1998, Integrin receptortargeted transfer peptides for efficient delivery of antisense oligodeoxynucleotides, J. Mol. Med., 76, 126–132. BARRON, L.G., MEYER, K.B. and SZOKA, F.C., JR, 1998, Effects of complement depletion on the pharmacokinetics and gene delivery mediated by cationic lipid– DNA complexes, Hum. Gene Ther., 9, 315–323. BASU, S. and WICKSTROM, E., 1997, Synthesis and characterization of a peptide nucleic acid conjugated to a D-peptide analog of insulin-like growth factor 1 for increased cellular uptake, Bioconjug. Chem., 8, 481–488. BELTINGER, C., SARAGOVI, H.U., SMITH, R.M., LESAUTEUR, L., SHAH, N., DEDIONISIO, L., CHRISTENSEN, L., RAIBLE, A., JAREYY, L. and GEWIRTZ, A.M., 1995, Binding, uptake and intracellular trafficking of phosphorothioatemodified oligodeoxynucleotides , J. Clin. Invest., 95, 1814–1823.

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BENIMETSKAYA, L., LOIKE, J.D., KHALED, Z., LOIKE, G., SILVERSTEIN, S.C., CAO, L., EL KHOURY, J., CAI, T.Q. and STEIN, C.A., 1997, Mac-1 (CD11b/CD 18) is an oligodeoxynucleotide-binding protein, Nat. Med., 3, 414–420. BENNETT, C.F., 1998, Antisense oligonucleotides: is the glass half full or half empty? Biochem. Pharmacol., 55, 9–19. BENNETT, C.F., CHIANG, M.Y., CHAN, H., SHOEMAKER, J.E. and MIRABELLI, C.K., 1992, Cationic lipids enhance cellular uptake and activity of phosphorothioate antisense oligonucleotides, Mol. Pharmacol., 41, 1023–1033. BIESSEN, E.A., VIETSCH, H., KuiPER, J., BusTERBOscH, M.K. and BERKEL, T.J., 1998, Liver uptake of phosphodiester oligodeoxynucleotides is mediated by scavenger receptors, Mol. Pharmacol., 53, 262–269. BONGARTZ, J.-P., AUBERTIN, A.-M., MILHAUD, P.G. and LEBLEU, B., 1994, Improved biological activity of antisense oligonucleotides conjugated to a fusogenic peptide, Nucleic Acids Res., 22, 4681–4688. BOUSSIF, O., LEZOUALC’H, F., ZANTA, M.A., MERGNY, M.D., SCHERMAN, D., DEMENEIX, B. and BEHR, J.P., 1995, A versatile vector for gene and oligonucleotide transfer into cells in culture and in vivo: polyethylenimine, Proc. Natl Acad. Sci. USA, 92, 7297–7301. CORRIAS, S. and CHENG, Y.C., 1998, Interaction of human plasma membrane proteins and oligodeoxynucleotides, Biochem. Pharmacol., 55, 1221–1227. DELONG, R., STEPHENSON, K., LOFTUS, T., ALAHARI, S.K., FISHER, M.H. and JULIANO, R.L., 1997, Characterization of complexes of oligonucleotides with polyamidoamine starburst dendrimers and effects on intracellular delivery, J. Pharm. Sci.. 86. 762–764. DEROSSI, D., CALVET, S., TREMBLEAU, A., BRUNISSEN, A., CHASSAING, G. and PROCHIANTZ, A., 1996, Cell internalization of the third helix of the Antennapedia homeodomain is receptor-independent, J. Biol Chem., 271, 18188–18193. DEROSSI, D., CHASSAING, G. and PROCHIANTZ, A., 1998, Trojan peptides: the penetration system for intracellular delivery, Trends Cell Biol., 8, 84–87. DOKKA, S., TOLEDO-VELASQUEZ, D., Sm, X., WANG, L. and ROJANASAKUL, Y., 1997, Cellular delivery of oligonucleotides by synthetic import peptide carrier, Pharm. Res., 14, 1759–1764. ELLIOTT, G. and O’HARE, P., 1997, Intercellular trafficking and protein delivery by a herpesvirus structural protein, Cell, 88, 223–233. Epand, R.M., SHAI, Y., SEGREAT, J.P. and ANANTHARAMAIAH, G.M., 1995, Mechanisms for the modulation of membrane bilayer properties by amphipathic helical peptides, Biopolymers, 37, 319–338. FATTAL, E., NIR, S., PARENTE, R.A. and SZOKA, F.C., JR, 1994, Pore-forming peptides induce rapid phospholipid flip-flop in membranes, Biochemistry, 33, 6721–6731. FAWELL, S., SEERY, J., DAIKH, Y., MOORE, C, CHEN, L.L., PEPINSKY, B. and BARSOUM, J., 1994, Tat-mediated delivery of heterologous proteins into cells, Proc. Natl Acad. Sci. USA, 91, 664–668. FILION, M.C. and PHILLIPS, N.C., 1997, Toxicity and immunomodulatory activity of liposomal vectors formulated with cationic lipids toward immune effector cells, Biochim. Biophys. Acta, 1329, 345–356.

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FISHER, T.L., TERHORST, T., CAO, X. and WAGNER, R.W., 1993, Intracellular disposition and metabolism of fluorescently-labeled unmodified oligonucleotides microinjected into mammalian cells, Nucl. Acids Res., 21, 3857–3865. FRANKEL, A.D. and PABO, C.O., 1988, Cellular uptake of the tat protein from human immunodeficiency virus, Cell, 55, 1189–1193. GOKHALE, P.C., SOLDATENKOV, V., WANG, F.H., RAHMAN, A., DRITSCHILO, A. and KASID, U., 1997, Antisense raf oligodeoxyribonucleotide is protected by liposomal encapsulation and inhibits Raf-1 protein expression in vitro and in vivo: implication for gene therapy of radioresistant cancer, Gene Ther., 4, 1289–1299. GOOD, L. and NIELSEN, P.E., 1998, Antisense inhibition of gene expression in bacteria by PNA targeted to mRNA , Nat. Biotechnol., 16, 355–358. GREBER, U.F., SINGH, I. and HELENIUS, A., 1994, Mechanisms of virus uncoating, Trends Microbiol, 2, 52–56. HANSS, B., LEAL-PINTO, E., BRUGGEMAN, L.A., COPELAND, T.D. and KLOTMAN, P.E., 1998, Identification and characterization of a cell membrane nucleic acid channel, Proc. Natl Acad. Sci. USA, 95, 1921–1926. HUGHES, J.A., AVRUTSKA, A.V., ARONSON, A. and JULIANO, R.L., 1996, Evaluation of adjuvants that enhance the effectiveness of antisense oligonucleotides, Pharm. Res., 13, 404–410. HUGHES, J.A., BENNETT, C.F., COOK, P.D., GUINOSSO, C.J., MIRABELLI, C.K. and JULIANO, R.L., 1994, Lipid membrane permeability of 2′-modified derivatives of phosphorothioate oligonucleotides, J. Pharm. Sci., 83, 597–600. JAIN, M.K., 1988, Introduction to Biological Membranes, New York: Wiley. JOLIOT, A., PERNELLE, C., DEAGOSTINI-BAZIN, H. and PROCHIANTZ, A., 1991, Antennapedia: homeobox peptide regulates neural morphogenesis, Proc. Natl Acad. Sci. USA. 88. 1864–1868. JULIANO, R.L., ALAHARI, S., YOO, H., KOLE, R. and CHO, M., 1999, Antisense pharmacodynamics: critical issues in the transport and delivery of antisense oligonucleotides, Pharm. Res., 16, 494–502. KASLOW, H.R. and BURNES, D.L., 1992, Pertussis toxin and target eukaryotic cells: binding, entry, and activation, FASEB J., 6, 2684–2690. KOTYK, A., 1985, Basic kinetics of membrane transport, Structure and Properties of Cell Membranes, G. Banga, ed., Boca Raton, FL: CRC Press. LEMAITRE, M., BAYARD, B. and LEBLEU, B., 1987, Specific antiviral activity of a poly(L-lysine)-conjugated oligodeoxyribonucleotide sequence complementary to vesicular stomatitis virus N protein mRNA initiation site, Proc. Natl Acad. Sci. USA, 84, 648– 652. LEONETTI, J.P., MECHTI, N., DEGOLS, G., GAGNOR, C. and LEBLEU, B., 1991, Intracellular distribution of microinjected antisense oligonucleotides, Proc. Natl Acad. Sci. USA, 88, 2702–2706. LETSINGER, R.L., ZHANG, G., SUN, D.K., IKEUCHI, T. and SARIN, P., 1989, Cholesterylconjugated oligonucleotides: synthesis, properties, and activity as inhibitors of replication of HIV in cell culture, Proc. Natl Acad. Sci. USA, 86, 6553–6556. LIEB, W.R. and STEIN, W.D., 1986, Non-Stokesian nature of transverse diffusion within human red cell membranes, J. Membr. Biol., 92, 111–119.

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LOKE, S.L., STEIN, C.A., ZHANG, X.H., MORI, K., NAKANISHI, M., SUBASINGHE, B., COHEN, J.S. and NECKER, L.M., 1989, Characterization of oligonucleotide transport into living cells, Proc. Natl Acad. Sci. USA, 86, 3474–3478. LORENZ, P., BAKER, B.F., BENNETT, C.F. and SPECTOR, D.L., 1998, Phosphorothioate antisense oligonucleotides induce the formation of nuclear bodies, Mol. Biol. Cell, 9, 1007–1023. MAHATO, R.I., TAKAKURA, Y. and HASHIDA, M., 1997, Development of targeted delivery systems for nucleic acid drugs, J. Drug Target., 4, 337–357. MARCUSSON, E.G., BHAT, B., MANOHARAN, M., BENNETT, C.F. and DEAN, N.M., 1998, Phosphorothioate oligodeoxyribonucleotides dissociate from cationic lipids before entering the nucleus, Nucl. Acids Res., 26, 2016–2023. MATTEUCCI, M., 1996, Structural modifications toward improved antisense oligonucleotides, Drug Discovery Des., 4, 1–16. NAGAHARA, H., VOCERO-AKBANI, A.M., SNYDER, E.L., HO, A., LATHAM, D.G., LISSY, N.A., BECKER-HAPAK, M., EZHEVSHY, S.A. and DOWDY, S.F., 1998, Transduction of full-length TAT fusion proteins into mammalian cells: TATp27Kip1 induces cell migration, Nat. Med., 4, 1449–1452. PARENTE, R.A., NIR, S. and SZOKA, F.C.J., 1990, Mechanism of leakage of phospholipid vesicle contents induced by the peptide GALA, Biochemistry, 29, 8720–8728. POLITZ, J.C., BROWNE, E.S., WOLF, D.E. and PEDERSON, T., 1998, Intranuclear diffusion and hybridization state of oligonucleotides measured by fluorescence correlation spectroscopy in living cells, Proc. Natl Acad. Sci. USA, 95, 6043–6048. PUGSLEY, A.P., 1996, Bacterial toxins deliver the goods, Proc. Natl Acad. Sci. USA, 93, 8155–8156. ROJAS, M., DONAHUE, J.P., TAN, Z. and LIN, Y.Z., 1998, Genetic engineering of proteins with cell membrane permeability, Nat. Biotechnol., 16, 370–375. ROJAS, M., YAO, S. and LIN, Y.Z., 1996, Controlling epidermal growth factor (EGF)stimulated Ras activation in intact cells by a cell-permeable peptide mimicking phosphorylated EGF receptor, J. Biol. Chem., 271, 27456–27461. SCHEULE, R.K., ST GEORGE, J.A., BAGLEY, R.G., MARSHALL, J., KAPLAN, J.M., AKITA, G.Y., WANG, K.X., LEE, E.R., HARRIS, D J., JIANG, C, YEW, N.S., SMITH, A.E. and CHENG, S.H., 1997, Basis of pulmonary toxicity associated with cationic lipid-mediated gene transfer to the mammalian lung, Hum. Gene Ther., 8, 689–707. SHOEMAN, R.L., HARTIG, R., 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 Nucl. Acid Drug Dev., 7, 291–308. SHOJI, Y., AKHTAR, S., PERIASAMY, A., HERMAN, B. and JULIANO, R.L., 1991, Mechanism of cellular uptake of modified oligodeoxynucleotides containing methylphosphonate linkages, Nucl Acids Res., 19, 5543–5550. SPILLER, D.G., GILES, R.V., GRZYBOWSKI, J., TIDD, D.M. and CLARK, R.E., 1998, Improving the intracellular delivery and molecular efficacy of antisense oligonucleotides in chronic myeloid leukemia cells: a comparison of streptolysin-O permeabilization, electroporation, and lipophilic conjugation, Blood, 91, 4738–4746.

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STEIN, C.A. and CHENG, Y.-C., 1993, Antisense oligonucleotides as therapeutic agents is the bullet really magical? Science, 261, 1004–1012. STEWARD, A., CHRISTIAN, R.A., HAMILTON, K.O. and NICKLIN, P.L., 1998, Coadministration of polyanions with a phosphorothioate oligonucleotide (CGP 69846A): a role for the scavenger receptor in its in vivo disposition, Biochem. Pharmacol., 56, 509–516. SUMMERTON, J., STEIN, D., HUANG, S.B., MATTHEWS, P., WELLER, D. and PARTRIDGE, M., 1997, Morpholino and phosphorothioate antisense oligomers compared in cell-free and in-cell systems, Antisense Nucl. Acid Drug Dev., 7, 63–70. SUMMERTON, J. and WELLER, D., 1997, Morpholino antisense oligomers: design, preparation, and properties, Antisense Nucl. Acid Drug Dev., 7, 187–195. TAKAKURA, Y., OKA, Y. and HASHIDA, M., 1998, Cellular uptake properties of oligonucleotides in LLC-PK1 renal epithelial cells, Antisense Nucl. Acid Drug Dev., 8, 67–73. THIERRY, A.R., RAHMAN, A. and DRITSCHILO, A., 1993, Overcoming multidrug resistance in human tumor cells using free liposomally encapsulated antisense oligodeoxynucleotides, Biochem. Biophys. Res. Commun., 190, 952–960. VIVES, E., BRODIN, P. and LEBLEU, B., 1997, A truncated HIV-1 Tat protein basic domain rapidly translocates through the plasma membrane and accumulates in the cell nucleus, J. Biol. Chem., 272, 16010–16017. WAGNER, E., 1998, Effects of membrane-active agents in gene delivery, J. Controlled Release, 53, 155–158. WAGNER, R.W., 1994, Gene inhibition using antisense oligodeoxynucleotides, Nature, 372, 333–335. WANG, S., LEE, R.J., CAUCHON, G., GORENSTEIN, D.G. and LOW, P.S., 1995, Delivery of antisense oligodeoxyribonucleotides against the human epidermal growth factor receptor into cultured KB cells with liposomes conjugated to folate via polyethylene glycol, Proc. Natl Acad. Sci. USA, 92, 3318–3322. WITTUNG, P., KAJANUS, J., EDWARDS, K., NIELSEN, P., NORDEN, B. and MALMSTROM, B.G., 1995, Phospholipid membrane permeability of peptide nucleic acid, FEBS Lett., 365, 27–29. WOLOSIN, J.M., GINSBERG, H., LIEB, W.R. and STEIN, W.D., 1978, Diffusion within egg lecithin bilayers resembles that within soft polymers, J. Gen. Physiol., 71, 93–100. WYMAN, T.B., NICOL, F., ZELPHATI, O., SCARIA, P.V., PLANK, C. and SZOKA, F.C., JR, 1997, Design, synthesis, and characterization of a cationic peptide that binds to nucleic acids and permeabilizes bilayers , Biochemistry, 36, 3008–3017. YAKUBOV, L.A., DEEVA, E.A., ZARYTOVA, V.F., IVANOVA, E.M., RYTE, A.S., YURCHENKO, L.V. and VLASSOV, V.V., 1989, Mechanism of oligonucleotide uptake by cells: involvement of specific receptors? Proc. Natl Acad. Sci. USA, 86, 6454–6458. ZELPHATI, O. and SZOKA, F.C., JR, 1996, Intracellular distribution and mechanism of delivery of oligonucleotides mediated by cationic lipids, Pharm. Res., 13, 1367– 1372. ZELPHATI, O. and SZOKA, F.C., JR., 1996b, Mechanism of oligonucleotide release from cationic liposomes, Proc. Natl. Acad. Sci. USA, 93, 11493–11498.

11 Pharmacokinetics of Oligodeoxynucleotides A.GOUYETTE

11.1 Introduction The ‘antisense’ concept (Zamecnik and Stevenson, 1978) is 20 years old and is still a rational and promising approach in developing oligonucleotides or analogues as therapeutic agents, with potential applications in oncology, in viral diseases and in gene-related disorders. These potential drugs have captured the imagination, since the compounds are rationally designed with supposedly high specificity to interfere with genetic information, from the gene to the protein, by intermediary alteration of RNA metabolism. In the past five years, we have learned a lot—but still not enough—on the fate of this new class of compounds, from basic interactions with their putative targets to tolerance and efficacy clinical trials in humans, through preclinical studies in animal models, in terms of pharmacokinetics and pharmacodynamics. Many publications have dealt, with considerable controversy (Crooke, 1996; Stein, 1996), with these compounds’ challenging mechanisms of action, specific and non-specific (Branch, 1998) and with their behaviour in the body, following different ways of administration and/or formulations (for a review, see Crooke, 1998). Their biological targets are also numerous: HIV (GEM® 91, Hybridon; AR177, Aronex Pharmaceuticals, etc.), protein kinase C (PKC-α, CGP 64128A or ISIS 3521), human c-raf-l kinase (CGP 69846A or ISIS 5132), Ha-ras (ISIS 2503), bcl-2 (G 3139), and thrombin (GS-522, Gilead Sciences), among others (see also Chapter 12). To bring these new active principles onto the market, different companies and academic research units have developed numerous analytical techniques (32P- or 33P- and 35S-radiolabelled oligonucleotides, liquid chromatography, capillary electrophoresis, fluorimetric assays, mass spectrometry, positron emission tomography, etc.) to study the stability of the natural phosphodiester oligonucleotides and their modified (phosphorothioates, 2′-O-methyl, cholesteryl, PNAs, etc.) (see also Chapter 2) counterparts (Uhlmann et al., 1997), their in vitro uptake in different cell types, their tissue distribution and their

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fate (biotransformation and elimination) in animals (mouse, rat, monkey) and in the human body. To improve the stability of these compounds vis-à-vis the nucleases, several galenic formulations have been developed (liposomes, nanoparticles, etc.) and modes of administration other than intravenous injection have been tested, such as subcutaneous, intraperitoneal, gastrointestinal and pulmonary routes (see Chapters 6 and 7). In this chapter, we shall not review the mechanisms of action of the oligonucleotides (see Crooke, 1996), but will focus on the pharmacokinetic data, in animal models and in humans, healthy volunteers or patients. 11.2 Pharmacokinetics Pharmacokinetics (Gouyette and Kerr, 1995), in brief, is a tool, referred to as ADME, used in the determination of the parameters which govern the absorption (A), the tissue distribution (D), the metabolism (M) or biotransformation and the elimination or excretion (E) processes, as a function of time. When the drug is injected intravenously, the bioavailability is equal to unity, by definition. But when other routes of administration are used, the bioavailability may vary from 0 to 1, depending on the stability of the principle (for example, in the gastric juice), on the first-pass metabolism in the liver and on the quantity which is absorbed from the injection or administration site. Therefore, when studying oligonucleotides, one must be very careful about those factors which dramatically influence the blood or plasma profiles and may lead to nontherapeutic concentrations or to major side-effects. Moreover, pharmacokinetics can apply to a single cell or a population of cells in vitro, to an isolated organ ex vivo, or to a whole body in vivo. However, before proceeding to pharmacokinetic studies, one must develop a validated analytical assay to quantitate the intact drug and, possibly, its metabolite(s) or degradation product(s), in blood, plasma, urine and in some tissues relevant to the potential target, whenever possible, with enough sensitivity to define precisely the elimination half-life and other pharmacokinetic parameters such as clearance. If a radiolabelled drug is to be used, we must ascertain that a separative technique is available to distinguish the intact drug from the degradation or metabolic products. Of importance is the development of non-invasive techniques to follow up the distribution of those compounds in vivo: in this area, positron emission tomography (PET scanning) seems to be a methodology of choice, since it also gives access to kinetic analysis.

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11.2.1 Analytical Chemistry At first, synthetic and post-synthetic radiolabelling strategies were developed to provide radioactive tracers amenable to animal tissue distribution studies and elimination balance quantitation: for example, 3H (Sands et al., 1994; Crooke et al., 1995, 1996) or 14C (Cossum et al., 1993) was incorporated during oligonucleotide synthesis, 35S was incorporated in the phosphorothioate derivatives (Agrawal et al., 1991; Zhang et al., 1995a; Broaddus et al., 1998), 5′ phosphate labelling was obtained through the use of 32P-y-ATP (Saijo et al., 1994; Nakada et al., 1996; Comas et al., 1997; Tenu et al., 1997; Etoré et al., 1998) and 33P was used in the study of AR177, or zintevir, in rats (Wallace et al., 1997). Then, the development of liquid chromatographic methods of separation of the intact oligonucleotide from its degradation products under the action of nucleases allowed the follow-up of blood or plasma levels as well as the analysis of the urinary extracts, using UV detection. Most of the separation procedures were based on ion exchange, since the natural oligonucleotides and the phosphorothioate analogues are polyanions (Bourque and Cohen, 1993; Chen et al., 1997; Gouyette, 1995; Nolting et al., 1997) but, in many cases, the limit of quantitation was not always appropriate for a proper determination of the elimination half-life. Some authors did extract the oligonucleotides by using anion-exchange solid-phase cartridges and purified the extracts on C18 reversephase cartridges (Phillips et al., 1997; Nicklin et al., 1998). Others developed high-performance liquid chromatographic methods, using columns packed with Dionex Nucleopack PA-100 and UV detection (Qian et al., 1997). In order to increase the sensitivity of the assays, fluorimetric probes linked to the oligonucleotides, prior to the administration or after extraction, have also been used (Giles et al., 1993). Then, the use of capillary electrophoresis with either UV or fluorimetric detection proved to be very efficient to separate the parent oligodeoxynucleotide from the sequential degradation products (Srivatsa et al., 1994; Crooke et al., 1996; Chen et al., 1997; Glover et al., 1997; Gray and Wickstrom, 1997). The development of electrospray (interfaced with capillary electrophoresis— Deroussent et al., 1995; Ni et al., 1996) or matrix-assisted laser desorption ionization (Van Ausdall and Marshall, 1998) mass spectrometry has allowed the analysis of oligonucleotides. Another original way to quantitate phosphodiester or phosphorothioate oligonucleotides was deigned by Deverre et al. (1997), based on a competitive enzyme hybridization assay, with a limit of quantitation of 900 pM, using 100 |il of plasma.

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11.2.2 Stability Stability measurements have been carried out in different ways. For example, Bruin et al. (1995) used capillary gel electrophoresis and matrix-assisted laser desorption ionization (MALDI) mass spectrometry to study the degradation of oligonucleotides, and discussed the factors influencing the kinetics of enzymatic digestion (specific nuclease concentration, antisense oligonucleotide structure, etc.). Deverre et al. (1997), using a competitive enzyme hybridization assay which seems to be highly specific for an intact 15-mer phosphodiester compound, found an apparent degradation half-life of ~30 min in mouse plasma; its phosphorothioate analogue was more stable with a degradation rate characterized by a half-life of ~1 h. 11.3 Chemistry of Oligonucleotides and Formulations Because the intrinsic instability of phosphodiester oligodeoxynucleotides in vivo is a drawback in their therapeutic use as drugs, many chemical modifications have been carried out to stabilize the oligonucleotide backbone (see Cantin and Woolf, 1993; Crooke and Bennett, 1996; Le Doan et al., 1996). Another approach was to protect the oligonucleotides from the nucleases by developing new formulations which also were aiming at a better membrane penetration (see Chapter 10), either liposomes (Juliano and Akhtar, 1992; Tari et al., 1996) or polyalkylcyanoacrylate nonoparticles where the negative charges of the oligonucleotides were associated with hydrophobic cations (Fattal et al., 1998). In another approach, polycationic substances (diethylaminoethyl dextran, Lipofectin®, etc.) were designed to interact with the oligonucleotide polyanions, which could improve the pharmacokinetic properties of those DNA fragments (Crooke, 1995; Steward et al., 1998). In order to target antisense cholesterol-linked oligonucleotides to specific tissues or cell types (de Smidt et al., 1991), the lipoproteins were utilized as transport vehicles, resulting in an increased elimination half-life (T½ = 9–11 min) compared to the control oligonucleotide (T½<1 min). 11.4 Cellular Pharmacokinetics The uptake of oligodeoxynucleotides (see also Chapters 9 and 10) has been reported to be an active process, with saturable kinetics (Loke et al., 1989), but the uptake of phosphodiester oligonucleotides is somewhat inefficient (Spiller and Tidd, 1992). Giles et al. (1993) showed that chimeric

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(methylphosphonodiester/ phosphodiester) oligonucleotides may have increased cellular import and access to the intracellular compartments. Zhao et al. (1993), using fluorescein-conjugated oligodeoxynucleotides, found that phosphorothioates had the highest cell binding and uptake. At the same time, they observed that the oligonucleotides were associated more with dead cells than with living cells by a factor of 50. By the use of confocal microscopy, they could confirm that the oligonucleotides were localized in the cytoplasm and that there was little nuclear uptake by 4 h. Crooke et al. (1995) studied the uptake, subcellular distribution and metabolism of ISIS 2105 (Afovirsen) in a variety of cell lines, and found that ISIS 2105 and other phosphorothioates are internalized and distributed in a time-, temperature-, concentration-, sequence- and cell line-dependent manner, with some influence of the culture medium also. They concluded that in vitro experiments are not likely to be predictive of what could happen in vivo. Hawley and Gibson (1996) described the interaction of oligodeoxynucleotides with mammalian cells and found, as others (Hefeneider et al., 1992; Zani et al., 1995), that different proteins could play a role as carriers or receptors for oligonucleotides to cross the cell membrane, besides pinocytosis and fluid phase endocytosis, the mechanisms changing according to cell type. As oligonucleotides are being developed for disease treatment, such as gliomas, Engelhard et al. (1996), using fluorescein-labelled compounds, followed the incorporation by immunofluorescence microscopy and flow cytometry. They also showed that uptake was dependent on the cell line used in the experiments, although uptake in tumor (glioma) cells was proven in vivo. Corrias et al. (1997) also focused their work on the study of the bioavailability of antisense (phosphodiester, phosphorothioate and cholesteryl) oligonucleotides in three different human neuroblastoma cell lines. At high doses, all the oligonucleotides caused a necrotic lysis of plasma membranes (no apoptotic cell death). As usual, phosphorothioates had the higher stability; combination of phosphorothioate links and lipid conjugation (as well as cholesteryl derivatization) was the most effective for intracellular accumulation. However, the actual amount of antisense molecules found inside the neuroblastoma cells was low in all conditions tested. Those observations were consistent in all publications. Lebleu et al. (1997) reviewed the major problems in the use of synthetic oligonucleotides in cell cultures: instability, passage through biological barriers, binding to serum and cellular proteins (oligonucleotides are polyanions). They also provided some ideas to improve the oligonucleotide delivery: for example, they proposed the use of poly(L-lysine) as a cationic polymer to deliver biologically active oligomers. Cell targeting can also be achieved through encapsidation in antibody-conjugated liposomes. But there was still a major barrier to bypass: the efficient escape from the endocytic compartment. Etoré et al. (1998) studied the internalization and the distribution of phosphorothioates in vascular smooth muscle cells. They found that the amount

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of protein-bound oligonucleotide species could be as high as 50% of the total amount internalized in cells. But it is still difficult to measure the fractions in the different organelles. 11.5 Preclinical Pharmacokinetics In preclinical development, Nolting et al. (1997) characterized the hepatic clearance of backbone-modified (phosphorothioate and methylphosphonate) oligonucleotides in the isolated perfused rat liver (for 2 h). The charged 15-mer , phosphorothioate was eliminated slowly from the liver: and an extraction ratio of . with a steadystate volume The phosphorothioate was highly protein-bound (80%). The oligonucleotide biliary concentration was below the limit of detection. By confocal microscopy, it was possible to localize the highest concentrations in the sinusoids and the walls of blood vessels, regions in which Kupfer cells are predominant. One of the first pharmacokinetic and distribution studies, after intravenous or intraperitoneal injection to mice, was made by Agrawal et al. (1991), using a [35S]-labelled 20-mer phosphorothioate, complementary to the HIV tat splice acceptor site (nucleotides 5349–5368). About 30% of the administered dose was recovered in the urine, within 24 h, as degraded products. The radioactivity tracer was found in stomach, heart and intestine, as well as chain length extension compounds, not identified so far. In mice, the pharmacokinetics and tissue distribution of ISIS 3466 (a 20-mer phosphorothioate) were studied following intraperitoneal or intravenous injections. The oligonucleotide was rapidly cleared from the circulation in the normal mice. The highest concentrations were measured in kidney and liver; brain had a very low concentration. Intact oligonucleotide was detected after 48 h. When the oligonucleotide was complexed with DOTMA, or N-[1-(2, 3dioleyloxy)propyl]-N, N, N-trimethylammonium chloride, the uptake and distribution in normal mice were not affected. But DOTMA significantly increased the oligonucleotide cellular uptake by LOX ascites in an intraperitoneal/intraperitoneal model (Saijo et al., 1994). Another biodistribution study was carried out by Sands et al. (1994) using a tritium-labelled 20-mer nucleotide phosphodiester and its phosphorothioate analogue. The dose was 6 mg/kg, injected intravenously. Relative concentrations were as follows: kidney>blood>heart>liver>lung>spleen (radiolabel in spleen peaked at 1 h and remained elevated for 24 h). At 2 h, the concentration in all organs, except spleen, was equal to that in blood. For the phosphorothioate, kidney uptake was high for 24 h (autoradiographic studies). No intact compound could be detected in the urine (excreted compounds were degradation products). However, in rats, Iversen et al. (1994) found that a 27-mer oligodeoxynucleotide complementary to the rev gene mRNA of HIV-1 was excreted unchanged in the urine (electrophoretic analysis), with a complete

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elimination in the urine within 3 days. They characterized the plasma profile by a min and . There two-compartment model with appears to be a contradiction between a complete elimination in 72 h and half-lives longer than 24 h! A 36-mer phosphorothioate oligonucleotide (targeting the tat gene of HIV-1) was studied in rats (Boado et al., 1995). The reported pharmacokinetic ml/min per kg; steady-state volume of parameters were: clearance, ml/kg; distribution half-life, min; and distribution, min. elimination half-life, Tissue distribution was described in tumor-bearing mice, after injection of a 15-mer oligonucleotide targeting the translation/initiation of the c-myc mRNA (Plénat et al., 1995), by autoradiography and immunohistochemistry, as well as with fluorescence microscopy. The findings can be summarized as follows: (a) within minutes, oligonucleotides permeate all cells and tissues with the exception of erythrocytes and intervertébral discs; (b) concentration of oligonucleotides is higher within the connective tissue cells than in the interstitial matrix; (c) after uptake, oligomers partition throughout all of the cellular compartment, including, at the highest intracellular concentrations, in the nuclei; (d) oligonucleotides penetrate easily the tumor cell compartments, oligonucleotide diffusion being impeded by the extracellular matrix. GEM® 91 (a 25-mer phosphorothioate targeting HIV-1 gag mRNA) was injected to rats, as a bolus of 30 mg/kg (Zhang et al., 1995a). The half-lives were ; . The main estimated to be: elimination process was renal excretion, with 26.7 ± 6.5% of the radioactivity ). recovered in the urine, within 24 h, compared with faecal excretion ( During the initial 30 min, the highest concentrations were found in the kidney, liver, spleen, lungs, and heart. Another pharmacokinetic study was carried out in cynomolgus monkeys (Lee et al., 1995) with an oligonucleotide (GS-522) developed as an inhibitor of thrombin. After a constant intravenous infusion (0.1, 0.3 and 0.5 mg/kg per min) for 60 min or a bolus injection (11.25 and 22.5 mg/kg), the half-lives were: min and min. The apparent steady-state volume of distribution (Vss) was superimposable with the blood volume of the monkey. Almost similar results were found with ISIS 3082 (a 20-mer phosphorothioate that inhibits the expression of mouse intercellular adhesion molecule-1) and analogues (ISIS 9045, ISIS 9046, ISIS 9047), where little intact drug could be recovered in the urine or faeces for any analogue. The metabolism of ISIS 3082 was similar to that reported for other phosphorothioates. After 2 h, most of the radioactivity in plasma was due to metabolites but, in tissues, intact ISIS 3082 was present for much longer times (Crooke et al., 1996). During the development of an animal model to study the effect of photoreceptorderived debris accumulation on the normal function of the retina in vivo, Rakoczy et al. (1996) described the uptake, distribution and persistence of an antisense oligonucleotide injected into the vitreous of 7-week-old RCS-rdy +

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rats. Following intravitreal injection, penetration of the oligonucleotide was observed in the ganglion cell layer in 2 h and in the photoreceptor and pigment epithelial layers 3 days later. It was also demonstrated that the fluoresceinlabelled oligonucleotide present in the retinal pigment epithelial cells was not degraded and retained its original 19-mer length. Deverre et al. (1997), using their competitive enzyme hybridization assay, determined the pharmacokinetics of a 15-mer phosphodiester oligonucleotide, after intravenous injection of 120 nmol/kg to mice. They found a very short halflife of ~4.8 min, an approximate area under the plasma-time curve (AUC) of 114 ng min/ml, and a clearance (CL) of about 4.4 l/kg per min, for the unchanged drug. When two phosphorothioate bounds were introduced in the oligonucleotide, the pharmacokinetic parameters were: half-life of 7.4 min, AUC of 188 ng min/ ml and CL of 2.7 l/kg per min. After replacement of all phosphodiester bonds by phosphorothioates, the AUC increased by a factor of 29 (AUC = 3257 ng min/ ml), the clearance decreased by a factor of 22 (CL = 0.2 l/ kg per min), but the elimination half-life was not significantly modified (5.2 min). Cellular distribution of phosphorothioate oligodeoxynucleotides in normal rodent tissues was also studied in vivo using three histological methods: immunohistochemistry, direct fluorescence microscopy and autoradiography (Butler et al., 1997). Proximal tubule cells in the kidney and Kupfer and endothelial cells in the liver were among the most heavily labelled, at all doses and time-points. At 2 h post-injection, the oligonucleotides were detectable in the extracellular matrix, although by 24 h the label was predominantly intracellular; they were not detected in erythrocytes, cartilage, compact bone and skeletal muscle. In spleen, white pulp was negative, whereas cells surrounding the sinusoids and nucleated cells in the red pulp were strongly positive. In nude mice, DeLong et al. (1997) also studied the fate of 15-mer modified (phosphorothioate, phosphorodithioate and methylphosphonate) 14Coligodeoxynucleotides complementary to the AUG region of K-ras, after intravenous injection to nude mice harbouring a K-ras-dependent human pancreatic tumor. Plasma concentration profiles appeared to be biphasic, with a rapid distribution characterized by a half-life, T½(α), of 1 min or less and an elimination half-life, T½(β), in the range of 24–35 min. The apparent volumes of distribution were 6.3 ml (phosphorothioate), 3.2 ml (phosphorodithioate), and 4.8 ml (methylphosphonate), compared with 3.6 ml, the volume of distribution for glucose (as a fluid-phase marker). Tissue distribution was highest in kidneys, followed in decreasing relative values by liver, spleen, tumor and muscle. Some intact oligonucleotide was detectable in all tissues studied, including tumor. Kidney and liver appeared to be the main clearance organs. Geary et al. (1997) focused their work on two phosphorothioate oligonucleotides designed as inhibitors of PKC-α (ISIS 3521 or CGP 64128A) and C-raf kinase (ISIS 5132 or CGP 69846A), before entering phase I/II clinical trials (see also Chapter 12). Their findings suggest that the pharmacokinetics of

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phosphorothioate oligodeoxynucleotides is independent of sequence and that the pattern of distribution to organs is similar across species and independent of the route of administration. For example, the plasma clearance of ISIS 3521 was 14. 5 ml/min per kg in mouse, 1.6 ml/min per kg in rat, and 2.1 ml/min per kg, in cynomolgus monkey, at comparable doses (0.6–1.0 mg/kg), although the relationship between dose and exposure of tissues varies non-linearly. The toxicological properties of phosphorothioate oligodeoxynucleotides were described by Henry et al. (1997), such as prolongation of clotting time, complement activation, proximal tubule degeneration and hepatotoxicity. Again, a pharmacokinetic and distribution study, in nude mice, of a 20-mer phosphorothioate DNA-methyl-transferase antisense oligodeoxynucleotide (Qian et al, 1997) displayed the same features concerning: (a) the amount of drug found in tissues (kidney>liver>tumor>lung>muscle>brain); (b) the elimination halflife, varying between 46 min (at 30 mg/kg) and 240 min (at 300 mg/kg). Wallace et al. (1997) found that AR177 (zintevir, a 17-mer oligonucleotide, with just two phosphorothioate bonds, one at the 5′-end and one at the 3′-end), which has anti-HIV activity in vitro, displayed a very long half-life, either in blood (367 h) or in plasma (271 h), with distribution in the liver (40%), in the bone marrow (17%) and even in the brain cortex (15%), 8 h after intravenous injection to rats. These authors also report that more than 90% of the [33P]radioactivity in the tissues represented intact AR177. In contrast with previous reports, those results were attributed to the tight binding of AR177 to tissues. With new and more specific analytical procedures, the degradation products, chain-shortened oligonucleotides, could be identified by capillary electrophoresis and electrospray mass spectrometry (Phillips et al., 1997). Positron emission tomography (PET) is also a very potent tool for in vivo imaging to study tissue distribution and kinetics. This technique was used by Tavitian et al. (1998) to trace the fate of an 18-mer oligonucleotide complementary to nucleotides 1209–1227 of the env sequence of the Lilly and Steeves strain of murine SFFV Friend erythroleukemia virus. PET scan was performed with 18F (fluorine)-labelled oligonucleotides at the 3′-end (which has no effect on the hybridization with its target complementary sequence). Then, these authors compared the body distribution of the phosphodiester oligonucleotide and the phosphorothioate and 2′-O-methyl-RNA analogues in baboons. They observed that, in fact, the kinetics are highly variable with the nature of the oligonucleotide backbone. They could measure the concentration of [18F]-labelled metabolites in plasma and confirmed that the oligonucleotides were mainly distributed in kidneys and liver, the phosphodiester being rapidly excreted in the bladder while the phosphorothioate was eliminated more slowly in the urine but higly concentrated in the kidney tissues. The 2′-O-methyl-RNA analogue has a behaviour which falls between the phosphodiester and the phosphorothioate (urinary excretion and kidney concentration). This technique should be preferred to whole-body autoradiography.

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Other modes of administration of antisense oligonucleotides have been used to deliver those drugs locally. A pharmacokinetic study has been reported (Leeds et al., 1998) after intravitreal administration of ISIS 2922, a 21-mer phosphorothioate that has shown antiviral activity against the cytomegalovirus, by inhibition of the expression of the major immediate-early gene. It has undergone phase III studies as a treatment for CMV-induced retinitis. Concentrations in the vitreous were proportional to the dose administered (11, 57 or 115 µg/eye), which ranged between 80 nM and 1.5 µM, 3 days after dosing. The uptake by the retina was reported to be saturated with average (n = 4, at the 57 µg dose) and concentrations of (n = 3, at the 115 µg dose). Here again, shorter oligonucleotides were detected in the vitreous and retina, according to the electrophoretic profiles. Subcutaneous, intra-peritoneal and intra-tracheal (pulmonary) routes were also investigated (Nicklin et al., 1998). Bioavalability was estimated by comparison of the intravenous administration in rats: subcutaneous (~31%), intra-peritoneal (~28%) and gastrointestinal (<2%), the pumonary bioavailability being dosedependent (3.2% at 0.06 mg/kg, 16.5% at 0.6 mg/kg and ~40% at 8 mg/kg). New formulations, such as nanoparticles (Nakada et al., 1996), may protect oligonucleotides from rapid degradation in plasma or tissues and act as a drug reservoir for prolonged delivery. 11.6 Clinical Pharmacokinetics Human pharmacokinetics of GEM® 91 were studied in six HIV-infected patients, with the [35S]-labelled 25-mer oligodeoxynucleotide (Zhang et al., 1995b). The antisense oligonucleotide was infused intravenously over 2 h at a dose of 0.1 mg/ kg. Plasma samples were monitored for total radioactivity and were analysed by autoradiography after gel electrophoresis. The urinary concentrations were determined by liquid scintillation counting (total radioactivity) and the urine collections were also analysed by ion-exchange HPLC. Based on total radioactivity plasma levels, the concentrations decreased and biexponentially with half-life values of . The mean AUC was 4387 ± 370 ng h/ml, the mean per kg, and the mean steady-state volume of clearance kg. The mean concentration distribution was estimated to be . The gel electrophoretic analysis at the end of the infusion was indicated the presence of intact GEM® 91 (up to 6 h after the end of the infusion) and lower molecular weight ‘metabolites’ in plasma. Cumulative excretion in the urine over 96 h was evaluated to 70.4 ± 6.7%, with a range between 50.4 and 93. 3%. At 24 and 48 h, the corresponding figures were 49.2 ± 6.8% (range, 35.6–81. 2%) and 60.7 ± 6.1% (range, 46.9–89.5%), respectively. The greater part of radioactivity in the urinary extracts corresponded to degradation products.

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During an open-label, multiple-dose safety clinical phase 1A study in HIVpositive patients, in France, we (Sereni et al., 1999) followed the pharmacokinetics of GEM® 91 as repeated doses (intravenous 2 h infusion) were increased (0.1, 0.3, 0.5 and 1.0 mg/kg, in the first part including 24 patients, then from 1.0 to 2.0 and 2.5 mg/kg per day in 18 patients). The quantitation of intact GEM® 91 was assessed by anion-exchange HPLC and UV detection. The pharmacokinetic parameters were calculated by curve-fitting according to a onecompartment model, with zero-order infusion (for 2 h) and first-order elimination. The mean elimination half-life was 55 ± 6 min at 1.0 mg/kg, 79 ± 11 min at 2.0 mg/kg and 93 ± 21 min at 2.5 mg/kg. The corresponding peak concentrations were 7.5 ± 1.9 µg/ml, 19.0 ± 5.1 µg/ml and 27.4 ± 2.6 µg/ml, and the total clearance was found to be 65.3 ± 17.7 ml/min, 42.6 ± 14.1 ml/min and 30.4 ± 4.6 ml/min, respectively. The apparent volume of distribution was determined as 5.1 ±1.01 at 1.0 mg/kg, 4.7 ± 1.1 l at 2.0 mg/kg and 4.0 ± 0.5 l at 2. 5 mg/kg. Taking these results with the study at lower doses where the pharmacokinetic parameters at the 1.0 mg/kg dose were superimposable with those of the second study (e.g. elimination halflife 56.6 ± 5.6 min; ), we could conclude that the pharmacokinetics of GEM® 91 in HIV patients is non-linear, the peak concentrations increasing in a quadratic manner with the dose. At the same time, the elimination half-life was increasing concomitantly with the decrease of the total clearance. In a phase I safety study, Glover et al. (1997) followed the pharmacokinetics of a 20-mer antisense phosphorothioate (ISIS 2302) in healthy male volunteers in a rising-dose study (from 0.06 to 2.0 mg/kg, infused over 2 h). Plasma elimination half-life of the intact molecule was 53–54 min, and that of the ‘total oligonucleotides’ was 67–74 min. Non-linear changes in AUC and steady-state volume of distribution were observed. Oligonucleotides co-migrating with n−1, n −2, n−3 chain-shortened versions of ISIS 2302 were detected in early samples of plasma. There was no accumulation after repeated dosing. 11.7 Conclusions The pharmacokinetics of oligonucleotides, more specifically the phosphorothioates, are now quite well characterized: the half-life in plasma is rather short although tissue distribution is high in kidneys and liver as well in spleen, but low in skeletal muscle and brain. It appears that pharmacokinetics are dose-dependent in animals and in patients, but unaffected by multiple dosing. The degradation processes are linked to the activity of (both 3′- and 5′-) exonucleases. Some higher molecular weight compounds have been detected, but their structure has never been defined (elongation products?). This information has been made available along with the development of new and specific analytical procedures (capillary electrophoresis, electrospray mass spectrometry or ‘hyphenated’ methods, such as liquid chromatography-mass

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spectrometry or capillary electrophoresis—mass spectrometry) or non-invasive tissue distribution studies by PET scanning. Different routes of administration can be used, except oral administration (although some authors claimed that 5′and 3′-modified—hairpins or loops—oligonucleotides could be made orally bioavailable), but one must keep in mind that pharmacokinetics are dosedependent and could be sequencedependent in some cases. References AGRAWAL, S., TEMSAMANI, J. and TANG, J.Y., 1991, Pharmacokinetics, biodistribution and stability of oligodeoxynucleotide phosphorothioates in mice, Proc. Natl Acad. Sci. USA, 88, 7595–7599. BOADO, R.J., KANG, Y.-S., Wu, D. and PARDRIDGE, W.M., 1995, Rapid plasma clearance and metabolism in vivo of a phosphorothioate oligodeoxynucleotide with a single, internal phosphodiester bond, Drug Metab. Disposition, 23, 1297–1300. BOURQUE, AJ. and COHEN, A.S., 1993, Quantitative analysis of phosphorothioate oligonucleotides in biological fluids using fast anion-exchange chromatography, J. Chromatogr., 617, 43–49. BRANCH, A.D., 1998, A good antisense molecule is hard to find, Trends Pharmacol. Sci., 23, 45–50. BROADDUS, W.C., PRABHU, S.S., GILLIES, G.T., NEAL, J., CONRAD, W.S., CHEN, Z.-J., FILLMORE, H. and YOUNG, H.F., 1998, Distribution and stability of antisense phosphorothioate oligonucleotides in rodent brain following direct intraparenchymal controlled-rate infusion, J. Neurosurg., 88, 734–742. BRUIN, G.J.M., BÔRNSEN, K.O., HÛSKEN, D., GASSMANN, E., WIDMER, H.M. and PAULUS, A., 1995, Stability measurements of antisense oligonucleotides by capillary gel electrophoresis, J. Chromatogr. A, 709, 181–195. BUTLER, M., STECKER, K. and BENNETT, C.F., 1997, Cellular distribution of phosphorothioate oligodeoxynucleotides in normal rodent tissues, Lab. Invest., 77, 379–388. CANTIN, E.M. and WOOLF, T.M., 1993, Antisense oligonucleotides as antiviral agents: prospects and problems, Trends Microbiol, 1, 270–276. CHEN, S.-H., QIAN, M., BRENNAN, M. and GALLO, J.M., 1997, Determination of antisense phosphorothioate oligonucleotides and catabolites in biological fluids and tissue extracts using anion-exchange high-performance liquid chromatography and capillary gel electrophoresis, J. Chromatogr. B, 692, 43–51. CORRIAS, M.V., GUARNACCIA, F. and PONZONI, M., 1997, Bioavailability of antisense oligonucleotides in neuroblastoma cells: comparison of efficacy among different types of molecules, J. Neuro-Oncol., 31, 171–180. COSSUM, P.A., SASMOR, H., DELLINGER, D., TRUONG, L., CUMMINS, L., OWENS, S.R., MARKHAM, P.M., SHEA, J.P. and CROOKE, S.T., 1993, Disposition of the 14C-labeled phosphorothioate oligonucleotide ISIS 2105 after intradermal administration to rats, J. Pharmacol Exp. Ther., 267, 1181–1190. CROOKE, R.S., GRAHAM, M.J., COOKE, M.E. and CROOKE, S.T., 1995, In vitro pharmacokinetics of phosphorothioate antisense oligonucleotides, J. Pharmacol. Exp. Ther., 275, 462–473.

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CROOKE, S.T., 1995, Delivery of oligonucleotides and polynucleotides, J. Drug Targeting, 3, 185–190. CROOKE, S.T., 1996, Progress in antisense therapeutics, Med. Res. Rev., 16, 319–344. CROOKE, S.T., 1998, Basic principles of antisense therapeutics, in Crooke, S.T. (ed.), Handbook of Experimental Pharmacology: Antisense research and application, Berlin, Heidelberg: Springer-Verlag. CROOKE, S.T. and BENNETT, C.F., 1996, Progress in antisense oligonucleotide therapeutics, Annu. Rev. Pharmacol. Toxicol., 36, 107–129. 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., 1996, Pharmacokinetic properties of several novel oligonucleotide analogs in mice, J. Pharmacol. Exp. Ther., 277, 923–937. DELONG, R.K., NOLTING, A., FISHER, M., CHEN, Q., WICKSTROM, E., KLIGSHTEYN, M., DEMIRDJI, S., CARUTHERS, M. and JULIANO, R.L., 1997, Comparative pharmacokinetics, tissue distribution, and tumor accumulation of phosphorothioate, phosphorodithioate, and methylphosphonate oligonucleotides in nude mice, Antisense Nucl. Acid Drug Dev., 7, 71–77. DEROUSSENT, A., LE CAËR, J.-P., ROSSIER, J. and GOUYETTE, A., 1995, Electrospray mass spectrometry for the characterization of the purity of natural and modified oligodeoxynucleotides, Rapid Commun. Mass Spectrom., 9, 1–4. DE SMIDT, P.C., LE DOAN, T., DE FALCO, S. and VAN BERKEL, T.J.C., 1991, Association of antisense oligonucleotides with lipoproteins prolongs the plasma halflife and modifies the tissue distribution, Nucl. Acids Res., 19, 4695–4700. DEVERRE, J.-R., BOUTET, V., BOQUET, D., EZAN, E., GRASSI, J. and GROGNET, J.-M., 1997, A competitive enzyme hybridization assay for plasma determination of phosphodiester and phosphorothioate antisense oligonucleotides, Nucl. Acids Res., 25, 3584–3589. ENGELHARD, H., NARANG, C., HOMER, R. and DUNCAN, H., 1996, Urokinase antisense oligodeoxynucleotides as a novel therapeutic agent for malignant glioma: in vitro and in vivo studies of uptake, effects and toxicity, Biochem. Biophys. Res. Commun., 227, 400–405. ETORÉ, F., TENU, J.-P., TEIGER, E., ADNOT, S., LONCHAMP, M.-O., PIROTSKI, E. and LE DOAN, T., 1998, Sequence dependency of the internalization and distribution of phosphorothioate oligonucleotides in vascular smooth muscle cells, Biochem. Pharmacol., 55, 1465–1473. FATTAL, E., VAUTHIER, C., AYNIE, I., NAKADA, Y., LAMBERT, G., MALVY, C. and COUVREUR, P., 1998, Biodegradable polyalkylcyanoacrylate nanoparticles for the delivery of oligonucleotides, J. Controlled Release, 53, 137–143. GEARY, R.S., LEEDS, J.N., HENRY, S.P., MONTEITH, D.K. and LEVIN, A.A., 1997, Antisense oligonucleotide inhibitors for the treatment of cancer: 1. Pharmacokinetic properties of phosphorothioate oligodeoxynucleotides, Anti-Cancer Drug Des., 12, 383–393. GILES, R.V., SPILLER, D.G. and TIDD, D.M., 1993, Chimeric oligodeoxynucleotide analogues: enhanced cell uptake of structures which direct ribonuclease H with high specificity, Anti-Cancer Drug Des., 8, 33–51. GLOVER, J.M., LEEDS, J.M., MANX, T.G., AMIN, D., KISNER, D.L., ZUCKERMAN, I.E., GEARY, R.S., LEVIN, A.A. and SHANAHAN W.R., JR, 1997, Phase I safety

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and pharmacokinetic profile of an intercellular adhesion molecule-1 antisense oligodoxynucleotide (ISIS 2302), J. PharmacolL Exp. Ther., 282, 1173–1180. GOUYETTE, A., 1995, Final analytical and pharmacokinetic reports on phase 1A of GEM® 91 (Hybridon/ANRS Gemini Program), Internal Report, Hybridon Inc., Worcester, MA. GOUYETTE, A. and KERR, D.J., 1995, Principles of pharmacokinetics, in Peckham, M., Pinedo, H.M. and Veronesi, U. (eds), Oxford Textbook of Oncology, Vol. 1, pp. 852–861, Oxford, New York, Tokyo: Oxford Medical Publications. GRAY, G.D. and WICKSTROM, E., 1997, Rapid measurement of modified oligonucleotide levels in plasma samples with a fluorophore specific for singlestranded DNA, Antisense Nucl. Acid Drug Dev., 7, 133–140. HAWLEY, P. and GIBSON, I., 1996, Interaction of oligodeoxynucleotides with mammalian cells, Antisense Nucl. Acid Drug Dev., 6, 185–195. HEFENEIDER, S.H., CORNELL, K.A., BROWN, L.E., BAKKE, A.C., MCCOY, S.L. and BENNETT, R.M., 1992, Nucleosomes and DNA bind to specific cell-surface molecules on murine cells and induce cytokine production, Clin. Immunol. Immunopathol., 63, 245–251. HENRY, S.P., MONTEITH, D. and LEVIN, A.A., 1997, Antisense oligonucleotide inhibitors for the treatment of cancer: 2. Toxicological properties of phosphorothioate oligodeoxynucleotides, Anti-Cancer Drug Des., 12, 395–408. IVERSEN, P.L., MATA, J., TRACEWELL, W.G. and ZON, G., 1994, Pharmacokinetics of an antisense phosphorothioate oligodeoxynucleotide against rev from human immunodeficiency virus type 1 in the adult male rat following single injections and continuous infusion, Antisense Res. Dev., 4, 43–52. JULIANO, R.L. and AKHTAR, S., 1992, Liposomes as a drug delivery system for antisense oligonucleotides, Antisense Res. Dev., 2, 165–176. LEBLEU, B., ROBBINS, L, BASTIDE, L., VIVES, E. and GEE, J.E., 1997, Pharmacokinetics of oligonucleotides in cell culture, in Oligonucleotides as therapeutic agents, pp. 47–59, Chichester: Wiley (Ciba Foundation Symposium 209). LE DOAN, T., ETORÉ, F., MA, D.D.F. and TENU, J.-P., 1996, Perspectives d'une chimiothérapie plus sélective avec les oligonucleotides antisens, Ann. Méd. Interne, 147, 542–552. LEE, W.A., FISHBACK, J.A., SHAW, J.-P., BOCK, L.C., GRIFFIN, L.C. and CUNDY, K.C., 1995, A novel oligodeoxynucleotide inhibitor of thrombin. II. Pharmacokinetics in the cynomolgus monkey, Pharm. Res., 12, 1943–1947. LEEDS, J.M., HENRY, S.P., BISTNER, S., SCHERRILL, S., WILLIAMS, K. and LEVIN, A.A., 1998. Pharmacokinetics of an antisense oligonucleotide injected intravitreally in monkeys, Drug Metab. Disposition, 26, 670–675. LOKE, S.L., STEIN, C.A., ZHANG, X.H., MORI, K., NAKANISHI, M., SUBASINGHE, C. and COHEN, J.S., 1989, Characterization of oligonucleotide transport into living cells, Proc. Natl Acad. Sci. USA, 86, 3474–3478. NAKADA, Y., FATTAL, E., FOULQUIER, M. and COUVREUR P., 1996, Pharmacokinetics and biodistribution of oligonucleotide absorbed onto poly (isobutylcyanoacrylate) nanoparticles after intravenous administration in mice, Pharm. Res., 13, 38–43. NI, J., POMERANTZ, S.C., ROZENSKI, J., ZHANG, Y. and MCCLOSKEY, J.A., 1996, Interpretation of oligonucleotide mass spectra for determination of sequence using

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electrospray ionization and tandem mass spectrometry, Anal. Chem., 68, 1989– 1999. NICKLIN, P.L., BAYLEY, D., GIDDINGS, J., GRAIG, S.J., CUMMINS, L.L., HASTEWELL, J.G. and PHILLIPS, J.A., 1998, Pulmonary bioavailability of a phosphorothioate oligonucleotide (CGP 64128A): comparison with other delivery routes, Pharm. Res., 15, 583–591. NOLTING, A., BELONG, R.K., FISHER, M.H., WICKSTROM, E., POLLACK, G.M., JULIANO, R.L. and BROUWER, K.L.R., 1997, Hepatic distribution and clearance of antisense oligonucleotides in the isolated perfused rat liver, Pharm. Res., 14, 516–521. PHILLIPS, J.A., GRAIG, S.J., BAILEY, D., CHRISTIAN, R.A., GEARY, R. and NICKLIN, P.L., 1997, Pharmacokinetics, metabolism, and elimination of a 20-mer phosphorothioate oligodeoxynucleotide (CGP 69846A) after intravenous and subcutaneous administration, Biochem. Pharmacol., 54, 657–668. PLÉNAT, F., KLEIN-MONHOVEN, N., MARIE, B., VIGNAUD, J.-M. and DUPREZ, A., 1995, Cell and tissue distribution of synthetic oligonucleotides in healthy and tumor-bearing nude mice, Am. J. Pathol., 147, 124–135. QIAN, M., CHEN, S.-H., VON HOFE, E. and GALLO, J.M., 1997, Pharmacokinetics and tissue distribution of a DNA-methyltransferase antisense (MT-AS) oligonucleotide and its catabolites in tumor-bearing nude mice, J. Pharmacol. Exp. Ther., 282, 663– 670. RAKOCZY, P.E., LAI, M.C., WATSON, M., SEYDEL, U. and CONSTABLE, I., 1996, Targeted delivery of an antisense oligonucleotide in the retina: uptake, distribution, stability, and effect, Antisense Nucl Acid Drug Dev., 6, 207–213. SAIJO, Y., PERLAKY, L., WANG, H. and BUSCH, H., 1994, Pharmacokinetics, tissue distribution, and stability of antisense oligodeoxynucleotide phosphorothioate ISIS 3466 in mice, Oncol. Res., 6, 243–249. SANDS, H., GOREY-FERET, L.J., COCUZZA, A.J., HOBBS, F.W., CHIDESTER, D. and TRAINOR, G.L., 1994, Biodistribution and metabolism of internally 3H-labeled oligonucleotides. I. Comparison of a phosphodiester and a phosphorothioate, Mol. Pharmacol., 45, 932–943. SERENI, D., TUBIANA, R., LASCOUX, C., KATLAMA, C., TAULERA, O., BOURQUE, A., COHEN, A., DVORCHIK, B., MARTIN, R.R., TOURNERA, C., GOUYETTE, A. and SCHECHTER, P.J., 1999, Pharmacokinetics and tolerability of intravenous trecovirsen (GEM 91) an antisense phosphorothioate oligonucleotide, in HIV-positive patients, J. Clin. Pharmacol., 39, 47–54. SPILLER, D.G. and TIDD, D.M., 1992, The uptake kinetics of chimeric oligonucleotide analogues in human leukaemia MOLT-4 cells, Anti-Cancer Drug Des., 7, 115–129. SRIVATSA, O.S., BATT, M., SCHUETTE, J., CARLSON, R.H., FITCHETT, J., LEE, C. and COLE, D.L., 1994, Quantitative capillary gel electrophoresis assay of phosphorothioate oligonucleotides in pharmaceutical formulations, J. Chromatogr. A, 680, 469–477. STEIN, C.A., 1996, Antitumor effects of antisense phosphorothioate c-myc oligodeoxynucleotides: a question of mechanism, J. Natl Cancer Inst., 88, 391–393. STEWARD, A., CHRISTIAN, R.A., HAMILTON, K.O. and NICKLIN, P.L., 1998, Coadministration of polyanions with a phosphorothioate oligodeoxynucleotide (CGP 69846A): a role for the scavenger receptor in its in vivo disposition, Biochem. Pharmacol., 56, 509–516.

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TARI, A., KHODADADIAN, M., ELLERSON, D., DEISSEROTH, A. and LOPEZBERESTEIN, G., 1996, Liposomal delivery of oligodeoxynucleotides, Leukemia Lymphoma, 21, 93– 97. TAVITIAN, B., TERRAZZINO, S., KÜHNAST, B., MARZABAL, S., STETTLER, O., DOLLÉ, F., DEVERRE, J.-R., JOBERT, A., HINNEN, F., BENDRIEM, B., CROUZEL, C. and Di GIAMBERARDINO, L., 1998, In vivo imaging of oligonucleotides with positron emission tomography, Nat. Med., 4, 467–471. TENU, J.-P., ETORÉ, F. and LE DOAN, T., 1997, Une méthode simple pour étudier le contenu cytosolique en oligonucleotides dans les cellules. A simple method for the study of the cytosolic content of oligonucleotides in cells, Comptes-Rendus l'Acad Sci. Paris, Sci. de la Vie/Life Sci., 320, 477–486. UHLMANN, E., PEYMAN, A. and WILL, D.W., 1997, Antisense: chemical modifications, in Encyclopedia of Cancer, vol. X, pp. 64–81, London and New York: Academic Press. VAN AUSDALL, D.A. and MARSHALL, W.S., 1998, Automated high-throughput mass spectrometry analysis of synthetic oligonucleotides, Anal Biochem., 256, 220–228. WALLACE, T.L., BAZEMORE, S.A., HOLM, K., MARKHAM, P.M., SHEA, J.P., CHAUDARY, N. and COSSUM, P.A., 1997, Pharmacokinetics and distribution of a 33P-labeled antihuman immunodeficiency virus oligonucleotide (AR177) after single- and multipledose intravenous administration to rats, J. Pharmacol. Exp. Ther., 280, 1480–1488. ZAMECNIK, P.C. and STEVENSON, M.L., 1978, Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide, Proc. Natl Acad. Sci. USA, 49, 280–284. ZANI, M., LAVITRANO, M., FRENCH, D., LULLI, V., MAIONE, B., SPERandIO, S. and SPADAFORA, C., 1995, The mechanism of binding exogenous DNA to sperm cells—factors controlling the DNA uptake, Exp. Cell Res., 217, 57–64. ZHANG, R., DIASIO, R.B., LU, Z., LIU, T., JIANG, Z., GALBRAITH, W.M. and AGRAWAL, S., 1995a, Pharmacokinetics and tissue distribution in rats of an oligodeoxynucleotide phosphorothioate (GEM 91) developed as a therapeutic agent for human immunodeficiency virus type-1, Biochem. Pharmacol., 49, 929–939. ZHANG, R., YAN, J., SHAHINIAN, H., AMIN, G., LU, Z., LIU, T., SAAG, M.S., JIANG, Z., TEMSAMANI, J., MARTIN, R.R., SCHECHTER, P., AGRAWAL, S. and DIASIO, R.B., 1995b, Pharmacokinetics of an anti-human immunodeficiency virus antisense oligodeoxynucleotide phosphorothioate (GEM 91) in HIV-infected subjects, Clin. Pharmacol. Ther. , 58, 44–53. ZHAO, Q., MATSON, S., HERRERA, C.J., FISHER, E., Yu, H. and KRIEG, A.M., 1993, Comparison of cellular binding and uptake of antisense phosphodiester, phosphorothioate, and mixed phosphorothioate and methylphosphonate oligonucleotides, Antisense Res. Dev., 3, 53–66.

PART FIVE Pharmacological Activity

12 Antisense as a Novel Therapy for Cancer B.P.MONIA

12.1 Novel Approaches for Anticancer Therapy Available cancer therapies still exhibit very limited value against most solid cancer types, and inevitably result in the development of multidrug-resistant tumors. Because of this, an urgent need exists for therapeutic alternatives to identify compounds with better tolerability at efficacious doses that are directed at defined, disease-relevant molecular targets. The progress made in understanding the molecular basis of mammalian cell transformation has led to unifying concepts of abrogated growth regulation in cancer cells. It is now well recognized that many products of ‘cancer genes’ encode proteins that regulate normal mitogenesis and apoptosis, and that the carcinogenic process is a progressive disorder of signal transduction (Croce, 1987; Alitalo and Schwab, 1988; Bos, 1989; Bishop, 1991; Rabbitts, 1994; Weinberg, 1994). In fact, many of the genes that are mutated or lost in cancer cells, including both oncogenes and tumor suppressor genes, encode proteins that are crucial regulators of intracellular signal transduction (Croce, 1987; Alitalo and Schwab, 1988; Bos, 1989; Bishop, 1991; Rabbitts, 1994; Weinberg, 1994). This conceptual framework has provided a basis for the development of novel anticancer strategies and therapeutic modalities aimed at inhibiting cancer growth either by blocking mitogenic signal transduction or by specifically inducing apoptosis of cancer cells. Although these approaches have not yet been clinically validated, these strategies are likely to identify anticancer agents with fewer undesirable side-effects and greater efficacy than standard chemotherapeutic agents. Specific inhibition of cancer-causing gene products can in principle be accomplished by appropriately designed small molecules, provided that the chosen targets display reasonable enzymatic functions (e.g. inhibitors of some protein kinases, extracellular matrix-degrading proteases, farnesyltransferases). However, a large proportion of putative cancer-causing or cancer-associated oncoproteins either do not have intrinsic enzymatic functions, such as various transcription

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Table 12.1 Antisense oligonucleotides currently in clinical trials for cancer Drug

Target

Cancer type under Company evaluation

Status

G-3139

Bcl-2

Genta

Phase II

ISIS 352l/

PKC-α ISI641A

Isis/Novartis

Phase II

ISIS 5132/ ODN698A ISIS 2503

C-raf kinase

Isis/Novartis

Phase II

Isis

Phase I

C-myb

Lymphoma, prostate, melanoma Solid tumors (wide-range), lymphoma Solid tumors (wide-range) Solid tumors (wide-range) Leukemia (CML)

Phase II

PKA (RIα)

Colon

University of Pennsylvania Hybridon

GEM 231

Ha-ras

Phase I

factors (e.g. myc, jun, fos), cell death suppressors (e.g. Bcl-2 or Bcl-X) and adaptor proteins (e.g. SH2 and SH3 proteins), or their overall structure and enzymatic functions are highly conserved (e.g. multigene families of proteins such as protein kinases, GTP-binding proteins), thereby eliminating the possibility of achieving acceptable specificity. It is for these reasons that antisense therapeutics holds such great promise as a more effective modality for treating cancer. Antisense action is based on inhibiting the synthesis of a particular protein using synthetic oligonucleotides that bind to protein-encoding RNA, thereby preventing RNA function. A number of mechanisms have been demonstrated by which antisense oligonucleotides (ASOs) exert their inhibitory effects on mRNA function. These include inhibition of splicing, inhibition of protein translation, and most commonly, reduction of steady-state mRNA levels through the utilization of RNase H enzymes in cells (Chiang et al., 1991; Crooke, 1993; Monia et al., 1993; Hodges and Crooke, 1995; Crooke and Bennett, 1996; Baker et al., 1997). Since ASOs can inhibit gene expression by targeting virtually any region within RNA transcripts (including 5′/3′ untranslated sequences and introns), and due to the degeneracy of the genetic code, it is relatively easy to design an antisense compound that specifically inhibits a member of a multigene family (Chiang et al., 1991; Crooke, 1993; Monia et al., 1993; Hodges and Crooke, 1995; Crooke and Bennett, 1996; Baker et al., 1997). Furthermore, the synthesis of virtually any class or type of protein can be inhibited through an antisense approach. This includes proteins that are fairly easy to target using traditional methods (e.g. enzymes and receptors) as well as proteins that are very difficult to obtain inhibitors against (e.g. adaptor proteins, structural proteins). The increasing interest in antisense-based approaches for the treatment of cancer is reflected by the growing number, and steady progression, of cancer

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clinical trials which are under way to evaluate the initial potential of this approach (Table 12.1). Preclinical data supporting these trials have been substantial and encouraging. Progress has also been made in the synthetic capabilities of ASOs which not only has allowed tests to be carried out in animal models more easily, but also has made the development of ASOs more commercially attractive. Here, I will attempt to summarize some of the progress that has been made using antisense approaches against a selection of molecular targets that are relevant to cancer therapeutics, focusing in greatest depth on the ras multigene family. 12.2 Antisense Approaches for Cancer 12.2.1 Bcl-2 The Bcl-2 gene, first identified in B-cell lymphoma, is a member of a family of genes encoding protein products that reside within the mitochondrial membrane and function as key regulators of programmed cell death (apoptosis). It is generally believed that in order for tumor cells to survive, they must escape the normal governing processes that control cell death. Bcl-2 functions to protect cells from programmed cell death. Overexpression of Bcl-2 is common in several cancers, in particular nonHodgkin's lymphoma, and is believed to contribute significantly to decreased sensitivity to chemotherapeutic agents (Reed, 1995). In non-Hodgkin's lymphoma, a chromosomal translocation event whereby the immunoglobulin G heavy chain region on chromosome 14 becomes juxtaposed to the Bcl-2 gene on chromosome 18 results in overexpression of Bcl-2 in the majority of follicular lymphomas and some high-grade lymphomas (Reed, 1995). Bcl-2 overexpression contributes to tumorigenesis by preventing the natural cell death process of B-cells, as well as the cell death processes induced by chemotherapeutic agents. An 18-mer phosphorothioate (G-3139) antisense oligodeoxynucleotide targeting the translational initiation codon of the Bcl-2 gene was shown to inhibit the growth of lymphoma cells in SCID mice (Gotten et al., 1994; Webb et al., 1997). Continuous infusions of G-3139 for 21 days at a dose of 5 mg/kg per day resulted in complete eradication of lymphomas in animals. Based on these findings, G-3139 was selected for phase I clinical trials against lymphoma. A phase I study of G-3139 was initiated at the Royal Marsden Hospital (Surrey, UK), recruiting patients with non-Hodgkin's lymphoma that expressed high levels of Bcl-2 (Webb et al., 1997). G-3139 was administered to nine patients as a daily subcutaneous infusion for 14 days. The starting dose was 0.125 mg/kg per day; this was increased to 2 mg/kg per day by the end of the study. No drug-

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related toxicities were seen at these dose levels, except for one patient who had a local inflammatory reaction. Reduced levels of Bcl-2 protein expression in circulating lymphoma cells were demonstrated in two out of five patients evaluated. Two patients experienced symptomatic relief, and two additional patients with lowgrade lymphomas experienced tumor shrinkage (one partial response and one complete remission). Interestingly, eight of these patients subsequently received further chemotherapy with various conventional regimens and six achieved partial remissions. It was suggested that sustained inhibition of Bcl-2 protein production using G-3139 restored chemosensitivity of lymphomas. Recent studies have been reported indicating that G-3139 may have utility as an anticancer drug for treating other cancer types beyond lymphoma. Jansen et al. (1998) reported that administration of G-3139 (continuous two-week infusion at 5 mg/kg per day) reduces Bcl-2 expression in vivo and significantly improves the chemosensitivity of human melanoma cells to dacarbazine. Antisense therapy resulted in tumor cell apoptosis in SCID mice which was augmented following combination treatment with dacarbazine. Administration of control oligonucleotides had no effect on any of these endpoints. G-3139 has also been shown to effectively block the growth of androgen-independent prostate tumors in SCID mice (M. Gleave, personal communication). Again, reduction of Bcl-2 expression in tumor was demonstrated and control oligonucleotides were without effect. These types of studies have resulted in the initiation of Phase I trials evaluating the potential of G-3139 as a treatment modality for both malignant melanoma and prostate cancer (B.Jansen and M.Gleave, personal communications). 12.2.2 C-myb Downregulation of the C-myb transcription factor occurs during differentiation of haematopoietic cells, and C-myb protein expression appears to be necessary for the proliferation of these cells in vitro (Westin et al., 1982; Clarke et al., 1988). Inhibition of the colony-forming ability of normal bone progenitor cells has been demonstrated using a phosphorothioate antisense oligodeoxynucleotide (18-mer targeted to codons 2–7) against C-myb (Calabretta et al., 1991). This oligonucleotide also reduced growth of primary acute myelogenous (AML) and chronic myelogenous (CML) leukemia cultures and proliferation of a T-cell leukemia cell line in vitro (Calabretta et al., 1991; Anfossi et al., 1989). In addition, the C-myb antisense was shown to inhibit the growth of K562 erythroleukemia cells in SCID mice and to prolong survival of animals (Ratajczak et al., 1992). Based on this type of preclinical activity, an ex vivo bone marrow purging study was initiated at the University of Pennsylvania with eight patients. Human stem cells were incubated with the C-myb antisense for 24 h prior to marrow cryopreservation. Patients were reinfused with their marrow following chemotherapy. One patient failed to engraft and four out of six patients

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displayed normal leukocyte counts three months after engraftment. In follow-up, one patient was in haematological remission at 18 months, a second had 80% normal metaphases at two years, and a third exhibited a minor response. In a parallel study, 17 CML patients (four in chronic state and 13 with blast crisis) received systemic infusions of the C-myb oligonucleotide continuously for seven days followed by one week’s drug-free period. No drug-related toxicities were reported and one patient with CML in blast crisis appeared to revert to chronic stage, surviving for 14 months (Gewirtz, 1997). These preliminary results suggest that the C-myb phosphorothioate antisense oligodeoxynucleotide may have utility for the treatment of haematological malignancies. 12.2.3 raf Kinase The raf family of gene products encodes serine/threonine-specific protein kinases which play a pivotal role in mitogenic signalling events (Rapp, 1991; Howe et al., 1992; Daum et al., 1994). Three known ra/isoforms are known to exist in cells: A-raf, B-raf and C-raf. C-raf kinase associates with ras and transmits signals downstream of ras in the mitogen-activated protein (MAP) kinase pathway (Rapp, 1991; Howe et al., 1992; Daum et al., 1994). In addition, C-raf has been shown to associate with Bcl-2 and thus may play a role in the regulation of apoptosis (Wang et al., 1994). These data suggest that inhibitors of C-raf kinase may be of value in controlling diseases associated with abnormal cell proliferation. To identify antisense oligodeoxynucleotides capable of inhibiting human Craf gene expression, a series of phosphorothioate oligodeoxynucleotides were designed and tested for reducing C-raf mRNA levels in A549 lung carcinoma cells (Monia et al., 1996a). Oligonucleotides used in this analysis targeted various regions of the C-raf mRNA including the 5′-untranslated region (UTR), the translation initiation AUG, the coding region, and the 3′-UTR, and all were 20 bases in length. Other groups have reported on the activities of antisensedesigned oligodeoxynucleotides targeted to the translation initiation AUG of human C-raf kinase (Kasid et al., 1989; Soldatenkov et al., 1997). Reductions in C-raf mRNA levels were observed following treatment with only a small subset of the Oligonucleotides targeting various regions of the C-raf mRNA (Monia et al., 1996a). Furthermore, for those oligonucleotides which did promote reduced C-raf mRNA levels, the degree of activity varied greatly. The most potent antisense inhibitor identified from this screen was ISIS 5132, which targets the 3′-UTR of the C-raf message. The sequence requirements for inhibiting C-raf gene expression have been examined thoroughly in vitro by comparing the dose-dependent effects of ISIS 5132 with a series of ‘mismatched’ oligonucleotides containing between one and seven mismatches within the ISIS 5132 sequence (Monia et al., 1996b). Melting temperatures (Tm) along with corresponding dissociation constants (Kd) were

252 ANTISENSE AS A NOVEL THERAPY FOR CANCER

determined under cell-free conditions for each of these oligonucleotides with a 20-mer oligoribonucleotide (RNA) complementary to ISIS 5132. As expected for Watson-Crick hybridization, affinity decreased as the number of mismatches contained within the ISIS 5132 sequence increased. The IC50 for ISIS 5132mediated reduction of C-raf mRNA levels in A549 tumor cells in culture is approximately 100 nM (Monia et al., 1996a). As expected, none of the mismatched oligonucleotides were as potent as ISIS 5132 in inhibiting C-raf mRNA expression. Furthermore, inhibition of C-raf mRNA expression diminished gradually with an increase in the number of mismatches within the ISIS 5132 sequence. Incorporation of a single mismatch resulted in a two-fold loss in potency, and no activity was observed for oligonucleotides containing more than four mismatches. In addition to sequence-specificity, ISIS 5132 has been shown to be extremely specific with respect to the target that is inhibited as well. ISIS 5132 treatment fails to inhibit the expression of other raf isoforms, other signalling molecules (e.g. ras), and housekeeping gene products. ISIS 5132-mediated inhibition of C-raf expression has been shown to exert very significant effects on downstream signalling events and cell proliferation (Monia et al., 1996a; Monia, 1997; Schulte et al., 1996; Xu et al., 1998). Inhibiting the expression of a single raf isoform (i.e. C-raf) can abrogate the MAP kinase phosphorylation cascade in response to specific growth factors and cytokines (Monia, 1997, Schulte et al., 1996; Xu et al., 1998). This includes the inhibition of ERK MAP kinase stimulation by growth factors and cytokines and of JNK MAP kinase stimulation by TNF-alpha. Antisense inhibition of C-raf expression has also been shown to modulate regulation of apoptosis in epithelial cells and to inhibit tumor cell proliferation in cell culture (Monia et al., 1996a; Xu et al., 1998). ISIS 5132 has displayed attractive activity in vivo in animal models of cancer. Administration of ISIS 5132 to mice bearing subcutaneously implanted tumors resulted in a time-dependent reduction in C-raf mRNA levels in tumor as determined by Northern blot analysis (Monia et al., 1996a). However, no effects on C-raf RNA levels were observed in tumor following administration of a control oligonucleotide. Moreover, ISIS 5132 displays attractive antitumour activity against a range of tumor types in tumor xenograft models in the doserange of 5–20 mg/kg per day, depending on the tumor type examined. Tumor types that have displayed sensitivity to ISIS 5132 treatment include Calu-1 nonsmall cell lung carcinoma, Mia Paca II and Pane 1 pancreatic carcinomas, and SQ20B laryngeal carcinoma (Gokhale et al., 1999; B.Monia, N.Dean, unpublished results). In addition, ISIS 5132 administration has been shown to sensitize tumors to ionizing radiation in animal models (Gokhale et al., 1999). These results have led to the initiation of clinical trials to evaluate the antitumour activities of ISIS 5132 against a range of tumor types. Clinical trials on the evaluation of ISIS 5132 for the treatment of solid tumors have progressed to Phase II. In Phase I, ISIS 5132 was tested in a multicentre trial using a number of different administration protocols to address issues

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 253

relating to drug safety, pharmacokinetics, proof-of-mechanism, and activity. Doses were escalated from 0.5 to 6.0 mg/kg during the course of the trial. ISIS 5132 was found to be extremely well tolerated in patients. Moreover, treatment with ISIS 5132 has been shown to produce a pronounced depletion of C-raf mRNA levels in peripheral blood mononuclear cells (PBMCs) in most of the patients treated at doses greater than or equal to 2.5 mg/kg on a thrice-weekly schedule (O’Dwyer et al., 1998). Furthermore, evidence of antitumour activity has been observed for ISIS 5132 (O’Dwyer et al., 1998). Based on these promising clinical results, phase II studies of ISIS 5132 have been initiated in which the drug is being evaluated against a range of tumor types, both as monotherapy and in combination with traditional anticancer drugs. 12.2.4 Protein Kinase C-α Protein kinase C (PKC) was identified originally as a serine/threonine kinase involved in mediating intracellular responses to a variety of growth factors, hormones and neurotransmitters (Nishizuka, 1992). Molecular cloning studies have revealed that PKC exists as a family of at least 11 closely related isoforms, which are subdivided on the basis of certain structural and biochemical similarities (Nishizuka, 1992). Considerable experimental evidence exists for a role for PKCs in some abnormal cellular processes including cancer. Antisense oligonucleotides have been identified that specifically target individual members of the PKC family for the purposes of using them as laboratory tools and as drugs (Dean et al., 1994). Isoform-specific antisense inhibitors against PKCs have been used extensively to determine isoform function in a variety of biological processes (Dean et al., 1994, 1996; Liao et al., 1997; Traub et al., 1997; Dean and Griffey, 1997). Antisense inhibitors have been identified against murine PKC-α that inhibit expression of PKC-α in normal mouse tissues (Dean and McKay, 1994). ISIS 4189, the lead murine-specific PKC-α antisense inhibitor, was identified in cell culture screens and shown to inhibit expression murine PKC-α in a sequencespecific and target-specific manner (Dean and McKay, 1994). Administration of ISIS 4189 to mice promoted a dose-dependent and isoform-specific reduction in PKC-α mRNA levels in liver, with an ID50 of approximately 20 mg/kg. Scrambled control oligonucleotides failed to reduce PKC-α mRNA levels in liver. The effects of a lead human-specific PKC-α antisense phosphorothioate oligodeoxynucleotide has been examined in animal tumor models. ISIS 3521 has been shown to suppress the growth of U-87 glioblastoma tumors in nude mice following daily administration at a dose of 2 mg/kg (Yazaki et al., 1996). Administration of a scrambled control oligonucleotide had no effect on the growth of tumor in these studies. In addition, administration of ISIS 3521 was found to prolong significantly the survival of animals bearing U-87 glioblastoma

254 ANTISENSE AS A NOVEL THERAPY FOR CANCER

tumors relative to controls. Levels of ISIS 3521 and the scrambled control oligonucleotide within tumor tissue were determined by capillary gel electrophoresis, and both were found to be present at approximately 2 µM. ISIS 3521 also reduced levels of PKC-α protein in tumor without affecting the expression levels of other PKC isoforms. Additional studies have also demonstrated that ISIS 3521 is effective at inhibiting the growth of other tumors in tumor xenograft models including MDA-231 breast carcinoma and Calu-1 non-small cell lung carcinoma (N.Dean, B.Monia, unpublished results). Based on the available biological evidence implicating PKC in the pathogenesis of certain solid tumor types, and the antitumour activity displayed by ISIS 3521 in nude mouse tumor xenograft models, a Phase I trial of ISIS 3521 was initiated for the treatment of cancer. A variety of tumor types were evaluated in the Phase I trial, which was completed recently. In one trial, ISIS 3521 was administered as a continuous 21-day infusion and the cycle was repeated if the drug was well tolerated and the tumor did not progress (Sikic et al., 1997). In a preliminary report of the trial, one patient with colon cancer displayed stabilization of previously rising carcino embryonic antigen (CEA) during a fourmonth treatment, and one ovarian cancer patient displayed stabilization of an enlarging abdominal mass for four months. No grade 3 or grade 4 toxicities were observed. Based on promising clinical trial results in phase I studies, phase II studies have been initiated examining the antitumour properties of ISIS 3521 both alone and in combination with traditional anticancer drugs against a wide range of tumor types. 12.2.5 Protein Kinase A Overexpression of the RIoc subunit of cAMP-dependent protein kinase (PKA) has been demonstrated in human cancer cell lines, in primary tumors, in cells following transformation with Ki-ras or transforming growth factor-α, and upon stimulation of cell growth following growth factor or phorbol ester treatment (Lohmann, 1972; Cho-Chung, 1990). Conversely, decreased expression of RIα correlates with growth inhibition induced by cAMP analogues in a broad spectrum of human cancer lines (Cho-Chung, 1990). Based on these observations, ChoChung and colleagues investigated the potential of antisense inhibitors targeted against RIα as anticancer agents. These investigators identified three 21-base phosphorothioate oligodeoxynucleotides targeted to various sites within the coding region of the RIα mRNA that each produced antiproliferative activity in vitro against a variety of human cancer lines, whereas control oligonucleotides were without effect (Yokozaki et al., 1993; Tortora et al., 1991). These results prompted studies on the in vivo antitumour potential of RIα antisense oligonucleotides (Nesterova and Cho-Chung, 1995). A single subcutaneous injection (1 mg/mouse) of any one of the three lead RIα antisense molecules produced impressive and sustained antitumour activity against

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 255

subcutaneously implanted LS-174T human colon carcinoma tumors in nude mouse xenograft models (Cho-Chung, 1990). In addition, antisense treatment resulted in an impressive reduction of RIα protein levels in tumor that persisted three days beyond the single oligonucleotide injection. Interestingly, antitumour activity persisted for several days beyond the time in which RIα protein suppression ceased (four to five days). A two-base mismatched control oligonucleotide displayed no antitumour activity and no effects on RIα protein levels in vivo. This unexpected finding may have a significant impact on the application of antisense therapy for cancer, since it suggests that antisense treatment may require relatively infrequent dosing to maintain antitumour activity. Based on these findings, a phase I clinical trial has been initiated investigating the potential of RIα-directed antisense oligonucleotides for the treatment of cancer. Reports on the design of these trials or their progress have not yet been made available. 12.3 Antisense as a Novel Anticancer Approach against ras ras has been the focus of intense research ever since its identification as the first human oncogene in 1982 (Lacal and Tronick, 1988). Much of this research has focused on the discovery of different ras isoforms and the elucidation of the mechanisms by which ras proteins function in normal cells and promote malignancy in cancer. However, despite intense research on ras since 1982, very few anticancer drugs that are known to act by inhibiting ras function have entered the clinic for the treatment of human cancer. Furthermore, very little information has been generated on the cellular functions of different ras isoforms in cells. The latter point is due mostly to the difficulties associated with generating isoformspecific inhibitors using traditional approaches. Three different ras genes (Ki-ras, Ha-ras, and N-ras) have been identified and characterized in mammalian tissues, ras genes can acquire transforming potential through a number of mechanisms, the best characterized being the acquisition of single base point mutations in their coding regions that result in amino acid substitutions in critical GTP regulatory domains of the protein. These mutations abrogate the normal function of ras, thereby converting a normally regulated cell protein to one that is constitutively active. Such deregulation of normal ras protein function is believed to contribute to the transforming activity of ras gene products (Lacal and Tronick, 1988; Bishop, 1987; Bos, 1989; Vogelstein et al., 1988). 12.3.1 Discovery of ras Antisense Inhibitors We have focused our attention initially on the discovery of antisense inhibitors against the human Ha-ras and Ki-ras isoforms. To identify antisense

256 ANTISENSE AS A NOVEL THERAPY FOR CANCER

oligonucleotides capable of inhibiting expression of these isoforms, a series of phosphorothioate oligodeoxynucleotides were designed and tested for inhibition of the appropriate ras isoform (Bennett et al., 1995). In both cases, oligonucleotides 15 to 20 bases in length were targeted to mRNA sequences comprising the 5′-untranslated regions, coding regions (including codons 12 and 61), and the 3′-untranslated regions. Two cell lines were chosen for these studies: the T24 bladder carcinoma cell line, which expresses a mutation-bearing Ha-ras mRNA (codon12, GGC→GTC), and the SW480 colon carcinoma cell line, which expresses a mutant Ki-ras mRNA (codon-12, GGT→GTT) (Reddy, 1983; Bos et al., 1986). Cells were treated with oligonucleotides at a concentration of 200 nM in the presence of cationic lipid to enhance cell uptake efficiency in vitro (Bennett et al., 1992). Inhibition of Ha-ras and Ki-ras mRNA expression was observed for only a subset of the oligonucleotides that were tested. The degree of inhibition of the two different ras gene products varied depending on the mRNA target site and the particular ras message. For example, the 5′-untranslated region, including the AUG site of Ha-ras mRNA, was very sensitive to inhibition with antisense oligonucleotides, whereas oligonucleotides targeted to the 3′-untranslated region of this message were without effect. In contrast, oligonucleotides targeted to the AUG site of Ki-ras mRNA were poor inhibitors of Ki-ras expression whereas the 5′-untranslated region was very sensitive to antisense activity. Interestingly, for both target mRNAs, oligonucleotides designed to hybridize with codons 12 or 61 were effective at inhibiting expression of the respective mRNAs targets, suggesting that mutantspecific inhibition of ras mRNA expression is feasible (Bennett et al., 1995; Monia et al., 1992). In subsequent studies, we have taken a similar approach for the discovery of antisense inhibitors against the human N-ras isoform (L.Cowsert, unpublished results). In these experiments, we have found that the most effective antisense inhibitors against N-ras were targeted to the 3′-untranslated region of the N-ras mRNA. In addition to measuring the effects of antisense oligonucleotides on ras mRNA levels, we have also demonstrated that these compounds are effective inhibitors of ras protein synthesis and reduce steady-state levels of ras protein in cells (Xu et al., 1998; Cowsert, 1997). As expected, based on the predicted halflife of ras proteins in cells (Cuadrado et al., 1993), a 50% reduction of steady-state ras protein levels following initiation of oligonucleotide treatment requires a period of time between 12 and 20 h. These results are remarkably consistent from cell type to cell type. 12.3.2 Specificity of ras Antisense Inhibitors The structures of the three ras isoforms (Ha-ras, Ki-ras, and N-ras) at the protein level are virtually identical throughout the protein, except for a short region at the carboxy terminus (Lacal and Tronick, 1988; Bishop, 1987; Bos, 1989). Thus,

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protein targeting drugs which can selectively target different ras isoforms without affecting the function of other G-proteins have not been described. However, because of the redundancy of the genetic code and the presence of noncoding (untranslated) sequences, highly related proteins are often encoded by highly diverged mRNA sequences. Therefore, it should be possible to design antisense inhibitors to block expression of one particular isoform with minimal consequences to related isoforms. To demonstrate isoform-specific inhibition of ras gene expression using antisense technology, oligonucleotides that were specifically designed to hybridize with either the Ha-ras mRNA or the Ki-ras mRNA were evaluated for isoformspecificity. ISIS 2503, an active 20-base phosphorothioate targeted to the Ha-ras mRNA AUG region, is complementary to the AUG region of the Ki-ras message in only 9 of 20 bases and, therefore, would not be expected to bind efficiently to Ki-ras mRNA (Lacal and Tronick, 1988; Bishop, 1987; Bos, 1989). Similarly, ISIS 6957, an active 20-base phosphorothioate targeted to the 5'untranslated region of Ki-ras mRNA, is complementary to the Ha-ras mRNA in only 4 of 20 bases, and therefore should not affect Ha-ras mRNA expression (Bennett et al., 1995). Cells treated with each of these oligonucleotides were analysed for Ha-ras and Ki-ras mRNA expression by Northern analysis. ISIS 2503 reduced Ha-ras mRNA levels to virtually undetectable levels without affecting Ki-ras mRNA levels, whereas ISIS 6957 inhibited Ki-ras mRNA expression without affecting Ha-ras mRNA levels (Figure 12.1A). We have also demonstrated isoform-specific reduction of Ha-ras and Ki-ras protein levels for these oligonucleotides (Figure 12.1B). ras genes often acquire their tumor-promoting properties by single base point mutations in their coding regions. Since the function of normal ras isoforms may be important for normal cell survival, inhibition of expression of the mutated ras gene in tumors may be preferred without affecting expression of the nonmutated ras isoforms. Saison-Behmoaras et al. (1991) have demonstrated inhibition of a mutant form of Ha-ras using a 9-base phosphodiester antisense oligonucleotide linked to an acridine intercalating agent. Chang et al. (1991) have also demonstrated selective targeting of a mutant Ha-ras message in which a mutation at codon 61 was targeted and methylphosphonate oligodeoxynucleotides were employed. Studies from our laboratory have demonstrated similar antisense specificity targeting the Ha-ras EJ bladder carcinoma point mutation (GGC→GTC) at codon 12 using phosphorothioate oligodeoxynucleotides (Bennett et al., 1995; Monia et al., 1992). In our studies, we demonstrated that mutation-specific inhibition can be achieved with phosphorothioate oligodeoxynucleotides, but that oligonucleotide affinity and concentration were critical to maintaining the selectivity. Oligonucleotides ranging in length between 5 and 25 bases targeted to Ha-ras codon 12 were tested for overall activity and point mutation selectivity. Oligonucleotides < 15 bases in length were inactive, whereas all oligonucleotides greater in length displayed good activity with potency correlating directly with oligonucleotide chain length

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Figure 12.1 Isoform-specific inhibition of ras mRNA and protein expression in tumor cell lines. T24 cells were treated with the indicated Ha-ras-specific (ISIS 2503) or Ki-rasspecific (ISIS 6957) antisense oligonucleotide (200 nM) and cell lysates were analysed for Ha-ras and Ki-ras mRNA levels 30 h post-treatment. A, Northern blot analysis of Haras and Ki-ras mRNA levels. B, Western blot analysis of Ha-ras and Ki-ras protein levels using isoform-specific antisera

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(affinity). However, selective inhibition of mutant Ha-ras expression did not increase with oligonucleotide chain length, but required a specific length between 15 and 19 bases. The maximum selectivity observed for inhibition of mutant Ha-ras expression relative to normal Ha-ras was achieved with a 17-mer oligonucleotide (ISIS 2570) (Bennett et al., 1995; Monia et al., 1992). Point mutation-specific targeting of Ki-ras oncogenes has also been demonstrated using phosphorothioate antisense oligonucleotides (Bennett et al., 1995). In this study, 15 bases was shown to be the optimal length for selectively targeting a codon 12 mutation (GGT→GTT) within the Ki-ras gene of SW480 colon carcinoma cells. Treatment of cells with the 15 base antisense oligonucleotide had no effect on Ki-ras mRNA levels in cells expressing nonmutated Ki-ras, whereas Ki-ras mRNA expression was completely suppressed in SW480 cells. Furthermore, no inhibition of Ha-ras gene expression was observed in SW480 cells following treatment with the Ki-ras 15 base oligonucleotide. These types of study demonstrate that point mutationspecific and isoform-specific inhibition of both Ha-ras and Ki-ras mRNA expression in tissue culture is possible through the use of properly designed antisense oligonucleotides. 12.3.3 Cellular Responses Resulting from Inhibition of ras Gene Expression Based on the generally accepted function of the MAP kinase signalling pathway in the transduction of extracellular signals that promote cellular proliferation and survival, the expected outcome of inhibiting ras expression using antisense oligonucleotides is modulation of downstream kinase and transcriptional activity, and attenuation of cellular proliferation and/or promotion of cell death, provided that inhibiting a single isoform of the ras multigene family is sufficient to promote these types of response. We and others have investigated these types of effect in various nontransformed and tumor cell lines and have found that inhibition of a single ras isoform in cells is sufficient to abrogate downstream cell signalling pathways and impede cellular proliferation (Monia, 1997; Xu et al., 1998; Bennett et al, 1995; Chen et al., 1996; Sharpe et al., 1999). Moreover, we have generated results supporting the conclusion that different ras isoforms possess unique functions in cells that often appear to be cell-type specific. We have measured the ability of ras antisense inhibitors to block stimulation of ERK activity in response to specific stimuli, and to modulate activation of specific transcription units. As shown in Figure 12.2, ERK stimulation by PDGF, angiotensin II, and TGF(3 can be blocked either partially or completely in vascular smooth muscle cells following treatment with an isoform-specific antisense inhibitor against Ha-ras. However, Ha-ras inhibition does not affect the ability of phorbol ester to stimulate ERK activity in these cells. Interestingly, antisense inhibition of Ki-ras in these cells also blocked PDGF and TGFP

260 ANTISENSE AS A NOVEL THERAPY FOR CANCER

Figure 12.2 Inhibition of MAP kinase stimulation by antisense oligonucleotides targeted to Ha-ras and Ki-ras. Serum-starved vascular smooth muscle cells were treated with the indicated Ha-ras or Ki-ras antisense oligonucleotides, with a control (scrambled) oligonucleotide, or were left untreated. 30 h following oligonucleotide treatment, cells were stimulated with the indicated agents for 10 min and analysed for MAP kinase activity against myelin basic protein substrate using ERK1/ERK2 immunoprecipitation assays. Quantitation was by phosphorimage analysis

stimulation of ERK activity, but did not affect the degree of ERK stimulation by angiotensin II. Similar to the effects displayed by the Ha-ras inhibitor, Ki-ras inhibition did not affect phorbol ester-mediated stimulation of ERK in vascular smooth muscle cells. These results are consistent with the conclusion that, at least for the cells described above, protein kinase-C stimulation of ERK activity occurs in a ras-independent manner. We have made similar observations in other cell types on the ability of ras antisense inhibitors to block transcriptional activation of specific genes (e.g. Cfos, cell adhesion molecules) in response to growth factors, cytokines and phorbol esters (B.Monia, unpublished results). These results demonstrate that, at least for some cell signalling pathways, different ras isoforms possess unique functions in signal transduction. The antiproliferative effects of ras antisense inhibitors have been investigated by a number of groups and it has been shown that these inhibitors can block the proliferation of a variety of tumor types in cell culture (Monia, 1997; Bennett et al., 1995; Saison-Behmoaras et al., 1991; Chen et al., 1996). We have previously reported that ISIS 2503, a phosphorothioate antisense inhibitor targeted against

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human Ha-ras, inhibits proliferation of the Ha-ras transformed T24 bladder carcinoma line in a dose-dependent and oligonucleotide sequence-specific manner, displaying an IC50 for this effect that is very similar to the IC50 for inhibiting Haras mRNA and protein expression (Monia, 1997; Bennett et al., 1995; Chen et al., 1996). We have also demonstrated apoptotic responses in T24 cells in vitro following administration of ISIS 2503. Again, these effects are highly sequencespecific and correlate well with reduction of target gene expression. In general, we observed that the time required to induce apoptosis by these oligonucleotides is significantly longer than the time required to inhibit proliferation (B.Monia, unpublished studies). Our results on T24 cell proliferation are consistent with a report demonstrating the antiproliferative effects of ISIS 2503 against tumor cell lines in vitro (Chen et al., 1996). Interestingly, it was also shown in that report that a Ki-ras antisense inhibitor (ISIS 6957, described above) displays no significant antiproliferative effects against the T24 tumor line, but is a potent antiproliferative agent against normal diploid fibroblasts, whereas the Ha-ras oligonucleotide has no effects on proliferation against this cell type. Neither oligonucleotide affected the proliferation of a bladder carcinoma tumor line (J-82) that does not contain a ras mutation. It will be interesting to determine whether an N-ras- specific antisense inhibitor, or a combination of isoform-specific ras inhibitors, can block proliferation of this tumor cell line. Similar isoform-specific antiproliferative effects have been observed against other cell lines using ras antisense inhibitors (B.Monia, unpublished results). Antisense inhibitors against specific ras family members have been used to demonstrate additional isoform-specific functions for these proteins. For example, Yan et al. (1997b) have reported that treatment of transformed colon epithelial cells with Ki-ras antisense oligonucleotides can prevent the upregulation of carcinoembryonic antigen (CEA), an important marker of malignancy, in human colon cancer, whereas a Ha-ras antisense inhibitor does not affect CEA upregulation. In a related study, the same group of investigators have employed antisense technology to demonstrate that Ki-ras (but not Ha-ras-) is critical for the proper maturation (glycosylation) of PJ integrins in colon epithelial cells (Yan et al., 1997a). Thus, antisense targeting of ras family members appears to be a viable approach for determining the roles of different ras isoforms in tumor cell signalling, transformation, tumor progression, and proliferation. 12.3.4 Antitumour Activity of ras Antisense Oligonucleotides in Animal Models The obvious extension of the types of studies described above is to test the feasibility of using antisense inhibitors against ras family members to prevent tumor growth in vivo. A number of studies have been reported describing in vivo

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antitumour effects of antisense oligonucleotides targeted to various cell signalling molecules that are very consistent with an antisense mode of action (Cotter et al., 1994; Jansenef et al., 1998; Gewirtz, 1997; Moniaetal., 1996a, 1996b; Gokhale et al., 1999; Yazaki et al., 1996; Nesterova and Cho-Chung, 1995; Bennett et al., 1995; Cowsert, 1997). One of the important observations that have been made in all these studies, as well as in other studies using different in vivo models, is that, despite the fact that cationic lipids or other transfection techniques are generally required for efficient oligonucleotide uptake in cell culture, simple saline formulation of oligonucleotides is all that is normally required to produce antisense effects in vivo following systemic administration. Although the mechanisms underlying these observations are not well understood, they obviously indicate that the mechanisms of macromolecular uptake by cells in animals are very different from cellular uptake mechanisms in culture. Antisense approaches against ras isoforms have been successfully employed to prevent the growth of a number of human tumor types in animal models (Table 12.2). Initial studies focused on the utilization of vector-mediated antisense RNA methods designed to inhibit expression of Ki-ras (Mukhopadhyay et al., 1991; Zhang et al., 1993; Georges et al., 1993). Intratracheal delivery of the Kiras antisense constructs was shown to prevent the growth of human lung tumors in an orthotopic mouse model. These studies demonstrated that Ki-ras is essential not only for initiation of tumor growth, but also for maintenance of the malignant phenotype in this particular tumor model. Antisense oligonucleotides targeted against ras isoforms have also been successfully employed to prevent the growth of human tumors in mouse models (Table 12.2). Activity has been demonstrated against a wide variety of tumor types including tumors that express mutations in Ha-ras alleles, Ki-ras alleles, or tumors that only express normal (unmutated) ras. In all these studies, appropriate control oligonucleotides were shown to exert little or no antitumor activity, supporting the conclusion that the antitumor activity displayed by these Table 12.2 Examples in which antisense approaches against ras isoforms have been successfully employed to prevent tumor growth in animals ras target Tumor

Tissue of origin ras genotype

Ki-ras

H460a

Lung

Ha-ras Ha-ras Ha-ras Ha-ras

Engineered (T24) T24 A549 BEL-7402

Bladder Bladder Lung Liver

Ki-ras mutated

Reference

Mukhopadhyay et al. (1991) Zhang et al. (1993) Georges et al. (1993) Ha-ras mutated Gray et al. (1993) Ha-ras mutated Schwab et al. (1994) Ki-ras mutated Bennett et al. (1995) Normal Liao et al. (1997)

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ras target Tumor

Tissue of origin ras genotype

Reference

Ha-ras

Pancreas Breast Lung Colon Pancreas

Ki-ras mutated Ki-ras mutated Unknown Ki-ras mutated Ki-ras mutated

Cowsert (1997)

Ha-ras

Mia PaCall MDA.MB231 H69 SW620 Panel

Ki-ras

CaLul U87

Lung Glioblastoma

Ki-ras mutated Unknown

MDA.MB231

Breast

Ki-ras mutated

Monia, B. (unpublished) Monia, B. (unpublished)

oligonucleotides is through an antisense mechanism of action. One of the most interesting observations from these studies has been the differential sensitivity displayed by different tumor types in vivo against isoform-specific ras antisense inhibitors. In some cases, antitumor activity can be demonstrated by targeting either Ha-ras or Ki-ras, suggesting that both isoforms play an essential function in the growth of those particular tumor types. However, other tumor types have been shown to be preferentially sensitive to oligonucleotides targeted to a particular ras isoform. For example, antisense oligonucleotides targeted against Haras are far more potent than Ki-ras antisense oligonucleotides in preventing the growth of MDA-MB231 tumors in mouse xenograft models (Cowsert, 1997). Moreover, although isoform-specific tumor sensitivity sometimes correlates with ras mutation status, a number of notable exceptions exist. For example, antisense inhibitors targeted against human Ha-ras have been reported to exert potent antitumour effects against tumor types know to contain mutations in Ki-ras genes (e.g. Mia Paca II and Pane I pancreas). Studies attempting to determine the mechanisms of isoform-specific antitumour activity of ras antisense oligonucleotides are under way in which in vitro and in vivo models are being employed. Nevertheless, studies of this nature demonstrate that antisense targeting of specific ras family members can produce potent antitumour effects in animals, and that antisense compounds may represent a novel class of drugs for the treatment of human malignancies in the clinic. 12.4 Conclusions and Future Prospects The studies described in this chapter demonstrate that antisense technology can be successfully employed for modulating the expression of oncogenic gene products to control cancer growth in preclinical cancer models. Furthermore, a number of antisense inhibitors that have been identified in preclinical studies have progressed to phase I and phase II clinical trials for cancer, and early indications on these trials are encouraging. One of the attractive features that

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antisense technology offers is that it can be used to validate rapidly a molecular target in a particular disease process and simultaneously provide the drug for clinical investigation. This may be best illustrated by the fact that one of the first anticancer drugs to enter the clinic that is specifically designed to inhibit ras function in tumors is a Ha-ras targeted antisense oligonucleotide. This is despite the fact that ras has been known to play a role in human tumorigenesis for over 20 years, and that heroic efforts have been made over this period of time in attempting to discover novel ras-specific anticancer drugs using traditional pharmaceutical approaches. Nevertheless, it is clear that we are only at the earliest stages of understanding and exploiting antisense technology to serve both therapeutics and basic research. Phosphorothioate oligodeoxynucleotides have performed well as first-generation antisense drugs preclinically as well as in the clinic. However, this chemistry may limit the full potential of antisense drugs for therapeutics. For example, phosphorothioate oligodeoxynucleotides possess distinct pharmacokinetic properties (e.g. oral bioavailability, duration of action) that may limit their usefulness as therapeutic agents. Second-generation oligonucleotide chemistries (see also Chapters 2, 3 and 4) are emerging to improve hybridization properties, duration of action, cell permeation, and efficacy, and to reduce toxicity and production costs. In addition, formulation research (see also Chapters 6 and 7) should provide important discoveries to advance further the utility of antisense drugs in the clinic. Thus, antisense technology has progressed rapidly over recent years and continues to do so. Furthermore, the possibility that antisense approaches will play an important role in the future to advance our current understanding of the molecular events underlying human malignancies and to provide a novel arsenal of anticancer drugs is very encouraging. Acknowledgements The author would like to thank a number of research colleagues who have contributed significantly to the conclusions discussed in this chapter: Frank Bennett, Nicholas Dean, and Andrew Dorr of Isis Pharmaceuticals; Rosanne Orr of the CRC Centre for Cancer Therapeutics (UK); and Doriano Fabbro of Novartis Pharmaceuticals (Switzerland). The author would also like to acknowledge Mrs Kim Alexis for her outstanding assistance in the preparation of this chapter. References ALITALO, K. and SCHWAB, M., 1988, Adv. Cancer Res., 46, 235-242. ANFOSSI, G., GEWIRTZ, A.M. and CALABRETTA, B., 1989, Proc. Natl Acad. Sci. USA, 86, 3379-3384.

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BAKER, B.F., LOTT, S.S., CONDON, T.P., CHENG-FLOURNOY, S., LESNIK, E.A., SASMOR, H.M. and BENNETT, C.F., 1997, J. Biol. Chem., 272, 11994-12000. BENNETT, C.F., CHIANG, M.-Y., CHAN, H., SHOEMAKER, J.E. and MIRABELLI, C.K., 1992, Mol. Pharmacol., 41, 1023-1033. BENNETT, C.F., DEAN, N., ECKER, D.J. and MONTA, B.P., 1995, in Agrawal, S., éd., Methods in Molecular Medicine: Antisense therapeutics, pp. 13-46, New York: Humana. BISHOP, J.M., 1987, Science, 235, 305-306. BISHOP, J.M., 1991, Cell, 64, 235-248. BOS, J.L., 1989, Cancer Res., 49, 4682-4689. BOS, J.L., VERLAAN-DE VRIES, M., MARSHALL, C.J., VERLAAN, D.V., MARSHALL, C.J., VEENEMAN, G.H., VAN BOOM, J.H. and VAN DER EB, A.J., 1986, Nucl. Acids Res., 14, 1209-1217. CALABRETTA, B., SIMS, R.B., VALTIERI, M., CARACCIOLO, D., SZCZYLIK, C., VENTURELLI, D., RATAJCZAK, M., BERAN, M. and GEWIRTZ, A.M., 1991, Proc. Natl Acad. Sci. USA, 88, 2351-2355. CHANG, E.H., MILLER, P.S., CUSHMAN, C., DEVADAS, K., PIROLLO, K.F., Ts'o, P.O. and Yu, Z.P., 1991, Biochemistry, 30, 8283-8286. CHEN, G., OH, S., MONIA, B.P. and STAGEY, D.W., 1996, J. Biol. Chem., 271, 28259– 28265. CHIANG, M.-Y., CHAN, H., ZOUNES, M.A., FREIER, S.M., LIMA, W.F. and BENNETT, C.F., 1991, J. Biol. Chem., 266, 18162-18171. CHO-CHUNG, Y.S., 1990, Cancer Res., 50, 7093-7100. CLARKE, M.F., KUKOWSKA-LATALLO, J.F., WESTIN, E., SMITH, M. and PROCOWNICK, E., 1988, Mol. Cell Biol., 8, 884-892. COTTER, F.E., JOHNSON, P., HALL, P., POCOCK, C., AL MAHDI, N., COWELL, J.K. and MORGAN, G., 1994, Oncogene, 9, 3049-3055. COWSERT, L.M., 1997, Anti-Cancer Drug Des., 12, 359-371. CROCE, C.M., 1987, Cell, 49, 155-156. CROOKE, S.T., 1993, Annu. Rev. Pharmacol, 32, 329-376. CROOKE, S.T. and BENNETT, C.F., 1996, Annu. Rev. Pharmacol. Toxicol., 36, 107– 127. CUADRADO, A., CARNERO, A. and LACAL, J.C., 1993, in Lacal, J.C. and McCormick, F., eds, The ras Superfamily ofGTPases, pp. 119-153: Boca Raton, FL: CRC Press. DAUM, G., EISENMANN-TAPPE, I., FRIES, H.-W. and TROPPMAIR, J., 1994, Trends Biol. Sci., 19, 279-283. DEAN, N.M. and GRIFFEY, R.H., 1997, Antisense Nucl. Acid Drug Dev., 1, 229-233. DEAN, N.M. and McKAY, R., 1994, Proc. Natl Acad. Sci. USA, 91, 11762-11766. DEAN, N.M., McKAY, R., CONDON, T.P. and BENNETT, C.F., 1994, J. Biol. Chem., 269, 16416-16424. DEAN, N.M., McKAY, R., MIRAGLIA, L., GEIGER, T., MULLER, M., FABBRO, D. and BENNETT, C.F., 1996, Biochem. Soc. Trans., 24, 623-629. GEORGES, R.N., MUKHOPADHYAY, T., ZHANG, Y., YEN, N. and ROTH, J.A., 1993, Cancer Res., 53, 1743-1750. GEWIRTZ, A.M., 1997, Anti-Cancer Drug Des., 12, 341-358.

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GOKHALE, P.C., MCRAE, D., MONIA, B.P., BAGG, A., KARMAN, A., DRITSCHILO, A. and KASID, U., 1999, Antisense Nucl. Acid Drug Dev., 9, 191–201. GRAY, G.D., HERNANDEZ, O.M., HEBEL, D., ROOT, M., POW-SANG, J.M. and WICKSTROM, E., 1993, Cancer Res., 53, 577–584. HODGES, D. and CROOKE, S.T., 1995, Mol. Pharmacol, 48, 905–918. HOWE, L.R., LEEVERS, S.J., GOMEZ, N., NAKIELNY, S., COHEN, P. and MARSHALL, C.J., 1992, Cell, 71, 335–342. JANSEN, B., SCHLAGBAUER-WADL, H., BROWN, B.D., BRYAN, R.N., VAN ELSAS, A., MULLER, M., WOLFF, K., EICHLER, H.-G. and PEHAMBERGER, H., 1998, Nat. Med., 4, 232–234. KASID, U., PFEIFER, A., BRENNAN, T., BECKETT, M., WEICHSELBAUM, R.R., DRITSCHILO, A. and MARK, G.E., 1989, Science, 243, 1354–1356. LACAL, J.C. and TRONICK, S.R., 1988, in Reddy, E.P., Skalka, A.M. and Curran, T., eds, The Oncogene Handbook, p. 257, Elsevier Science. LIAO, D.-F., MONIA, B.P., DEAN, N. and BERK, B.C., 1997, J. Biol. Chem., 272, 6146– 6150. LIAO, Y., TANG, Z.Y., LIU, K.D. et al, 1997, J. Cancer Res. Clin. Oncol, 123, 25–39. LOHMANN, E.G., 1972, Curr. Topics Cell Reg., 5, 99–133. MONIA, B.P., 1997, in Applied Oligonucleotide Technology, eds, C.Stein and A.Krieg, New York: Wiley. MONIA, B.P., JOHNSTON, J.F., ECKER, D.J., ZOUNES, M.A., LIMA, W. and FREIER, S.M., 1992, J. Biol. Chem., 267, 19954–19962. MONIA, B.P., JOHNSTON, J.F., GEIGER, T., MULLER, M. and FABBRO, D., 1996a, Nat. Med., 2, 668–675. MONIA, B.P., LESNIK, E., GONZALEZ, C., LIMA, W.F., McGEE, D., GUINOSSO, C.F., KAWASAKI, A.M., COOK, P.D. and FREIER, S.M., 1993, J. Biol. Chem., 268, 14514– 14522. MONIA, B.P., SASMOR, H., JOHNSTON, J.F., FREIER, S.M., LESNIK, E.A., MULLER, M., GEIGER, T., ALTMANN, K.-H., MOSER, H. and FABBRO, D., 1996b, Proc. Natl Acad. Sci. USA, 93, 15481–15484. MUKHOPADHYAY, T., TAINSKY, M., CAVENDER, A.C. and ROTH, J.A., 1991, Cancer Res., 51, 1744–1754. NESTEROVA, M. and CHO-CHUNG, Y.S., 1995, Nat. Med., 1, 528–533. NISHIZUKA, Y., 1992, Science, 258, 607–614. RABBITTS, T.H., 1994, Nature, 372, 143–149. RAPP, U.R., 1991, Oncogene, 6, 495–500. RATAJCZAK, M.Z., KANT, J.A., LUGER, S.M., HIJIYA, N., ZHANG, J., ZON, G. and GEWIRTZ, A.M., 1992, Proc. Natl Acad. Sci. USA, 89, 11823–11827. REDDY, E.P., 1983, Nature, 220, 1061–1063. REED, J.C., 1995, Curr. Opin. Oncol., 1, 541-546. SAISON-BEHMOARAS, T., TOCQUE, B., REY, I., CHASSIGNOL, M., THUONG, N.T. and HELENE, C., 1991, EMBO J., 10, 1111–1118. SCHULTE, T.W., BLAGOSKLONNY, M.V., ROMANOVA, L., MUSHINSKI, J.F., MONIA, B.P., JOHNSTON, J.F., NGUYEN, P., TREPEL, J. and NECKERS, L.M., 1996, Mol. Cell Biol., 16, 5839–5845. SCHWAB, G., CHAUANY, C. and DUROUX, I., 1994, Proc. Natl Acad. Sci. USA, 91, 10460–10467.

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SHARPE, C.C., DOCKRELL, M., SCOTT, R., NOOR, M.I., COWSERT, L.M., MONIA, B.P. and HENDRY, B.M., 1999, j. Am. Soc. Nephrol, 10, 1186–1192. SIKIC, B.I., YUEN, A.R., HALSEY, J., FISHER, G.A., PRIBBLE, J.P., SMITH, R.M. and DORR, A., 1997, Proc. Am. Soc. Clin. Oncol., 16, 212a. SOLDATENKOV, V.A., DRITSCHILLO, A., WANG, F.-H., OLAH, Z., ANDERSON, W.B. and KASID, U., 1997, Cancer J. Sci. Am., 3, 13–20. TORTORA, G., YOKOZAKI, H., PEPE, S., CLAIR, T. and CHO-CHUNG, Y.S., 1991, Proc. NatlAcad. Sci. USA, 88, 2011–2015. TRAUB, O., MONIA, B.P., DEAN, N.M. and BERK, B.C., 1997, J. Biol. Chem., 272, 31251–31257. VOGELSTEIN, B., FEARON, E.R., HAMILTON, S.R., KERN, S.E., PREISINGER, A.C., LEPPERT, M., NAKAMURA, Y., WHITE, R., SMITS, A.M. and Bos, J.L., 1988, N. Engl. J. Med., 319, 525–532. WANG, H.-G., MIYASHITA, T., TROPPMAIR, S., SATO, T., TORIGOE, T., KRAJEWSKI, S., TANAKA, S., HOVEY, L., TROPPMAIR, J., RAPP, R., READ, R.U. and REED, J.C., 1994, Oncogene, 9, 2751–2756. WEBB, A., CUNNINGHAM, D., COTTER, F.E., CLARKE, P.A., DI STEFANO, F., CORBO, M. and DZIEWANOWSKA, Z., 1997, Lancet, 349, 1137–1141. WEINBERG, R.A., 1994, CA Cancer J. Clin., 44, 160–168. WESTIN, E.H., GALLO, R.C., ARYA, S.E., EVA, A., SOUZA, L.M., BALUDA, M.A., AARONSON, S.A. and WONG-STAAL, F., 1982, Proc. Natl Acad. Sci. USA, 79, 2194– 2199. XU, X., VANDERZIEL, C., BENNETT, C.F. and MONIA, B.P., 1998, J. Biol. Chem., 273, 33230–33238. YAN, Z., CHEN, M., PERUCHO, M. and FRIEDMAN, E., 1997a, J. Biol. Chem., 272, 30928– 30936. YAN, Z., DENG, X., CHEN, M., XU, Y., AHRAM, M., SLOANE, B.F. and FRIEDMAN, E., 1997b, J. Biol. Chem., 272, 27902–27907. YAZAKI, T., AHMAD, S., CHAHLAVI, A., ZYLBER-KATZ, E., DEAN, N.M., RABKIN, S.D., MARTUZA, R.L. and GLAZER, R.I., 1996, Mol. Pharmacol., 50, 236–242. YOKOZAKI, H., BUDILLON, A., TORTORA, G., MEISSNER, S., BEAUCAGE, S.L., MIKI, K. and CHO-CHUNG, Y.S., 1993, Cancer Res., 53, 868–872. ZHANG, Y., MUKHOPADHYAY, T. and DONEHOWER, L.A., 1993, Human Gene Ther., 4, 451–460.

13 Modulation of Inflammatory Processes with Antisense Oligonucleoticles C.F.BENNETT

13.1 Introduction The inflammatory process is vital for the survival of higher eukaryotic organisms. Although it is a tightly regulated system, failures in the checks and balances can occur, resulting in disease. With the exception of rare genetic deficiencies such as leukocyte adhesion disorders due to defects in [32 integrin (Anderson and Springer, 1987), patients with X-linked hyper-IgM syndrome due to defects in CD40L (Aruffo et al., 1993) or an autosomal recessive form of severe combined immunodeficiency due to a mutation in zap-70 (Chan et al., 1994), the underlying cause of the diseases is unknown. Historical approaches for regulating the immune system utilized either general cytotoxic compounds or glucocorticoids. While these agents clearly provide benefit to the patient, they also expose the patient to undesirable risks because of the nonspecific nature of their activity. The identification of cyclosporin A and, more recently, FK-506 and rapamycin demonstrated that it was possible to attenuate immune responses without causing generalized myelosuppression. These findings, combined with the explosion in our understanding of how the immune system functions, have opened up tremendous opportunities for the treatment of inflammatory disorders. Unfortunately, identification of new chemical entities that selectively inhibit specific pathways in immune cell function has been difficult, with few selective inhibitors forthcoming. Many investigators and companies have relied on monoclonal antibodies or expressed protein products to validate targets, and also as therapeutic approaches. In fact several of these products are on the market, such as OKT3, GM-CSF and various interferons. There is still a need for alternative strategies to identify selective inhibitors of proteins that are thought to play important roles in regulating an immune response. Antisense oligonucleotides represent such an alternative approach for inhibiting the function of proteins thought to be important in regulating immune cell function. In contrast to more conventional approaches, the target for antisense oligonucleotides is the RNA that codes for the protein, rather than the

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protein product itself. Identification of a lead antisense oligonucleotide can be a rapid process, enabling validation of a molecular target within a couple of months. One of the advantages to antisense oligonucleotides is that the subcellular location of the protein product does not matter. The RNA products that code for these proteins are all, in theory, equally accessible to the antisense oligonucleotide. Therefore, intracellular as well as extracellular targets may be selected. In contrast, monoclonal antibodies or other protein products are primarily useful for extracellular targets. Another advantage of antisense oligonucleotides is that they do not appear to be antigenic, which limits the longterm application of monoclonal antibody products. This chapter will focus primarily on intercellular adhesion molecule 1 (ICAM-1) as one example of how antisense oligonucleotides can be used to target an mRNA encoding a protein that plays a central role in immune regulation. Other examples of the use of antisense oligonucleotides to suppress immune responses will be highlighted. Previous reviews have discussed in vitro application of antisense oligonucleotides (Crooke, 1992; Crooke and Bennett, 1996; Bennett, 1993; Bennett and Crooke, 1996), therefore the focus of this chapter will primarily be on recent in vivo applications. 13.2 ICAM-1, a Case Study Extravasation of leukocytes from the circulation through post-capillary venules into tissues is a carefully orchestrated process involving production of soluble chemotactic factors at sites of inflammation, adhesion of leukocytes to vascular endothelium, and diapedesis between endothelial cells (Springer, 1990a, 1994; Butcher, 1991; Ebnet et al., 1996). At least three distinct steps can be identified in leukocyte emigration: reversible adhesion or rolling on vascular endothelial cells, activation of leukocytes resulting in firm adhesion and diapedesis, each mediated by cell-cell adhesion. The initial rolling steps appear to be mediated by selectins (E-, L- and P-selectin, Table 13.1) expressed on either vascular endothelial cells or on leukocytes interacting with specific carbohydrate structures expressed on the cognate cell type (Bevilacqua, 1993; Kansas, 1996). Several papers have been published suggesting that vascular cell adhesion molecule 1 (VCAM-1) interacting with VLA4 can also mediate rolling on endothelial cells (Berlin et al., 1995; Alone et al., 1995). Leukocytes can become activated by a variety of chemotactic factors, either soluble or cell-associated (Ebnet et al., 1996; Zimmerman et al., 1996). Best characterized are neutrophils, which upon activation undergo marked shape changes, translocate MAC-1 from intracellular granules to the cell surface, shed L-selectin and increase affinity of integrins for their ligands, among other changes. Firm adhesion is mediated by the (32 integrins LFA-1 and MAC-1 binding to ICAM-1 and ICAM-2 on endothelial cells and the pi integrin VLA4

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binding to VCAM-1. Activation of P2 integrins LFA-1 and MAC-1 on neutrophils is thought Table 13.1 Endothelial cell-leukocyte adhesion molecules Cell adhesion molecule

Other names

Gene family

Expression pattern

Counterreceptor

E-selectin

ELAM-1, CD62E

Selectin

Sialyl lewis X

L-selectin

CD62L

Selectin

P-selectin

PADGEM-1, CD62P

Selectin

ICAM-1

CD54

Immunoglobul in

ICAM-2

CD 102

Immunoglobul in

ICAM-3

ICAM-R, CD50

Immunoglobul in

Induced on endothelial cells Constitutively expressed on most leukocytes Stored in Weibel-Palade bodies of endothelial cells and αgranules of platelets. Expressed on cell surface after cell activation Activated endothelial cells, keratinocytes, fibroblasts, Blymphocytes, monocytes, etc. Constitutively expressed on endothelial cells induced on activated lymphocytes, platelets Constitutively expressed on lymphocytes Constitutively expressed on most leukocytes

ICAM-4

Immunoglobul in

Sialyl lewis X expressed on GlyCAM-1, MadC AM- 1, Sialyl lewis X expressed on PSGL-1

LFA-1, MAC-1, fibrinogen, hylarounic acid, rhinovirus

LFA-1, MAC-1

LFA-1, αdp2 integrin LFA-1

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 271

Cell adhesion molecule

Other names

Gene family

Expression pattern

Counterreceptor

MadC AM- 1

Mucosal addressin, MECA-367, MECA-89

Immunoglobul in and mucin

Expressed on high endothelial venules in Peyers patches, mesenteric lymph nodes, endothlelial cells of lamina propria and spleen

α4β7 integrin, L-selectin

Table 13.1 (cont'd) Cell adhesion molecule

Other names

Gene family

Expression pattern

Counterreceptor

PEC AM- 1

CD31

Immunoglobuli n

Constitutively expressed on endothelial

VCAM-1

CD 106

Immunoglobuli n

Induced on endothelial cells, dendritic

LFA-1 (αLβ2)

CD11a/CD18

Integrin

MAC-1 (αMβ2)

CD11b/CD 18

Integrin

P150/95 (αxβ2)

CD11c/CD18

Integrin

Constitutively expressed on most leukocyte populations Expressed on monocytes, NK cells and neutrophils (stored in secondary granules) Constitutively expressed on most leukocytes, higher level of expression on monocytes

PEC AM- 1, αvβ3 integrin cells, platelets, monocytes and neutrophils VLA4 (α4βl) and α4β7 cells, smooth muscle cells ICAM-1, ICAM-2, ICAM-3, ICAM-4 ICAM-1, ICAM-2, fibrinogen, iC3b, factor X

iC3b, fibrinogen

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Cell adhesion molecule

Other names

Gene family

Expression pattern

Counterreceptor

αdβ2

CD11d/CD18

Integrin

ICAM-1, ICAM-3

VLA4 (α4β1)

CD49D/CD29

Integrin

α4β7

LPAM-1

Integrin

Constitutively expressed on most leukocytes Constitutively expressed on lymphocytes and monocytes Constitutively expressed on lymphocytes

VCAM-1, fibronectin

MadCAM- 1, VCAM-1, fibronectin

to be required for firm adhesion and transmigration (Butcher, 1991; Springer, 1990b). Passage of leukocytes through endothelial monolayer is predominantly mediated by ICAM-1/LFA-1 interactions (Furie et al., 1991; Oppenheimer-Marks et al., 1991), although contributions by PECAM-1 have also been suggested (Newman, 1997). In addition to playing a role in the migration of leukocytes to sites of inflammation, most adhesion molecules are also capable of signalling leukocytes and endothelial cells (Altmann et al., 1989; Kuhlman et al., 1991; Damle et al., 1992, 1994; Waddell et al., 1995; Lo et al., 1991). In the case of lymphocytes, both LFA-1 and VLA4 provide co-stimulatory signals required for productive response to antigens. Inhibiting these co-stimulatory signals has been shown to attenuate the response of the lymphocyte. Binding of leukocytes to endothelial cell has also been shown to activate the endothelial cells, facilitating the emigration process (Doukas and Pober, 1990; Durieu-Trautmann et al., 1994; Pfau et al., 1995; Karmann et al., 1996). ICAM-1 is expressed at low levels on resting endothelial cells and can be markedly upregulated in response to inflammatory mediators such as TNF-α, interleukin 1 and interferon-y. In addition to endothelial cells ICAM-1 can be induced on a wide variety of cell types such as fibroblasts, smooth muscle cells, epithelial cells and monocytes. ICAM-1 binds the leuko-integrins LFA-1 (Marlin and Springer, 1987) and Mac-1 (Diamond et al., 1991) and has been reported to bind fibrinogen and hylarounic acid (Table 13.1). In addition, ICAM-1 is the receptor for the major group of rhino viruses (Staunton et al., 1989; Marlin et al., 1990). Because of the fundamental role adhesion molecules play in initiating and propagating an immune response, there has been much interest in identifying inhibitors. Monoclonal antibodies have been used to demonstrate proof of concept in a variety of preclinical pharmacological models, as well as in early clinical studies (Kavanaugh et al., 1994; Isobe et al., 1992; Podolsky et al., 1994; Winn et al., 1993; Orosz et al., 1992; Winn and Harlan, 1993; Doerschuk

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et al., 1990; Haug et al., 1993; Wegner et al., 1990). Identification of selective small molecular weight inhibitors of cell adhesion is highly desirable; unfortunately, efforts to identify such compounds have not met with large success. Therefore, there is still a need for alternative approaches. We have used antisense oligonucleotides to inhibit the expression of a variety of endothelial cell adhesion molecules and are currently evaluating them for activity in preclinical pharmacology models as well as in the clinic. ICAM-1 will serve as an example of how antisense oligonucleotides can be used to regulate expression of adhesion molecules, while results obtained with other adhesion molecules will serve to exemplify additional observations. We have tested over 100 different oligonucleotides of various chemistries for effects on ICAM-1 expression. The most effective first-generation phosphorothioate oligodeoxynucleotides identified targeted specific sequences in the 3′-untranslated region of the human ICAM-1 mRNA, ISIS 1939 and ISIS 2302 (Chiang et al., 1991; Bennett et al., 1994). Both ISIS 1939 and ISIS 2302 inhibit ICAM-1 expression by an RNase H-dependent mechanism of action (Bennett et al., 1994). Because ISIS 1939 was very pyrimidine rich (90% C and T), there was concern that this oligonucleotide may produce undesirable effects through interaction with other molecules (both RNAs and non-RNA). Because of these concerns, ISIS 2302 was selected for additional studies. ISIS 2302 will selectively inhibit ICAM-1 expression in a variety of cell types (Bennett et al., 1994; Miele et al., 1994; Nestle et al., 1994). Both sense and a variety of scrambled control oligonucleotides fail to inhibit ICAM-1 expression, including a two-base mismatch control (Bennett et al., 1994; Miele et al., 1994; Nestle et al., 1994). Treatment of endothelial cells with ISIS 2302 blocked adhesion of leukocytes, demonstrating that blocking expression of ICAM-1 will attenuate adhesion of leukocytes to activated endothelial cells (Bennett et al., 1994). ISIS 2302 will also block a one-way mixed lymphocyte reaction when the antigenpresenting cell is pretreated with ISIS 2302 to downregulate ICAM-1 expression prior to exposure to the lymphocyte (T.Vickers et al., unpublished data). Thus, ISIS 2302 is capable of blocking both leukocyte adhesion to activated endothelial cells and co-stimulatory signals to T lymphocytes: both activities were predicted based on previous studies with monoclonal antibodies to ICAM-1. 13.2.1 Pharmacology of ICAM-1 Antisense Oligonucleotides 13.2.1.1 Proof of mechanism Much has been written concerning the non-antisense effects of phosphorothioate oligodeoxynucleotides (Stein, 1995, 1996; Wagner, 1994) (see also Chapter 9).

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With this in mind, can the pharmacological activity observed with an oligonucleotide be ascribed to an antisense effect, rather than the non-antisense effects? Although it is difficult to conclude unequivocally that all the pharmacological activity reported for antisense oligonucleotides is due to antisense effects, with proper controls and experimental design it has been possible to build a strong case that the ICAM-1 antisense oligonucleotides are producing the described pharmacological activity by an antisense mechanism of action. Multiple lines of evidence support this conclusion. 1 In cell culture, the oligonucleotides were identified after screening multiple oligonucleotides all capable of hybridizing to ICAM-1 mRNA. The oligonucleotides used for pharmacological evaluation were identified as being the most effective at inhibiting ICAM-1 expression in cell culturebased assays (Chiang et al., 1991; Bennett et al., 1994). This exercise of screening multiple oligonucleotides for direct inhibition of ICAM-1 expression, identifying the most potent inhibitors, results in compounds which have a larger signal-to-noise ratio. In that phosphorothioate oligodeoxynucleotides will produce non-antisense effects at higher concentrations or doses, identification of potent compounds enables the use of the oligonucleotides at doses which do not produce the non-antisense effects. 2 The ICAM-1 antisense oligonucleotides will selectively reduce ICAM-1 protein in multiple cell types with a wide range of stimuli including interleukin-1 α and β, tumor necrosis factor-α, IFN-γ, bacterial endotoxin and phorbol esters (Chiang et al., 1991; Bennett et al., 1992, 1993, 1994; Miele et al., 1994; Nestle et al., 1994; Stepkowski et al., 1994; Baker et al., 1997). It is unlikely that the oligonucleotides are interfering with a central signalling pathway or receptor-ligand interaction, as these agents induce ICAM-1 expression by different signalling mechanisms (Strassman et al., 1994; Ohh et al., 1994; Read et al., 1995; Wertheimer et al., 1992; Cornélius et al., 1993). 3 For the ICAM-1 antisense oligonucleotides which target the 3′-untranslated region of ICAM-1 mRNA it is possible to demonstrate a selective reduction in mRNA which appears to be due to RNase H (Chiang et al., 1991; Bennett et al., 1994). 4 The antisense oligonucleotides are species-specific, as would be predicted based on poor conservation of sequence between different species. 5 In several in vivo experiments it has been possible to demonstrate a reduction in ICAM-1 mRNA or protein following treatment with the ICAM-1 antisense oligonucleotide (Bennett et al., 1996, 1997; Kumasaka et al., 1996; ChristofidouSolomidou et al., 1997). For chronic models it is difficult to conclude unequivocally that reductions in ICAM-1 by the oligonucleotides are a direct antisense effect, as the oligonucleotides could also affect expression of cytokines which induce ICAM-1 expression or

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 275

activation of cells which release the cytokines. However, we have demonstrated selective reduction in ICAM-1 expression in acute models when ICAM-1 is directly induced by bacterial endotoxins (Kumasaka et al., 1996). It is highly unlikely that these effects would be due to a non-antisense mechanism, as the reductions in ICAM-1 expression were detected 2 to 4 h after stimulation. 6 In several in vivo models the effects of the ICAM-1 antisense oligonucleotide are similar to the effects produced with ICAM-1 monoclonal antibodies (Stepkowski et al., 1994; Kumasaka et al., 1996; Katz et al., 1995). 7 The ICAM-1 antisense oligonucleotide produces the expected pharmacology in vivo for an agent inhibiting ICAM-1 expression. 13.2.1.2 Human xenografts ISIS 2302 is selective for human ICAM-1 mRNA, limiting its application for in vivo pharmacology studies. To test the pharmacology of the human-specific antisense oligonucleotide, we have resorted to experimental models in which human tissue is xenografted in immunocompromised mice. One model examined the role of ICAM-1 in metastasis of human melanoma cells to the lung of mice. ICAM-1 is expressed at high levels of advanced primary melanomas and melanoma metastasis (Johnson et al., 1989; Natali et al., 1990). Treatment of human melanoma cells with either TNF-α or IFN-γ prior to injection into nude mice results in a significant increase in the number of lung métastases and ICAM-1 expression (Miele et al., 1994). To address whether ICAM-1 played a role in the increase in the number of lung metastases, the melanoma cells were pretreated with ICAM-1 antisense oligonucleotides prior to treatment with cytokines. Pretreatment of the melanoma cells with the ICAM-1 antisense oligonucleotides reduced the number of lung metastases, while an irrelevant control oligonucleotide failed to decrease the number of metastases (Miele et al., 1994). The rank order potency for inhibition of ICAM-1 expression correlated with the rank order potency for inhibition of ICAM-1 expression for the ICAM-1 antisense oligonucleotides. The mechanism by which ICAM-1 contributes to the development of lung metastasis is not clear. One possibility is that adhesion of leukocytes to circulating melanoma cells results in embolism in the microvasculature of the lung. Also, ICAM-1 is known to activate leukocytes which could release a variety of proteases and other mediators, enhancing colonization of the melanoma cells in lung tissue. A second study addressed the role of ICAM-1 in an experimental model of cytotoxic dermatitis (lichen planus). In this model human skin is grafted onto SCID mice (Yan et al., 1993; Murray et al., 1994). When the human tissue becomes engrafted, heterologous lymphocytes are injected into the graft which migrate into the epidermis (epidermaltropism) and produce a cytotoxic

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interaction between effector lymphocytes and epidermal cells (ChristofidouSolomidou et al., 1997). Migration of the lymphocytes into the epidermis was linked to expression of ICAM-1 in the epidermis. Systemic administration of ISIS 2302 inhibited ICAM-1 expression in the human graft and decreased the migration of lymphocytes into the epidermis and subsequent lesion formation. Scrambled control oligonucleotides failed to attenuate the responses. These data demonstrate that an ICAM-1 antisense oligonucleotide administered systemically can attenuate an inflammatory response in the skin. 13.2.13 Rodent allografts Because of the lack of conservation between human, mouse and rat ICAM-1 mRNAs in the 3′-untranslated region, where ISIS 2302 hybridizes, it was necessary to identify rat- and mouse-specific antisense oligonucleotides. ISIS 3082 and ISIS 9125 are 20-base phosphorothioate oligodeoxynucleotides which hybridize to an analogous region in the 3′-untranslated region of murine and rat ICAM-1 mRNA, respectively. Similarly to ISIS 2302, ISIS 3082 and ISIS 9125 selectively inhibit ICAM-1 expression in mouse or rat cells by an RNase Hdependent mechanism (Stepkowski et al., 1994, 1998). Previous studies have demonstrated that monoclonal antibodies to ICAM-1 prolong heterotopic cardiac allograft survival in mice (Isobe et al., 1992). ISIS 3082 was tested in the same model to determine whether an ICAM-1 antisense oligonucleotide would prolong cardiac allografts (Stepkowski et al., 1994). Treatment of recipient C3H mice with ISIS 3082 for 7 or 14 days by continuous intravenous infusion resulted in a dose-dependent prolongation of C57BL/10 cardiac allograft survival. Maximal effects occurred between 5 and 10 mg/kg per day. Treatment of recipient mice with 5 mg/kg per day for 14 days increased cardiac allograft survival from 7.7 ± 1.4 days to 23.0 ±7.5 days. Similar results were obtained with two additional strain combinations. Two control phosphorothioate oligodeoxynucleotides failed to prolong cardiac allograft survival. ISIS 3082 was either additive or synergistic with anti-lymphocyte serum, brequinar or rapamycin in prolonging cardiac allograft survival. Similarly to previous reports using monoclonal antibodies to ICAM-1 and LFA-1 (Isobe et al., 1992), the combination of ISIS 3082 and a monoclonal antibody to LFA-1 increased survival of the cardiac allograft to greater than 150 days. These results suggest that the combination of an LFA-1 monoclonal antibody and an inhibitor of ICAM-1 (either an antibody or antisense oligonucleotide) induces donorspecific transplantation tolerance. In the mouse model of cardiac allograft rejection, the combination of ISIS 3082 plus cyclosporin A attenuated the effect of each agent when given alone. This apparent antagonism between ISIS 3082 and cyclosporin A was unique to the mouse heterotopic heart model. ISIS 9125 (the rat-specific oligonucleotide) is synergistic with cyclosporin A in rat kidney and heart allograft models

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(Stepkowski et al., 1998). ISIS 2302 does not attenuate the effects of cyclosporin A in a primate kidney transplant model (S. Stepkowski, unpublished). Finally, in a twoway mixed lymphocyte reaction, ISIS 2302 did not reverse the inhibitory effects of cyclosporin A (Bennett and Condon, 1998). These data suggest that the apparent lack of synergy between cyclosporin A and ISIS 3082 is unique to the mouse cardiac allograft model. In that the effects of cyclosporin A in mice are variable, these results are not unexpected. ISIS 3082 also prolonged survival of mouse islet cell allografts, demonstrating that the effects are not restricted to the heart (Katz et al., 1995). In both the cardiac allograft model and the islet cell allograft models, the effects of ISIS 3082 were as good as or better than an ICAM-1 monoclonal antibody. The rat ICAM-1 antisense oligonucleotide ISIS 9125 prolongs survival of rat cardiac and kidney allografts in a dose-dependent manner (Stepkowski et al., 1998). The effects of the oligonucleotide were more pronounced in the kidney allograft model, which is consistent with the pharmacokinetics of phosphorothioate oligodeoxynucleotides in that the kidney is the major organ of disposition (Cossum et al., 1993; Agrawal et al., 1991; Crooke et al., 1996). There are several mechanisms by which the ICAM-1 antisense oligonucleotides may prolong survival of allografts, such as: 1 inhibition of ICAM-1 expression on endothelial cells of the graft, preventing leukocyte infiltration into the graft tissue 2 inhibition of ICAM-1 expression on either professional or non-professional antigen presenting cells in the graft tissue 3 inhibition of ICAM-1 expression on recipient lymphocytes or NK cells. We have performed several experiments to address this question. The most revealing data were generated in the rat renal allograft model, in which treatment of the donor animal or perfusion of the graft at time of harvest with ISIS 9125 resulted in prolongation of the kidney allograft survival, suggesting that the effects of the oligonucleotide are on the donor tissue rather than the recipient. 13.2.1.4 Renal ischaemia A series of independent studies have used an ICAM-1 antisense oligonucleotide to decrease acute renal injury following ischaemia in rats and following isografting (Haller et al., 1996; Dragun et al., 1998a, 1998b). They identified a 20 base phosphorothioate oligodeoxynucleotide, targeting the 3′-untranslated region of rat ICAM-1 mRNA. This oligonucleotide was shown to inhibit ICAM-1 expression in rat cells in a sequence-specific manner. Using a cationic lipid formulation of the oligonucleotide (see also Chapters 7 and 10) infused into the femoral vein, they demonstrated decreased ICAM-1 protein expression following ischaemic injury and decreased leukocyte infiltrate (Haller et al.,

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1996). The ICAM-1 antisense oligonucleotide also preserved renal function as blood urea nitrogen and serum creatine were reduced in the antisense oligonucleotide treated group 12 to 24 h after injury compared to saline or the control oligonucleotide. These studies were extended to evaluate the protective effect of the ICAM-1 antisense oligonucleotide in a rat model mimicking the ischaemia which occurs during kidney transplantation (Dragun et al., 1998a, 1998b). In kidney allografts, both ischaemia-reperfusion injury and immune response against the donor tissue contribute to loss of graft function. To dissect the role reperfusion injury plays in loss of kidney function, the authors re-transplanted the kidney, after a period of cold and warm ischaemia, either back into the original animal (Dragun et al., 1998b) or into a syngeneic recipient (Dragun et al., 1998a). Similarly to the studies of Stepkowski et al.. (1998), donor animals were pretreated with the antisense oligonucleotides before tissue harvesting. The ICAM-1 antisense oligonucleotide decreased ICAM-1 expression, leukocyte infiltration and tubular necrosis in a sequence-specific manner. Pretreatment with the antisense oligonucleotide increased long-term survival of the animals and improved renal function at all time points examined. These data suggest that inhibition of ICAM-1 expression or function protects against ischaemia-reperfusion injury in kidney and could be useful in enhancing the function of kidney grafts. 73.2.7.5 Colitis Increased expression of ICAM-1 has been detected in both ulcerative colitis and Crohn's disease (Koizumi et al., 1992; Schuermann et al., 1993). The murinespecific ICAM-1 antisense oligonucleotide ISIS 3082 was evaluated in dextran sulphate model of colitis in mice (Bennett et al., 1997). Administration of 5% dextran sulphate in the drinking water of mice for five to seven days produces a colitis, which persists up to six weeks upon discontinuing administration (Okayasu et al., 1990). Mice treated with dextran sulphate for seven days exhibited increased ICAM-1 expression on endothelial cells in the submucosa and in lymphoid structures, demonstrating that ICAM-1 was expressed in inflamed colon tissue. ICAM-1 was also detected on mucosal leukocytes infiltrating in the tissue. The localization of the ICAM-1 antisense oligonucleotide in normal and diseased colon tissue was determined using a rhodamine-labelled oligonucleotide. In normal tissue the ICAM-1 oligonucleotide was localized in the lamina propria and to a lesser extent in epithelial cells in normal mice. The distribution of rhodaminelabelled ISIS 3082 in mice with colitis changed in that epithelial cells accumulated significantly more of the oligonucleotide compared to normal animals (Bennett et al., 1997). Treatment of mice with ISIS 3082 decreased ICAM-1 expression and leukocyte infiltration into the colon of dextran sulphate-treated mice. ISIS 3082 was effective in preventing the development of colitis when administered

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prophylactically, and also in attenuating existing colitis. The optimal dose for preventing development of colitis was between 0.3 andl.Omg/kg per day, while approximately 10-fold higher concentrations were required to attenuate existing disease. Several control oligonucleotides were also evaluated in the model and found to have minimal effects. Thus ICAM-1 antisense oligonucleotides could be of value in treating inflammatory bowel disease. 13.2.2 Toxicology of ICAM-1 Antisense Oligonucleotides The human ICAM-1 antisense oligonucleotide ISIS 2302 has been evaluated for both acute and chronic toxicities in primates and mice (Henry et al., 1996, 1997). In addition the murine specific antisense oligonucleotide ISIS 3082 has been evaluated for exaggerated pharmacological toxicities in mice (Bennett et al., 1997; Hendy et al., 1997). ISIS 2302 contains a 1 base mismatch for the same region in cynomolgus monkey ICAM-1 mRNA, and accordingly is approximately two-fold less effective in inhibiting ICAM-1 expression in cynomolgus monkey cells compared to human cells (unpublished data). Therefore, ISIS 2302 is capable of inhibiting ICAM-1 expression in cynomolgus monkey tissues, albeit at higher doses than would be effective for humans. In both the mouse and monkey studies there was no evidence for toxicities which could be attributed to exaggerated pharmacology. These results were not unexpected, in that genetic deletion of the ICAM-1 gene in mice does not result in any marked phenotypic changes (Sligh et al., 1993; Xu et al., 1994). The observed toxicities were common to other phosphorothioate oligodeoxynucleotides discussed elsewhere in this volume. Briefly, in the monkey studies dose-dependent prolongation in aPTT and evidence for oligonucleotide accumulation in proximal tubular epithelial cells were observed. In the mice studies, both ISIS 2302 and murine analogue, 3082, caused dosedependent increases in spleen and liver weights and mononuclear cell infiltrates in several organs, although the magnitude of changes was smaller than observed for some phosphorothioate oligodeoxynucleotides (Monteith et al., 1997). Other changes noted were increases in circulating monocytes in the 100 mg/kg dose groups and increases in liver enzymes in the serum at the same dosage level (Bennett et al., 1997). These data suggest that at pharmacologically relevant doses, the ICAM-1 antisense oligodeoxynucleotides are well tolerated with repeat administration. 13.2.3 Clinical Studies with ISIS 2302 A phase 1 safety assessment of ISIS 2302 in normal volunteers was recently completed (Glover et al., 1997b). The results of this study demonstrated that ISIS 2302 was well tolerated in normal volunteers, with no adverse events reported.

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The pharmacokinetics (see also Chapter 11) of ISIS 2302 in man was similar to the pharmacokinetics in cynomolgus monkeys. ISIS 2302 is currently being evaluated for efficacy in five different clinical indications: rheumatoid arthritis, psoriasis, acute renal transplant rejection, Crohn's disease and ulcerative colitis. ISIS 2302 has demonstrated preliminary evidence of efficacy and safety in a 20 patient, double-blind, placebo-controlled trial in patients with active Crohn’s disease (Yacyshyn et al., 1998). Patients received 13 2 h i.v. infusions (0.5, 1, or 2 mg/kg in cohorts of 4, 4, and 12 patients, respectively) over 26 days, and were followed for six months. At the end of the treatment period, 1 of 5 placebotreated patients, a patient already in remission at baseline, and 7 of 15 ISIS 2302treated patients were in remission (CDAI<150). At the end of the study (month 6), 5 of the 7 ISIS 2302 remitters were still in remission. Patients treated with ISIS 2302 reduced their steroid use, suggesting that the drug provided a steroidsparing effect in this patient population. Similarly to the murine colitis studies, a decrease in ICAM-1 expression in colon biopsies was demonstrated, suggesting that the drug was working by an antisense mechanism of action. ISIS 2302 was well tolerated, with the only important adverse effect being a transient (2–4 h), dose-related increase in aPTT (~10 s at 2 mg/kg) with each infusion, as observed in monkeys and normal volunteers (Glover et al., 1997a), without evidence of increased GI bleeding, A confirmatory, 300 patient, pivotal quality Crohn’s disease study is in progress, as are smaller studies investigating subcutaneous dosing and shorter courses of i.v. therapy. 13.2.4 Second- and Third-generation Chemistry Four major objectives for an antisense oligonucleotide medicinal chemistry programme are to increase potency, decrease toxicity, alter the pharmacokinetics and reduce costs (for chemistry of oligonucleotides, see also Chapters 2, 3 and 4). It is gratifying to see that there have been tremendous advancements in all four areas. A large number of different chemically modified oligonucleotides and derivatives have been evaluated for activity against ICAM-1. Two types of approach have been taken: maintaining an oligodeoxynucleotide segment or ‘gap’ to support RNase H activity (see also Chapter 1) (Monia et al., 1993) or utilization of uniformly modified oligonucleotides which do not support RNase H activity (Chiang et al., 1991; Baker et al., 1997). Surprisingly, among the most potent oligonucleotides identified to date are oligonucleotides uniformly modified on the 2′ position of the sugar, such as 2′-fluoro or 2′-methoxyethyl. These oligonucleotides do not support RNase H activity, yet are 10- to 20-fold more potent than the best phosphorothioate oligodeoxynucleotide which supports RNase H (Baker et al., 1997). These results demonstrate that it is not necessary to induce RNA turnover to obtain potent antisense oligonucleotides. In general, we have observed a correlation between increase in hybridization affinity for the target RNA and antisense activity in cell culture. However, it should be kept in

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mind that methods used to introduce oligonucleotides into cells, such as cationic lipids or electroporation, may skew the data towards one type of chemistry. For example, we have observed that oligonucleotides with reduced or no charge interact poorly with cationic lipids, yet are very effective at inhibiting ICAM-1 expression when electroporated or microinjected into cells. Thus, direct comparison of different chemistries using cationic lipids as the only means for enhancing cellular delivery may bias towards highly charged species. In addition to increasing potency, we have found that several modifications will decrease the class-specific toxicity of phosphorothioate oligodeoxynucleotides. For example, both 5 substituted pyrimidines and 2′-sugarmodified oligonucleotides produce less polyclonal B cell proliferation than unmodified oligodeoxynucleotides (Boggs et al., 1997; Zhao et al., 1995; Krieg et al., 1995). The 2′-methoxyethyl modification also appears to decrease potential for acute toxicities in primates, namely increases in aPTT and complement activation (Monteith et al., manuscript submitted). The in vivo pharmacokinetics for several oligonucleotide modifications have been described (Crooke et al., 1996; Agrawal et al., 1995; Zhang et al., 1995a, 1996; Pardridge, 1995). Results from these studies demonstrate that it is possible to change the tissue distribution of oligonucleotides with different chemical modifications. In addition, more stable oligonucleotide analogues have been identified which would allow for less frequent administration of the drug. 13.3 Other Examples 13.3.1 Other Endothelial-Leukocyte Adhesion Molecules We have taken a similar approach as described for ICAM-1 to identify antisense oligonucleotides targeting VCAM-1, PECAM-1 and E-Selectin (Bennett et al., 1994). In each case, targeting several sites on the respective mRNAs with antisense oligonucleotides resulted in the identification of phosphorothioate oligodeoxynucleotides which would selectively inhibit expression of the respective adhesion molecules in different species. One of the antisense oligonucleotides targeting human E-selectin, ISIS 4730, provides some interesting insights into the molecular mechanism of action of phosphorothioate antisense oligodeoxynucleotides (Condon and Bennett, 1996). Treatment of human umbilical vein endothelial cells with ISIS 4730 inhibits the synthesis of Eselectin in a dose- and sequencespecific manner. Following treatment of cells with ISIS 4730, a novel lower molecular weight transcript was induced, which was due to RNase H cleavage of the pre-mRNA, trapping the last intron in the cleavage product. The resulting transcript was stable and appeared to remain in the cell nucleus. These results demonstrate that phosphorothioate

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oligodeoxynucleotides are capable of binding to the pre-mRNA in the cell nucleus prior to RNA processing. This result opens up interesting opportunities for the use of oligonucleotides to regulate RNA maturation. Recently, the effects of a porcine E-selectin antisense oligodeoxynucleotide have been evaluated in a model of septic shock (Goldfarb et al., 1999). Pretreatment of pigs with 10 mg/kg of the E-selectin antisense oligonucleotide attenuated the drop in cardiac output, and increased peripheral vascular resistance due to endotoxin administration. In addition, the E-selectin antisense oligodeoxynucleotide prevented endotoxin-induced neutropenia, presumably due to inhibiting neutrophil margination. A control oligonucleotide had no effect on these parameters. These results demonstrate that an E-selectin antisense oligonucleotide can attenuate acute inflammatory changes. We have found that human VCAM-1 is especially sensitive to the nonantisense effects of oligonucleotides. In cell culture-based experiments, specific VCAM-1 oligonucleotides are approximately five-fold more effective at inhibiting VCAM1 expression than control oligonucleotides. Other adhesion molecules exhibit a greater selectivity than this. The non-antisense effects are not limited to phosphorothioate oligodeoxynucleotides, as more potent phosphodiester and heterocyclic modified oligonucleotides also inhibit VCAM-1 expression with a similar selectivity. The reason why VCAM-1 appears to be more prone to the non-antisense effects of oligonucleotides than other molecules is currently not known. VCAM-1 antisense oligodeoxynucleotides have been evaluated for pharmacological activity in several animal models. Although they have shown activity in the models, they are either equal to or less effective than the ICAM-1 antisense oligonucleotide. 13.3.2 Interleukin 1 Receptor Perhaps one of the first demonstrations of in vivo efficacy for an antisense oligonucleotide was a study performed by Burch and Mahan (1991) in which they identified an oligodeoxynucleotide targeting human and murine type 1 interleukin 1 receptor. They demonstrated that subcutaneous injection of a phosphorothioate oligodeoxynucleotide targeting murine IL-1 receptor inhibited IL-1-induced neutrophil influx into skin by 37%. To obtain this response, it was required to treat the mice at least 48 h prior to the injection with IL-1, presumably to downregulate existing receptors (Burch and Mahan, 1991). The human-specific antisense oligonucleotide, which contains five mismatched bases to the murine antisense oligonucleotide, did not modify neutrophil influx. The authors did not demonstrate an effect of the antisense oligonucleotide on receptor expression in murine tissue to determine whether the degree of receptor inhibition correlated with decrease in neutrophil influx. As there is evidence for spare IL-1 receptors (Dinarello, 1994), it is possible that the antisense

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oligonucleotide was more effective than it would appear to be based on neutrophil influx. 13.3.3 NF-κB NF-κB is a member of a family of transcription factors which regulate expression of a large number of gene products including immunoglobulin κ, interleukin-2 receptor, GM-CSF, E-selectin, ICAM-1, VCAM-1, interleukin 1, interleukin 6, interleukin 8, TNF, etc. (Baeuerle and Henkel, 1994). There are several members of the NF-κB or Rel family which form either homodimers or heterodimers. NFκB is a p50–p65 heterodimer which forms a trimeric complex with 1κB (inhibitor of κB) in resting cells. Upon activation, 1κB becomes phosphorylated and subsequently degrades, releasing NF-κB from the complex. NF-κB translocates into the nucleus where it activates transcription of a variety of gene products. Two groups have independently utilized antisense oligonucleotides targeting the p65 subunit of NF-κB to inhibit the growth of tumor cells in mice (Narayanan et al., 1993; Higgins et al., 1993; Kitajima et al., 1992). More recently, Neurath et al. (1996) have demonstrated that antisense oligonucleotides to the p65 subunit reverses established colitis in mice. A single intravenous injection or intracolonic application of a p65 antisense oligodeoxynucleotide reversed clinical symptoms in mice with TNBS-induced colitis and reversed ongoing intestinal inflammation determined by histology. Macrophages isolated from the intestine of the antisense oligonucleotide-treated mice produced significantly lower amounts of IL-1, IL-6 and TNF-α. The p65 antisense oligodeoxynucleotide was also found to be effective in reversing clinical and histological scores in IL-10 deficient mice which develop chronic intestinal inflammation. In each case the mismatched phosphorothioate oligodeoxynucleotides failed to exhibit activity. Although the authors did not directly show an effect of systemic or local administered oligonucleotide on p65 levels within cells in the tissue, the data indicate that the oligonucleotide was producing these dramatic effects by an antisense mechanism of action. Previously it has been demonstrated that some of the earlier pharmacological activity described for p65 antisense oligonucleotides may be due to nonantisense effects (Maltese et al., 1995): the oligonucleotides used in the colitis study did not contain the four consecutive guanines which contributed in part to the non-antisense effects. These studies also demonstrate that short-term suppression of an ongoing inflammatory response may provide long-term benefit. Similar observations have been made for ICAM-1 and TNF-α monoclonal antibodies as well as the ICAM-1 antisense oligonucleotide (Kavanaugh et al., 1994; Yacyshyn et al., 1998; Elliott et al., 1993, 1994).

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13.3.4 Adenosine Receptors The findings that asthmatics are more sensitive to adenosine challenge than normal subjects (Church and Holgate, 1986; Cushley et al., 1983), combined with the detection of increased levels of adenosine in the bronchoalveolar lavage fluid of asthma patients (Driver et al., 1993), suggest that inhibitors of adenosine may be beneficial in the treatment of asthma. Nyce and Metzger (1997) used antisense oligonucleotides to demonstrate a role of adenosine in a rabbit model of asthma. Administration by aerosilazation of a phosphorothioate oligodeoxynucleotide directed against the adenosine Al receptor increased the concentration of adenosine by at least a factor of 10 required to reduce lung compliance by 50%. The mismatched control oligonucleotide failed to produce an effect. Receptor binding assays demonstrated a 2.5-fold reduction in adenosine Aj receptors but no effect on adenosine A2 or bradykinin B2 receptors. To demonstrate further a specific antisense effect, the authors treated animals with an oligonucleotide designed to bind to the bradykinin B2 receptor. Similarly to the adenosine At antisense, the bradykinin B2 antisense oligonucleotide selectively reduced expression of the bradykinin B2 receptor. Finally, the authors used dust mite challenge to demonstrate that inhibition of adenosine A1 receptor expression is effective in preventing decrease in pulmonary compliance in response to an allergen. 13.3.5 Nitric Oxide Synthetase At least three distinct gene products have been described for nitric oxide synthetase (NOS): an endothelial constitutive NOS, a neuronal form and an inducible form (Nathan and Xie, 1994). All three forms of NOS are present in the kidney (Bachmann et al., 1995; Mohaupt et al., 1994). Experimental evidence suggests that nitric oxide and its metabolic product peroxynitrite contribute to the pathophysiology in acute renal failure (Yu et al., 1994). The commonly used NOS inhibitors fail to discriminate among the three different isoforms, therefore it is difficult to ascertain which isoforms contribute to ischaemic kidney disease. Antisense oligonucleotides are an ideal tool to determine the role of different NOS enzymes in mediating kidney disease. Noiri et al. (1996) used a phosphorothioate oligodeoxynucleotide to inhibit the expression of inducible NOS (iNOS) and evaluate the role of this enzyme in ischaemic renal failure. The antisense iNOS oligonucleotide, but not control oligonucleotides, preserved kidney function when pretreated 10 h before surgically induced ischaemic injury in rats. The authors validated that the oligonucleotide reduced iNOS expression by Western blot analysis and immunohistochemistry. Thus these data provide direct evidence that the iNOS

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isoform is responsible for producing the excess nitric oxide in ischaemic reperfusion injury resulting in kidney epithelial damage. 13.4 Regulation of Immune Response by Non-antisense Mechanisms Most, if not all, phosphorothioate oligodeoxynucleotides will produce some degree of immune activation which is not attributable to a specific antisense effect (Monteith et al., 1997). Krieg et al. (1995) reported that phosphorothioate oligonucleotides containing a CpG motif flanked on the 5′-side by two purines and on the 3′-side by two pyrimidines were especially effective in activating murine B lymphocytes. These studies have been extended to elucidate the mechanisms by which phosphorothioate oligodeoxynucleotides activate immune cells (Yi and Krieg, 1998; Yi et al., 1996, 1998) and to characterize which immune cells are activated by related sequence motifs (Jakob et al., 1998; Ballas et al., 1996; Stacey et al., 1996). In addition to B-lymphocytes, NK cells, dendritic cells and macrophages are activated by bacterial DNA or oligonucleotides with specific sequence motifs. Upon activation cells release a variety of cytokines, most prominent being interferon-y, IL-6, IL-12, GM-CSF and TNF-α. This group of cytokines promotes differentiation of T lymphocytes to a T helper-1 immune (Thl) response (Romagnani, 1997; Mosmann and Sad, 1996). Thl cells are involved in cell-mediated immune responses such as cytotoxic reactions and delayed-type hypersensitivity. In contrast, the cytokines IL-4, IL-5 and IL-13 promote differentiation of T cells to a Th2 phenotype which is implicated in allergic inflammatory response typified by IgE antibody production and eosinophil proliferation. This sequence-specific, non-antisense effect of oligonucleotides is currently being exploited to enhance immune response as a vaccine adjuvant and to promote tumor immunity, among other uses (Roman et al., 1997; Chu et al., 1997; Davis et al., 1998). The ability of certain oligonucleotides to promote selectively a Thl immune response can also be useful for the treatment of allergic diseases such as asthma, which is thought to be the result of an abnormal Th2 immune response to allergens. To test this hypothesis directly, Broide et al. (1998) evaluated the effects of immunonostimulatory DNA in a murine model of allergen-induced airway hyper-responsiveness. The DNA inhibited airway eosinophilia and reduced hyper-responsiveness to inhaled methacholine. The DNA was effective when administered systemically or by intranasal instillation. A single administration of the immunostimulatory DNA was as effective as daily corticosteroid administration in reducing airway inflammation and IL-5 production. Thus oligonucleotides with the appropriate immunostimulatory sequences may be useful not only as adjuvants but also in disease where redirection of the immune response away from a Th2 response may be desirable.

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13.5 Conclusions With an increased understanding of how the immune system functions under normal conditions and a greater appreciation of how dysregulation of immune response contributes to a variety of inflammatory diseases, a large number of potentially therapeutically useful molecular targets are being identified. For intracellular targets, antisense oligonucleotides represent one of the most direct routes to address the role of a given protein in normal and abnormal immune responses. In addition, antisense oligonucleotides have utility for inhibiting the expression of extracellular targets, and offer some advantages over other approaches. However, it should be kept in mind that not all cell types within a tissue will be equally sensitive to the effects of the antisense oligonucleotide, due to the pharmacokinetic behaviour of specific oligonucleotide chemistries (Cossum et al., 1993; Agrawal et al., 1991; Zhang et al., 1995; Plenat et al., 1995; Rifaj et al., 1996; Butler et al., 1997). Therefore, as with any other technology, careful experimental design and interpretation of the results are required. We and others have demonstrated that phosphorothioate oligodeoxynucleotides targeting ICAM-1 exhibit broad anti-inflammatory activity in a variety of animal models (Stepkowski et al., 1994; Bennett et al., 1996, 1997; Kumasaka et al., 1996, Christofidou-Solomidou et al., 1997; Katz et al., 1995; Haller et al., 1996). The effects of the oligonucleotides were sequence-specific, and in many instances direct effects of the oligonucleotide on tissue expression of ICAM-1 have been demonstrated. These findings support the conclusion that the oligonucleotides were acting by an antisense mechanism of action. Based on pharmacological activity, safety profile, pharmacokinetic behaviour and medical need, we are currently developing a parenteral formulation of the ICAM-1 antisense oligodeoxynucleotide in several inflammatory diseases. Data from a Crohn’s disease study, in which an ICAM-1 antisense oligonucleotide was given by intravenous infusion, are very encouraging, suggesting that the oligonucleotide has beneficial effects in this group of patients. To obtain broad utility for chronic inflammatory diseases, it is clear that more convenient dosage forms are needed. Both second-generation chemistries and advanced formulations appear to meet this need. Although it is still in its infancy, the outlook for the application of antisense technology towards the treatment of human diseases is very promising. References AGRAWAL, S., TEMSAMANI, J. and TANG, J.Y., 1991, Proc. Natl Acad. ScL USA, 88, 7595–7599. AGRAWAL, S., ZHANG, X., Lu, Z., ZHAO, H., TAMBURIN, J.M., YAN, J., CAI, H., DIASIO, R.B., HABUS, I., JIANG, Z., IYER, R.P., Yu, D. and ZHANG, R., 1995, Biochem. Pharmacol., 50, 571–576.

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14 Oligonucleotides as Antiparasite Compounds J.J.TOULMÉ

14.1 Introduction Human parasitic diseases constitute major infectious pathologies responsible for millions of deaths every year, mainly of children, all over the world. In contrast to the recent progress in the development of new drugs and vaccines for bacterial and viral diseases, most of the antiparasite drugs still in use today were discovered in the early decades of this century. These drugs were identified by an empirical approach and the mode of action remains unknown for many of them. For some parasitic diseases the situation is worse now than it was 15 years ago. For instance, about 25% of Leishmania strains are resistant to antimonials in India; the advent of chloroquine resistance in Plasmodium falciparum led to a major public health problem. New technologies should help in identifying new compounds of interest through either rational drug design against parasite-specific targets or combinatorial strategies. Moreover, the information arising from the systematic sequencing of entire genomes—several parasitic protozoan organisms will be fully sequenced in the next three to five years—will provide a wealth of new potential targets which will be characterized and explored. ‘Oligonucleotide therapies’ which make direct use of the encoded genetic information offer new ways to design potential therapeutic agents and to create powerful tools to investigate gene function in parasites. The antisense strategy, which rests on the selective recognition of an RNA region by a complementary sequence, has been used extensively over the past 15 years, leading to numerous successful studies (see Hélène and Toulmé, 1990; Stein and Cheng, 1993; Toulmé, 1992 for reviews). Promising results have been obtained in several fields, in particular for cancer and viral diseases. The development of several major viruses has been controlled in vitro in cultured cells. These include influenza virus (Zerial et al., 1987), herpes virus (Kulka et al., 1993), and human immunodeficiency virus (Matsukura et al., 1989). In a pioneering work published in 1978, Zamecnik and Stephenson demonstrated that they could control the in vitro multiplication of the Rous sarcoma virus. Animal

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studies have also been performed along these lines. In 1993, Offensperger et al. reported that an antisense oligonucleotide complementary to the 5′ region of the pre-S-gene was able to inhibit the replication of duck hepatitis B virus in infected Pekin ducks. The PDA recently approved the application for an antisense phosphorothioate oligonucleotide for the treatment of cytomegalovirus-induced retinitis in AIDS patients (Crooke, 1998). Far fewer studies have been devoted to the us e of oligonucleotides against parasites, even though these pathogens plague the world and are responsible for considerable morbidity and mortality. This is certainly not related to any particular problem which would make the antisense strategy inappropriate for fighting parasites, but rather reflects the limited interest in pathologies that essentially reside in underdeveloped countries. 14.2 Design of Antisense Oligonucleotides for Antiparasite Use The design of antisense oligonucleotides against parasite targets faces the same requirements as for other uses; the limitations are very similar to those encountered for antiviral oligonucleotides. Due to the presence of nucleases in the parasite, in the host cell, in the fluid in which the parasites multiply or even in the growth medium (in the case of in vitro studies), unmodified oligodeoxynucleotides are of limited interest. Numerous derivatives have been synthesized which fulfil the criterion of nuclease resistance (see Chapter 2). Several of these oligonucleotide analogues have been used to block the expression of parasite genes (Toulmé et al., 1997a). Phosphorothioates were by far the most exten sively studied oligonucleotide derivatives, both in cell-free studies and in cultured cells. They displayed a limited toxicity at the concentration of interest but were shown to exhibit non-specific effects in different instances. This means that the observed inhibition of protein synthesis or the antiparasitic effect was largely sequenceindependent: a noncomplementary control phosphorothioate sequence was as efficient as the antisense one at inhibiting in vitro translation of Leishmania mRNA (Compagno et al., 1999). This also accounted for most if not all of the in vitro antimalarial activity of phosphorothioate oligomers (Clark et al., 1994), a situation similar to that observed with the human retro virus HIV-1 (Matsukura et al., 1987; Zelphati et al., 1994). This is probably related to the non-selective binding of the oligomer to the parasite surface proteins and/or to the host membrane receptor(s) which mediate Plasmodium infection (see Part III). Other nuclease-resistant oligoribonucleotides, morpholino derivatives—2′-O-methyl phosphorodiamidates (Figure 14.1)—have also been successfully used against the amastigote stage of Leishmania mexicana. The selectivity of the antisense oligomer is crucial, and constitutes one of the major potential interests of this strategy. Two different situations should be

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Figure 14.1 Chemically-modified antisense oligonucleotides. (a) Nuclease résistant analogues (from left to right): phosphorothioate (PS), 2′-O-methyl, ribonucleotide (OMe), morpholino,phosphorodiamidate (MF). These derivatives bind to the target RNA with a lower (PS) or a higher affinity (OMe, MF) than unmodified DNA. Only PS oligomers elicit RNase H activity. PS, OMe or MF analogues of a 16-mer complementary to the miniexon RNA of L. amazonensis displayed a specific leishmanicidal activity, (b) Selectively binding complementary (SBC) A and T analogues. 2, thio thymine and 2, amino adenine are proposed to form a single hydrogen bond hence a weak pair whereas they give rise to extra-stable base pairs with unmodified adenine and uracile residues (Kutyavin et al., 1996)

considered. Firstly, the inhibition needs to be strictly restricted to the target gene. This is a key point when, for instance, oligonucleotides are used for unravelling a cascade of genes involved in a metabolic pathway, and in general if one wants to uncover the function of the target gene. A particular case is constituted by the validation of a given gene as a therapeutic target. Secondly, the oligonucleotide should not exhibit an effect on the expression of any gene of the host organism, but may lead to valuable parasiticidal properties following binding to multiple sites in the pathogen. Indeed, even though RNA is the intended target, one cannot exclude the interaction of the oligonucleotide with other molecules. This is well documented for phosphorothioate analogues which are known to bind strongly to proteins (Stein and Cheng, 1993) (see also Chapter 9). A large number of non-antisense effects (but which may be sequencedependent) have been reported. This is generally easily revealed by the use of several control sequences (scrambled, inverted or mismatched oligomers, as well as mutated target RNA) (Toulmé et al., 1996a). This kind of non-antisense effect might be of interest if restricted to the parasite.

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Table 14.1 Minimal size of antisense oligonucleotides (calculated according to equations 14.1 and 14.2) containing only A and T (n(AT)) or only G and C (n(GC)) to ensure the unicity of the target in the human genome and in that of Plasmodium falciparum, Leishmania major and Trypanosoma brucei. The genome size (N) and the fraction of AT pairs (f) are given

Human P. falciparum L. major T. brucei

N

f

n (AT)

n (GC)

8×109

0.6 0.8 0.3 0.5

19 19 10 13

15 8 16 13

2.8×107 3.5×107 2.5×107

The selectivity of an oligonucleotide at the gene level is primarily driven by the length of the antisense sequence. The unicity of a target for an oligomer in a given genome is dictated by the size and the base composition of both the antisense sequence and the genome of interest (Hélène and Toulmé, 1990). The probability Po of finding a sequence of n nucleotides with a, t, g and c adenine, thymine, guanine and cytosine residues, respectively, in a genome containing a fraction f of AT pairs is given by: (14.1) The number Q of identical sequences of n nucleotides in a genome composed of N bases is given by: (14.2) The minimal lengths n that an oligonucleotide should have to find statistically a unique target (Q ≤ 1) in the genomes of Plasmodium falciparum, Leishmania major or Trypanosoma brucei are indicated in Table 14.1. These numbers can be compared to that for the human genome. These calculations assume a statistical distribution of base pairs, which of course is not correct. Refined probability can be obtained if nearest neighbour frequencies are known. This might substantially change the results; for instance, the dinucleotide CpG is under-represented in eukaryotes (Hélène and Toulmé, 1990). The numbers given in Table 14.1 have been calculated taking account of the DNA complexity. As only a part of DNA is transcribed, the values at the RNA level are lower. Of course, if one wants a specific effect against a parasite gene without detrimental effect on its host, the highest n value should be used: for a sequence containing exclusively Gs and Cs a 15-mer should be used against a P. falciparum gene in a human context, but one needs a 16-mer if L. major gene is targeted. It is worth mentioning that lengthening the antisense sequence does not guarantee an increased specificity. It might actually result in the opposite effect, as longer sequences will generate more stable mismatched hybrids with nontarget RNAs: any extra stability might be a source of undesired effects (Larrouy et al., 1995). Conversely, the shortest antisense sequence predicted to ensure statistically the target unicity might not lead to the inhibition of gene expression, due to a low binding constant. Indeed, we never observed in vitro translation

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inhibition with an A-T rich antisense 10-mer targeted to the Leishmania miniexon RNA, probably due to the low stability of such RNA—oligonucleotide complexes. The other limitation to the antisense strategy is the poor uptake of oligonucleotides by live cells (see also Chapters 9 and 10). The different derivatives designed to increase the membrane penetration of eukaryotic cells (oligomers conjugated to polycations (Lemaître et al., 1987) or hydrophobic tails (Boutorin et al., 1989)) can be used against parasites. One can also take advantage of peculiarities; for instance, Leishmania develops inside the macrophage, a cell type known for high phagocytic activity. Indeed, the in vitro delivery of leishmanicidal drugs by liposomes was shown to lead to increased efficacy (Alving et al., 1978; New et al., 1978). Oligomers covalently linked to a palmitate chain for ensuring their association with low density lipoproteins were reported to display an enhanced leishmanicidal activity compared to free oligomers (Mishra et al., 1995). A similar improvement was observed with oligonucleotides encapsulated into liposomes (see also Chapter 7) coated with maleylated bovine serum albumin, a ligand for macrophage scavenger receptors which ensured a selective delivery to the host cell (Chaudhuri, 1997). It is also of interest to note that the membrane permeability of infected cells might be changed compared to non-infected ones. It has been reported that Plasmodiuminfected erythrocytes permitted the entry of oligonucleotides, whereas noninfected ones did not (Rapaport et al., 1992). A similar conclusion was reached in the frame of a study on antisense oligonucleotides against the Friend retrovirus (Ropert et al., 1993, 1996). Interestingly, these alterations of the membrane permeability contribute to the selectivity of anti-parasite oligomers as the oligonucleotide is preferentially taken up by infected cells. This might also reduce the intracellular concentration of the oligonucleotide in non-infected cells compared to infected ones, thus reducing the cytoxicity towards the former. However, even if the oligonucleotide accumulates into the host cell, it has to cross the parasite membrane. Protozoan parasites, either intracellular like Leishmania or extracellular like Trypanosoma brucei, are generally characterized by a low endocytotic activity. Therefore the delivery of oligonucleotides inside parasites would require specific strategies. In particular, targeting intracellular parasites implies that two membranes should be penetrated successively—the host cell and the parasite membranes—suggesting that ideally a two-step delivery vehicle should be designed. 14.3 Antiparasite Effects of Antisense Oligonucleotides The first studies with antisense oligonucleotides against RNA from parasites aimed at determining whether a sequence found at the 5′ end of messenger RNA coding for variant surface glycoproteins (the surface proteins of African trypanosomes) was of general occurrence in trypanosomatids. Indeed, such a

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sequence was also found in a few mRNA from Trypanosoma cruzi, suggesting a common mechanism of mRNA synthesis (Agabian, 1990; Borst, 1986). Two teams independently performed in vitro translation of trypanosome mRNA in the presence of oligonucleotides complementary to part of this 39 nucleotide long sequence termed mini-exon or spliced leader. Anti-mini-exon sequences were shown to inhibit the protein synthesis directed by every trypanosome mRNA, either in wheat germ extract (Cornelissen et al., 1986) or in rabbit reticulocyte lysate (Walder et al., 1986). The inhibition of translation was selective as, firstly, anti-mini-exon oligonucleotides had no effect on the in vitro translation of non-related mRNA such as brome mosaic virus mRNA and, secondly, non-complementary oligonucleotides had no effect on the translation of trypanosome mRNA. Moreover, the mini-exon sequence varies from one trypanosome species to the other; oligomers targeted to the T. brucei mini-exon had a limited effect on the in vitro translation of T. vivax mRNA and vice versa (Cornelissen et al., 1986; Verspieren et al., 1990). Therefore these results demonstrated that trans-splicing, the mechanism by which the mini-exon sequence is acquired by mature mRNA, is a general characteristic of gene expression in trypanosomatids (see section 14.3.2). Several studies have then been performed, almost exclusively devoted to two types of protozoan parasites: Plasmodium, the causative agent of malaria, and kinetoplastidaes (trypanosomes and leishmania). 14.3.1 Antisense Oligonucleotides against Plasmodium Plasmodium falciparum is the most virulent strain of the four species responsible for human malaria. This parasite undergoes a complex life cycle alternating between vertebrate and insect hosts. Upon infection by an infected female mosquito, the sporozoite is transported to the target cell—a hepatocyte—by the blood circulation. After having matured and developed, the parasite can invade erythrocytes. There it grows in a parasitophorous vacuole, where it divides to form erythrocytic merozoites which are liberated when the mature stage of infected cells (schizonts) bursts. Erythrocytic merozoites can re-invade red blood cells. Studies with antisense oligonucleotides have been performed both in cell-free translation extracts and with the erythrocytic stage in culture. Unmodified oligodeoxynucleotides were targeted to mRNA encoding for two enzymes involved in the biosynthesis of dTMP on the one hand and the salvage of purines (as Plasmodium cannot carry out de novo purine synthesis) on the other hand. Therefore dihydrofolate reductase—thymidylate synthase (DHFR-TS) and hypoxanthine—guanine phosphororibosyl transferase (HPRT) control key pathways for DNA and folate synthesis and constitute targets of interest; indeed, malaria was formerly treated by pyrimethamine, an antifolate now ineffective due to the emergence of resistant strains. Antisense oligomers complementary to

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the initiator AUG of the DHFR-TS and HPRT genes were able to block in vitro translation in rabbit reticulocyte lysate (Dawson et al., 1993; Sartorius and Franklin, 1991). However, only unusually long sequences (>30 nucleotides) were efficient against the DHFRTS mRNA. Moreover it was reported that a 49-mer complementary to the DHFR-TS coding region was the most efficient antisense sequence and that the inhibition occurred through an RNase H-independent mechanism (Sartorius and Franklin, 1991). This is at variance with the numerous studies demonstrating that, in the absence of induced cleavage of the target mRNA, complementary oligonucleotides cannot block elongating ribosomes unless a covalent link is created between the antisense sequence and the target RNA (Toulmé, 1992; Toulmé and Tidd, 1998). This might indicate that the long (49 nt) anti-DHFR-TS oligomer forms a non-conventional RNA—DNA complex which is not unwound by the ribosome or that the target region presents some peculiar feature (for instance a strong translational pause site) making it sensitive to complementary bound sequences. Indeed, we recently observed RNase Hindependent translation inhibition by an oligomer complementary to a retroviral frame-shifting signal, i.e. to an RNA sequence traversed by elongating ribosomes (Le Tinévez, Chabas and Toulmé, unpublished). Conflicting results have been reported about the effect of antisense oligonucleotides on the in vitro proliferation of Plasmodium falciparum. The first paper in the field (Rapaport et al., 1992) described an antimalarial effect of phosphorothioate antisense sequences complementary to the initiation region of translation of the DHFR-TS mRNA and of a message coding for surface antigens of the erythrocytic merozoite. But the specificity was low, and at least one mismatched sequence demonstrated an activity similar to antisense ones. Nevertheless, a chloroquine-resistant strain was as susceptible to phosphorothioate oligomers as sensitive ones. It was then confirmed by Clark et al. (1994) that phosphorothioate oligonucleotides exerted an antimalarial effect dependent on oligomer length, concentration and time of addition to the culture but independent of the sequence: anti-DHFR-TS, sense and random oligonucleotides displayed an anti-Plasmodium effect of the same amplitude. More recent results also described non-specific activity of several different phosphorothioate oligonucleotides (Ramasamy et al., 1996), in particular with oligomers complementary to the choline phosphate cytidylyl transferase mRNA (Yeo and Vial, personal communication). The sequence-independent activity of these oligomers was essentially ascribed to a polyanion effect which inhibits red blood cell invasion, probably due to the binding of the oligomers to membrane proteins, thus interfering with the interaction of merozoite to erythrocyte receptors. A similar effect was recently reported with dextran sulphate, whereas the addition of cationic liposomes abolished the effect of phosphorothioate oligonucleotides (Kanagaratnam et al., 1998). In this study the antimalarial activity was observed at 10 µM of an antisense 18-mer targeted to the MSA-2 mRNA, coding for a surface antigen. In this latter case, no effect was observed at concentration below 0.5 µM, i.e. a concentration

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at which Barker et al. (1996) reported sequence-dependent inhibition of the growth of P. falciparum using anti-DHFR-TS oligomers. In the same study these authors described sequence-independent effects at concentrations higher than 1 µM. Therefore one may surmise that only genes sensitive to low oligonucleotide concentration will respond specifically to antisense phosphorothioate oligomers. If a higher concentration (>1 µM) is required to turn off protein synthesis—for instance, if a non-accessible RNA sequence or a highly abundant message is targeted—one essentially sees a ‘chemistry-driven’ anti-malarial effect due to the well-known non-specific binding of phosphorothioate oligomers to proteins. The two effects might eventually co-exist: P. falciparum proliferation was inhibited by a phosphorothioate 18-mer antisense to the AUG initiator region of the superoxide dismutase (SOD) mRNA as well as by control sequences, but only the antisense sequence induced a reduction of SOD synthesis (Dives, personal communication). A slightly different strategy was successfully used against P. falciparum demonstrating that sequence-dependent effect can be achieved with phosphorothioate sequences. Hammerhead ribozymes bear a catalytic RNA motif flanked by two oligonucleotide stretches complementary to a pre-selected RNA. Upon binding of the ribozyme wings to the intended RNA target site the catalytic core is brought in the vicinity of the substrate, leading to a selective cleavage of the target RNA, hence the inactivation of the corresponding message. Chemically synthesized ribozymes made of an RNA core inserted between two wings containing 10 phosphorothioated RNA residues were targeted to the Plasmodium mRNA coding for the carbamoyl—phosphate synthetase (Flores et al., 1997). The Plasmodium viability was reduced by about 50% following exogenous delivery of these ribozymes to parasite cultures (at 0.5 µM). Control sequences (either ribozymes with non-complementary flanks or the antisense sequence composed of the two wings in tandem without the catalytic motif) displayed a modest effect (5–15% reduction), even though these molecules contained 20 to 28 phosphorothioate ribonucleotides. This supported a sequence-dependent anti-plasmodial activity of phosphorothioate sequences at low concentration (≤0.5 mM), and indicated that some genes which do not respond to antisense oligomers on a sub-micromolar concentration range could be efficiently targeted with ribozymes. 14.3.2 Antisense Oligonucleotides against Trypanosomatids Trypanosomatids are protozoans which comprise several species responsible for severe or even fatal diseases in human beings. Trypanosoma cruzi is the pathogen responsible for Chagas disease in South America; in Africa sleeping sickness results from infection by Trypanosoma brucei. Different species of Leishmania are responsible for cutaneous, muco-cutaneous or visceral diseases

PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 301

in a large inter-tropical area. Transmitted by insects, these parasites multiply intracellularly (Leishmania, T. cruzi) or in the bloodstream (T. brucei). The expression of genes in these organisms involves a trans-splicing step between two pre-RNA species: one contains the coding region whereas the second one brings a cap structure at the 5′ end of the so-called mini-exon sequence, which is unique for a given trypanosomatid species. This results in the presence of a 39 nt long common sequence at the 5′ end of every mature mRNA (Agabian, 1990; Ullu and Tschudi, 1991) which has been extensively used as a target for oligonucleotides (see Toulmé et al. (1997a) for a review). It was tempting to target this mRNA region, as a single complementary sequence can prevent the synthesis of every protein of the parasite. Moreover, this sequence is present neither in the human (the physiological host) nor in the mouse genomes (a model host). The potential interest of this strategy firstly demonstrated with T. brucei mRNA was then confirmed for other species (Cornelissen et al., 1986; Walder et al., 1986). Anti-mini-exon oligonucleotides were also shown to prevent the translation of Leishmania (Pascolo et al., 1993) or Crithidia mRNA (Gabriel et al., 1987) in cell free extracts. The anti-mini-exon strategy led to the in vitro inhibition of protein synthesis from Ascaris suum and Haemoncus contortus (Bektesh et al., 1988). It was then demonstrated that a trans-splicing mechanism generated a 22 nucleotide long spliced-leader on a subset of mRNA from these worms, and that such a process was also used in Fasciola hepatica (Davis et al., 1994) and Schistosoma mansoni (Rajkovic et al., 1990), two flat worms responsible for human diseases. Trans-splicing might be an important form for gene expression in early metazoa. Therefore, this makes the anti-miniexon approach potentially valuable also for controlling the development of nematodes and trematodes which comprise major human pathogenic parasites. The studies on trypanosomatids were further extended from cell-free extracts to cultured parasites. In a series of studies to investigate the splicing mechanism in trypanosomes, Tschudi and Ullu made use of antisense oligomers to delineate accessible regions in the pre-mini-exon RNA and to shed some light on the role played by U2, U4 and U6 snRNAs (Tschudi and Ullu, 1990; Ullu and Tschudi, 1993). In this work, lysolecithin permeabilized trypanosomes were used to allow oligonucleotide entry into the cell. However, no such treatment was necessary to observe antisense properties: a nonamer (Acr9mer) complementary to the T. brucei mini-exon sequence, which was shown to reduce selectively the protein synthesis in a cell-free assay, induced drastic morphological changes of cultured procyclics (the trypanosome stage corresponding to the insect form) upon addition to the growth medium. Large vacuoles appeared in the cells after 24–30 h incubation and parasites exhibited a limited mobility (Verspieren et al., 1987; Verspieren et al., 1988). Then the cells took a round shape, suggesting a loss of osmotic pressure control, and subsequently died. A high (>100 µM) oligonucleotide concentration was required to observe such effects, probably due to the presence of the mini-exon sequence on every mRNA in the cell leading to a high intracellular concentration of the target.

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A non-modified (phosphodiester) backbone was used but the anti-mini-exon Acr9mer was linked at its 3′ end to an acridine derivative which aimed at increasing the affinity of this short oligomer for the complementary RNA sequence: the intercalation of the dye in the oligonucleotide/RNA duplex was shown by physicochemical measurement to stabilize the sense/antisense complex (Toulmé et al., 1986). Moreover, this acridine residue brought two additional benefits. Firstly, it prevented the attack of the oligomer by 3′ exonucleases which constitute the major activity responsible for the degradation of the oligonucleotides (Verspieren et al., 1987). (However, the experiment was performed in a serum-free medium to reduce the amount of nucleases.) Secondly, the acridine ring promoted the uptake of the oligonucleotide in the parasite, probably through hydrophobic interactions between the dye and the cell membrane (Toulmé et al., 1997a). The presence of the dye was crucial for the killing of the parasites: the unconjugated 9-mer did not induce any change on cultured trypanosomes. Reduced affinity, shortened lifetime and limited uptake might account for this lack of effect, but the first reason is less likely as longer unmodified complementary oligomers did not show any activity either. The trypanocidal property of Acr9mer was sequence-dependent and therefore probably reflected a true antisense effect: non-complementary acridineconjugated 9-mers and a shortened antisense sequence (Acr6mer) with a low affinity for its target did not display any effect (Verspieren et al., 1987). These negative results ruled out a direct effect of the acridine residue which might have been released following the degradation of the oligonucleotide by intracellular nucleases. These experiments pointed out the requirement for chemically modified oligonucleotides. Indeed, unmodified oligomers complementary to the mini-exon RNA of Leishmania amazonensis were without any effect on either promastigotes (the free stage) or amastigotes (the intracellular form of the parasite). Phosphorothioate derivatives, in which a non-bridging oxygen of the phosphodiester bond was substituted by a sulphur atom, were shown to exhibit an increased nuclease resistance. These oligomers bind to the complementary RNA sequence, although with a reduced affinity compared to regular DNA, and elicit RNase H activity (Hélène and Toulmé, 1990; Toulmé and Tidd, 1998) (see also Chapter 1). The addition, to infected murine peritoneal macrophages in culture, of a 16mer phosphorothioate oligonucleotide (16PS) targeted against the mini-exon sequence of Leishmania amazonensis cured about 40% of infected cells after a 48 h treatment at 25 µM (Ramazeilles et al., 1994). The cured cells displayed highly fragmented and empty parasitophorous vacuoles. In some cases parasite remnants were detected. For those cells which contained more than one live leishmania, the parasitic load was considerably reduced: about 15% of treated cells contained a single parasite compared to 3% for untreated ones, whereas less than 10% of treated macrophages were infected by more than 10 parasites compared to 40% for the control of untreated ones. We were never able to cure

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more than 50% of the cells, even at high oligonucleotide doses. This might be related to the multiplicity of infection when the oligonucleotide is added, or to the properties of the host cell (the phagocytic activity, for instance), but not to the susceptibility of the parasite itself. In particular one can exclude that some leishmania were resistant to the oligonucleotide treatment. The leishmanicidal effect fulfilled the criteria expected for an antisense mechanism: (i) the parasiticidal efficiency was dose-dependent, (ii) shorter sequences which bind with a lower affinity to the target RNA exhibited a reduced effect, and (iii) non-complementary phosphorothioate 16-mers (inverted, random, scrambled, sense) did not induce such an effect (Ramazeilles et al., 1994). Not only anti-mini-exon sequences have been evaluated: oligonucleotides complementary to the intron part of the mini-exon pre-RNA were also shown to induce a lethal effect on L. amazonensis, demonstrating firstly that antisense oligonucleotides can reach the nucleus of the parasite (Ramazeilles et al., 1994), and secondly that they can interfere with trans-splicing (and/or transport of the mature mRNA). The uptake of oligonucleotides by live cell is a limiting step as discussed in a previous section. In a preliminary attempt to improve the efficacy of antiminiexon sequences, PS oligonucleotides conjugated to a palmitate chain were delivered to the cultured cells associated with low density lipoproteins (Mishra et al., 1995). Forty per cent of infected macrophages were cured at 2.5 µM oligonucleotide, i.e. at a 10-fold lower concentration than naked PS oligomers. This improved efficacy was strictly dependent on the oligonucleotide sequence and was ascribed to an increased uptake of the antisense oligonucleotide. But negative results were obtained with delivery vehicles such as poly L-lysine or polyethylenimine which proved to be efficient with other target cells (Toulmé and Bourget, unpublished results). The properties of other derivatives were tested with respect to Leishmania. In particular, 2′-O-methyl, oligoribonucleotides (OMe) were re-evaluated in order to determine the role of RNase H in the effect of anti-mini-exon oligomers. These enzymes were shown to play a key role in the antisense effect, in particular when the target sequence is located downstream of the initiation AUG codon (see Toulmé and Tidd, 1998, for a review). Both fully modified 2′-Omethyl and OMe/PO/OMe sandwich 16-mers were used. In sandwich oligomers, the central part made of phosphodiester DNA is flanked by OMe stretches, thus restricting the RNase H activity to the RNA region bound to the central PO part of the oligonucleotide (for PO windows larger than five nucleotides). Contrarily to a previous report (Toulmé et al., 1997a), OMe and sandwich 16-mers induced the selective killing of L. mexicana amastigotes in cultured macrophages (Bourget and Toulmé, unpublished results). A similar result was obtained with morpholino, phosphorodiamidate derivatives (Bourget et al., unpublished). As neither 2′-O-methyl nor morpholino analogues elicit RNase H activity, this

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demonstrated that the leishmanicidal properties of anti-mini-exon oligomers are controlled by an RNase H-independent mechanism. The use of reactive oligonucleotides is a way to circumvent the possible requirement of RNase H activity. In this case the oligomer is tethered to a chemical agent which, upon binding of the carrier oligonucleotide, induces a chemical modification of the RNA target. This strategy was used against the mini-exon sequence of trypanosomatids. An alkylating group borne by an oligonucleotide complementary to the T. brucei mini-exon led to the expected covalent cross-link (Boiziau et al., 1991). In the case of Leishmania a 12-mer carrying a psoralen derivative (Psol2mer) induced an RNA—oligonucleotide photo-adduct upon UV irradiation, which was responsible for a specific inhibition of in vitro protein synthesis in rabbit reticulocyte lysate (Pascolo et al., 1994). This experiment also demonstrated a restricted access of the target sequence, suggesting that the L. amazonensis mini-exon RNA adopted a secondary (tertiary) structure (see below). 14.3.3 Other Parasites A limited number of studies involving antisense sequences have been performed in the framework of investigations dealing with the control of the development of other parasites. These include the targeting of host genes which play a role in the infection. One example is provided by a study about the effect of the bovine casein kinase II (CKII), serine/threonine protein kinase, on the permanent proliferation of Theileria, a protozoan parasite, in bovine lymphoblastoid cells (Shayan and Ahmed, 1997). It was reported that the expression of the CKII a subunit is closely related to the presence of the parasites in the host cell. The treatment of the infected cells by buparvaquone, a theilericidal drug, leads to the inhibition of the CKII a mRNA expression. Conversely, an antisense oligonucleotide complementary to the host CKII a mRNA reduced [3H]thymidine incorporation by Theileria-infected cells by about 50%. These results were confirmed in an independent study performed with a 30-mer complementary to the translation initiation region. On the contrary, antisense oligonucleotides targeted to the CKII a of the parasite did not show a specific effect: both sense and antisense sequences inhibited the proliferation of the parasite (Chaussepied and Langsley, personal communication). In this latter study the authors used unmodified oligonucleotides. In order to reduce the degradation by nucleases, foetal calf serum—preheated at 65°C instead of 56°C —was added (at only 2% instead of 10%), allowing the development of Theileria-infected bovine lymphocytes in culture. As discussed previously, the uptake of antisense sequences is of key importance. Limited information about this topic is available in the case of parasites. The uptake and compartmentalization of phosphorothioate oligomers have been studied on the trematode Schistosoma mansoni. A minor fraction of

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the oligonucleotide accumulated in the tegumental coat of the worm (Tao et al., 1995). The uptake of 35S-labelled oligonucleotides was time-dependent, reaching a maximum after about 40 h. The oligomer was shown to be more stable in nuclei than in cytosol fractions. 14.4 RNA Structures are Valid Targets for Regulatory Oligonucleotides In an effort to optimize the antisense sequence against mini-exon RNAs, a series of phosphodiester oligonucleotides complementary to trypanosome or leishmania mini-exon sequences were evaluated both for affinity for the target and for inhibition of in vitro protein synthesis. A fair relationship between the two parameters was obtained: as expected, the longer the oligomer, the more important is the inhibitory effect. Thermal elution of filter-bound oligonucleotide —RNA complexes (Toulmé et al., 1996a) allowed one to demonstrate that the critical temperature of half elution (i.e. the affinity) was proportional to the logarithm of l+x (where l is the length and x is the GC content of the oligonucleotide). However, the two investigated sequences complementary to L. amazonensis mRNA exhibited an abnormally low affinity for their target, suggesting a restricted access of this RNA region (Verspieren et al., 1990). This was confirmed by the fact that Pso12mer, a psoralen oligonucleotide conjugate, exhibited a very weak photo cross-linking efficiency with the full length miniexon RNA compared to that observed with the 5′ half of this sequence (Pascolo et al., 1994). This can be explained by a competition between intramolecular RNA folding and intermolecular oligonucleotide-RNA complex formation. This constitutes a very general problem for antisense applications: even if RNA is a single-chain nucleic acid, more than 50% of a messenger RNA is doublestranded (Wyatt and Tinoco, 1993) due to intramolecular pairing between complementary stretches. Predicting structure for long RNA molecules is far from accurate. Therefore, targeting an ‘open’ region with an antisense sequence is essentially a hit-and-miss process. Systematic screening of a target mRNA from the 5′ to the 3′ end has been undertaken, and methods have been devised to identify non-structured RNA portions used (Milner et al., 1991: Monia et al., 1996). In the case of the Leishmania mini-exon there is almost no freedom for shifting due to the short length of the target. It was recently confirmed by RNase mapping that this RNA fragment adopted a non-perfect hairpin structure in which most of the sequence was engaged (Figure 14.2; Compagno et al., 1999). It was also demonstrated that the precursor of the miniexon RNA in trypanosomatids folds into stem-loop structures (Lecuyer and Crothers, 1993). Therefore targeting either the mature or the unspliced mini-exon RNA implies that secondary structures have to be taken into account. Designing strategies to this aim is of general interest, as the number of RNA structures playing a key role in gene expression is growing. These RNA motifs

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Figure 14.2 Sequence of the mini-exon RNA from Leishmania amazonensis (Miller et al., 1986). In addition to 7mGppp, the terminal part of the 5′ sequence contains four modified nucleosides (in brackets) designed as the ‘cap 4 structure’ (Freistadt et al., 1987). Moreover, the sixth residue is a 2′-O-methyl riboadenosine (Perry et al., 1987). The target sequence of the phosphorothioate oligonucleotide 16PS which shows leishmanicidal properties (Ramazeilles et al., 1994) extends from G(8) to G(23). The L. amazonensis mini-exon secondary structure, deduced from enzymatic and chemical footprinting studies (Compagno and Toulmé, 1999), shows that for binding to the miniexon RNA 16PS has to compete with intramolecular RNA interactions

are frequently selectively recognized by proteins which contribute to regulatory processes. Numerous functional RNA structures have been identified in viruses; the trans activating response RNA element of the human immunodeficiency virus is one of the many examples available (Gait and Karn, 1993). Ligands able to bind to such structured regulatory RNA domains would allow interference with the biological processes that they mediate. Several possibilities have been considered to this end (Figure 14.3; see Toulmé et al. (1996b) for a review): (i) designing oligomers able to invade the RNA structure, (ii) forming triple helices on double-stranded RNA regions of appropriate sequence, (iii) selecting oligonucleotides able to recognize the folded RNA structure. The design of RNA structure invaders can be achieved in two different ways. Firstly, minimizing the energy required for unfolding a large enough portion of the RNA takes advantage of structural peculiarities (bulges, internal or apical loops). The interest of this approach has been demonstrated for the TAR RNA

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Figure 14.3 Strategies for targeting RNA structures. (1) High-affinity oligomers such as SBC oligonucleotides can strand-invade RNA hairpins, i.e. shift the equilibrium towards the unfolded RNA (Compagno and Toulme, 1999). (2) Oligopyrimidines or oligopurines can form triple helical structures following binding to the double-stranded stem of an RNA hairpin. This requires an oligopyrimidine-oligopurine stem. A similar approach generates ‘double hairpin’ complexes from clamp oligonucleotides (Pascolo and Toulme, 1996; see text). (3) Aptamers can be selected from randomly synthesized DNA or RNA libraries. The structure of the complex between the aptamer and the target RNA is not known a priori and may involve non-canonical interactions

element of HIV-1 and for a portion of the H-Ras mRNA (Ecker et al., 1992; Lima et al., 1992) (see also Chapter 12). The binding of an oligonucleotide to an RNA double-strand would be favoured if we were able to design modified nucleic acid bases (let's say A′ and T ′) which would result in an increased stability of hybrid pairs compared to normal RNA pairs (i.e. A′-U, T′-A>AU) and which could not pair with each other (A-U >> A′-T′). This last criterion prevents intramolecular folding of the antisense sequence. 2-aminoadenine and 2-thio thymine (Figure 14.1) satisfy these conditions and have been shown to strand-invade a double-stranded DNA (Kutyavin et al., 1996). The process is favoured both kinetically and thermodynamically. Complementary oligonucleotides containing these modified A and T residues, called selectively binding complementary (SBC), have been shown to invade the Leishmania miniexon RNA hairpin and to bind with a 50-200-fold higher affinity than the normal base (NB)-containing oligomer with either a phosphodiester or a phosphorothioate backbone (Compagno et al., 1999). A complementary 25-mer was a much more efficient inhibitor of in vitro translation in the SBC than in the NB version: half inhibition was observed at 0. 08 µM with 25SBC whereas 25NB induced only a 25% decrease of Leishmania

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protein synthesis at 1 µM. These SBC oligomers retained the ability to elicit RNase H activity. However, SBC 15-mers displayed only a modest improvement of the leishmanicidal properties of phosphorothioate anti-mini-exon oligonucleotides, indicating that the target RNA structure did not constitute the major limitation to the antisense efficacy in the cell. Triple-stranded structures can be formed from a purine—pyrimidine doublestrand. The third strand binds to the purine strand of the double helix through socalled Hoogsteen hydrogen bonds. Clamp oligomers have been designed which first anneal with a single-stranded homopurine region giving rise to a WatsonCrick double helix and secondarily fold back on themselves to generate a local triple helix (Giovannangeli et al., 1991). The use of clamp oligonucleotides has been extended to the recognition of hairpin motifs in which the stem is a homopurinehomopyrimidine duplex, leading to a double hairpin complex (Brossalina et al., 1993; Brossalina and Toulmé, 1993). Similar complexes have been observed by targeting the Leishmania mini-exon hairpin: the antisense oligomer was designed to form 10 base-pairs with nucleotides 5–14 (Figure 14.2). A four T connector allowed the 15 nt long 3′ portion of the antisense to fold back on the duplex and on the double-stranded stem of the mini-exon hairpin, thus forming a 16 triplet triple helix. The third strand (the 3′ part of the oligomer) was derived from the ‘pyrimidine motif’, i.e. was designed to form TA*T and C-G*C+ triplets; six inverted pairs were read by either G or T to minimize the destabilization. Both electrophoretic mobility shift assays and footprinting studies demonstrated that a double hairpin complex was formed (Pascolo and Toulmé, 1996). However, this required an acidic pH and therefore could not be evaluated with respect to translation inhibition. More appropriate sequences or the use of modified bases which allow the formation of stable triple helices at physiological pH can be considered (Povsic and Dervan, 1989). Recently, combinatorial approaches have been blossoming to identify molecules of interest in randomly synthesized libraries. In the case where the library is made of nucleic acids, up to 1015 different sequences can be screened at a time in a so-called ‘SELEX’ experiment to generate ‘aptamers’ (Ellington and Conrad, 1995; Gold et al., 1995). Either RNA or DNA sequences have been characterized which recognize different target molecules (proteins, antibiotics, etc.) with both high affinity and high selectivity. Such a strategy has been used against nucleic acids, and in particular against structured DNA or RNA motifs. It has been reported that DNA oligomers obtained through an in vitro selection experiment were able to bind to a structure derived from the DNA version of the Leishmania mini-exon sequence (Mishra et al., 1996; Mishra and Toulmé, 1994). The candidates were designed according to the model described above for clamp oligomers: a fixed region complementary to the single-strand at the bottom of the hairpin anchored the oligonucleotide onto the target through Watson Crick base-pairing, whereas the 3′ part constituted the randomized region. In vitro selection led to the identification of aptamers whose 3′ sequence interacted with the target hairpin through uncharacterized bonds, contributing to the stabilization

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of the hairpinaptamer complex. Interestingly, the aptamers selected against the ‘DNA miniexon hairpin' were shown to recognize the RNA version of this structure and to prevent selectively the in vitro translation of a luciferase mRNA in which the target hairpin was inserted upstream of the initiator AUG (Le Tinévez et al., 1998). Similar results were obtained in the frame of a selection performed in a DNA library using a non-perfectly structured DNA motif as a target (Boiziau et al., 1997). More recently RNA and DNA libraries have been successfully screened to identify aptamers against a 59 nt long RNA motif (the TAR element) involved in the trans-activation of transcription of the HIV RNA (Boiziau et al., 1999; Toulmé et al., 1997b, Ducongé and Toulmé, 1999). In both cases the selection identified a class of candidates which adopted a stem-loop structure, the top of which displayed a consensus octameric sequence, complementary to the apical part of the TAR RNA element. The TAR RNA—aptamer complexes were able to give rise to six base pairs through loop-loop interactions. Such ‘kissing’ hairpin complexes were previously characterized for natural RNA and were shown to be responsible, for instance, for the control of some plasmid replication in E.coli (Tomizawa, 1990). In the case of the anti-TAR sequences the affinity of the aptamers for the RNA hairpin did not reside only in the six Watson-Crick pairs; short oligomers containing the sequence complementary to the TAR loop, unable to fold into a hairpin, exhibited a 50-fold reduced binding constant (Boiziau et al., 1999). The stem region, next to the aptamer loop, probably acted as a scaffold to pre-organize the bases complementary to the target loop in such a way that a minimal reorganization (i.e. a minimal thermodynamic cost) was required for complex formation. Interestingly, the aptamers selected in DNA and RNA libraries did not lead to the same consensus sequence: even though six base pairs can potentially be formed in both TAR RNA-DNA and TAR RNA-RNA aptamer complexes, the complementary motif is shifted by one nucleotide in the former case compared to the latter one (Boiziau et al., 1999; Ducongé and Toulmé, 1999). It has also been demonstrated that the aptamers identified are highly dependent on the ionic conditions: the consensus sequence surrounding the six base motif was different for a selection performed at high (10 mM) or low (3 mM) magnesium (Boiziau et al., 1999; Sekkai et al., unpublished results). The aptamers selected in 10 mM Mg2+ could not bind at 3 mM, demonstrating the key role played by the bases next to the interacting nucleotides to minimize electrostatic repulsions between the two partners in loop-loop complexes. Other classes of high-affinity ligands have been isolated for which the interaction pattern is not identified yet. Similar experiments have been undertaken using the mini-exon RNA of Leishmania or its precursor as a target.

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14.5 Conclusion The investigations undertaken so far with antisense oligonucleotides in the world of parasites underline the high potential of this strategy. Despite the small number of organisms studied and the limited sampling of RNA targets, antisense oligonucleotides have been shown to constitute promising antiparasite molecules. Two major groups of protozoan parasites—Plasmodium falciparum and the trypanosomatids, Trypanosoma brucei and Leishmania amazonensis— were demonstrated to be sensitive to antisense oligonucleotides. For the trypanosomatids the parasiticidal activity of oligonucleotides targeted to the mini-exon RNA sequence exhibited the characteristics of true antisense effects; in particular, non-complementary sequences did not induce a lethal consequence. For Plasmodium, even though the results are still controversial, it is clear that a significant part of the anti-malarial properties of phosphorothioate oligonucleotides are sequenceindependent and probably result from interference with the invasion process of the red blood cell by the parasite. Whatever the mechanism, for T. brucei, L. amazonensis and P. falciparum antisense oligonucleotides allowed one to control the multiplication of the pathogens. However, independently of any further problems, the cost of oligonucleotides and the geographical area in which these parasites constitute a major health problem make it unlikely that antisense oligomers will be considered as therapeutic agents of interest. Up to now the potential of antisense oligonucleotides as tools in molecular genetics of parasites has not been demonstrated. The non-specific effects of phosphorothioate analogues hamper their use to dissect gene function in Plasmodium. The use of other oligonucleotide analogues (many are available) might circumvent this problem. The target worked out in trypanosomatids—the universal mini-exon sequence—did not allow the selectivity of the antisense approach to be explored in these organisms. (The author is not aware of any attempt to turn down the expression of a single gene in trypanosomatids by antisense oligonucleotides.) Moreover, no in vivo experiment has been reported yet, in contrast to viral pathologies, for instance, for which model studies and even clinical trials have been performed. Recently, however, ‘nucleic acid therapies’ have demonstrated an interest in the field of parasitology. The role of paraflagellar rod (PFR)—a large structure contained in the flagellum—in the motility of the trypanosomes has been demonstrated with the help of an antisense construct. The PFR is made of two closely related proteins: PFR-A and PFR-C. A trypanosome transformant obtained with the PFR-A gene in the antisense orientation led to a reduced expression of PFR-A at both the mRNA and protein levels (Bastin et al., 1998). No effect was seen on PFR-C expression. The transformed cells grew normally but were paralysed. However, this effect was due to the integration of the antisense construct at one of the two PFR-A loci. No phenotype was observed when the

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antisense construct was targeted to another site, indicating that position in the genome and not antisense production was critical in this case. Double-stranded (ds) RNA was also shown to give rise to genetic interference in trypanosomes: the in vivo simultaneous expression of sense and antisense RNA led to the selective degradation of the homologous RNA sequence, thus invalidating the expression of the corresponding gene. It was demonstrated that dsRNA homologous to the 5′ untranslated region of the α-tubulin message led to morphological alteration of the parasites (Ngo et al., 1998). Either strand (sense or antisense) did not individually induce the same changes. It was demonstrated that dsRNA-mediated RNA degradation takes place in the cytoplasm, and might be associated with mRNA translation. The enzymes involved in such a process, which has also been described in nematodes and plants, are not identified and the physiological function of ds RNA is still unknown, but might constitute a mechanism for regulating gene expression in ancient eukaryotic organisms. This adds one more opportunity to use the genetic information itself to interfere selectively with the expression of a gene of interest. Acknowledgements The author wishes to thank C.Bourget, S.Chabas, M.Chaussepied, D.Compagno, D.Dives, F.Ducongé, G.Langsley, R.Le Tinévez, D.Sekkai, H.Vial and H.J.Yeo for sharing unpublished results. The research projects developed in the author's laboratory are supported by INSERM, the Conseil Régional Aquitaine, the Pôle Médicament Aquitaine, the Agence Nationale de Recherche sur la SIDA and the European Community (Biotechnology Programme). References AGABIAN, N., 1990, Trans splicing of nuclear pre-mRNAs, Cell, 61, 1157-1160. ALVING, C.R., STECK, E.A., CHAPMAN, W.L., WAITS, V.B., HENDRICKS, L.D., SWARTZ, G.M. and HANSON, W.L., 1978, Therapy of leishmaniasis: superior efficacies of liposome-encapsulated drugs, Proc. Natl Acad. Sci. USA, 75, 2959–2963. BARKER, R.H., METELEV, V., RAPAPORT, E. and ZAMECNIK, P., 1996, Inhibition of Plasmodium falciparum malaria using antisense oligodeoxynucleotides, Proc. Natl Acad. Sci. USA, 93, 514–518. BASTIN, P., SHERWIN, T. and GULL, K., 1998, Paraflagellar rod is vital for trypanosome motility [letter], Nature, 391, 548. BEKTESH, S., VAN DOREN, K. and HIRSH, D., 1988, Presence of the Caenorhabditis elegans spliced leader on different mRNAs and in different genera of nematodes, GenesDev., 2, 1227–1283. BOIZIAU, C., BOUTORINE, A.S., LOREAU, N., VERSPIEREN, P., THUONG, N.T. and TOULMÉ, J.J. , 1991, Effect of antisense oligonucleotides linked to alkylating

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316 OLIGONUCLEOTIDES AS ANTIPARASITE

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PHARMACEUTICAL ASPECTS OF OLIGONUCLEOTIDES 317

ZERIAL, A., THUONG, N.T. and HÉLÈNE, C., 1987, Selective inhibition of cytopathic effect of type A influenza viruses by oligodeoxynucleotides covalently linked to an intercalating agent, Nucl. Acids Res., 15, 9909–9919.

Index

α4β7 (LPAM-1) 265 α-anomeric oligonucleotides 51–2 αdβ2(CD11d/CD18) 265 A-raf 247 AAT motifs 37 abasic sites 56 absorbance (OD) 60 absorption (A) 227 accessible site, p53–248 18 acridine 57, 58, 294 acute myelogenous leukaemia 246 acute renal transplant rejection 274 acyloxyalkyl 82 acyloxyaryl 82 adamantoyl chloride 45 adaptor proteins 244 addressin 265 adenosine Al and A2 receptors 278 adenosine receptors 277–8 2′, 5′-adenylate-dependent RNase L 47 adhesion of leukocytes 264–8 adhesion molecules, table 265–6 ADME 227 aggregation 151 AIDS patients 38, 234–5 albumin 133 2′-O-alkyl-oligoribonucleotide/ phosphodiester 17 2′-O-alkylation 16, 41, 52 alkylation, post-synthesis 83 alkylphosphonates 48–9 2′-O-alkylribonucleotides 47, 52 all-phosphorothiaote modification 38 allergic diseases 277–8 allergic inflammatory response 279

alpha-anomeric compounds, translation blocking 12–13 alpha-anomeric oligonucleotides 51–2 alpha-globin gene 17 9-amino-6-chloro-2-methoxy-acridine 5 8 2′-aminoadenine 300 2′-O-aminoalkylribonucleoside phosphoroamidites 58 Γ-aminobutyroyloxyethyl (GABOE) 81 2′-aminonucleoside triphosphates 46 amphipathic helix peptides 118, 219–20 amyloid precursor protein (APP) 119 analysis of oligonucleotides 59–68 analytical chemistry 227–8 angiotensinogen 155, 156 animal models cytotoxic dermatitis in SCID mice 270 mouse cardiac allograft rejection 271 mouse tumor xenograft 249 rodent allografts 270–2 SCID mice cytotoxic dermatitis 270 G3139 antisense inhibition of lymphoma 245–6 tat gene of HIV-l 231 anion-exchange HPLC 63, 235 anionic lipidic vesicles 148 anionic lipids, flip-flop 153 anionic liposomes 147, 149 Antennapaedia peptide 116, 118–19 Antennapaedia protein 99, 113, 219 anti-c-myc oligonucleotides 150 anti-c-myc phosphorothioate 148 anticancer therapy 101, 150, 243–59 antigene 101–3 triplex, schema 36 318

Index

antigene strategy, comb-type copolymers 172–91 anti-H2K antibody 162 anti-HER2 Fab’ 152, 162 anti-HIV 103 antimalarial activity 291–2 antimicrobials 101 anti-mini-exon oligonucleotides 291 structure 298 anti-myb oligonucleotides 161 antiparasite compounds 286–303 anti-ras oligonucleotide 139 antisense, schema 36 antisense cancer therapy 101, 150, 243–59 approach against ras 250–8 Bcl-2 245–6 C-myb 246–7 clinical trials of oligonucleotides 244 conclusions and future prospects 259 protein kinase A 250 protein kinase C-α 249–50 raf kinase 247–8 antisense chimeric structures 50, 53–5 antisense effect 207–10, 217–18 intracellular locations 205 antisense inhibition gene expression mRNA destruction 14–21 steric blocking 12–14 PNA translation 99 antisense inhibitors 251–4 analogues, structure 288 antisense oligonucleotides (ASOs) 244, 263–80 antiparasite use 286–303 clinical studies 38 coupled to a KDEL peptide 115 defined 35 delivery in vitro 201–20 cell surface oligonucleotide-binding proteins 202–4, 216 delivery reagents 206–7 experimental considerations 207–10 intracellular compartmentalization 205–6 ras, antitumour activity 257–8 gene expression inhibition 3 mechanisms of action 3–21

319

RIα antitumour activity 250 antisense ribozymes, triplex 39–40 anti-telomerase 101 apoptosis, CpG motifs 12 apoptosis in epithelial cells 248 aptameric binding 3 aptameric effects, G-quartet oligonucleotides and analogues 7–10 aptamers 299, 301 schema 36 aPTT 273–4 AR-177 38, 226, 228, 233 A-raf 247 arylphosphonates 48–9 Ascaris suum 294 asialoglycoprotein-PLL conjugates 184–5 asthma 277–8 model 279 attenuation of angiotensinogen protein 155– 6 AUC 232 AUG initiation coding region 101, 247 AUG initiation codon 14, 99, 155, 156 AUG region of K-ras 233 automated synthesis on solid support 86 autoradiography 64, 231–2 β1 VLA4 integrins 264 β2 integrins LFA-1 264, 266–7 B lymphocytes 245 murine 278–9 B-cell activation 10 CpG motifs 11 B-cell lymphoma 245 B-cells, murine 278–9 β-globin gene 13 β-globinmRNA 17 B-raf 247 β-thalassaemia 13 Bcl-2 155 antisense approaches for cancer 226, 244–6 Bcl-X 244 bcr-abl antisense oligomers 18, 20 leukaemia cell inhibition 6 bcr-abl mRNA 18, 150 Beaucage's reagent 48, 52, 87, 89

320 INDEX

Beer’s law 60 beta-cyanoethyl group 44 beta-globin gene, splice sites 13 beta-globin mRNA 17 beta-thalassemia 13 bicyclo-[3.2.1]-DNA 53 biotin 57 2, 3-bis(oleoyl)propyl trimethyl ammonium chloride (DOTMA) 231 bladder carcinoma 139 boranophosphate, structure 48 bovine casein kinase II (CKII) 297 bradykinin B2 receptors 278 breast carcinoma 6, 152, 162, 249 brochoalveolar fluid, adenosine receptors 277–8 Brome mosaic virus mRNA 291 buparvaquone 297 C+GC motifs 37 C5 propynyl pyrimidines 55–6, 157 C5 trifluoroacetyl-protected aminoalkenyl pyrimidine 58 C7-aminoalkynyl-7-deazapurine neucloside phosphoramidites 58 c-myb antisense approaches for cancer 246–7 smooth muscle cell hyperplasia inhibition 7, 8 c-myc 8, 13, 18, 19, 20 antisense oligodeoxynucleotide 16 breast and lung cancer cell line inhibition 6 mRNA 231 smooth muscle cell hyperplasia inhibition 7 C-raf 247 C-raf kinase 226, 233, 247 C-raf mRNA 247–8 calcein molécules 159, 160 cancer 128, 140, 148 anticancer therapy 101, 150, 243–59 cancer cells, telomerase 101 capillary gel electrophoresis (CGE) 64–5, 228 carboxyesterase activation 80, 85 carcinoembryonic antigen (CEA) 250, 257

cardiac allografts 271 CaSki cells 151, 155 cationic amphiphilic molecule 135 cationic hydrophobic peptides 129 cationic lipids 43, 112, 136, 152, 206–7, 214, 218, 275 delivery mechanisms 218–19 cationic liposomes 147, 149, 150–7 intracellular delivery and distribution 151–3 PEG modified 152, 162 pharmacological efficacy, in vitro/in vivo 139, 154–7 cationic molecules, hydrophobic peptides 129 cationic peptides 112 cationic polyaminoacids 112 cationic polystyrene nanoparticles 132, 136 cationic porphyrin delivery vehicles 59 CD4, phosphorothioate oligodeoxynucleotide analogue binding 7 CD4 monoclonal-antibody-targeted liposomes 160 CD molecules 265–6 cell, schema 36 cell adhesion molecules, table 265–6 cell proliferation arrest, deoxynucleoside release 4–6 cell surface heparin-binding proteins 202–4 cell surface oligonucleotide-binding proteins 202–4, 216 cellular capture of oligonucleotides 153 cellular pharmacokinetics 229-30 cellular uptake 54, 58, 103, 113, 128, 213– 15 cetyltrimethylammonium bromide (CTAB) 135, 137, 139 CG motif 41 CGP 64128A 139, 156, 226, 233, 249–50 CGP 69846A 226, 233, 247 CHEMS (cholesterol hemisuccinate) 157, 160, 164 chimeric formacetal-phosphodiester oligonucleotides 50 chimeric oligomers 41, 53–5 CHO cell line 155

INDEX 321

cholesterol(3-imidazol-lyl propyl) carbamate 157 cholesterol 57, 136, 138, 147, 148, 159, 160, 161, 163, 209 cholesterol hemisuccinate (CHEMS) 157, 160, 164 cholesterol-coupled oligonucleotides 58, 161, 214, 229 3′-cholesteryl oligonucleotides 58 5′-cholesteryl oligonucleotides 58 choline phosphate cytidyl transferase mRNA 292 chromatography 59, 63, 132 chronic myelogenous leukaemia 246 cell line 150, 246 chronic myeloid leukaemia 18, 20 circular dichroism 175–7 G-quartet identification 7 clinical pharmacokinetics 234–6 clinical trials lymphoma 24 non-Hodgkin’s lymphoma 38, 245–6 oligonucleotides, antisense cancer therapy 244 CMV retinitis, cytomegalovirus 234 colitis 272–3 colon carcinoma 249 colon carcinoma cell line 251 comb-type copolymer 172–91 as cell specific DNA carrier 184–91 as stabilizer for DNA duplex and triplex 172–84 combinatorial chemistry 301 competitive enzyme hybridization assay 228–9, 232 conjugates, types 57–9, 229 conjugation cationic lipids or peptides 112 with cholesterol 58, 161, 214, 229 fusogenic peptides 117–22 internucleoside linkages 57–8 lipophilic molecules 58 oligonucleotides 56–9, 229 positively charged polypeptides 58 copolymers, comb-type copolymers 172– 91 copolymers of poly(l-lysine), with polysaccharide 174

CpG motifs 11, 210, 278, 289 apoptosis 12 immune stimulation 3, 10–11 tyrosine kinase inhibition 11–12 Crithidia 294 Crohn’s disease 38, 272, 274 CTAB (cethyltrimethylammonium bromide) 135. 137. 139 2-cyanoethyl phosphoramidite chemistry 52 cyclosporin A 271 cytokines see interleukins and other named cytokines cytomegalovirus (CMV) 38 CMV retinitis 234 cytoplasm 43, 113, 129, 148 schema 36 cytosine phosphate guanine see CpG cytotoxic dermatitis, SCID mice 270 DDAB (dimethyldioctadecyl ammonium bromide) 151, 152, 155 DEAE-dextran 136, 137 7-deazaguanine, GGGG-motifs 55, 56 degradation, RNA 12 delivery peptides 111–27, 217, 219–20 dendritic cells 279 2′-deoxy-2′-fluoro oligonucleotide 52 (3′S, 5′R)-2′-deoxy-3′, 5′-ethano-beta-Dribofuranosyladenine 53 deoxynucleoside release, cell proliferation arrest 4–6 2′-deoxynucleoside triphosphates 45–6 deoxynucleosides 41–2 monodeoxynucleotide hydrolysis 5 dephospho oligonucleotide analogues 50–1 dextran sulphate 272–3, 292 1, 2-diacyl-3-trimethylammonium propane (DOTAP) 151, 152, 158, 159 dialkylsilyl internucleoside linkages 50 diapedesis 264 dicyanoimidazoles 44 diethylaminoethyl dextran, lipofectin(r) 229 dihydrofolate reductase-thymidylate synthase (DHFR-TS) 291 diisopropylsiloxane likages 50

322 INDEX

dilauroylphosphatidylcholine (DLPC) 151 dimer stability, biological media 81–3 4′, 4′-dimethoxytrityl (Dmt) group 45 dimethyldioctadecyl ammonium bromure (DDAB) 151, 152, 155 dimyristoylphosphatidylglycerol (DMPG) 147 dinitrophenyl 57 1, 2-dioleoyl-3-succinylglycerol (1, 2DOSG) 159 2, 3-dioleoyl)propyl trimethyl ammonium chloride (DOTMA) 231 dioleyl phosphatidyl ethanolamine (DOPE) 151, 153, 154, 155, 156, 157, 158, 159, 160, 162, 164 2, 3-dioleyloxy-N-(sperminecarboxamino) ethyl-N, N-dimethyl-1-propanaminium trifluoroacetate (DOSPA) 152 1, 2-dipalmitoyl-3-succinylglycerol (1, 2DSPG) 159, 160 dipalmitoyl-Dl-alpha-phosphatidyl-Lserine (DPPS) 163 dipalmitoylphosphatidylcholine (DPPC) 147 dipalmitoylsuccinylglycerol (DPSG) 162 DLPC (dilauroylphosphatidylcholine) 151 DMPG (dimyristoylphosphatidylglycerol) 147 DNA bacterial, methylation 11 chemical structure 98 melting curves 61–2 schema 36 DNA carriers, antigène strategy, comb-type copolymers 172–91 DNA mimics see peptide nucleic acids (PNA) DNA mini-exon hairpin 299–300 DNA receptor protein, 30 kDa 202 DNA-PNA chimeras 53–5 5′-DNA-PNA chimeras 41, 54 DOGS (polyamines) 136, 151, 173–84, 206 DOPE (dioleyl phosphatidyl ethanolamine) 151, 153, 155, 156, 157, 158, 159, 160, 162, 164 DOSPA (2, 3-dioleyloxy-N(sperminecarboxamino)ethyl-N, N-

dimethyl-1-propanaminium trifluoroacetate) 152 DOTAP (1, 2-diacyl-3trimethylammonium propane) 151, 152, 158, 159 DOTMA (2, 3-bis(oleoyl)propyl trimethyl ammonium chloride) 231 double-stranded (ds) RNA, PNA-dsDNA schematic drawing 102 DPPC (dipalmitoylphosphatidylcholine) 147 DPPS (dipalmitoyl-Dl-alpha-phosphatidylL-serine) 163 DPSG (dipalmitoylsuccinylglycerol) 159, 160, 162 drug carriers, polymeric nanoparticles 128– 45 DU 145 prostate cancer cells 206 E-selectin antisense oligonucleotide 265, 275–6 egg phosphatidylcholine 147 ELAM-1 (E-selectin) 265 electropermeabilization 128 electrophoresis 59, 64 CGE 64–5 PAGE, slab gels 64 electrophoretic mobility shift assays 64–8 electroporation 14, 112, 214 intracytoplasmic delivery 4 electrospray ionization (ESI) 65 electrospray mass spectrometry (ESI-MS) 65–6, 228, 234 elimination or excretion (E) 227 emulsion polymerization 131, 133 encapsulation 137, 149–1 3′, 5′-end capped modification, plus protection at internal pyrimidines 38 5′-end conjugates 57 3′-end conjugates 57 3′-end-capped modification 38 3′, 5′-end-capped modification 38 end-capping 38, 41 endocytosis 4, 42, 111, 112, 113, 115, 116, 128, 129, 146, 148, 152, 157, 161, 163, 207, 213 endonuclease degradation 5, 15, 39, 41

INDEX 323

endosomal membrane penetration 205, 218 endosomes 4, 43, 112, 152, 153, 218 endothelial-leukocyte adhesion molecules 265–6, 275–6 env mRNA complementary oligonucleotide 158 env sequence, SFFV virus 234 enzymatic synthesis of oligonucleotides 45 enzymolabile groups 80–3 dimers, stability 81–3 hydrolysis, mechanism 81 structure 80–1 epidermal growth factor receptors (EGFR) 162, 164 A431 cell inhibition 6 ERK MAP kinase stimulation 248, 255 erythrocytic merozoite 291–2 erythroleukaemia cells, K562 206, 246 Escherichia coli 16–17, 100, 101 23S RNA 100 ESI-MS, mass spectrometry, oligonucleotide analysis 65–6 ethylcellulose 134 ethylcellulose nanospheres 134 europium, single-stranded RNA 59 exocytosis 4 5′-exonucleases 42 3′-exonucleases 15, 48, 57, 140 degradation 5, 39, 41, 42, 53 extinction coefficient 60 farnesyltransferases 243 Fasciola hepatica 294 FGF signal sequence 220 fibrinogen 203 fibronectin-antisense phosphorothioate oligonucleotides 153 analogue binding 7 flip-flop of anionic lipids 153 fluid phase endocytosis see endocytosis fluid phase pinocytosis 4 fluorescein 57, 92, 120, 153 fluorescence correlation spectroscopy 214 fluorescence labelling 46, 92–3, 163–4 fluorescence microscopy 43, 93, 122, 140, 153, 231–2

fluorescence resonance energy transfer (FRET) 153 fluorescently-labelled nanoparticles 138 fluorescently-labelled oligomers 4, 59, 148, 205–6. 209. 213–14. 250 2′-fluoro oligonucleotides 52 2′-fluoronucleoside triphosphates 46 folate 161–2, 164, 291 folate receptor 162 footprinting studies 37, 50, 301 formacetal linkages 50 formacetal-phosphodiester oligonucleotides 50 fos Friend retrovirus 158, 234, 290 fusion 117, 151 fusion protein 163 fusogenic liposomes 163–4 fusogenic peptides 117–22, 214 fusogenic proteoliposomes 163–4 G-3139 155, 226, 244–6 antisense oligonucleotides, antilymphoma clinical trials 38, 244–6 G-quartet oligonucleotides 203, 210 aptameric effects 7–10 defined 7 formation, relA antisense effector 9 intermolecular 8 G-SATE (glucuronoyl-S-acylthioethyl) 81 G-tetrad see G-quartet GABOE (Γ-aminobutyroyloxyethyl) 81 GALA 219–20 galanin receptor 119 antisense downregulation 99 gap-mers 38, 41 GAPDH mRNA 19 GEM-92 38 GEM-132 38 GEM-231 244 GEM(r)-91 226, 232, 234–5 GGC motifs 37 glioblastoma 249, 258 gliomas 230 glucuronic acid with glucuronoyloxyethyl (GOE) 81 glucuronoyl-S-acylthioethyl (G-SATE) 81

324 INDEX

glyceraldehyde 3-phosphate dehydrogenase 19 P-glycoprotein 150 glycoproteins, variant surface 290 glycosylated PLL 173 GM-1 160 GM-CSF 275, 279 GOE (glucuronic acid with glucuronoyloxyethyl) 81 Gps0193 38 GS-522 226, 232 GTG2TG3TG3TG3T, HIV1 integrase inhibition 9 GTP regulatory domains 251 GTP-binding proteins 244 H9 cells 161 H mechanism. non-RNase 13 H-phosphonate method 44-5, 48, 57 H-ras (ISIS 2503) 226, 300 Ha-ras 138, 226, 251, 258 oncogenes 139 haemagglutinating virus of Japan (HVJ) 163–4 Haemonchus contortus 276, 294 hairpin motifs 299–300 hairpin ribozymes 35 hammerhead consensus sequence, conserved nucleotides 36 hammerhead ribozymes 35, 56, 293 Hep-G2 cells 139, 155, 204 heparin-binding integrin 203 heparin-binding protein 202–4 phosphorothioate oligodeoxynucleotide analogue binding 7 hepatitis B virus 154–5 herpes virus 220 heterocyclic bases, modification 55–6 hexagonal structure 153, 159 hexitol nucleic acids (HNA) 53 high-performance liquid chromatography (HPLC) 84, 91, 228, 235 anion-exchange 63, 235 oligonucleotide analysis 62–3 reversed-phase 62–3, 85, 89

HIV-1 envelope glycoprotein (gp120) 9, 204 mutation 10 phosphorothioate oligodeoxynucleotide analogue binding 7 HIV-1 gp41 fusogenic peptide 118 HIV-1 infection anti-HIV drugs 38, 103, 226 pharmacokinetics 234–5 HIV-1 integrase inhibition, GTG2TG3TG3TG3T 9 HIV-1 replication inhibition 8, 160–1 S-T2G4T2 octamer 9 HIV-1 tat gene 231 HIV-1 Tat protein 113, 116, 117 HIV-1 transmembrane gp41 glycoprotein 114 HIV-1 vpr gene 8 HL60 cells 206 HLA class I antigen, immunoliposomes directed 160 homopurine-homopyrimidine duplex 180, 300 Hoogsteen base pairing 42, 101, 102, 178– 9, 210, 300 structure 37 HPLC see high-performance liquid chromatography HPRT (hypoxanthine-guanine phosphororibosyl transferase) 291 human c-raf-1 kinase 226, 233, 247 human IgE, dissociation constants 10 human IgG, dissociation constants 10 human papillomavirus 119, 155 human xenografts 269–70 HVJ-liposomes 163–4 hyaluronic acid 187–91 hybrid arrest of translation, defined 12 hydrogen bonds, Hoogsteen base pairing 300 hydrolysis, mechanism 80–1 hydrophobic peptides 129 3′-hydroxylacceptor 46 hypochromicity 61 hypoxanthine-guanine phosphororibosyl transferase (HPRT) 291

INDEX 325

ICAM-1 (CD54) antisense oligodesoxyribonucleotide 156 antisense oligonucleotide 154, 264–75 ICAM-2 (CD 102) 265 ICAM-3 (ICAM-R, CD50) 265 IgE 10 antibody production 279 IGF-1 receptor 217 IgG 10 IL-1 277 IL-2 277 IL-4 279 IL-5 279 IL-6 277, 279 IL-8 277 IL-12 279 IL-13 279 imidazole-4-carboxamide 55, 56 immune response 278–9 immune stimulation 210 CpG motifs 3, 10–11 immunoglobulin secretion 10 immunoliposomes 146–8, 160–4 directed to HLA class I antigen 160 inflammatory bowel disease 38 inflammatory processes 263–80 modification with antisense oligonucleotides 263–80 influenza virus 154, 155 influenza virus haemagglutinin 113, 219 influenza virus haemagglutinin fusogenic peptide 117–18 initiation codon 12–14, 99, 155, 156, 296 initiator AUG 14, 99, 155, 156, 296 integrins 264–7 β1 VLA4 264 β2 LFA-1 264, 266–7 β2 sub-family 216–17 phosphorothioate oligodeoxynucleotide analogue binding 7 intercellular adhesion molecule-1 (ICAM-1) 232 interferon treatment 47 interferon-gamma 140, 279 interleukins IL-1 receptor 276–7

regulation by NF-kappaB 277 internucleotide linkage 79 boronated 50 conjugation 57–8 intracellular compartmentalization 113 intracellular delivery and distribution 42– 3, 58 138, 151–3 pharmacological efficacy 154–7 intracytoplasmic delivery 4, 6 intramolecular folding 298 intrathecal administration 119 intratumoral administration 139 intravitreal injection 232 introns 244 3′, 3′, 5′, 5′-inversions 46–7 ion spray 66 ISIS-1570 154 ISIS-1939 267–8 ISIS-2105 229 ISIS-2302 38, 235, 267–70, 273–4 ISIS-2503 38, 226, 244, 256, 300 ISIS-2922 28, 234 ISIS-3082 27, 232, 270–1 ISIS-3466 231 ISIS-3521 38, 139, 156, 226, 233, 244, 249–50 ISIS-4189 249 ISIS-4730 275 ISIS-5132 38, 226, 233, 244, 247–8 ISIS-5320 38 ISIS-9045 232 ISIS-9046 232 ISIS-9047 232 ISIS-9125 154, 270–1 isothermal titration calorimetry (ITC) 179– 80 JNK MAP kinase stimulation by TNF-α 248 K562 erythroleukaemia cells 206, 246 K-ras-dependent human pancreatic tumor 233 KDEL peptide, antisense oligonucleotide (ASOs) 115 keratinocytes 5, 111 Ki-ras 250, 258

326 INDEX

kidney transplant model 272 kidney transplant treatment 38 kissing hairpin complexes 299–300 L-olingomers 117 laryngeal carcinoma 248 laser-induced fluorascence (LIF) 65 lectin 117, 165 Leishmania 293–300 L. amazonensis 288, 295 L. major 289 L. mexicana 287, 295 leukaemia acute myelogenous leukaemia 246 chronic myelogenous leukaemia 246 chronic myeloid leukaemia 18, 20 leukemia cell inhibition, bcr-abl antisenseoligomers 6 leukaemia cell lines 6, 12–13, 16–20, 148– 50, 150, 161, 246 leukocytes 264, 267 adhesion 264–8 LFA-1 integrins 264, 266–7 ligation 46 lipid bilayer, interactions of oligonucleotides 215–16 lipids, cationic 43, 112, 136, 152, 206–7, 214, 218, 275 lipofectamine 13 lipofectin® 154, 229 lipofectin™ 43, 139, 155, 156, 207 lipofection, cationic liposomes 4, 14 liposomes 42, 112, 128, 129, 290 anionic 147–50 cationic 150–7 containing N-stearoylcysteamine 160 delivery of ONs 146–65 fusogenic 163–4 immunoliposomes 146–8, 160–4 intracellular delivery and distribution 151–3 ON delivery 146–65 PEG-coated modified 152, 162, 214 pH-sensitive 146, 149, 157–60 pharmacological efficacy, in vitro/vivo 154–7 poly(glycidol)-modified 159

proteoliposomes 163–4 liver sinusoidal endothelial cells 187 locked nucleic acid (LNA) 53 loop structures D-loop 39 P-loop 37 loop-loop complexes 301 low density lipoproteins 296 LPAM-1 266 lutetium, single-stranded DNA 59 lymphoma B-cell lymphoma 245 clinical trials 24 non-Hodgkin’s lymphoma 38, 245–6 lysine, poly(L-lysine) 113, 116–17, 120, 164, 173–91, 214, 230 lysine pentapeptide, extended by nuclear localization signal 117 lysolecithin 294 lysosomes 43, 138 degradation 129, 157, 163 MAC-1 (CD11b/CD18) 203–4, 216–17, 266 macrophages 159, 279 MadCAM-1 265 magnesium ions 35 malaria 291 MALDI (matrix-assisted laser desorption ionization) 65, 66–7, 228 MALDI-TOF 89 MAP kinase, inhibition by antisense ONs 255 MAP kinase phosphorylation cascade 248 mass spectrometry 59, 90 oligonucleotide analysis 65–7 oligonucleotide degradation 228–9 matrix-assisted laser desorption ionization (MALDI) 65, 66–7, 228 -TOF 89 MCF-7/ADR cells 150 MDA-MB231 tumors 249 mdr (multidrug résistance) 150 melanoma 119 metastasis 269–70 melting temperature (Tm) 16, 48, 49, 50, 51, 54, 60, 61, 116

INDEX 327

membrane-active peptides 219–20 18-mer phosphorothioate (G-3139) 155, 226, 245–6 metabolism (M) 227 metastasis 140 human melanoma 269–70 2′-O-methoxyethoxy 52 2′-O-methyl nucleoside triphosphates 46 2′-O-methyl oligoribonucleotides (OMe) 287, 296 morpholino phosphorodiamidates 287 splicing inhibition 13 2′-O-methyl RNA 16, 41, 52, 208, 234 methylene 48 methylphosphonamidites 49 methylphosphonate 15, 16, 48–9, 83, 147 compounds 215 HPLC 62 nuclease resistance 41 oligodeoxynucleotide protection 5 oligodeoxynucleotides 150, 214–15 oligonucleotides 230 phosphodiester linkage replacement 5 methylphosphonodiester/phosphodiester chimeric antisense oligodeoxynucleotides 50, 53–5 microinjection 43, 112, 128, 214 microparticles 128–41 polylactide-co-glycolide (PLGA) 134, 140 microscopy, fluorescence microscopy 43, 93, 122, 140, 153, 231–2 minimal volume entrapment (MVE) 148, 150 ‘mismatched’ oligonucleotides 288 modification on heterocyclic bases 55–6 2′-modified oligoribonucleotides 55–6 molecular beacon 205 monensin 205–6 monoclonal antibodies Tat basic cluster 120 to ICAM-1 154 to Mac (-1) 203–4 morpholino compounds 215–16, 288 translation blocking 12–13 morpholino oligonucleotide 296 exon skipping 13–14 mouse cardiac allograft rejection 271

mouse tumor xenograft 249 mRNA, schema 36 mRNA degradation 43 5′-untranslated initiation codon region 12 multidrug resistance (mdr) 150 multiple emulsion (water-oil-water) solvent evaporation method 131, 133 murine B lymphocytes 278–9 MVE (minimal volume entrapment) 148, 150 myc probe 13 see also c-myc myeloma cells 155 N4, N4-ethano-5-methyl-cytosine 56 N4-(3-acetamidopropyl)-cytosine 56 N-(alpha-trimethylammonioacetyl) didodecyl-D-glutamate chloride (TMAG) 151 N-ras 250 N-stearoylcysteamine 160 nanocapsules 129, 132, 134 nanoparticles 42, 112, 128–41, 158 biodegradability 130 cationic polystyrene 132, 136 as drug carriers 128–41 fluorescently-labelled 138 in vivo studies 139 ON adsorption, in vitro stability 137 ON association 134–5 ON delivery, rationale 129–30 ON loaded cell interactions 138 in vitro pharmacological activity 138– 9 poly(alkylcyanoacrylates) (PACA) 229 poly(dialkylmethilidene malonate) 132 polylactic acid–PEG 136 preformed polymers 133–4 preparation, monomer polymerization 130–3 Stealth 130 nanospheres 129, 133, 134 neo gene 158 neointima formation 164 neurotensin-receptor 99

328 INDEX

NF-kappaB 277 tumor cell adhesion inhibition 8 NIH 3T3 cells 158 nitric oxide synthetase (NOS) 278 5-nitroindole 56 3-nitropyrrole 56 NK cells 203, 279 NMR, oligonucleotides 59, 67–8 non-antisense mechanisms 278–9 non-Hodgkin’s lymphoma 38, 245–6 G-3139 clinical trials 38, 245–6 non-RNase H mechanism 13 non-small cell lung carcinoma 6 NFS, protecting group on nucleobases 86, 87–9 nuclear accumulation of oligonucleotides 4 nuclear lamina nuclear localization signal (NLS) 120, 148 nuclear magnetic resonance 59, 67–8 nuclear pores 113 nuclease-resistant ON analogues 15 nucleases, degradation 111, 146 nucleic acid binding receptor-1 (NABR1) 202, 204 nucleic acids delivery, peptides 112–14 uptake, delivery vehicles 111–12 nucleobases conjugation 57–8 protecting groups 86, 87–9 nucleolytic degradation, deoxyribonucleoside release 3 nucleoside-2′-phosphoramidite building blocks 47 nucleoside-3′-beta-cy anoethyl N, Ndiisopropylphosphoramidite 44 nucleoside-3′-H-phosphonate 45 nucleoside-3′-phenylphosphonamidites 49 nucleoside-5′-O’phosphoramidites 47 nucleoside-5′-O’succinyl-supports 47 nucleus 43, 153 schema 36 ODNs see oligodeoxynucleotides 4-(2, 3-bis-oleoyloxy-propyl)-lmethyl-1H-imidazole 157

2, 3-bisoleoylpropyl trimethyl ammonium chloride (DOTMA) 154, 156, 157 2′, 5′-oligoadenylates 46, 47 oligodeoxynucleotides (ODNs) 38–9, 175, 247 analogues, nuclease-resistant 15 containing CPG motifs 10–11, 210, 278, 289 immune stimulation 3 ODN-698A 244 pharmacokinetics 226–36 phosphorothioates 232–3, 246, 271 oligolysine 117 oligonucleotide(s) 35–68 2′, 5′-linkages 46–7 2′-O-methyl-oligoribonucleotides 287 3′, 3′-/5′, 5′-end inversions 46–7 α-anomeric 51–2 analysis see oligonucleotide analysis as antiparasite compounds 286–303 antisense, mechanisms of action 3–21 carriers 208 conjugates 229 3′-end 57 5′-end 57 types 58–9 delivery by liposomes 146–65 dephospho linkages 50–1 design 39 in vivo 21 mismatched 288 modifications 43 2′-modified 52 alpha-anomeric 51–2 conjugates 56–9 degree 40–2 heterocyclic bases 55–6 universal base and abasic sites 56 internucleoside linkages, nucleobases and ribose 57–8 peptide nucleic acids 52–5 phosphorus 47–50 alkylphosphonates and arylphosphonates 48–9 phosphorothioates 47–8 sterically locked nucleic acid analogues 53 sugar modified 53

INDEX 329

sugar moiety 51–3 synthesis and properties 42–59 nanoparticle association 134–9 2′, 5′-oligoribonucleotides, synthesis 47 ON-cholesterol conjugates 229 ON-peptide conjugates 111–27, 220 peptide-mediated delivery 111–27 prodrugs 79–97 receptors and transporter proteins 216– 17 synthetic 111–12 unmodified 3′, 5′-phosphodiester linkages 42–6 chemical synthesis 44–5 enzymatic synthesis and ligation 45–6 see also antisense oligonucleotide analysis 59–68 electrophoretic techniques 64–5 CGE 64–5 PAGE on slab gels 64 HPLC analysis 62–3 anion-exchange 63, 235 reversed-phase 62 mass spectrometry 65–7 ESI-MS 65–6 MALDI 66–7 NMR 67–8 UV spectroscopy 60–2 DNA melting curves 61–2 quantification 60 see also oligonucleotide(s) oligonucleotide-DEAE-nanoparticle complexes 137 oligonucleotide-binding proteins, cell surface 202–4, 216 oligonucleotide-loaded nanoparticles 138– 9 2′, 5′-oligoribonucleotides, synthesis 47 oncogenes 5, 128, 139 open loops 18–19 opsonization 159, 165 ovarian cancer 156, 250 p53 antisense oligodeoxynucleotides 18 p53 mRNA 17, 20 p53–248, accessible site 18 p64 Myc protein 13, 14

p65 antisense oligodeoxynucleotide 277 P150/95 266 p210 Bcr-Abl protein 150 32P end-labelled oligomers 209 33P-pdT 16 139 PACA (polyalkylcyanoacrylate) 131, 134– 5, 229 PAGE 139 slab gels 64 2, 3-bis-palmitoyl-propyl-pyridin-4-ylamine 157 palmitoylhomocysteine (PHC) 157, 162 4-(2, 3-bis-palmitoyloxy-propyl)-1 methyl-1H-imidazole 157 pamamycin 57 pancreatic carcinomas 248 pancreatic tumor, K-ras-dependent 233 parasitic diseases 286–303 parasitophorous vacuole 291 particulate carriers 158 particulars delivery systems 112, 128 PCR, RLPCR (reverse ligation-mediated polymerase chain reaction) 15, 21 PE (phosphatidyl-ethanolamine) 157 PEC AM-1 266, 275 PEG see polyethyleneglycol PEG-modified cationic liposomes 152, 162 PEG-PE (polyethyleneglycolphosphatidylethanolamine) 151 Penetratin 118 peptide bond, PHONAs 55 peptide coupling, strategies 114–16 peptide nucleic acids (PNA) 39, 41, 47, 53– 5, 98–104, 215 chemical structure 54, 98 delivery 112–14 drug development 99 future 103–4 homopyrimidine 37 HPLC 62 oligomers 13 pharmacology 103 PNA-DNA chimeras 54 peptide-oligonucleotide conjugates 111– 27, 220 peptide-mediated delivery of oligonucleotides 111–27 peptides 217, 219–20

330 INDEX

synthesis 115 peptidyl transferase centre 100 pH-sensitive liposomes 146, 149, 157–60 pharmacodynamics 79 pharmacokinetics 226–36 ADME 227 cellular 229–30 clinical 79, 234–6 oligodeoxynucleotides 226–36 oligonucleotides 275 preclinical 230–4 and tissue distribution of liposomes 154 pharmacological efficacy in vivo 154–7 Phase I and II trials 38 PHC (palmitoylhomocysteine) 157, 162 phenoxazine 55 phenoxazine-substituted oligonucleotides 56 phenyl-S-acylthioethyls (Me-, tBu- or PheSATE) 80–1 PHONAs, peptide bond 55 phosphatidyl-ethanolamine (PE) 157 phosphatidylserine 148, 163 3′, 5′-phosphodiester linkages, unmodified oligonucleotides 42–6 phosphodiester oligodeoxynucleotides 228–9 phosphodiester oligonucleotides 214–15, 232, 234, 300 natural 86 3′-phosphodiesterase activity 5 phosphodiesterases 4, 83, 116 degradation 5 phospholipid flip-flop 153, 215 phosphoramidate, structure 48 phosphoramidite building blocks 44, 48, 87 method 44, 45 phosphorodiamidate derivatives 296 phosphorothioates 6, 47–8, 53 2′-O-methyl-oligoribonucleotides 287 18-mer (G-3139) 155, 226, 244–6 all-phosphorothiaote modification ‘gap-mer’ modification 38 antiproliferative effects 6 backbone 8, 208, 300 oligodeoxynucleotides 232-3, 246, 271 analogues 228, 231, 288

extracellular aptameric effects 6-7 oligonucleotides 4, 14, 208, 215-16, 228, 230 phosphorodithioate 15 ribonucleotides 293 side effects 40 structure 48 phosphorous environment modification 47-50 second generation pro-oligonucleotides 86 photolabile linker, solid support 86 PIBCA (polyisobutylcyanoacrylate) 135, 139 PIHCA (polyisohexylcyanoacrylate) 135, 136, 137, 138, 139 pinocytosis 158, 203 pivaloyl 45 pivaloyloxymethyl (POM) 79–80, 82, 83, 85, 91 PKC-alpha see protein kinase C plasma-time curve (AUC) 232 Plasmodium falciparum 289, 291–3 Plasmodium-infected erythrocytes 291 PLGA (polylactide-co-glycolide) microparticles 134, 140 PLL see poly (L-lysine) PNA see peptide nucleic acids polyacrylamide gel electrophoresis (PAGE), slab gels 64 polyacrylamide gels 139 polyalkyl chains 58 poly(alkylcyanoacrylates) (PACA) 131, 134, 135 nanoparticles 229 polyamines (DOGS) 136, 151, 173–84, 206 polyaminoacids 112 polyanionic molecules 217 polyanions 148 polyarginine 117 poly(beta-hydroxybutyrate) 133 polycations, comb-type 172–91 poly(dialkylmethylidene malonate), nanoparticles 132 poly(ethylene oxide) 185 polyethyleneglycol (PEG) 146, 150, 151, 159, 160, 162 -coated cationic liposomes 152, 162

INDEX 331

derivatives 130 polyethyleneglycol (PEG)– phosphatidylethanolamine (PEG-PE)) 151 polyethyleneimine 112, 296 poly(glycidol)-modified liposomes 159 polyisobutylcyanoacrylate (PIBCA) 135, 139 polyisohexylcyanoacrylate (PIHCA) 135, 136, 137, 138, 139 poly(L-arginine) 173 poly(L-lysine) 113, 116–17, 120, 173, 173– 91, 214, 230 PLL-g-Dex 174 polylactic acid 133, 134 -PEG nanoparticles 136 polylactide 134 polylactide-co-glycolide (PEGA) microparticles 134, 140 polylysine 164 see also poly(L-lysine) 3′-and 5′-polylysine-modified oligonucleotides 117, 174 polymeric drug carriers 128–45 polymeric nanoparticles 128–45 polymers biodegradable 140 preformed 133–4 polymethylmethacrylate 145 polyornithine 117 polysaccharide side chains comb-type DNA carrier 184–91 PLL-G-Dex 174 polystyrene 132, 134 polystyrene nanoparticles 132, 136 POM (pivaloyloxymethyl) 79–80, 82, 83, 85, 91 porphyrin delivery vehicles 59 positron emission tomography (PET) 227, 234 pre-mRNA 294 steric blockade 3, 13 preclinical pharmacokinetics 230–4 prion protein 10 prodrug approach 79–97 professional antigen presenting cells pro-oligonucleotides 79–97 alkylation synthesis 84

limitations 85 cell uptake 92 first models 83–6 stability in biological media 83–5 SATE, masking groups 89–92 second generation 86–92 stability in biological media 90–2 synthesis 111 protecting groups on nucleobases 86, 87–9 protein kinase A 250 protein kinase C (PKC) isoenzymes 156 phosphorothioate oligodeoxynucleotide analogue binding 6–7 PKC-α 139, 156, 226, 233, 249–50 protein kinases 244 proteins 154 aptameric binding 3 inhibition 101 phosphorothioate oligodeoxynucleotide analogue binding 6 30 kDa protein 202 47 kDa protein production 14 schema 36 viral 113 proteoliposomes 163–4 pseudoisocytosine 55 psi2neo cells 158 psoralen, conjugation 59 psoralen derivatives 57 psoriasis 38, 274 PTT 273–4 quantification of oligonucleotides 60 rabbit reticulocyte lysate 291, 296 raf kinase, antisense approaches for cancer 247–8 ras 250–8 antisense approach 250–1 antisense inhibitor specificity 252–4 antisense inhibitors 251–2, 255 antitumour activity 257–8 gene expression inhibition, cellular responses 254–7 on nanoparticles 139

332 INDEX

ras genes, (Ki-ras, Ha-ras and N-ras) 251 rat αl (I) collagen gene promoter receptor-mediated endocytosis see endocytosis receptors 216–17 counter-receptors 265–6 relA antisense effector, G-quartet formation 9 release of deoxynucleosides 4–6 renal ischaemia 272 renin-angiotensin system 156 resonance energy transfer (FRET) 153 reticuloendothelial system 112, 220 retinitis, cytomegalovirus 234 reverse ligation-mediated polymerase chain reaction (RLPCR) 15, 21 reversed-phase HPLC 62–3, 85, 89 rheumatoid arthritis 38, 204, 274 rhodamine-labelled lipids 153 rhodamine-labelled oligomers 121–2, 206 ribonuclease H see RNase H ribose, conjugation 57–8 ribosomes 292 schema 36 ribozymes 17, 293 for antisense, design criteria 39–40 development 35 hairpin 35 hammerhead 35, 56, 293 schema 36 RLPCR (reverse ligation-mediated polymerase chain reaction) 15, 21 mRNA 3′-fragment detection 21 RNA, double-stranded (ds) RNA 102 RNA folding 39–40, 298 RNase, mRNA ablation 3 RNase H 208, 244, 274 mRNA degradation 12, 47–8 non-RNase H mechanism, peptide chain elongation inhibition 13 non-targeted cleavage 18 partial oligonucleotide/RNA activation 40 RNase H-independent mechanism 99, 205 RNase L, 2′, 5′-adenylate-dependent 47, 116 rodent allografts 270–2 rodent xenografts 249 rolling on endothelial cells 187

Rous sarcoma virus 35 Rp configuration 45, 47, 49 S-acetylthiomethyl (SATM) 80–1 S-acylthioethyl see SATE 35S-labelled oligodeoxynucleotide 234–5 35S-labelled phosphorothioate oligonucleotide 231 S-T2G4T2 octamer, HIV–1 replication inhibition 9 α-sarcin loop 100 SATE (S-acetylthioethyl) 85, 86, 88–92 dimer protection, gastric juice stabilisation 82–3 pro-oligonucleotides 79–81, 82, 83 SATM (S-acetylthiomethyl) 80–1 scavenger receptors 217 Schistosoma mansoni 294, 297 schizonts 291 SCID mice cytotoxic dermatitis 270 G3139 antisense inhibition of lymphoma 245–6 selectins 264, 265 selectively binding complementary A and T analogues (SBC) 288, 300 SELEX 301 Sendai virus 163 serine/threonine protein kinase 249 enzyme activity 11–12 SH2 and SH3 proteins 244 sialyl lewis X 265 sinusoidal endothelial cells 187 SKVLB cells 150 smooth muscle cell hyperplasia inhibition, c-myb 7, 8 sodium azide 148, 201 solid support, photolabile linker 86 Sp configuration 47, 48, 49 Sp-isomer 45 spermidine 179 spermine 176–7, 179 splice sites, pre-mRNA 13 stability measurements of oligonucleotide degradation 228

INDEX 333

pro-oligonucleotides in biological media 90–2 starburst dendrimers 207–8 Stealth nanoparticles 130 stem-loop 155, 299 steric blocking mechanism gene expression 12–14 pre-mRNA 3 sterically locked nucleic acid analogues 53 streptolysin 4, 13, 14, 15, 17, 18, 19, 20, 112 reversible plasma membrane permeabilization 12 substance P (SP)/neurokinin-l receptor 156 sugar modifications 51–3 superoxide dismutase (SOD) 293 suramin 203 surface plasmon resonance (SPR) 180 SV40 T-antigen nuclear localization SW480 colon carcinoma cells 251 3′, 3′-switches 47 synthetic polymers 45–6 T4 DNA ligase 45–6 T7 polymerase 45–6 T helper-1 immune (Thl) response 279 T lymphocytes 279 TAR RNA element 300–2 Tat basic domain 119 TAT motifs 37 Tat peptides internalization promotion 121 translocation, structure-activity relationship 121 Tat polybasic sequence 220 Tat protein 116 Tat protein chimeras 220 Tb(DPA)33-compound 163 TCAACGTT 11 telomerase anti-telomerase 101 cancer cells 101 temperature, melting (Tm) 16, 48, 49, 50, 51, 54, 60,61, 116 tetraethylthiuram disulphide 48 texaphyrins 59 Th2 phenotype 279

β-thalassaemia 13 Theileria 297 2-thio thymine 300 3′-thioformacetal 47, 50 5′-thioformacetal 50 5′-thiophosphoryl 46 thrombin 226, 232 thrombin binding aptamer 9 time-of-flight (TOF), mass detection 66–7 tissue distribution (D) 227 TMAG (N-(alphatrimethylammonioacetyl) didodecyl-D-glutamate chloride) 151 TNF-α 203–4, 269, 277, 279 leukotriene B4 203 toxicity of cationic lipids 112, 218 toxicology antisense oligonucleotides 273 phosphorothioate oligodeoxynucleotides 233 toxins, cytoplasm entry 219 transcription 103 schema 36 transfer of oligonucleotides from endosomal to cytoplasmic compartments 205, 218 transferrin 112, 117, 185 translation, scheme 36 translation inhibition 12 transmembrane transport of oligonucleotides 213–20 transplantation tolerance 272 transporter proteins 216–17 triethylammonium acetate 62 triple helix 300 triplex 299 for antisense, design criteria 39–40 triplex DNA, triplex-forming ONs (TFOs) 172–84 Trypanosoma brucei 289, 293–7 Trypanosoma cruzi 291, 293–7 tryptophan residue 118, 119 (CGT[C]GA) tyrosine kinase inhibition 11– 12 ulcerative colitis 38, 272–4 universal base, defined 56

334 INDEX

5′-untranslated initiation codon region 12– 13 mRNA degradation 12 UV spectroscopy 60, 89 G-quartet identification 7 nucleic acid quantification 60 psoralen conjugation 59 UV-melting curves 59, 61–2 variant surface glycoproteins 290 vascular cell adhesion molecule 1 (VCAM-1) 264, 266, 275–6 vesicular stomatitis virus 119 viral infections 47, 128, 130, 140, 154, 158 vitamin E 58 vitravene 38 Watson-Crick double helix 35, 37, 39, 42, 46, 53, 54, 56, 101, 300–1 wheat germ extract 17 xenografts 269–70 Xenopus oocytes 12, 17 zintevir (AR177) 38, 226, 228, 233