Principles of Molecular Neurosurgery

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Principles of Molecular Neurosurgery

Progress in Neurological Surgery Vol. 18

Series Editor

L. Dade Lunsford

Pittsburgh, Pa.

Principles of Molecular Neurosurgery

Volume Editors

Andrew Freese Minneapolis, Minn. Frederick A. Simeone Philadelphia, Pa. Paola Leone Camden, N.J. Christopher Janson Camden, N.J.

96 figures, 12 in color, and 25 tables, 2005

Basel · Freiburg · Paris · London · New York · Bangalore · Bangkok · Singapore · Tokyo · Sydney

Andrew Freese, MD, PhD

Frederick A. Simeone, MD

Department of Neurosurgery, University of Minnesota School of Medicine MMC 96, 420 Delaware St., S.E., Minneapolis, MN 55455 (USA)

University of Pennsylvania, Simeone Neuroscience Center, Pennsylvania Hospital, 800 Spruce St., Philadelphia, PA 19107 (USA)

Paola Leone, PhD

Christopher Janson, MD

Cell and Gene Therapy Center, UMDNJ-Robert Wood Johnson Medical School, Division of Neurosurgery, 401 Haddon Ave., Rm 390, Camden, NJ 08103 (USA)

Cell and Gene Therapy Center, UMDNJ-Robert Wood Johnson Medical School, Division of Neurosurgery, 401 Haddon Ave., Rm 390, Camden, NJ 08103 (USA)

Library of Congress Cataloging-in-Publication Data Principles of molecular neurosurgery / volume editors, Andrew Freese ... [et al.]. p. ; cm. – (Progress in neurological surgery, ISSN 0079-6492 ; v. 18) Includes bibliographical references and indexes. ISBN 3-8055-7784-2 (hard cover : alk. paper) 1. Nervous system–Diseases–Treatment. 2. Molecular neurobiology. 3. Nervous system–Surgery. [DNLM: 1. Nervous System Diseases–therapy. 2. Gene Therapy–methods. WL 140 P9573 2005] I. Freese, Andrew. II. Series. RC349.8.P75 2005 616.8–dc22 2004024871 Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® and Index Medicus. Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection and dosage set forth in this text are in accord with current recommendations and practice at the time of publication. However, in view of ongoing research, changes in government regulations, and the constant flow of information relating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important when the recommended agent is a new and/or infrequently employed drug. All rights reserved. No part of this publication may be translated into other languages, reproduced or utilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying, or by any information storage and retrieval system, without permission in writing from the publisher. © Copyright 2005 by S. Karger AG, P.O. Box, CH-4009 Basel (Switzerland) www.karger.com Printed in Switzerland on acid-free paper by Reinhardt Druck, Basel ISSN 0079–6492 ISBN 3–8055–7784–2

Contents

IX Dedication X Acknowledgements XI Series Editor’s Note Lunsford, L.D. (Pittsburgh, Pa.) XIII Foreword Anderson, W.F. (Los Angeles, Calif.) 1 Introduction Freese, A. (Minneapolis, Minn.); Janson, C.; Leone, P. (Camden, N.J.); Simeone, F.A. (Philadelphia, Pa.) The Spine and Spinal Cord 5 The Molecular Basis of Intervertebral Disc Degeneration Leo, B.M.; Walker, M.H. (Charlottesville, Va.); Anderson, D.G. (Philadelphia, Pa.) 30 Genetics of Degenerative Disc Disease Kurpad, S.N.; Lifshutz, J. (Milwaukee, Wisc.) 37 Gene Therapy for Degenerative Disc Disease Kim, J.; Gilbertson, L.G.; Kang, J.D. (Pittsburgh, Pa.)

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52 Bone Morphogenetic Proteins. Spinal Fusion Applications Bomback, D.A.; Grauer, J.N. (New Haven, Conn.) 65 Cellular and Gene Therapy Approaches to Spinal Cord Injury Steinmetz, M.P.; Liu, J.K.; Boulis, N.M. (Cleveland, Ohio) 104 Neural Stem Cell Transplantation for Spinal Cord Repair Iwanami, A.; Ogawa, Y.; Nakamura, M.; Kaneko, S. (Tokyo/Osaka); Sawamoto, K. (Osaka); Okano, H.J.; Toyama, Y.; Okano, H. (Tokyo/Osaka) Functional and Restorative Molecular Neurosurgery 124 Contemporary Applications of Functional and Stereotactic Techniques for Molecular Neurosurgery House, P.A.; Rao, G.; Couldwell, W. (Salt Lake City, Utah) 146 Xeno-Neurotransplantation Schumacher, J.M. (Miami, Fla.) 154 Adeno-Associated Viral Vectors for Clinical Gene Therapy in the Brain Samulski, R.J.; Giles, J. (Chapel Hill, N.C.) 169 Molecular Mechanisms of Epilepsy and Gene Therapy Telfeian, A. (Lubbock, Tex.); Celix, J. (Seattle, Wash.); Dichter, M. (Philadelphia, Pa.) 202 Emerging Treatment of Neurometabolic Disorders Brady, R.O.; Brady, R.O., Jr. (Bethesda, Md.) 213 Gene Therapy for Parkinson’s Disease Hadaczek, P.; Daadi, M.; Bankiewicz, K. (San Francisco, Calif.) 246 Simplifying Complex Neurodegenerative Diseases by Gene Chip Analysis Scherzer, C.R.; Gullans, S.R. (Cambridge, Mass.); Jensen, R.V. (Middletown, Conn.) 258 Molecular Pathology of Dementia. Emerging Treatment Strategies Gouras, G.K. (New York, N.Y.)

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270 Expanding the Role of Deep Brain Stimulation from Movement Disorders to Other Neurological Diseases Leone, M.; Franzini, A.; Broggi, G.; Bussone, G. (Milano) 284 Molecular Mediators of Pain Chaudhary, P.; Burchiel, K. (Portland, Oreg.) 322 Gene Transfer in the Treatment of Pain Fink, D.; Mata, M. (Ann Arbor, Mich.); Glorioso, J.C. (Pittsburgh, Pa.) Neurovascular Disorders 336 Gene Discovery Underlying Stroke Barone, F.C. (King of Prussia, Pa.); Read, S.J. (Macclesfield) 377 Molecular Mediators of Hemorrhagic Stroke Macdonald, R.L. (Chicago, Ill.) 413 Advances towards Cerebrovascular Gene Therapy Watanabe, Y.; Heistad, D.D. (Iowa City, Iowa) 439 Ex vivo Gene Therapy and Cell Therapy for Stroke Kondziolka, D. (Pittsburgh, Pa.); Sheehan, J. (Charlottesville, Va.); Niranjan, A. (Pittsburgh, Pa.) Neuro-Oncology 458 Neurosurgical Applications for Polymeric Drug Delivery Systems Wang, P.P.; Brem, H. (Baltimore, Md.) 499 Immunotherapy Strategies for Treatment of Malignant Gliomas Harshyne, L.; Flomenberg, P.; Andrews, D.W. (Philadelphia, Pa.) 521 Glioma-Genesis. Signaling Pathways for the Development of Molecular Oncotherapy Kapoor, G.S.; O’Rourke, D.M. (Philadelphia, Pa.) 557 Oncolytic Viral Therapy for Glioma Lamfers, M.L.M.; Visted, T. (Charlestown, Mass.); Chiocca, E.A. (Columbus, Ohio) 580 Molecular Neurosurgery in the Pituitary Gland. Gene Transfer as an Adjunctive Treatment Strategy Castro, M.G.; Jovel, N.; Goverdhana, S.; Hu, J.; Yu, J.; Ehtesham, M.; Yuan, X.; Greengold, D.; Xiong, W.; Lowenstein, P.R. (Los Angeles, Calif.).

Contents

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624 Stem Cells for Targeting CNS Malignancy Yip, S. (Vancouver); Sidman, R.L. (Boston, Mass.); Snyder, E.Y. (Boston, Mass./La Jolla, Calif.) 645 Author Index 646 Subject Index

Contents

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Dedication

This book is dedicated to the memory of Drs. Ernst and Elisabeth Freese, brilliant scientists and wonderful parents who deciphered the chemical basis of mutagenesis, the engine of evolution and God’s way of making us better. We also dedicate the section on functional and restorative molecular neurosurgery to the memory of Anne Janson, a victim of Alzheimer’s Disease, and all the other patients who suffer from this terrible disease which robs the mind of its memories and dignity, as well as all children affected by neurodegenerative diseases – for those who have died, they are not forgotten; and for those who are living, may they soon have the promise of a cure. The section on Oncology is dedicated to the memory of Jack Geary, a victim of cancer. Finally, we dedicate the section on the spine and spinal cord to the memory of Anthony Simeone, M.D., whose devotion to his patients and family will always be remembered. The Editors Andrew Freese Frederick A. Simeone Paola Leone Christopher Janson

IX

Acknowledgements

We gratefully acknowledge the dedicated, diligent work of Jackie Alutis in helping produce this book, and the valuable contributions of Joanne Coughlin and Marcia Freese as well. Without them, this book would not have been possible. In addition, we acknowledge the support of the CNS Gene Therapy Consortium in the production of this book. Otto Freese produced the front cover illustration, for which we are grateful as well. The Editors Andrew Freese Frederick A. Simeone Paola Leone Christopher Janson

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Series Editor’s Note

Changing the Paradigm of Neurosurgery

During the last decade of the 20th century and the first years of the 21st century, neurosurgery has been part of an enormous paradigm shift. While we previously concentrated on dealing with the removal or management of structural masses (blood clots, aneurysms, brain tumors, spinal bone spurs, ruptured discs), the future of neurosurgery lies in the application of a wide variety of new knowledge. Loosely termed ‘molecular’, this new knowledge can be applied widely to the diagnosis, management, and possible prevention of serious neurological illness. As such, we practitioners and surgeons must embrace this new knowledge and attempt to implement it in the current practice of neurosurgery. The future of neurosurgery is molecular, minimally invasive, and multidisciplinary. For the first time, we will be bringing the emerging data from the laboratory and beginning to apply it to clinical problems, rather than the reverse, the old paradigm of trying something in the operating room and then going back to the laboratory to see why it did or didn’t work. Volume 18 of Progress in Neurological Surgery is an elegant compilation of current and emerging knowledge related to the influence of molecular neurosurgery on both the present and the future. Drs. Freese, Simeone, Leone, and Janson have accumulated a wealth of information which can be applied to the spinal column and spinal cord disorders, functional and restorative brain surgery, neurovascular disorders and neuro-oncology. The author list is impressive, and the story that is presented should entice the reader to glimpse the

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future of neurosurgery, which is rapidly descending on us. I congratulate the current authors for their excellent collaboration, and believe the readership will enjoy this new volume which does, indeed, represent progress in neurological surgery. L. Dade Lunsford, MD The University of Pittsburgh School of Medicine, Pittsburgh, Pa., USA

Series Editor’s Note

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Foreword

There are not many advantages to becoming ‘senior’, an euphemism for being an old man. But there are a few, and two of them are exemplified for me by this volume. The first is in seeing how scientific/medical progress can mushroom over a span of decades. As I think back on my training, in the 1950s and early 1960s, and then look through this fascinating book, I am in awe at the progress. Yes, I have been intimately involved in gene therapy and the genetic basis of disease my whole career, but the laying out of the application of these technologies to the single field of neurosurgery leaves me in wonder. Using gene therapy to treat malignant gliomas is revolutionary enough, but scanning the chapters herein reveals the use of gene therapy and genetic approaches for degenerative disc disease, for spinal cord injuries, for epilepsy, for Parkinson’s and other neurodegenerative diseases, for the treatment of pain, for stroke as well as for malignancies. And not just genetic approaches: neurosurgery now embraces protein therapy, cell therapy, oncolytic viral therapy, immunotherapy, stem cell transplantation, and xeno-neurotransplantation. Truly a glorious story of scientific/medical progress! The second advantage of growing old is seeing the successes of the many young physician investigators that one has trained. Everywhere that I travel, there seems to be someone who had passed through my laboratory over the past 40 years. The joy of hearing the stories of successful, productive careers is wonderful. But this book brings an added touch. Andrew Freese is the son of one of the men that played a significant role in my career. When I interviewed at NIH in 1963 for a position of Research Associate (following my medical

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training), I narrowed my choices to two: Marshall Nirenberg (who was in the middle of deciphering the genetic code) and Ernst Freese, who was doing fascinating work in genetic model systems. This was one of the toughest decisions of my career. I found Dr. Freese (I still cannot call him by his first name although he has asked me to for years!) to be a brilliant scientist and marvelous human being. So much so that, although I joined Marshall Nirenberg and helped in the final genetic code decipherment, I maintained a long and fruitful friendship and scientific mentorship with Dr. Freese. Thus, it was with extraordinary pleasure that I received a letter from Dr. Freese’s son, Andrew, to write the Foreword for this book. As I think back to all the advice and knowledge that I received from Dr. Freese over so many years, I am honored and humbled to add my name to a volume that is dedicated to his memory by his son. W. French Anderson, MD USC Keck School of Medicine, Los Angeles, Calif., USA

Foreword

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 1–4

Introduction Andrew Freesea, Christopher Jansonb, Paola Leoneb, Frederick A. Simeonec a Department of Neurosurgery, University of Minnesota School of Medicine MMC 96, Minneapolis, Minn., bCell and Gene Therapy Center, UMDNJ-Robert Wood Johnson Medical School, Division of Neurosurgery, Camden, N.J., cDepartment of Neurosurgery, University of Pennsylvania, Philadelphia, Pa., USA

Until recently, Neurosurgery has been a surgical discipline largely focused on ablative approaches to diseases affecting the nervous system – clipping aneurysms, evacuating hematomas, removing disc herniations, decompressing the stenotic spine, extirpating tumors, lesioning the neostriatum, and others. However, as physician-scientists have begun to unravel the molecular basis of many neurological disorders, a paradigm shift has begun to occur – one that promises to dramatically alter the face and texture of Neurosurgery, and convert it into a field that not only ablates diseased tissues and structures, but also restores and improves function within the nervous system. Thus, we believe the advent of a variety of molecular and cellular technologies will have a marked impact on what neurosurgeons, neurologists, psychiatrists, neuroradiologists, and other medical practitioners focused on the nervous system can offer patients. Indeed, distinctions among these medical disciplines will begin to fade, as psychiatric diseases increasingly find neurosurgical solutions, and neurosurgical diseases ‘molecularize’ into nonsurgical, neurological approaches. As the field of medicine in general continues to hone in on molecular interventions, in parallel, the field of Neurosurgery will convert itself from a macromolecular discipline to one that relies increasingly on molecular approaches to improve the outcomes of patients. To hopefully assist our colleagues in medical and surgical disciplines dealing with the nervous system in anticipating this future, we believe that a book focused on the current principles of molecular Neurosurgery is needed, and we have, therefore, attempted to bring together a

variety of superb contributors who can further shed light on this topic in this compilation of chapters. In this edition, several categories of neurological diseases are examined. It is important to note that the chapters are not meant to be an exhaustive compilation of facts detailing the entire field of molecular neurosurgery, but instead are meant to highlight some of the most exciting advances. Thus, this book attempts to spark the imagination of its readers as much as it tries to recite and explain exhaustive details of the most recent research work and discoveries. If there is one fact of which we are certain, it is that this book will be obsolete within several years of its publication, and a number of promising technologies outlined therein will have proven to be failures and subsequently will have been abandoned. Similarly, undoubtedly a number of approaches that are only briefly skimmed or even inadvertently omitted in this book will prove to be extraordinarily successful and important. We cannot predict which will succeed and which will fail. However, we are certain that Neurosurgery faces an exciting future as these types of approaches continue to evolve and ultimately allow us to better care for our patients. The first section of the book, encompassing the first six chapters, addresses disorders of the spine and spinal cord. The chapter by Leo et al. focuses on understanding the molecular and biochemical alterations associated with degenerative disc disease, a problem that has a huge financial and emotional impact on a large proportion of our society. Only by understanding the molecular basis of disc disease can we ever hope to meaningfully intervene prior to the development of symptoms and the progressive cycle of incapacitation that results from degenerative disease. Kurpad and Lifshutz discuss the genetics of disc and spinal degeneration, as there is clear evidence of a genetic predisposition to this disease process, and a variety of gene defects and altered protein profiles have been associated with it. Once a better understanding of the genetic alterations, and subsequent molecular and biochemical disruptions that occur in degenerative spine disease can be garnered, then one can begin to envisage meaningful interventions. The chapter by Kim et al. outlines one exciting area of investigation, focused on genetic intervention using different gene transfer technologies to introduce therapeutic gene products directly into the disc to retard degeneration or promote restoration of normal function. Although ideally one would want to intervene in the spine before the emergence of axial back pain and/or radicular symptoms, surgical intervention will still be required in a number of patients who need fusion procedures. To optimize the success of this type of surgery, however, molecular approaches to enhance fusion need to be developed and promise to significantly improve outcomes from fusion procedures. The chapter by Bomback and Grauer enumerates these approaches. Although degenerative spine disorders have an enormous impact

Freese/Janson/Leone/Simeone

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on our society, it is those patients who have traumatic spinal cord injuries that frequently suffer the most. Relatively little can be done currently for these patients, but enormous progress is being made in understanding the molecular basis of spinal cord injury and hopes for restoration of function. The last two chapters (Steinmetz et al. and Iwanami et al.) in this section on the spine thus discuss a variety of molecular and cellular interventions that are being developed to improve outcomes in patients with spinal cord injury. The second section of the book, encompassing 11 chapters, addresses functional and metabolic disorders of the nervous system, and restorative approaches to these diseases. In the chapter by House et al., an overview of the field is provided with an emphasis on targeting structures within the nervous system using contemporary neurosurgical techniques. Schumacher as well as Samulski and Giles identify some exciting technological advances that allow molecular and functional intervention in the nervous system using cellular transplantation and gene therapy approaches, selecting two unique and promising systems, one based on xenotransplantation, and the other, a promising gene transfer system based on adeno-associated virus, both of which have already been used in clinical trials for human neurological diseases. Although these two chapters focus on a specific tissue source and viral vector, respectively, they also discuss alternatives and principles underlying cell transplantation and viral vectormediated gene transfer. Telfeian et al. discuss current concepts regarding the molecular basis of epilepsy and seizure disorders, identifying promising research approaches to these diseases. Since pharmacological approaches to epilepsy are often suboptimal, it is likely that cellular and genetic intervention will allow more directed and efficacious therapy to the tissue source of the aberrant electrical activity. Brady and Brady Jr. discuss the advent of molecular intervention for neurometabolic diseases, starting with the successful development of enzyme replacement therapy, and then branching out to exciting new advances in gene and cell therapy. The following four chapters address a variety of neurodegenerative diseases, including Parkinson’s disease, Alzheimer’s disease, and others, with a focus on further elucidating the molecular bases of these diseases, and then improving neurosurgical approaches to them. Chaudhary and Burchiel as well as Fink et al. then focus on pain, and include a thorough evaluation of the molecular mediators of pain, and subsequently novel genetic and surgical interventions for patients with intractable pain disorders. The third section of the book, encompassing four chapters, focuses on neurovascular disorders. The first two chapters (Barone/Read and Macdonald) identify different genes and molecular mediators associated with stroke, an understanding of which may identify potential genetic and molecular targets for improved clinical intervention. The chapter by Watanabe and Heistad focuses on genetic intervention in stroke, including an evaluation of preclinical studies,

Introduction

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with the hope of developing neurosurgical or endovascular delivery systems to minimize damage to neural tissue associated with stroke. Kondziolka et al. then discuss the advent of cellular transplant therapy for stroke, including some results of a recent clinical neurosurgical trial. The fourth and final section, encompassing the last six chapters, addresses the field of neuro-oncology, with a neurosurgical perspective. The chapter by Wang and Brem provides a thorough overview of novel polymeric and related intracranial drug delivery systems for chemotherapeutic agents as adjuvant therapy following surgical extirpation of brain tumors. Harshyne et al. discuss immunotherapy strategies for malignant brain tumors, and their likely impact on developing a more global approach to a disease process that usually extends beyond the typical resection margins of surgery, virtually assuring recurrence. The chapter by Kapoor and O’Rourke identifies common molecular signaling events involved in gliomagenesis, and their relevance for developing targeted approaches to intervene in these biochemical sequences and their role in progression to a more malignant tumor phenotype. Lamfers et al. then focus on viral gene therapy approaches to malignant brain tumors, including an evaluation of failures of clinical trials of gene therapy in the past, and opportunities for improvement in the future. The chapter by Castro et al. further provides an overview of the potential for genetic intervention in brain tumors, but in a particular subset, pituitary adenomas, in which focal gene expression may well have a significant impact clinically, and may someday be the primary modality of treatment for these typically benign tumors which nonetheless cause significant morbidity. The final chapter by Yip et al. gives an exciting glimpse into the potential for stem cell technology to seek out malignancies in the nervous system, and selectively destroy them. It is our hope that by providing an overview of the developing interface between molecular biology and clinical Neurosurgery, we will further stimulate the imaginations of clinicians and scientists, and provide additional impetus for aggressive investigation of these and related technologies. Andrew Freese, MD, PhD Professor and Vice Chairman of Neurosurgery Director of Spinal Neurosurgery University of Minnesota 420 Delaware Street, S.E. Suite D429, Mayo Memorial Building Minneapolis, MN 55455 (USA) Tel. ⫹1 612 624 2471, Fax ⫹1 612 624 0644, E-Mail [email protected]

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The Spine and Spinal Cord Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 5–29

The Molecular Basis of Intervertebral Disc Degeneration Brian M. Leoa, Matthew H. Walker a, D. Greg Andersonb a Department of Orthopaedic Surgery, University of Virginia, Charlottesville, Va., bDepartment of Orthopaedic Surgery, Thomas Jefferson University, Philadelphia, Pa., USA

Introduction

Low back pain is an endemic problem in Western societies leading to significant morbidity [1–3]. Not only does back pain account for much individual suffering, but the societal costs of time lost from work, for treatment, and for compensation of lost wages numbers in the billions of dollars annually. It has been estimated that up to 80% of the population experiences some form of back pain over the course of their lives, making this a leading health concern [1–5]. Although the etiologies are many, intervertebral disc degeneration appears to be the leading cause for chronic axial low back pain [6]. Significant strides have been made in understanding the molecular basis for disc degeneration. Despite this, the currently available treatment modalities for disc-related spinal pain continue to focus on alleviating symptoms rather than addressing the underlying cause of degeneration. It is likely that clinical outcomes for patients with painful intervertebral disc degeneration would improve if therapies were developed that could slow, halt, or reverse this process. Histopathological changes classified as ‘degeneration’ have been recognized as early as the second decade of life and are known to progress through a series of stages that have been quantified histologically [7–10] and characterized noninvasively using magnetic resonance imaging [7, 9, 11, 12]. Initially, the degenerative process begins asymptomatically in the nucleus pulposus (NP) with cell loss and matrix alteration. This leads to an inability to retain water and results in slight disc space narrowing. As this process progresses, the outer annulus fibrosis (AF) becomes increasingly disorganized, losing its normal lamellar arrangement and leading to diminished mechanical strength. Tissue fissures

and clefts begin in the inner AF and progress outwards towards the periphery, ultimately culminating in a loss of mechanical integrity [13]. Mechanical stresses are progressively transferred to the surrounding vertebral endplates causing microfractures and marginal osteophyte formation. Cytokines, produced within the disc, leach out, leading to the ingrowth of nerve and vascular elements that may play a role in the etiology of spinal pain [14–17]. The secondary bony changes serve to stiffen the spinal segment and restabilize the spine. Interestingly, although this sequence happens throughout life in essentially all humans, there are significant variations in the degree of symptoms noted by different people. Equally perplexing is the poor correlation between the degree of degenerative changes noted on imaging studies and the presence of symptoms of spinal pain [18]. Clearly, we have much to learn regarding the relationship between spinal degenerative disease and various pain syndromes. In recent years, researchers have attempted to understand the genetic and molecular aspects of disc degeneration in order to determine the etiology of degeneration and to identify stages in this process where therapeutic intervention would be beneficial. There has been a growing enthusiasm in the concept of developing tissue engineering strategies that can alter the course of the degenerative process and address the underlying disease process. This chapter will review the normal disc development and structure as well as the changes that occur during degeneration on the biomechanical, biochemical and ultrastructural levels. By gaining a better understanding of the cellular and molecular biology of the disc, the various strategies that are being discussed or studied to combat disc degeneration can be evaluated in a rational manner.

Embryological Development of the Disc

Although the embryology of the human disc has not been well studied directly, animal models have contributed significantly to our understanding of the development of the intervertebral disc and the axial skeleton. The development of the spine begins during the third week of gestation in a phase termed ‘gastrulation.’ It is during this phase that the ectoderm, mesoderm, and endoderm are formed; embryological layers, which eventually give rise to all tissues of the body. Differentiation of these tissues begins with the formation of the primitive streak. Associated with the primitive streak is a primitive node surrounding a small invagination known as the primitive pit, located slightly caudally from the midline of the embryo. Cells in the primitive pit, known as prenotochordal cells, migrate cephalad to the prechordal plate and contribute to the cell layers forming the notochordal plate. As these cells proliferate and detach from the endoderm, they form a solid cord of cells that becomes

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The developing embryo Primitive node Primitive streak

Prenotochordal cells migrating

Fig. 1. The developing embryo has a primitive streak extending caudad from the primitive node. Arrows approximate the migration of the prenotochordal cells cephalad to the prechordal plate.

the definitive notochord and forms the basis of the axial skeleton (fig. 1). Concurrently, the vertebral column and outer portion of the intervertebral discs form from an aggregation of mesenchyme surrounding the notochord. This process involves the medial migration of somatic mesoderm from the ventromedial portions of the somites, the sclerotomes. In a process called ‘segmentation,’ a pattern of alternating light and dark bands becomes evident in the mesenchymal column by the time the embryo reaches 5 mm in length [19] (fig. 2). The dark bands contain cells with a greater nuclear density than those of the light bands and are the precursors of the intervertebral discs, termed the perichordal discs. The light bands are the precursors of the vertebral bodies [19]. By the time the embryo reaches 12.5 mm, the perichordal disc becomes trilaminar with a denser middle region surrounded by lighter regions both cranially and caudally [20]. At this point, the outer mesenchymal cells begin to arrange themselves in a lamellar manner with their long axis parallel to the long axis of the embryo [19]. This outer lamellar zone darkens as it becomes populated with fibroblasts forming the primitive AF [19]. The lighter inner cell mass is formed primarily from notochordal cells and embryonic cartilage and can be well seen by the 40–50 mm stage [19]. Cells in the AF begin to deposit collagen fibers in the outer region of the perichordal disc by the 20–40 mm stage [19]. With continued growth, the outer AF becomes progressively more fibrous and less cellular, while the inner AF becomes fibrocartilaginous and retains a high cell density [21]. Growth of the disc is hypothesized to occur due to increasing lamellar thickness, as opposed to an increase in the number of lamellar layers; the number of lamellae remain

Molecular Basis of Intervertebral Disc Degeneration

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Precursor of vertebral body

Notochord Precursor of intervertebral disc – perichordal disc

Fig. 2. The mesenchymal column segments into light and dark bands. The dark bands contain cells with a greater nuclear density and represent the precursors to the intervertebral discs, the perichordal discs. The light bands represent the precursors to the vertebral bodies.

fairly constant throughout development, and in lumbar discs constitutes twelve to sixteen layers [19, 21]. The lamellar fibers in these alternating layers of the AF are oriented at oblique angles to each other, a fashion designed to optimally dissipate multidirectional stresses on the disc. The NP continues to develop from both the intervertebral expansion of the notochord and the growth of primitive cartilage. As the NP region develops, the ground substance softens leading to a loosely arranged matrix [19, 22]. The notochordal cells of the NP play a key role in cell division and matrix formation. As the matrix of the NP becomes looser, the once compact mass of notochordal cells is broken up into cellular clusters in a loose network known as the ‘chorda reticulum’ [23]. The NP continues to expand in size during fetal development and early postnatal life with an 18-fold expansion in the number of notochordal cells before becoming quiescent [19]. Although the appearance of the NP gradually changes to become more fibrous, resembling the transition zone, the embryological notochordal cells remain active producers of matrix material until the end of the first decade of life, at which time a ‘notochordal’ NP can no longer be defined due to the increased collagen content and loss or metaplasia of notochordal cells [19, 24]. Insults to the developing fetus can have severe consequences to organ systems undergoing rapid development at the time of the insult. The time period for the most rapid development of the lumbar vertebral canal is between 12 and 32 weeks in utero [116]. A recent retrospective study by Jeffrey et al. [25]

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Atlas 7 Cervical vertebrae

Axis Cervical curvature

Thoracic curvature

12 Thoracic vertebrae

5 Lumbar vertebrae Lumbar curvature Sacrum (5 fused vertebrae) Coccyx (4 fused vertebrae) Sacral curvature

Fig. 3. Schematic of the human spine. (Reproduced with permission [117].)

showed that low birth weight, low placental weight, low socioeconomic class, and smoking during pregnancy can have detrimental effects on the size of the lumbar vertebral canal and may predispose these people to future spinal problems. Structure and Anatomy

The human spine contains twenty-three intervertebral discs (fig. 3). The size of the discs generally increases in both height and diameter as one moves

Molecular Basis of Intervertebral Disc Degeneration

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Fig. 4. Micrograph of rabbit intervertebral disc histology. # ⫽ Outer AF; * ⫽ inner AF; arrowhead ⫽ transition zone; NP ⫽ nucleus pulposus.

more caudally within the spine. The disc itself is a component of a complex biomechanical system composed of the bony vertebral endplate, the cartilaginous endplate, the AF, and the NP. This complex organ functions to allow motion and to dissipate stress within the spinal column. Similar to other connective tissues, the disc is composed mostly of extracellular matrix with a complex array of structural and water-binding proteins and a relative paucity of cells. Each disc can be further subdivided into four regions: the outer annulus, inner annulus, transition zone, and NP (fig. 4). The outer annulus is composed of highly organized, directionally oriented collagenous lamellae running at alternating 30-degree angles to the long axis of the spine. The collagen in this region of the disc is mostly type I and includes fibrils that insert into the vertebral bodies. The cell population in the outer annulus is primarily fibroblastic. The inner AF is larger and more fibrocartilaginous, containing less collagen and lacking the lamellar architecture of the outer AF. Collagen in this region is mostly type II. In addition, the inner AF contains a higher proportion of large proteoglycan aggregates. The cell population in the inner AF has characteristics of both fibroblasts and chondrocytes. The transition zone is a distinct, thin acellular fibrous layer that separates the inner AF from the NP. The NP contains an amorphous matrix of highly hydrated proteoglycans embedded in a loose network of collagen. Like the inner AF, the collagen in the NP is mostly type II. The cell population of the NP is sparse and unevenly distributed with more cells present in the central regions of the NP than at the periphery. At least two distinct cell populations are recognized in the NP in early life. The first is a small round cell resembling a chondrocyte. The second is much larger and has a vacuolated appearance described by Virchow [22] as

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Table 1. Collagen composition of the intervertebral disc. (Reproduced with permission from [116]) Type

Predominant location

Percent of total collagen (%)

Fibril-forming collagens I II III V XI

Annulus Annulus and nucleus Annulus Annulus and nucleus Annulus and nucleus

0–50 0–50 ⬍5 1–2 1–2

Short helix collagens VI IX XII

Annulus and nucleus Annulus and nucleus Annulus

5–20 1–2 ⬍1

‘physoliferous’ (or ‘bubble-bearing’), containing prominent cellular processes and intracellular glycogen deposits. This cell type is thought to be of notochordal origin. As mentioned, in humans, these large, notochordally derived cells tend to disappear (or become rare) by adolescence, leaving scattered chondrocyte-like cells in their place [7, 8, 26, 27]. A recent study by Kim et al. [28] demonstrated the migration of endplate chondrocytes into the NP region of the disc, which might account in part for the shift in cell populations within the NP region. The disc matrix consists primarily of collagens and proteoglycans, but varies significantly between regions of the disc. Collagen cross-linking gives the disc substantial tensile strength, while highly hydrated proteoglycans give the disc stiffness and resistance to compression [27]. Collagens account for 60% of the dry weight of the AF, with type I collagen being the most abundant type (80%); the NP, on the other hand, contains up to 20% collagen with a predominance of type II collagen [7, 29, 30]. Additional collagen types are also present throughout the disc in much smaller quantities. For example, other fibrillar collagens such as type V and type XI are found in greatest concentrations in areas with high type I collagen and type II collagen, respectively [27]. Nonfibrillar collagens found in the disc include short, helical collagens, specifically types VI (up to 10% in the annulus and 15% in the nucleus), IX, and XII [27] (table 1). Proteoglycans comprise a small proportion of the outer AF, but become increasingly abundant as one moves toward the more hydrated central regions of the disc, accounting for 50% of the dry weight of the NP. Large proteoglycan aggregates consist of central hyaluronan molecules with multiple attached

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Fig. 5. Schematic of a proteoglycan molecule with a central core of hyaluronic acid and attached chains of aggrecan.

proteoglycan side chains predominantly of aggrecan. The connection between the proteoglycan side chains and the hyaluronan backbone is stabilized by small link proteins. The proteoglycan molecules contain numerous sulfated polysaccharide molecules leading to a strong net negative charge. It is this net negative charge that serves to attract water within the disc (fig. 5). The relative proportion of collagens and proteoglycans changes throughout life and with disc degeneration. Age-related changes lead to a decline in the amount of large proteoglycan aggregates in the NP, thus resulting in diminished water-binding capacity. In addition, there is an increase in the proportion of nonaggregated proteoglycans and a shift in the composition of sulfated polysaccaride side chains leading to the diminished structural properties of the disc [31]. The vertebral endplate is a specialized structure that contains both a bony and a cartilaginous portion. The bony endplate is made up of cortical bone, which is thicker around the periphery of the disc and thinner in the central region. Adjacent to the bony endplate are specialized capillaries, which are the primary source of nutrient exchange to the disc. The bony endplate is covered by a layer of hyaline cartilage that forms a barrier between the vertebral body and the disc and limits solute transport into and out of the disc. With age, the cartilaginous endplate undergoes progressive calcification, a process which diminishes its diffusional capacity and may lead to a nutritional crisis within the disc [32]. In addition to aging, a number of environmental and genetic factors have been linked to disc degeneration possibly by altering the disc by limiting endplate diffusion. Smoking, in particular, causes shrinkage of the vascular

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buds adjacent to the endplate that undoubtedly has a negative impact on disc nutrition [33–35]. The intervertebral disc is innervated only in its outermost portion. Small unmyelinated and encapsulated nerve endings have been found on the surface of the outer annulus, and small free nerve endings may penetrate the outermost layers of the annulus. The recurrent nerve of Luschka (sinuvertebral nerve), formed from small branches of the lumbar ventral ramus, provides this sensory innervation to the disc and is likely to be responsible for the discogenic pain. In addition to supplying the outer annulus, these branches also supply the posterior longitudinal ligament and ventral dural tube. Nerve endings have not been found in the inner annulus or NP region of normal human discs [27].

Disc Nutrition

Most of the normal intervertebral disc is avascular, depending on the diffusion for nutrient and waste exchange. Although small blood vessels can be found on the surface of the annulus and may penetrate a short distance into the outer layers of the disc, the central region has cells which can lie 6–8 mm from the closest blood supply, making the disc the largest avascular organ in the human body [27]. Unfortunately, the diffusional capacity of the disc is relatively poor even in the nonpathological state, and is further limited by aging and degenerative changes. Studies comparing the transport of small molecules into the disc in exercised and anesthetized dogs have shown no differences between the two groups, suggesting that simple diffusion rather than a physiological ‘pump’ mechanism is responsible for small molecule exchange within the disc [35]. These results have been confirmed in motionless [36] and mobilized spinal segments [37]. Recently it has been recognized that the supply of simple nutrients is a crucial factor regulating the density of disc cells. Diffusion chamber studies have suggested that glucose supply rather than oxygen is the major factor regulating cell viability within the disc [38]. Smoking probably promotes disc degeneration via a nutritional mechanism although a direct toxic insult to disc cells is also possible [39]. In the largely avascular environment of the disc, cells can survive with little oxygen [38]. However, under anaerobic conditions disc cells produce lactate as a by-product of metabolism (fig. 6), thus leading to an acidic pH. Normal disc tissue has a mildly acidic pH of 6.9–7.2, but under conditions of stress (degenerative disc disease) the disc pH can be as low as 6.1 [35]. It is known that an acidic pH leads to the inhibition of proteoglycan and collagen synthesis and may, therefore, contribute to matrix deterioration [40]. However, matrix degradation is likely to be dependant on many other factors, as suggested by

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Glucose ATP 1 ADP Glucose-6-phosphate 2 Fructose-6-phosphate ATP 3

ADP

Fructose-1,6-bisphosphate 4

5

Glyceraldehyde-3-phosphate 6

Dihydroxyacetone-phosphate

2 NAD ⫹Pi

2 NADH ⫹2 H 2 1,3-bis-phosphoglycerate ADP

7

ATP

Enzyme key 2 3-Phosphoglycerate 1. Hexokinase 2. Phosphoglucoisomerase 3. Phosphofructokinase 4. Aldolase 5. Triose-P-isomerase 6. Glyceraldehyde-3-Pdehydrogenase 7. Phosphoglycerate kinase 8. Phosphoglycerate mutase 9. Enolase 10. Pyruvate kinase 11. Lactate dehydrogenase

8 2 2-Phosphoglycerate 9 2 Phosphoenolpyruvate ADP 10

ATP

11

2 Pyruvate

2 Lactate

2 NADH ⫹ 2 H

2 NAD

Fig. 6. The glycolytic pathway. Anaerobic metabolism leads to lactate as a by-product, an acid that can lower the pH in the environment of disc cells.

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Compressive force causes disc bulging Vertebral body

Disc

Vertebral body

Fig. 7. Disc in compression (intervertebral disc between two vertebral bodies). Compressive forces on one side of the disc lead to disc bulging, with these forces being converted to tensile hoop stresses by the AF. On the opposite side, the disc fibers stretch.

studies demonstrating a poor correlation between measured oxygen or lactate levels and the grade of degeneration [41].

Disc Biomechanics

Intervertebral discs in humans have evolved to withstand the significant forces of an upright posture. When healthy, the normal disc can withstand forces greater than the surrounding bone, which fractures prior to the disruption of the disc. By dissipating the large compressive forces in the spine, generated as the result of musculature activity and vigorous physical situations, the disc serves to protect the surrounding spine from trauma. Forces up to 17,000 N have been estimated in lumbar discs during heavy lifting activities [42] (fig. 7). To dissipate these loads, the disc converts compressive forces to tensile stresses in the outer annulus by exerting a hydrostatic pressure via the interstitial fluid within the disc. However, the tensile properties vary within different regions of the annulus leading to a ‘biphasic phenomenon’ during loading. Because fibers in the anterior/outer regions are stiffer than those in posterolateral/inner regions of the annulus, the stiffer outer layers convert compressive loads into hoop stresses while the inner layers act as a ‘shock absorber’ [27]. The high tensile modulus of the normal annulus helps to prevent disc bulging. During aging and in the degenerating disc, the swelling pressure of the NP decreases and the stiffness of

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the AF increases [43, 44]. This results in poor load dissipation and increased stress transfer to the bony elements of the spine [43–46]. Several studies have shown that pathological loading of the spine may play a role in disc degeneration [47–49]. Hadjipavlou et al. [50] demonstrated disc degeneration following a 30-degree torsional injury to the spine in an animal model. This stress led to early degenerative changes in the disc including an increase in phospholipase A2 and a decrease in NP volume by 60–90 days. Following the onset of degeneration, increased levels of calcitonin gene-related peptide and vasoactive intestinal peptide were found within the ganglion, supporting the association of disc degeneration with spinal pain [51]. Researchers have shown, in animal models, that discs loaded statically are more prone to degeneration when compared to those loaded cyclically. However, a recent investigation by MacLean et al. [52] demonstrated that dynamic loading (12.6 N with a frequency of 0.2 Hz for 2 h) in rats led to decreased collagen type I and II gene expression in the AF, and an increase in the catabolic genes collagenase and stromelysin. In the NP, these mechanically induced changes in gene expression were less significant [52]. In addition, the magnitude and frequency of loading serve to affect the rate of degeneration [53]. For example, Kasra et al. [54] demonstrated that rabbit intervertebral disc cells in the AF and the NP respond to high-freqenecy (20 Hz) and highamplitude (1.7 MPa for annular cells and up to 3.0 MPa for nuclear cells) loading by increasing collagen synthesis and decreasing collagen degradation when subjected to 3 days of loading. Nuclear cells demonstrated significantly less collagen degradation as the load increased in amplitude from 1.0 to 3.0 MPa [54]. These contrasting studies suggest a role in mechanical loading for both degenerative and regenerative phenomenon in the disc suggesting a complex interaction between mechanical stress and metabolism within the disc. Segmental spinal instability associated with disc degeneration has been quoted as a cause of low back pain. Although there is some controversy in the literature regarding the relationship between disc degeneration, annular fissures and nonlinear segmental instability, several researchers have documented an increased range of axial rotation in degenerative discs. Mimura et al. [55] studied flexionextension forces in human lumbar cadaveric spines and found that the range of motion in flexion-extension decreased while axial rotation increased in degenerative spines. Krismer et al. [56] documented similar findings and correlated increased axial instability with fissure formation in the outer annulus.

Etiology of Disc Degeneration

Disc degeneration is undoubtedly a multifactorial process involving both environmental and genetic contributions. Some environmental factors thought

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Table 2. Factors that have been implicated to cause intervertebral disc degeneration.

Possible etiologies of disc degeneration 1. Heavy lifting 2. Vibration 3. Immobilization 4. Trauma (torsion) 5. Smoking 6. Diabetes 7. Vascular disease 8. Genetics 9. Infection

to contribute to disc degeneration include: demanding physical activities, such as heavy lifting; vibration (experienced while driving or operating machinery); immobilization, and repetitive torsional loads. Other environmental factors that affect disc metabolism include smoking, poor glycemic control and vascular disease [57] (table 2). In spite of the many environmental factors linked to disc degeneration, it is felt that genetic influences play the predominant role in early and perhaps more symptomatic disc disease. The genetic contribution is supported by family studies, case-control studies, and twin studies as well as the identification of certain genetic linkages and single gene defects leading to degenerative disc disease. Scapinelli [58] identified disc degeneration as a familial trait. In a case-control study, Matsui et al. [59] documented an increased incidence of disc degeneration among the family members of patients requiring lumbar surgery. Disc degeneration has been found to be significantly more prevalent in siblings of patients with disc degeneration than in random population samples [60]. Twin studies by Sambrook et al. [61] have shown a strong heritable component to disc degeneration in both the cervical and lumbar regions. Genetic studies have identified molecular defects contributing to disc degeneration in certain subgroups of patients. Videman et al. [62] compared MRI-documented disc degeneration in Finnish twins with alleles of the vitamin D receptor and found an increased risk of disc degeneration with two specific vitamin D receptor alleles. In a study of Japanese women, Kawaguchi et al. [63] found that women with smaller numbers of tandem repeats in the aggrecan gene had more severe disc degeneration. Annunen et al. [64] identified mutations in the type IX collagen ␣-2 gene which causes a single codon substitution (tryptophan for glutamine) leading to disc degeneration in 4% of the Finnish back pain population. Another defect in the ␣-3 chain of type IX collagen was shown to be associated with an elevated risk of disc degeneration [65]. Certain alleles of the matrix metalloproteinase-3 (MMP-3) gene

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have been linked with disc degeneration in elderly Japanese patients [69]. The environmental risk factor obesity has been shown to act synergistically with an allele of the COL9A3 gene, leading to a high rate of early degenerative disc disease [70].

Genesis of Back Pain

Exactly how disc degeneration relates to back pain is poorly understood. Some patients with minimal morphological changes in the disc complain of chronic back pain while others with significant changes note minimal symptoms. Many factors including structural changes in the spine, soluble mediators and nerve/vessel ingrowth into the outer annulus have all been hypothesized to be a cause of chronic spinal pain [57]. Studies have shown that mononuclear cells infiltrating along the margins of herniated discs express inflammatory mediators such as interleukin-1 (IL-1), intracellular adhesion molecule-1, lymphocyte function-associated antigen and basic fibroblast growth factor [68]. These mediators may contribute to persistent inflammation and pain and the induction of neovascularization [68]. Sang-Ho et al. [69] studied the expression of mRNA of various cytokines and chemokines in herniated lumbar discs and noted an association between the expression of IL-8 and radicular pain produced by back extension, suggesting IL-8 as a possible target for symptomatic treatment. Alterations in the disc can lead to changes in the alignment and the mechanical milieu of the vertebral bodies, facet joints, spinal ligaments and muscles, producing a complex and poorly understood biomechanical environment that may contribute to spinal pain. It is known that the degenerating disc is capable of producing a spectrum of cytokines and chemical mediators that are capable of stimulating pain in the surrounding nerve endings and inducing blood vessel and nerve ingrowth into annular defects in the disc [57]. For example, tumor necrosis factor-␣ (TNF-␣), a proinflammatory cytokine, has been shown to be a key pain mediator in neurogenic pain following disc herniation. Onda et al. [70] demonstrated, using electrophysiological testing, that an antibody to tumor necrosis factor-␣ partially blocked the neurogenic response suggesting that tumor necrosis factor-␣ blockage may have a therapeutic role in treating sciatica. In addition, Nygaard et al. [71] studied leukotriene and thromboxane levels in herniated intervertebral discs and found significantly higher levels of leukotrienes B4 and thromboxanes B2 in noncontained as compared to contained disc herniation; they suggested that these inflammatory mediators play a role in discogenic pain and sciatica and thus were possible targets for therapeutic intervention.

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Degenerative Events: Apoptosis, Degradative Enzymes and Inflammatory Cytokines

In order to develop rational therapies that can slow or reverse the degenerative process, it is first necessary to understand the molecular events leading to disc degeneration. In recent years, investigators have identified candidate molecules and cellular processes such as apoptosis, or programmed cell death, which appear to play a prominent role in disc degeneration. Although the current molecular understanding of disc degeneration is relatively crude, knowledge in this area is expanding rapidly. Cell viability has been shown to be affected by age, with a larger proportion of necrotic cells present in older individuals. In fetal and infantile intervertebral discs approximately 2% of NP cells are necrotic; this number increases steadily to 50% by adolescence and 80% in elderly humans [57]. In addition, apoptosis, or programmed cell death, appears to play a prominent role in the disc, with higher rates of apoptosis present in older individuals [72]. High rates of apoptosis have also been recognized in herniated disc fragments [73, 74]. This process appears to be related in part to the expression of Fas and the Fas ligand, which trigger an intercellular cascade leading to programmed cell death when a Fas-bearing cell comes into contact with a Fas ligand-carrying cell. Normal disc cells do not appear to express the Fas receptor, but do up-regulate this membrane-bound protein shortly after the onset of experimental disc degeneration [75]. As mentioned previously, static compression of intervertebral discs can lead to degeneration. A recent study by Ariga et al. [76] showed that static loading of mouse intervertebral discs resulted in higher numbers of apoptotic cells in the cartilaginous endplate; the number of apoptotic cells increased with the load. Inhibitors of mitogen-activated protein kinase and p38 significantly increased the number of apoptotic cells in the loaded discs. Certain growth factors, such as insulin-like growth factor-1 (IGF-1) and platelet-derived growth factor can exert an anti-apoptotic effect on cultured disc cells suggesting a possible mechanism for the apoptosis of cells within the disc [76, 77]. Some researchers have suggested that degeneration represents an alteration in the disc cellular homeostasis, with the balance tipping away from anabolic events and towards disc catabolism. Antoniou et al. [78] found decreased levels of aggrecan and type II collagen production by old and degenerated disc cells when compared to younger nondegenerated disc cells. Aguiar et al. [79] found that NP cells were able to up-regulate their production of proteoglycans when cocultured with notochordal cells, which are found in high concentrations only in younger humans. The effect appeared to be due to the presence of a soluble mediator produced by the notochordal NP cells and may explain the onset of degeneration shortly after these notochordal cells disappear within the disc.

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In addition to soluble mediators, direct cell-to-cell communication probably contributes to the behavior of the disc cells, as gap junctions and connexin proteins that have been identified within the disc [80]. A host of inflammatory, degradative, and catabolic factors have been identified that may play a role in disc degeneration. These include proteolytic and degradative enzymes, oxygen free radicals, nitric oxide, ILs, and prostaglandins. Proteolytic enzymes involved in disc degeneration include cathepsin, lysozyme, aggrecanase, and several MMPs [81–86]. A positive correlation between the level of MMPs-1, -2, -3 and -9 and the grade of disc degeneration has been documented [81, 83, 86]. Similarly, Melrose et al. [85] found higher levels of lysozyme in older and degenerative discs. Membrane-damaging oxygen-derived free radicals and nitric oxide have been observed in cultured disc cells [87]. Herniated discs have also been shown to express inducible nitric oxide synthetase and produce nitric oxide [82]. These molecules have the potential to cause direct chemical injury to cell membranes and matrix proteins. Collagens and fibronectin, for example, are known to undergo cleavage or form high-molecular-weight complexes following exposure to superoxides and other oxygen-derived free radicals [87] and may accumulate as lipoprotein complexes within the matrix [88, 89]. Other disc macromolecules undergo complex glycation reactions to form sugar-amino acid by-products that may interfere with normal cell-matrix interactions [90]. Inflammatory cytokines such as IL-1 have been shown to play a major role in articular cartilage degeneration and may play a role in disc degeneration as well. Cell culture experiments have demonstrated that rabbit disc cells increase their rate of caseinolytic activity in response to IL-1 [91]. IL-1 has been shown to decrease the rate of proteoglycan synthesis by the disc, an effect that could be blocked by an IL-1 receptor antagonist [92]. Other effects of IL-1 include inducing increased expression of stromelysin-1, a matrix degradation protease, and an increased production of prostaglandin E2, an inflammatory mediator [93]. Other mediators produced by disc cells include IL-6, nitric oxide, and prostaglandin E2 [94, 95]. Herniated disc fragments are capable of producing very high levels of phospholipase A2, an enzyme critical in the production of prostaglandins and leukotrienes, which are important mediators of inflammation and pain [96–98]. As the matrix of the disc undergoes degeneration, many of the macromolecules are only partially broken down. These nonfunctional molecules accumulate within the disc matrix and may be seen as lipofusion or amyloid by light microscopy [88] or dense granular material when viewed with the electron microscope [99–101]. Some by-products, such as fibronectin, appear to build up within the disc during degeneration. Of interest is the role of bioactive fragments of fibronectin which may promote tissue degeneration [102–104] (fig. 8).

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a

b

c Fig. 8. a Degenerative disc histology. b Degenerative changes in the AF with chondroid nests 16 weeks after injection of FN-f (the amino terminal fragment of fibronectin). c Higher powered micrograph of NP degeneration with a loss of cells and matrix disarray 4 weeks after FN-f injection.

Growth factor decline has also been implicated in disc degeneration. For instance, the IGF-1 receptor appears to decrease in older animals leading to a decrease in the IGF-1-dependent proteoglycan synthesis and perhaps the expression of an IGF-1-binding protein [105]. Therapeutic interventions with growth factors remain an active area of interest. Biological Approaches to Disc Degeneration

A thorough understanding of the molecular aspects of intervertebral disc degeneration is fundamental to the development of rational biological therapies

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Replication-deficient virus carrying a therapeutic gene

Growth factor RNA

Cell

Fig. 9. Schematic of virally mediated gene transfer. A desired gene is inserted into a viral carrier that is taken up by the cell. Viruses depend on host cells for replication; this process increases the expression of the desired gene.

aimed at slowing or stopping this process. A large number of factors have been elucidated that contribute to the process of degeneration and it is likely that the interplay of many different pathways contribute to the end result, making therapeutic intervention a challenge. However, by understanding the molecular basis of the disc degeneration, researchers hope to target specific steps in the degenerative process, thus producing a therapeutic benefit for patients with degenerative disc disease. Because growth factors and stimulatory cytokines can stimulate both cell division and metabolism, these molecules have been seen as good targets for therapeutic intervention. Epidermal growth factor and transforming growth factor-␤ (TGF-␤) were shown to produce a 5-fold increase in the metabolic activity of cultured NP cells [106], and were more effective than fibroblast growth factor or IGF-1. Osteogenic protein-1 has been shown to overcome the degradative effects of IL-1 on cultured disc cells [107]. TGF-␤ also has the added benefit of promoting the ‘chondrogenic phenotype’ by increasing the production of type II collagen and proteoglycan, an effect that is desirable in the inner regions of the disc [108]. Despite the promising results in vitro, direct injections of recombinant proteins into the disc does not appear to be a viable solution to disc degeneration due to the limited half-life of these molecules in vivo [109]. Gene therapy has generated a high level of interest for achieving a longterm solution to disc degeneration. Nishida et al. [108] transduced rabbit NP cells in vivo with an adenovirus carrying the TGF-␤ gene driven by a cytomegalovirus promotor and observed a 2-fold increase in proteoglycan

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production at the one week time point (fig. 9). Although encouraging, the authors noted that the long-term expression of a therapeutic gene might be difficult using the viral vector systems due to the possibility of immunological activity against viral antigens. Additionally, it is unclear whether a degenerating disc in metabolic disarray would be able to respond to growth factors in the same manner as the normal disc cells. Investigations are currently ongoing to determine the optimal genes and transduction mechanisms for gene therapy within the disc. Another attractive therapeutic option being investigated currently is the use of a cell-based therapy using either mature cells (chondrocytes or disc cells) or pluripotent cells (stem cells). Gruber et al. [110] successfully studied autologous disc cells transplanted into the intervertebral disc of the sand rat (Psammomys obesus). These authors demonstrated that autografted cells were able to exhibit morphologies similar to native disc cells and were able to survive at least 33 weeks in vivo. Others have suggested that stem cells, which are able to differentiate into multiple cell types, including chondrocytes, may be an ideal vehicle for cell-based therapy. These cells could be treated with therapeutic genes ex vivo prior to implantation and thus used to deliver therapeutic genes to the disc and participate in the repair process. Although encouraging, such strategies have yet to be successfully achieved in a reasonable model of degenerative disc disease. Early tissue engineering approaches have evaluated cellular scaffolds for the delivery of cells to the disc. The advantage of a cell scaffold is that the therapeutic cells are maintained at the implantation site and are provided with a three-dimensional environment necessary for division and migration within the disc [111]. Perka et al. [112] found that NP cells suspended in fibrin-alginate beads and fibrin beads were capable of proliferating and producing extracellular matrix. Lee et al. utilized cells transduced with the TGF-␤ gene in a ‘pellet culture’ system as an alternative to alginate bead microspheres and achieved a native cell phenotype capable of producing type II collagen and proteoglycan [111]. Some scaffolds may not be as effective as previously hoped. Alini et al. [113] demonstrated problems with proteoglycan retention when using scaffolds of type I collagen and hyaluronan seeded with bovine NP and AF cells. In contrast, Sato et al. [114] found that allografted AF cells placed in an atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS-scaffold) were able to proliferate, retain type II collagen mRNA, and actually decrease the narrowing of intervertebral disc space in Japanese white rabbits [115]. Much more research is needed to determine the role of cell scaffolds and cellular therapies in treating intervertebral disc degeneration.

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Conclusion

The intervertebral disc is a part of a complex mechanical system, pivotal in the dissipation of forces in the spinal column. Aging, environmental and molecular-genetic factors all contribute to the degeneration of the disc and its ultimate mechanical failure. Recently, researchers around the world have begun to understand the molecular basis of intervertebral disc degeneration. This understanding forms the basis for the design of biological therapies aimed at slowing or reversing the degenerative process. With time, these early efforts may lead to a shift in treatment options from those focusing on symptomatic relief to those aimed at correcting the underlying disease process. Although promising, these early attempts are far from achieving the goal of tissue regeneration. Much work remains to define the ability of gene- and cell-based strategies to produce a biologically desirable result and to apply these clinically for the benefit of patients. Fortunately, the underlying molecular basis of this ubiquitous disease process is rapidly becoming clear.

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Ohshima H, Urban JP: The effect of lactate and pH on proteoglycan and protein synthesis rates in the intervertebral disc. Spine 1992;17:1079–1082. Bartels EM, Fairbank JC, Winlove CP, Urban JP: Oxygen and lactate concentrations measured in vivo in the intervertebral discs of patients with scoliosis and back pain. Spine 1998;23:1–7. Cholewicki J, McGill SM, Norman RW: Lumbar spine loads during the lifting of extremely heavy weights. Med Sci Sports Exerc 1991;23:1179–1186. Acaroglu ER, Iatridis JC, Setton LA, Foster RJ, Mow VC, Weidenbaum M: Degeneration and aging affect the tensile behavior of human lumbar anulus fibrosus. Spine 1995;20:2690–2701. Panagiotacopulos ND, Knauss WG, Bloch R: On the mechanical properties of human intervertebral disc material. Biorheology 1979;16:317–330. Fujita Y, Duncan NA, Lotz JC: Radial tensile properties of the lumbar annulus fibrosus are site and degeneration dependent. J Orthop Res 1997;15:814–819. Ishihara H, Urban JP: Effects of low oxygen concentrations and metabolic inhibitors on proteoglycan and protein synthesis rates in the intervertebral disc. J Orthop Res 1999;17:829–835. Kroeber MW, Unglaub F, Wang H, et al: New in vivo animal model to create intervertebral disc degeneration and to investigate the effects of therapeutic strategies to stimulate disc regeneration. Spine 2002;27:2684–2690. Natarajan RN, Ke JH, Andersson GB: A model to study the disc degeneration process. Spine 1994;19:259–265. Sandover J: Dynamic loading as a possible source of low-back disorders. Spine 1983;8:652–658. Hadjipavlou AG, Simmons JW, Yang JP, et al: Torsional injury resulting in disc degeneration. I. An in vivo rabbit model. J Spinal Disord 1998;11:312–317. Hadjipavlou AG, Simmons JW, Yang JP, Bi LX, Simmons DJ, Necessary JT: Torsional injury resulting in disc degeneration in the rabbit. II. Associative changes in dorsal root ganglion and spinal cord neurotransmitter production. J Spinal Disord 1998;11:318–321. MacLean JJ, Lee CR, Grad S, Ito K, Alini M, Iatridis JC: Effects of immobilization and dynamic compression on intervertebral disc cell gene expression in vivo. Spine 2003;28:973–981. Ching CT, Chow DH, Yao FY, Holmes AD: The effect of cyclic compression on the mechanical properties of the intervertebral disc: An in vivo study in a rat tail model. Clin Biomech (Bristol, Avon) 2003;18:182–189. Kasra M, Goel V, Martin J, et al: Effect of dynamic hydrostatic pressure on rabbit intervertebral disc cells. J Orthop Res 2003;21:597–603. Mimura M, Panjabi MM, Oxland TR, Crisco JJ, Yamamoto I, Vasavada A: Disc degeneration affects the multidirectional flexibility of the lumbar spine. Spine 1994;19:1371–1380. Krismer M, Haid C, Behensky H, Kapfinger P, Landauer F, Rachbauer F: Motion in lumbar functional spine units during side bending and axial rotation moments depending on the degree of degeneration. Spine 2000;25:2020–2027. Buckwalter JA, Boden SD, Erye DR, Mow VC, Weidenbaum M: Intervertebral disk aging, degeneration, and herniation; in Buckwalter JA, Einhorn TA, Simon SR (eds): Orthopaedic Basic Science – Biology and Biomechanics of the Musculoskeletal System, ed 2. Rosemont, American Academy of Orthopaedic Surgeons, 2000, pp 557–566. Scapinelli R: Lumbar disc herniation in eight siblings with a positive family history for disc disease. Acta Orthop Belg 1993;59:371–376. Matsui H, Kanamori M, Ishihara H, Yudoh K, Naruse Y, Tsuji H: Familial predisposition for lumbar degenerative disc disease. A case-control study. 1998. Bijkerk C, Houwing-Duistermaat JJ, Valkenburg HA, et al: Heritabilities of radiologic osteoarthritis in peripheral joints and of disc degeneration of the spine. Arthritis Rheum 1999;42:1729–1735. Sambrook PN, MacGregor AJ, Spector TD: Genetic influences on cervical and lumbar disc degeneration: A magnetic resonance imaging study in twins. Arthritis Rheum 1999;42:366–372. Videman T, Leppavuori J, Kaprio J, et al: Intragenic polymorphisms of the vitamin D receptor gene associated with intervertebral disc degeneration. Spine 1998;23:2477–2485. Kawaguchi Y, Osada R, Kanamori M, et al: Association between an aggrecan gene polymorphism and lumbar disc degeneration. Spine 1999;24:2456–2460. Annunen S, Paassilta P, Lohiniva J, et al: An allele of COL9A2 associated with intervertebral disc disease. Science 1999;285:409–412.

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65 66

67

68 69 70

71 72 73 74 75 76 77 78

79 80

81 82

83

84 85 86

87

Paassilta P, Lohiniva J, Goring HH, et al: Identification of a novel common genetic risk factor for lumbar disk disease. JAMA 2001;285:1843–1849. Takahashi M, Haro H, Wakabayashi Y, Kawa-uchi T, Komori H, Shinomiya K: The association of degeneration of the intervertebral disc with 5a/6a polymorphism in the promoter of the human matrix metalloproteinase-3 gene. J Bone Joint Surg Br 2001;83:491–495. Solovieva S, Lohiniva J, Leino-Arjas P, et al: COL9A3 gene polymorphism and obesity in intervertebral disc degeneration of the lumbar spine: Evidence of gene-environment interaction. Spine 2002;27:2691–2696. Doita M, Kanatani T, Harada T, Mizuno K: Immunohistologic study of the ruptured intervertebral disc of the lumbar spine. Spine 1996;21:235–241. Sang-Ho A, Yoon-Woo C, et al: mRNA expression of cytokines and chemokines in herniated lumbar intervertebral discs. Spine 2002;27:911–917. Onda A, Yabuki S, Kikuchi S: Effects of neutralizing antibodies to tumor necrosis factor-alpha on nucleus pulposus-induced abnormal nociresponses in rat dorsal horn neurons. Spine 2003;28: 967–972. Nygaard O, Mellgren S, Osterud B: The inflammatory properties of contained and noncontained lumbar disc herniation. Spine 1997;22:2484–2488. Gruber HE, Hanley EN Jr: Analysis of aging and degeneration of the human intervertebral disc. Comparison of surgical specimens with normal controls. Spine 1998;23:751–757. Park JB, Chang H, Kim KW: Expression of Fas ligand and apoptosis of disc cells in herniated lumbar disc tissue. Spine 2001;26:618–621. Park JB, Kim KW, Han CW, Chang H: Expression of Fas receptor on disc cells in herniated lumbar disc tissue. Spine 2001;26:142–146. Anderson DG, Izzo MW, Hall DJ, et al: Comparative gene expression profiling of normal and degenerative discs: Analysis of a rabbit annular laceration model. Spine 2002;27:1291–1296. Ariga K, Yonenobu K, Nakase T, et al: Mechanical stress-induced apoptosis of endplate chondrocytes in organ-cultured mouse intervertebral discs. Spine 2003;28:1528–1533. Gruber HE, Norton HJ, Hanley EN Jr: Anti-apoptotic effects of IGF-1 and PDGF on human intervertebral disc cells in vitro. Spine 2000;25:2153–2157. Antoniou J, Steffen T, Nelson F, et al: The human lumbar intervertebral disc: Evidence for changes in the biosynthesis and denaturation of the extracellular matrix with growth, maturation, aging, and degeneration. J Clin Invest 1996;98:996–1003. Aguiar DJ, Johnson SL, Oegema TR: Notochordal cells interact with nucleus pulposus cells: Regulation of proteoglycan synthesis. Exp Cell Res 1999;246:129–137. Gruber HE, Ma D, Hanley EN Jr, Ingram J, Yamaguchi DT: Morphologic and molecular evidence for gap junctions and connexin 43 and 45 expression in annulus fibrosus cells from the human intervertebral disc. J Orthop Res 2001;19:985–989. Crean JK, Roberts S, Jaffray DC, Eisenstein SM, Duance VC: Matrix metalloproteinases in the human intervertebral disc: Role in disc degeneration and scoliosis. Spine 1997;22:2877–2884. Furusawa N, Baba H, Miyoshi N, et al: Herniation of cervical intervertebral disc: Immunohistochemical examination and measurement of nitric oxide production. Spine 2001;26: 1110–1116. Kanemoto M, Hukuda S, Komiya Y, Katsuura A, Nishioka J: Immunohistochemical study of matrix metalloproteinase-3 and tissue inhibitor of metalloproteinase-1 human intervertebral discs. Spine 1996;21:1–8. Konttinen YT, Kaapa E, Hukkanen M, et al: Cathepsin G in degenerating and healthy discal tissue. Clin Exp Rheumatol 1999;17:197–204. Melrose J, Ghosh P, Taylor TK: Lysozyme, a major low-molecular-weight cationic protein of the intervertebral disc, which increases with ageing and degeneration. Gerontology 1989;35:173–180. Roberts S, Caterson B, Menage J, Evans EH, Jaffray DC, Eisenstein SM: Matrix metalloproteinases and aggrecanase: Their role in disorders of the human intervertebral disc. Spine 2000;25: 3005–3013. Oegema T: The role of proteinases and other degradative mechanics in idiopathic low back pain; in Weinstein JN, Gordon SL (eds): Low Back Pain: A Scientific and Clinical Overview. Rosemont, American Academy of Orthopaedic Surgeons, 1996.

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88 Ishii T, Tsuji H, Sano A, Katoh Y, Matsui H, Terahata N: Histochemical and ultrastructural observations on brown degeneration of human intervertebral disc. J Orthop Res 1991;9:78–90. 89 Yasuma T, Koh S, Okamura T, Yamauchi Y: Histological changes in aging lumbar intervertebral discs. Their role in protrusions and prolapses. J Bone Joint Surg Am 1990;72:220–229. 90 Hormel SE, Eyre DR: Collagen in the ageing human intervertebral disc: An increase in covalently bound fluorophores and chromophores. Biochim Biophys Acta 1991;1078:243–250. 91 Shinmei M, Kikuchi T, Yamagishi M, Shimomura Y: The role of interleukin-1 on proteoglycan metabolism of rabbit annulus fibrosus cells cultured in vitro. Spine 1988;13:1284–1290. 92 Maeda S, Kokubun S: Changes with age in proteoglycan synthesis in cells cultured in vitro from the inner and outer rabbit annulus fibrosus. Responses to interleukin-1 and interleukin-1 receptor antagonist protein. Spine 2000;25:166–169. 93 Rannou F, Corvol MT, Hudry C, et al: Sensitivity of anulus fibrosus cells to interleukin 1 beta. Comparison with articular chondrocytes. Spine 2000;25:17–23. 94 Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Evans CH: Herniated cervical intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2. Spine 1995;20:2373–2378. 95 Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Donaldson WF III, Evans CH: Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2. Spine 1996;21:271–277. 96 Franson RC, Saal JS, Saal JA: Human disc phospholipase A2 is inflammatory. Spine 1992;17: S129–S132. 97 Gronblad M, Virri J, Tolonen J, et al: A controlled immunohistochemical study of inflammatory cells in disc herniation tissue. Spine 1994;19:2744–2751. 98 Saal JS, Franson RC, Dobrow R, Saal JA, White AH, Goldthwaite N: High levels of inflammatory phospholipase A2 activity in lumbar disc herniations. Spine 1990;15:674–678. 99 Trout JJ, Buckwalter JA, Moore KC: Ultrastructure of the human intervertebral disc. II. Cells of the nucleus pulposus. Anat Rec 1982;204:307–314. 100 Yasuma T, Makino E, Saito S, Inui M: Histological development of intervertebral disc herniation. J Bone Joint Surg Am 1986;68:1066–1072. 101 Yasuma T, Arai K, Suzuki F: Age-related phenomena in the lumbar intervertebral discs. Lipofuscin and amyloid deposition. Spine 1992;17:1194–1198. 102 Oegema TR Jr, Johnson SL, Aguiar DJ, Ogilvie JW: Fibronectin and its fragments increase with degeneration in the human intervertebral disc. Spine 2000;25:2742–2747. 103 Trout JJ, Buckwalter JA, Moore KC, Landas SK: Ultrastructure of the human intervertebral disc. I. Changes in notochordal cells with age. Tissue Cell 1982;14:359–369. 104 Hollander AP, Heathfield TF, Webber C, et al: Increased damage to type II collagen in osteoarthritic articular cartilage detected by a new immunoassay. J Clin Invest 1994;93:1722–1732. 105 Okuda S, Myoui A, Ariga K, Nakase T, Yonenobu K, Yoshikawa H: Mechanisms of age-related decline in insulin-like growth factor-I dependent proteoglycan synthesis in rat intervertebral disc cells. Spine 2001;26:2421–2426. 106 Thompson JP, Oegema TR Jr, Bradford DS: Stimulation of mature canine intervertebral disc by growth factors. Spine 1991;16:253–260. 107 Takegami K, Thonar EJ, An HS, Kamada H, Masuda K: Osteogenic protein-1 enhances matrix replenishment by intervertebral disc cells previously exposed to interleukin-1. Spine 2002;27:1318–1325. 108 Nishida K, Kang JD, Gilbertson LG, et al: Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: An in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene. Spine 1999;24:2419–2425. 109 Nishida K, Gilbertson LG, Robbins PD, Evans CH, Kang JD: Potential applications of gene therapy to the treatment of intervertebral disc disorders. 110 Gruber HE, Johnson TL, Leslie K, et al: Autologous intervertebral disc cell implantation: A model using Psammomys obesus, the sand rat. Spine 2002;27:1626–1633. 111 Sato M, Asazuma T, Ishihara M, et al: An experimental study of the regeneration of the intervertebral disc with an allograft of cultured annulus fibrosus cells using a tissue-engineering method. Spine 2003;28:548–553.

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112 Perka C, Arnold U, Spitzer RS, Lindenhayn K: The use of fibrin beads for tissue engineering and subsequential transplantation. Tissue Eng 2001;7:359–361. 113 Alini M, Li W, Markovic P, Aebi M, Spiro RC, Roughley PJ: The potential and limitations of a cell-seeded collagen/hyaluronan scaffold to engineer an intervertebral disc-like matrix. Spine 2003;28:446–454. 114 Sato M, Asazuma T, Ishihara M, et al: An atelocollagen honeycomb-shaped scaffold with a membrane seal (ACHMS-scaffold) for the culture of annulus fibrosus cells from an intervertebral disc. J Biomed Mater Res 2003;64A:248–256. 115 Yung LJ, Hall R, Pelinkovic D, et al: New use of a three-dimensional pellet culture system for human intervertebral disc cells: Initial characterization and potential use for tissue engineering. Spine 2001;26:2316–2322. 116 Buckwalter JA, Einhorn TA, Simon SR (eds): Orthopaedic Basic Science – Biology and Biomechanics of the Musculoskeletal System, ed 2. Rosemont, AAOS, 2000, p 550. 117 Ashton-Miller JA, Schultz AB: Biomechanics of the human spine; in Mow BC, Hayes WC (eds): Basic Orthopaedic Biomechanics. Philadelphia, Lippincott-Raven, 1997, pp 353–393.

D. Greg Anderson, MD Department of Orthopaedic Surgery Thomas Jefferson University 925 Chestnut St., 5th Floor Philadelphia, PA 19107 (USA) Tel. ⫹1 267 339 3623, E-Mail [email protected]

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Genetics of Degenerative Disc Disease Shekar N. Kurpad, Jason Lifshutz Department of Neurosurgery, Medical College of Wisconsin, Milwaukee, Wisc., USA

Introduction

Back and neck pain are some of the most common conditions for which patients seek medical attention. 80% of the population has reported this complaint at some point during their lifetime, with 5% having chronic pain [8, 14]. Back pain is the most frequent cause of activity limitation in patients under the age of 45 and is a common cause of disability and medical cost [8, 14]. While there are several causes of back pain, degenerative and mechanical disorders of the spine and intervertebral discs encompass the most common reasons. Traditionally, degeneration has been considered to be a mechanical phenomenon; current studies suggest that genetic and biochemical mechanisms may play a much larger role than is generally appreciated. Topics examined in this review include a brief description of the normal anatomy and biochemistry of the intervertebral disc. In addition, the disc complex is examined from a cellular, biochemical, and genetic basis. We conclude with a look toward the future, and how these new developments may be used in translational research in going from bench to bedside.

Biochemistry of the Intervertebral Disc

The adult intervertebral disc is an avascular fibrocartilaginous complex that links the adjacent vertebrae of the spine. Each disc is made of a gelatinous nucleus pulposus surrounded by a laminated annulus fibrosus [3, 5–8, 14]. There is no definitive interface between these two regions, and it is referred to in the literature as the ‘transition zone’ [8, 14]. The adult disc is comprised of

poorly characterized cells surrounded by an exhaustive extracellular matrix. There are generally two types of cells, fibrocytes in the outer annulus and chondrocytes in the remaining layers [3, 5–8, 14], which are thought to be better equipped to withstand the avascular environment of the disc. The role of these cell populations is to synthesize, maintain and repair the matrix of the disc. The matrix of the disc is a framework of polar macromolecules bound with water, mainly collagen fibrils and proteoglycans, which are glycosaminoglycans such as chondroitin sulfate and keratin sulfate attached to a protein core. Collagens are important in conferring tensile strength to the disc, and appear to play a role in the genetic predisposition toward spinal degeneration. They make up approximately 70% of the annulus but only 20% of the nucleus pulposus. In the annulus, collagen is found in tightly packed fibrils arranged in specific lamellae. The majority of collagen found in this region is type I, with smaller amounts of types II, III, V, and IX also being present. Within the nucleus pulposus, 85% of the collagen is of type II, with smaller amounts of VI and IX also being found [3, 7, 8, 11, 14]. Proteoglycans absorb water, conferring both stiffness and resilience to the disc; they are present within the lamellae of the collagen fibrils and are found in the nucleus pulposus in their greatest concentrations. The proteoglycan aggregate is made up of a central glycosaminoglycan hyaluronate filament that is attached to various proteoglycans via linker proteins [7, 8, 14]. Intervertebral discs are situated between the cartilaginous endplates of adjacent vertebrae. The endplates are initially composed of hyaline cartilage, produced by chondrocytes; later in life, this is replaced in part by calcified cartilage. Collagen fibers of the annulus attach directly to the endplates. This is a site susceptible to mechanical failure, especially when exposed to shear forces [8, 11, 14]. At birth the intervertebral discs have an abundant vascular supply. As one ages, the discs generally lose this vascular supply (usually by 2 years of age), a process accelerated by the degeneration and calcification of the vertebral endplate, and subsequently derive their nutrients and eliminate their waste through the process of diffusion, which is driven by the high osmotic pressure within the discs plus the hydrostatic pressure acting on the discs. This process requires a high intrinsic water content, provided by the proteoglycan attraction.

Molecular Biology of Disc Degeneration

Diffusion The main characteristic of disc degeneration is the loss of the hydrostatic properties of the disc. At birth, the nucleus pulposus contains 85–90% water. With aging, this percentage drops to approximately 70%. This drop in water content occurs with changes in the proteoglycan extracellular matrix of the disc.

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A decrease in the turgor of the disc causes the disc to lose its ability to resist compression and provides resilience to the spine. The diffusion of disc homeostatic processes may become impaired in this dehydrated state due to quantitative and qualitative changes in the extracellular matrix [8, 14]. Diffusion may become impaired by other means that decrease blood flow to the spine/disc. Blood flow to the disc space regresses as aging proceeds and other concomitant factors such as diabetes and vascular disease may impair the vascular supply to the vertebral endplate [8, 14]. Calcification of the vertebral endplate may have a detrimental effect on the diffusive processes necessary for the nutrition of resident cells. Lack of adequate oxygen supply to cellular structures of the disc may lead to anaerobic glycolysis, lactate production, a decrease in the pH, and ultimately breakdown and damage to the extracellular matrix. This pH change causes poor hydrostatic pressure, and hence further deterioration in the diffusion process, with its related consequences [3, 5, 7, 8, 14]. Cellular Activity During the early phases of degeneration, there is thought to be a proliferation of cells within the annulus, with a metaplasia of these cells into chondrocytes [7, 8, 14]. As degeneration continues, diffusion through the disc declines, leading to anaerobic metabolism and cell death. With the loss of support cells, synthesis and maintenance of the disc matrix disappears with decreased water content and hence a decrease in the diffusion of essential nutrients. Biochemical Changes Proteoglycans Both quantitative and qualitative changes are seen in the extracellular matrix of the aging disc as it degenerates. First, the amount of proteoglycans decreases in the degenerating disc. This decrease in proteoglycans is generally an early sign of degeneration. The mucopolysaccharide complexes in young discs are made up primarily of chondroitin sulfate A and C side chains, which are strongly hydrophilic [8, 14]. As the disc ages, these large molecules break down into smaller ones, such as chondroitin sulfate B and keratin sulfate, which do not have the water-storing capacity of types A and C chondroitin sulfate, and subsequently lead to disc dehydration [8, 14]. Keratin sulfate, in particular, has been found to be a marker of disc degeneration in surgical and pathological specimens [5]. Disc dehydration eventually results in impaired diffusion and further disc degeneration [14]. Collagen As previously mentioned, the intervertebral disc is composed mostly of type I and type II collagen [3, 5–8, 14]. Type I collagen is found primarily in

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the annulus fibrosus and type II collagen in the nucleus pulposus. In early degeneration, the location and types of collagen do not change. However, there is an increase in the amount of collagen found within both the annulus and nucleus. There also may be an increase in collagen type III, V, and VI [3, 7, 8, 14]. As degeneration continues, there are qualitative changes seen in the type of collagen in various portions of the disc. For example, there is an increase in the amount of collagen type I within the nucleus. In addition, there is a loss of collagen type II at the endplates. Further changes that are seen include the formation of collagen types IV and X, and changes in the post-translational modification of these new fibrils. These larger collagen fibrils are thought to be weaker than their narrow counterparts, which may lead to tearing and disruption [8, 14]. Young and healthy discs have been found to have active matrix formation and denaturation of collagen type II. Studies on the role of collagen in disc degeneration show a decrease in the levels of aggrecan and type II procollagen formation and a general increase in type II collagen degeneration and type I synthesis. Type IX collagen has recently been reported to be present in both the aging and degenerated disc, whose formation may represent a compensatory repair process. A molecular defect in type IX collagen has been discovered as a contributing factor toward disc degeneration, specifically a conversion of the codon for glutamate to tryptophan in the COL9A2 gene. This genetic polymorphism was found in approximately 10% of patients with disc disease, and MRI correlated an association of the genetic defect with radial tears within the disc. In addition, another genetic mutation in collagen formation, the TRP3 allele, has been found in 12% of patients studied with disc degeneration. Inflammation There are several inflammatory mediators which are present within the degenerative disc. Some of those that are present include nitric oxide, interleukin-6 (IL-6), IL-8 [2] (which has been associated with radiculopathy), prostaglandin E2, and a family of enzymes known as matrix metalloproteinases (MMPs) [3–13]. IL-6 and Prostaglandin E2 seem to exert their effects by inhibiting proteoglycan synthesis. They appear to be under the influential control of IL-1, which is both a central mediator of the inflammatory process, and a direct toxin to the proteoglycan matrix. The principal cause of the release of IL-1 is yet to be determined [4, 7, 9]. The role of extracellular matrix degeneration in the disc disease process is becoming better understood. MMPs are proteinases that degrade at least one component of the extracellular matrix; they are secreted in a latent form and

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require activation for proteolytic activity. Their activity is inhibited by specific tissue inhibitors. This family of enzymes can be divided into four groups; collagenases, stromelysins, gelatinases and membrane metalloproteinases [3, 4, 7, 8, 14]. Recent studies suggest that this family of proteases, in particular MMP-1, MMP-2, MMP-3 and MMP-9, play a significant role in the degradation and degeneration of the intervertebral disc [3–5, 7–12]. Recent studies showed that increases in the mRNA in MMP-1 and MMP-3 have been reported in cell populations of discs cultured with IL-1, IL-12 and tumor necrosis factor-␣. Further evidence of the role of MMPs is demonstrated in their activity in the degeneration of aggrecan in degenerated discs. Finally, changes in the role of cathepsins, fibromodulin, and fibronectin are being described and elucidated in the degenerative disease process [8, 14]. A therapeutic goal is to find potential inhibitors of these enzymes. Some experimental work has targeted the enzymes with specifically designed inhibitors. Other inhibitors being investigated include hydroxamic acid derivatives, tetracyclines and quinolones [8, 14]. Vertebral Endplates There are a variety of biological changes that occur in the degeneration of the vertebral endplates, which contribute, at least in part, to this disease phenomenon. As the endplate ages, it becomes calcified and replaced by bone. This calcification and bone formation impedes the vascular supply to the disc and accelerates the negative diffusion process described above. In addition, these bony changes within the endplate lead to an unequal distribution of loadsharing forces across the disc complex, which further accelerates the degenerative process. There is currently a paucity of biological study on endplate changes in degenerative disease. However, recent work has described differences in the proteoglycan composition on degenerated endplates, the contribution of MMP-3 in endplate degeneration, and a possible role for apoptosis in this disease process.

Genetics in Disc Disease

The role of genetics in degenerative disc disease is still largely unknown. Any genetic defect affecting collagen synthesis, proteoglycan synthesis, or growth and development of resident support cells (e.g., chondrocytes) would be expected to impact the rate of disc degeneration. As described above, Annunen et al. reported on the role of the COL9A2 gene in disc degeneration in a cohort of patients in Finland. In their report, this gene, which codes for a portion of the type IX collagen of the disc, was screened and found to have common polymorphisms in patients with intervertebral disc disease, associated with

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abnormal formation of the collagen 3 chain. Gruber and Hanley have described the role of apoptosis in both age and degenerative changes which related to a decrease in the amount of cells in the disc, and it appears likely that genetic factors could contribute toward a susceptibility to this process. Fibroblast-derived growth factors may regulate proteolytic activity in herniated discs. Nagano et al. studied degenerated disc in the rat model and showed that that normal structures were replaced with scattered chondrocytes with fibroblast growth factor-like activity and receptors [7]. New studies of the canine disc have demonstrated that both epidermal growth factor and transforming growth factor are associated with matrix synthesis and cell proliferation within the disc, and could represent another genetic risk factor if such factors are deficient.

Conclusion

Degenerative spine disease is a common problem for which neurosurgical patients seek treatment. New technology and advances in basic science has led to a better and more thorough understanding of the molecular biology of this disease process. With this new information, we may be able to develop novel and minimally invasive therapeutics for this very common disease entity. The role of genetics has until recently been underemphasized in the etiology of disc disease, but as more genetic influences are recognized, the number of targets for in vivo and ex vivo gene transfer will increase and new therapies will become available.

References 1 2 3 4 5 6 7 8

Ahn S-H, et al: mRNA expression of cytokines and chemokines in herniated lumbar intervertebral discs. Spine 2002;27:911–917. Burke JG, et al: Spontaneous production of monocyte chemoattractant protein-1 and interleukin-8 by the human lumbar intervertebral disc. Spine 2002;27:1402–1407. Crean JKG, et al: Matrix metalloproteinases in the human intervertebral disc: Role in disc degeneration and scoliosis. Spine 1997;22:2877–2884. Doita M, et al: Influence of macrophage infiltration of herniated disc tissue on the production of matrix metalloproteinases leading to disc resorption. Spine 2001;26:1522–1527. Fujita K, et al: Neutral proteinases in human intervertebral disc – Role in degeneration and probable origin. Spine 1993;18:1766–1773. Furusawa N, Baba H, et al: Herniation of cervical intervertebral disc – Immunohistochemical examination and measurement of nitric oxide production. Spine 2001;26:1110–1116. Goupille P, Jayson M, et al: Matrix metalloproteinases: The clue to intervertebral disc degeneration? Spine 1998;23:1612–1626. Guiot B, Fessler R: Molecular biology of degenerative disc disease. Neurosurgery 2000;47: 1034–1040.

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9

10 11 12 13 14

Kang J, et al: Toward a biochemical understanding of human intervertebral disc degeneration and herniation – Contributions of nitric oxide, interleukins, prostaglandin E2 and matrix metalloproteinases. Spine 1997;22:1065–1073. Matsui Y, et al: The involvement of matrix metalloproteinases and inflammation in lumbar disc herniation. Spine 1998;23:863–869. Nemoto O, et al: Matrix metalloproteinase-3 production by human degenerated intervertebral disc. J Spinal Disord 1997;10:493–498. Roberts S, Caterson B, et al: Matrix metalloproteinases and aggrecanase – Their role in disorders of the human intervertebral disc. Spine 2001;25:3005–3013. Takao T, Iwaki T: A comparative study of localization of heat shock protein 27 and heat shock protein 72 in the developmental and degenerative intervertebral discs. Spine 2002;27:361–368. Vaccaro A, Betz R, Zeidman S (eds): Principles and Practice of Spine Surgery. Mosby Publisher, 2003, chaps 6, 28.

Shekar N. Kurpad, MD, PhD Department of Neurosurgery, Medical College of Wisconsin 9200 West Wisconsin Avenue, Milwaukee, WI 53226 (USA) Tel. ⫹1 414 805 3666, E-Mail [email protected]

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Gene Therapy for Degenerative Disc Disease Joseph Kim, Lars G. Gilbertson, James D. Kang Department of Orthopaedic Surgery, University of Pittsburgh Medical Center, Pittsburgh, Pa., USA

Introduction

Degenerative disc disease (DDD) is a chronic process that can clinically manifest in multiple disorders, such as idiopathic back pain, disc herniation, radiculopathy, myelopathy, and spinal stenosis. It is a significant source of patient pain and morbidity, utilizing a large portion of health care resources [2, 3, 5, 15, 34]. The available treatment options for the clinical manifestations of DDD include conservative measures such as bed rest, anti-inflammatory drugs, analgesia, and physical therapy. This approach is usually effective in alleviating symptoms within 2 months in the majority of cases. However, when conservative methods fail, invasive surgical procedures such as discectomy, instrumentation, or fusion, with their inherent complication risks and expense, may be required. These treatment modalities focus on the clinical symptoms of intervertebral disc (IVD) degeneration without addressing the underlying pathological processes occurring early in the course of degeneration. However, recent advances in molecular biology may result in the development of novel therapies that target the ongoing physiological changes that occur in this disease. Although the pathophysiology of DDD is not completely understood, an insult to the disc or its supporting structures initially leads to a cascade of cellular changes that may promote either healing or further disc degeneration. One major contributing factor includes the progressive decline in aggrecan, the primary proteoglycan of the nucleus pulposus [1, 7, 24]. At the biochemical level, aggrecan homeostasis is altered by various combinations of decreased synthesis and increased breakdown. Kang et al. [13] demonstrated increased levels of matrix metalloproteinases in degenerated human discs compared to

normal, nondegenerated controls. These enzymes are known to contribute to net proteoglycan loss by increasing its degradation. With reductions in proteoglycan content of the intervertebral matrix, the nucleus pulposus dehydrates, decreasing both disc height and its load-bearing capacity [8, 32, 33]. This may directly affect biomechanical function by altering the loads experienced by the facet joints, leading to degenerative changes. Although disc degeneration most probably evolves in response to a complex interplay of multiple biochemical and biomechanical factors [10], the ability to restore proteoglycan content may have therapeutic benefit by increasing disc hydration and potentially improving biomechanics. The ability to increase proteoglycan synthesis in the IVD was demonstrated by Thompson et al. [30] who showed that the exogenous application of human transforming growth factor (TGF)-1 to canine disc tissue in culture stimulated in vitro proteoglycan synthesis. The authors suggested that growth factors might be useful for the treatment of disc degeneration. Subsequent studies with other growth factors such as insulin-like growth factor-1 (IGF-1), bone morphogenic protein-2 (BMP-2), and osteogenic protein-1 also exhibited the ability to up-regulate proteoglycan content in IVD cells [23, 29]. However, due to the relatively brief half-life of these factors, practical application of growth factor therapy to chronic conditions such as DDD would necessitate repeated administrations. Consequently, efforts were directed at developing approaches to induce endogenous synthesis of growth factors via gene therapy such that genetically modified disc cells manufacture the desired growth factors on a continuous basis.

Overview of Gene Therapy

The definition of gene therapy has become quite broad. The term was previously used to describe replacement of a defective gene with a functional copy by means of gene transfer. The diseases originally targeted for gene therapy were classic, heritable genetic disorders. The term now defines therapy involving the transfer of genes encoding therapeutic proteins into cells to treat any disease [26]. Genetically altered cells are made into factories producing disease-altering proteins. These proteins affect not only the metabolism of the cells from which they were made, but they can also affect the metabolism of adjacent nongenetically altered cells via paracrine mechanisms (fig. 1). Successful gene therapy for DDD will depend on efficient transfer of genes to target cells with sustained expression. With few exceptions, naked DNA is not taken up and expressed by cells and consequently, vectors are necessary to package and insert genes into cells in such a way that the genetic

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DNA coding for growth factor

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Fig. 1. The DNA encoding the growth factor of interest is constructed into a viral vector that is rendered incapable of replication. The vector is then exposed to host cells, attaches to their surface, and is then internalized. The released genetic information can then either travel to the nucleus, where it may become integrated into the host genome or remain episomal. It then commandeers the normal protein-making machinery of the cell and produces large quantities of the transgene.

information can be expressed. The two broad categories of vectors are viral and nonviral. The most commonly used nonviral vectors are liposomes. These phospholipid vesicles deliver genetic material into a cell by fusing with the cell’s phospholipid membrane. Liposome vectors are simple, inexpensive, and safe. Their drawbacks are transient expression of the transgene, cytotoxicity at higher concentrations, and low efficiency of transfection. Other nonviral methods of gene delivery include DNA-ligand complexes and the biolistics or penetration of cells with a ‘gene gun.’ These vectors are nonpathogenic and relatively inexpensive to construct. However, the overall transfection efficiency of nonviral vectors is generally inferior to that of viral-mediated gene transfer. Thus, most current studies involving gene therapy employ viral vectors. The most commonly used viral vectors are retroviruses, herpes simplex viruses, adeno-associated viruses, and adenoviruses; although, there are likely to be many other naturally occurring viruses which could be adapted for gene transfer. Viruses are frequently rendered incapable of replication prior to gene therapy application in an effort to make them less pathogenic. The various viral vectors and their advantages and disadvantages are discussed elsewhere in this volume, and will not be discussed further.

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There are two fundamental approaches to delivering exogenous genes with vectors to target cells within the body. The first is the direct, in vivo method in which the gene-carrying vector is directly injected into the patient. The second approach, known as ex vivo gene therapy, involves removing target cells from the body, genetically altering them in vitro, and then reimplanting them in the body. There are advantages and disadvantages to both of these approaches that depend on the anatomy and physiology of the target organs, the pathophysiology of the disease being treated, the vector of choice, and safety considerations [9].

Biology of the IVD

The IVD is an avascular organ consisting of nucleus pulposus cells scattered within an extracellular matrix. This gel-like inner core is encircled by an outer annulus fibrosis. The highly differentiated, nondividing cells of the nucleus pulposus are responsible for matrix synthesis. The matrix of a healthy nucleus pulposus is normally rich in proteoglycans and type II collagen, with a water content of over 85% by volume in juveniles, decreasing to approximately 70–75% in adults, and decreasing even further with aging and degeneration [11, 12]. This progressive loss of matrix and water content reflects the inability of the IVD for self-repair, as demonstrated by Bradford et al. [6]. The avascular disc receives the majority of its nutrition via passive diffusion through the cartilaginous endplates. The lack of a direct vascular supply results in low oxygen tension within the disc and causes the cells of the nucleus pulposus to undergo anaerobic metabolism. The ensuing high lactate concentration and subsequent low environmental pH most likely inhibit matrix repair. The avascularity of the IVD, though limiting its potential for repair and regeneration, does confer a distinct advantage in the context of gene therapy application. Early research attempting to characterize the IVD demonstrated an autoimmune response when subjects were exposed to their own nucleus pulposus tissue, suggesting that the IVD is an immune-privileged site exempt from prior exposure to the host immune system [4].

Adenoviral (Ad) Vectors for Gene Therapy to the IVD

Vectors based on adenoviruses have been frequently used in gene therapy studies for the IVD due to their ability to efficiently transduce highly differentiated, nondividing cells such as the cells of the nucleus pulposus. However, successful gene therapy depends not only on efficient gene transfer, but also on the expression of transgene for sufficiently long periods of time. The duration

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of gene expression following adenoviral (Ad) transfer to an immunocompetent animal is limited in most organs and tissues by immune reactions to viral proteins and to foreign proteins encoded by the transgene [17, 31, 36]. Specifically, expression for longer than 12 weeks has been difficult to achieve in musculoskeletal tissues following Ad delivery due to brisk immune responses. On the other hand, sustained gene expression can occur for longer periods of time in an immune-privileged site such as the IVD. Kang et al. [14] demonstrated positive expression of the marker genes lacZ and luciferase in the rabbit lumbar disc one year after adenovirus-mediated transduction. A corollary experiment attempting to characterize the immune response in the same study showed no production of neutralizing antibody to viral proteins in 3 of 6 rabbits after intradiscal injections of Ad vectors carrying the luciferase marker gene. The positive immune response in the other 3 rabbits was presumably due to the leakage of virus from an injected disc. Importantly, all 6 rabbits, including the 3 with circulating antibodies, had positive transgene expression several weeks after injections. On histological review, there was no evidence of cellular infiltration, increased vascularity, fibrosis, or other hallmarks of an immune response. Furthermore, intradiscal expression of luciferase was apparent for up to at least 42 days in rabbits whose immune system had been deliberately primed by subcutaneous inoculations with Ad proteins 2 weeks prior to intradiscal vector-gene injections (fig. 2). This implied that circulating antibodies do not reach the IVD in significant quantities to exert an immune response against the transduced disc cells. These findings further confirmed the IVD as being an immune-privileged organ and demonstrated the feasibility of using Ad vectors to achieve efficient, sustained expression of foreign genes.

Comparative Studies of Intradiscal Gene Therapy

With the success of in vitro studies demonstrating an increase in proteoglycan synthesis in IVD cells treated with the exogenous administration of growth factors, efforts were directed towards stimulating endogenous synthesis of these proteins by IVD cells with the use of gene therapy. Nishida et al. [22] reported the first successful in vivo gene transfer to the IVD in 1998, using an Ad vector to deliver the lacZ marker gene to the rabbit lumbar disc. The authors were able to demonstrate sustained transgene production with no significant reduction in the expression for up to 3 months after transduction (fig. 3). Followup studies revealed evidence of continued foreign gene expression even at one year [14]. Notably, the rabbits used in these studies showed no signs of systemic illness in response to the Ad vector and its transgene synthesis. In addition, no histological changes suggesting a cellular immune response were observed.

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Fig. 2. a–c Plots on the left show sequential production of specific antibodies for Ad proteins in peripheral blood. Bar charts on the right show luciferase activity at 42 days postintradiscal injection of Ad-luciferase. a In Group A, 3 rabbits produced little or no antibody to Ad proteins in the peripheral blood after injection of Ad-luciferase into the lumbar IVD. In the remaining 3 rabbits, antibody was produced within 3 weeks, presumably due to the leakage of virus from injected discs. b In Group B, all rabbits exhibited significantly increased production of antibody within 2 weeks after simultaneous subcutaneous and intradiscal injection of Ad-luciferase. c In Group C, the rabbits were immunized by subcutaneous injections of Ad-luciferase 2 weeks prior to the intradiscal gene therapy. All rabbits exhibited increased production of antibody in the peripheral blood by the time of the intradiscal injection. a–c As shown in the bar charts (right), all rabbits from the three groups exhibited significant amounts of intradiscal transgene expression. There was no correlation between the neutralizing antibody titer and intradiscal transgene expression at 6 weeks postintradiscal injection by Pearson correlation analysis (p 0.395).

Encouraged by these results with marker proteins, successful in vivo transduction of the IVD with a putative therapeutic gene was soon accomplished [21]. Using Ad vector, the gene for human TGF-1 was delivered. This study demonstrated a 30-fold increase in active TGF-1 synthesis and a 5-fold increase in total TGF-1 production in discs injected with the Ad-growth factor construct (fig. 4). Biological modulation was also documented by a 100%

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increase in proteoglycan synthesis (fig. 5). Assays for TGF-1 production and proteoglycan synthesis were performed with experimental discs that had been injected with Ad vector carrying only the luciferase marker gene. These viral control discs demonstrated no increase in production of TGF-1 or proteoglycan, indicating that the increases in the TGF-1 experimental group were a result of transgene expression and not a nonspecific response to the Ad vector. As in the previous studies, no signs of local or systemic immune response were noted. Additional in vitro studies with cultured human nucleus pulposus cells yielded similar results. Successful transduction of the lacZ marker gene delivered via Ad vectors was achieved with human cells from degenerated discs [19]. Similar experiments with retroviral delivery of marker genes resulted in a smaller percentage of transduction [25], perhaps due to the minimal mitotic activity of the IVD cells. The response of human cells from degenerated discs to Ad-mediated delivery of TGF-1 was assessed; increased expression of TGF-1, as well as increased proteoglycan and collagen synthesis, was demonstrated in cells receiving gene therapy as compared to controls [20]. Of note, cells receiving the Ad-TGF-1 construct showed increased proteoglycan and collagen synthesis when compared to cells receiving exogenous TGF-1 protein, presumably in response to the sustained expression of this growth factor. Interestingly, the viral dose required to increase proteoglycan synthesis was significantly less than that required for 100% transduction of the cells, perhaps highlighting the ability of a transduced cell to influence the biological activity of nongenetically altered neighboring cells. The concept that successfully transduced cells appear to exert a paracrine-like effect on their nontransduced neighboring cells implies that significant alteration in protein synthesis can be achieved with a small number of transduced cells [9]. A better understanding of this paracrine effect with TGF-1 gene transfer may enable the use of decreased viral loads to achieve a therapeutic effect, thereby minimizing potential viral toxicity. These experiments were also performed with a viral control, which further established that the increase in biological activity was the result of the delivered genetic material and not of the Ad vector. Subsequent in vitro studies with other growth factors such as BMP-2 and IGF-1 documented the potential of Ad delivery of these factors to increase the proteoglycan synthesis in a viral dose-dependent manner [18, 35]. Tissue inhibitor of metalloproteinase-1 also demonstrated the same ability [34] following Ad vector delivery. Tissue inhibitor of metalloproteinase-1 is an endogenous inhibitor of matrix metalloproteinases, which are enzymes capable of degrading the extracellular matrix of the IVD [27]. This finding established a second gene therapy strategy to modify the disrupted balance of synthesis and catabolism occurring in the degenerated IVD, namely, inhibition of

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Fig. 4. a Active TGF-1 production in rabbit nucleus pulposus tissue one and 6 weeks after in vivo injection of Ad-TGF-1, as compared to intact control discs and discs injected with saline and Ad-luciferase. b Total (active and latent) TGF-1 production in rabbit disc tissue one and 6 weeks after in vivo injection. There were no significant differences in either active or total TGF-1 production at one week and 6 weeks, indicating that the therapeutic gene expression was sustained. * A significant increase over corresponding intact, saline, and viral (Ad-luciferase) control groups (p 0.05).

matrix degradation with ensuing net increases or stabilization of proteoglycan content. Considering the potential adverse effects of viral vectors, studies have been undertaken to develop strategies to minimize viral loads while maintaining the same biological effects. Experiments with combination gene therapy involving TGF-1, IGF-1, and BMP-2 suggested that these growth factors are synergistic in amplifying matrix synthesis [18]. Ad delivery of a single growth factor increased proteoglycan synthesis by a range of 180–295%, whereas combination gene therapy with two agents resulted in increases of 322–398%. When all three growth factors were combined, proteoglycan synthesis was increased by 471% (fig. 6). It remains to be determined if combination gene Fig. 3. a–g Qualitative analysis of intradiscal lacZ transgene expression up to and including one year after injection of Ad-lacZ into lumbar intervertebral discs of adult New Zealand white rabbits. Serial histological sections were stained with X-Gal and counter-stained with eosin. Representative sections of lumbar discs at 3 weeks (a, b), 6 weeks (c, d), and 24 weeks (e, f ) postinjection are shown. All of the discs injected with Ad-LacZ exhibited positive X-Gal staining. [Original magnifications: a, c, e. 40; b, d, f. 200.] At 52 weeks postinjection, positive X-Gal staining was observed in the discs from two of three rabbits. However, the intensity of positive staining was less than in discs from the other time periods (g). [Original magnification: g. 600].

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Ad/TGF-1

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therapy with both an anabolic growth factor and a catabolic inhibitor such as tissue inhibitor of metalloproteinase-1 will have a similar synergistic effect.

Areas of Ongoing Research

For the continued progress of gene therapy for DDD toward successful human clinical trials, it is critical to rigorously test the proposed gene therapy strategies in animal models of disc degeneration that closely simulate the human condition. A number of models have been proposed. Disc degeneration occurs spontaneously in some species, such as the nonchondrodystrophic beagle and the sand rat [28]. Other species require artificial interventions to bring about degenerative changes within a reasonable time frame. The annular stab model of degeneration in the New Zealand white rabbit has been well described in the literature by Lipson and Muir [16]. In previous gene therapy studies with this model, our group found that a 3 mm incision of the anterior annulus allowed escape of nuclear material from the disc, with subsequent loss of viral

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Fig. 6. Proteoglycan synthesis in human intervertebral disc cells treated with different combinations of therapeutic Ad vectors (Ad-TGF-1, Ad-IGF-1, Ad-BMP-2). All groups showed significant increase in synthesis compared to saline and viral (Ad-luciferase) control groups (* p 0.05).

injections directed at the nucleus pulposus. There was also concern that degenerative changes induced by the full thickness 3 mm annular incision were too abrupt, in contrast to the gradual changes that occur in the human condition. For these reasons, we modified this technique to produce a puncture injury using a 16-gauge hypodermic needle. Extensive MRI and histological data have shown that the needle puncture model produces gradual and consistent degenerative changes that closely parallel human disc degeneration. The MRI analysis revealed progressive loss of mean nucleus pulposus signal intensity of stabbed lumbar discs as a function of time from puncture surgery (fig. 7). Importantly, there was no MRI evidence of spontaneous recovery of any of the degenerated discs. Histological examinations of punctured discs revealed cracks and clefts within the nucleus as well as delamination and infolding of the annulus. In addition, clusters of notochordal cells were readily apparent in healthy discs but were sparse in discs that had been punctured (fig. 8). Further validation of the puncture model was achieved by the demonstrated loss of mean water content from 85 to 70% twenty-four weeks after needle injury.

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Fig. 8. a Healthy L5–6 rabbit disc. Clusters of notochordal cells are apparent. b Degenerated L4–5 rabbit disc 24 weeks after puncture surgery. Nuclear displacement occurred, accompanied by infolding of the contralateral inner annulus towards the direction of nuclear displacement. Clusters of notochordal cells are sparse.

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Future Directions

The potential of gene therapy to alter the biological processes occurring in the degenerated IVD disc has been clearly established. The next step in this development will be to assess the feasibility of transducing degenerated rabbit discs, using needle-stab modeling as described above, with marker and therapeutic genes. Additional in vivo studies in this model will help to clarify the potential benefits and toxicity of gene therapy. Because other vectors are now available which can transduce cartilagenous cells, most notably adeno-associated vector, expanding this experimental methodology to other vector systems will be important. The basic science of the effects of growth factors and catabolic inhibitors in the biological processes and mechanical functioning of the spine also needs to be further elucidated, to determine which factors are best to promote growth of collagen and proteoglycans. Biochemical studies are necessary to delineate the relationship between viral concentration, transgene synthesis, and protein expression in the disc space. Despite the hurdles that remain, gene therapy to alter the course of IVD degeneration holds much clinical promise, and will continue to stimulate future investigations. References 1 2 3 4 5 6 7 8 9 10 11 12 13

Adler JH, Schoenbaum M, Silberberg R: Early onset of disk degeneration and spondylosis in sand rats (Psammomys obesus). Vet Pathol 1983;20:13–22. Anderson J: Back pain and occupation; in Jayson MIV (ed): The Lumbar Spine and Back Pain. London, Churchill Livingstone, 1987, pp 2–36. Anderson JA: Epidemiological aspects of back pain. J Soc Occup Med 1986;36:90–94. Bobechko: Auto-immune response to nucleus pulposus in the rabbit. J Bone Joint Surg Br 1965; 47:574–580. Borenstein D: Epidemiology, etiology, diagnostic evaluation, and treatment of low back pain. Curr Opin Rheumatol 1992;4:226–232. Bradford DS, Cooper KM, Oegema TR Jr: Chymopapain, chemonucleolysis, and nucleus pulposus regeneration. J Bone Joint Surg Am 1983;65:1220–1231. Buckwalter JA: Aging and degeneration of the human intervertebral disc. Spine 1995;20: 1307–1314. Butler D, Trafinow JH, Andersson GB, McNeil TW: Discs degenerate before facets. Spine 1990; 15:111–113. Evans CH, Robbins P: Possible orthopaedic applications of gene therapy. J Bone Joint Surg Am 1995;77:1103–1113. Garfin SR: The intervertebral disc: Disc disease – Does it exist? in Weinstein JN (ed): The Lumbar Spine. Philadelphia, W.B. Saunders, 1990, pp 369–380. Hallen A: Hexosamine and ester suphate content of the human nucleus pulposus at different ages. Acta Chem Scand 1958;12:1869–1872. Hallen A: The collagen and ground substance of the human nucleus pulposus at different ages. Acta Chem Scand 1962;16:705–709. Kang JD, Georgescu HI, McIntyre-Larkin L, Stefanovic-Racic M, Donaldson WF 3rd, Evans CH: Herniated lumbar intervertebral discs spontaneously produce matrix metalloproteinases, nitric oxide, interleukin-6, and prostaglandin E2. Spine 1996;21:271–277.

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Kang JD, Boden SD: Orthopaedic gene therapy. Spine. Clin Orthop 2000;379S:256–259. Kraemer J: Natural course and prognosis of intervertebral disc diseases. International Society for the Study of the Lumbar Spine, Seattle, Washington, June 1994. Spine 1995;20:635–639. Lipson SJ, Muir H: 1980 Volvo Award in Basic Science: Proteoglycans in experimental intervertebral disc degeneration. Spine 1981;6:194–210. McCoy RD, Davidson BL, Roessler BJ: Expression of human interleukin-1 receptor antagonist in mouse lungs using recombinant adenovirus: Effects on vector induced inflammation. Gene Ther 1995;2:437–442. Moon S, Nishida K, Gilbertson LG, Hall RA, Robbins PD, Kang JD: Biologic response of human intervertebral disc cell to gene therapy cocktail. San Francisco, Orthopaedic Research Society, 2001. Moon SH, Gilbertson LG, Nishida K, Knaub M, Muzzingro T, Robbins PD, Evans CH, Kang JD: Human intervertebral disc cells are genetically modifiable by adenovirus-mediated gene transfer. Spine 2000;25:2573–2579. Moon SH, Nishida K, et al: Proteoglycan synthesis in human intervertebral disc cells cultured in alginate beads; exogenous TGF-1 vs adenovirus-mediated gene transfer of TGF 1 cDNA. Orlando, Florida Orthopaedic Research Society 2000; (Abstr 1061). Nishida K, Kang JD, Gilbertson LG, Moon SH, Suh JK, Vogt MT, Robbins PD, Evans CH: Modulation of the biologic activity of the rabbit intervertebral disc by gene therapy: An in vivo study of adenovirus-mediated transfer of the human transforming growth factor beta 1 encoding gene. Spine 1999;24:2419–2425. Nishida K, Kang JD, Suh JK, Robbins PD, Evans CH, Gilbertson LG: Adenovirus-mediated gene transfer to nucleus pulposus cells. Implications for the treatment of intervertebral disc degeneration. Spine 1998;23:2437–2442; discussion 2443. Osada R, Oshima H, Ishihara H: Autocrine/paracrine mechanism of insulin-like growth factor-1 secretion, and the effect of insulin-like growth factors-1 on proteoglycan synthesis in bovine intervertebral discs. J Orthop Res 1996;14:690–699. Pearce RH, Grimmer BJ, Adams ME: Degeneration and the chemical composition of the human lumbar intervertebral disc. J Orthop Res 1987;5:198–205. Reinke J, et al: Transfer of therapeutic genes to human chondrocytes-like cells of lumbar disc prolapse (abstract 56). Annual Meeting of International Society for the Study of the Lumbar Spinel, Singapore, 1997. Robbins PD, Ghivizzani SC: Viral vectors for gene therapy. Pharmacol Ther 1998;80:35–47. Roberts S, Caterson B, Menage J, Evans EH, Jaffray DC, Eisenstein SM: Matrix metalloproteinases and aggrecanase: Their role in disorders of the human intervertebral disc. Spine 2000; 25:3005–3013. Silberberg R, Aufdermaur M, Adler JH: Degeneration of the intervertebral disks and spondylosis in aging sand rats. Arch Pathol Lab Med 1979;103:231–235. Takegami K, Thonar EJ, An HS, Kamada H, Masuda K: Osteogenic protein-1 enhances matrix replenishment by intervertebral disc cells previously exposed to interleukin-1. Spine 2002;27: 1318–1325. Thompson JP, Oegema TR Jr, Bradford DS: Stimulation of mature canine intervertebral disc by growth factors. Spine 1991;16:253–260. Tripathy SK, et al: Immune responses to transgene-encoded proteins limit the stability of gene expression after injection of replication-defective adenovirus vectors. Nat Med 1996;2: 545–550. Urban JP, McMullin JF: Swelling pressure of the intervertebral disc: Influence of proteoglycan and collagen contents. Biorheology 1985;22:145–157. Urban JP, McMullin JF: Swelling pressure of the lumbar intervertebral discs: influence of age, spinal level, composition, and degeneration. Spine 1988;13:179–187. Waddell G: Low back pain: A twentieth century health care enigma. Spine 1996;21: 2820–2825. Wallach CJ, Sobajima S, Watanabe Y, Gilbertson LG, Kang JD: Gene transfer of the catabolic inhibitor TIMP-1 increases measured proteoglycans in human intervertebral disc cells. International Society for the Study of the Lumbar Spine, Cleveland, Ohio, 2002.

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Yang Y, et al: Cellular immunity to viral antigens limits E1-deleted adenoviruses for gene therapy. Proceedings of the National Academy of Sciences of the United States of America 1994; 91:4407–4411.

James D. Kang, MD Assistant Professor of Orthopaedic Surgery and Neurological Surgery Division of Spinal Surgery, University of Pittsburgh Medical Center Department of Orthopaedic Surgery, Liliane Kaufmann Building 3471 Fifth Avenue, Suite 1010, Pittsburgh, PA 15213 (USA) Tel. 1 412 605 3241, Fax 1 412 687 3724, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 52–64

Bone Morphogenetic Proteins Spinal Fusion Applications

David A. Bomback, Jonathan N. Grauer Department of Orthopaedics and Rehabilitation, Yale University School of Medicine, New Haven, Conn., USA

Overview of Bone Morphogenetic Proteins (BMPs)

The clinical track record of autogenous iliac crest bone graft makes it the current ‘gold standard’ for spinal arthrodesis. However, autograft utilization is accompanied by a number of limitations. For example, pseudoarthrosis rates vary from 5 to 35% [1], which prolongs recovery from surgery and leads to potential complications such as graft migration, instability, or even spinal cord impingement. Moreover, chronic donor site pain has been reported in up to 25% of patients who undergo removal of iliac crest material for spinal autografting. The availability of donor bone from a given patient may also be limited secondary to prior graft harvest or poor bone quality. Finally, an additional operative site increases blood loss, operative time, and cost [1]. Such limitations have prompted investigations into a variety of bone graft alternatives. The goals of such efforts are aimed at eliminating donor-site pain and increasing union rate with a product that is virtually limitless in supply. Such alternatives can be classified as either bone graft extenders or substitutes. Graft extenders, when added to autogenous bone, allow for arthrodesis of a greater number of levels or the use of less autograft and yield a fusion rate equal to or superior to that of autograft alone. Graft substitutes completely replace autogenous bone yet allow for comparable or increased fusion rates compared with the autograft [2]. In order to understand the biological application of bone graft alternatives, one must be familiar with the basic terminology. Osteoconduction is the ability of a material to behave as a scaffold for the ingrowth of new host bone. Osteoinduction is defined as the capability of initiating de novo bone formation

by inducing osteoblastic precursor stem cells to differentiate into mature boneforming cells. An ideal bone graft substitute must possess both of these characteristics. Osteogenesis simply refers to the ability of graft cells to directly form bone. Only autogenous bone graft and bone marrow aspirates possess osteogenic properties [3]. In 1965, Urist [4] made the observation that implanted devitalized bone was capable of inducing a cellular response resulting in new bone formation. His laboratory subsequently demonstrated that proteins extracted from the organic component of bone were responsible for such a behavior [5, 6]. Implantation of this bone matrix protein mixture into animals resulted in a multitude of cellular events including mesenchymal cell infiltration, cartilage formation, vascular ingrowth, bone formation, and bony remodeling [7]. Urist thus coined the term ‘bone morphogenetic protein’ (BMP). Over time, such extracts and proteins have become exploited and modified to induce fusions.

Biology of Spinal Fusion

The goal of spinal arthrodesis using decortication and autogenous bone graft is the development of a well-formed fusion mass bridging one bony surface to another. In order to achieve such an endpoint, a specific set of events needs to occur. First, osteoprogenitor cells must enter the fusion bed. Decortication of host bone enables cells to exit the bone marrow and enter the fusion environment. Next, osteoprogenitor cells differentiate into osteoblast precursors and ultimately mature osteoblasts, depositing new bone matrix. Finally, bony remodeling of the fusion mass occurs according to Wolff’s law (i.e., remodeling occurs in response to physical stresses; bone is deposited in sites subjected to stress and resorbed from sites of little stress), resulting in a stable fusion mass able to withstand physiological stress [3]. To study the many variables which affect bone formation and fusion, animal models have been developed. One such approach is the New Zealand white rabbit posterolateral lumbar fusion model, which has been validated and extensively studied [8]. Histological analysis reveals that maturation of the spine fusion mass occurs first in the ‘outer zone’ (adjacent to the transverse processes) followed by the ‘central zone’ (between transverse processes). This temporal and spatial sequence would be expected postdecortication because osteoprogenitor cells from the marrow must travel a longer distance to reach the central zone. In each region, inflammatory, reparative, and remodeling histological phases of bone healing can be observed [3]. In further evaluating the process, mRNA expression of various BMPs has been

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shown to occur at different times and at different locations during fusion. Such findings suggest unique roles for specific BMPs during spine fusion and indicate the potential for clinical applications of these proteins. A full understanding of the process of BMP expression during fusion has not yet been achieved, although limited gene expression studies following induction with BMP have been performed.

Molecular and Cellular Mechanisms of Action of BMPs

The BMPs are dimeric molecules belonging to the transforming growth factor-␤ superfamily based on amino acid homology [9, 10]. BMP molecules are multifunctional proteins that exhibit both autocrine and paracrine effects. They act by binding to specific serine-threonine kinase receptors present on the surface of undifferentiated mesenchymal stem cells. The receptors then transduce a signal via a group of G-proteins known as Smads, which in turn activate genes in the nucleus of the cell related to the osteoblast phenotype [11]. When applied in vivo, BMPs induce undifferentiated mesenchymal stem cells to switch from a fibrogenic to an osteogenic pathway of development, culminating in mature bone with normal marrow cavities [12]. The activity of BMPs is tightly controlled and self-limiting. Outside the cell, inhibitory proteins (e.g., noggin, chordin, follistatin) can bind specific BMPs, thus preventing their binding to cell surface receptors [11, 13, 14]. Furthermore, intracellular BMP transcription and translation is regulated by a combination of signal-transducing and inhibitory Smad proteins. BMPs can themselves up-regulate the expression of these extracellular antagonists and intracellular inhibitors, suggesting a negative feedback autoregulation cycle. As a result of all of these regulatory mechanisms, bone induction is tightly limited and bone overgrowth is avoided [11].

Research and Clinical Use of BMPs

Several BMP preparations have been, and are currently being investigated preclinically and clinically for use in spinal arthrodesis. These BMP products include recombinant human BMPs (rhBMPs) and demineralized bone matrices (DBMs). The two rhBMPs which have been most investigated are rhBMP-2 (Medtronic Sofamore Danek, Memphis, Tenn., USA) and rhBMP-7 also known as osteogenic protein-1 or OP-1 (Stryker Biotech, Hopkinton, Mass., USA). They are highly purified single proteins produced by recombinant DNA biotechnology. Others include BMP-9 [9, 15] and growth and differentiation

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factor-5 [16]. Small amounts of BMP may also be present in some DBM preparations. Examples of these formulations, which are derived from human allograft bone, include, but are not limited to, Grafton (Osteotech, Eatontown, N.J., USA), Dynagraft (Gen-Sci Regeneration Laboratories, Calif., USA), and Osteofil (Regeneration Technologies, Fla., USA). However, concentration of BMPs in DBM is not thought to be sufficient for it to be a complete bone graft substitute in spinal applications [17, 18]. In addition, a more concentrated DBM preparation, bovine bone-derived BMP extract or bBMPx (Sulzer Orthopedics Biologics, Denver, Colo., USA), has been investigated [10, 19]. Theoretically, this may offer more osteogenic potential than the standard human DBM preparations.

Preclinical Studies with Specific Classes of BMP

rhBMP-2 The first preclinical interbody cage study using rhBMP-2 was by Sandhu et al. [20]. L4-L5 retroperitoneal anterior lumbar interbody fusions were performed in a sheep model. Cylindrical, threaded titanium fusion cages were filled with either iliac crest autograft or rhBMP-2. The 6-month follow-up results demonstrated a 100% fusion rate for the rhBMP-2 group compared with a 37% fusion rate for the autograft controls. A similar study in a goat model using titanium BAK fusion cages (Spinetech, Minneapolis, Minn., USA) packed with either autograft or rhBMP-2 yielded a 95% fusion rate in the rhBMP-2 and a 48% fusion rate in the autograft group [21]. Finally, another study utilizing allograft bone dowels for anterior interbody fusion in nonhuman primates showed that dowels filled with rhBMP-2 resulted in a 100% fusion rate at 6 months, as opposed to the 33% fusion rate seen in the autograft-filled dowels [22]. Numerous posterolateral fusion studies have been reported in the past decade, with similar conclusions. Schimandle et al. [23] noted a 100% fusion rate for rhBMP-2 in a rabbit posterolateral fusion model, while autograft controls fused only 42% of the time. In addition, fusions in the rhBMP-2 animals were biomechanically stronger and stiffer than autograft fusions. A canine model demonstrated 100% rhBMP-2 fusions and 0% autograft fusions at 12 weeks [24]. Use of rhBMP-2 as an autograft enhancer also has been studied in a canine model; in that model, gross specimens and CT scans demonstrated significantly increased fusion mass volume 6 months after surgery in rhBMP-2 autograft dogs when compared with autograft alone dogs [25, 26]. Martin et al. [27] established the systemic effects of ketorolac on posterolateral spine fusion and then tested rhBMP-2’s ability to overcome such inhibition. First, the investigators demonstrated an autograft fusion rate of 35% with

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IV ketorolac pump infusion as compared to an autograft fusion rate of 75% with IV saline infusion. Fusion rates subsequently increased to 100% in an autograft/rhBMP-2 group with IV ketorolac infusion. In a related study, Silcox et al. [28] demonstrated that the inhibitory effect on fusion of systemic nicotine could be overcome with rhBMP-2. These investigators administered nicotine to rabbits via mini-osmotic pumps. They subsequently performed single-level posterolateral fusions on these rabbits comparing autograft alone to autograft mixed with rhBMP-2 to allogeneic DBM mixed with BMP-2. They achieved a 100% fusion rate in the autograft/rhBMP-2 group, a 64% fusion rate in the DBM/rhBMP-2 group and a 0% fusion rate in the autograft alone group. rhBMP-7 (OP-1) Cook et al. [29] reported the first spinal application of OP-1 using a canine posterolateral fusion model. Radiographical and histological examination revealed solid fusion for the OP-1 group 6 weeks after surgery. The autograft group attained comparable fusion rates but not until 26 weeks postsurgery. No fusions were observed for negative controls (i.e., no implant material or carrier alone). Grauer et al. [30] have studied comparative intertransverse lumbar fusion in the New Zealand white rabbit model. Study groups were autograft alone, carrier alone, or OP-1 with carrier. Manual palpation and biomechanical testing at 5 weeks confirmed a 0% fusion rate in the carrier group, a 63% fusion rate in the autograft group, and a 100% fusion rate in the OP-1 group. At 5 weeks, histology revealed more mature bone in the OP-1 group. Cunningham et al. [31] studied a skip-level posterolateral canine model using autograft alone, autograft and OP-1, or OP-1 alone. Statistically significant differences in the rate of fusion between the autograft alone and the OP-1-containing specimens were noted at all timepoints studied (4, 8 and 12 weeks postoperatively). Only a few other studies reporting the use of OP-1 in interbody fusion have been reported. Magin and Delling [32] compared OP-1, autograft alone, and an osteoconductive hydroxyapatite bone graft alternative using a posterior lumbar interbody fusion sheep model. At 4 months time, OP-1 animals had an 80% fusion rate with a 60% increase in bone formation compared to the other groups. Cunningham et al. [33] studied a sheep thoracic spine model using threaded fusion cages (BAK devices) placed thoracoscopically. BAK cages packed with OP-1 had fusion rates equivalent to those packed with autograft and to autograft bone dowel alone, suggesting that OP-1 is as effective as autograft in obtaining interbody fusion. Similar to the rhBMP-2 nicotine study above, Patel et al. [34] tested the ability of OP-1 to overcome the inhibitory effects of nicotine in a rabbit posterolateral fusion model. L5-L6 fusions were performed using either iliac crest autograft or OP-1. Nicotine was administered to all animals via a subcutaneous

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mini-osmotic pump. The fusion rate was 25% for the nicotine-exposed autograft group and 100% for the nicotine-exposed OP-1 group at 5 weeks, demonstrating the latter’s ability to overcome the negative effects of nicotine in this model. DBM

Grafton DBM was shown to be effective as a graft extender with autograft in a rabbit posterolateral spine fusion model. It demonstrated equal effectiveness with autograft in a 1:1 or 3:1 dosing ratio. However, it never demonstrated fusion rates greater than the autograft alone [1]. In a canine posterior fusion model, Cook et al. [35] evaluated fusion in 9 adult mongrel dogs at 6, 12, and 26 weeks. Four sites on each animal received implants consisting of DBM gel, DBM gel with allograft, allograft alone, or autograft alone. Radiographical studies demonstrated that the autograft sites had achieved fusion by 26 weeks postoperatively. Conversely, the DBM gel alone and with the allograft demonstrated some new bone formation but did not achieve fusion by 26 weeks. Mechanically, the autograft sites demonstrated torsional stability significantly greater than all other fusion sites. Histological analysis confirmed the radiographical and mechanical findings. The results indicate that the DBM gel alone or with the allograft is inferior to the autograft. Only sparse reports are available regarding utility of bBMPx in spinal arthrodesis, but the limited information appears encouraging. Boden et al. [36] demonstrated a dose response with bBMPx in the rabbit intertransverse process fusion model. bBMPx mixed with collagen and DBM achieved fusion rates of 50–100% depending on dose. Autograft fusion rates were 62%, and DBM with collagen alone were only 17%. More recently, a posterolateral fusion model was studied in nonhuman primates comparing bBMPx delivered in DBM to the autograft alone. Efficacy data demonstrated an autograft fusion rate of 21%; the bBMPx displayed a dose response in which 3 mg per side gave twice the fusion rate as that of the autograft [19]. Clinical Studies Using BMPs

The preclinical data cited above paved the way for completed, ongoing, and future human trials with these three differentiation factors. rhBMP-2 In 1996, 14 patients with single-level lumbar degenerative disc disease were enrolled in a prospective randomized nonblinded controlled trial to test interbody cage with BMP treatment versus bone autograft. All patients received

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Bridging bone

Fig. 1. Patient one-year status postinterbody fusion with Infuse (BMP-2 on absorbable collagen sponge) in an LT Cage (image courtesy of Medtronic Sofamor Danek, Memphis, Tenn., USA).

a tapered titanium interbody fusion device (LT Cage, Medtronic Sofamor Danek, Memphis, Tenn., USA) filled with either rhBMP-2 or iliac crest autograft. All 11 patients randomized to the rhBMP-2 group were fused radiographically at 6 months, while one of the 3 patients in the autograft group had a nonunion at one-year follow-up. Given the small numbers, the differences were not statistically significant [37], although the clinical results were considered excellent with rhBMP-2 compared to other interventions. A variety of clinical trials followed this pilot study, which have demonstrated efficacy in varied clinical applications of BMPs. Anterior lumbar interbody fusion rates performed either open or laparoscopically using rhBMP-2-filled interbody fusion cages have been shown to be equivalent to those with autograft-filled cages [37] (fig. 1). When rhBMP-2 was used with machined allografts (bone dowels) for anterior lumbar interbody fusion, it yielded higher fusion rates, superior improvement in pain and function, and a greater likelihood of returning to work compared with autograft-filled dowel controls [38]. It should be noted, however, that heterotopic bone within the spinal canal was noted in patients enrolled in an rhBMP-2 posterior lumbar interbody fusion or PLIF trial. There were no neurological sequelae reported, but the study was halted prior to completion [39]. Further investigation is warranted to define appropriate safety parameters, given the concern about bony overgrowth. Finally, the first human trial investigating rhBMP-2 as an adjunct to posterolateral intertransverse arthrodesis has recently been reported. Twentyfive patients undergoing lumbar arthrodesis were randomized based on the arthrodesis technique: autograft/Texas Scottish Rite Hospital (TSRH) pedicle

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screw instrumentation (n ⫽ 5), rhBMP-2/TSRH (n ⫽ 11), and rhBMP-2 only without internal fixation (n ⫽ 9). On each side, 20 mg of rhBMP-2 were implanted. The patients had single-level disc degeneration, grade 1 or less spondylolisthesis, mechanical low back pain with or without leg pain, and failure of nonoperative treatment for at least 6 months. The radiographical fusion rate was 40% (2/5) in the autograft/TSRH group and 100% (20/20) with rhBMP-2 group with or without TSRH internal fixation (p ⫽ 0.004). In addition, statistically greater and quicker improvement in patient-derived clinical outcome was measured in the rhBMP-2 groups [40]. These results strongly suggest that BMP treatment augments the efficacy of spinal fusion in the setting of autographs, with or without internal fixation. All interbody clinical trials with rhBMP-2 demonstrated less blood loss compared to autograft controls. In addition, the incidence of donor site pain in those patients who underwent bone graft harvest was 30–40% at 2 years [37]. rhBMP-7 (OP-1) All OP-1 human trials have involved uninstrumented posterolateral intertransverse process fusion in the setting of degenerative spondylolisthesis. An early Australian study [41] placed autograft on one side and OP-1 on the contralateral side. The 6-month follow-up noted bone formation to be equal or greater on the OP-1 side (assessed by CT scan) as compared with the autograft side. Although this was encouraging, it is difficult to interpret the results of such studies with different bone graft materials on different sides, as one side may affect the other. An initial safety and efficacy study in the United States compared autograft alone to autograft augmented with OP-1 for posterolateral arthrodesis. Sixteen patients with degenerative lumbar spondylolisthesis and spinal stenosis were randomized to each treatment arm. At 6-month follow-up, 75% of patients in the OP-1/autograft group were radiographically fused, whereas only 50% in the autograft only group were fused [42] (fig. 2). A subsequent study of similar design is underway, in which patients receive either iliac crest autograft alone or OP-1 alone [43]. The 6-month results for 36 enrolled patients have shown a clinical success rate 32% higher in the OP-1 group than in the autograft group [44]. No OP-1-related adverse events have been observed to date. DBM At the present time, there have been no prospective clinical trials with human DBM products in the spine literature. A clinical trial is underway in Switzerland testing bBMPx in a posterolateral lumbar fusion but results are not yet available [19].

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Bridging bone

Fig. 2. Patient one-year status postdecompression and posterolateral fusion with OP-1 alone for degenerative spondylolisthesis.

Future Directions in Spinal Fusion Research

Gene therapy may play an active role in future preclinical and clinical trials with various BMPs. Gene-based therapies attempt to deliver specific genes, known as transgenes, to target cells to change the existing physiological state or disease process [45]. Genes encoding for factors in the osteogenic cascade are either inserted into the patient’s own cells that exist at the fusion site (in vivo) or into cells that have been removed and will be reimplanted at the site of fusion (ex vivo) [3]. Once these cells are in place, the transgene produces a protein that initiates the bone-formation cascade. Hence, it is the activity and half-life of the transgene itself that is the limiting temporal factor for the presence of osteoinductive stimulatory signals at the fusion site [3]. This therapy might allow for potentially longer expression of BMP activity and thus perhaps an increased window of time for bone formation. Boden et al. [46] have reported successful use of gene therapy techniques in an athymic rat posterolateral spine fusion model. The gene encoding for anosteoinductive intracellular signaling protein named LIM mineralization protein-1 was identified and cloned [47]. It appears to be regulated by BMP-6 and to function very early in the cascade of events leading to de novo bone formation [46].

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Using an ex vivo gene therapy strategy, the LIM mineralization protein-1 gene was transfected into the harvested bone marrow cells of athymic rats and then reimplanted at appropriate posterolateral fusion sites. Successful arthrodesis was achieved in 100% of the sites receiving cells containing the LIM mineralization protein-1 gene and 0% of the sites receiving control cells [46]. In vivo gene therapy methodology has also been reported. In two separate studies, an adenovirus vector containing either rhBMP-2 or rhBMP-9 was injected percutaneously into the lumbar paraspinal musculature of athymic rats. Both studies reported successful arthrodesis at the experimental sites without any evidence of canal or neuroforaminal compression [9, 48]. Future techniques may include delivery of genetic material via nonviral means (e.g., liposomes, gene gun therapy) or with newer viral vectors (e.g., adeno-associated virus) demonstrating less immunogenicity. All of these promising techniques, however, are certainly associated with greater expense and there are concerns of oncogenesis and bony overgrowth. Although gene therapy may allow for higher levels of osteoinductive proteins to be expressed for longer time periods, it is unknown if high fusion rates can be achieved with one-time applications. In addition, potentially increased bone production might compromise the safety of these implants. Vector design must incorporate gene regulation techniques and spine-specific targeting strategies before human clinical trials can be safely conducted [45]. In terms of local application of recombinant BMP products at the time of surgery, the use of carrier systems with BMP recombinant proteins is still in evolution. Carriers for BMP in spine fusion are used to increase the retention of these differentiation factors at the fusion site while at the same time providing an osteoconductive matrix on which bone formation can occur. Four major categories of carriers are used for BMP delivery: inorganic materials (e.g., hydroxyapatite, tricalcium phosphate), synthetic polymers (e.g., polylactide, polyglycolide), natural polymers (e.g., collagen formulations), and composites of the above three materials [49]. Carrier efficacy is both site specific and species specific. The dosing of BMP products with their associated carriers is currently under investigation. At the present time, it is unclear if the optimal dose or the optimal delivery system has been established, and if delayed-release products can successfully compete with the theoretical advantages of gene transfer.

Conclusions

The ability of BMPs to promote, extend, or enhance spinal fusion is attracting interest in both the basic science and clinical settings. Although autograft currently remains the ‘gold standard’ for initiating spine fusion in the clinical

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arena, osteoinductive differentiation factors combined with osteoconductive matrices are being investigated in preclinical and clinical trials. Results of these early clinical investigations indicate that rhBMP may be an acceptable, safe bone graft alternative. However, current numbers are small and follow-up is still of relatively short duration. Longer follow-up and additional studies are, therefore, needed to test acute application and long-term application of osteoinductive factors. Newer gene therapy techniques have not yet been introduced into clinical trials, but preliminary animal study results are promising. References 1 2

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David Bomback, MD 400E 71st Street Apt. #51 New York, NY 10021 (USA) Tel. ⫹1 212 472 2143, Fax ⫹1 212 774 2779, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 65–103

Cellular and Gene Therapy Approaches to Spinal Cord Injury Michael P. Steinmetz, James K. Liu, Nicholas M. Boulis Department of Neurosurgery S31, Cleveland Clinic Foundation, Cleveland, Ohio, USA

Introduction

Acute spinal cord injury (SCI) is relatively uncommon, affecting about one in 40 patients who present to a major trauma center [1]. It is estimated that there are 11,000 new cases per year or 40 cases per million population (National Spinal Cord Injury Databank, 2001). Despite this relatively low incidence, these injuries pose serious problems for the patients, their families, and society in general [2]. Mortality from SCI is estimated to range from 4.4 to 16.7% for those that survive the initial injury and receive treatment [3]. Aside from the obvious physical damage, there may be serious psychological effects on both the patients and their families. The financial burden to the patient, the health care system, and society is great in terms of both direct and indirect costs (i.e., lost income and productivity) [4]. In 1990, it was estimated that the cost to the United States of caring for all SCI patients was USD 4 billion annually [5]. Clinical therapy for acute SCI is sparse and often disappointing. The clinician is limited to surgical decompression (if appropriate), IV methylprednisolone, and acute and long-term SCI rehabilitation. Various research protocols are also in progress. Despite these therapies, the prognosis for SCI remains dismal. The failure of functional recovery following SCI is multifactorial. Axonal regeneration requires neuroprotection, neuronal cell-body stimulation, the need to overcome local inhibitors at the injury site, and finally reconnection of neuronal pathways (both ascending and descending) essential for functional recovery. Current research is focused on each of these areas. Both genetic and

cellular therapies are emerging as strategies to overcome barriers to neural regeneration. This chapter will review past and current research with genetic and cellular therapeutic options.

Cellular Therapies

There are many evolving cellular therapeutic strategies for SCI. The focus of this therapy is on neuroprotection, remyelination, and regeneration. Cellular therapies include endogenous and transplanted stem cells, fetal tissue transplants, allo- and xenografted Schwann cells and olfactory ensheathing cells (OECs) as well as autologous macrophages. Each area of research has uncovered difficulties unique to individual cellular approaches. These include the ethical and moral concerns raised by embryonic stem (ES) cells and fetal grafts, as well as the potential need for immunosuppression after cellular transplants.

Models of SCI

There are many animal models of SCI available to the researcher. The most popular animals for these injury paradigms include the rat and mouse. Both are fairly inexpensive and are readily available. The mouse model has the further advantage of being used for transgenic experiments. Methods of experimental SCI entail complete spinal cord transection, partial transection, contusion, and compression [6]. Complete transection involves the complete disruption of the spinal cord. The main advantage of this model is that all tracts are transected; therefore, any axons demonstrated on retrograde labeling (see below) are due to regeneration and not from sparing (not destroyed during the experimental injury). Partial transection models utilize animals in which only certain tracts are cut (e.g., the rubrospinal tract). This leaves the contralateral tract available for comparison [6]. The absence of complete paraparesis and urinary retention renders these animals easier to care for (i.e., one does not need to manually express the bladder in those rats who have only a unilateral rubrospinal transection). A serious disadvantage of these models is the potential for confusing spared and regenerating axons. Contusion and compression models reflect more accurately the SCIs that generally occur in humans [6]. Methods available for contusion and compression models include weight-drop and clip-compression strategies. Because the lesions in these models are even less discrete, they have an even greater potential for confusion between axon sparing and true regeneration.

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Recovery Assays

Immunohistochemical, electrophysiological, and behavioral assays can all be used to measure spinal cord recovery [6]. Axons tracers are used to follow tracts in the spinal cord (both anterograde and retrograde). Anterograde tracers include biotinylated dextran amine and the cholera toxin B subunit. These tracers are applied to the cell body and then transported anterograde and may be used to identify axons regenerating at the injury site. Retrograde tracers are taken up by axons and transported back to the cell body. These may then be placed distal to the injury site to assess regeneration at a site of injury. Examples include Flouro-Gold and the cholera toxin B subunit. Electrophysiological tests may assess axon integrity, both in vivo and in vitro. Examples of in vivo tests include somatosensory evoked and motor potentials. Isolated spinal cords (in vitro) may also be assessed neurophysiologically [6]. Finally, behavioral tests are available to evaluate the neurological recovery of the injured animal. These include open field test of locomotion, such as the BBB; sensory tests, such as sensing and removing a piece of tape from a paw; and other tests or motor and skilled behavior, such as walking across a wire grid or narrow beam.

Cellular Therapies for SCI

Stem Cells Neural stem cells (NSCs) are defined by their ability to generate neural tissue (both neuronal and glial), their ability to self-renew, and their pluripotency. Pluripotency refers to a cell’s ability to generate a variety of lineages through cell division [7]. Progenitor cells have a more restricted fate (fig. 1). For example, neural progenitors differentiate into all the neuronal cells of the central nervous system (CNS), but not the glia. NSCs may be isolated from adult and fetal brain and also embryonic tissue. Cells may be harvested from the adult subventricular zone, hippocampus [7], the fetal telencephalon, or the inner cell mass of blastocyst-stage embryos [8]. These cells are then grown in cell culture in the presence of a high concentration of mitogens such as fibroblast growth factor (FGF) or epidermal growth factor [7]. After several rounds of division, the cells are exposed to either media with the mitogens withdrawn or to a new substrate. Different substrates can drive stem cell differentiation into specific lineages (e.g., oligodendrocyte). Immunostaining for specific marker antigens can be used to identify these lineages. Cells may be infected with a replication-incompetent retroviral vector

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Cell type Pluripotent embryonic stem cell self-renewing

Multipotent stem cells self-renewing

Neural progenitor cells limited self-renewal

Committed progenitor cells no self-renewal Neuronal

Glial

Differentiated cells no self-renewal

Neuron

Glial

Fig. 1. The progression of cell development from a pluripotent, self-renewing, embryonic stem cell to differentiated neurons and glia.

encoding lacZ in order to assess clonal relationships of progeny and identify them in situ following transplantation [9]. NSCs The adult CNS has a limited capacity to repair itself after injury. This is due in part to the inability to generate new neurons and the inability to initiate

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Exogenous stem cells

1.

2.

Syrinx

Syrinx

a Exogenous factor

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1.

Central canal

Syrinx

Potential stem cells

Central canal

Syrinx

b Fig. 2.a Exogenous stem cell transplantation. 1. Exogenous stem cells have been transplanted into a syrinx cavity following a chronic SCI. 2. Following transplantation, the stem cells have populated the cavity and migrated into the spinal cord parenchyma. b Endogenous stem cells. 1. The endogenous stem cells of the spinal cord probably reside in the region of the central canal. 2. Following injury or factor injection, these cells are stimulated to divide and migrate into the area of injury (in this case a syrinx cavity). Some cells also migrate into the cord parenchyma.

functional axonal regrowth. The ability to transplant multipotent NSCs may overcome the former. As such, significant enthusiasm has focused on the application of NSCs for the repair of focal neural tissue destruction, including that which is seen with SCI. These stem cells may be isolated from embryonic or adult brain tissue of a variety of species, including mouse, rat, and human [7, 10]. They may also be derived from the mouse and human ES cells [8, 11–13] derived from nonneural embryonic tissue. These cells are stable through multiple passages in vitro without loss of their multipotentiality [14]. Multipotentiality or ‘pluripotency’ refers to the NSC’s ability to differentiate into a variety of lineages, including neuronal, oligodendrocytic, or astrocytic phenotypes. These stem cells have been shown to survive transplantation into the CNS and also have the ability to migrate. Thus, these cells may be transplanted into the injured CNS with the potential to repair specific regions. In addition to the ability to transplant exogenous stem cells, it may be possible to induce endogenous multipotential cells to ‘self-repair’ after injury or disease [14] (fig. 2).

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Recent evidence also suggest that NSCs may be derived from bone marrow and umbilical cord blood, thus serving as another source of both exogenous and endogenous stem cells, but residing outside the CNS [15]. Both in vivo and in vitro studies have demonstrated neuronal and glial differentiation; furthermore, these cells (i.e., bone marrow or umbilical cord blood) have the potential to be delivered systemically for the treatment of CNS pathology as opposed to direct transplantation into the CNS [15]. The use of marrow-derived stem cells may permit the clinician to harvest autologous stem cells, amplify them in vitro and then transplant them back into the patient. This may obviate the need for chronic immunosuppression and its inherent morbidity. Experimentally, autologous bone marrow-derived stem cells have been used to regenerate infarcted myocardium [16]. Further progress may provide the same opportunity for the nervous system. Exogenous NSCs Experiments have demonstrated that NSCs demonstrate significant survival, migration, and differentiation. NSCs undergo area-specific differentiation following transplantation into the CNS [9, 17, 18]. It appears that these cells have the capacity to respond appropriately to local signals in the developing CNS [14]. It may be that the local environment is the predominant determinant of the differentiated fate of the engrafted cells. When pluripotent NSCs are transplanted into the injured spinal cord, the engrafted cells differentiate only into astrocytes, and the temporal progression of that differentiation is markedly retarded [19, 20]. The mechanism regulating this transformation is unknown. Successful neuronal replacement may, therefore, require transplanting NSCs already committed to a neuronal lineage to avoid local environmental cues defining a glial lineage. Neural restricted precursors (NRPs) are an exciting alternative to multipotent NSCs. These cells are committed to a neuronal lineage at the time of isolation, and have been isolated from embryonic CNS tissue, ES cells, and multipotential NSCs [11] (fig. 1). These cells have been transplanted into the adult rat spinal cord. Neuronal maturation was observed, but was significantly retarded. It appears that additional modification of the grafts and/or the host environment will be needed for mature neuronal differentiation [14]. The intrinsic state of NSCs at the time of transplant, like the host environment, may also be important [21]. There appear to be differences between neural progenitor cells isolated from different brain regions [22, 23]. When NRPs derived from embryonic spinal cord are grafted into the subventricular zone (SVZ), the cells were observed to migrate extensively and generate mature neurons of various neurochemical and morphological phenotypes [24]. When NRPs isolated from the SVZ are engrafted back to the SVZ, they demonstrate less migratory and differentiation potential [25]. Spinal cord-derived NRPs were

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also observed to differentiate into a neuronal phenotype expressing choline acetyltransferase in brain areas where endogenous choline acetyltransferasepositive neurons have not been found. In this instance, therefore, intrinsic characteristics of the transplanted NRPs committed them to a restricted phenotype even after ectopic engraftment [14]. This finding suggests that one may need to isolate precursors from specific CNS regions for proper functional neural replacement [14]. Many studies have demonstrated successful transplantation of NSCs and NRPs into the CNS, but the physiological function of these grafts has not been completely elucidated. McDonald et al. [26] demonstrated functional improvement (locomotor) after transplantation of NSCs derived from ES cells into a rat spinal cord in a contusion injury model. ES cell embryoid bodies were derived from the D3 cell line (mouse) [27] at the 4⫺/4⫹ stage (i.e., 4 days without, then 4 days with retinoic acid) for transplantation. These cells were transplanted as cell aggregates directly into the syrinx (fig. 2a) 9 days after the experimental SCI. Two weeks after transplantation, labeled ES cells [Bromodeoxyuridine (BrdU), or mouse-specific antibodies M2, EMA, or Thy 1.1/1.2] were identified in situ. Cells were found to be filling the syrinx cavity, but also as far as 8 mm away from the syrinx edge in either the rostral or caudal direction. 43% of these cells were found by immunohistochemistry to be oligodendrocytic and 19% were found to be astrocytic. Many of the ES-derived oligodendrocytes were immunoreactive for myelin basic protein. 8% demonstrated neuronal staining (neuron-specific nuclear protein, NeuN). One month following transplantation, a difference of 2 points on the BBB scale was observed (7.9 vs. 10) between treated animals and sham controls. The difference in the BBB score reflected the ability to mobilize with partial hind limb weight-bearing and coordination as opposed to no hind limb weight-bearing or coordination. It is unclear what factors were responsible for the improved functional score following transplantation. The ES cells may have remyelinated the injured axons or provided neurotrophic or tissue-sparing effects. Furthermore, ES-derived neurons may have matured and made functional connections with injured spinal tracts. The rapidity of locomotor improvement and the observation that most ES cell-derived cells were oligodendrocytes positive for myelin basic protein make remyelination the most probable cause of recovery. The fact that oligodendrocyte precursors transplanted into chemical lesions have previously been associated with remyelination and improved axonal conduction creates further precedence for this explanation [28]. Various goals underlie the rationale for NSCs or NRPs spinal cord transplantation. As previously discussed, it is unlikely that transplanted stem cells will be able to completely recapitulate the injured ascending and descending tracts of the spinal cord (fig. 3e). Although stem cell-derived neurons have been

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NSC

Trophic factors

a

NSC

Trophic factors

Native glial cell

b

Ex vivo Fibroblast gene transfer

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Trophic factors

NSC precursor

Glia

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NSC

e Fig. 3. Various mechanisms of NSC graft-induced recovery following spinal cord injury. a NSCs may secrete various trophic factors that induce regrowth of spinal cord axons. b NSCs may induce native CNS support cells (e.g., glial cells) to secrete trophic factors that lead to axonal regrowth. c Fibroblasts implanted following ex vivo gene transfer may secrete trophic factors, leading to axonal regrowth. d NSCs that have been stimulated to become oligodendrocyte or glial precursors are transplanted into the spinal cord. Following further differentiation, remyelination of damaged axons is initiated. e A common misconception of NSC transplantation is that NSCs completely recapitulate a new axon, replacing the damaged axon. It is highly unlikely that this occurs following NSC transplantation.

shown to possess ion channels similar to native neurons and are capable of generating action potentials, no study has clearly shown that these cells integrate into host circuitry and create functional synapses. It is more likely that these grafts provide neuroprotection (tissue sparing), trophic support, or remyelination, although direct evidence is speculative (fig. 3a). Furthermore, stem cell grafts may provide effective tissue bridges that permit or promote the passage of endogenous regenerating axons. Endogenous Stem Cells The use of exogenous stem cells necessitates a grafting procedure and possibly immunosuppression. The use and manipulation of endogenous stem cells may obviate these cumbersome and potentially hazardous interventions. It is now widely accepted that neurogenesis occurs in the adult CNS. This process has been demonstrated in the hippocampus and the SVZ [29–32]. Lois and Alvarez-Buylla [31] labeled the brains of adult male mice with [3H]thymidine. Proliferating, hence, dividing cells were localized almost exclusively to the SVZ. To test the fate of these cells, the SVZ of labeled brains were isolated and grown

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in culture. By six cell divisions, the explants had generated an outgrowth of flat glial cells (GFAP positive) and cells containing processes growing on the glial monolayer. These cells were determined to be neurons due to their immunoreactivity to neuronal markers (MAP-2, NF, and NSE) and absence of staining for glial markers (GFAP). Explants stained with neuron-specific antibodies were processed for autoradiography to detect the presence of [3H]thymidine in the cell nuclei. It was found that approximately 84% of neurons were labeled with [3H] thymidine. These results indicate that proliferating cells do exist in the adult SVZ in vivo and these cells possess the ability to generate neurons and glia. Eriksson et al. [29] demonstrated similar results in the adult human dentate gyrus. They examined the hippocampus and SVZ of patients who had succumbed to cancer and had received an intravenous infusion of BrdU before they died. The results of these experiments demonstrated that in all BrdU-treated patients, the granule cell layer contained BrdU-positive cells, which also doublestained with neuron-specific markers (e.g., NeuN). Furthermore, the SVZ also contained BrdU-positive cells, but these did not colabel with neuron-specific markers. It is believed that these too are progenitor cells, but they must first migrate from the SVZ before they differentiate. It is believed that the cellular substrate for this neurogenesis is the endogenous stem cell [33]. The mechanisms that induce and control this process are unknown. It may be that one can manipulate these endogenous cells to replace damaged neural tissue following injury. Indeed some important observations have been made. Johansson et al. [34] demonstrated that multipotential NSCs migrate to the area of injury after dorsal funiculus sectioning. However, similar to cellular transplants, most differentiated into astrocytes. After prolonged administration of BdrU to the spinal cord of rats, a substantial number of ependymal cells lining the central canal were labeled and few were seen outside the spinal cord central canal ependyma (due to the lack of an SVZ in the spinal cord). After an incision (one day following injury) was made in the dorsal funiculus at T2, there was a 50-fold increase in the proliferating ependymal cells. Electron microscopy demonstrated the cell division to be asymmetric. To demonstrate the fate of these ependymal cells, lesions were made in animals that had received an injection with a fluorescent marker called Dil that labels the ependymal cells prior to the lesion. Dil-labeled cells were abundantly seen in the injury site within one week after the lesion. These cells demonstrated immunoreactivity to GFAP, but neither ␤-tubulin nor O4, confirming them to be astrocytes, and not neurons or oligodendrocytes. Therefore, local cues will have to be overcome so that neurons are formed rather than glia, which may exacerbate scarring. It is also postulated that since there is little neurogenesis occurring in the mature spinal cord, the number of endogenous stem cells may be inappropriate for the replacement of tissue following injury.

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Remyelination of the Spinal Cord Glial precursors have been found throughout the developing and adult CNS. A large proportion of these are oligodendrocyte precursors. When adult rats are pulse-labeled with BrdU, 70–75% of BrdU-labeled cells found in the spinal cord and cortex express NG2, a marker for oligodendrocyte precursors [35, 36]. The exact function of these cells is unknown, but one may speculate that these cells remyelinate the CNS following injury. It has been demonstrated that the numbers of these oligodendrocyte precursors are markedly increased following SCI and demyelination [37–39]. It is hypothesized that these glial precursor cells may be transplanted into the injured spinal cord to remyelinate axons and promote functional recovery. As previously mentioned, when multipotential NSCs are grafted into the injured CNS, only a fraction differentiates into oligodendrocytes. Therefore, stimulating oligodendrocyte lineage commitment prior to transplantation may be necessary to achieve effective remyelination. Grafts of this type are called oligospheres. When oligospheres are transplanted into the myelin-deficient spinal cord, significantly larger areas of myelination were demonstrated compared to neurosphere transplantation [13, 40]. It appears that astrocytes also play an important role during myelination. Therefore, glial restricted precursors may prove more effective for remyelination than oligospheres due to their ability to differentiate into both astrocytes and oligodendrocytes after transplantation [41]. The efficacy of these glial restricted precursors for functional myelination is still in question. Although the use of endogenous and transplanted stem cells has demonstrated some remyelination of axons, no study has clearly demonstrated ‘functional’ myelination after this cellular therapy. As with other NSC grafting strategies, the therapeutic mechanism of oligospheres remains unknown. As with the other grafts, neuroprotective or trophic mechanisms may contribute (fig. 3b). In vitro studies have demonstrated partial electrophysiological recovery of remyelinated axons [42]. However, there has not been electrophysiological evidence of recovery in the live animal [14]. Although few studies have demonstrated functional recovery following stem cell therapy, the field is advancing rapidly. The ability to replace lost neural elements (i.e., neurons and glia) is paramount to neural regeneration following injury. Non-Stem Cell Strategies Fetal Tissue After SCI, a gap often exists at the site of injury. There is often a cyst or syrinx cavity in the spinal cord. Therefore, a bridge may be necessary to permit adequate neural regeneration for both spinal and supraspinal projections. Fetal

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tissue transplants have been demonstrated to provide a permissive condition for axonal regrowth and provide such a bridge. After complete spinal cord transection in the newborn rat or kitten, fetal spinal cord tissue transplanted into the site of injury has allowed some restoration of supraspinal projections and improvement in the locomotor function [43, 44]. In a transection model of adult SCI, transplantation of fetal tissue at the lesion permitted axonal regeneration into the graft, but not beyond the graft-host interface [45–48]. The failure to achieve significant numbers of graft-spanning axons has remained an obstacle to most studies involving tissue grafts for the treatment of SCI. Some have demonstrated that the exogenous administration of brain-derived neurotrophic factor (BDNF) or neurotrophin-3 (NT-3) increases supraspinal axonal growth into the transplant fetal tissue grafts and prevents the atrophy of axotomized supraspinal neurons [49]. It is also problematic that most studies utilizing tissue bridges employ acute models of SCI. These models may not be clinically relevant. Often a syrinx cavity does not develop until the subacute or chronic phase of SCI. Coumans et al. [50] demonstrated that if a fetal tissue transplant and neurotrophin administration is delayed 2–4 weeks after a complete SCI in the rat, axonal regrowth from both propriospinal and supraspinal neurons is increased within the transplant and the host cord caudal to the lesion. These animals also demonstrated significant improvement in locomotion, including recovery of weight-supported plantar stepping on both treadmill and over-ground tasks such as stair climbing. In summary, although fetal tissue transplants have shown some success as tissue bridges, experiments remain hampered with the distal host-graft interface. Some studies have demonstrated axonal presence across the host-graft interface. The numbers of these axons is sparse and may actually represent ‘axonal sparing’ and not regeneration. OECs Axonal regrowth into a site of injury following cellular grafting is plagued by the inability of those axons, which have entered the graft, to cross the hostgraft interface. Therefore, a cell, which may enable axons to re-enter the CNS, may be useful to overcome this barrier to regeneration. Olfactory axons continue to re-enter the olfactory bulb throughout adult life. The entry point is associated with special glial cells known as OECs [51–54]. Investigators have demonstrated that OECs transplanted into the spinal cord mediate the re-entry of regenerating dorsal root axons into the spinal dorsal horn and the injections also increased axon growth into Schwann cell-filled guidance channels [55, 56]. As opposed to other cellular grafts, transplantation of OECs facilitated axonal growth past the host-graft interface. This may be due to the migratory capacity of these cells. In a study by Li and Raisman [57], regenerating axons were

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demonstrated to re-enter the distal host corticospinal tract up to 10 mm caudal to the injection site. These regenerating axons are covered by peripheral myelin formed by the OEC cell. Schwann Cells Because the environment of the peripheral nervous system (PNS) is permissive for regeneration, Schwann cell transplants have also been used as a strategy for the treatment of experimental SCI. These cells may be neuroprotective and have been demonstrated to secrete various growth factors. Theoretically, these cells are also able to form myelin around spared and regenerating axons. As with other cellular grafts, investigators have found that following the transplantation of cultured Schwann cells, the cells integrate into the host tract glial structure [57, 58]. These cellular grafts greatly increase axon sprouting in lesions of the corticospinal tract, but few axons were found to re-enter the distal tract [59]. In contrast, other investigators have demonstrated axonal growth beyond the graft. Schwann cells transplanted after a moderate contusion of the rat thoracic spinal cord permitted propriospinal and supraspinal axons reaching 5–6 mm beyond the graft. A modest improvement in hind limb locomotor performance was detected at 8–11 weeks after injury [60]. Nonetheless, the limited growth beyond the graft, even in this experiment, suggests that recovery was likely to be due to neuroprotection or remyelination of spared axons rather than axonal regeneration. Schwann cells have also been seeded into mini-channels that have been used as bridges. When this transplantation technique is combined with exogenous neurotrophin administration, axonal growth was demonstrated into the graft and into the distal spinal cord, albeit for a limited distance [61]. In summary, these studies demonstrate that both OECs and Schwann cell transplants may be useful to induce axonal regeneration and remyelination after SCI. Although some studies have demonstrated some functional improvement following Schwann cell or OEC transplants, it is unclear if the improvement is due to neuroprotection, trophic factor secretion, or remyelination (see above). The myelination that has been observed has not been demonstrated to be functional nor has it been quantified. While some studies have demonstrated axonal growth into the distal spinal cord, the amount of regrowth is not highly significant, and is unlikely to account for a significant amount of functional improvement following SCI. Macrophages Macrophage recruitment and stimulation are among the earliest events in the multifactorial process of tissue healing. This observation has led to the

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hypothesis that stimulating an appropriate inflammatory response could encourage a cascade of events necessary for regeneration and repair [62]. Because of the immune-privileged status of the CNS, a restricted inflammatory response is seen following injury. This restriction may contribute to the poor regenerative capacity of the CNS compared to the PNS [63–65]. Macrophages, previously exposed in vitro to regenerating peripheral nerve segments, have been shown to induce axonal regrowth in completely transected rat optic nerves [64]. This observation drew attention to the potential for stimulated macrophages to play a role in spinal cord repair. Macrophages stimulated via exposure to peripheral nerve segments in vitro and then re-implanted at the site of a complete SCI in the rat induce partial recovery of motor function [62]. The fact that this function was lost after retransection proved that the recovery was not due to intrinsic spinal cord reflex pathways. Anterograde labeling demonstrated continuity of nerve fibers across the transection site. The authors of this study hypothesize that activated macrophages may provide cytokines, growth factors, and other wound-healing factors [41, 66, 67]. These factors may control the astrocytic response seen after injury, thus reducing the glial scar known to inhibit axonal regeneration [68]. Stimulated macrophages may accelerate processes that normally occur relatively slowly in the injured CNS [62]. Because the activated macrophage strategy is aimed at upstream processes in the injury cascade, this one intervention may then affect numerous downstream events. Given the complexity of SCI pathophysiology, multifactorial therapies of this kind may ultimately prove the most effective. Furthermore, because autologous cells are utilized, many of the ethical and immunological difficulties inherent in other cellular therapies are absent.

Molecular Therapies for SCI

Concepts of Gene Transfer A variety of investigators have pursued gene transfer as a means of inducing neuroprotection and axonal regeneration in the injured spinal cord. As with cellular therapies, a variety of potential strategies exist for spinal cord gene transfer. Transfer can be affected with viral and nonviral vectors. In vivo strategies, which entail gene transfer directly into injured cord parenchyma, and ex vivo strategies, which entail gene transfer into cells that are subsequently grafted, have both been proposed. The best method for gene delivery remains debated. An effective method must accomplish four basic steps. Gene delivery is initiated when a vector binds to the host cell. The cell membrane constitutes the first barrier to gene delivery.

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Efforts are ongoing in our laboratory to create neurotrophic vectors that will target specific cell types. Once past the cell membrane, a vector must provide protection from degradation in the cytoplasm. If the vector enters through endocytosis, lysosomal fusion may result in enzymatic degradation of the transgene. An effective delivery method must either avoid entry into the cell via endocytosis thus preventing lysosomal degradation, or allow entry into the cell via an alternative pathway. The next barrier to gene delivery is the nuclear membrane. In order for most transgenes to be expressed, they must enter the nucleus. The nuclear membrane serves as a relatively effective barrier against foreign entry into the nucleus [69]. Early in the development of gene therapy strategies, entry into the nucleus was limited to mitotically active host cells, and thus naturally occurring breakdown of the nuclear membrane [70–72]. Recent advances have involved the use of nuclear localization signals and viral vectors to overcome this barrier. The final step of gene delivery is expression of the transgene. Because recovery from SCI will require significant amounts of time, gene-based approaches to SCI require long-term gene expression. The duration of expression can vary depending on the vector being used, from transient to extended expression. Long-term expression is usually accomplished through integration of the transgene into the host DNA. Integration may be accomplished through a variety of methods depending on the vector being used. While an effective gene therapy delivery system is able to accomplish these four basic steps, an ideal vector should not be a source of pathogenicity to the host cell. Thus an ideal vector must be nontoxic and elicit little, if any, immune response in the host. Vectors for gene therapy can be divided into two main groups: viral and nonviral gene therapy. Here we will discuss the advances that have been made in each category of gene therapy delivery. Non-Viral Gene Therapy Nonviral gene therapy poses several advantages over viral gene therapy. The main advantage is the lack of pathogenicity of nonviral vectors. The simplest method to provide a transgene for a host is the delivery of naked DNA. In 1980, Capecchi [69] was able to successfully microinject DNA via glass micropipettes directly into the nuclei of host cells in vitro, although DNA expression could not be detected when DNA was injected into the cytoplasm. These results emphasize the importance of overcoming the nuclear membrane as a barrier to transgene delivery. In 1990, Wolff et al. [73] showed that both DNA and RNA transgenes could be effectively expressed when injected into mouse skeletal muscle. However, injection into other major organs, such as the liver, spleen and brain, resulted in relatively inefficient transgene

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expression. In order to increase the efficiency of gene delivery while retaining the simplicity of naked DNA delivery, researchers have developed methods to augment transgene uptake into the host cell. One such method is utilizing electropermeabilization to enhance the uptake of plasmid DNA. Electrically mediated gene transfer has proven effective for gene delivery to murine melanoma cells [74]. Lin et al. [75] designed an intrathecal electroporation probe to be used following intrathecal injection of plasmid DNA. This device greatly enhanced transgene uptake into the spinal cord. Unfortunately, expression of the transgene was transient, greatly diminishing after 2 weeks. To further increase the uptake of DNA in the host cell, researchers have combined plasmid DNA with nonviral carriers. One method utilized by Yang et al. [76] involved coating the transgene onto fine gold particles. In vivo particle bombardment was shown to be effective in a variety of major organs in both rats and mice. Another method of delivery involved combining plasmid DNA with cationic lipid-forming lipoplexes [77]. Variant forms of lipoplexes can improve the efficiency of transfer. The addition of cationic polymers, such as poly-L-lysine or protamine, to the DNA/liposome complex can greatly enhance transgene delivery through a number of methods. The polymers enhance lipoplex endocytosis, provide protection from nuclease activity and enhance transgene entry into the host cell nucleus [78]. Another common addition to lipoplexes is dioleoylphosphatidylethanolamine. Dioleoylphosphatidylethanolamine is a neutral lipid that is capable of destabilizing the lysosomal membrane, permitting the release of the plasmid into the host cell cytoplasm, thus reducing lysosomal degradation of the transgene [79, 80]. An alternative to combining plasmid DNA with cationic lipids is the use of cationic polymers. Cationic polymers have been found to be far more effective than their lipid counterparts at condensing DNA. One such polymer being used is polyethylenimine. Polyethylenimine also acts as a proton sponge, which causes osmotic disruption of the lysosome, rescuing the transgene from enzymatic degradation. Protection of the delivered gene allows for the greater transfection efficiency, which has allowed for polyethylenimine to become one of the most efficient synthetic delivery systems available [81]. Transposons have also emerged as an effective method of delivering plasmid DNA into the host cell. Transposons are naturally occurring elements capable of integrating foreign plasmids into the host cell DNA with the help of two enzymes, integrase and transposase. Transposons have proven to be an extremely effective transgene delivery system for their ability to integrate into the host genome and allow for long-term expression. Transposons can also specifically direct the site of transgene integration [82].

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Viral Vectors Given the limitations present in using nonviral vectors, viral vectors have emerged as the most efficient form of gene therapy delivery. Viral vectors present a variety of advantages over nonviral vectors, including increased efficiency of transfection as well as extended transgene expression. The drawbacks of using viral vectors include the immune response induced as well as constraints on transgene size. Nonetheless, the overall transfection efficiency of viral over nonviral vectors has led researchers to develop novel viral vectors that minimize their limitations while maintaining their effectiveness. Here, we will discuss the four main types of viral vectors being used for gene therapy delivery. Retroviral Vectors Researchers have used retroviral vectors for the purpose of gene delivery for a relatively long time. Retroviruses are advantageous for gene delivery because they allow integration of the transgene into the host genome. This allows for the expression of the transgene for the life of the host cell. Early retroviral studies were successful in using oncoretroviruses such as the Moloney murine leukemia virus for in vivo transfection [83, 84]. The problem posed by these early retroviral vectors was the inability to infect nondividing cells; a problem also faced when using nonviral vector delivery [85, 86]. Thus, focus has turned to lentiviruses, a form of retroviruses that are able to infect nondividing cells. Retroviruses replicate with the help of a preintegration complex that replicates viral RNA through a DNA intermediate, which allows for integration into the host genome. The preintegration complex in oncoretroviruses is believed to be excluded by the nuclear membrane while the matrix protein in lentiviruses contains a nuclear-targeting component which allows for transport of the transgene into the host nucleus, explaining the ability of lentiviruses to infect nondividing cells [87–89]. Human immunodeficiency virus type 1 (HIV-1) is a well-known member of the lentivirus family, which was found to be able to infect nondividing macrophages [90, 91]. HIV-based lentivirus vectors are capable of transfecting liver and muscle tissue, sustaining expression of the transgene for over 6 months [92]. In order to increase the range of transfection, the membranes of lentivirus vectors were modified to contain envelop proteins from different viruses. One common virus whose envelope was used for this purpose was the vesicular stomatitis virus G (VSV-G) [93]. An obvious concern in the use of HIV-1 as vehicle for gene therapy delivery is the inadvertent infection of the host. This has led to the development of attenuated forms of the virus. Attenuation of lentiviruses can be accomplished by eliminating accessory viral genes without hindering the transfection efficiency of the vector. HIV requires several basic genes for function. In addition to structural genes gag, pol, and env, HIV-1 genome contains two regulatory genes, tat and

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rev, and four accessory genes. nef, vif, vpr, vpu [94]. Researchers have attempted to create attenuated HIV vectors that can eliminate as many regulatory and accessory genes as possible to allow for minimal pathogenicity while maintaining transfection efficiency. Zufferey et al. [95] first introduced the attenuated HIV vector by deleting accessory genes vif, vpr, vpu, and nef as well as the structural gene env. They showed that this first generation-attenuated vector was able to retain its transfection ability in nondividing cells. Further studies by Kim et al. [96] demonstrated that the tat gene is not necessary for HIV-1 transfection in nondividing cells in vitro. Tat is a strong transcriptional activator of HIV-1, but Kim showed that this function might be replaced by inserting a human cytomegalovirus promoter into the HIV genome. A third generation lentivirus vector was created which, containing only the gag, pol, and env genes, was shown to be successful in transfecting neurons in vivo [97]. In addition to these attenuated viruses, Zufferey et al. [98] also developed self-inactivating lentiviruses through deletions in the 3⬘ long terminal repeats of the HIV genome. Using self-inactivating viruses decreases the possibility of recombination with wild-type virus, further rendering the vector safe for gene delivery. An alternative to attenuation is the use of nonprimate lentiviruses such as feline immunodeficiency virus. Feline immunodeficiency viruses are unable to infect human cells, but when pseudotyped with VSV-G, transfection of nondividing human cells is possible [99]. Herpes simplex Virus Vectors Herpes simplex virus 1 (HSV-1) is a member of the human herpes viruses. HSV-1 became an attractive vector for the delivery of therapeutic transgenes for several reasons. Like lentivirus, HSV-1 is able to infect nondividing cells. The HSV-1 genome is composed of 152-kb double-stranded DNA, which allows the insertion of large transgenes and general ease of genetic manipulation. Lastly, HSV-1’s most distinguishing characteristic as a viral vector is its ability to establish latent infection in neurons [100]. Like lentivirus, HSV-1 must be attenuated to prevent viral replication in the host. One method to accomplish this was the creation of defective viral vectors. One example is amplicons, which contain the transgene to be delivered flanked by viral recognition sequences. The absence of any genes encoding viral proteins reduces the potential for an inflammatory response to these proteins and prevents replication. However, in order for the transgene to be packaged in an HSV-1 coat, the viral genes for replication and packaging must be provided in trans through a helper virus. Thus HSV-1 vectors may be produced by transfecting cells with amplicon and either cotransfecting with helper virus DNA or superinfecting with HSV [101]. Geller and Breakefield [102] demonstrated the use of such a defective HSV-1 vector to deliver the Escherichia coli

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lacZ gene to neurons in vitro. Originally, Geller used an HSV-1 temperaturesensitive mutant, ts K, as the helper virus in HSV-1 vector production to avoid cell damage. However, because ts K was later found to revert to wild type, an HSV-1 deletion mutant which also effectively packages the amplicon was substituted [103]. An alternative to amplicons is the creation of recombinant viruses. The HSV-1 genome contains three main classes of genes; immediate early genes (IE), early (E) and late (L) genes. Mutational analysis revealed that most of these genes were nonessential for viral replication in cell cultures. The development of HSV-1 deletion mutants has allowed the effective delivery of reporter genes into postmitotic cells [104]. Research is continuing to define deletions capable of further reducing the potential for wild-type reversion of HSV recombinants. Adenovirus Adenovirus holds several advantages over its viral counterparts as a transgene delivery vehicle. Adenovirus is comprised of a 36-kb double-stranded DNA genome, allowing for a large area of transgene manipulation [105]. Several generations of attenuated adenovirus have been created in an attempt to decrease viral toxicity while maintaining efficiency of infectivity. First generation adenovirus was created by deleting the E1 gene, which is necessary for viral gene expression and replication. These viruses were used to successfully deliver the cystic fibrosis transmembrane conductance regulator gene into the lungs of nonhuman primates [106]. The attenuated virus was able to induce transgene expression, though only for a limited time. Transient gene expression is one of the major obstacles to the application of adenovirus. The lack of extended expression may be secondary to the immune response initiated by the low level expression of the remaining viral genes. Such an immune response may precipitate the destruction of transfected cells eliminating transgene expression [107–109]. Immunosuppression in parallel with adenoviral administration prolongs transgene expression [110]. An alternative approach was to completely eliminate viral gene expression. Thus, subsequent generations of adenoviruses were developed each with a larger deletion of viral genes. Second and third generation viruses include deletions in the E1, E2a, as well as the E4 genes. These further-attenuated forms of adenovirus caused less inflammation and allow for longer transgene expression [111–116]. The most advanced generation of adenoviral vectors involve removal of all viral genes. These ‘gutless’ vectors lack all viral genes with the exception of the inverted terminal repeats and packaging sequences required for inclusion into the vector. The lack of viral genes allows insertion of transgenes up to 28 kb in size. In order for this vector to be packaged, a helper virus is needed to provide viral genes in trans. This helper virus lacks the inverted terminal repeats and

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packaging sequences, thus inhibiting it from being packaged into the virus particle [117, 118]. These gutless vectors have allowed for high levels of gene expression with little immune response. Adeno-Associated Virus Adeno-associated virus (AAV) is a 4.7-kb single-stranded DNA virus. Unlike the other systems discussed, AAV is not infectious to humans in its wildtype form. In fact, wild-type AAV is naturally attenuated requiring a helper virus to provide the necessary genes before initiating replication [119]. The AAV vector itself contains no viral genes with the exception of the 125-bp AAV terminal repeats flanking the transgene in question. In order for the vector to be packaged into the AAV vector, viral genes rep and cap are provided in trans through the use of a helper plasmid. This helper plasmid, much like the helper plasmid used in gutted adenoviral production, contains the necessary viral genes for replication and encapsidation, but lacks the terminal repeats necessary to package the helper plasmid itself into the viral vector. AAV is capable of gene delivery to terminally differentiated cells, with minimal inflammatory response and resulting long-term gene expression. More recent studies have been able to further reduce the potential for toxicity of AAV by applying the use of a truncated adenoviral genome rather than a virus to supply the necessary helper genes. This eliminates the risk of contamination of viral preparations by infectious helper virus [120]. Another advantage of AAV is the ability to integrate into the host genome. AAV has been shown to be capable of specific integration into chromosome 19. This capacity may be responsible for the vector’s prolonged transgene expression [121]. Integration along with the lack of a significant immune response has allowed for AAV-delivered transgene expression for up to 18 months [122]. Kaplitt et al. [123] were the first to demonstrate the use of AAV for delivery of transgenes into postmitotic cells in vivo. Much recent work in viral gene therapy has focused on the use of AAV due to its nonpathogenic properties. One of the major drawbacks of AAV is the limited genome size. The AAV genome is not able to accommodate transgenes greater than 4.7 kb in size [124]. Recent work by Sun et al. [125] has provided a strategy to overcome this handicap by utilizing the ability of AAV to heterodimerize. A large single transgene is split and packaged into two separate AAV vectors and coinfected into the host cell. Once inside the cell, heterodimerization occurs which allows for expression of the original transgene. Therapeutic Transgenes The application of gene therapy to SCI depends on the existence of genes capable of stimulating neuroprotection, remyelination, or regeneration. Because

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neurotrophic factors possess these properties, they have attracted the most enthusiasm for application to SCI. A great deal of focus has surrounded members of the ‘classic’ neurotrophin family, including nerve growth factor (NGF), BDNF, and NT-3. Different neurotrophic factors show varying degrees of effectiveness in promoting regeneration of the spinal cord. The difference in effectiveness of these neurotrophic factors can be accounted for by the presence or absence of receptors for these factors on varying types of neurons. For example, in the adult rat lumbar dorsal root ganglia (DRG), receptors for NGF, known as TrkA, are found predominantly in small, unmyelinated neurons, which enter into the dorsal horn from the DRG. Oudega and Hagg [126] have shown that continuous infusion of NGF into the spinal cord following peripheral nerve transection and insertion of a peripheral nerve graft promoted re-entry of sensory axons into the denervated dorsal columns. Further studies by Ramer et al. [127] confirmed NGF’s ability to promote sensory axon growth into the spinal cord. The axons responding to NGF are positive for calcitonin gene-related peptide, a marker for small, umyelinated peptidergic axons. In contrast, TrkB and TrkC receptors, which bind BDNF and NT-3 respectively, are found mainly in DRG neurons possessing thick, myelinated axons [128], which form most of the ascending fibers of the dorsal columns. The scarcity of TrkB receptors within the dorsal horn may explain the failure of spinal BDNF administration to promoted sensory axon growth into the spinal cord [127]. In contrast, BDNF has been shown to promote motor axon growth, illustrating that different neurotrophic factors exert their effects on different neuronal subtypes [129]. Unlike BDNF, NT-3 has been shown to be able to promote sensory axon growth into the spinal cord. Oudega and Hagg [130] showed that continuous infusion of NT-3 into the spinal cord promotes regeneration of dorsal column sensory axons into the spinal cord. In a separate experiment, NT-3 was infused into the spinal cord at the site of a crush lesion. NT-3 was shown to stimulate axonal regrowth in the region of the lesion and distally, without the use of a peripheral nerve graft. NT-3 is also the only neurotrophic factor capable of promoting the growth of corticospinal axons. Axonal sprouting has been observed following a single injection [131]. Glia cell line-derived neurotrophic factor (GDNF) is a neurotrophic factor, which belongs to the cytokine rather than the neurotrophin family. Once thought to be specific for the protection of dopaminergic neurons, GDNF has been proven effective in protecting from motor neuron death following axotomy [132]. Although GDNF was not shown to have an effect on the regrowth of lesioned dorsal column axons, it is more effective than NGF on stimulating axonal growth into the spinal cord [127, 133]. In contrast to the growth-inducing abilities of neurotrophic factors, anti-apoptotic proteins have been utilized in an effort to protect neuronal

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degeneration following injury. The Bcl-2 family of proto-oncogenes is a group of apoptosis-regulating proteins. Two proteins from this family, Bcl-2 and Bcl-XL, play an anti-apoptotic role and are believed to exert their actions by preventing the release of cytochrome C from the mitochondria, an important step in the apoptosis pathway [134]. Overexpression of Bcl-2 into embryonic sensory neurons was able to prevent death following deprivation of certain growth factors, while Bcl-XL has been shown to prevent death in primary neuronal cultures when delivered via adenoviral gene transfer [135, 136]. In addition, Bcl-xL has also been shown in vivo to prevent apoptotic death of cholinergic neurons following axotomy [137]. Thus anti-apoptotic proteins have become a reasonable addition to the library of therapeutic transgenes capable of protecting neuron survival. Viral Gene Delivery to the Spinal Cord While direct spinal cord injection of viral vectors carrying therapeutic transgenes is the most efficient method of introducing transgenes into the spinal cord, several complications have arisen from this method of injection. Liu et al. [138] attempted to inject recombinant first generation adenovirus expressing the lacZ gene under control of the cytomegalovirus promoter directly into the T7–8 levels of spinal cord in adult Sprague-Dawley rats. Transgene expression was effective after one week, but quickly diminished thereafter, almost completely disappearing by 2 months postinjection. This down-regulation is most likely due to the intense immune response elicited with the injection of adenoviral vectors. Our laboratory observed a cellular infiltrate in spinal cords 7 days after adenoviral injection (fig. 4). Immunohistochemistry suggested that this response was predominantly gliotic although a variety of mononuclear cells were also observed. In animals immunsuppressed with cyclosporin A, ␤-gal transgene staining remained robust up to 2 months postinjection. The immune response can be partly attributed to a specific reaction in response to the early generation adenovirus and partly attributed to the nonspecific immune reaction in response to the trauma induced by spinal cord injection. The former can be partly solved using the now available ‘gutless’ adenoviral vectors. Because these vectors lack viral genes, the host cannot present the viral gene products on major histocompatibility complexes. However, an immune response can still be mounted against the viral capsid, which is itself immunogenic. In addition, the application of gutless vectors does not eliminate the problem of the trauma of direct injection. The presence of a significant immune response to direct spinal cord injection and the potential trauma of this approach has spurred the search for alternatives. Direct intraparenchymal spinal cord and brain injection of early generation adenoviral vectors results in a mononuclear inflammatory infiltrate,

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a

b

c

Fig. 4. Photomicrographs demonstrating local inflammatory response following adenoviral injection into the T-8 spinal cord. a At 7 days post-PBS (vehicle) injection, no infiltrate surrounding the cannula tract (arrow) is seen. b At 7 days post-lacZ adenovirus injection, inflammatory infiltrate is revealed (arrow). c At 7 days post-NGF adenovirus injection, a mild inflammatory infiltrate (arrow) is found surrounding the cannula tract.

eliminating transgene expression [139, 140]. Researchers, including us, have thus turned to peripheral injections as an alternative. In theory, this approach should remove the viral capsid from the spinal cord, hence eliminating the potential for damage through an immune response to the viral coat proteins. This approach also avoids direct trauma to the spinal cord. Earlier studies have shown that replication-defective adenoviruses have the ability to undergo retrograde transport following injection into the CNS [141, 142]. Kuo et al. [142] used adenoviral vectors containing the lacZ gene under the control of the Rous sarcoma virus promoter for retrograde axonal tracing studies. Kuo demonstrated staining at the site of injection as well as several sites distal to the injection. These studies led our laboratory to evaluate the retrograde transport of vectors injected into the PNS and its projection areas. Attenuated adenoviral vectors with deletions in the E1a, E1b, and E3 viral genes expressing lacZ under the Rous sarcoma virus promoter were injected into the sciatic nerve, foot pad, and anterior tibialis muscle of adult rats. Histological examination of the spinal cord revealed ␤-gal staining (transgene expression) occurring predominantly in neuronal cells with large cell bodies (fig. 5). Staining was also present in the DRG. This phenomenon, which we call ‘remote delivery’, was significantly greater in spinal cords following injection into the sciatic nerve in comparison with foot pad and intramuscular injection [140]. These studies demonstrated that vectors based on viruses that had not previously been considered neurotrophic were capable of penetrating the CNS from peripheral inoculation sites.

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Fig. 5. ␤-Galactosidase staining of motor neuron cell bodies from rat spinal cord. Animals were injected into the sciatic nerve with adenovirus carrying a lacZ reporter gene 7 days prior to histology.

In order to confirm that retrograde axonal transport is responsible for the remote delivery phenomenon, we studied the effects of intraneural colchicine on adenoviral vector transport. Colchicine inhibits tubulin polymerization and hence disrupts axonal transport. Intrasciatic colchicine injection inhibits remote adenoviral and AAV gene delivery following sciatic injection in a dose-dependant fashion implicating retrograde axonal transport in this process [143]. Spinal cord gene expression following peripheral injections did not trigger the inflammatory response observed following direct injections. Because the termination of gene expression is linked to the inflammatory response, this discovery led to the hope that remote delivery might prolong transgene expression. Nonetheless, spinal cord transgene expression following remote delivery followed the same time course as direct injection deteriorating within 3 weeks of injection [140]. Both chronic dexamethasone and cyclosporine treatment stimulated higher levels of gene expression in the lumbar spinal cord and DRG and prolonged gene expression following remote adenoviral injection [144]. Nonetheless, no sign of cell death could be detected in parallel with the disappearance of transgene expression. Together, these findings suggested that an inflammatory response to the vector at the site of injection was shutting off

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gene expression through a promoter level noncytolytic mechanism. Because the neurons of interest appeared to remain healthy after the disappearance of transgene expression, we conducted repeated sciatic nerve injection of adenoviral vectors in the hope of boosting gene expression. While repeated injection resulted in prolonged gene expression at the site of injection, spinal cord gene delivery was not boosted. Following initial injection, gene expression in the nerve was found predominantly in the perineurium without a significant inflammatory response at early timepoints. In contrast, gene expression at the repeat-injection sites occurs within phagocytic infiltrative cells noted shortly after injection. This immune response is likely to reduce the available titer of vector for remote delivery and may inhibit axonal uptake. Glatzel et al. [145] were also able to use adenovirus for the delivery of transgenes into the spinal cord. Though they were unable to show spinal cord expression of the transgene following intramuscular injection, they were able to demonstrate staining following adenoviral sciatic nerve injection. In their experiment, injection directly into the DRG resulted in a much higher efficiency of gene transfer compared to sciatic nerve injections, without an increase in neurological side effects. In addition, they evaluated the role that the immune response had on eliminating gene expression. Rag-1⫺/⫺ mice, which lack differentiated B and T cells, were transfected with adenoviral vectors containing the lacZ transgene. ␤-Gal expression could be seen for up to 102 days without any signs of deterioration, which further confirms the role of the immune response as the rate-limiting step in the duration of transgene expression. Because AAV vectors induce prolonged gene expression with a minimal inflammatory response, attention has turned to their application to spinal cord gene transfer. AAV vectors were demonstrated to successfully allow for transgene expression following direct injection into the cervical enlargement of adult rat spinal cord [146]. Thus, attention turned to using AAV as a vector for delivery given the lack of an immune response generated by administration of AAV [124]. Glatzel injected recombinant AAV delivering enhanced green fluorescent protein (EGFP) into the DRG of L4/L5. Expression of EGFP was detectable up to 52 days postinjection without any signs of deterioration [145]. Our laboratory has also observed excellent transduction of cervical spinal cord neurons following direct spinal cord injection (fig. 6). The next step with AAV, as with adenovirus, was to utilize AAV’s property of retrograde transport to allow for the remote delivery of transgene into the spinal cord following indirect injection in the PNS. Kaspar et al. [147] have demonstrated retrograde transport of AAV-delivered GFP from the hippocampus and striatum to the entorhinal cortex and substantia nigra. Our laboratory has observed retrograde delivery of AAV following peripheral nerve injection in mice at a variety of locations [143].

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Fig. 6. Motor neuron GFP expression 3 weeks after AAV.GFP injection of the cervical spinal cord in SOD1 mutant mice.

Lentiviral vectors have also proven useful for transgene delivery to the spinal cord. Mazarakis et al. [148] was able to demonstrate that equine infectious anemia virus pseudotyped either with rabies-G envelope or VSV-G can effectively deliver transgenes into the rat spinal cord following intraspinal injection. Following injection, the spinal cord failed to show any significant cell damage and only a mild degree of inflammation, confirming the low toxicity of lentiviral vector delivery. Mazarakis also showed that rabies-G pseudotyped lentivirus, but not VSV-G, is able to undergo retrograde transport following injection into rat gastrocnemius muscle. Transgene expression was detected at 3 weeks following lentiviral delivery, but is believed to persist for much longer time periods based on observations in other parts of the CNS. Prior to this study, lentiviral vectors have not been shown to undergo retrograde transport. This new capacity for retrograde transport is likely to be secondary to innate properties of the rabies-G protein to convey axonal uptake and transport [149]. Our laboratory has achieved retrograde delivery to cervical spinal cord motor neurons through injection into the brachial plexus using the rabies-G pseudotyped equine infectious anemia virus carrying a lacZ reporter gene. Spinal cord stained 3 weeks following brachial plexus injection of the virus showed transgene expression, confirming retrograde transport (fig. 7). The properties of the rabies-G envelope combined with a low immune toxicity should provide several new possibilities for using lentiviruses as potential vectors for transgene delivery into the spinal cord. In addition to making ‘remote delivery’ possible, direct injection of this vector into the spinal cord should make gene delivery to

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Fig. 7. ␤-Galactosidase staining of motor neuron cell bodies in the ventral horn of spinal cord from mice injected with lacZ expressing rabies-G pseudotyped equine infectious anemia virus. The mice were injected with 4 ⫻ 108 PFU titer of EIAV.RabG.lacZ into the brachial plexus. The spinal cord was stained 3 weeks postinjection.

both the site of injection as well as upstream projection areas possible. This may prove useful for trophic factor delivery to upper motor neurons projecting into the site of injury. Therapeutic Animal Models Several animal models have been used to test the therapeutic potential of the viral vectors discussed above. One animal model is the delivery of GDNF into transgenic superoxide dismutase 1 (SOD1) mice. Transgenic SOD1 mice contain a mutation in the Cu/Zn SOD1 gene on chromosome 21, mimicking a form of amyotrophic lateral sclerosis (ALS) found in 20% of all ALS cases. GDNF has been shown to demonstrate an overwhelmingly potent ability to protect motor neuron survival compared to other neurotrophic factors, and is thus an ideal candidate for use in ALS animal models [132]. Acsadi et al. [150] delivered GDNF via intramuscular adenoviral vector injection into SOD1 mice. This adenoviral vector contained the rat GDNF

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cDNA under a cytomegalovirus promoter followed by an internal ribosomal entry signal and an EGFP cDNA (AVR-GDNF). The virus was injected at a titer of 5 ⫻ 109 plaque forming units (PFUs) into the anterior tibialis, gastrocnemius, quadriceps, and paraspinal muscles of 5- to 7-day-old SOD1 mice. Control animals were injected with virus containing lacZ as the reported gene in order to confirm uptake of the virus into the muscle and retrograde transport into the spinal cord via ␤-gal staining. ELISA analysis was used to confirm the level of GDNF in the muscles, measuring the GDNF expression at 3 months postinjection to be 454 ⫾ 268 pg/mg (mean ⫾ SE), and at 4 months postinjection to be 180 ⫾ 106 pg/mg. GDNF levels in untreated mice were at undetectable levels. Importantly, injections in neonatal animals appeared to induce a longer lasting period of gene expression. This effect may be secondary to limited immune recognition in the neonate. SOD1 mice treated with AVR-GDNF showed a clear delay in the development of ALS symptoms. Untreated mice developed symptoms (e.g., hind limb tremor, slowing of movements) at 106.2 ⫾ 2.71 (mean ⫾ SD) days of age compared to treated mice, which developed symptoms at 116.1 ⫾ 8.6 days of age. Injection with AVR-GDNF also increased the lifespan of SOD1 mice following onset of symptoms by 8 days compared to untreated SOD1 mice, increasing lifespan overall by an average of 14 days. To quantitatively measure the effect of the disease on mice, the ability of the mice to maintain their balance on a rotating rod (RotaRod) was measured. RotaRod performance started to decline in SOD1 mice compared to wild-type mice following 8 weeks of age. The study showed that SOD1 mice treated with AVR-GDNF showed a significantly slower decline in performance compared to untreated SOD1 mice. Finally, the effect of GDNF was demonstrated in motor neuron counts of the spinal cord anterior horn 2, 3, and 4 months postinjection compared to untreated SOD1 mice. AVR-GDNF demonstrated an ability to prolong large motor neuron (⬎20 ␮m) survival for up to 2 months, after which motor neuron survival declined in a similar fashion to that found in untreated SOD1 mice. A similar study conducted by Manabe et al. [151] also delivered an adenoviral vector-containing GDNF into SOD1 mice. In this study, adenovirus at 108 PFU was injected into the gastrocnemius muscle of SOD1 mice, once a week starting at 35 weeks of age. Quantitative measurements of disease included evaluation of clinical grade, unilateral movement in a circular cage, and RotaRod performance at 35, 40, 42, and 46 weeks of age. Although there was not a significant difference between adenoviral vector-containing GDNFtreated mice compared to untreated mice, there was a tendency of improvement in the treated animals. In addition, large motor neuron survival was evaluated via hematoxylin and eosin staining as well as immunohistochemistry for p-Akt positive large motor neurons indicative of apoptotic death. Both means of

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evaluation showed significant preservation of motor neurons in adenoviral vector-containing GDNF-treated SOD1 mice. The difference in the survival outcome between these two studies may be derived from the age of the animal at the time of treatment. Wang et al. [152] were able to demonstrate the neuroprotective effects of AAV vector GDNF gene delivery. Once again, SOD1 mice were used as animal models. In order to differentiate GDNF that is transgenically expressed from those being endogenously expressed, an AAV vector containing a transgene expressing a GDNF-FLAG fusion protein was developed. GDNF-FLAG can be easily determined via immunofluorescence staining with polyclonal rabbit anti-FLAG antibodies. The AAV-GDNF was injected into the gastrocnemius and triceps brachii muscles of all the four limbs of SOD1 mice at 9 weeks of age. At 110 days of age (7 weeks postinjection), GDNF levels in AAV-GDNFtreated mice was 7,985.0 ⫾ 874.0 pg/mg, an increase of ⬎120-fold compared to untreated mice. AAV-GDNF-treated mice also showed considerable preservation of the gastrocnemius muscle, showing little evidence of neutrogenic atrophy and weighing nearly one and a half times more than untreated mice. Retrograde transport of GDNF was demonstrated via FLAG staining of the spinal cord. Nissl staining of the motor neurons in the lumbar spinal cord showed significant protective effects of AAV-GDNF in the side of the cord ipsilateral to AAV-GDNF injection. Finally, AAV-GDNF demonstrated similar effects as adenovirusdelivered GDNF on RotaRod testing. Performance on the RotaRod deteriorated after 12 weeks of age in SOD1 mice compared to wild-type mice. AAV-GDNF was able to delay the onset of motor deficits as well as slow the deterioration of performance on the RotaRod. The onset of motor deficit in AAV-GDNF treated mice was 114.0 ⫾ 4.0 days compared to 101.3 ⫾ 5.4 days in untreated mice. Lifespan in treated mice was increased by a mean of 16.6 ⫾ 4.1 days; an improvement remarkably similar to the one found in AVR-GDNF-treated SOD1 mice in the study conducted by Acsadi et al. [150]. Despite the delay in the onset of symptoms and increase in lifespan, AAV-GDNF-treated mice showed no difference in the duration of disease when compared to untreated mice. The significantly higher level of expression of GDNF in these experiments compared to those found in the AVR-GDNF experiments lends a great deal of promise to the use of AAV as an ideal vector for delivery of therapeutic transgenes. Further experiments are necessary to test whether administering the transgene at an earlier age can affect the duration of disease [152]. GDNF’s ability to protect motor neuron following axotomy was also illustrated by Baumgartner and Shine [153]. Adenoviral vectors were created containing expression cassettes for BDNF, GDNF, NGF, or ciliary neurotrophic factor. Adenoviral vectors were injected into the gastrocnemius, flexor longus digitorum, and tibialis PFU titer. Adenoviruses carrying a lacZ transgene and

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adenovirus void of the transgene, as well as a virus-free vehicle, were injected into the same hind limb muscles as controls. Retrograde transport of virally delivered transgenes was confirmed with ␤-gal staining in the lumbar spinal cord motor neurons. In order to study the neuroprotective effects of the growth factors, 2 days following injection the sciatic nerve was severed. Seven days postaxotomy, the lumbar spinal cord segments were removed and it was found that only animals treated with GNDF-containing adenovirus showed a significant difference in preserving neuron survival when compared to empty adenovirus or vehicle-treated animals. At 2 days postaxotomy, animals treated with GDNF showed preservation of 70% of its motor neurons compared to 44% seen in vehicle controls. The neuroprotective effect of GDNF proved to be transient, showing no difference from control animals at 42 days postaxotomy. These results further demonstrate the potent ability of GDNF in neuroprotection as well as setting the stage for the use of neurotrophic factors for protection following SCI. Smith and his colleagues [154] have been able to utilize adenoviral vectors for the delivery of neurotrophic factors to induce functional recovery of axons into the dorsal root entry zone. Recombinant adenoviral vectors were created containing transgenes encoding for FGF2, NGF, L1 cell adhesion molecule, or ␤-galactosidase (LacZ). Sprague-Dawley rats were treated with triple crush lesions at the L4 and L5 dorsal roots. Under natural conditions, peripheral nerve regeneration is halted at the dorsal root entry zone, the CNS border. In rats injected with a 7.5 ⫻ 106 PFU/␮l titre of adenovirus carrying either NGF or FGF2 16 days following rhizotomy, large numbers of calcitonin gene-related peptide-positive axon fibers can be seen growing into the dorsal horn compared to uninfected rats. In addition to histological analysis, Smith was able to demonstrate functional recovery following NGF or FGF2 administration. Rats treated with NGF or FGF2 showed recovery of nociception as evaluated using a plantar heat test. Furthermore, recovery of proprioception was evaluated using a grid runway test. None of the neurotrophic factors administered was able to induce any recovery of proprioception, indicating specific targeting by NGF and FGF2. Ex vivo Gene Transfer The use of cell grafts that express a therapeutic transgene is an alternative to in vivo gene transfer. This method, known as ex vivo gene transfer, involves genetically modifying cells in vitro to express a gene of interest, and then transplanting the cell graft into the host. One advantage of this method is the potential to verify transgene expression in the desired cells before transplantation into the host. Another major advantage is the ability of the transplanted cell to produce a long-term steady-state therapeutic level of transgene, a problem commonly encountered with the use of viral or nonviral vectors (fig. 8).

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Retroviral vector

Retroviral vector

Fibroblast

Spinal cord

NGF Spinal cord

NGF

b

NGF Spinal cord

a Fig. 8.a Ex vivo gene transfer. Following in vitro infection with a retroviral (or an alternative virus) vector carrying a therapeutic transgene, the fibroblasts are grown in culture. Verification of the gene expression can be confirmed before transplantation of the fibroblast into animal spinal cords. b In vivo gene transfer. Retroviral vectors carrying the therapeutic transgene are directly injected into the spinal cord of animal models.

The first consideration in ex vivo gene transfer is the type of cell to be used for grafting. Ease of infection in vitro and the viability of the cells themselves, as well as their effects on the viability of the cells around them once grafted into the host, are all factors that can mitigate the choice of cell type. For these reasons,

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primary fibroblasts have been a popular choice for ex vivo experiments. Not only are fibroblasts easily sustained in vitro and well accepted into the CNS, fibroblasts naturally secrete collagen and fibronectin, which provide a conducive environment for neurite growth. Also, since fibroblasts are nonneuronal and nonglial in origin, they are void of any growth-inhibiting molecules that may prohibit neuronal growth. The earliest studies using fibroblasts to provide therapeutic transgenes to the CNS came from Rosenberg et al. [155], who were able to modify fibroblasts by infecting them with mouse NGF cDNA via a retroviral vector. The cells were then grafted into the brain following surgical lesion between the fimbria and the fornix. Two weeks after graft implantation, the animals were sacrificed and stained for choline acetyltransferase, an indication of survival of cholinergic cell bodies. It was found that animals receiving the NGF-secreting cell graft preserved 92% of their cholinergic cells compared to only 49% in control animals. These experiments set the stage for using fibroblasts for ex vivo gene transfer. Fibroblasts producing NGF have been transplanted into the spinal cord in an attempt to induce neuronal growth. Such cell grafts were found to be viable and producing NGF for up to one year following transplantation [156]. The grafts heavily induced growth of sensory axons, verified by calcitonin generelated peptide staining, proving the grafts’ ability to induce growth of a specific axonal type. The ability of fibroblast grafts to induce axonal growth following acute SCI was also observed. NGF-producing fibroblasts were implanted into the spinal cord following spinal dorsal hemisection lesions. The cell grafts were able to induce growth of not only sensory axons, but also of motor axons, albeit to a lesser extent [157]. The injury-evoked responsiveness to NGF provides important insight into the selective use of NGF as a neurotrophic factor in SCI. Similar results were obtained in studies in which NGF grafts were implanted 1–3 months following spinal cord lesion to study the effects on the chronically injured spinal cord. While sensory fibers were noted to regenerate, no motor neuron response was observed in the chronically injured spinal cord. Following SCI neurotrophin receptors are hypothesized to increase [158]. This upregulation is most likely transient, explaining the lack of motor neuron response following chronic injury [159]. The above experiments were carried out using early generation retrovirus to genetically modify the cell grafts. Retroviral vectors can be effective because they integrate into the host genome and allow for long-term gene expression. Alternative vectors have also been utilized and proved to be successful. Lentiviral vectors, retroviral vectors capable of gene transfer to terminally differentiated cells, have been successfully used to modify fibroblasts [160, 161]. Liu et al. [162] were also able to use adenoviral vectors to modify fibroblasts. Though ex vivo neurotrophic gene transfer to fibroblasts has shown promise, the use of alternative cell lines as well as other neurotrophins are also

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capable of inducing axonal growth in the CNS. Because of their possible advantage for autologous grafting, skin fibroblasts have been attempted for use as cell grafts. Early attempts at using skin fibroblasts were unsuccessful in producing prolonged transgene expression [163]. OECs have also been used as implants for ex vivo gene transfer. Primary OECs have been modified with both adenoviral and lentiviral vectors, with grafts allowing for transgene expression for up to one and 4 months respectively [164]. Cell grafts that have been modified to produce NT-3 have also been shown to promote corticospinal axon growth when grafted into the spinal cord following hemisection lesion [165]. Conclusion

Despite many years of research in the field of SCI and regeneration, the prognosis for those who have sustained an SCI remains dismal. Few therapeutic strategies are available to the clinician. The barriers to effective neural regeneration, and hence functional recovery, are multifactorial. These barriers include the lack of an intrinsic cellular response to divide and regenerate, the need to overcome inhibitory barriers at the injury site, and the need to recapitulate the native circuitry following injury. Advances in both genetic and cellular therapy for SCI have begun to unravel some of the difficulties with functional neuronal recovery. Stem cells, both exogenous and endogenous, have the capability to differentiate into various CNS lineages. These cells may, therefore, provide neurogenesis, neuroprotection, trophic support, and/or remyelination. Furthermore, they may provide effective tissue bridges for regenerating axons. Stem cell therapy may supplant existing tissue or cell transplant paradigms and their inherent shortcomings. The fields of molecular neurobiology and genetic therapy are rapidly advancing. This therapeutic strategy will grant clinicians the ability to alter the function of intrinsic or extrinsically placed cells. These cells may then produce growth factors or other growth-permissive and neural-protective proteins capable of supporting the injured spinal cord. These therapies may be delivered systemically without the need to access the CNS surgically. Despite the grim prognosis of SCI, cellular and genetic therapies continue to provide the hope of recovery following an SCI. In experimental animal models of SCI, both are providing evidence of functional recovery. This work lays the groundwork for human clinical trials. References 1

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125 Sun L, Li J, Xiao X: Overcoming adeno-associated virus vector size limitations through viral DNA heterodimerization. Nat Med 2000;6:599–602. 126 Oudega M, Hagg T: Nerve growth factor promotes regeneration of sensory axons into adult rat spinal cord. Exp Neurol 1996;140:218–229. 127 Ramer M, Priestley J, McMahon S: Functional regeneration of sensory axons into the adult spinal cord. Nature 2000;403:312–316. 128 McMahon S, Armanini M, Ling L, Phillips H: Expression and coexpression of Trk receptors in subpopulations of adult primary sensory neurons projecting to identified peripheral targets. Neuron 1994;12:1161–1171. 129 Jakeman L, Wei P, Guan Z, Stokes B: Brain-derived neurotrophic factor stimulates hindlimb stepping and sprouting of cholinergic fibers after spinal cord injury. Exp Neurol 1998;154:170–184. 130 Oudega M, Hagg T: Neurotrophins promote regeneration of sensory axons in the adult rat spinal cord. Brain Res 1999;818:431–438. 131 Schnell L, Schneider R, Kolbeck R, Barde Y, Schwab M: Neurotrophin-3 enhances sprouting of corticospinal tract during development and after adult spinal cord lesions. Nature 1994;367:170–173. 132 Henderson C, Phillips H, Pollock R, Davies A, Lemeulle C, Armanini M, Simpson L, Moffet B, Vandlen R, Koliatsos V, Rosenthal A: GDNF: A potent survival factor for motor neurons present in peripheral nerve and muscle. Science 1994;266:1062–1064. 133 Bradbury E, Khemani S, King V, Priestley J, McMahon S: NT-3 promotes growth of lesioned adult rat sensory axons ascending in the dorsal columns of the spinal cord. Eur J Neurosci 1999;11:3873–3883. 134 Kluck R, Bossy-Wetzel E, Green D, Newmeyer D: The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apoptosis. Science 1997;275:1132–1136. 135 Allsopp T, Wyatt S, Paterson H, Davies A: The proto-oncogene bcl-2 can selectively rescue neurotrophic factor-dependent neurons from apoptosis. Cell 1993;73:295–307. 136 Matsuoka N, Yukawa H, Ishii K, Hamada H, Akimoto M, Hashimoto N, Miyatake S: Adenovirusmediated gene transfer of Bcl-xL prevents cell death in primary neuronal culture of the rat. Neurosci Lett 1999;270:177–180. 137 Blomer U, Kafri T, Randolph-Moore L, Verma I, Gage F: Bcl-xL protects adult septal cholinergic neurons from axotomized cell death. Proc Natl Acad Sci USA 1998;95:2603–2608. 138 Liu Y, Himes B, Moul J, Huang W, Chow S, Tessler A, Fischer I: Application of recombinant adenovirus for in vivo gene delivery to spinal cord. Brain Res 1997;768:19–29. 139 Byrnes A, Rusby J, Wood M, Charlton H: Adenovirus gene transfer causes inflammation in the brain. Neuroscience 1995; 66:1015–1024. 140 Boulis N, Turner DE, Dice J, Bhatia V, Feldman E: Characterization of adenoviral gene expession in spinal cord after remote vector delivery. Neurosurgery 1999;45:131–137. 141 Ghadge G, Roos R, Kang U, Wollmann R, Fishman P, Kalynych A, Barr E, Leiden J: CNS gene delivery by retrograde transport of recombinant replication-defective adenoviruses. Gene Ther 1995;2:132–137. 142 Kuo H, Ingram D, Crystal R, Mastrangeli A: Retrograde transfer of replication deficient recombinant adenovirus vector in the central nervous system for tracing studies. Brain Res 1995;705:31–38. 143 Boulis NM, Willmarth N, Song D, Feldman E, Imperiale M: Intraneural colchicine inhibition of adenoviral and adeno-associated viral vector remote spinal cord gene delivery. Neurosurgery 2003;52:381–387. 144 Turner D, Noordmans A, Feldman E, Boulis N: Remote adenoviral gene delivery to the spinal cord: Contralateral delivery and reinjection. Neurosurgery 2001;48:1309–1316. 145 Glatzel M, Flechsig E, Navarro B, Klein M, Paterna J, Bueler H, Aguzzi A: Adenoviral and adenoassociated viral transfer of genes to the peripheral nervous system. Proc Natl Acad Sci USA 2000; 97:442–447. 146 Peel A, Zolotukhin S, Schrimsher G, Muzyczka N, Reier P: Efficient transduction of green fluorescent protein in spinal cord neurons using adeno-associated virus vectors containing cell type-specific promoters. Gene Ther 1997;4: 16–24. 147 Kaspar B, Erickson D, Schaffer D, Hinh L, Gage F, Peterson D: Targeted retrograde gene delivery for neuronal protection. Mol Ther 2002;5:50–56. 148 Mazarakis N, Azzouz M, Rohll F, Ellard F, Wilkes F, Olsen A, Carter E, Barber R, Baban D, Kingsman S, Kingsman A, O’Malley K, Mitrophanous K: Rabies virus glycoprotein pseudotyping

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of lentiviral vectors enables retrograde axonal transport and access to the nervous system after peripheral delivery. Hum Mol Genet 2001;10:2109–2121. Tsiang H: Evidence for an intraaxonal transport of fixed and street rabies virus. J Neuropathol Exp Neurol 1979;38:286–299. Acsadi G, Anguelov R, Yang H, Toth G, Thomas R, Jani A, Wang Y, Ianakova E, Mohammad S, Lewis R, Shy M: Increased survival and function of SOD1 mice after glial cell-derived neurotrophic factor gene therapy. Hum Gene Ther 2002;13:1047–1059. Manabe Y, Nagano I, Gazi M, Murakami T, Shiote M, Shoji M, Kitagawa H, Setoguchi Y, Abe K: Adenovirus-mediated gene transfer of glial cell line-derived neurotrophic factor prevents motor neuron loss of transgenic model mice for amyotrophic lateral sclerosis. Apoptosis 2002;7:329–334. Wang L, Lu Y, Muramatsu S, Ikeguchi K, Fujimoto K, Okada T, Mizukami H, Matsushita T, Hanazono Y, Kume A, Nagatsu T, Ozawa K, Nakano I: Neuroprotective effects of glial cell linederived neurotrophic factor mediated by an adeno-associated virus vector in a transgenic animal model of amyotrophic lateral sclerosis. J Neurosci 2002;22:6920–6928. Baumgartner B, Shine H: Neuroprotection of spinal motor neurons following targeted transduction with an adenoviral vector carrying the gene for glial cell line-derived neurotrophic factor. Exp Neurol 1998;153:102–112. Romero M, Rangappa N, Garry M, Smith G: Functional regeneration of chronically injured sensory afferents into adult spinal cord after neurotrophin gene therapy. J Neurosci 2001;21:8408–8416. Rosenberg M, Friedmann T, Robertson R, Tuszynski M, Wolff J, Breakefield X, Gage F: Grafting genetically modified cells to the damaged brain: Restorative effects of NGF expression. Science 1988;242:1575–1578. Tuszynski M, Peterson D, Ray J, Baird A, Nakahara Y, Gage F: Fibroblasts genetically modified to produce nerve growth factor induce robust neuritic ingrowth after grafting to the spinal cord. Exp Neurol 1994;128:1–14. Tuszynski M, Gabriel K, Gage F, Suhr S, Meyer S, Rosetti A: Nerve growth factor delivery by gene transfer induces differential outgrowth of sensory, motor, and noradrenergic neurites after adult spinal cord injury. Exp Neurol 1996; 137:157–173. Ernfors P, Henschen A, Olson L, Persson H: Expression of nerve growth factor receptor mRNA is developmentally regulated and increased after axotomy in rat spinal cord motor neurons. Neuron 1989;2:1605–1613. Grill R, Blesch A, Tuszynski M: Robust growth of chronically injured spinal cord axons induced by grafts of genetically modified NGF-secreting cells. Exp Neurol 1997;148:444–452. Englund U, Ericson C, Rosenblad C, Mandel R, Trono D, Wictorin K, Lundberg C: The use of a recombinant lentiviral vector for ex vivo gene transfer into the rat CNS. Neuroreport 2000;11: 3973–3977. Ericson C, Wictorin K, Lundberg C: Ex vivo and in vitro studies of transgene expression in rat astrocytes transduced with lentiviral vectors. Exp Neurol 2002;173:22–30. Liu Y, Himes B, Tyron B, Moul J, Chow S, Jin H, Murray M, Tessler A, Fischer I: Intraspinal grafting of fibroblasts genetically modified by recombinant adenoviruses. Neuroreport 1998;9:1075–1079. Palmer TD, Rosman G, Osborne W, Miller A: Genetically modified skin fibroblasts persist long after transplantation but gradually inactivate introduced genes. Proc Natl Acad Sci USA 1991; 88:1330–1334. Ruitenberg M, Plant G, Christensen C, Blits B, Niclou S, Harvey A, Boer G, Verhaagen J: Viral vector-mediated gene expression in olfactory ensheathing glia implants in the lesioned rat spinal cord. Gene Ther 2002;9:135–146. Grill R, Murai K, Blesch A, Gage F, Tuszynski M: Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury. J Neurosci 1997;17:5560–5572. Nicholas M. Boulis, MD Department of Neurosurgery S31, Cleveland Clinic Foundation 9500 Euclid Ave, Cleveland, OH 44195 (USA) Tel. ⫹1 216 444 5188, Fax ⫹1 216 445 1466, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 104–123

Neural Stem Cell Transplantation for Spinal Cord Repair Akio Iwanami a–c, Yuto Ogawa a–c, Masaya Nakamura b,c, Shinjiro Kaneko a–c, Kazunobu Sawamoto c,d, Hirotaka James Okano a,c, Yoshiaki Toyama b,c, Hideyuki Okano a,c,d Departments of aPhysiology and bOrthopaedic Surgery, Keio University School of Medicine, Shinjuku, Tokyo, cCore Research for Evolutional Science and Technology, Japan Science and Technology Corporation, Osaka and dDepartment of Neuroanatomy, Osaka University Graduate School of Medicine, Suita, Osaka, Japan

Introduction

Ever since the neuroanatomist Cajal [1] reported in the 1920s that the mature central nervous system (CNS) cannot generate new neurons, it was believed that the mammalian CNS does not have the capacity for repair after injury. Nevertheless, in the 1970s, preliminary work in neural transplantation was done which indicated that the neural tissue obtained from fetal rat brain survived, reconstructed neuronal networks, and reversed motor abnormalities when grafted into the animal model of Parkinson’s disease. With the discovery of the potential for functional brain transplantation, interest in neural transplantation escalated sharply and the field of ‘Functional Neurosurgery’ was born. During the 1980s, clinical transplantation trials for Parkinson’s disease attempted to replace dopaminergic neurons with transplants of dopamine-producing cells of various derivations. Clinical application of neural tissue transplantation is still practically limited by the lack of availability of donor fetal brain or spinal cord tissue. In recent years, however, it has become evident that the developing and even the adult mammalian CNS contains self-renewing, multipotent neural stem cells (NSCs), which can be harvested as a source of material for grafting. Recent technological advances developed for the identification, isolation, and expansion of

NSCs raises the enormous potential of therapeutic applications for nervous system disorders [2]. Studies of neural progenitor cells or NSCs are broadly divided into studies on the activation of endogenous NSCs in situ, or studies involving transplantation of NSCs isolated from the brain or spinal cord. At least within the spinal cord, therapeutic strategies using activation of endogenous NSCs are not expected to be practical, because endogenous NSCs appear to proliferate but differentiate only into astrocytes after spinal cord injury (SCI) [3–5]. On the other hand, there are many reports demonstrating the transplantation potential of neural progenitor cells for various CNS disorders or trauma. These cells have not only shown the reconstruction of neuronal networks, but have also rendered functional recovery in animal models. In this chapter, we will introduce recent progress in transplantation, especially as it pertains to practical issues of timing. In addition, we will discuss the future prospects for their clinical application.

Definition and Selective Culture of NSCs

NSCs have been defined as neural cells with the potential to self-renew and generate all three major cell types of the CNS: neurons, astrocytes, and oligodendrocytes. The existence of mammalian NSCs was first suggested by researchers such as Altman, Bayer, Kaplan and others starting in the 1960s, but it was not until the 1990s that these cells were demonstrated in humans and were isolated in culture. Enormous progress has been made in studies on the biological properties of NSCs and their localization in the body over the last decade since Reynolds and Weiss and coworkers [6, 7] developed the neurosphere technique, a selective culture technique for NSCs in 1992. They cultured CNS cell populations including NSCs derived from the mouse embryonic striate body and spinal cord in serum-free culture medium containing insulin, transferrin, selenium, progesterone, and cell division-promoting epidermal growth factor or fibroblast growth factor 2. Although many of these cells could not survive in serum-free medium, the cells surviving in this unusual environment could be grown as floating cell aggregates or ‘neurospheres’. When the neurosphere was dissociated into single cells and cultured in the same medium, they formed the neurospheres again, indicating a self-renewing ability. Moreover, the differentiation into the three neural cell types was driven by growth factor withdrawal, demonstrating multipotency. Thus, the neurosphere technique was shown to expand multipotent, self-renewing NSCs for many passages without apparent phenotypic change (fig. 1) [8]. Subsequently, other culture techniques, such as a low-density monolayer technique, were developed by

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Fig. 1. Neurosphere method. The major breakthrough for research on stem cell biology of the CNS was the development of the clonogenic expansion of NSCs in floating culture, called neurosphere culture, within a serum-free medium containing epidermal growth factor EGF and/or fibroblast growth factor 2 or FGF2. A neurosphere derived from a single cell is capable of generating the major three lineages of the CNS, i.e., neurons, astrocytes, and oligodendrocytes, indicating the multipotency of the neurosphere-initiating cell, upon the differentiation assay. If the neurosphere is dissociated into single cells, each cell starts to form a secondary neurosphere again with high frequency. From [6].

Davis and Temple [9] and a high-density monolayer technique was developed by Gage and his coworkers [10]. Selective Markers of NSCs

Even though selective culture methods for NSCs have been established, specific markers for NSCs have not been identified. Instead, highly selective markers of NSCs are known. These include the intermediate filament Nestin [11], the RNA-binding protein Musashi1 [12] identified by our group, and RC2 (i.e., marker of radial glia). These markers are strongly expressed in NSCs; however, they are also expressed in intermediate progenitor cells such as neuronal and glial progenitor cells. Therefore, they are not 100% specific for NSCs. Since cell populations expanded by the neurosphere technique include neural progenitor cells that have differentiated to some degree, it is currently impossible to completely discriminate NSCs from partially differentiated progenitor cells using a positive marker.

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Existence and Localization of NSCs in Adult Mice and Humans

Reynolds and Weiss and coworkers [6, 7, 13] demonstrated the existence of NSCs in the embryonic mouse striate body and spinal cord and successfully cultured them. In 1996, Gritti et al. [14] showed that multipotent, self-renewing NSCs also exist in the adult mouse striatum, indicating that NSCs exist not only in the embryonic but also in the adult mouse brain. Furthermore, Eriksson et al. [15] demonstrated that neurogenesis also occurred in the adult human brain. That study stained postmortem brain samples from cancer patients with 5-bromodeoxyuridine (BrdU) treatment and demonstrated that neurons incorporating BrdU were present in the hippocampal dentate gyrus. Using Nestin and Musashi1 as markers, the collaborative team of Goldman’s group [16] at Cornell University and our own research group showed that NSC-like cells were present around the lateral ventricles of intractable epileptics who had undergone temporal lobectomy. In support of the studies by Eriksson et al. [15], these observations also indicate the existence of NSCs in the adult brain. Their locations correspond to sites of neurogenesis in the granule cell layer of the hippocampal dentate gyrus, the subventricular zone facing the lateral ventricles, and/or the ependymal layer. Recent studies have also suggested the existence of NSCs in the parenchyma of the adult cerebral cortex and spinal cord [10, 17]. Although CNS injury leads to the proliferation of endogenous NSCs, these cells are not usually capable of self-repair. While recent results suggest that forebrain damage due to ischemia could be recovered by activating endogenous NSCs to induce de novo neurogenesis [18, 19], such a strategy has not yet been successful in the injured spinal cord. This failure is presumably because there are few endogenous NSCs in the adult spinal cord or because their differentiation into neuronal cells is inhibited by some mechanism, which remains to be elucidated. Studies are in progress throughout the world in two major areas of research to develop therapeutic strategies for CNS injuries and disorders: first, the activation of endogenous NSCs and second, the transplantation of harvested NSCs. This chapter primarily addresses transplantation therapy using neuronal precursor cells or NSCs.

In situ Identification and Effective Isolation of NSCs

The effective isolation, culture, and expansion of NSCs are essential in considering the clinical application of transplantation therapy, and it is necessary to develop appropriate methods to achieve these purposes. As described above, it is feasible to expand NSCs by the methods represented by the

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neurosphere technique. However, we can define the neurosphere-initiating cells as NSCs retrospectively only after the formation of the neurosphere. It has been difficult to prospectively identify them in the early stages of culture and impossible to do so in situ. Thus, for experimental purposes we have made transgenic mice that express enhanced green fluorescent protein (EGFP) under the control of the Nestin gene promoter or enhancer to isolate Nestin-positive cells by a fluorescence-activated cell sorter according to the intensity of EGFP expression and fluorescence [20]. We found that the activity of the isolated cells as NSCs correlates well with the intensity of fluorescence; a more intensely fluorescent group of cells had a higher formation rate of neurospheres, showing self-renewing ability and multipotency even in low-density culture. This finding is significant not only because it became possible to prospectively identify NSCs in a ‘living state’ by GFP fluorescence using fluorescence-activated cell sorter, but also because NSCs can be concentrated by this method without using growth factors as in conventional methods [8]. Recently, it is feasible to perform transplantation therapy with a new source of NSCs in place of embryonic tissue transplantation.

Shifting from Neural Tissue Transplantation to Neural Precursor Cell Transplantation

Studies on Transplantation for Parkinson’s Disease Studies on transplantation therapy for CNS disorders have been more advanced for Parkinson’s disease than for other diseases because of the earlier establishment of animal models. In 1979, Björkund and Stenevi [21] reported that rats with experimental Parkinson’s disease recovered from symptoms after transplantation of embryonic rat midbrain tissue into their striata. Later, numerous studies reported results including symptomatic recovery following transplantation of fetal cells of different derivations, and clinical trials also started. In fact, some patients transplanted with fetal tissue have achieved symptomatic relief for more than 10 years and have been demonstrated by PET to have cell transplants functioning effectively [22]. However, fetal tissue transplantation posed many problems such as low engrafting rates and the need for as many as five to ten fetal midbrains for a unilateral striatum transplant. Because of these practical and ethical problems, it was hoped that new donor cells would be developed. Against this background, in 1996, Svendsen et al. [23, 24] transplanted rat neural progenitor cells and human-derived cells shortly afterwards into the striata of model rats with Parkinson’s disease (6-OH-dopamine-administered rats), and reported successful engrafting of the transplants. They reported that although many of the transplanted cells

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differentiated into glia, a few tyrosine hydroxylase-positive cells were observed, with modest functional improvement. By taking advantage of the NestinEGFP system, we tried to isolate neural progenitor cells from the fetal ventral mesencephalic region. In fact, we obtained a strongly GFP-positive undifferentiated cell population from the fetal ventral mesencephalon of the NestinEGFP mouse by fluorescence-activated cell sorter, which was transplanted into the striatum body of rat models of Parkinson’s disease. We demonstrated the differentiation of the cell transplants into dopaminergic neurons, with recovery from symptoms of Parkinson’s disease [25]. Studies on Transplantation for SCI Experiments on transplantation of nervous system cells for SCI started in 1980 with peripheral nerve transplantation by Aguayo and his coworkers [26]. Then in 1993, Bregman et al. [27] reported that both immature and adult rats in which the thoracic spinal cords had been partially transected and which were transplanted with a fetal spinal cord showed elongation of injured axons with functional recovery, which was predominant in the immature rats. These studies indicated that the introduction of an appropriate environment into the injured site could cause injured axons to regenerate. In addition, other reports described limited spinal cord regeneration including the promotion of the regeneration of injured axons by neurotrophic factors [28] and the identification of axonal growth inhibitors [29]. These studies indicated that regeneration of the injured spinal cord might really be possible. Although researchers first focused on the effectiveness of fetal spinal cord transplantation for SCI [30–32], as with Parkinson’s disease, donor tissue shortage and ethical problems precluded the practical clinical application of this approach. As a result of remarkable advances in neuroscience in recent years, NSCs also have stepped into the limelight as a new transplant material in the field of the spinal cord repair. In 1999, McDonald et al. [33] developed elaborate sequential culture conditions that differentiated mouse ES cells into NSCs in vitro, and transplanted them into the traumatic cavity of rat models of thoracic spinal cord contusion injury. They reported that the engrafted cells could differentiate into neurons, astrocytes, and oligodendrocytes, and that the model rats improved in lower limb motor function to a greater degree than the control group. More recently, Vacanti et al. [34] transplanted gels packed with adult rat-derived NSCs into rat models of thoracic spinal cord transection, with similar results. Our group has looked specifically at time-dependent changes in the cavity microenvironment after SCI and achieved excellent results. On the ninth post-traumatic day during the subacute stage between the immediate post-traumatic stage and the chronic stage during which glial scarring of the injured site progressed, we transplanted fetal rat spinal cord-derived

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NSCs into the cavity region of rat models of cervical spinal cord contusion injury and observed differentiation of the transplanted cells into neurons, forming synapses with host axons, with recovery of motor function [35, 36] (see Strategies of Transplantation of Fetal Spinal Cord-Derived NSCs for SCI). Other Reports on NSC Transplantation Other studies reported that the transplantation of NSCs into the cerebral ventricle of myelin basic protein-deficient dysmyelinated shiverer mice caused engrafting of myelin basic protein-positive cells [37]. Moreover, transplantation of rat hippocampus-derived NSCs into the growing retina of rats resulted in the appearance of cells expressing molecular markers and having the morphology of Mueller, amacrine, bipolar, horizontal, and photoreceptor cells and astrocytes [38]. These experiments, unlike the above-described ones, have a drawback in that there was no testing of functional improvement. However, based on these results, hopes are mounting for future experiments on their clinical application. Aims of Neural Progenitor Cell Transplantation We consider that the above-described studies on neural precursor cell transplantation had two broad aims: first, to allow NSC transplants to appropriately proliferate and differentiate, replace lost neurons, reform synapses, and induce remyelination; second, to activate endogenous NSCs by the trophic effects of the grafted cell, to induce the differentiation in the desired direction and repair injured neural tissue. As described above, NSCs have now been shown to exist in a number of separate locations, such the fetal and adult brain, spinal cord, and retina [39]. As reported by Kempermann et al. [40], some stimuli appear to activate endogenous NSCs to increase the generation of neurons and glia. Unfortunately, the manner and mechanism of this activation remain to be elucidated. In contrast, the transplantation of exogenous NSCs is aimed at activating endogenous NSCs through neurotrophic factors or some signal to participate in the mechanism of repair and regeneration of lost tissue. In line with these aims, we describe below the present status and future prospects of NSC transplant-based regenerative medicine for the injured spinal cord.

Strategies of Transplantation of Fetal Spinal Cord-Derived NSCs for SCI

Properties of Endogenous NSCs of the Spinal Cord When the spinal cord is injured, Nestin-positive cells, derived from vigorously proliferating cells near the central canal adjacent to the injured site,

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Fig. 2. Properties of endogenous NSCs of the spinal cord. Recent studies have shown that there are endogenous NSCs in the adult spinal cord near the central canal. However, these cells differentiate into astrocytes, but not into neurons or oligodendrocytes after SCI.

migrate to the area of the injured site and differentiate into astrocytes [3]. The study of Johansson et al. [4] in 1999 showed the existence of NSCs in the adult spinal cord near the central canal. After SCI, these endogenous NSCs proliferate [5] and differentiate mostly into astrocytes, but not neurons or oligodendrocytes. Since endogenous astrocytes eventually form a glial scar around the wound cavity in a time-dependent manner after injury, the regeneration, elongation and remyelination of damaged axons is entirely disturbed (fig. 2). For repairing injured spinal cord, therefore, neuronal replacement therapy remains the most promising therapeutic strategy. Optimal Timing of NSPCs Transplantation For the purpose of transplantation into the injured spinal cord, we cultured NSCs obtained from 14-day gestational age rat spinal cord, using the neurosphere technique. When these cells were induced to differentiate in vitro, they differentiated into neurons, astrocytes, and oligodendrocytes. About 50% of the cells formed astrocytes and 5% of the total into neurons [Nakamura, pers. commun.]. Thus, it seems highly unlikely that the transplantation of NSCs without any contrivance results in the repair and regeneration of neurons and oligodendrocytes that have been lost through axonotemesis or apoptosis.

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NSCs obtained from adult CNS showed extensive differentiation potential when transplanted into an adult neurogenic site (i.e., the hippocampal dentate gyrus) [41]. It is well known that the surrounding microenvironment greatly influences the survival and differentiation of engrafted neural progenitor cells. To address the optimal time of transplantation, we investigated the post-traumatic changes of the microenvironment within the injured spinal cord. In the injured spinal cord, the expression of mRNA for various proinflammatory cytokines [e.g., tumor necrosis factor-, interleukin (IL)-1, IL-1, IL-6] peaked 6–12 h after injury and remained elevated until the fourth day [42]. Hart and coworkers [43] have reported the same results. Since these proinflammatory cytokines are known to have biphasic actions, both neurotoxic and neurotrophic, their action within the injured spinal cord requires careful interpretation. Extremely high expression at least within 7 days after injury is thought to be neurotoxic, representing a microenvironment unfit for the survival of NSC transplants. Johe and coworkers [44] have reported that platelet-derived growth factor, ciliary neurotrophic factor, and thyroid hormone (T3) instructively induced the fetal rat hippocampus-derived NSCs to differentiate into neurons, astrocytes, and oligodendrocytes, respectively. Taga and coworkers [45] reported that leukemia inhibitory factor and bone morphogenic protein-2 promote the differentiation of fetal mouse neuroepithelium-derived NSCs into astrocytes. These reports both described members of the IL-6 superfamily (e.g., ciliary neurotrophic factor, leukemia inhibitory factor) and suggest that a signal mediated by the gp130 subunit of the cytokine receptors induces the differentiation of NSCs into astrocytes. During the acute inflammatory phase immediately after SCI, under the condition in which there are high levels of IL-6, NSC transplants are difficult to engraft, but also easy to differentiate into astrocytes if they engraft. We have found that the expression of the anti-inflammatory cytokine transforming growth factor -1 (TGF-1) did not increase immediately after injury, but gradually increased with a peak on the fourth day after injury [42], suggesting that TGF-1 acts to alleviate the inflammatory situation. These observations on the survival and differentiation of NSC transplants indicate that the optimal time of the transplantation is probably not immediately after injury. However, if too much time passes after injury, a glial scar forms around the injured site and inhibits the regeneration of axons; therefore, we considered the optimal time of transplantation to be 7–14 days after trauma (fig. 3). In addition, the benefits of NSC transplantation at this timepoint could also result from microvascular regeneration in the host, considering previous findings from fetal neural tissues transplanted into the cerebral cortex [46, 47]. Correspondingly, a recent report indicates that the formation of new

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Acute phase Subacute – chronic phase Inflammation SCI

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Fig. 3. Optimal timing of neural stem/progenitor cells (NSPCs) transplantation based on changes in the microenvironment within the injured spinal cord. We consider the optimal timing of NSPCs transplantation to be 7–14 days after trauma, when the lesion site is neither inflammatory and neurotoxic, nor surrounded by glial scar.

vessels occurs most actively 7–14 days after a contusion injury to the rat spinal cord [48]. Fetal Rat Spinal Cord-Derived Cells for Rat SCI: Delayed Transplantation Based on the above considerations, we made models of quantitative cervical cord contusion injury by performing C4–5 laminectomy on adult rats and allowing a 35-gram weight to stand still on the exposed dura for 15 min. On the ninth day after injury, we transplanted fetal rat spinal cord-derived neural progenitor cells that had been cultured and expanded by the neurosphere technique and labeled with BrdU in and around the injured site (fig. 4). The transplanted cells survived in the host spinal cord and differentiated into neurons, astrocytes, and oligodendrocytes at the 5-week timepoint after transplantation (fig. 5a–c). To investigate the properties of new neurons derived from donors in more detail, we took advantage of the fact that the 1.1-kb promoter element of the T-1 tubulin gene is only active in cells of the neuronal lineage (including neuronal progenitors and postmitotic neurons), and not those of the glial lineage (fig. 6a, b) [49–52]. Here, we used rats that had been treated with transplanted neurospheres derived from the fetal spinal cords (E14.5) of T-1-EYFP transgenic rats. By

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9 days after injury Fetal spinal cord-derived NSPCs transplantation

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Fig. 4. Delayed transplantation of NSPCs after SCI. We made models of quantitative cervical cord contusion injury by performing C4–5 laminectomy on adult rats and allowing a 35-gram weight to stand still on the exposed dura for 15 min. On the ninth day after injury, we transplanted fetal rat spinal cord-derived NSPCs that had been cultured and expanded by the neurosphere technique and labeled with BrdU, in and around the injured site.

a

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Fig. 5. Survival and differentiation of the engrafted NSPCs. The NSPC transplants had survived in the host spinal cord and differentiated into neurons, astrocytes, and oligodendrocytes at the 5-week timepoint after transplantation. a Hu (neuronal marker) and 5-bromodeoxyuridine (BrdU) double-positive cells (brown: Hu; blue: BrdU). b GFAP (marker of astrocytes) and BrdU double-positive cells (brown: GFAP; blue: BrdU). c CNPase (marker of oligodendrocytes) and BrdU double-positive cells (brown: CNPase; blue: BrdU). Scale bar 5 m.

injecting BrdU intraperitoneally, we could label cells that had divided after the BrdU injection. The presence of postmitotic neurons that were double positive for BrdU-labeling and EYFP expression demonstrated that donor-derived progenitor cells underwent mitotic neurogenesis within the host spinal cord (fig. 6c).

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Fig. 6. The advantage of using T-1-EYFP Tg rats. a k-1 transgene. b The expression of EYFP in the fetal T-1-EYFP Tg rat brain. As the promoter element of T-1 tubulin gene is only active in cells of the neuronal lineage (including neuronal progenitors and postmitotic neurons), and not those of the glial lineage, the EYFP-positive region is coincident with the region stained with -III tubulin (neuronal marker). c The proof of neurogenesis by the transplanted NSPCs. By injecting BrdU intraperitoneally, we could label cells that had divided after the BrdU injection. The presence of postmitotic neurons that were double positive for BrdU labeling and EYFP expression demonstrated that donor-derived progenitor cells underwent mitotic neurogenesis within the host spinal cord.

Five weeks after transplanting neurospheres derived from the fetal spinal cords of T-1-EYFP transgenic rats, donor-derived EYFP-positive neurons extended their axons within the host spinal cord (fig. 7a). We found that T-1-EYFP-positive neurons were surrounded by synaptophysin-immunopositive sites, a presynaptic marker (fig. 7b). Furthermore, we observed EYFPpositive presynaptic structures with presynaptic vesicles that were connected with EYFP-negative postsynaptic structures with postsynaptic densities by immnoelectron microscopic studies (fig. 7c). We also found EYFP-negative presynaptic structures that were connected with EYFP-positive postsynaptic structures. Interestingly, we found some cases in which EYFP-positive neurons had formed a synapse with host motor neurons at the injury site. In addition, compared with the control group (which received an injection of only culture fluid on the ninth day after injury), the transplantation group showed a greater degree of functional recovery as demonstrated by the pellet retrieval test (fig. 8) [32]. These results indicate that if NSCs are transplanted in the subacute phase, neither in the acute phase after SCI nor in the chronic phase characterized by marked glial scarring, they can engraft and contribute to

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T-1-EYFP

Synaptophysin

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c Fig. 7. Neurons derived from transplanted cells extend their axon into the host spinal cord. a Five weeks after transplanting neurospheres derived from the fetal spinal cords of T-1-EYFP transgenic rats, donor-derived EYFP-positive neurons extended their axons within host spinal cord. Scale bar 50 m. b T-1-EYFP-positive neurons were surrounded by synaptophysin-immunopositive sites that are well-characterized pre-synaptic markers. Scale bar 5 m. c EYFP-positive presynaptic structures with presynaptic vesicles that were connected with EYFP-negative postsynaptic structures with postsynaptic densities. Immunoelectron microscopic studies. Scale bar 0.2 m.

some degree to the repair of the injured site. It is important to consider the following three possibilities to explain these data: (1) the transplanted cells may have differentiated into neurons, which formed synapses with neurons above and below the injured site; (2) the transplanted cells may have differentiated into oligodendrocytes, which might have remyelinated the axons that had been demyelinated by the injury; (3) the transplanted cells may have released some neurotrophic factors, which inhibited neuronal death, induced neuronal protection, or activated endogenous NSCs to repair the injured site (fig. 9). The actual functions of the transplanted NSCs are yet to be elucidated and require further study. Bregman and coworkers [53] transplanted fetal rat spinal cord tissue into two groups of rats with SCI immediately after and 2 weeks after injury, and compared the two groups in terms of their anatomical features and the degree of functional recovery. They found that the group receiving transplants 2 weeks after injury showed better regeneration of the injured axons and better lower limb functional recovery compared with the group receiving transplants

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Number of pellets 90 80 70 60 50 40 30 20 10 0

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ope ()

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Fig. 8. Transplantation of NSPCs improved functional recovery. a Pellet retrieval test. Rats could obtain pellets only with their forelimbs (arrows). b Results of pellet retrieval test. *p 0.01. The p value was determined using a Mann-Whitney U-test.

Synapse formation

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Remyelination of growing axons

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Trophic support for axonal regeneration

Fig. 9. Mechanism of functional recovery by the grafts. a The transplanted NSPCs differentiated into neurons, which formed synapses with neurons above and below the injured site. b The transplanted NSPCs differentiated into oligodendrocytes, which might have remyelinated the axons that had been demyelinated by the injury. c The transplanted NSPCs released some neurotrophic factors, which inhibited neuronal death, induced neuronal protection, or activated endogenous NSCs to repair the injured site.

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immediately after injury. These results also strongly support the effectiveness of the delayed transplantation paradigm.

Clinical Applications of Human NSC Transplantation

As described above, transplant experiments with NSCs have been conducted throughout the world. Needless to say, the ultimate goal of these experiments is the human clinical application of NSC transplantation. As the culture techniques for human NSCs become established, transplant experiments in the clinic are under consideration. After the first demonstration by Svendsen et al. of transplantation into Parkinson’s models (see Studies on Transplantation for Parkinson’s Disease), Flax et al. [54] expanded NSCs from the human fetal telencephalon by the neurosphere method, transplanted them into the brains of newborn mice, and reported that they differentiated into neurons, astrocytes, and oligodendrocytes. Brüstle et al. [55] similarly transplanted NSCs cultured from the human fetal brain into the fetal rat cerebral ventricles and reported that differentiation and engrafting occurred in the rat forebrain, midbrain, and hindbrain. In 2001, Ourednik et al. [56] transplanted human fetal NSCs into the fetal monkey cerebral ventricles, and reported that some of the engrafted cells differentiated into neurons and glia, while the remaining cells engrafted and remained undifferentiated. This report holds promise in the sense that the transplantation of human NSCs into primates is essential as a preliminary step toward clinical application. It is hoped that, for SCI, experiments in transplantation of human NSCs in primate models will also yield good results.

Future Prospects

There are still many problems to solve before NSC transplantation finds clinical application. For further improvement of the transplantation therapy, more efficient techniques to isolate NSCs are needed. In contrast to the experiments with mice, for the purpose of clinical application, the collaborative team of Goldman’s group [51, 57] and our group introduced the gene for a fluorescent protein into adult human hippocampal cells by the lipofection method, and successfully isolated human NSCs. We also prepared adenoviruses expressing EGFP under the control of the Nestin enhancer or the Musashi1 promoter for gene transfer, and succeeded in effectively isolating NSCs from human fetal brain tissue [58]. Using the same strategy as for hematopoietic stem cells, an effective method was developed for effectively isolating NSPCs using antibodies to cell surface antigens [59].

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Another problem is how to further examine the mechanisms of the autoregulation of NSC differentiation. If a favorable environment were created for endogenous NSCs, they might migrate rapidly to injured or degenerated sites to self-repair these sites, and the need for heterologous neural cell transplantation would be reduced. The key to solving this problem may be neurotrophic factors or activation of the immune system. Recently, new concepts on the plasticity of NSCs have emerged. For example, Kondo and Raff [60] have shown that oligodendrocyte precursor cells acquire multipotency similar to NSCs after the manipulation of culture conditions. Clarke et al. [61] have shown that adult ROSA26 mouse-derived NSCs, which have been transplanted into the embryonic chicken amniotic cavity and the mouse blastocyst, differentiate into ectodermal, mesodermal, and endodermal tissues and cells. These observations suggest that NSCs have the potential for differentiation similar to ES cells, and depending on their environment, sometimes differentiate into non-neural cells. On the other hand, other studies reported that non-neural bone marrow stromal cells, which are mesenchymal stem cells, differentiated into neurons in vitro [62], or migrated to the cerebrum and cerebellum to differentiate into astrocytes after transplantation into the neonatal mouse cerebral ventricle [63]. Furthermore, a study has reported that bone marrow stromal cells, which have been transplanted into the injured rat spinal cord one week after injury, bridge the epicenter of the injury in association with immature astrocytes, thus serving as guiding strands for regenerating axons, causing significant functional recovery [64]. More intriguingly, marrow stromal cells, previously thought to differentiate into mesenchymal lineages such as osteocytes, chondrocytes, and adipocytes, could be induced to generate ectoderm-derived CNS cells. However, it remains in doubt whether this so-called transdifferentiation actually occurs constantly in vivo. Many other related problems with SCI remain unsolved. In addition to considering NSC transplantation, it is necessary to create a permissive microenvironment within the site of SCI. This may entail making a biological or other physical scaffold to facilitate the elongation of regenerating axons into the traumatic cavity [65], eliminating axonal growth inhibitors (e.g., Nogo [26], myelin-associated glycoprotein [66], semaphorin [67], chondroitin sulfate) that continue to be released from post-traumatic glial scar tissue, or concomitantly using neurotrophic factors (e.g., neurotrophin-3, brain-derived neurotrophic factor) [68–70] to create a permissive environment for grafting. The most important fundamental problem continues to be how to regenerate the chronically injured spinal cord, in terms of neuronal cell body and axonal growth in patients with existing or long-standing injuries. More than 99% of patients with SCI, numbering more than 100,000 in Japan and almost 250,000

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in the USA, are patients with long-standing injuries. Without their recovery, there can be no success in the treatment of SCI. References 1 2 3

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Kondo T, Raff M: Oligodendrocyte precursor cells reprogrammed to become multipotential CNS stem cells. Science 2000;289:1754–1757. Clarke DL, Johansson CB, Wilbertz J, Veress B, Nilsson E, Karlstrom H, Lendahl U, Frisen J: Generalized potential of adult neural stem cells. Science 2000;288:1660–1663. Woodbury D, Schwarz EJ, Prockop DJ, Black IB: Adult rat and human bone marrow stromal cells differentiate into neurons. J Neurosci Res 2000;61:364–370. Kopen GC, Prockop DJ, Phinney DG: Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA 1999;96:10711–10716. Hofstetter CP, Schwarz EJ, Hess D, Widenfalk J, El Manira A, Prockop DJ, Olson L: Marrow stromal cells form guiding strands in the injured spinal cord and promote recovery. Proc Natl Acad Sci USA 2002;99:2199–2204. Teng YD, Lavik EB, Qu X, Park KI, Ourednik J, Zurakowski D, Langer R, Snyder EY: Functional recovery following traumatic spinal cord injury mediated by a unique polymer scaffold seeded with neural stem cells. Proc Natl Acad Sci USA 2002;99:3024–3029. Cai D, Deng K, Mellado W, Lee J, Ratan R, Filbin M: Arginase I and polyamines act downstream from cyclic AMP in overcoming inhibition of axonal growth MAG and myelin in vitro. Neuron 2002;35:711–719. De Winter F, Oudega M, Lankhorst AJ, Hamers FP, Blits B, Ruitenberg MJ, Pasterkamp RJ, Gispen WH, Verhaagen J: Injury-induced class 3 semaphorin expression in the rat spinal cord. Exp Neurol 2002;175:61–75. Olson L, Widenfalk J, Josephson A, Greitz D, Klason T, Kiyotani T, Lipson A, Ebendal T, Cao Y, Hostetter C, Schwartz E, Prockop D, Manson S, Jurban M, Lindqvist E, Lundströmer K, Nosrat C, Brene S, Spenger C: Experimental spinal cord injury models: Prospective and repair strategies; in Ikada Y, Oshima N (eds): Tissue Engineering for Therapeutic Use, ed 5. Amsterdam, Elsevier Science BV, 2001, pp 21–36. Grill R, Murai K, Blesch A, Gage FH, Tuszynski MH: Cellular delivery of neurotrophin-3 promotes corticospinal axonal growth and partial functional recovery after spinal cord injury. J Neurosci 1997;17:5560–5572. Liu Y, Kim D, Himes BT, Chow SY, Schallert T, Murray M, Tessler A, Fischer I: Transplants of fibroblasts genetically modified to express BDNF promote regeneration of adult rat rubrospinal axons and recovery of forelimb function. J Neurosci 1999;19:4370–4387.

Dr. Hideyuki Okano Department of Physiology, Keio University School of Medicine, Shinjuku Tokyo 160-8582 (Japan) Tel. 81 3 5363 3747, Fax 81 3 3357 5445, E-Mail [email protected]

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Functional and Restorative Molecular Neurosurgery Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 124–145

Contemporary Applications of Functional and Stereotactic Techniques for Molecular Neurosurgery Paul A. House, Ganesh Rao, William Couldwell Department of Neurological Surgery, University of Utah Medical Center, Salt Lake City, Utah, USA

Introduction

Tremendous advances have been made in understanding the molecular basis of many neurological diseases. Although the molecular biology of brain tumors and neurodegenerative diseases has become better understood, utilizing this information to achieve improved therapeutic results remains a challenge. In some neurological diseases, the dysfunction of specific neuroanatomic sites is primarily responsible for a disease process, for example, degeneration of dopaminergic neurons of the substantia nigra pars compacta in Parkinson’s disease. Other diseases are more diffuse; for example, a glioblastoma multiforme (GBM) may have tumor cells several centimeters away from the primary tumor focus. Each of these scenarios calls for a different type of treatment approach, either delivering therapy locally or diffusely. In this chapter, we discuss strategies that are currently under investigation for delivery of drug or molecular-cellular treatments.

Basic Treatment Approaches to Neurological Disease

Neuro-Oncology Treatment of brain tumors typically involves some combination of surgical resection, radiotherapy, and chemotherapy. Surgical resection has been shown to improve survival in certain tumors such as GBM, whereas others are definitively treated by radiation, such as germinoma. Still others, such as

oligodendroglioma, respond very favorably to chemotherapy. Despite advances in these classes of treatment, the survival for patients with most primary brain tumors remains poor. The survival rate for GBM, the most common primary brain tumor, has not improved in over a decade. Improved strategies for treating GBM must address the diffuse nature of intrinsic brain tumors. Tumor cells spread widely along white matter tracts and can be found on the contralateral hemisphere from a primary tumor focus. Definitive therapy for primary brain tumors will require treatments such as well-targeted inactivation of aberrantly expressed oncogenes or re-establishment of the activity of lost or nonfunctioning tumor suppressor genes. Neurodegenerative Diseases Neurodegenerative diseases such as Alzheimer’s disease involve a continuous loss of neurons. In Alzheimer’s disease, diffuse loss of neurons in the cortex as well as basal structures such as the locus ceruleus and nucleus basalis is characteristic. The same widespread loss of neurons holds true for Parkinson’s disease, in which dopaminergic neurons are depleted, and Huntington’s disease in which striatal neurons are lost. In the case of these disorders, dysfunction of specific neuroanatomic structures must be addressed. Other neurodegenerative diseases such as amyotrophic lateral sclerosis involve degeneration of anterior horn cells throughout the spinal cord, providing a unique therapeutic challenge. Definitive therapy for all of these disorders is likely to involve grafting of cells to restore function, along with approaches to deliver local trophic and growth factors. Spinal Cord Injury Molecular underpinnings of the normal healing process following spinal cord injury suggest that a variety of steps in the healing cascade may be amenable to intervention. While some forms of intervention for spinal cord injury, such as corticosteroids, can be delivered systemically, future therapies will involve proteins and small molecules that need to be delivered locally. Although not classically considered in the realm of functional neurosurgery, the need for targeted local therapy in the spinal cord may expand the role of the functional neurosurgeon. At least three types of axon-inhibiting molecules present in the myelin of the injured spinal cord have now been characterized. Two separate types of myelin-associated glycoproteins named Nogo and MAG, along with oligodendrocyte-myelin glycoprotein, have been shown to inhibit growth cones [1–3]. It has been demonstrated that blocking Nogo-A with a monoclonal antibody (IN-1) leads to enhanced long fiber tract regeneration in spinal cord injury models [4]. Because diffusely blocking Nogo-A leads to sprouting of uninjured

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axons in the intact central nervous system (CNS) [5], it is likely that blocking antibodies will need to be delivered in a focused manner to patients with injured spinal cord to avoid diffuse axonal sprouting. Similarly, blocking the receptors to these glycoproteins or blocking the signaling cascade they induce will likely be treated best in a focused manner. Neurosurgeons will be required to develop and implement these technologies in the clinic.

New Approaches to Drug Delivery in the Brain

Gene Therapy Advances in understanding the genetic aberrations leading to GBM have provided new therapeutic targets. For example, loss of the tumor suppressor phosphatase and tensin homolog has been shown to be an important event in the development of GBM [6–9]. Restoration of the function of such tumor suppressor genes has shown promise in vitro. Typically, these therapeutic genes are delivered via adenoviral or retroviral vectors. Transduction of functional phosphatase and tensin homolog into glioma cell lines has been shown to reduce the proliferation of tumor cells [10]. Other successes have been obtained by introducing herpes simplex virus type 1 thymidine kinase into glioma cell lines and inducing cytotoxicity with gancyclovir or other prodrugs [10–15]. Although many promising therapies have been successful in the laboratory, improvement in patients has been significantly less dramatic. Translating in vitro successes to human patients remains a challenge and there are risks inherent to this type of treatment [16–18]. Antisense gene therapy is being developed for therapy in which overexpression of cancer-promoting genes (e.g., oncogenes) plays a role in tumor progression. The simplest antisense constructs utilize an oligonucleotide sequence in complementary orientation to a target gene, and the antisense cDNA binds to a target DNA or mRNA and prevents transcription or translation. Antisense therapy has been used on glioma targets with some success. For example, matrix metalloproteinase-9 (MMP-9) has been shown to be important for cell migration and invasion of gliomas. Antisense constructs targeted against MMP-9 in both in vitro and in vivo models have shown regression of tumor growth [19]. Similar preclinical results have been obtained with the delivery of antisense constructs directed against epidermal growth factor receptor gene, which is upregulated in gliomas. Another new antisense therapy involves the use of short interfering RNA, which can bind to mRNA and cause degradation, known as RNA interference [20]. The major obstacle to gene therapy relates to inefficient delivery to the CNS. While many gene therapy techniques utilizing adenoviral or retroviral

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Fig. 1. Localization of GPi with CED.

vectors have been successful in cell lines, targeted delivery in the clinical setting remains quite difficult. The past failures of cancer gene therapy are mainly due to the poor delivery of the gene to tumor cells, and the method of manual injection of vector-producing cells limits the distribution of these cells [14]. The development of newer vectors such as recombinant adeno-associated virus (AAV) has provided hope for in vivo delivery. AAV is based on a nonpathogenic, replication-defective virus and has been used successfully for efficient and sustained gene transfer to proliferating and differentiated cells without a detectable immune response or toxicity [21–23]. AAV has been shown to be effective for long-term delivery of genes at biologically relevant levels in both the CNS and intramuscularly [22, 24–27]. There may be limitations to this vector, although it has safety advantages over other adenoviral or retroviral vectors [22, 23, 28]. Convection-Enhanced Delivery One of the more promising techniques for clinical drug delivery to the brain is convection-enhanced delivery (CED) or high-flow microinfusion. CED involves placement of an infusion catheter directly into the brain parenchyma and relies on bulk flow (as opposed to passive diffusion) through the CNS parenchyma, thus bypassing the blood brain barrier. Although technical issues remain (e.g., cannula size, location of the infusion pump, cellular damage caused by high flow rates), there are distinct advantages over conventional

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Fig. 2. Targeted brainstem delivery with CED.

intravenous chemotherapy, intraventricular delivery, or drug-impregnated polymer-based therapy. CED allows for significantly higher concentrations of pharmacotherapy to be delivered to a larger volume of brain tissue, with important applications in neurodegenerative and neuro-oncological diseases. Stereotactically implanted catheters may be targeted at specific structures. For example, high-flow microinfusion of the caudate with biotinylated dextran has been performed successfully with very little spillover into the adjacent structures [29]. Similar perfusion of the globus pallidus has also been achieved (fig. 1) [30]. Indirect targeting through axonal tracts also has been shown by infusion of the striatum in rats, with subsequent identification of the infusate in the substantia nigra pars compacta [29]. These findings have therapeutic implications for the treatment of neurodegenerative diseases such as Parkinson’s or Huntington’s. CED also shows promise for diffuse neoplastic disease as there is spread of the infusate along white matter tracts. It can be used relatively safely in the cerebral hemispheres, brainstem, and spinal cord (fig. 2) [31]. Preclinical testing has demonstrated a survival advantage in C6 glioma-bearing rats treated with BCNU or toptecan delivered via CED [32, 33]. This observation has been extended to human trials, which take advantage of the overexpression of transferrin, interleukin-13, or interleukin-14 receptors on glioma cells. By linking a toxic compound (such as Pseudomonas exotoxin) to a ligand specific for these receptors, glioma cells can be targeted for specific destruction [34, 35]. CED lends itself nicely to this technique, and recent trials have shown some promise for this route of delivery.

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Stem Cell Therapy Stem cell therapy has been touted as a potential treatment for neurodegenerative diseases such as Parkinson’s disease, Huntington’s disease, and Alzheimer’s disease. It is now well established that neural stem cells possess the ability to differentiate into any of the various cell types in the CNS. Transplantation of fetal dopamine cells into rat models of Parkinson’s disease was successfully started decades ago [36–39]. These experiments showed that transplanted dopaminergic cells could survive and function in vivo. Fetal cell transplantation has been performed in humans with some success, and in some cases caused a huge improvement in Parkinsonian symptoms [40]. There are, however, serious difficulties with current fetal dopamine cell transplants. First, recovery of these cells from aborted fetuses is expensive and although it is legal, there are ethical considerations in ramping up production of these cells from primary sources. Further, the survival rate of these transplants can be quite poor with the majority of cells undergoing apoptosis in the absence of immunosuppression. Embryonic stem cells (ES cells) offer a more attractive source of dopaminergic cells, as these are available from any number of established ES cell lines and could be genetically engineered to match a host through new techniques of somatic cell nuclear transfer. Transplantation of ES cells is still problematic regarding control of cell growth and differentiation, as well as having a sufficient quantity of cells to transplant. In rat models of Parkinson’s disease many ES cells will not survive in situ, and up to 20% will differentiate into lethal teratomas [41]. These problems are being addressed by allowing some cellular differentiation to occur in vitro prior to transplantation. Investigators are currently attempting to develop cell lines of dopamine-producing ES cells using gene transfer with prodopaminergic genes (e.g., Nurr1) or treatment with soluble signaling factors (e.g., epidermal growth factor, insulin-like growth factor). Also, it may be possible to develop specific neuronal cells from other stem cells located elsewhere in the body (e.g., blood, bone marrow, skin) for purposes of autotransplantation. Growth Factors as a Therapeutic Strategy Growth factors used to promote functional restoration of neurons that are affected in neurodegenerative diseases include glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor, brain-derived growth factor, and insulinlike growth factor-1. GDNF in particular has shown clinical promise for neural repair. Described as the most potent neurotrophic factor for motoneurons, it was tested heavily beginning in the mid 1990s. In vitro studies showed that GDNF promoted the survival of motoneurons [42–44]. However, because of difficulties with delivery, short half-life of the recombinant protein, and various inflammatory

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effects, clinical trials met with poor success and side effects [22, 45]. The combination of growth factor genes and viral vectors such as AAV has restored hope for its use in neurodegenerative disorders. For example, an AAV-GDNF construct injected intramuscularly into mice has been shown to result in sustained expression of transgenic GDNF and is delivered via retrograde transport to spinal motoneurons [22]. This type of therapy holds promise for the treatment of motor neuron diseases such as amyotrophic lateral sclerosis, because GDNF expression in the muscles of transgenic amyotrophic lateral sclerosis mice has improved their survival [24]. Molecular Therapies for Spinal Cord Injury Neurotrophins have been investigated for their ability to allow regenerating axons to cross the area of an injured spinal cord. For example, neurotrophin-3 promotes axon sprouting through the gray matter in lesioned spinal cords when delivered continuously via a fibroblast-producing cell line [46]. Functional neurosurgeons will need to become involved in the development of new drug delivery systems to provide such sustained levels of neurotrophins to the sites of injury. As with the blocking of inhibiting epitopes in the injured spinal cord, delivery of neurotrophins to the injured spinal cord could possibly utilize CED [47]. Osmotic pumps have been used in experimental animals to deliver some of these small molecules and could perhaps be adapted for the purpose in human subjects. A variety of biomaterials are being developed that might not only deliver the needed concentration gradients but also provide a permissive substrate for axon regrowth [48]. The transplantation of stem cells of multiple lineages also holds promise in providing permissive microenvironments for spinal cord regeneration.

Novel Surgical Techniques for Spinal Cord Injury

While a progressive understanding of the molecular biology of spinal cord injury will provide new avenues to aid recently injured patients, those with preexisting spinal cord deterioration suffer from a host of secondary complications for which neuro-augmentative surgery could provide functional improvement. Many augmentative technologies and techniques would also benefit patients with progressive degenerative disease. For example, loss of bowel and bladder control is often cited as one of the most disabling complications of diplegia or tetraplegia. Indeed, chronic hydronephrosis and secondary infections are often the ultimate cause of death for those who are disabled. Anterior sacral root stimulation combined with dorsal rhizotomy to treat the neurogenic bladder is the most widely used neurosurgical method employing a neuroprosthetic device to

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augment bladder function. This implantation technique yields full continence in the majority of patients [49] and has been found to be cost effective [50]. This system has limitations, however, including side effects on sexual function, prompting further investigations into mechanisms that allow specific stimulation of coordinated bladder emptying as well as continence. In the decerebrate cat, stimulation of the dorsolateral funiculus within the lower thoracic spinal cord (T9–T13) has been shown to produce coordinated bladder contraction with decreased urethral sphincter tone [51]. If a coordinated control center can be found in the human spinal cord, it may be possible to produce a microelectrode system for bladder control. The functional neurosurgeon, already adept at microelectrode recording and targeting, will be needed to help overcome technical hurdles and make such a system possible. Although therapy to treat sequelae of spinal cord injury may seem like a small goal compared to the ultimate goal of restoring total spinal cord function, it may be a more achievable goal and would definitely enhance quality of life until true spinal cord repair is possible, which may take decades of further work and refinement. Advancing bionic technologies also present an area where neurosurgical expertise could lead to the development of new human-prosthetic interfaces, which would greatly augment the functional capacities of those with disabilities. Commercial examples of advancements in this area include the ‘iBot Mobility System’ (Independence Technology, LLC) which incorporates gyroscopic guidance into a wheelchair design, allowing the system to climb stairs and balance on two wheels. A similar capability-expanding peripheral device in development is called ‘Robowalker’ (Yobotics, Inc.). This exoskeleton-type device could greatly enhance the mobility of those with lower extremity weakness or limited leg control. These technologies demonstrate how sophisticated movements can be controlled using only a limited amount of input information, namely, the leaning body weight of a patient lacking full mobility. Other devices, which rely on tracking eye movement to initate and control movements, are in development for the patient with spinal cord injury.

Neuro-Prosthetic Therapies

For patients with severe neurodegenerative disorders or severe CNS injury, one factor limiting the utilization of new forms of augmentative technology is the difficulty in providing communication between the device and the injured CNS. Providing simple two-dimensional directional control has been achieved by direct implantation of a microelectrode into the human motor cortex (fig. 3) [52]. This system provides a brain-computer interface by having the computer learn to recognize firing patterns of motor cortex associated

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2 mm Fig. 3. One device which has been designed to serve as a brain-machine interface is the Utah Microelectrode Array. A 100 electrode array is pictured above next to a penny for size comparison. The electrode tips have been machined to penetrate neocortex to layer IV. An electron micrograph of the 4 ⫻ 4 mm array is below.

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with direction-specific movement. A similar system utilizing a vastly increased number of microelectrodes allowed a monkey to remotely control a robot arm in a three-dimensional reaching task [53]. These experiments outline a strategy that may be employed in the future to extend cognitive control of artificial limbs or mobility-extending devices to patients with very high cervical cord injuries. A host of technical difficulties must still be overcome to allow even rudimentary cognitive control of artificial limbs, but there is certainly reason for hope [54]. Current technology allows the simultaneous recording of approximately one hundred neurons through implanted microelectrodes or microelectrode arrays [55]. Perhaps surprisingly, real-time analysis of only fifty to one hundred motoneurons was sufficient to reproduce the three-dimensional arm movements previously discussed. The first devices capable of directly recording electrical information from the brain were developed in the 1950s as external electroencephalography recorders. These devices provide limited spatial resolution but are noninvasive. Through ‘bio-feedback’, patients can operate simple one-variable devices in controlled situations. The signal resolution from surface recordings, however, is too limited to provide driving information for even the simplest artificial limb system. Subdural electrode grids, already widely used as monitoring devices in the evaluation of epilepsy, deliver finer spatial resolution than superficial devices. The possibility of implanting subdural grids was a strategy utilized to provide the first cortical stimulation systems designed to deliver visual information to the blind [56]. Such systems are able to provide enhanced communication with the CNS compared with surface EEG. Yet, each subdural electrode is affected by many thousands of neurons and spatial resolution is still too limited to drive most useful artificial limb systems. One benefit of limited resolution is the need to transfer only a limited amount of information to a recording/interpreting computer. Progress on telemetry systems now allows continuous neuronal recordings to be obtained from an entirely implanted system, although limited to only two electrodes [57]. Specially designed integrated circuits and telemetry devices continue to be developed, but so far, such devices are not able to provide continuous telemetry of implanted multielectrode subdural systems. To provide the cortical spatial resolution needed to drive an artificial limb or replace sensory information in a detailed manner, several varieties of highdensity microelectrode systems are in development. The Utah microelectrode array provides 100 electrode contacts at 400-micron spacing [58]. A microelectrode system developed at the University of Michigan also provides multiple sites of electrode contacts along each electrode [59]. Each system utilizes silicon semiconductor fabrication processes producing a robust interface with high biocompatibility.

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As the ability to communicate with the CNS becomes progressively refined, a host of prosthetic devices will be introduced for clinical application. The cochlear implant system is a good example of a device that has been developed in the past to restore a sense in a functional way [60]. Prosthetics designed to restore vision to the blind are in development and often receive much attention from the popular press [61]. It may also become possible to restore somatosensory function to those utilizing artificial limbs. Interpreting neuronal coding signals offers hope to those with little ability to control their bodies or outside situations. Another application of these devices is implantable stimulators, which have been designed to abort epileptic activity [62]. It may even become possible to artificially replicate portions of the CNS function, such as hippocampal input [63], through implantable/programmable neurostimulator devices, or to simulate axonal connections between brain regions with coordinated stimulators in more than one brain region.

Intraoperative Navigation and Imaging

The application of intraoperative navigation has continued to evolve with new technology. Routine intraoperative navigation is now employed at most surgical centers, and many systems are commercially available. Most of these systems utilize archived data sets (CT/MRI/angiography) to provide target localization. The most important advances made over the past decade have been with the application of intraoperative imaging to provide real-time feedback to the operating surgeon. This is especially important for those instances in which shifts of important structures occur which render archived data inaccurate, such as in the resection of large intra-axial tumors. Refinements in real-time imaging of intracranial tumors are valuable to neurosurgeons in maximizing resections in a safe manner. The most contemporary method of imaging refinement is in the application of CT [64] and MRI to the operating room environment. The superior soft-tissue resolution of MRI over CT has made it the preferred intraoperative imaging machine in most institutions. The advantages of intraoperative MRI (iMRI) will be likely to make this an ubiquitous feature in neurosurgical operating rooms. Since their introduction into surgical practice in the mid 1990s, iMRI systems have allowed the delineation of the lesion, including ‘under the surface’ vision, and obtained real-time feedback of the extent of resection and the position of any residual tumor tissue. High-performance computing has extended the capabilities of iMRI with multimodal information and three-dimensional reconstructions [65]. One of the major issues surrounding the use of intraoperative magnets is the safety and ease-of-use considerations for the surgeon, nurses,

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anesthesiologist, and patient. Safe working environment demands the use of MR-compatible instruments, head holders, and anesthesia equipment with most machines. These safety issues have been well reviewed recently by Russell [66]. Improvements in Surgical Outcomes In contemporary series, both low- and high-field iMRI have had a positive impact on patient care, maximizing tumor resection, and shortening length of stay. In a report by Schulder and Carmel [67], iMRI-guided resection of tumors in 112 patients resulted in additional tumor removal in 36%. In another 31%, imaging confirmed that the goals of surgery had been attained, so potentially harmful further dissection in and around the brain was avoided. iMRI offers the possibility of further tumor removal during the same surgical procedure in case of tumor remnants, increasing the rate of complete tumor removal. The effects of brain shift can be compensated by using intraoperative imaging data for updating. This capability is especially important in cases involving intrinsic tumor surgery (especially low-grade tumors), and in skull-base tumors in which direct surgical view is not possible (e.g., large pituitary tumors with suprasellar extension). Most current systems combine the advantages of intracranial computerassisted cranial navigation with real-time or intermittent intraoperative imaging to verify location and tumor resection status. Various other indications for the use of iMRI as a surgical adjunct include iMRI-guided instillation of phosphorous-32 for cystic craniopharyngiomas [68], monitoring resection of epilepsy foci, and resection of vascular lesions (AVM and cavernous malformations). Combining iMRI with a comprehensive neuronavigation environment with the use of ultrasound, cortical stimulation, and navigation system-guidance of biopsy probes, instruments, and endoscopy has been described [69]. iMRI has been used with planned adjuvant radiosurgical treatment [70]. The emerging use of combining functional MRI with diffusion-weighted imaging to provide the anatomical detail of cortical and subcortical white matter tracts will enhance safe and complete resections of tumors adjacent to eloquent regions of the brain. Costs of Imaging Technology A concern of many centers is the cost involved with the establishment of an iMRI program. The Department of Neurosurgery at the University of Minnesota recently published a retrospective cost comparison of the costs and benefits of brain tumor resection in a conventional operating room and those associated with the iMRI suite [71]. A comparison of the length of stay,

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Fig. 4. The low-field Odin 0.12 T Polestar system. This unit may be swung into the operative field at any time imaging is required. Photo courtesy of Odin company.

hospital charges and payments, hospital direct and indirect costs, readmission rates, repeat resection interval, and net health outcome was performed between the patients cared for in the two operating environments. The authors noted a reduced length of stay, reduced repeat-resection interval, and reduced hospital charges and costs. Other centers, such as the University of Cincinnati College of Medicine in Ohio, have utilized a shared-resource MRI, in which the suite functions to provide both neurosurgical and diagnostic procedures in a single unit [72]. The open low-field (0.3 T) Hitachi unit is used for diagnostic studies when not being used for neurosurgical cases. The ability to perform diagnostic procedures in a shared unit has been a cost-effective solution for this particular institution. iMRI System Options There are several different options for iMRI application in the contemporary operating room environment. These include the use of low- or high-magnetic field strength units. There are also different solutions to the layout of the operating room and the concessions made to be able to image in the OR environment. The most common iMRI systems are designated low-field systems. Systems include a Siemens (Erlangen, Germany) 0.2-Tesla Magnetom Open unit [73, 74] or Odin 0.12-Tesla Polestar system [67, 69, 75, 76] (fig. 4). These systems have

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Fig. 5. High-field intraoperative MRI machine in use at the University of Calgary is a ceiling-mounted system on rails. Photo courtesy of University of Calgary, Department of Neurosurgery.

gained widespread use and experience in multiple centers, largely for reasons of reduced cost and ease of implementation with minimal operating room renovations necessary. Room shielding requirements are minimized, and some systems require no modification with the use of a portable shielding apparatus that may be brought over the patient and machine when in use. One of the early iterations of a low-field designated intraoperative unit was the GE Signa SP system, which enables operating within the open magnet [77]. Such a system has the advantage of performing continuous real-time or periodic imaging. The openconfiguration MRI installed at the Brigham and Women’s Hospital in Boston has been in use since the mid 1990s [78]. Since that time neurosurgeons at that center have gained experience with over 500 craniotomies and 100 biopsies. The advantage of such a system is that it allows real-time imaging; disadvantages are somewhat restricted surgeon and patient positioning and the necessity to utilize MRI-compatible instruments. Higher field strength magnets are increasing in popularity, to enable acquisition of improved quality images. They also enable the use of expanded MR capabilities such as MR spectroscopy and functional MRI. There are several systems available. One well-designed system is the moveable high-field (1.5 T) magnet that is located on a roller system fixed to the ceiling as employed at the University of Calgary, Canada [79] (fig. 5). This configuration is similar to the operating microscope and other surgical adjuncts, with

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MR technology moved to and from the patient as needed. Their system has been used to monitor a variety of neurosurgical procedures, including tumors, epilepsy, AVMs and other vascular malformations, and some cervical spine disorders. Another clever adaptation for the use of a high-field unit is the recent introduction of the Siemens 1.5 T intraoperative magnet (fig. 6). The machine is a standard 1.5 T MRI with functional MRI and MR spectroscopy capability. The room is designed to accommodate the 1.5 T machine with minimal disruption to the standard neurosurgical operating environment, including the use of standard operating instruments and microscope, which are located outside the 5 Gauss line. This enables the use of standard neurosurgical instrumentation, microscope, and image guidance systems. The patient is placed on a mobile operating table which is then rotated to fit on to the gantry of the MRI when imaging is desired. The machine has capabilities for intraoperative MR spectroscopy and functional MRI. There is no impediment to the operating surgeon, and operative positioning is independent of the scanning. Future Considerations Future developments in imaging will include more use of advanced MRI capabilities such as spectroscopy and functional MRI for intraoperative decision-making [80]. Also several centers are now planning for the adaptation of higher field strength magnets (e.g., 3 T) to the operating room environment. The introduction of MR-compatible robotic surgery with integration of robotic technology to the MR environment is an area that will help to revolutionize the future of neurosurgery, including the ability to locate and target a variety of deep structures in the brain. When combined with advances in viral gene Fig. 6.a The current high-field MRI Siemens system, demonstrating the ability of the surgeons to operate outside the 5 G line and use standard surgical instruments and microscope. Photo courtesy of Christopher Nimsky, MD, Department of Neurosurgery, Erlangen, Germany. b Schematic representation of the operating room layout for use of the Siemens 1.5 T machine with surgeons, nurses, and anesthetists positioned outside the 5 G line. c Pictures of the operating table rotating into position for intraoperative image acquisition. d(i) An example of image quality. Pre- and postresection T1-weighted images of a patient with a pituitary macroadenoma. d(ii) The use of iMRI facilitates glioma resection. Shown are comparative pre- and intraoperative images demonstrating the use of both T1-enhanced and T2 images to assess extent resection. e Standard image-guidance systems may also be employed in conjunction with intraoperative imaging. In this picture, the registration fiducials for a standard image guidance system are pictured.

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transfer, stem cell engineering, drug delivery devices, and neuroprosthetics, these complimentary technologies will allow precise targeting and delivery of molecular neurosurgical drugs to the brain and spinal cord. References 1 2 3 4 5

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onset of progressive degeneration in a rat model of Parkinson’s disease. Exp Neurol 1999;160: 205–214. Wright JF, Qu G, Tang C, Sommer JM: Recombinant adeno-associated virus: Formulation challenges and strategies for a gene therapy vector. Curr Opin Drug Discov Devel 2003;6:174–178. Lieberman DM, Laske DW, Morrison PF, Bankiewicz KS, Oldfield EH: Convection-enhanced distribution of large molecules in gray matter during interstitial drug infusion. J Neurosurg 1995; 82:1021–1029. Lonser RR, Corthesy ME, Morrison PF, Gogate N, Oldfield EH: Convection-enhanced selective excitotoxic ablation of the neurons of the globus pallidus internus for treatment of parkinsonism in nonhuman primates. J Neurosurg 1999;91:294–302. Laske DW, Youle RJ, Oldfield EH: Tumor regression with regional distribution of the targeted toxin TF-CRM107 in patients with malignant brain tumors. Nat Med 1997;3:1362–1368. Bruce JN, Falavigna A, Johnson JP, Hall JS, Birch BD, Yoon JT, Wu EX, Fine RL, Parsa AT: Intracerebral clysis in a rat glioma model. Neurosurgery 2000;46:683–691. Kaiser MG, Parsa AT, Fine RL, Hall JS, Chakrabarti I, Bruce JN: Tissue distribution and antitumor activity of topotecan delivered by intracerebral clysis in a rat glioma model. Neurosurgery 2000;47:1391–1398; discussion 1398–1399. Recht L, Torres CO, Smith TW, Raso V, Griffin TW: Transferrin receptor in normal and neoplastic brain tissue: Implications for brain-tumor immunotherapy. J Neurosurg 1990;72:941–945. Joshi BH, Leland P, Asher A, Prayson RA, Varricchio F, Puri RK: In situ expression of interleukin-4 (IL-4) receptors in human brain tumors and cytotoxicity of a recombinant IL-4 cytotoxin in primary glioblastoma cell cultures. Cancer Res 2001;61:8058–8061. Gage FH, Brundin P, Strecker R, Dunnett SB, Isacson O, Bjorklund A: Intracerebral neuronal grafting in experimental animal models of age-related motor dysfunction. Ann NY Acad Sci 1988;515:383–394. Perlow MJ, Freed WJ, Hoffer BJ, Seiger A, Olson L, Wyatt RJ: Brain grafts reduce motor abnormalities produced by destruction of nigrostriatal dopamine system. Science 1979;204: 643–647. Bjorklund A, Stenevi U: Reconstruction of the nigrostriatal dopamine pathway by intracerebral nigral transplants. Brain Res 1979;177:555–560. Freed WJ, Perlow MJ, Karoum F, Seiger A, Olson L, Hoffer BJ, Wyatt RJ: Restoration of dopaminergic function by grafting of fetal rat substantia nigra to the caudate nucleus: Long-term behavioral, biochemical, and histochemical studies. Ann Neurol 1980;8:510–519. Freed CR, Greene PE, Breeze RE, Tsai WY, DuMouchel W, Kao R, Dillon S, Winfield H, Culver S, Trojanowski JO, Eidelberg D, Fahn S: Transplantation of embryonic dopamine neurons for severe Parkinson’s disease. N Engl J Med 2001;344:710–719. Freed CR: Will embryonic stem cells be a useful source of dopamine neurons for transplant into patients with Parkinson’s disease? Proc Natl Acad Sci USA 2002;99:1755–1757. Yan Q, Matheson C, Lopez OT: In vivo neurotrophic effects of GDNF on neonatal and adult facial motor neurons. Nature 1995;373:341–344. Oppenheim RW, Houenou LJ, Johnson JE, Lin LF, Li L, Lo AC, Newsome AL, Prevette DM, Wang S: Developing motor neurons rescued from programmed and axotomy-induced cell death by GDNF. Nature 1995;373:344–346. Henderson CE, Phillips HS, Pollock RA, Davies AM, Lemeulle C, Armanini M, et al: GDNF: A potent survival factor for motoneurons present in peripheral nerve and muscle. Science 1994;266: 1062–1064. Yuen EC: The role of neurotrophic factors in disorders of peripheral nerves and motor neurons. Phys Med Rehabil Clin N Am 2001;12:293–306, viii. Grill RJ, Blesch A, Tuszynski MH: Robust growth of chronically injured spinal cord axons induced by grafts of genetically modified NGF-secreting cells. Exp Neurol 1997;148:444–452. Lonser RR, Gogate N, Morrison PF, Wood JD, Oldfield EH: Direct convective delivery of macromolecules to the spinal cord. J Neurosurg 1998;89:616–622. Hench LL, Polak JM: Third-generation biomedical materials. Science 2002;295:1014–1017. Brindley GS, Polkey CE, Rushton DN, Cardozo L: Sacral anterior root stimulators for bladder control in paraplegia: The first 50 cases. J Neurol Neurosurg Psychiatry 1986;49:1104–1114.

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Wielink G, Essink-Bot ML, van Kerrebroeck PE, Rutten FF: Sacral rhizotomies and electrical bladder stimulation in spinal cord injury. 2. Cost-effectiveness and quality of life analysis. Dutch Study Group on Sacral Anterior Root Stimulation. Eur Urol 1997;31:441–446. Fedirchuk B, Shefchyk SJ: Effects of electrical stimulation of the thoracic spinal cord on bladder and external urethral sphincter activity in the decerebrate cat. Exp Brain Res 1991;84: 635–642. Kennedy PR, Bakay RA, Moore MM, Adams K, Goldwaithe J: Direct control of a computer from the human central nervous system. IEEE Trans Rehabil Eng 2000;8:198–202. Wessberg J, Stambaugh CR, Kralik JD, Beck PD, Laubach M, Chapin JK, Kim J, Biggs SJ, Srinivasan MA, Nicolelis MA: Real-time prediction of hand trajectory by ensembles of cortical neurons in primates. Nature 2000;408: 361–365. Nicolelis MA: Actions from thoughts. Nature 2001;409(suppl):403–407. Guillory KS, Normann RA: A 100-channel system for real time detection and storage of extracellular spike waveforms. J Neurosci Methods 1999;91:21–29. Dobelle WH, Quest DO, Antunes JL, Roberts TS, Girvin JP: Artificial vision for the blind by electrical stimulation of the visual cortex. Neurosurgery 1979;5:521–527. Nieder A: Miniature stereo radio transmitter for simultaneous recording of multiple single-neuron signals from behaving owls. J Neurosci Methods 2000;101:157–164. Nordhausen CT, Rousche PJ, Normann RA: Optimizing recording capabilities of the Utah Intracortical Electrode Array. Brain Res 1994;637:27–36. Hoogerwerf AC, Wise KD: A three-dimensional microelectrode array for chronic neural recording. IEEE Trans Biomed Eng 1994;41:1136–1146. Rauschecker JP, Shannon RV: Sending sound to the brain. Science 2002;295:1025–1029. Maynard EM: Visual prostheses. Annu Rev Biomed Eng 2001;3:145–168. Fanselow EE, Reid AP, Nicolelis MA: Reduction of pentylenetetrazole-induced seizure activity in awake rats by seizure-triggered trigeminal nerve stimulation. J Neurosci 2000;20:8160–8168. Berger T: World’s first brain prosthesis revealed. New Scientist 2003, March 12. Broggi G, Ferroli P, Franzini A, Dones L, Marras C, Marchetti M, Maccagnano E: CT-guided neurosurgery: Preliminary experience. Acta Neurochir Suppl 2003;85:101–104. Jolesz FA, Talos IF, Schwartz RB, Mamata H, Kacher DF, Hynynen K, McDannold N, Saivironporn P, Zao L: Intraoperative magnetic resonance imaging and magnetic resonance imaging-guided therapy for brain tumors. Neuroimaging Clin N Am 2002;12:665–683. Russell L: Intraoperative magnetic resonance imaging safety considerations. Norton Healthcare, Louisville, KY. Schulder M, Carmel PW: Intraoperative magnetic resonance imaging: Impact on brain tumor surgery. Cancer Control 2003;10:115–124. Hall WA, Liu H, Truwit CL: Intraoperative MR-guided instillation of phosphorus-32 for cystic craniopharyngiomas: Case report. Technol Cancer Res Treat 2003;2:19–24. Tuominen J, Yrjana SK, Katisko JP, Heikkila J, Koivukangas J: Intraoperative imaging in a comprehensive neuronavigation environment for minimally invasive brain tumour surgery. Acta Neurochir Suppl 2003;85:115–120. Schulder M, Jacobs A, Carmel PW: Intraoperative MRI and adjuvant radiosurgery. Stereotact Funct Neurosurg 2001;76:151–158. Hall WA, Kowalik K, Liu H, Truwit CL, Kucharezyk J: Costs and benefits of intraoperative MR-guided brain tumor resection. Acta Neurochir Suppl 2003;85:137–142. McPherson CM, Bohinski RJ, Dagnew E, Warnick RE, Tew JM: Tumor resection in a sharedresource magnetic resonance operating room: Experience at the University of Cincinnati. Acta Neurochir Suppl 2003;85:39–44. Nimsky C, Ganslandt O, Gralla J, Buchfelder M, Fahlbusch R: Intraoperative low-field magnetic resonance imaging in pediatric neurosurgery. Pediatr Neurosurg 2003;38:83–89. Nimsky C, Ganslandt O, Tomandl B, Buchfelder M, Fahlbusch R: Low-field magnetic resonance imaging for intraoperative use in neurosurgery: A 5-year experience. Eur Radiol 2002;12:2690–2703. Kanner AA, Vogelbaum MA, Mayberg MR, Weisenberger JP, Barnett GH: Intracranial navigation by using low-field intraoperative magnetic resonance imaging: Preliminary experience. J Neurosurg 2002;97:1115–1124.

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Schulder M, Sernas TJ, Carmel PW: Cranial surgery and navigation with a compact intraoperative MRI system. Acta Neurochir Suppl 2003;85:79–86. Vitaz TW, Hushek S, Shields CB, Moriarty T: Intraoperative MRI for pediatric tumor management. Acta Neurochir Suppl 2003;85:73–78. Nabavi A, Gering DT, Kacher DF, Talos IF, Wells WM, Kikinis R, Black PM, Jolesz FA: Surgical navigation in the open MRI. Acta Neurochir Suppl 2003;85:121–125. Sutherland GR, Kaibara T, Louw DF: Intraoperative MR at 1.5 Tesla – Experience and future directions. Acta Neurochir Suppl 2003;85:21–28. Liu H, Hall WA, Truwit CL: The roles of functional MRI in MR-guided neurosurgery in a combined 1.5 Tesla MR-operating room. Acta Neurochir Suppl 2003;85:127–135.

William T. Couldwell, MD, PhD Department of Neurological Surgery, University of Utah Medical Center Suite 3B409, 30 North 1900 East Salt Lake City, UT 84132-2303 (USA) Tel. ⫹1 801 581 6908, Fax ⫹1 801 581 4385, E-Mail [email protected]

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Xeno-Neurotransplantation James M. Schumacher Center for Movement Disorders, University of Miami School of Medicine, Miami, Fla., USA

Introduction

Though limited, the human nervous system has some capability to regenerate or repair damaged or degenerated cells. This inherent biological mechanism is not sufficient to reverse the devastating effects of neurodegenerative conditions such as Parkinson’s, Alzheimer’s, and Huntington’s disease. Strategies to replace degenerative neuronal systems have included transplantation of human embryonic fetal cells, embryonic stem cells, adult ‘stem’ cells, genetically modified somatic cells, viral-assisted gene transfer, and crossspecies cell transplants (i.e., xenotransplantation). Cell replacement therapies for neurodegenerative diseases were considered for human application after Parkinson’s disease (PD)-like motor deficits in animal models of the disease were ameliorated using transplanted embryonic dopamine cells. Fetal allogenic neuronal transplants have been shown to effect functional and behavioral recovery in a variety of animal models of neurodegenerative disease [1, 12, 15]. Using PD as a target syndrome, several investigations have been performed in humans. The first human neurotransplantation for PD was performed in 1988 [17]. Since then, over 300 patients worldwide have been transplanted with human tissue. Open-label studies suggested efficacy of transplantation and resulted in many cases in graft survival and increased dopamine utilization in the striatum [16]. Until recently, however, none of these surgical studies were done with adequate controls. Recently, two controlled studies have been performed using solid grafts in patients with advanced PD. Neither study demonstrated statistically powered efficacy [6, 7]. In the first study the endpoint was the Global Rating Scale. This scale is a subjective account of how the patient feels after surgery. The second study looked at the Unified Parkinson’s Disease Rating Scale (UPDRS), Part III motor ‘off scores.’

Table 1. Milestones of xeno-neurotransplantation 1890

Thompson. Cat cerebral cortex into brains of adult dogs. (No survival.)

1917

Dunn. Rat neonatal cerebral cortex into adult rat brain.

1921

Shirai. First description of brain immunoprivilege and xenografts.

1979

Bjorklund. Fetal rat brain to adult rat brain.

1985

Isacson. Fetal rat brain into adult rat model of Huntington’s and Parkinson’s.

1995

Schumacher. Fetal pig dopaminergic cells into a Parkinson’s patient.

Although results were uneven, there were patients within these studies and in previous open-label studies who have shown remarkable improvement in their condition and decreased need for pharmacological dopamine replacement. PET fluorodopa studies have also confirmed restoration of dopamine in the striatum of previously depleted areas. Patients who improved the most from transplantation were those who had a large difference between their ‘on’ and ‘off ’ UPDRS scores. Meta-analyses of the published open-label studies demonstrate that neurotransplantation is a very promising work in progress [12–14]. In order to demonstrate benefit and adequate dopamine cell survival, nearly 10 fetuses (3–5 human fetuses per putamen of embryonic age 8–10 weeks) are required for transplantation in any given patient. The logistics of obtaining this quantity of human tissue are prohibitive, notwithstanding the ethical considerations involved. Hence, the search for alternate cell sources has led investigators to cross-species transplants (xenotransplantation) and embryonic stem cells. In the case of xenotransplantation, neuroblasts from other species such as pigs could provide unlimited, screenable, precisely incubated cells for transplantation. Early attempts at cross-species neurotransplantation were unsuccessful due to immune rejection. With the introduction of cyclosporine and other immunosuppressive agents, however, successful xenotransplantation has become possible (table 1). Xenotransplantation is a beneficial laboratory tool in animal models of neurodegenerative disease. When host animals undergo immunosuppression, cross-species transplantation of embryonic dopaminergic cells has shown similar results to allografts [11, 19]. In addition, the unique antigenicity of the graft and host allows specific antibody cell labeling. In this

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manner, graft survival, outgrowth and target specificity can be clearly defined. Several studies using embryonic porcine dopaminergic transplants into animal models of PD have been successful. Porcine tissue has been chosen for most studies because it is plentiful (up to 20 fetuses/liter), similar phenotypically to human, and has been used extensively in human medicine (i.e., cardiac valves, insulin). Human trials using porcine tissue have now been performed to treat PD, Huntington’s disease and epilepsy. Recent trials of neurotransplantation and the safety and immunological concerns of xeno-neurotransplantation are discussed below.

Immunology of Neural Xenografts

Although the central nervous system has a higher degree of immunoprivilege than other systems such as the heart, lung, liver and kidney, immunological reactions are a concern in allografts and especially xenografts. Factors that determine successful graft-host integration include the donor tissue embryonic stage, phylogenetic distance between donor and host, method of transplantation, preparation of graft (i.e., solid vs. suspension), host site, and method of immunosuppression. Several factors contribute to the immunological privilege in the host. In xenotransplantation, the lack of major histocompatibility complex (MHC) class I and II antigens is probably most important in graft survival. Cytokines are an important factor in graft cell death. These antigens can be induced in either system by influx of cytokines in the face of the inflammatory response of transplantation trauma [22]. Subsequent T-cell and macrophage activation and cell death is deactivated to a major extent by treatment with cyclosporine immunosuppression. Continued immunosuppression is important in that several other cytokines such as interleukin-2, -4, and -10 are induced for up to 30 days after transplantation [5, 18]. Anti-C5 (complement) antibody treatment has been found to inhibit cell death in xenografts. Further graft survival is seen with the combination of C5 inhibitor, cyclosporine methylprednisolone and azathioprine [2]. In the adult brain, MHC antigens are restricted to endothelial cells. Solid grafts contain intact endothelial cells and supporting cells and for this reason cell suspension grafts are favored over solid tissue pieces. The role of MHC 1 in tolerance induction has been shown to be an effective strategy in animals of xenotransplantation [18]. The antibody against the graft cell surface antigen is thought to promote tolerance by inhibiting T-cell induction. This technique is not as effective as cyclosporine but may provide an adjunct to immunosuppression and graft protection.

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Safety Issues in Xenotransplantation

Acute, type I graft rejection is generally not seen in neurotransplantation or xeno-neurotransplantation. Patterns of rejection are mild when compared with that seen in major organ transplantation. In neurotransplantation, T-cell and macrophage-mediated rejection is seen over days and weeks. This response is greatly reduced with cyclosporine and other modifiers of immune response. In experimental animal models and human studies, no adverse side effects in the host have been seen. Either in animals or in human safety studies, no systemic immune effect has been documented. Local pathological effects of the inflammatory cell response have been observed. Immunosuppressants carry their own hazards of use including increased risk of infection and renal damage. Of special concern in cross-species transplantation is the risk of transmission of viral nucleic acid sequences (i.e., porcine endogenous retrovirus). To date, no transmission of porcine endogenous retrovirus from animal to man has been observed. Risk of transmission of virus or bacteria between species (xenozooinosis) is possible. Animals and their tissues must be carefully screened and monitored to avoid this problem. Prophylactic antibiotics are given before and after transplantation.

Xeno-Neurograft Procedures in Humans

Clinical trials of xenotransplantation in humans have been quite limited. Our study in transplanting porcine dopaminergic mesencephalic cells into patients with PD was the first human study of xeno-neurotransplantation [20], and will be discussed below. Subsequently, other studies for Huntington’s disease and epilepsy have been performed and these also will be briefly discussed. Patient Selection Patients selected for xenotransplantation were affected with advanced PD, were failing medical treatment with L-dopa, and were having ‘on-off’ motor fluctuations but were still responsive to L-dopa. Patients were screened using the Core Assessment Protocol in Intracerebral Transplantation (CAPIT protocol; UPDRS ⫹ time testing). Patients with dementia, poor medical condition, or serious comorbidity were excluded. Fluorodopa PET scans and cranial MRIs were performed before surgery and at 6 months and one year after transplantation. Preparation of Embryonic Porcine Ventral Mesencephalon Tissue Donor animals from a Yorkshire porcine herd were screened by serology for pathogen exposure, tested for parasites, and isolated. Embryonic tissue was

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prepared by dissection of the ventral mesencephalon region from embryonic day 25–28 fetuses. Cells were then trypsonized to prepare a cell suspension at 50,000 cells/␮l. In our initial study, some cells were treated with an F(ab⬘)2 fragment of a monoclonal antibody directed against MHC I. This technique has been shown to promote xenograft survival without pharmacological immunosuppression [8, 18]. Viability prior to transplantation was assessed by acridine orange staining and screen by gram stain for bacteria. Aliquots of cell suspensions were cultured for dopaminergic cells using antibody to tyrosine hydoxylase. Patient’s blood mononuclear cells were obtained, archived and tested for porcine endogenous retrovirus. Preoperative Preparation In our study, 6 patients were loaded preoperatively with cyclosporine (15 mg/kg) 12 h prior to surgery. Six other patients were transplanted with cells that had been treated with the monoclonal antibody. All patients received perioperative antibiotics. Surgical Procedure Patients underwent the procedure with local anesthesia and MRI/CT-guided stereotaxy. Eighty microliter volumes of suspension were transplanted unilaterally in the striatum along three separate 5 mm tracts. One tract was placed in the caudate head and two in the mid and posterior putamen. Postoperative Evaluation MRI after one week showed evidence of the tracts in the striatum. Standard adverse event reporting, chemistry and blood testing was done per protocol. Cyclosporin levels were followed in those immunosuppressed patients. CAPIT testing was performed at 6 months and one year after surgery as was PET scanning and MRI. Clinical Results in Xenotransplantation In the original human study of CNS xenotransplantation of porcine cells, no adverse effects were seen in the 10 patients evaluated. None of the patients’ disease worsened in the year following surgery. In the medication ‘off’ state, 3 patients improved by ⬃30%. As a group the CAPIT scores improved by 19%. No significant change was seen in PET scans [20]. Another study with PD patients has been recently reported where bilateral ‘solid piece’ grafts were placed. Half the patients were given sham burr holes as a control. The patients that received xenografts improved 28% and the sham patients 23%. This study failed to show significant group improvement in CAPIT scores and showed a remarkable sham placebo effect [10]. Phase I

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safety trials also have been performed using porcine fetal neural cells in Huntington’s disease. On evaluation after one year following transplantation, no adverse events were seen. No significant deterioration and no improvement in functional capacity were seen [9]. A small group of patients with frontal lobe epilepsy had GABAergic porcine grafts transplanted in the seizure focus, and this study is still ongoing.

Future Direction in Xeno-Neurotransplantation

Studies in both animal models and in humans have shown that transplanted neurons across species barriers can survive and establish functional axonal and synaptic contact with the immunosuppressed host. Neurotransmitters can be replenished and neuronal circuitry re-established. Preclinical studies in animals have clearly shown that pathological and behavioral deficits can be reversed using xenografts. The value of having an unlimited supply of selected neuronal cells for transplantation cannot be underestimated. Xenografts can be carefully screened for the disease and selected for the function and precise embryonic age. The greatest obstacle in xenotransplantation is still graft rejection. This obstacle is somewhat offset by modern methods of immunosuppression, but it is not yet optimized. Novel strategies are underway to improve cell survival. Transgenic pigs have been genetically engineered to express human cell surface markers. These cells are less immunogenic and promote graft survival [3, 4]. Critics of neurotransplantation have cited problems or adverse events in recent controlled human studies using human fetal material. As a group, these studies failed to meet their endpoint of statistical significance in improvement. Of particular concern, dyskinesia was observed during defined ‘off’ periods in some patients that were transplanted [6]. This phenomenon may represent unregulated dopamine production by the graft or may reflect more ‘on’ time with dyskinesia. Those patients did have ‘on’ dyskinesia prior to transplantation and were successfully treated with medication and in 2 patients with globus pallidus stimulation. Although xenografts are not as viable as human allografts, we believe that studies should continue, in particular to engineer hybrid human-porcine cell lines that may show less immunogenicity and greater in vivo activity. The utility of the unlimited supply and possibility of tissue screening make xenografts an important resource. Our current level of understanding of the immune system limits the use of xenografts as a treatment in human disease. These studies also provide the information that will make transplantation with allografts or embryonic stem cells more successful.

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Ultimately, the answer to curing neurodegenerative disease is not only in the protection of native cells but in reconstruction of damaged and lost neuronal circuitry. Pharmacological therapy cannot provide the neurotransmitter and signal regulation needed at the cellular level. This regulation can only be provided by cell configurations at the synaptic level. Until we can determine how to promote native regeneration and regrowth of neural elements, our best strategy for neurodegeneration will incorporate aspects of cellular replacement through transplantation.

References 1 2 3 4

5 6 7 8 9 10 11 12 13

14 15 16

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Bjorklund A, Stenevi U, et al: Cross species neural grafting in the rat model of Parkinson’s disease. Nature (Lond) 1982;298:652–654. Cicchetti F, Fodor W, et al: Immune parameters relevant to neuroxenograft survival in the primate brain. Xenotransplantation 2003;10:41–49. Deacon P, Schumacher J, et al: Histological evidence of fetal pig neural cell survival after transplantation into a patient with Parkinson’s disease. Nat Med 1997;3:350–352. Deacon T, Fodor W, et al: Xenotransplantation of transgenic fetal pig dopamine neurons to rats and systemic prevention of host compliment-mediated cell lysis. Abstr Soc Neurosci 1998; 24:1056. Duan W: Immunological and inflammatory responses against intrastriatal neural grafts in the rat. Thesis, Lund University, 1997. Fahn S, Freed C, Breeze W: Transplantation of embryonic dopamine neurons for severe Parkinson’s disease. New Eng J Med 2001;334:710–719. Fahn S, Green P, et al: Double blind controlled trial of human embryonic dopaminergic tissue transplants in advanced Parkinson’s disease: Clinical outcomes. Neurology 1999;52:A405. Faustman D, Coe C: Prevention of xenograft rejection by masking donor HLA class 1 antigens. Science 1991;252:1700–1702. Fink J, Schumacher J, Elias S, Isacson O: Porcine xenographs in Parkinson’s disease and Huntington’s disease patients: Preliminary results. Cell Transplant 2000;9:273–278. Freeman T: Porcine xenografts in patients with Parkinson’s disease. Abstract: American Association of Neurological Surgeons 2003. Freeman T, Wojak J, et al: Cross-species intracerebral grafting of embryonic swine dopaminergic neuron. Prog Brain Res 1998;78:473–477. Isacson O, Bjorklund L: Parkinson’s disease. Interpretations of transplantation study are erroneous. Nature Neurosci 2001;4. Isacson O, Deacon P, Pakzaban P: Transplanted xenogenic neural cells in neurodegenerative disease models exhibit remarkable axonal targets specificity and distinct growth patterns of glial and axonal fibres. Nat Med 1995;1:1189–1194. Isacson O, Deacon T, Schumacher J: Immunobiology and neuroscience of xenotransplantation and neurological disease. San Diego, Academic Press, 1998, pp 365–387. Isacson O, Dunnett S, Bjorklund A: Graft-induced recovery in an animal model of Huntington’s disease. Proc Natl Acad Sci USA 1986;83:2728–2732. Kordower J, Freeman T, et al: Neuropathological evidence of graft survival and striatal reinnervation after the transplantation of embryonic mesencephalic tissue in a patient with Parkinson’s disease. New Eng J Med 1993;332:1118–1124. Lindvall O, Widner H, et al: Transplant of fetal dopamine neurons in Parkinson’s disease. Ann Neurol 1992;31:155–165. Pakzaban P, Deacon T, Isacson O: A novel mode of immunoprotection of neuroxenotransplants: Masking of donor major histocompatibility complex class 1 enhances transplant survival in the CNS. Neuroscience 1995;65:983–986.

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Pakzaban P, Isacson O: Neuroxenotransplantation: Reconstruction of neuronal circuitry across species barriers. Neuroscience 1994;62:989–1001. Schumacher J, Ellias P, et al: Transplantation of embryonic porcine mesencephalic tissue in patients with PD. Neurology 2000;54:1042–1050. Shirai Y: Transplanting rat sarcoma in adult heterogenous animals. Jap Med World 1921;1:14–15. Widner H, Lindhval O (eds): Basic and Clinical Aspects of Neuroscience, vol 5. Heidelberg, Springer, 1993, pp 63–74.

James M. Schumacher, MD Center for Movement Disorders University of Miami School of Medicine, Miami, FL 33136 (USA) Tel. ⫹1 305 243 4675, Fax ⫹1 305 243 3337, E-Mail [email protected]

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Adeno-Associated Viral Vectors for Clinical Gene Therapy in the Brain R. Jude Samulski, Jennifer Giles Gene Therapy Center, Department of Pharmacology, University of North Carolina at Chapel Hill, N.C., USA

Introduction

A number of recombinant viral vectors have been engineered for gene transfer to the brain. The vectors in widest use for neuroscience applications include herpes virus vectors, adenovirus vectors, and lentiviral vectors, in addition to the parvovirus vector, adeno-associated virus (AAV). Despite its relatively small capacity of ⬍5 kb, AAV vectors have gained wide acceptance as a preferred vector for gene transfer to the central nervous system (CNS), due to its advantages of neurotrophism, nonpathogenicity, and stable in vivo gene expression. AAV is one of the few viral vectors that have already proven itself as a gene transfer vector for functional genomics as well as clinical applications in gene transfer to the human brain. AAV is a nonpathogenic virus that is not associated with any human viral syndrome or disease. It depends on the presence of a helper virus, such as adenovirus or herpes virus, for replication. The wild-type AAV (wtAAV) has a 4.68-kb single-stranded DNA genome comprised of capsid (cap) and replication (rep) open reading frames flanked by inverted terminal repeats. Three structural proteins, VP1, VP2, and VP3, are encoded by the single cap gene using alternative splicing and alternative start codons. The AAV virion is composed of VP1, VP2, and VP3 at a ratio of 1:1:10, respectively. The rep gene codes for four overlapping proteins involved in AAV DNA replication and the control of AAV gene expression. The two larger rep proteins, Rep78 and Rep68, are controlled by the p5 promoter and are needed for viral DNA replication,

while the smaller Rep52 and Rep40 proteins are transcribed from the p19 promoter and serve to facilitate the accumulation of single-stranded virus. The inverted terminal repeats are the only cis-acting elements required for AAV replication, packaging, integration, and rescue [1].

Production of Clinical-Grade AAV Vector

Recombinant AAV (rAAV) is an increasingly important gene therapy vector. Perhaps most beneficially, wtAAV is innocuous and has a known integration site at chromosome 19qter13.4 [2]. Long-term transgene expression is facilitated by the ability of rAAV to persist in vivo episomally and possibly also by integrating into the host genome. This long-term persistence is further enhanced by the fact that AAV does not induce a cell-mediated immune response in the host [3]. Adding to its appeal as a therapeutic vector, rAAV has been shown to infect both dividing and nondividing cells in a broad range of tissues, including muscle, liver, brain, and retina [4]. The rAAV plasmid is constructed by replacing the entire AAV coding genome with a transgene expression cassette flanked by the viral inverted terminal repeats. The rAAV plasmid is then used to transfect cells concurrently with a helper virus infection and an AAV helper plasmid that contains the rep and cap genes needed to supply the Rep and Cap proteins in trans. This cotransfection procedure allows efficient rescue and encapsidation of the rAAV genome from the recombinant vector plasmid [5] and production of rAAV vectors that can then be used for gene therapy delivery. It is important to note that the same protocol can be used for production of any of the AAV serotypes or modified vectors. An Ad-free AAV production system, using cotransfection of plasmid encoding the Ad helper genes, is a recent development that allows quick, easy generation of rAAV vectors for typical lab-scale use [6]. In order to generate the high quantities of virus that will be needed for clinical applications, cell lines engineered to produce AAV vectors or alternative methods of transfection must be developed. Inducible cell lines for AAV production are a current focus of virology research. These cell lines, that contain integrated copies of some or all of the AAV genes needed for packaging, utilize a variety of approaches to provide the helper genes and a vector in the host genome [7, 8]. While there are many advances being made in the development of gene delivery systems targeting the CNS, production of clinical grade viral vectors continues to be a bottleneck in the progression of these therapies to the clinic. Production of viral vectors under conditions that satisfy FDA Good Laboratory Practices and Good Manufacturing Practice guidelines introduces a wide range of testing and facility modifications not needed for research-grade vectors. All

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steps in the process must be thoroughly documented, from the plasmid and mammalian cells used to produce the virus to the final viral vector itself. Quality control and release assays for vectors to be used in the clinic are extensive, and include, to name but a few, ELISA assays to determine the physical titer of the virus, quantitative PCR to determine the genomic titer, determination of the infectious titer, assays for contamination by the mammalian cell line used to generate the virus, silver stain/Coomassie blue gel staining to assay for protein contamination, and Western blot to assess the ratio of capsid proteins VP1, VP2, and VP3. The unprocessed ‘bulk harvest,’ which includes the mammalian cells, media, and unpurified virus, undergoes a series of quality control assays as well. Those include tests for sterility, mycoplasma, and replicationcompetent AAV and adenovirus. After a series of purification steps, the final vector preparation is assayed for sterility, bacteria, fungi, endotoxin, and residual DNA. One of the unfortunate consequences of the need for adherence to the above regulations is a shortage in facilities that are able to produce clinicalgrade reagents for gene therapy trials. The National Institutes of Health (NIH) have designated two U.S. national vector labs for the production of viral vectors for the clinic, one at Indiana University that specializes in retroviral vectors and one at Baylor University specializing in adenoviral vectors. These facilities have succeeded in establishing the technical expertise required to generate viral vectors and the resources to pay for extensive testing of the final product. Unfortunately, a number of novel vectors that do not fall under the mainstream production procedure require expertise typically located in the labs that derive these new systems. In the case of AAV, a Human Applications Lab (HAL) at the University of North Carolina at Chapel Hill (UNC) is an academic facility which has succeeded in producing AAV for a clinical trial. The successment implementation of the recent Canavan’s disease clinical trial highlights the need for such a facility to produce reagents for Phase I clinical trials. With fewer than 1,000 children in the USA affected by the disease, it is not an attractive target for private industry, while most academic institutions do not have Good Laboratory Practices facilities. Facilities like the HAL at UNC fills a critical gap between clinicians interested in gene therapy applications for rare genetic disorders and the patients who have so much to gain through these pioneering clinical trials. The amount of vector necessary to treat 21 human patients, on the order of milliliters of the final product, has been scaled up from the quantities that are more typical of experiments in animal models, on the order of microliters, and it is expected that further scale-up for other clinical trials will be possible in the future, especially as technical advances in large-scale AAV production at our center are achieved.

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AAV Serotypes for Gene Transfer

AAV exists naturally as a variety of serotypes that have immunologically unique properties. To date, a total of eight mammalian serotypes have been discovered and tested as viral vectors [9–15]. AAV serotype 1 or AAV-1 was the first to be isolated and characterized. Although isolated from the rhesus monkey, epidemiological data indicates that it frequently infects humans as well; however, it has yet to be recovered from a human sample. AAV serotypes 2, 3, and 5 are human parvoviruses, and have a high level of infection in the general population, as shown in epidemiological studies. The most unique AAV serotype on the nucleic acid level, AAV serotype 4, was isolated from the African green monkey and is rarely found in humans. It has, however, demonstrated the ability to infect human cells in vitro [16]. AAV serotype 6 is not serologically unique, and is more than 99% homologous to AAV-1 in its capsid proteins at the amino acid level. An analysis of AAV-1 and AAV-2 nucleic acid sequences suggests that AAV-6 is the product of a recombination event between these two serotypes [10]. Most recently, AAV serotypes 7 and 8 were isolated from nonhuman primates. As would be suggested by the serological uniqueness of the AAV serotypes, comparison of their capsids shows that they are indeed heterogeneous. Initial studies to evaluate the different AAV serotypes as gene delivery vectors indicated that serotypes have unique tropisms and differing transduction efficiencies depending on the cell type transduced, when compared to AAV-2 or against each other [17]. Until recently, the majority of the research conducted using AAV-based vectors implored AAV-2. This serotype historically has been used to study critical steps in AAV DNA replication, site-specific recombination, and AAV viral gene expression [18]. For this reason, it was only natural to extend upon this base of knowledge in the early development of AAV vectors. Only after extensive use of AAV-2 vectors in vivo and the identification of limitations in efficient transduction did attention turn to the other serotypes. Each can be distinguished by the efficiency of transduction for specific target tissue when compared to traditional AAV-2 vectors. For example, AAV serotype 5, but not AAV-2, binds to the apical surface of airway epithelia and facilities gene transfer [12, 13]. AAV-1 appears more robust in muscle cells while AAV-4 has been suggested to infect primarily ependymal cells when introduced into the mouse brain [12, 13]. AAV serotypes 7 and 8 have been shown to have muscle (AAV-7) and liver-specific tropism (AAV-8). Surprisingly, only ninety amino acid differences exist between these two serotypes, strongly suggesting that the capsid domain responsible for tissue tropism can be narrowed down and eventually identified. Understanding these differences and the capsid regions required for tropism for cells of the CNS will be critical for developing effective therapies for neurometabolic disorders.

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Fig. 1. Ribbon structure of AAV-2 VP3.

The above evidence clearly points to the benefits of exploiting the natural and unique tropisms of AAV serotypes other than AAV-2 to increase AAVmediated gene transfer efficiency in different cell types. In order to achieve the most efficient gene delivery to the CNS, an approach that takes advantage of different serotypes will be needed.

Crystal Structure of AAV and the Future of Specific Targeting with AAV Vectors

The structure of parvovirus capsid proteins is now known. The structure of six autonomous parvoviruses and one dependo-virus have been solved: B19 [19], canine parvovirus (CPV) [20], feline panleukopenia virus (FPV) [21], Galleria mellonella densovirus (an insect parvovirus) [22], Aleutian Mink Disease parvovirus [23], minute virus of mice [24], and AAV-2 [25]. Sequence alignment of the capsid genes of B19 and CPV/FPV shows only 23% amino acid identity [26], yet these capsid proteins share extensive basic structural motifs. The virions of these viruses are made up of sixty subunits, with the smallest capsid protein making up the majority of the virion. They all share the eight B-barrel motif with looped out regions between barrels (fig. 1) [20, 21, 26]. It is possible to change parvovirus tropism by swapping key capsid amino acids. CPV and FPV share 98% sequence similarity within the capsid-coding region [27]. However, these viruses have different host range infectivity [28]. Using recombinants of CPV and FPV capsid sequences, Parrish et al. [28] were

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BC1/C0 Loop

HI Loop

GH12/13 Loop

GH10/11 Loop

GH2/3 Loop

I Loop

Fig. 2. Ribbon diagram of AAV-2 VP3. Regions in bold from AAV-1, -2, -3, -7, and -8 alignment are highlighted in blue, many are surface-displayed and may reflect muscle versus liver tropism differences. They are labeled according to Xie et al. [25].

able to map specific functions to epitopes on the capsid including determinants of host range infectivity. Refinement of this work defined the determinants of host range to amino acids 93 and 323 of VP2. Coding sequence of these amino acids were introduced into FPV, which could then replicate in canine cells [29]. These data support the ability to interchange epitopes and tropism between parvoviruses. A comparison of parvovirus capsid structures indicates that they are quite similar. AAV-2 VP3 has eight B-barrel motifs that are separated by looped out regions (fig. 2) [26]. The known differences between AAV-2 and the other serotypes may provide the information essential for understanding receptor binding and entry step of AAV vectors. Similarly to the differences found between CPV and FPV, amino acid sequences in loops 3 and 4 may explain the differences in cellular receptors used by AAV2 and the other serotypes. The functional domains can be identified by regions of viral capsid homology. All serotypes of AAV have unique tropisms based on epitopes present on the virion shell. However, the epitopes responsible for those tropisms are not well understood. In the future it will be beneficial to understand which domains on the virion are responsible for each serotype’s unique tropism, to improve targeting of specific cell types. The virions of all serotypes of AAV are assembled from a homologous set of precursor capsid proteins. The alignment of amino acid sequence of the capsid proteins illustrates the degree of homology between each serotype [12], and

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the domains within the capsid sequence with high or low levels of homology have been identified [30]. Areas of low homology are of special interest since they may hold the key to the determinants of the serotypes. Alignment of AAV serotypes elucidates those domains that share the lowest degree of amino acid homology. Prior to the availability of the crystal structure of AAV-2, we exchanged domains between serotypes 1, 3, 4, and 5 from amino acid 440–603 (AAV-2 numbering) and AAV-2 (fig. 2). This domain occupies almost all of the GH looped out domain, the largest and most variable domain between all serotypes, including the recently discovered AAV-7 and AAV-8 as well as the most autonomous parvoviruses. Using pair-wise comparisons this domain has the highest level of homology between AAV-2 and AAV-3 (70%) and the least homology between AAV-4 and AAV-5 (10%). Additionally, comparisons of the autonomous CPV and the FPV shows that amino acid substitutions that resulted in species-jumping from feline to canine are located in the homologous GH loop. This leads us to make the assumption that those domains that are surfacelocalized and have low homology between the AAV serotypes may be responsible for tropism differences between AAV serotypes. This information will also direct future chimeric vector design in order to incorporate phenotypes specific to vector application. Determining which amino acids are surface-displayed is essential for understanding tropism. To approach this problem in a rational way, the newly resolved AAV-2 crystal structure will be essential. The usefulness of the crystal structure has been demonstrated with the positioning of targeting insertions into the adenovirus knob HI loop. For the autonomous parvoviruses a wealth of information has been revealed through comparisons of the amino acid sequences and the crystal structure with respect to surface-display and tropism. The availability of the crystal structure now provides a specific road map to rational structural/functional analysis.

Delivery of Gene Therapy to the Brain

In spite of the tremendous growth in the field of gene therapy and the numerous clinical trials currently underway, relatively few applications target disorders of the CNS. This is due in large part to the complexity of the brain and its circuitry, which is intolerant to even mild inflammation or toxicity. Limited access to the brain itself makes direct injection difficult. Additionally, the protection afforded to the CNS by the blood-brain barrier hinders global delivery of viral vectors through venous injection or cerebrospinal fluid. One of the first experiments in rodents to demonstrate the utility of rAAV vectors in vivo was aimed at transduction of brain tissue into rats [31]. Many of

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Step 1 Receptor binding FGFR

HSPG

␣v␤5

H+

␣v␤5

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Step 2 Nuclear entry

Step 3 DNA template

Fig. 3. Diagram of potential rate-limiting steps in efficient AAV transduction.

the recent advances in the understanding of rAAV vectors have come about through the need to better understand in vitro and in vivo transduction. Although several recent studies have shown great promise in terms of duration of transgene expression in vivo, there has been a shortfall in transduction efficiency, which was unexpected, based on previous results in vitro [32]. High transduction efficiency is of particular importance in the treatment of global neurometabolic disorders which require gene delivery to every affected cell in order to be therapeutically useful. Regardless of the serotype, all of the AAV vectors follow three basic steps for productive infection (fig. 3). First, receptor binding to the cell membrane is required; second, internalization and nuclear entry; and third, DNA template formation. After receptor binding, internalization, and nuclear entry, AAV virions uncoat and release a single-stranded DNA template, which must convert to a duplex intermediate before transcription can ensue. The efficiency of forming the complementary strand can significantly impact vector transduction [33, 34].

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a

wtAAV (ssDNA)

b

Duplex AAV (scDNA)

Fig. 4. Self-complementary AAV packages both strands.

There are two possible mechanisms by which single-stranded AAV genomes can be converted to duplex templates. The first mechanism relies on the reannealing of two single-stranded genomes of different polarity (⫹ and –). Since AAV packages both strands with equal efficiency, this polarity may offer a viable mechanism for solving the duplex template requirement. The second (and usual) mechanism by which single-stranded AAV genomes can be converted to duplex templates involves DNA replication. AAV productive infection relies on the 145-bp hairpin terminal repeat and a self-priming mechanism for viral DNA replication [35]. The terminal repeat exists as double-stranded DNA duplex ‘T’ shaped structure and serves as an origin of replication for the singlestranded viral template [35]. The single-stranded viral template and the terminal repeat hairpin structures are required to form a duplex intermediate [36]. Second-strand synthesis is a rate-limiting step for rAAV transduction. Evidence supporting this conclusion was found in experiments correlating the induction of transgene expression with the conversion of the single-stranded virion DNA to the duplex. Generation of a duplex DNA template is required before transcription can ensue. Careful analysis of this process has now determined unique proviral intermediates (monomer, dimer, concatemeric structures, and circular molecules), all of which are derived from input single-stranded viral DNA [3, 37–48]. The dimer length of vector molecules originally characterized comprise duplex monomers, which are covalently linked at one end and are identical to substrates characterized for wtAAV [34, 36]. The characteristic lag of vector gene expression after infection in nondividing cells correlates with the formation of these duplex DNA intermediates [34, 36, 40, 41, 47, 49]. Double-stranded vectors, (fig. 4) rather than the naturally packaged singled-stranded molecule, could bypass the rate limiting step of second-strand synthesis. Recently, we have generated a novel double-stranded AAV (dsAAV) vector and demonstrated that steps which influence traditional single-stranded AAV

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transduction (i.e., availability of host DNA pol) were not required for dsAAV vectors, supporting the importance of the duplex intermediate as a rate-limiting step in AAV transduction [50]. The use of dsAAV may be particularly useful in the treatment of global CNS disorders that necessitate very high transduction rates. At present, AAV vectors in vivo appear to have very little toxicity or immune consequences after vector transduction [51]. Increased understanding of the biology of AAV has led to the generation of preclinical data for the treatment of neurological disorders and in the case of Canavan’s disease and Parkinson’s disease, Phase I clinical trials.

Treatment of Focal Brain Disorders with rAAV Vectors

Huntington’s Disease Like many other neurological disorders, Huntington’s disease (HD) is caused by the degeneration of specific cell groups within the CNS. By directly targeting these cell groups with gene transfer to express neurotrophic factors, this degeneration may be reversed or avoided altogether. HD is manifested as an array of motor, cognitive, and psychiatric disturbances caused by the degeneration of medium-sized spiny neurons in the striatum and cerebral cortex. Glial cell-line-derived neurotrophic factor has been shown to prevent the loss of striatal neurons in animal models of HD [52–56]. In an animal model of HD, bilateral injections of a rAAV viral vector containing the glial cell-line-derived neurotrophic factor transgene into the striatum showed marked protection of striatal neurons and prevention of behavioral disturbances [57]. Because HD is passed on through an autosomally dominant gene, persons who have not yet sustained any neurological damage could be identified as having the disease and receive gene therapy, making it possible to avoid the debilitating effects of the disease altogether. Seizure Disorders Gene therapy with AAV also has been tested for the treatment of focal seizure disorders. A study by Haberman et al. [58] shows that the delivery of N-methyl D-aspartic acid receptor antisense using an AAV-derived vector can modulate seizure disorder in vivo. However, it was also shown that using different promoters to drive the transgene expression resulted in completely opposite physiological effects, increasing seizure sensitivity as opposed to reducing it. In order to work around this effect, the gene for the inhibitory neuroactive peptide, galanin, was delivered to cells using an AAV-derived vector, and constitutively secreted through the use of the fibronectin signal sequence. Importantly, the choice of promoter to drive the transgene did not impact the

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decrease in seizure sensitivity [59]. This approach provides a new option for the long-term control of focal seizure disorders. Parkinson’s Disease Another devastating neurological disorder, Parkinson’s disease, is characterized by the degeneration of the substantia nigra pars compacta and consequently, reduced dopamine in the striatum. The resulting lack of inhibition of the subthalamic nucleus (STN) contributes to the motor abnormalities typical of the disease. Deep brain electrical stimulation of the STN has been shown to effectively reduce symptoms of Parkinson’s disease. One alternative approach for the treatment of Parkinson’s using gene therapy seeks to achieve the same effect biochemically. GABA, the brain’s major inhibitory transmitter, can be generated by two isoforms of glutamic acid decarboxylase. Stereotactic injection of rAAV carrying the glutamic acid decarboxylase transgenes into the STN of adult parkinsonian rats resulted in neuroprotection of the STN and a reduction in the excitatory phenotype of the disease. Robust expression of the transgene was seen up to 5 months after injection, with no significant immune response [60]. One of the two clinical trials currently underway that utilize rAAV vectors to treat neurological disorders uses this vector in human patients.

Global Gene Delivery with rAAV Vectors

Canavan’s Disease The original clinical trial in which rAAV was first used as a vector for gene delivery in humans is for the treatment of Canavan’s disease. This study, led by Dr. Paola Leone at UMDNJ-Robert Wood Johnson Medical School, was the first gene transfer clinical trial to use viral vectors to treat a neurodegenerative disorder. Canavan’s disease is an inherited disease, with autosomal recessive inheritance, that shortens life expectancy to a few years. Symptoms are generally first seen within the first 6 months of life and include megalocephaly and developmental delays. As the disease progresses, mental retardation, spasticity and cortical blindness develop, culminating in seizures and childhood death. A lack of the enzyme aspartoacylase (ASPA) that hydrolyzes N-acetylaspartic acid (NAA) into L-aspartate and acetate causes the damage associated with Canavan’s [61, 62]. It is thought that the accumulation of metabolic precursors such as NAA, the function of which remains undetermined, is toxic and leads to neurological damage [63]. It also appears that high levels of NAA affect the phenotype of developing myelinating cells through a complex set of

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gene expression effects which are currently being worked out [Leone, pers. commun.]. This clinical trial is intended to retard the damage of elevated NAA levels by injecting rAAV carrying the aspartoacylase gene into the brain. Preliminary data suggest beneficial effects of treatment on the biochemical and clinical level, with an absence of adverse events. dsAAV and Improved Global CNS Delivery? Because global neurometabolic disorders require long-term transgene expression, and in cases of intrinsically expressed gene products, transduction of nearly every affected cell, the development of less intrusive delivery methods is a key step in getting these treatments into the clinic. A recent study by Fu et al. demonstrated the utility of the dsAAV vector for global CNS distribution in a mouse model. An IV injection of 4 ⫻ 1011 particles of dsAAV2-expressing green fluorescent protein preceded by an injection of 12.5% mannitol showed a global distribution of the transgene in the brain and spinal cord 4–8 weeks after injection. No green fluorescent protein expression was seen using dsAAV2 in 12.5% mannitol or with the viral vector without mannitol. Additionally, no green fluorescent protein expression was seen when ssAAV was injected intravenously preceded by the injection of mannitol. Through the use of mannitol to transiently open the blood-brain barrier in conjunction with dsAAV to increase transduction efficiency, minimally invasive intravenous injections may provide another effective route to global gene delivery to the CNS, in addition to intraparenchymal injection protocols.

Conclusion

Safe and efficient delivery of corrective gene therapy to the CNS using rAAV vectors has great promise for the treatment of neurological disorders. An array of recent developments including advances in production, the elucidation of the rAAV crystal structure, and newly discovered serotypes will facilitate further clinical applications. While the CNS presents unique challenges to the development of effective therapies, the devastating nature of the diseases being targeted should serve as an impetus to overcome the inherent hurdles.

Acknowledgements Thanks to the members of the University of North Carolina, Chapel Hill (UNC) Gene Therapy Core.

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R. Jude Samulski, PhD Gene Therapy Center, Department of Pharmacology CB# 7352, 7119 Thurston Bowles, University of North Carolina at Chapel Hill Chapel Hill, NC 27599–7352 (USA) Tel. ⫹1 919 962 3285, Fax ⫹1 919 966 0907, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 169–201

Molecular Mechanisms of Epilepsy and Gene Therapy Albert Telfeiana, Juanita Celixb, Marc Dichterc a

Division of Neurosurgery, Texas Tech University Medical Center, Lubbock, Tex., Department of Neurosurgery, University of Washington, Seattle, Wash., c Department of Neurology, University of Pennsylvania, Philadelphia, Pa., USA b

Introduction ‘Extreme remedies are appropriate for extreme diseases…’ Hippocrates (460–370 B.C.)

Epilepsy is a neurological disorder that afflicts 1–2% of the general population and encompasses a variety of disorders with seizures [1]. To best understand where we must go in the treatment of epilepsy, it is necessary to understand first where we have failed. The prognosis for seizure control in epilepsy with medication is good in ⬃60% of patients, and up to 40% of individuals suffer from intractable pharmacoresistant epilepsy. There are over twenty different anti-epileptic drugs available to the neurologist or neurosurgeon to treat seizures, but patients not controlled on monotherapy with the first anti-epileptic agent have only a 10% chance of being controlled with additional anti-epileptic agents, even when using medications that work by diverse mechanisms [2]. Newer is not necessarily better in terms of drug regimens. Only a small minority (⬍5%) of patients refractory to traditional drug therapy has been reported to become seizure free with a new generation anti-epileptic drug [2]. The more we understand about the genetic basis of this disease, the more naïve it appears that a single drug tailored to a specific channel or neurotransmitter receptor will effectively cure epilepsy. In fact, it appears that the better drugs have a wider basis in their mechanisms of action. Most clinically efficacious anti-epileptic drugs possess a combination of various properties. While there have been advances in the drugs available to control the seizures associated

with epilepsy, to date there is no effective therapy for the prevention of epilepsy. Generally, one third of all cases of epilepsy have a potential cause, the most obvious being trauma, tumors, and stroke, but treatment seems to have done little to prevent the process of increased intrinsic excitability, synchronization, or synaptic connectivity that may be responsible for the development of seizures. Here, we concentrate on how a better understanding of the molecular mechanisms of epilepsy may guide future therapies, such as gene therapy, for this disease.

Review of Pharmacotherapy in Epilepsy

Anti-epileptic drug therapy is the first-line treatment for epilepsy. Until the 1990s, there were only a handful of drugs available to treat the various seizure disorders. The earliest drugs used to control seizures (e.g., bromides, phenobarbital, valproic acid) were identified inadvertently. Later, a more scientific approach utilized animal models of epilepsy against which potential antiepileptic drugs were tested. The maximal electroshock (MES) model is used to evaluate agents for the ability to decrease seizure severity and to identify those drugs with efficacy in treating generalized tonic-clonic or partial seizures. The pentylenetetrazole (PTZ) model is utilized to test for agents that increase the seizure threshold and exert possible anti-absence seizure properties. While the early anti-epileptic drugs identified by these models were understood to function via action at the neural membrane or synapse, the animal models could not elucidate the important mechanisms of action. It was not until the introduction of more modern electrophysiological and pharmacological research techniques that the effects of anti-epileptic drugs at the neural membrane and synapse were determined. The diligent study of anti-epileptic therapies lead to the discovery of the principle mechanisms of action of the clinically efficacious drugs used to treat seizure disorders. In general, anti-epileptic agents control the initiation, maintenance, or propagation of epileptiform discharges through augmented inhibition, suppressed excitation, or modulation of action potential ion current, with effects at the level of localized neurons to entire neural networks. Typically, anti-epileptic drugs act on one of four classes of neuron ion channels: ␥-aminobutryic acid (GABAA) ligand-gated chloride channels, glutamate ligand-gated sodium and calcium channels (NMDA, N-methylD-aspartate; AMPA, ␣-amino-3-hydroxy-5-methyl-4-isoxazoleproprionic acid; and kainate), voltage-gated sodium channels, and voltage-gated T-type (low threshold) calcium channels. Most of the agents with activity in the MES model function through use-dependent inactivation of voltage-gated sodium

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channels. Drugs with efficacy in the PTZ model function via blockade of T-type calcium channels or augmentation of GABAA receptor-mediated chloride channels. Several of the recently developed anti-epileptic drugs that have been introduced during the past decade have new or additional mechanisms of action, targeting pre- and postsynaptic membrane-bound receptors and enzymes that function in the metabolism of neurotransmitters. Still, there are a few anti-epileptic agents whose mechanisms of action remain largely unknown (e.g., topiramate, felbamate). As we continue to make advances in cellular and molecular biology and gain a greater understanding of the excitability and synchronization of neurons in circuit, a fuller appreciation for the mechanisms of action of the efficacious anti-epileptic drugs will undoubtedly become more apparent.

Traditional Pharmacotherapy

Phenytoin, carbamazepine, and valproic acid are traditional anti-epileptic drugs with primary actions at the voltage-gated sodium channel. Phenytoin acts at the sodium channel in a voltage- and frequency-dependent manner. It preferentially binds to and stabilizes the channel in the open inactive state. Selective blockade of the inactive sodium channel prevents release of excitatory amino acid neurotransmitters, particularly glutamate and aspartate, delays channel recovery time, and slows propagation of electrical discharges. There is evidence that sustained, high-frequency repetitive firing plays a major role in neuron excitability, and that phenytoin functions to limit this repetitive firing [3]. As sodium channels may be more susceptible to blockade when they are in the open inactive state, preferential binding of phenytoin to the inactivated voltagegated sodium channel of the depolarized neuron during seizure activity is the likely mechanism through which phenytoin acts to limit sustained repetitive firing and terminate seizure activity. At high drug doses, phenytoin has additional clinically significant actions, including voltage-gated calcium channel blockade and decreased presynaptic glutamate release. The anti-epileptic properties of carbamazepine, initially developed as a tricyclic antidepressant, and valproic acid were fortuitously discovered. Similar to phenytoin, carbamazepine and valproic acid both function in a voltage- and frequency-dependent manner to bind the inactive sodium channel and delay recovery. Valproic acid also indirectly facilitates the inhibitory activity of GABA via increased synaptic neurotransmitter levels, while carbamazepine’s additional mechanisms of action include inhibition of norepinephrine reuptake, decrease in intracellular cAMP levels through interaction with adenosine receptors, and possible potentiation of GABA inhibition through interaction with the

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GABAB receptor. The clinical importance of these additional mechanisms of action remains to be determined. The voltage-gated calcium channels consist of at least four types, L-type, T-type, N-type, and P-type, with differing levels of voltage activation and inactivation. Ethosuximide is an anti-epileptic drug with activity at the T-type calcium channel, a low threshold voltage-gated channel. Ethosuximide functions primarily at the T-type channels of thalamic neurons, reducing the calcium current and interrupting the 3 Hz spike/wave thalamocortical action potentials typical of absence seizures. Activity at GABA ligand-gated chloride channels is the primary mechanism of action of the most well known anti-epileptic drugs, barbiturates and benzodiazepines. The GABAA receptor complex contains multiple modulatory sites for site-specific interaction with various agents. The GABA receptor subtypes have differential developmental and regional expression and varying sensitivities to the diverse ligands. Phenobarbital is one of the oldest antiepileptic drugs. At the GABAA receptor chloride channel, it potentiates the activity of GABA through enhanced duration of channel opening. At high drug doses, the barbiturates also interact with the N-type voltage-gated calcium channels to block the calcium influx and prevent release of excitatory amino acid neurotransmitters. Benzodiazepines also function at the GABAA receptor, where they increase the frequency of chloride channel opening without affecting the duration of opening.

Advances in Pharmacotherapy

The recent introduction of new generation anti-epileptic drugs illustrates our increased understanding of the multiple interrelated mechanisms by which synchronous excitatory electrical activity can be initiated, maintained or propagated, and enables a more rational approach to pharmacotherapy in epilepsy. While some of the newer agents were discovered using the MES and PTZ animal models of epilepsy, others were rationally designed to produce a specific molecular effect. Many newer agents have multiple mechanisms of action and predominantly function via metabotropic or ionotropic channels, similar to the traditional anti-epileptic drugs. Lamotrigine is a new generation drug that functions at the presynaptic open inactive voltage-gated sodium channel to delay channel recovery and decrease excitatory amino acid release. It may also interact with the GABA receptor, and it has efficacy in treating certain seizure types that cannot be explained by its primary mechanism of action. Unexplained therapeutic effects are common with many of the new generation agents. Gabapentin, which was developed based on the theoretical role of

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GABA in epileptogenesis, is a novel drug formed by the linkage of a cyclohexyl group to GABA. This modification allows it to easily penetrate the blood-brain barrier (BBB). As a structural analog of the endogenous inhibitory neurotransmitter, gabapentin was designed to function via GABA-mimetic activity, but the drug does not interact with GABA receptors in the CNS. Neither does it interact with voltage-gated sodium or calcium channels, nor with NMDA ligandgated ion channels. It is unclear through which mechanism or combination of mechanisms gabapentin exerts its anti-epileptic effect, but it does indeed have anti-epileptic properties. A novel class of second generation anti-epileptic drugs is active in modulating local levels of GABA. Altered intracellular metabolism of endogenous neurotransmitter and inhibition of reuptake from the synaptic cleft are the mechanisms by which these new agents augment levels of inhibitory GABA within the brain and ultimately alter the balance of excitation and inhibition. Vigabatrin is an irreversible inhibitor of intracellular GABA transaminase, the protein responsible for the metabolic degradation of GABA. Inhibition of GABA degradation allows an increase in the local concentration of GABA and potentiation of its physiological role in limiting excitatory neural activity in the brain. Tiagabine increases synaptic GABA through blockade of neuronal and glial GABA reuptake from the synaptic cleft. Two of the new generation drugs were designed based on the hypothesized role of glutamate neurotransmission in seizure generation. These agents employ a novel mechanism of action with activity at the NMDA ligand-gated calcium channel. The NMDA receptor is believed to play a fundamental role in the initiation and propagation of epileptiform activity. NMDA receptormediated blockade of the calcium channel results in inhibition of neuronal hyperexcitability, with the potential to not only control seizure activity, but to modulate the underlying epileptogenic defect. In practice, the NMDA receptor antagonists have not proven efficacious in controlling seizure activity, and the incidence of adverse effects with this class of drugs limits their clinical usefulness. The elucidation of the basic mechanisms of action of the anti-epileptic drugs and the development of novel agents has afforded us a greater appreciation for the complex molecular mechanisms underlying neural excitability and synchronization in epileptogenesis. While these drug discoveries allow clinicians to move from the empirical treatment of epilepsy to a more rational approach to seizure control, they may not have a significant impact on the effective treatment of intractable seizures. An approach to epilepsy treatment based on innovative understandings of epileptogenic activity and novel avenues of investigation holds the greatest promise for the future of epilepsy research and the ultimate development of a cure.

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The Future of Pharmacotherapy in Epilepsy

The future of pharmacotherapy in epilepsy will be based on an appreciation of the molecular mechanisms that result in abnormal synchronized excitatory electrical activity in the brain. Understanding the basic principles of initiation, maintenance, and propagation of seizure activity and their relationship to the factors that influence susceptibility to spontaneous recurrent seizures offers the potential for fundamental shifts in the paradigms of therapy in epilepsy. Integration of our knowledge of pharmacogenetics, drug resistance mechanisms, drug delivery systems, and cellular and gene therapy will allow us to develop a more powerful approach to drug therapy in epilepsy. The most promising strategies in pharmacotherapy should aim to propel us beyond treatment of the symptoms of epilepsy, namely suppression of seizures, to prevention or cure of this devastating neurological disease.

Pharmacogenetics: A New Approach to Drug Therapy

An increased understanding of both the molecular mechanisms of epilepsy and the mechanisms of action of the anti-epileptic drugs has made it increasingly possible to move away from the empirical treatment of epilepsy and employ a more rational approach to pharmacotherapy. Yet our considerable knowledge of the etiology of epilepsy, including the underlying neuropathological processes, electrophysiology and biochemistry of a seizure, and specific therapeutic and adverse effects of each anti-epileptic drug has not enabled clinicians to anticipate an individual patient’s response to a particular anti-epileptic agent. The individual response to a specific drug is still empirical. As a result, patients with refractory epilepsy undergo multiple trials of single or combination therapy with significant risk of CNS or systemic toxicity and severe idiosyncratic reactions. A novel approach to enhanced drug therapy in epilepsy is based upon our understanding of genetic polymorphism in drug metabolism. Genetic variations in drug pharmacokinetics are a likely factor in refractory seizures due to either lack of anti-epileptic drug efficacy or intolerable side effects. Genetic differences may influence both the pharmacokinetics and pharmacodynamic effects of a particular anti-epileptic drug. Abnormal drug metabolism can be due to genetic polymorphisms that result in altered metabolic enzymes. Rapid metabolizers may have low plasma concentrations of a drug despite appropriate dosing and good adherence to therapy, and essentially appear to be refractory to medical management. Slow metabolizers can experience high plasma concentrations at normal drug dosages with resultant CNS and systemic toxicity that limits the use of an anti-epileptic drug.

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The effects of genetic polymorphisms of cytochrome P450 isoenzymes on phenytoin metabolism have been studied extensively. In adult patients with epilepsy, impaired CYP2C9 or CYP2C19 isoenzymes were associated with altered phenytoin hydroxylation, which resulted in dramatically increased plasma drug concentrations even at normal or low phenytoin doses [4]. Molecular studies revealed two G to A point mutations in the CYP2C19 gene as the cause of the defect in the functional CYP2C19 protein (de Morias et al., 1994a,b), and multiple amino acid variants in the CYP2C9 protein have been described (Kaminsky et al., 1992). Identification of the genetic mutations in CYP2C19 allows for widespread genotyping by simple polymerase chain reaction restriction fragment length polymorphism for the defective isoenzyme. Identification of genetic polymorphisms in anti-epileptic drug metabolism has broad implications for the effective drug treatment of epilepsy. Knowledge of an individual’s genotype has the potential to allow for tailored drug therapy and effective control of seizures in someone who was once considered medically refractory due to aberrant drug metabolism. Idiosyncratic hypersensitivity reactions may also be influenced by alterations in metabolic enzymes. It has been proposed that CYP450 anti-epileptic drug bioactivation results in reactive metabolites that mediate the chemical modification of detoxifying enzymes. The aberrantly modified enzyme leads to deficient detoxification of anti-epileptic drug and a subsequent increase in the availability of bioactivated drug. A host-dependent immune response is believed to have a role in the complex series of events that lead to a hypersensitivity reaction. Further characterization of the pathways involved may allow for genotyping studies that will identify patients at risk for adverse effects with anti-epileptic drug therapy, and permit clinicians to take a truly rational approach to pharmacotherapy in epilepsy.

Pharmacoresistance in Epilepsy

Of patients who are refractory to first-line conventional anti-epileptic drugs, less than 5% will attain good seizure control with use of the newer agents, and a patient whose seizures cannot be adequately controlled with one anti-epileptic drug has only a 5–10% chance of controlling their seizures with multiple anti-epileptic agents [2]. In this population, multiple trials of drugs with differing mechanisms of action do not improve the chance of a positive response. Given the character of global resistance to different anti-epileptic drugs with varying mechanisms of action, it is unlikely that acquired alterations in the multiple receptors upon which anti-epileptic drugs act can adequately explain

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pharmacoresistance. It is instead hypothesized that pharmacoresistant epilepsy may be due to altered permeability of anti-epileptic drugs across the BBB and that the mechanisms whereby drug access to the brain is limited are most likely nonspecific [5]. It is further proposed that nonspecific drug resistance mechanisms may represent adaptive changes in the epileptic brain [5]. As we continue in search of an effective drug treatment for pharmacoresistant epilepsy, it is crucial that we appreciate the full nature of drug resistance mechanisms and the specific character of those mechanisms in the epileptogenic brain. Our understanding of epilepsy continues to evolve and the pursuit of an effective drug treatment for epilepsy continues to advance. But if we are to truly make breakthroughs in the pharmacotherapy of epilepsy we must move beyond the traditional line of investigation. An appreciation of the mechanisms of pharmacoresistance in epilepsy is essential to the development of better epilepsy therapy. The discovery of cellular membrane proteins that function in chemotherapy-resistant neoplasms has advanced our understanding of drug resistance in epilepsy. Extensive biochemical study of the molecular character of these proteins has elucidated the mechanisms by which drugs are denied access to or extruded from the intracellular space, and overexpression of multidrug transporters in neoplastic cells has been shown to correlate with resistance to chemotherapeutic drugs. Multidrug transporters in the BBB have been described and alterations in these drug transporters are one proposed mechanism of drug resistance in epilepsy. P-glycoprotein and members of the multidrug resistance-associated protein (MRP) family are the primary drug transporters identified to have a role in drug-resistant epilepsy. P-glycoprotein is a transporter expressed in endothelial cells of the BBB that functions in the active transport of lipophilic molecules from the intracellular space into the vascular space. Studies of epileptogenic tissue surgically removed from patients with intractable seizures show an overexpression of P-glycoprotein at the epileptic focus [6] and experiments in animals provide evidence for the role of P-glycoprotein in regulating the brain concentrations of multiple anti-epileptic drugs [7, 8]. The family of proteins known as MRP also functions in the transport of lipophilic molecules at the BBB. These proteins are normally expressed in various tissue types throughout the body. In the brain, MRP expression is found normally in capillary endothelial cells. Studies in human brain tissue have shown the abnormal expression of MRP in neurons and glia. In surgically resected lesional tissue from patients with refractory epilepsy due to hippocampal sclerosis, focal cortical dysplasia, and dysembryoplastic neuroepithelial tumors, abnormal MRP expression was demonstrated in reactive astrocytes, dysplastic neurons, and capillary endothelium [9], suggesting a physiological basis for drug resistance.

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While the overexpression of multidrug transporters in the brain has implications for the effective control of seizure activity with an anti-epileptic drug, the inability to adequately control seizure activity may have etiological implications for drug-resistant epilepsy. A recent study of seizure-induced expression of P-glycoprotein in rodents demonstrates that both acute and chronic epileptic activity significantly increases the level of P-glycoprotein mRNA [10]. These findings suggest that uncontrolled seizures may contribute to pharmacoresistant epilepsy. For the subpopulation of patients with a genetic predisposition to drugresistant epilepsy, the lack of effective pharmacotherapy to adequately control seizure activity may be especially detrimental. The overexpression of drug transporters in an epileptic focus appears to promote pharmacoresistant epilepsy by limiting local access of anti-epileptic drugs. In the future, novel pharmacotherapeutic approaches to epilepsy may include the development of anti-epileptic drugs that are not substrates for membrane permeability proteins and the adjunctive use of multidrug transport inhibitors with traditional anti-epileptic drugs. The lipophilic nature of most anti-epileptic drugs enhances their ability to penetrate the brain, but may inadvertently promote drug resistance in those patients that overexpress multidrug transporters. The development of new drug delivery systems that effectively deliver anti-epileptic agents to the brain, but avoid the potential to promote multidrug transporter-mediated pharmacoresistant epilepsy, is an area that deserves attention.

Epilepsy: An Autoimmune Disorder?

As the scientific community continues to search for the mechanisms that underlie intractable epilepsy, our understanding of the complexity of factors that influence the development of seizures continues to expand. In the late 1970s, based on empirical evidence from children with epilepsy who received immunoglobulin for recurrent upper respiratory tract infections, the theory of an immunological component of epilepsy was revisited. A decrease in the frequency and severity of seizures was observed in this population. This prompted a flourish of research in the area of immunological mechanisms in the central nervous system. Research has revealed new evidence that certain refractory seizure disorders may be autoimmune mediated. The presence of specific antibodies has been identified in Rasmussen’s encephalitis, noninflammatory focal epilepsy, and pediatric ‘catastrophic’ epilepsy and research has shown these antibodies to be directed towards the GluR3 subtype of the AMPA glutamate receptor [11]. Characterization of the antibodies indicates that some interact with the GluR3

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receptor at a site distant from the glutamate binding site and function in channel opening and neuronal activation [12]. This provided the first evidence of autoimmune-mediated activation of a metabotropic ion channel receptor in the central nervous system. Further studies demonstrated the influence of IgG immunoreactivity and complement activation on neuronal death [13]. Cortical dysfunction via immune-mediated mechanisms may be due to the combination of excitotoxic overactivation of the neuronal glutamate receptor and activation of complement leading to neuron injury. It is hypothesized that Rasmussen’s encephalitis is an autoimmune disease that results in neuronal damage and subsequent development of seizures. Animal studies revealed that rabbits immunized with a portion of the GluR3 protein demonstrated production of anti-GluR3 antibodies and development of a Rasmussen’s type disorder characterized by inflammatory cerebral lesions and recurrent seizures. In a study of pediatric patients with Rasmussen’s encephalitis, serum GluR3 autoantibodies were identified and titers were correlated with seizure frequency [14]. In both studies, a response to plasma exchange to remove circulating anti-GluR3 antibodies was demonstrated. In subsequent studies, a positive response to the use of intravenous immunoglobulin in patients with the adult-onset variant of Rasmussen’s encephalitis was observed [15, 16]. In separate animal studies, though, mice immunized with the GluR3 peptide displayed significant pathological brain abnormalities, but no seizure activity [17, 18], lending support to the hypothesis that production of anti-GluR3 autoantibodies may be necessary for the development of excitotoxic and complement-mediated neuronal damage in Rasmussen’s encephalitis, but is not sufficient to play a primary role in development of epileptic seizures. A causative autoimmune mechanism has also been hypothesized for the intractable epilepsy associated with West’s syndrome, Lennox-Gastaut syndrome, and Landau-Kleffner syndrome. While most of the support comes from noncontrolled studies, small case series, or single case reports, there is evidence that immunotherapy with intravenous immunoglobulin can completely control seizures in a portion of these patients. There are multiple factors that may contribute to the development of epilepsy as a result of autoimmune-mediated neuronal activity. The presence of autoantibodies could have a role in modulating epileptic activity independent of glutamate receptor activation. Immunoreactivity-associated epilepsy is seen in Hashimoto’s and viral encephalitis where it is mediated by autoantibodies against voltage-gated potassium channels (Lang and Vincent, 1996). Animal studies have shown that mice immunized with GluR3B peptide demonstrate production of anti-ssDNA antibodies at levels similar to those seen in the mouse model of systemic lupus erythematosus [17, 18]. It has been documented that systemic lupus erythematosus patients exhibiting high levels of anti-ssDNA

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antibodies can experience seizures, but it is not known through what mechanism anti-DNA reactivity influences epileptogenesis. As would be anticipated in a complex disease process that results in epileptic activity, genetic factors are also likely to influence susceptibility to autoimmune seizure development. Many autoimmune disorders, such as systemic lupus erythematosus and juvenile onset diabetes mellitus, are associated with genetic variations in MHC gene expression. There is evidence of an increased incidence of Rasmussen’s encephalitis in patients with specific human leukocyte antigen haplotypes. While the precise mechanisms whereby autoantibodies alter neural circuits and influence seizure activity are being elucidated, there is evidence that treating appropriately selected patients with immunotherapy is beneficial in reducing seizure frequency and improving function. In select patients with Rasmussen’s encephalitis, treatment with intravenous human immunoglobulin or protein A immunoadsorption resulted in decreased seizure frequency, improved cognitive function, and improvement on SPECT imaging [15]. Plasma exchange and intravenous IgG are beneficial in patients with antibodyassociated Hashimoto’s or viral encephalitis and epilepsy. Despite the lack of definitive data as to the precise role of autoantibodies in seizure development, empirical evidence does support a possible role for immunotherapy in select types of intractable epilepsy. The influence of immunological mechanisms in certain epilepsy syndromes provides support for the multifactorial nature of seizure etiology and encourages researchers to consider novel approaches to pharmacotherapy in epilepsy.

Neuroprotection: Preventing Epilepsy?

Traditionally, pharmacological neuroprotection is a concept primarily associated with acute neurodegeneration due to cerebral ischemia or traumatic brain injury. The goal of drug therapy in these settings is to restore the normal biochemical environment and protect neurons from the cytotoxic effects of inflammation and hyperexcitability. Anti-thrombotic, thrombolytic, and anti-inflammatory agents are used in stroke and head injury to prevent the sequelae of such insults to the brain. Recent studies of anticoagulation with unfractionated heparin following ischemia and brain trauma in animal models show a decrease in lesion size and improvement in motor and cognitive deficits [19]. Unfractionated heparin is believed to have not only anti-coagulant, but anti-oxidant, anti-inflammatory, anti-excitatory, and neurotrophic effects that may act synergistically to provide neuroprotection in acute brain insult. After traumatic brain injury, neuroprotective therapy generally focuses on prevention of secondary brain injury. Many pharmacotherapeutic agents are being

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investigated and a variety of promising therapeutic options have emerged, including glutamate receptor antagonists, calcium channel antagonists, and free radical scavengers. A common theme in neuroprotection is prevention of seizures in patients who are at increased risk for developing epilepsy. Potential causes of epilepsy include cerebrovascular disease, perinatal hypoxia or ischemia, infection, febrile seizures, tumors, congenital malformations, trauma, and status epilepticus. Underlying genetic factors may also have a role in determining seizure susceptibility after an insult. The ability of the anti-epileptic drugs to prevent epileptogenesis in at-risk patients is largely unknown. Clinical studies of phenytoin, phenobarbital, carbamazepine, and valproate have failed to show a protective effect in the development of epilepsy after head injury. Studies in animal models, though, have shown that valproate may be effective in preventing epilepsy. These findings emphasize the complex multifactorial nature of seizure development and emphasize the need for multifaceted treatment strategies in epilepsy. Identifying the fundamental mechanisms of epileptogenesis may allow us to develop therapies that target the underlying disease process and effectively alter the development or progression of epilepsy. There is undoubtedly a cascade of disparate events that occurs in the development of epilepsy. A primary insult in the setting of genetic susceptibility may lead to fundamental structural or biochemical changes that result in spontaneous seizures and epilepsy. Pharmacotherapeutic strategies that can alter the sequelae of brain injury, influence the process of epileptogenesis, prevent or terminate seizure activity, modify underlying pathology, or interfere with multidrug resistance mechanisms do have an essential role in the successful treatment of patients with epilepsy.

Defective Ion Channels: From Pathogenesis to Therapy

Further development of effective therapeutic strategies will depend upon elucidation of genetic factors that influence epilepsy and novel approaches to modulating genetic defects. Altered gene expression and mutated gene products are known to play an etiological role in epilepsy. Genetic mutations have been identified in some of the rare familial epilepsies as well as some types of idiopathic epilepsy. Many of the identified genes encode for ligand- or voltagegated channels. A variety of paroxysmal disorders are due to defective ion channels, or ‘channelopathies,’ including some of the myotonias and other neuromuscular disorders, and long QT-syndrome. Defects in sodium, potassium, calcium, chloride, or glycine-mediated channels have been identified in these disorders. Epilepsy models employing sporadic mutant animal strains, genetically

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engineered animals, and knockout animals, coupled with human studies have helped elucidate the role of ion channel defects in epilepsy. Ion channels have a recognized role in generating electrical currents and a channel defect could easily alter the balance of excitation and inhibition in neural networks, resulting in neuronal hyperexcitability and progression of epileptogenesis. All of the genes identified in the human familial epilepsies encode ion channels or auxiliary subunits. Genetic defects in ion channels not only contribute to idiopathic epilepsy, but may also participate in the development of post-traumatic epilepsy. Neuronal injury induces the expression of ion channels, and overexpression of abnormal ion channels may lead to epilepsy after a cerebral insult [20]. Recognition of some of the multiple genetic defects associated with epilepsy illustrates the incredible heterogeneity of genes that function in producing the epileptic phenotype. Just as the scientific community has adjusted its view of cancer etiology to emphasize a multifactorial basis, the approach to epilepsy research must consider the varied genetic and environmental factors that underlie the mechanisms of epileptogenesis. It is unlikely that a single mechanism is responsible for the development of seizures and progression to epilepsy. A singular approach to epilepsy therapy based on the premise that a single agent operating on a single mechanism can potentiate effective treatment is fundamentally limited. An appreciation for the diversity of genetic influences in seizure disorders will guide future therapeutic strategies.

Advances in Drug Delivery

The disappointing failure of the new anti-epileptic drugs to significantly improve outcomes in the pharmacotherapeutic treatment of epilepsy has prompted researchers to re-evaluate the current approach to seizure control and pursue innovative avenues of investigation. Traditional approaches to anti-epileptic drug development have focused on formulating new agents or improving existing agents. A novel avenue of drug development focuses on advanced delivery systems. From engineered drug reservoirs to implantable drug pumps and synthetic polymers, these new methods of drug delivery may prove successful in improving the efficacy while decreasing the systemic effects of anti-epileptic drugs. Moreover, new drug delivery systems may have applications beyond simple delivery of anti-epileptic drugs to delivery of cell and gene therapy agents. Special oral formulations of traditional anti-epileptic drugs have been developed and are in widespread clinical use. Some of these new forms of medication were designed for use in certain seizure settings or specific patient

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populations, while others were developed to achieve the elusive goal of pharmacotherapy with enhanced efficacy and decreased systemic toxicity. For emergency situations that require quick drug delivery and acute seizure exacerbations, such as occur in febrile seizures, traditional oral therapy is not an effective mode of delivery. Transmucosal administration of anti-epileptic drugs theoretically allows for faster drug delivery. A rectal gel formulation of diazepam is available and commonly used in terminating acute seizure activity. Nasal or buccal mucosa administration of anti-epileptic medication may also achieve rapid delivery. While there are no formulations currently approved for administration through these routes, the off-label use of liquid benzodiazepine formulations via nasal and buccal routes is common practice. Drug delivery through the respiratory mucosa is an attractive alternative mode of therapy. The administration of inhaled drugs is a daily practice in the induction of anesthesia and the treatment of acute asthma exacerbations. Recently, inhaled chemotherapeutic agents have been used to treat lung cancer. The engineering of micro- and nanoparticulate systems with an adhesive coating may allow for better delivery to the alveoli and greater uptake across the blood-air barrier, and holds promise for the development of an improved anti-epileptic drug delivery system. Drug administration in the pediatric population has been made easier with the introduction of syrups, sprinkles, and chewable formulations of certain anti-epileptic drugs. Depot forms of anti-epileptic drugs, such as transdermal patches or subcutaneous implants, may also prove effective in improving pharmacotherapy in the pediatric and other populations, but these drug formulations are still only theoretical. While new anti-epileptic drug formulations increase the treatment options available to patients with epilepsy and may improve adherence to therapy, they have not had a significant impact on the proportion of patients that achieve seizure control. Some researchers argue that the focal delivery of anti-epileptic agents holds the most promise for significantly improving seizure control in patients with epilepsy. This is an area of drug development that has gained much attention in recent years. The administration of a prodrug that is systemically inert and becomes activated at the seizure focus holds the potential to inhibit seizure activity with minimal systemic toxicity. The development of the prodrug DP-VPA is already underway [21]. This engineered drug is composed of valproic acid coupled to a phospholipid moiety that serves to inactivate the valproic acid. At the seizure focus, abnormal neuronal activity results in elevated activity of phospholipases, which function to cleave the phospholipid moiety and release the activated drug. Direct administration of anti-epileptic drugs into the CSF space provides for the possibility of better prevention of seizure activity and may have a role

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in termination of seizures. The use of an intraventricular or intrathecal catheter to deposit drug directly into the CSF bypasses one of the major problems in CNS drug delivery, penetration of the BBB, with the additional benefit of decreased systemic toxicity. Subcutaneous infusion pump implants are already in clinical use for the long-term intrathecal administration of analgesics for chronic pain control and anti-spasticity medications following brain injury. While the application of an intrathecal infusion pump system in the delivery of anti-epileptic drugs is still speculative, a collaborative effort to investigate the infusion of an NMDA antagonist via the intrathecal route is underway. Direct infusion of drugs into the CNS has proven successful in certain settings, but there are limitations to this therapy. The delivery of anti-epileptic drugs directly into a seizure focus is an innovative approach with immense potential to improve pharmacotherapy in intractable epilepsy. Microinjection and microinfusion systems have been used to investigate the effects of focal application of anti-seizure agents on seizure activity in animals. Local perfusion of an anti-epileptic drug directly into the seizure focus in an animal model of epilepsy was effective in attenuating ictal and interictal events. Pioneering work in the development of a computer-controlled drug delivery system coupled with a seizure detection device is being conducted (Stein et al., 2000). The automated system employs a seizure-prediction algorithm that activates a programmable infusion pump to deliver a predetermined amount of anti-epileptic drug directly into the seizure focus. Animal studies show the ability of such a system to shorten seizure duration and prevent subsequent seizures. The ability to prevent progression of partial to generalized seizures would prove invaluable to many patients who suffer from intractable epilepsy. More powerful detection algorithms hold the potential to deliver anti-epileptic agents into the seizure focus before a seizure is clinically evident. Engineered drug reservoirs such as liposomes and nanoparticles have broad applications as delivery systems and are currently under investigation for use in delivering anti-epileptic drugs to the CNS and directly to the seizure focus. These inert carrier vehicles direct agents to a target tissue via ligand-receptor binding, theoretically increasing potency at the target site while decreasing toxicity in other tissues. The specific delivery and penetration parameters of unmodified carriers vary depending upon the lipid composition of the vehicle. In contrast, modified carriers are tagged with a ligand or receptor that functions to increase vehicle affinity for a specific cell type, thus enhancing drug delivery to the desired site of action. These tags include antibodies, hormones, cytokines, toxins and engineered ligands. A major obstacle in the development of a system of drug delivery to the CNS is penetration of the BBB. The cerebral capillary endothelium and astrocyte foot processes that comprise this protective barrier play an important role

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in maintaining a constant chemical environment and shielding neurons from toxic agents. Hydrophilic and large molecules are normally excluded from the CNS by this impermeable barrier. Liposomes are generally unable to penetrate normal endothelial pores (2 nm). The tight junctions and lack of pores that characterize the cerebral endothelium present a significant impediment to the vehicle-mediated delivery of drugs to the CNS. Deliberate chemical disruption of the BBB is already used to gain direct access to the CNS for the delivery of chemotherapy agents, and researchers are investigating the use of electrical disruption as a similar means of gaining direct CNS access [Orr, 2000]. It is known that seizures disrupt the BBB, which holds interesting implications for the focal delivery of an anti-epileptic drug immediately preceding or at the onset of a seizure. Moreover, the methods of direct CNS delivery of anti-epileptic drugs may also apply to the delivery of cell and gene therapy agents. An alternative approach to disruptive penetration of the CNS proposes the use of active targeting of transport vectors to circumvent the protective nature of the BBB. Endogenous peptides, modified proteins, and monoclonal antibodies can be used to transport large, water-soluble molecules across the BBB and deliver therapeutic agents directly to targets in the CNS. A recent study demonstrated the CNS delivery of systemically administered vasoactive intestinal peptide coupled to a monoclonal antibody [Bikel et al., 2001]. The use of this approach to gain access across the BBB will depend upon the identification of appropriate targets within the CNS. Its value as an effective method of drug delivery in epilepsy will require the characterization of the seizure focus and the discovery of common targets. As researchers continue to elucidate the cellular and molecular nature of epileptogenic brain the prospects for development of effective drug delivery strategies to treat epilepsy will continue to improve. The use of polymers to deliver anti-epileptic drugs directly to the seizure focus is another method that has potential for seizure control in intractable epilepsy. A polymer is a complex of drug in a dissolvable matrix. As the matrix dissolves, drug is released into the immediate area. Polymers can be engineered to vary dissolution rates in response to changes in the chemical environment. The use of polymers as a strategy in drug delivery is being employed in the treatment of recurrent glioblastoma multiforme. A polymer composed of an alkylating agent is implanted in the tumor bed following resection and slowly releases the chemotherapy agent directly into the malignant tissue. The use of polymers in anti-epileptic drug delivery to the brain has been investigated in animal models and was shown to decrease or attenuate seizure activity [22]. While this promising delivery strategy is still in development, the potential requirement of multiple craniotomies may make its use impractical.

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As the cellular and molecular mechanisms underlying epilepsy are elucidated, it has become apparent that traditional strategies of pharmacotherapy will continue to fail until we fully appreciate the complex interplay of multiple factors that influence the development of treatment-resistant epilepsy. Innovative methods of enhanced drug delivery do indeed hold promise for effective treatment of intractable epilepsy, but even direct delivery of antiepileptic agents into a seizure focus does not attempt to modulate the fundamental physiological and structural changes that characterize epileptogenic brain. While the development of novel drug delivery systems is primarily directed towards improving the pharmacological treatment of epilepsy, these strategies have been extended to the delivery of cell and gene therapy agents in the hope of ultimately curing this multifaceted disease.

Current Alternatives to Pharmacotherapy

When pharmacotherapy fails to adequately control seizures there are a variety of adjunctive or alternative therapies available. While some may be considered extreme, in the patient with intractable epilepsy these nonpharmacological therapies offer the only possibility of a life free from seizures. Noninvasive adjunctive therapy is limited to the ketogenic diet. The options for invasive treatment of epilepsy are varied and include conventional surgical techniques, multiple subpial transection, implantation of a vagus nerve stimulator, gamma knife radiosurgery, and implantation of depth electrodes for deep brain stimulation. Dietary Treatment of Epilepsy The ketogenic diet has been utilized in the adjunctive treatment of refractory epilepsy for over 75 years. Its pattern of use in treating intractable seizures has varied and we are currently experiencing resurgence in the use of the ketogenic diet to control seizures that are resistant to pharmacotherapeutic strategies. The high-fat, low-protein, and low-carbohydrate diet produces a ketotic state similar to that seen in starvation. The diet has been evaluated using the MES and PET infusion models in a manner similar to anti-epileptic drugs. It has been shown that ketosis functions to increase the seizure threshold, but there is no evidence that it lessens the severity of seizures. The exact mechanisms whereby a ketotic state produces an anti-epileptic effect are unclear. Ketosis causes changes in overall brain energy metabolism, cell membrane lipid composition, localized cerebral pH, and brain water content, though the extent to which any one or a combination of these alterations influences seizures is not certain. The results of recent work in the biochemical basis of the ketotic effect suggest that alterations in brain excitatory amino acid metabolism

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may be primarily responsible for the anti-seizure property of the ketogenic diet. Altered metabolism has the overall effect of shifting neuronal amino acid equilibrium to favor glutamate production at the expense of aspartate production, enhance synthesis of GABA from the increased pool of glutamate, increase removal of glutamate from the synaptic cleft, and enhance release of glutamate in the form of glutamine into the vascular space. The ultimate result of these biochemical alterations is the ability to attenuate and possibly terminate the development of seizures. Some reports indicate the ketogenic diet to be at least as effective as the anti-epileptic drugs in controlling certain seizure types, and even exceeds pharmacotherapeutic efficacy in some cases. Studies of efficacy vary and indicate that 14–46% of children on the diet experience a greater than 50% reduction in seizure frequency and 7–54% of children achieve complete seizure control [23]. While this can represent a significant improvement in seizure activity, in practice it seldom offers freedom from the burden of daily debilitating seizures. What the ketogenic diet does offer is a new paradigm to think about the molecular mechanisms of epilepsy. A change in the primary metabolic substrate in the brain from glucose to ketones appears to alter the seizure threshold in one area of brain without affecting the normal global function throughout. The ketogenic diet has utility in treating seizures of multiple types and various etiologies. This lends to the theory that there are biochemical pathways common to the different seizure types and etiologies that make diverse seizure disorders responsive to a single therapy. Elucidation of the fundamental changes in cerebral energy metabolism that underlie the development of seizures holds major implications for the development of successful therapy for epilepsy. Surgery: A Cure for Epilepsy? For over a century, the surgical resection of epileptogenic lesions has proven curative in certain types of epilepsy. At present, it remains the only true cure for certain subtypes of this devastating disorder, but remains significantly underutilized. The remarkable advances in diagnostic tools available to evaluate the epileptogenic brain and localize seizure foci have enabled surgical intervention to play an increasingly important role in epilepsy therapy. The use of magnetic resonance imaging, PET and SPECT functional imaging, electrocorticography, and implanted depth electrodes allow better localization of seizure foci. Coupled with refined surgical techniques, localization-related refractory epilepsy may be particularly amenable to surgical therapy. While the standard of medical management of epilepsy defines intractable epilepsy as seizures that continue after 2 years of therapy with at least two first-line anti-epileptic drugs, it is not uncommon for the patient with refractory epilepsy to have been tried on numerous traditional and new

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generation anti-epileptic drugs in various combinations for more than 2 years. In the pediatric population there is an even greater reluctance to consider early surgical intervention, as the natural history of seizures in this population may be self-limited. The natural history of epilepsy is dependent upon several factors, including the clinical presentation, epilepsy syndrome, and etiology of epilepsy. The importance of recognizing self-limited disorders from those that are truly refractory becomes evident when one considers the cognitive and psychosocial consequences of intractable epilepsy. The ability to reduce the high morbidity that accompanies both temporal- and extra-temporal localization-related epilepsy provides a strong argument for early surgical intervention in children with intractable seizures of these etiologies. There are also nonlocalized epilepsy syndromes with severe seizures in which surgical intervention can have a significant impact on the progression of the devastating neurodevelopmental consequences. The capacity to actually affect the course of progression of a seizure syndrome is of considerable importance in the surgical therapy of intractable epilepsy. Anti-epileptic drugs may be of great benefit in controlling the seizures associated with a particular disorder, but they cannot offer any advantage in altering the natural history of the epilepsy. A 30–35% recurrence rate of seizures after the discontinuation of an anti-epileptic drug is not uncommon, while surgical therapy for certain seizure disorders can reduce or completely eliminate seizures in 70–90% of treated patients [24]. Surgical treatment of epilepsy is often considered radical and carries known risks. A rigorous presurgical evaluation is essential to identify those patients that will benefit from surgical intervention. Anatomical or functional hemispherectomy is generally reserved for patients with progressive devastating nonlocalizing epilepsies who are not candidates for a localized resection. If accomplished early enough, neuronal plasticity will allow for some recovery of function in selected areas. As previously indicated, this intervention can have a significant affect on the progression of neurodevelopmental consequences. Corpus callosotomy has benefit in treating frequent intractable drop attacks from both tonic and atonic seizures. Surgical intervention in this setting cannot confer freedom from seizures and is instead aimed at improving quality of life. Focal temporal cortical resection is effectively curative in appropriately selected patients with intractable complex partial seizures originating in the temporal lobe. Multiple surgical techniques have been employed, from amygdalohippocampectomy to en bloc resection. Focal extratemporal cortical resection is the most commonly employed surgical intervention in children. Presurgical evaluation is complicated in this population as the seizure focus is generally more difficult to localize in extratemporal as compared to temporal lobe epilepsy, and the focus may be located in a silent region of the brain and

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clinically apparent only after spread to adjacent areas. In patients with a seizure focus either completely or partially localized to eloquent cortex multiple subpial transection allows for the surgical manipulation of epileptogenic cortex without significant neurological deficit. The procedure is designed to disrupt horizontal neuronal connections without interrupting efferent cortical fibers. The successful surgical treatment of epilepsy depends on the identification of the appropriate surgical candidate. In all cases, early identification of suitable syndromes and timely surgical intervention offers the best possibility for a normal life. In the future, perhaps, surgical intervention in epilepsy will not be limited to removal of epileptogenic brain or mechanical disruption of neural connections, but will instead be utilized in the cellular and genetic therapy of epilepsy to fundamentally alter the pathological processes underlying this debilitating disorder. Vagus Nerve Stimulation In 1997, a new treatment modality for intractable epilepsy was introduced. Vagus nerve stimulation is FDA-approved for the adjunctive therapy of refractory partial seizures. Patients who are not candidates for epilepsy surgery or elect not to undergo intracranial surgery may benefit from the subpectoral implantation of a programmable pulse generator and left mid-cervical vagus nerve electrodes for continuous cyclical stimulation. Studies evaluating the mechanism of action of vagus nerve stimulation indicate it functions via immediate and long-term effects. Short-term changes in the nucleus of the solitary tract and its connections cause synchronization and desynchronization of electrical activity in the brain [Magnes et al., 1961; Peñaloza, 1964; Chase et al., 1967]. The solitary tract nucleus has projections to the parabranchial nucleus, hypothalamus, amygdala, infralimbic cortex, ventroposterior, intralaminar, and midline thalamic nuclei, insular cortex, dorsal raphe, and nucleus ambiguous. Long-term changes in cerebral blood flow and neurotransmitter concentrations have been documented [25, 26]. Increased noradrenergic and serotoninergic activity, which functions to increase the seizure threshold, has also been hypothesized to play a role [27]. Blinded randomized controlled trials of efficacy of vagus nerve stimulation are methodologically difficult, but longitudinal studies demonstrate a mean reduction in seizure frequency of 22–48%, which varies with intensity of stimulation, with some patients experiencing >75% reduction in seizures [28]. While this may represent a significant improvement in seizure status for an individual, vagus nerve stimulation does not confer complete seizure control. A novel approach to vagus nerve stimulation employs a transcutaneous stimulator for noninvasive therapy of refractory partial seizures [29].

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Gamma Knife Radiosurgery Advances in our understanding of the molecular mechanisms of epilepsy coupled with an increasing appreciation for the role of surgical treatment of intractable seizures has lead to the application of a variety of novel surgical techniques for control of seizures. Gamma knife radiosurgery allows for the precise irradiation of a specific target with minimal radiation effects on surrounding tissue. It is commonly used in the ablation of arteriovenous malformations and neoplastic lesions. In the early 1980s animal experiments established the role of ionizing radiation in restricting the spread of discharges in the epileptic brain. A role for radiosurgery in epilepsy therapy was first noted in a series of patients who underwent gamma knife surgery for the treatment of cerebral arteriovenous malformations and showed a concomitant improvement of seizures. Complete seizure control can be achieved after radiosurgery treatment of a lesion with seizures at presentation, and a significant decrease in seizure frequency can be seen in adults with low-grade astrocytoma and intractable epilepsy following conformal radiotherapy. Brachytherapy and conventional radiotherapy for low grade tumors with refractory seizures can also have a significant impact on seizure frequency. The success of gamma knife treatment of seizures associated with mass lesions essentially introduced the possibility of radiosurgery as effective therapy for focal epilepsy. Less than a decade ago the first patient with intractable mesial temporal lobe epilepsy (MTLE) was treated with gamma knife entorhinoamygdalohippocampectomy. Since then, studies evaluating radiosurgery instead of microsurgery for MTLE indicate it is an efficacious and safe treatment option that can reduce the morbidity associated with invasive surgical intervention [30]. The mechanism of action of gamma knife surgery is largely unknown. Computed tomography and MRI imaging show radiation-induced structural changes in the mesial temporal lobe, but the significance of these findings is unclear. Clinical studies of gamma knife surgery suggest improvement of seizures may represent an actual anti-epileptic effect independent of structural alterations. Using animal models it has been demonstrated that non-necrotizing doses of irradiation can improve seizures [31] and the anti-epileptic effect increases with increasing doses [32]. Biochemically, it is theorized that radiation inhibits protein synthesis, thus preventing maintenance of spontaneous bursting in neurons [33], and has a differential effect on the inhibitory GABA system and the excitatory amino acid system [31]. Developments in the techniques for noninvasive surgery provide novel treatment options for intractable epilepsy. Gamma knife radiosurgery offers the possibility of effective treatment for certain types of refractory seizures with minimal impact on normal brain function.

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Deep Brain Stimulation Brain stimulation is a novel therapeutic strategy for intractable epilepsy with the potential to effectively control seizures associated with certain epilepsy types in patients who are not candidates for surgical resection. Neuromodulation of brain structures by electrical stimulation has been used to treat neurological disorders of movement and chronic pain. In the treatment of Parkinson’s disease, high frequency stimulation of the thalamus, pallidum, and subthalamic nucleus produces the same effect as ablative neurosurgery. Previous attempts to influence seizure activity by electrical stimulation of deep brain structures have targeted the caudate nucleus, anterior thalamus, centromedian thalamic nucleus, posterior hypothalamus, and hippocampus. Results have been varied and limited by study design. Recent attention to deep brain stimulation in epilepsy therapy is focusing on the subthalamic nucleus as a target for stimulation. While the inhibitory effect of high-frequency stimulation of the subthalamic nucleus on the substantia nigra was initially based on the theory that electrical stimulation inhibits function, several studies provide evidence for the molecular mechanisms that underlie inhibition via electrical stimulation. High-frequency stimulation of subthalamic neurons has been shown to produce a long-lasting blockade of depolarization of voltage-gated channels [34]. Both spontaneous and induced epileptiform activity has been reduced or terminated by high-frequency cortical, subthalamic, and hippocampal stimulation, and this inhibition of activity occurs when neurons are depolarized [35]. There is also evidence that high-frequency stimulation may activate inhibitory GABAergic circuits in the basal ganglia and inhibit postsynaptic activity in the subthalamic nucleus [36, 37]. Direct inhibition of deep brain structures may not be the only effect of electrical stimulation. An excitatory effect of high-frequency stimulation has also been supported. Functional imaging provides evidence for activation of stimulated structures. Neurophysiological studies indicate the findings from microelectrode recordings of stimulated structures are inconsistent with the hypothesis that high-frequency stimulation inhibits the target structure [38, 39]. Stimulation of deep brain structures may also affect cortical activity through anti-dromic connections. Using animal models, stimulation of the subthalamic nucleus has demonstrated retrograde activation of the corticosubthalamic pathway, a major afferent projection to the subthalamic nucleus, evidenced by measurable cortical potentials [40, 41]. Preliminary studies in patients with epilepsy demonstrated evoked cortical potentials after subthalamic nucleus stimulation [38]. It is unclear precisely how retrograde cortical activation could suppress seizure activity. Anti-dromic activation of cortical interneurons may be the mechanism whereby cortical excitability is inhibited. Computer models of high-frequency stimulation suggest simultaneous neuronal excitation and

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inhibition may mediate the therapeutic effects of subthalamic nucleus stimulation in epilepsy [42]. Stimulation of the subthalamic nucleus in the treatment of certain intractable epilepsy types in humans has been successful in reducing seizures. Modulation of glutamatergic subthalamic output may influence cortical excitability by inactivation of the nigral control system as well as activation of cortical GABAergic inhibition, resulting in suppression of seizure activity. A greater understanding of the basic principles by which stimulation of deep brain structures can influence the endogenous control systems in the brain and modulate cortical excitability will promote the application of deep brain stimulation to certain intractable epilepsies.

From Drug Delivery to Gene Delivery

Novel drug delivery strategies are being applied to the cellular and genetic treatment of epilepsy. Cellular transplantation and delivery of genetic material hold the potential to not only effectively treat intractable seizures, but also offer the possibility of altering the fundamental defects that result in epilepsy. Cellular Therapy for Epilepsy Cell transplantation or replacement theoretically offers the possibility of providing a continuous endogenous supply of a deficient neuromodulator to a localized area of brain, essentially enabling the restoration of functional neuronal connections. The feasibility of this innovative approach to treating pharmacoresistant epilepsy has already been demonstrated. The intraventricular grafting of an adenosine-releasing synthetic polymer in an animal model of epilepsy was shown to significantly decrease seizure activity [43]. As an endogenous inhibitory neuromodulator, adenosine has the potential to influence the development of synaptic connections and alter the balance of excitation and inhibition in the epileptogenic brain. This pioneering work has important implications for cellular therapy of epilepsy. The grafting of stem cells or free cells from another species has the potential to establish operative neural circuits in areas of the brain that are intrinsically defective. Several studies have investigated cell transplantation in animal models of epilepsy. Neuronal grafts from fetal rats have been transplanted into rats modeling temporal lobe epilepsy and amygdala-kindled rats and demonstrated the ability to restore GABAergic interneuron connections and reduce epileptiform after-discharges and clinical seizures, respectively [44–46]. More recently, fibroblasts engineered to release adenosine and encapsulated into polymers were grafted into the ventricles of kindled rats. The grafted rats demonstrated a

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significant reduction in kindled seizure activity and significant suppression of after-discharges [47]. The transplantation of neuronal grafts as a therapeutic strategy in epilepsy holds tremendous promise for effectively treating this disorder. Currently, one of the major limitations of such a therapy is immunological rejection. As the brain is an immunologically privileged site, a host-versus-graft immunoreaction to transplanted cells can occur. The ability to utilize immature cells that are immunologically neutral is one possibility in circumventing this problem. Continued research in the areas of transplantation and neural stem cells is crucial and will undoubtedly lead to advances in the application of this treatment option. Strategies in Gene Therapy for Epilepsy Many concepts in drug delivery and cell therapy can be applied to gene therapy for epilepsy. What gene therapy offers as an epilepsy treatment strategy is the possibility of correcting the defect that underlies epileptogenic brain and essentially curing epilepsy. Just as we have adjusted our view of the pathogenesis of epilepsy to encompass a complex multifactorial etiology with genetic influence, we must also revise our approach to the treatment of epilepsy to include novel concepts in genetic therapy. The use of animal models in the study of the molecular mechanisms that underlie hyperexcitability and epileptogenesis has contributed significantly to our understanding of the genetic basis of epilepsy. Spontaneous epileptic mutants involving both mono- and polygenic inheritance allow researchers to progress from phenotype to genotype and identify many of the genes involved in the development of cortical hyperexcitability. The use of engineered transgenic mouse models permits a genotype to phenotype approach that can enable the elucidation of the critical steps in epileptogenesis and function in the systematic testing of pharmacological therapies in epilepsy. Through the use of genetic animal models we have gained tremendous insight into the genetic abnormalities that can influence the intrinsic excitability of epileptogenic brain, the mechanisms of altered synaptic transmission, and disruptions in neural networks. An estimated 40–50% of epilepsy and epilepsy syndromes are considered idiopathic and presumed to have a genetic basis [1]. Most of the epilepsy syndromes are not likely to be the result of a single genetic defect, but the outcome of multiple factors, including a genetic abnormality. Researchers have identified a number of genes with known causative roles in the pathogenesis of certain epilepsy syndromes. Many of these syndromes are associated with metabolic derangements or neurodegenerative disorders. The genetic defects in several of the progressive myoclonus epilepsy syndromes have already been elucidated. Unverricht-Lundoborg disease, also known as progressive myoclonic epilepsy type I, is due to truncation or unstable

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insertion in the gene encoding the protease cystatin B and MERRF, or myoclonic epilepsy associated with ragged-red fibers, results from defects in mitochondrial DNA. The genetic mechanisms underlying some of the familial epilepsies have also been identified, including autosomal dominant nocturnal frontal lobe epilepsy, benign familial neonatal convulsions, generalized epilepsy with febrile seizures, and episodic ataxia with partial epilepsy. In all of the familial epilepsies, the identified genes encode for entire cation channels or their subunits, further evidence that channelopathies may have an essential role in the development of seizure activity and progression to epilepsy. Continued research in the molecular basis of the epilepsies will undoubtedly elucidate additional mechanisms whereby genetic defects lend to the development of epilepsy. Elucidating the genetic mechanism underlying an epileptic disorder provides for the possibility of effective treatment by replacing either the defective DNA or abnormal protein product. Autosomal dominant nocturnal frontal lobe epilepsy is due to a genetic defect that results in a dysfunctional neuronal nicotinic acetylcholine receptor ␣4 subunit. Knowledge of this defect offers the possibility of a genetic therapy that could restore the CHRNA4 gene or replace the abnormal receptor subunit. In the most prevalent epilepsies where a single genetic defect is not the known etiology, gene therapy theoretically remains a reasonable treatment option. Similar to cellular therapy, genetic therapy in the setting of an unknown gene defect may prove therapeutic if utilized as a modality of drug treatment. The introduction of genetic material into the brain in this setting cannot correct underlying pathology, but it can provide a continuous source of neurotransmitter to normalize the balance between excitation and inhibition. Strategies in gene therapy have been applied to the treatment of cerebral neoplasia, neurodegenerative disorders, lysosomal storage disease, Parkinson’s disease, and stroke with varying success. As a therapeutic approach in epilepsy, gene therapy is an area of investigation still in its infancy. The successful delivery of genetic material into the brain is based on strategies borrowed from novel approaches used in drug delivery and cellular therapy. Penetration of the BBB remains a formidable challenge, but many of the strategies utilized in drug delivery, such as the coupling of modified proteins or monoclonal antibodies to transport vectors, as previously discussed, can be applied to the delivery of DNA. There are several methods whereby DNA can be delivered to cells in the brain. Both ex vivo and in vivo approaches to gene therapy are utilized in the delivery of genetic material to the CNS. Ex vivo methods are characterized by the introduction of transgenes into cells that are then grafted into the brain, while in vivo methods employ vectors to introduce transgenes directly

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into cells in the brain. The use of viral vectors and the development of engineered vectors, such as plasmids and peptide vehicles, represent the variety of techniques whereby genetic material is delivered to the CNS. Herpes simplex virus (HSV), adenovirus, adeno-associated virus (AAV), retrovirus, and lentivirus are the commonly used viral vectors in gene therapy. The neurotrophic nature of HSV makes it a good candidate for delivery of genetic material to the brain, but its long latency period and resultant transient expression of gene products presents the major obstacle to its effective use as a genetic vector. The therapeutic use of HSV as a genetic vector has been demonstrated in rodent models of neuroprotection in focal cerebral ischemia. The use of an engineered gene that encodes a herpes simplex enzyme designed to activate a prodrug, such as herpes simplex thymidine kinase, is another strategy in gene therapy utilizing the HSV. Adenovirus vectors are also in use in experimental models of gene therapy. Adenovirus is known to infect both dividing and nondividing cells, which permits its use in both rapidly dividing malignant brain tumors and neurological disorders of the postmitotic CNS, such as Parkinson’s disease and epileptic disorders that generally do not exhibit cell division. AAV shows great promise for effective use in the delivery of genetic material to the brain. In vivo studies utilizing recombinant AAV vectors have demonstrated long-term gene expression without evidence of infection or immune response, and in a mouse model of traumatic brain injury recombinant AAV was successfully delivered to the hippocampus. The feasibility of vector-mediated gene transfer into the epileptogenic brain has been demonstrated in both rats and humans. For example, the tremor rat is a genetic mutant that exhibits absence-like seizures and is used as a model of inherited epilepsy. This rat is now known to be an animal model for Canavan disease. A deletion of the aspartoacylase gene has been discovered in these animals and the resultant high levels of N-acetyl-aspartate are understood to be responsible for the epileptic seizures in tremor rats. Recent studies utilizing the intraventricular administration of a recombinant adenovirus carrying the rat aspartoacylase gene demonstrated significant inhibition of the generation of absence-like seizures in experimental animals [48]. In human studies, the effective use of viral vectors to mediate the transfer of genes into human epileptogenic brain slices has been shown [49]. Nonviral methods of delivery of genetic material to the brain offer an advantage over viral vectors, which can be limited by inadequate brain penetration and ineffective cell transfection. The use of liposome-packaged plasmids conjugated to a monoclonal antibody has been shown to successfully cross the BBB and access the microvasculature and parenchyma of the brain. Viral genetic material can also be packaged into liposomes, termed virosomes,

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for delivery to the brain. Virosomes containing AAV plasmids have been delivered into the ventricles in both primates and humans. Cationic vectors, such as lipospermines and polyethylenimine, can also be used to facilitate the transport of genetic material into the brain. The current body of research in genetic therapy demonstrates the potential for effective treatment of certain epilepsy disorders using a gene therapy approach. The transfer of genetic material into neurons in a seizure focus and the expression of inhibitory neurotransmitters or neuropeptides, membrane transporters, postsynaptic receptor subunits, or antisense sequences provide for the possibility of altering the path of signal transduction and inhibiting the initiation, propagation, or maintenance of seizure activity. Gene therapy has the potential to influence epileptogenesis due to a variety of causes, including neurological injury, defective ion channels, or altered levels of neurotransmitter or receptors. Further development of viral vectors that can be used to transfer therapeutic genes offers the hope of a cure for certain epilepsy disorders. Other avenues of investigation must focus on delivery of transgenes to the target tissue. The stereotaxic procedures of molecular neurosurgery provide a powerful method of delivery of genetic material to cerebral tissue. Currently, stereotaxic techniques are applied to neuroablative, neuroaugmentative, and neuroendoscopic procedures, as well as radiation dosing, anatomical-physiological correlation in neuroimaging, tumor biopsy and resective therapy, and restorative surgical therapy. Both point-in-space and volumetric techniques are utilized in stereotaxic procedures, but volumetric stereotaxis provides many advantages in molecular neurosurgery including localization of a target structure, conceptualization of the three-dimensional shape of a target structure, preoperative planning of surgical approach and trajectory, and positional differentiation of target and adjacent tissue. A stereotaxic neurosurgical approach to genetic therapy in epilepsy can be utilized to deliver a transgene-containing viral vector or genetically engineered cells to a seizure focus. When a focal seizure origin cannot be identified the global delivery of genetic material via the endovascular system can be accomplished using stereotaxic intraventricular or intraparenchymal injection, interstitial infusion, or catheter-mediated delivery of transgenic vectors. As the molecular mechanisms of the epilepsies are elucidated, it is becoming apparent that an aggressive approach to gene therapy in epilepsy must be pursued. The delivery of genetic material to cerebral tissue offers a therapeutic strategy that can alter the pathological basis underlying epileptogenesis in the human brain. The possibility of a cure for certain epilepsy disorders is on the horizon and we must continue to pursue innovative and novel approaches to gene therapy for this devastating neurological disorder.

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RNA Expression Profiling: Pharmacogenomics and Disease ‘Fingerprinting’

The analysis of mRNA expression in individual cells provides a strategy to compare the transcriptional profile of individual phenotypically characterized neurons. This approach has been implemented in human and experimental epilepsy models and in live as well as fixed cell types. The use of an oligo-dT primer and T7 RNA polymerase permits amplification of a broad population of expressed genes across many gene families. The size range and complexity of the amplified mRNA provides a comprehensive view of differential gene expression in single cells. Individual cell differences in gene expression could be used to develop new targets for epilepsy pharmacotherapy, ‘personalize’ treatment with existing drugs, or ‘fingerprint’ individuals for disease diagnostics. The concept of ‘personalized medicine’ is now within reach due to the landmark innovation of the biochip and the wealth of information created by the Human Genome Project. The massive amount of genomic information generated by sequencing efforts could only be exploited by using complex bioinformatics tools to comprehensively analyze systems at the DNA, RNA, and protein level. These bioinformatics tools together with the data available from RNA expression profiling using DNA chips has led to the comprehensive analyses of individual clinical samples in an attempt to describe disease and disease risk at the molecular level and integrate data to facilitate clinical decision making. Pharmacogenomics aims to optimize patient management by customizing and synthesizing drugs based on genetic variations in drug response. Its thrust is based on genome-based rational therapeutics that addresses interindividual variations or polymorphisms affecting metabolism, receptors, and absorption that can influence drug sensitivity, toxicity, and dosing. Potential benefits of pharmacogenomics include increasing efficacy and preventing adverse drug reactions, thus improving patient care and decreasing costs. All of these technologies are not yet in current clinical use and it is also too early to decide whether molecular ‘fingerprints’ or genomic profiles will have the diagnostic and prognostic power currently predicted. One approach that we have taken to investigate new targets for epilepsy pharmacotherapy has been to profile dentate gyrus granule cells from human epilepsy tissue. Mesial temporal lobe sclerosis is a common pathological finding in patients with medically intractable temporal lobe epilepsy. This disease is characterized by extensive cell loss in the hilus and the hippocampal CA1 and CA3 cell fields in addition to synaptic reorganization throughout the dentate gyrus. The dentate granule cells from hippocampal slices of patients diagnosed with medial temporal lobe sclerosis exhibit reduced synaptic inhibition with concomitant hyperexcitability. These physiological changes have been studied

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Control human dentate gyrus

a Epileptic human dentate gyrus

b GABA␣1 GABA␤1 NMDA1a GAD65 GluR1 GLT-1 HES S49

GABA␣2 GABA␤2 NMDA2a GAD67 GluR2 Netrin-1 INX EAAC1

GABA␣3 GABA␤3 NMDA2b GFAP GluR4 Netrin-2 CREB c-fos

GABA␣4 GABA␥2 NMDA2c pBS GluR5 Nestin OTX-1 c-jun

GABA␣5 GABA␥3 NMDA2d ␤-Actin GluR6 BF1 ARC AChE

GABA␣6 NOS VGAT GABA-T GluR7 BF2 4B2 NCAM

c Fig. 1. Radiolabeled amplified aRNAs are used as probes of small scale cDNA arrays containing candidate genes of interest. For each 32P-labelled aRNA from a cell, duplicated slot blots were used for each hybridization reaction. An mRNA expression profile could then be obtained. a Expression profile for autopsy control dentate gyrus granule cell. b Expression profile for medial temporal lobe epilepsy dentate gyrus granule cell. c Candidate genes and controls used.

relative to the hippocampi of patients with temporal lobe tumors in which the cell loss and synaptic reorganization are not seen. The synaptic reorganization of both excitatory and inhibitory systems in the dentate gyrus of the hippocampus may be an important mechanism that contributes to chronic limbic seizures. Of interest is the role of neurotransmitter receptors and their uptake sites in the generation of seizures in MTLE. Differences in gene expression in temporal lobe epilepsy have been reported from investigations on surgically removed hippocampi implicating an up-regulation in the expression of excitatory neurotransmitter receptor genes in a role in epileptogenesis. Increases in mGluR1 (Mathern 97, 98; Lynd 96; Blumcke 00), mGluR2 (Blumcke 96), NMDAR2a (Mathern 99), NMDAR2b

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Fig. 2. Harvesting single cells. Human hippocampi fixed in paraformaldehyde were stained with TA51, an antibody for neurofilament. First round synthesis of cDNA was done on the histologically sectioned tissue. Single cells were identified and removed from the dentate gyrus granule cell layer. A glass microelectrode is shown moving in to harvest a single granule cell. Conversion to double-stranded cDNA, then amplification of this cDNA as radiolabeled antisense mRNA was then performed.

(Mathern 99, 98) mRNA expression have all been reported in the dentate gyrus from hippocampal surgical specimens. These findings support the hypothesis that changes in hippocampal circuitry alter the postsynaptic gene expression in a way that contributes to chronic seizure. Our strategy has been to remove individual dentate gyrus granule cells from fixed specimens (fig. 1) of surgically removed hippocampi from patients with MTLE and autopsy hippocampi, stained with TA51, an antibody for neurofilament. Radiolabeled aRNA from these cells was used to probe cDNA arrays containing the GABAA ␣1–6, and ␤1–3 receptor subunits, mGluR1–6, NMDAR 1A-B, NMDAR2A-D receptor subunits, GAD65, GAD67, and VGAT. The relative intensity of each mRNA-cDNA hybrid is then quantified (fig. 2). Selective differences can be found at the level of gene expression in hippocampal dentate gyrus granule cells from MTLE patients compared to nonseizure autopsy controls. Reduced transcription of select receptors and increased expression of other subunits in MTLE may contribute to epileptogenesis. Although select differences in mRNA expression can be found in human epilepsy tissue, it is the level of functional receptor protein, and any associated regulatory component, which will determine the functional significance of these findings. Routine biochemical analysis (e.g., Western blots) cannot be

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performed at the single cell level in order to determine how protein expression correlates with mRNA expression. Immunohistochemistry is a more qualitative technique although it can be resolved at the single cell level. However, it cannot be used for more than two simultaneous antigens and is still not very specific for receptor subunits. It is likely, thus, that physiological or pharmacological analyses will be required to determine the functional significance of the expression differences. Despite these difficulties, ultimately, in order to understand epilepsy and develop highly targeted therapies, molecular characterization of individual neuronal cell types in critical areas of the involved CNS is likely to be necessary. References 1 2 3 4

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Hauser WA, Annegers JF, Kurland LT: Incidence of epilepsy and unprovoked seizures in Rochester, Minnesota: 1935–1984. Epilepsia 1993;34:453–468. Regesta G, Tanganelli P: Clinical aspects and biological bases of drug-resistant epilepsies. Epilepsy Res 1999;34:109–122. Macdonald RL, McLean MJ: Anticonvulsant drugs: Mechanisms of action. Adv Neurol 1986;44: 713–736. Mamiya K, Ieiri I, Shimamoto J, et al: The effects of genetic polymorphisms of CYP2C9 and CYP2C19 on phenytoin metabolism in Japanese adult patients with epilepsy: Studies in stereoselective hydroxylation and population pharmacokinetics. Epilepsia 1998;39:1317–1323. Loscher W, Potschka H: Role of multidrug transporters in pharmacoresistance to antiepileptic drugs. J Pharmacol Exp Ther 2002;301:7–14. Abbott NJ, Khan EU, Rollinson CM, et al: Drug resistance in epilepsy: The role of the blood-brain barrier. Novartis Found Symp 2002;243:38–47. Potschka H, Fedrowitz M, Loscher W: P-Glycoprotein-mediated efflux of phenobarbital, lamotrigine, and felbamate at the blood-brain barrier: Evidence from microdialysis experiments in rats. Neurosci Lett 2002;327:173–176. Sills GJ, Brodie MJ: Update on the mechanisms of action of antiepileptic drugs. Epileptic Disord 2001;3:165–172. Sisodiya SM, Lin WR, Harding BN, Squier MV, Thom M: Drug resistance in epilepsy: Expression of drug resistance proteins in common causes of refractory epilepsy. Brain 2002;125:22–31. Rizzi M, Caccia S, Guiso G, et al: Limbic seizures induce P-glycoprotein in rodent brain: Functional implications for pharmacoresistance. J Neurosci 2002;22:5833–5839. Mantegazza R, Bernasconi P, Baggi F, et al: Antibodies against GluR3 peptides are not specific for Rasmussen’s encephalitis but are also present in epilepsy patients with severe, early onset disease and intractable seizures. J Neuroimmunol 2002;131:179–185. Twyman RE, Gahring LC, Spiess J, Rogers SW: Glutamate receptor antibodies activate a subset of receptors and reveal an agonist binding site. Neuron 1995;14:755–762. He XP, Patel M, Whitney KD, Janumpalli S, Tenner A, McNamara JO: Glutamate receptor GluR3 antibodies and death of cortical cells. Neuron 1998;20:153–163. Andrews PI, McNamara JO: Rasmussen’s encephalitis: An autoimmune disorder? Curr Opin Neurobiol 1996;6:673–678. Leach JP, Chadwick DW, Miles JB, Hart IK: Improvement in adult-onset Rasmussen’s encephalitis with long-term immunomodulatory therapy. Neurology 1999;52:738–742. Villani F, Spreafico R, Farina L, et al: Positive response to immunomodulatory therapy in an adult patient with Rasmussen’s encephalitis. Neurology 2001;56:248–250. Levite M, Hermelin A: Autoimmunity to the glutamate receptor in mice – A model for Rasmussen’s encephalitis? J Autoimmun 1999;13:73–82.

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Levite M, Fleidervish IA, Schwarz A, Pelled D, Futerman AH: Autoantibodies to the glutamate receptor kill neurons via activation of the receptor ion channel. J Autoimmun 1999;13:61–72. Stutzmann JM, Mary V, Wahl F, Grosjean-Piot O, Uzan A, Pratt J: Neuroprotective profile of enoxaparin, a low molecular weight heparin, in in vivo models of cerebral ischemia or traumatic brain injury in rats: A review. CNS Drug Rev 2002;8:1–30. Westenbroek RE, Bausch SB, Lin RC, Franck JE, Noebels JL, Catterall WA: Upregulation of L-type Ca2⫹ channels in reactive astrocytes after brain injury, hypomyelination, and ischemia. J Neurosci 1998;18:2321–2334. Bialer M, Johannessen SI, Kupferberg HJ, Levy RH, Loiseau P, Perucca E: Progress report on new anti-epileptic drugs: A summary of the Fifth Eilat Conference (EILAT V). Epilepsy Res 2001;43: 11–58. Tamargo RJ, Rossell LA, Kossoff EH, Tyler BM, Ewend MG, Aryanpur JJ: The intracerebral administration of phenytoin using controlled-release polymers reduces experimental seizures in rats. Epilepsy Res 2002;48:145–155. DiMario FJ Jr, Holland J: The ketogenic diet: A review of the experience at Connecticut Children’s Medical Center. Pediatr Neurol 2002;26:288–292. Snead OC 3rd: Surgical treatment of medically refractory epilepsy in childhood. Brain Dev 2001;23:199–207. Henry TR, Votaw JR, Pennell PB, et al: Acute blood flow changes and efficacy of vagus nerve stimulation in partial epilepsy. Neurology 1999;52:1166–1173. Ben-Menachem E, Hamberger A, Hedner T, et al: Effects of vagus nerve stimulation on amino acids and other metabolites in the CSF of patients with partial seizures. Epilepsy Res 1995;20: 221–227. Rafael H, Moromizato P: Vagus stimulator for seizures. J Neurosurg 1993;79:636–637. Valencia I, Holder DL, Helmers SL, Madsen JR, Riviello JJ Jr: Vagus nerve stimulation in pediatric epilepsy: A review. Pediatr Neurol 2001;25:368–376. Ventureyra EC: Transcutaneous vagus nerve stimulation for partial onset seizure therapy. A new concept. Childs Nerv Syst 2000;16:101–102. Regis J, Bartolomei F, de Toffol B, et al: Gamma knife surgery for epilepsy related to hypothalamic hamartomas. Neurosurgery 2000;47:1343–1351; discussion 1351–1352. Regis J, Kerkerian-Legoff L, Rey M, et al: First biochemical evidence of differential functional effects following Gamma Knife surgery. Stereotact Funct Neurosurg 1996;66(suppl 1):29–38. Mori Y, Kondziolka D, Balzer J, et al: Effects of stereotactic radiosurgery on an animal model of hippocampal epilepsy. Neurosurgery 2000;46:157–165; discussion 165–168. Chalifoux R, Elisevich K: Effect of ionizing radiation on partial seizures attributable to malignant cerebral tumors. Stereotact Funct Neurosurg 1996;67:169–182. Beurrier C, Bioulac B, Audin J, Hammond C: High-frequency stimulation produces a transient blockade of voltage-gated currents in subthalamic neurons. J Neurophysiol 2001;85:1351–1356. Bikson M, Lian J, Hahn PJ, Stacey WC, Sciortino C, Durand DM: Suppression of epileptiform activity by high frequency sinusoidal fields in rat hippocampal slices. J Physiol 2001;531: 181–191. Iribe Y, Moore K, Pang KC, Tepper JM: Subthalamic stimulation-induced synaptic responses in substantia nigra pars compacta dopaminergic neurons in vitro. J Neurophysiol 1999;82:925–933. Kayyali H, Durand D: Effects of applied currents on epileptiform bursts in vitro. Exp Neurol 1991;113:249–254. Montgomery EB Jr, Baker KB: Mechanisms of deep brain stimulation and future technical developments. Neurol Res 2000;22:259–266. Dostrovsky JO: Immediate and long-term plasticity in human somatosensory thalamus and its involvement in phantom limbs. Pain 1999;suppl 6:S37–S43. Maurice N, Deniau JM, Glowinski J, Thierry AM: Relationships between the prefrontal cortex and the basal ganglia in the rat: Physiology of the corticosubthalamic circuits. J Neurosci 1998;18: 9539–9546. Maurice N, Deniau JM, Menetrey A, Glowinski J, Thierry AM: Prefrontal cortex-basal ganglia circuits in the rat: Involvement of ventral pallidum and subthalamic nucleus. Synapse 1998;29: 363–370.

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McIntyre CC, Grill WM: Selective microstimulation of central nervous system neurons. Ann Biomed Eng 2000;28:219–233. Boison D, Scheurer L, Tseng JL, Aebischer P, Mohler H: Seizure suppression in kindled rats by intraventricular grafting of an adenosine releasing synthetic polymer. Exp Neurol 1999;160: 164–174. Loscher W, Ebert U, Lehmann H, Rosenthal C, Nikkhah G: Seizure suppression in kindling epilepsy by grafts of fetal GABAergic neurons in rat substantia nigra. J Neurosci Res 1998;51: 196–209. Shetty AK, Turner DA: Fetal hippocampal grafts containing CA3 cells restore host hippocampal glutamate decarboxylase-positive interneuron numbers in a rat model of temporal lobe epilepsy. J Neurosci 2000;20:8788–8801. Shetty AK, Zaman V, Turner DA: Pattern of long-distance projections from fetal hippocampal field CA3 and CA1 cell grafts in lesioned CA3 of adult hippocampus follows intrinsic character of respective donor cells. Neuroscience 2000;99:243–255. Huber A, Padrun V, Deglon N, Aebischer P, Mohler H, Boison D: Grafts of adenosine-releasing cells suppress seizures in kindling epilepsy. Proc Natl Acad Sci USA 2001;98:7611–7616. Seki T, Matsubayashi H, Amano T, et al: Adenoviral gene transfer of aspartoacylase into the tremor rat, a genetic model of epilepsy, as a trial of gene therapy for inherited epileptic disorder. Neurosci Lett 2002;328:249–252. O’Connor WM, Davidson BL, Kaplitt MG, et al: Adenovirus vector-mediated gene transfer into human epileptogenic brain slices: Prospects for gene therapy in epilepsy. Exp Neurol 1997;148: 167–178.

Albert Telfeian, MD, PhD Neurosurgical Associates, LLP 3601 21st Street, Lubbock, TX 79410 (USA) Tel. ⫹1 806 797 2222, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 202–212

Emerging Treatment of Neurometabolic Disorders Roscoe O. Brady, Roscoe O. Brady, Jr. Developmental and Metabolic Neurology Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, Md., USA

Introduction

Metabolic disorders are caused by mutations in genes that result in harmful reductions of catalytic activity of any of the enzymes required for the normal ‘housekeeping’ functions of cells. Many of these conditions are characterized by the accumulation of deleterious amounts of nondegraded materials within lysosomes, and are commonly known as ‘lysosomal storage disorders.’ Lysosomes are subcellular organelles which contain a plethora of enzymes that are necessary for the biodegradation of subcellular materials. These enzymes are preferentially active under acidic conditions that are characteristic of the intraluminal milieu of lysosomes. Substances that undergo lysosomal biodegradation include glycogen, mucopolysaccharides, and the major class of lipids called sphingolipids. Because approaches to enzyme replacement therapy (ERT) and gene therapy are particularly advanced in the sphingolipid storage disorders, we shall limit this chapter primarily to considerations of these conditions, although the basic techniques of gene and ERT have wide applicability to many metabolic disorders of the brain. We shall indicate briefly what has been accomplished to date, and then describe approaches that we believe will be required for the effective treatment of patients in which neurological involvement is a prominent cause of morbidity and mortality.

Background

Sphingolipids have a portion of their common structure comprised of the long chain amino alcohol sphingosine (fig. 1a). In these lipids, a long-chain fatty acid

a

Sphingosine CH3-(CH2)12-CH⫽CH-CH-CH-CH2OH OH NH2 D-erythro-trans-2-amino-4-octadecene-1,3-diol

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Ceramide Sphingosine CH3-(CH2)12-CH⫽CH-CH-CH-CH2OH OH NH CH3-(CH2)22-C⫽O Fatty acid

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Glucocerebroside Sphingosine-Glucose Fatty acid

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Sphingomyelin Sphingosine-Phosphocholine Fatty acid

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Globotriaosylceramide [Ceramidetrihexoside (GB3)] Sphingosine-Glucose-Galactose-Galactose Fatty acid

f

Ganglioside GM2 Sphingosine-Glucose-Galactose-N-acetylgalactosamine Fatty acid

N-acetylneuraminic acid

Ganglioside GM1

g

Sphingosine-Glucose-Galactose-N-acetylgalactosamine-Galactose Fatty acid

N-acetylneuraminic acid

Fig. 1. Structures of pertinent sphingolipids. a Sphingosine; b Ceramide; c Glucocerebroside; d Sphingomyelin; e Globotriaosylceramide; f Ganglioside GM2; g Ganglioside GM1.

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is linked to the nitrogen atom covalently bound to carbon atom two of sphingosine, forming a moiety known as ceramide (fig. 1b). In all of the sphingolipid storage disorders (except Farber’s disease) in which ceramide itself is the principal accumulating substance, additional components are linked to the oxygen on carbon one of the sphingosine moiety of ceramide. The most prevalent metabolic storage disorder of humans is Gaucher’s disease. Here, the principal accumulating substance is glucocerebroside, comprised of ceramide to which a single molecule of glucose is linked by a ␤-glycosidic bond (fig. 1c). Another common sphingolipid storage disorder is Niemann-Pick’s disease. Here, the accumulating material is sphingomyelin (fig. 1d). Still another prominent condition is Fabry’s disease in which globotriaosylceramide (ceramidetrihexoside)(Gb3) accumulates in many organs (fig. 1e). Of particular significance to neuroscientists are Tay-Sach’s disease in which the ganglioside GM2 accumulates (fig. 1f ) and generalized gangliosidosis in which ganglioside GM1 is the major accumulating metabolite (fig. 1g). The nature of the metabolic abnormalities in the sphingolipid storage disorders was established 38 years ago by Brady et al. [1, 2] with the demonstration that the enzymatic defect in Gaucher’s disease was the insufficient activity of glucocerebrosidase, the enzyme that catalyzes the hydrolytic cleavage of glucose from glucocerebroside. There are three principal clinical phenotypes of Gaucher’s disease. The first is Type 1 (nonneuronopathic) Gaucher’s disease in which the CNS is not involved. The second is Type 2 (acute neuronopathic) Gaucher’s disease that is characterized by early and extensive CNS damage. The term neuronophagia is used in the context of Type 2 Gaucher, because of the widespread destruction of neurons by monocytes that are attracted into the brain from the circulation by cytokines that are elaborated by damaged neurons. The third is Type 3 (chronic neuronopathic) Gaucher’s disease in which signs of CNS involvement occur later than in Type 2 Gaucher patients. Neurological manifestations in Type 3 patients may be confined to horizontal, or less frequently, vertical gaze paresis. Some Type 3 patients also have progressive myoclonic epilepsy that is notoriously difficult to control. The identification of the enzymatic defects in Niemann-Pick’s disease [3], Fabry’s disease [4], generalized gangliosidosis [5], Tay-Sach’s disease [6], and Krabbe’s disease [7] followed soon after the elucidation of the enzymatic defect in Gaucher’s disease. This information was used to develop widely used enzymatic assays for the diagnosis [8], carrier detection [9] and prenatal identification of fetuses at risk for these conditions [10–12].

Development of ERT

A long period of time elapsed before effective treatment for any of these debilitating conditions became available. Once again, investigations into the

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nature of Gaucher’s disease were of paramount importance. Since glucocerebrosidase activity was less than normal in patients with this disorder, it appeared to be a fairly rudimentary task to purify this enzyme and determine whether its administration to Gaucher patients would be beneficial [13]. In order to minimize the possibility of sensitizing recipients to the exogenous protein, human placental tissue was initially used as the source of glucocerebrosidase. This treatment strategy, whereby exogenous enzyme is administered to a patient in whom a deficiency caused disease, was later termed ‘enzyme replacement therapy.’ These initial studies, using protein extracted from human tissue, were performed before the advent of recombinant DNA techniques made it possible to clone a gene and express the resultant protein without the need for biological source material. The first major impediment in establishing the effectiveness of this therapeutic approach was the difficulty in obtaining sufficient quantities of purified glucocerebrosidase to undertake clinical trials. Eventually, a limited amount of the enzyme was isolated in a sufficiently pure form that was believed not to be harmful if injected into patients with Gaucher’s disease. Small quantities of placental glucocerebrosidase were injected intravenously into 2 splenectomized patients with Gaucher’s disease. Percutaneous liver biopsy was performed before administering the enzyme and another the day after the injection. In both patients, the quantity of glucocerebroside in the postinfusion biopsy specimens was 26% less than that in the preinfusion biopsy samples [26]. Moreover, there was a long-lasting reduction of glucocerebroside in the blood [14]. An additional year was required to isolate enough enzyme to examine its effect in a third Gaucher patient. In this patient, only an 8% reduction of glucocerebroside occurred in the liver following the enzyme delivery, and there was no change in the blood level. Based on the amount of glucocerebroside in the biopsy samples from the third recipient, it was deduced that insufficient glucocerebrosidase had been administered to obtain a significant clearance of the accumulated glucocerebroside. Because of the difficulty in obtaining large quantities of the enzyme from the placenta with the original methods of purification, a new isolation procedure was developed by Furbish et al. [15] in the mid-1970s. It was found that glucocerebrosidase obtained by this procedure was inconsistently delivered to lipidstoring macrophages such as Kupffer cells in the liver. Macrophages have a lectin on their surface that has a particularly high affinity for mannose-terminal glycoconjugates [16]. In order to target glucocerebrosidase to these cells, the oligosaccharide side chains of glucocerebrosidase were trimmed with three exoglycosidases to produce mannose as the terminal sugar [17]. In experimental animals, this modification of glucocerebrosidase resulted in a 50-fold increase in the uptake of enzyme by Kupffer cells. A clinical trial was conducted in which

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190 IU of mannose-terminal glucocerebrosidase were injected weekly over a period of 6 months into 7 adults and one child with Type 1 Gaucher’s disease. Only the child showed any beneficial clinical response [18]. A dose-response study was carried out by performing percutaneous liver biopsies before and after injecting the enzyme over a wide dosing range. The quantity of enzyme that consistently produced a reduction of hepatic glucocerebroside was 60 IU/kg of body weight. When this amount of mannose-terminal placental enzyme was given to 12 adults with Type 1 Gaucher’s disease every 2 weeks for a period of 6 months, striking beneficial effects occurred in all of the recipients [19]. Based on these findings, mannose-terminal glucocerebrosidase was approved for the treatment of patients with Type 1 Gaucher’s disease by the U.S. Food and Drug Administration on April 5, 1991. Recombinant glucocerebrosidase was subsequently produced in Chinese hamster ovary cells. This product is biologically equivalent to placental glucocerebrosidase [20] and was approved for the treatment of Gaucher patients in the USA in 1994. ERT for Gaucher patients was later approved in 55 countries. At this time, more than 4,000 patients with Gaucher’s disease throughout the world are being treated by ERT, based on work originating back in the 1960s. Extension of this treatment to patients with neuronopathic forms of Gaucher’s disease is of great importance. Reduction of hepatosplenomegaly and skeletal improvement was universal in clinical trials in patients with Type 3 Gaucher’s disease, but improvement of the supranuclear gaze palsy manifested by these patients has been inconsistent [21]. ERT also has been examined in patients with Type 2 Gaucher’s disease. Again, systemic improvement occurred in infants, but no amelioration of the CNS impairment was evident [22]. This finding is not surprising since it has been known for many years that intravenously injected enzymes do not reach the brain because of the blood-brain barrier [23]. Alternative delivery strategies have, therefore, been explored for the treatment of patients with neuronopathic Gaucher’s disease.

Substrate Depletion

Inhibition of the formation of glucocerebroside (substrate depletion) was proposed a number of years ago as a therapeutic strategy for the treatment of metabolic storage disorders [24]. The effect of blocking glucocerebroside synthesis with N-butyl-deoxynojirimycin (NB-DNJ) has recently been examined in patients with Type 1 Gaucher’s disease, and some apparently salutary effects have been reported [25]. N-butyl-deoxynojirimycin is a small-molecular-weight compound that has been shown to reach the brain of experimental animals when given orally in large doses. A trial of N-butyl-deoxynojirimycin has been

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undertaken in patients with Type 3 Gaucher’s disease in whom the systemic manifestations are controlled by intravenous administration of mannose-terminal glucocerebrosidase. Abnormal saccadic eye movements of the recipients will be monitored as a critical clinical endpoint. This general approach to substrate inhibition is also relevant to other important lysosomal storage diseases such as Tay Sach’s and Sandhoff’s diseases, and successful studies in animal models have led to studies in human subjects. Advances in neurosurgical delivery may help to increase the effectiveness of the approach and offset the current limitations (e.g., toxicity, nontargeted delivery, insufficient penetration of target tissue in the CNS) of systemic dosing of substrate inhibitors.

Intracerebral Injection of Mannose-Terminal Glucocerebrosidase

It is likely that much, if not all, of the glucocerebroside that accumulates in neurons in the brain of patients with Type 2 Gaucher’s disease originates from the catabolism of larger sphingoglycolipids such as gangliosides (fig. 1f, g). Ganglioside turnover is most active during the neonatal period of life. Thereafter, it decreases to a constant level that is approximately 5% of the maximum velocity. Glucocerebrosidase activity in patients with Type 2 Gaucher’s disease is very low, usually in the range of 1–2% of normal [2]. It is conceivable that these patients might improve if glucocerebrosidase could be supplied to the brain during the neonatal period to catabolize glucocerebroside in neurons during this critical period of development. Investigators in the Surgical Neurology Branch of the National Institute of Neurological Disorders and Stroke (NINDS) have developed a technique called convection-enhanced delivery to deliver proteins in solution directly to the brain [27]. It was desirable to determine whether glucocerebrosidase could be delivered to the brain by this technique. An investigation was carried out to examine the safety of the procedure and the distribution of intracerebrally injected glucocerebrosidase in normal rats [28]. The procedure was found feasible and innocuous to the recipient animals. Glucocerebrosidase was carried by convective flow along white matter fiber tracts from the site of administration in the striatum to the cerebral cortex (fig. 2). The half-life of injected glucocerebrosidase in the brain was ⬃9 h, which is comparable to that in other major organs such as the liver following intravenous administration. The enzyme was specifically taken up by neurons, precisely the cells that appear to require it to prevent neuronophagia that is a hallmark of this condition (fig. 3). The reason for the selective delivery to neurons is believed to be due to a mannose lectin on their surface [29, 30] that is present in a lesser amount but is qualitatively similar to that on macrophages.

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Glucocerebrosidase diffusion in rat brain Enzymatic activity mU

aft

fore

Striatum Left hemisphere Control side

12 11 10 9 8 7 6 5 4 3 2 1

Injection site

fore

aft

Striatum Right hemisphere Infused side

Fig. 2. Distribution of human glucocerebrosidase in the brain of normal rats following intracerebral injection of the enzyme. [Reproduced from 28 with permission of Wiley-Liss, a subsidiary of John Wiley & Sons].

Fig. 3. Immunohistochemical staining of human glucocerebrosidase in neurons of normal rats following intracerebral injection of the enzyme. [Reproduced from 28 by permission of Wiley-Liss, a subsidiary of John Wiley & Sons].

The safety and intracerebral distribution of glucocerebrosidase will be examined in nonhuman primates. If the results of these investigations are favorable, it may be worthwhile exploring this approach for the treatment of patients with Type 2 Gaucher’s disease during the neonatal period. It is likely that a combination of intracerebral and intravenous administration of the enzyme will be necessary. Moreover, it may be useful to include a substrate-depleting agent in the treatment regimen to reduce glucocerebroside formation. If intracerebral administration of glucocerebrosidase proves beneficial in patients with

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neuronopathic Gaucher’s disease, it is likely that this approach will be extended to a number of other human metabolic disorders in which the CNS is involved. Again, intracerebral delivery will require neurosurgical expertise for clinical trials and will require the development of additional instrumentation and technologies to uniformly disperse the enzyme in the brain. Some areas for future development from a neurosurgical perspective might involve improved colloidal formulations of the enzyme solution as well as pumps and dispersion (injection) devices in the brain.

Gene Therapy

Gene therapy has been examined in patients with Type 1 Gaucher’s disease using retroviral transduction of bone marrow stem and progenitor cells. In the first recipient, no expression of the transgene was detected. In the second subject, expression of the transgene was detected over a period of several months [31]. Several steps are necessary before gene therapy for patients with Type 1 Gaucher’s disease becomes realistic. Among the initial goals that must be reached are: (1) more effective transduction of stem and progenitor cells with the gene of interest; (2) selective enrichment of transduced cells before their reintroduction into patients; (3) development of procedure(s) for the delivery and implantation of the transduced cells into the patient’s bone marrow, and (4) elimination of harmful effects of retroviruses including various forms of leukemia [32, 33] which have been attributed to gene therapy in the context of X-linked severe combined immunodeficiency. The use of self-inactivating lentiviral vectors has recently come under investigation. The principal advantages of lentivirus vectors are: (1) efficient integration into the genomes of target cells; (2) sustained long-term gene expression; (3) no apparent immune response, and (4) ability to infect nondividing cells such as neurons. Delivery of genes into nondividing cells with pseudotyped, high-titer, replication-defective HIV-1 vector has already been achieved [34]. This strategy was improved by the construction of a herpes simplex virus VP22 fusion protein that greatly increased the intercellular delivery of the test protein [35]. A logical extension of this investigation was the use of such a construct to deliver genes and their protein products to nondividing cells in the CNS [36]. Use of herpes simplex virus VP22 greatly enhanced the delivery of proteins between cells. Incorporation of the neuron-specific enolase promoter resulted primarily in the transduction of neurons within the CNS. These encouraging findings have led to the development of a lentivirus gene construct containing the human glucocerebrosidase gene and VP22. It is expected that the fusion product will assist in the intercellular transport of the

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therapeutic protein to a major portion of the brain. If this hypothesis is substantiated, it is expected to provide a significant impetus for serious consideration of gene therapy for patients with neuronopathic Gaucher’s disease. Whether the results of such investigations can be translated to other metabolic storage disorders remains to be established. The development and exploration of gene therapy in authentic animal analogs of human enzyme deficiency conditions should significantly accelerate our sense of the potential of this approach and hopefully reveal any unanticipated difficulties prior to their application to patients. In addition to lentivirus, a number of other viral vectors and nonviral gene delivery systems must be considered. As time goes by, the limitations of effective gene therapy are more related to technical obstacles that are gradually being overcome, rather than fundamental problems with the gene therapy approach. One can envision a future time in which the promise of ERT and gene transfer are fully realized through advances in neurosurgical delivery and improvements in vector design.

References 1 2 3

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Brady RO, Kanfer JN, Shapiro D: Metabolism of glucocerebrosides. II. Evidence of an enzymatic deficiency in Gaucher’s disease. Biochem Biophys Res Commun 1965;18:221–225. Brady RO, Kanfer JN, Bradley RM, Shapiro D: Demonstration of a deficiency of glucocerebrosidecleaving enzyme in Gaucher’s disease. J Clin Invest 1966;45:1112–1115. Brady RO, Kanfer JN, Mock MB, Fredrickson DS: The metabolism of sphingomyelin. II. Evidence of an enzymatic deficiency in Niemann-Pick disease. Proc Natl Acad Sci USA 1966;55: 366–369. Brady RO, Gal AE, Bradley RM, Martensson E, Warshaw AL, Laster L: Enzymatic defect in Fabry’s disease. Ceramidetrihexosidase deficency. N Engl J Med 1967;276:1163–1167. Okada S, O’Brien JS: Generalized gangliosidosis: Beta-galactosidase deficiency. Science 1968; 160:1002–1004. Kolodny EH, Brady RO, Volk BW: Demonstration of an alteration of ganglioside metabolism in Tay-Sach’s disease. Biochem Biophys Res Commun 1969;37:526–531. Suzuki K, Suzuki Y: Globoid cell leucodystrophy (Krabbe’s disease): Deficiency of galactocerebroside beta-galactosidase. Proc Natl Acad Sci USA 1970;66:302–309. Kampine JP, Brady RO, Kanfer JN, Feld M, Shapiro D: The diagnosis of Gaucher’s disease and Niemann-Pick disease using small samples of venous blood. Science 1967;155:86–88. Brady RO, Johnson WG, Uhlendorf BW: Identification of heterozygous carriers of lipid storage diseases. Am J Med 1971;51:423–431. Brady RO, Uhlendorf BW, Jacobson CB: Fabry’s disease: Antenatal detection. Science 1971;172: 174–175. Epstein CJ, Brady RO, Schneider EL, Bradley RM, Shapiro D: In utero diagnosis of NiemannPick disease. Am J Hum Genet 1971;23:533–535. Schneider EL, Ellis WG, Brady RO, McCulloch JR, Epstein CJ: Infantile (Type II) Gaucher’s disease: In utero diagnosis and fetal pathology. J Pediatr 1972;81:1134–1139. Brady RO: Sphingolipidoses. N Engl J Med 1966;275:312–318. Brady RO, Pentchev PG, Gal AE, Hibbert SR, Dekaban AS: Replacement therapy for inherited enzyme deficiency: Use of purified glucocerebrosidase in Gaucher’s disease. N Engl J Med 1974;291:989–993.

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Pentchev PG, Brady RO, Gal AE, Hibbert SR: Replacement therapy for inherited enzyme deficiency: Sustained clearance of accumulated glucocerebroside in Gaucher’s disease following infusion of purified glucocerebrosidase. J Mol Med 1975;1:73–78. Furbish FS, Blair HE, Shiloach J, Pentchev PG, Brady RO: Enzyme replacement therapy in Gaucher’s disease: Large-scale purification of glucocerebrosidase suitable for human administration. Proc Natl Acad Sci USA, 1977;74:3560–3563. Stahl PD, Rodman JS, Miller MJ, Schlesinger PH: Evidence for receptor-mediated binding of glycoproteins, glycoconjugates, and lysosomal glycosidases by alveolar macrophages. Proc Natl Acad Sci USA 1978;75:1399–1403. Brady RO, Furbish FS: Enzyme replacement therapy: Specific targeting of exogenous enzymes to storage cells; in Martonosi AT (ed): Membranes and Transport. New York, Plenum, 1982, vol 2, pp 587–592. Barton NW, Furbish FS, Murray GJ, Garfield M, Brady RO: Therapeutic response to intravenous infusions of glucocerebrosidase in a patient with Gaucher disease. Proc Natl Acad Sci USA 1990;87:1913–1916. Barton NW, Brady RO, Dambrosia JM, DiBisceglie AM, Doppelt SH, Hill SC, Mankin HJ, Murray GJ, Parker RI, Argoff CE, Grewal RP, Yu K-T: Replacement therapy for inherited enzyme deficiency – Macrophage-targeted glucocerebrosidase for Gaucher’s disease. N Engl J Med 1991;324:1464–1470. Grabowski GA, Barton NW, Pastores G, Dambrosia JM, Banerjee TK, McKee MA, Parker C, Schiffmann R, Hill SC, Brady RO: Enzyme therapy in Gaucher disease Type 1: Comparative efficacy of mannose-terminated glucocerebrosidase from natural and recombinant sources. Ann Intern Med 1995;122:33–39. Altarescu G, Hill S, Wiggs E, Jeffries N, Kreps C, Parker CC, Brady RO, Barton NW, Schiffmann R: The efficacy of enzyme replacement therapy in patients with chronic neuronopathic Gaucher’s disease. J Pediatr 2001;138:539–547. Prows CA, Sanchez N, Daugherty C, Grabowski GA: Gaucher disease: Enzyme therapy in the acute neuronopathic variant. Am J Med Genet 1997;71:16–21. Johnson WG, Desnick RJ, Long DM, Sharp HL, Krivit W, Brady B, Brady RO: Intravenous injection of purified hexosaminidase A into a patient with Tay-Sach’s disease. Birth Defects Orig Artic Ser 1973;IX:120–124. Radin NS: Inhibitors and stimulators of glucocerebroside metabolism. Prog Clin Biol Res 1982; 95:357–370. Cox T, Lachmann R, Hollak C, Aerts J, van Weekly S, Hrebicek M, Platt F, Butters T, Dwek R, Moyses C, Gow I, Elstein D, Zimran A: Novel oral treatment of Gaucher’s disease N-butyldeoxynojirimycin (OGT 918) to decrease substrate biosynthesis. Lancet 2000;355: 1481– 1485. Bobo RH, Laske DW, Akbasak A, Morrison PF, Dedrick RL, Oldfield EH: Convection-enhanced delivery of macromolecules in the brain. Proc Natl Acad Sci USA 1994;91:2076–2080. Zirzow GC, Sanchez OA, Murray GJ, Brady RO, Oldfield EH: Delivery, distribution and neuronal uptake of exogenous mannose-terminal glucocerebrosidase in the intact rat brain. Neurochem Res 1999;24:301–305. Burudi EM, Regnier-Vigouroux A: Regional and cellular expression of the mannose receptor in the post-natal developing mouse brain. Cell Tissue Res 2001;303:334–339. Schueler U, Kaneski C, Murray G, Sandhoff K, Brady RO: Uptake of mannose-terminal glucocerebrosidase in cultured human cholinergic and dopaminergic neuron cell lines. Neurochem Res 2002;27:325–330. Dunbar CE, Kohn DB, Schiffmann R, Barton NW, Nolta J, Esplin J, Pensiero M, Long Z, Lockey C, Emmons RVB, Cski S, Leitman S, Kreps CB, Carter C, Brady RO, Karlsson S: Retroviral transfer of the glucocerebrosidase gene into CD34⫹ cells from patients with Gaucher disease: In vivo detection of transduced cells without myeloablation. Hum Gen Ther 1998;9:2629–2640. Hacein-Bey-Abina S, von Kalle C, Schmidt M, Le Deist F, Wulffraat N, McIntyre E, Radford I, Villeval JL, Fraser CC, Cavazzana-Calvo M, Fischer A: A serious adverse event after successful gene therapy for X-linked severe combined immunodeficiency. N Engl J Med 2003;348: 255–256.

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Verma IM: A voluntary moratorium? Mol Ther 2003;7:141. Reiser J, Harmison G, Kluepfel-Stahl S, Brady RO, Karlsson S, Schubert M: Transduction of non-dividing cells using pseudotyped defective high-titer human immunodeficiency virus type 1 particles. Proc Natl Acad Sci USA 1996;93:15266–15271. Lai Z, Han I, Zirzow GC, Brady RO, Reiser J: Intercellular delivery of a herpes simplex virus VP22 fusion protein from cells infected with lentiviral vectors. Proc Natl Acad Sci USA 2000;97: 11297–11302. Lai Z, Brady RO: Gene transfer into the central nervous system in vivo using a recombinant lentivirus vector. J Neurosci Res 2002;67:363–371.

Roscoe O. Brady, MD Building 10 Room 3D04, National Institutes of Health 9000 Rockville Pike, Bethesda, MD 20892–1260 (USA) Tel. ⫹1 301 496 3285, Fax ⫹1 301 496 9480, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 213–245

Gene Therapy for Parkinson’s Disease Piotr Hadaczek, Marcel Daadi, Krystof Bankiewicz Molecular Therapy Laboratory, Department of Neurological Surgery, University of California, San Francisco, Calif., USA

Why Gene Therapy for Parkinson’s Disease?

The main existing pharmacological therapy for Parkinson’s disease (PD) centers on replacement of dopamine (DA) by administration of the DA precursor L-dopa. In many cases, agents that prolong the action of DA by preventing its breakdown are also used to potentiate L-dopa effects [1]. Current problems associated with L-dopa treatment include motor fluctuations and choreic or dystonic involuntary movements (dyskinesias), which are superimposed on underlying breakthrough symptoms of bradykinesia, rigidity, and postural instability [2]. With the inevitable progression of the disease, L-dopa loses its initial effects of symptom relief. The major limitations of L-dopa treatment are 3-fold: inability to achieve site-specific delivery, which results in unwanted side effects and limits the amount of drug which can be given [3]; nonsustained drug levels within the central nervous system (CNS), thought to contribute to unpredictable ‘on-off’ effects [4, 5]; and progressive degeneration of DA-secreting nerve cells during treatment [6]. Development of new therapeutic approaches to PD must address these inadequacies of L-dopa. Most importantly, L-dopa addresses the biochemical sequelae of PD but does not address the underlying causes. Therefore, a primary goal for therapy of PD is the development of neuroprotective therapy which will slow down and prevent the death of neurons in substantia nigra (SN). Gene therapy encompasses any technique whereby an absent or faulty gene is replaced by a working one, so that a cell can make the correct enzyme or protein and consequently eliminate the cause of the disease. As a result, gene transfer may serve as a compensation for missing or defective protein expression. Several features of PD make it particularly suited for a gene therapy-based approach to treatment: (1) The pathology of the disease has been well characterized (loss of dopaminergic neurons and degeneration of the nigrostriatal

circuitry); (2) the initial pathology is confined to a discrete location within the brain where stereotactic targeting is possible (i.e., global gene transfer is not required in early stages); (3) disease processes such as apoptosis occurring within the SN may be prevented with a gene transfer approach, and (4) established animal models are available for testing clinical efficacy, safety and prognostic assessments. Gene therapy models for PD have focused on two treatment strategies. One is the replacement of biosynthetic enzymes for DA synthesis. It has been hypothesized that the transfer of genes involved in DA production would help to ameliorate the direct motor symptoms of the disease by the sustained local delivery of this neurotransmitter. The biochemistry of the DA synthesis involves several enzymes and cofactors. The rate-limiting enzyme in DA production is tyrosine hydroxylase (TH), which converts the amino acid tyrosine to L-dopa. L-dopa is then converted to DA by the aromatic amino acid decarboxylase (AADC) [7]. Another cofactor that is essential for DA metabolism is 6-tetra-hydrobiopterin, the level of which is limited by the availability of the enzyme GTP-cyclohydrolase 1 (GTPCH-1) [8, 9]. In theory, these enzymes could be genetically manipulated to produce increased DA levels. Some studies have reported a benefit from such enzyme replacement therapy, but others have challenged the relevance of providing biosynthetic enzymes to a milieu in which the cells are dying and incapable of properly using DA in any case. Another treatment strategy is providing neurotrophic factors for protection and restoration of dopaminergic neurons, thereby preventing them from further degeneration. Within each of these separate strategies, both in vivo (direct transfer of the gene into brain) and ex vivo (transplantation of genetically engineered cells) approaches have been considered. In vivo Approach Neurodegenerative diseases like PD are chronic; therefore, treatment needs to be longlasting. This situation makes PD particularly suited to treatment with viral vectors, where a single application of a vector can result in prolonged, stable transgene expression of relevant enzymes or growth factors. In vivo gene therapy involves direct gene transfer into the host somatic cell via viral vectors or a liposome vehicle [10]. There are several advantages of direct gene transfer over cellular transplantation: (1) vector delivery may be less invasive for the synaptic circuitry and brain parenchyma; (2) there is limited risk of unregulated cellular proliferation; (3) tissue-specific delivery systems for regulatable transgene expression can be designed, and (4) multiple genes can be administered at the same time. If gene therapy is to become a truly practical mode of treatment of PD, the therapeutic gene will need to be expressed for a sustained length of time and it

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will require stable transduction and maintenance of gene expression in the targeted region of the brain and limited immune response to the vector and the target product. Various vector systems represent different features (table 1) that have to be carefully considered before clinical application. Both viral and nonviral vectors have become an important and powerful tool for gene delivery to the human nervous system. Neurodegenerative diseases like PD are suitable candidates for ‘molecular neurosurgery’ approaches because they are localized and specific regions of the brain are responsible for their development. Researchers have developed many different virus-based systems to manipulate subtle neuronal cell biochemistry and physiology. Crucial issues that need to be taken into consideration include transgene transduction efficiency, adverse tissue responses, targeting specificity, and regulation of transgene expression. Issues related to vector toxicity, long-term expression, gene regulation, vector production, CNS administration, and axonal transport will need to be addressed to develop an optimal gene delivery system for PD. Herpes Simplex Virus (HSV) – Based Vectors Herpes Simplex Virus 1 (HSV-1) has some features, which make it attractive as a vehicle for the delivery of therapeutic genes to the nervous system. HSV-1 is neurotropic and viral genomes persist as extrachromosomal elements. A neuronal-specific HSV promoter is capable of remaining active during viral latency, making HSV-based vector systems less susceptible to promoter silencing. Additional modifications to the viral genome (e.g., removal of genes responsible for the lytic cycle) reduce the cytotoxicity of the vector [11]. One technical advantage of the HSV genome for vector construction is that viral genes are almost entirely found as contiguous transcribable units, which makes their genetic manipulation relatively straightforward. The large-size viral genome of 152 kb permits insertion of a large size transgene and the ability to deliver multiple therapeutic genes via a single vector source. In general, HSV-based vector systems can be assigned to one of two major categories, either recombinant viral vectors or defective viral vectors. The recombinant HSV vectors carry a foreign gene in the native viral genome. They lack essential viral genes crucial for replication, but retain their ability to enter into the latency state within neuronal cells. The other type of HSV vector, the plasmid-based amplicon (‘defective’ HSV vector) contains approximately 1% of the HSV-1 genome and its backbone includes an eukaryotic plasmid modified by the addition of an HSV origin of replication (ori) and packaging sequence. This system requires a helper virus such as the wild-type HSV for high-level transgene expression and packaging [12]. Titers of amplicon stocks are typically lower than those of recombinant vectors (⬃106–107 units). Since defective HSV vector stock preparations may contain helper virus, the use of such vectors

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Table 1. Review of viral vectors used in gene therapy systems Hadaczek/Daadi/Bankiewicz

Vector

HSV

HSV amplicon

Ad

Minimal Ad ‘gutless’

Lentivirus (HIV)

AAV

Diameter

200 nm

200 nm

70–100 nm

70–100 nm

80 nm

20 nm

Size of viral genome

152 kb

minimal (only HSV replication and packaging origins)

30–40 kb

500 bp

7 kb

4.7 kb

Insert capacity

⬍30 kb

⬎50 kb

⬍8 kb

36 kb

7 kb

4.8 kb

Occurrence in cell

episomal

episomal

episomal

episomal

integrated in genome

episomal/ integrated in genome

Vector type

recombinant

defective

recombinant

defective

defective

defective

Viral contamination during production

yes

yes

yes

minimal

no

minimal

216

Immunogenicity

high

high

high

low

low

low

Titers (TU/ml)

1011

107

1012

107

107

1012

Major advantage

large capacity of transgene; high titers

large capacity of transgene; low immunogenicity/ toxicity

high titers

low immunogenicity/ toxicity; large capacity of transgene

ability to transduce dividing and nondividing cells

low immunogenicity/toxicity

Major limitation

transient expression/triggers immune response

low titers

triggers immune response/transient expression

tedious production

possible conversion to HIV-1; random genomic integration

small size of insert

in vivo may result in the expression of cytotoxic gene products from the helper virus, leading to neuropathological effects. Progress in reducing cytotoxicity includes improvements in the packaging system such as increasing the ratio between defective viral vector and helper; usage of helper virus with a larger deletion in IE3 (immediate early gene); using helper-free packaging systems; and improving purification of amplicon from helper virus. Studies using HSV vectors for gene therapy in PD have had mixed results. In a rat 6-hydroxydopamine (6-OHDA) PD model, HSV-based vectors containing the TH gene were delivered to the rat striatum and the animals appeared to demonstrate behavioral and biochemical recoveries for one year [13, 14]. Using the same animal model, neuroprotective effects on dopaminergic neurons have been demonstrated using glial derived neurotrophic factor (GDNF) and the apoptosis inhibitor Bcl2 with HSV-derived vectors. It was also shown that cotransfection of HSV-GDNF and HSV-Bcl2 had additive neuroprotective properties [15]. Both vector systems have shown reduction of amphetamine-induced rotations in the 6-OHDA rat model of PD. As mentioned, the main limitations of HSV systems include CNS cytotoxic effects and poor long-term gene expression, with limited number of cells expressing the transgene [13]. For these reasons, clinical use of HSV-1 appears to be impractical unless changes in vector design are implemented. Adenovirus Vectors Adenoviruses (Ad) have been a popular vehicle for gene transfer. Their attractive features include the capacity to accommodate large transgene inserts up to 36 kb and the ability to infect a wide variety of cell types and species (including postmitotic cells). The four main cell types in the brain which can be transduced by Ad vectors are neurons, astrocytes, oligodendrocytes, and ependymal cells [16, 17]. Recombinant Ad vectors have focused on deletions of E1, E2, E3, and E4 genes to reduce immunogenicity [11]. In humans, perhaps the most important quality is that Ad is not associated with any neoplastic disease and causes relatively mild, self-limiting illness in immunocompetent individuals (respiratory infection, keratoconjunctivitis, gastroenteritis). The development of Ad vectors of first, second and third generations are all based on deletions of one or more of these genes. Replication-deficient Ad vectors are propagated on special cell lines that provide functions of the early transcription units. The first-generation vectors (lacking E1) can still induce substantial inflammation, despite being replicantdeficient. Both viral proteins and therapeutic proteins were found to be targets for immune attack. Despite the lack of E1, viral proteins are expressed on firstgeneration vectors at levels sufficient to elicit a T-cell response [18]. Unfortunately, in addition, even the therapeutic gene will often be recognized as foreign by the

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host. To bypass such an immune response, use of tissue-specific promoters has been proposed [19]. To improve the utility of Ad vectors for gene therapy, investigators have further modified the virus by mutating or deleting regions E2-E4 (second- and third-generation Ad vectors) [20–23]. Ads have not gained widespread use because of their inconsistent performance, probably due to the instability of the deleted vector genome [24]. Deletion of all Ad protein-coding sequences is possible with fully deleted Ad vectors (minimal or ‘gutless’ Ad vectors). The only Ad sequences that need be retained are ⬃500 bp of cis-acting DNA elements, including the viral inverted terminal repeats located at each end of the genome and the viral packaging signal. Current methods for producing gutless Ad involve its coreplication in the presence of a second helper virus that provides replicative functions. The advantages of this system are increased cloning capacity (⬃37 kb), increased safety, and potentially reduced immune responses due to elimination of viral sequences. The episomal nature of Ad often means that ultimately the transgene will be expelled from the cytosol during cell division. However, the genome may persist as an episome in nondividing cells (neurons) with sustained transgenic expression for longer than one year [25]. Nevertheless, repeat vector administration is probably required in order to boost transgene expression levels to initial levels. Unfortunately, in most cases administration to immunocompetent individuals results in the formation of anti-Ad neutralizing antibodies (directed at the vector capsid) which presents a significant barrier to vector readministration [26–28]. In vivo use of Ad vectors in PD animal models has focused on delivery of either Ad-TH or Ad-GDNF. When Ad vector encoding the TH gene was introduced into the striatum of 6-OHDA-lesioned rats, a reduced frequency of apomorphine-induced rotational behavior was observed [29]. TH expression, being confined predominantly to astrocytes, was demonstrated only for 1–2 weeks following gene transfer and an inflammatory response with gliosis was detected. More recent experiments with Ad vector encoding TH under the control of the repressible tetracycline regulatory system (‘tet off’) also showed that this vector mediates synthesis of TH in striatal cells. Transgene expression was observed in a large proportion of cells for at least 17 weeks, resulting in a significant overall reduction of apomorphine-induced rotation for at least 30 days. However, after 6 weeks, the pre- and postinjection outcomes were comparable [30]. In studies with multisite partitioned delivery of Ad-TH, Leone et al. [31] showed a correlation between the numbers of TH-immunoreactive cells and the loss of apomorphine-induced rotation, with a near-linear relationship between TH expression and phenotypical recovery. Those data suggested that only a fraction of striatal cells need to be transduced in order to exert phenotypical effects. Neuroprotective effects on dopaminergic neurons have been demonstrated when Ad-GDNF vectors were delivered [32, 33], with increased survival of SN

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dopaminergic neurons and preservation of dopaminergic innervation to the striatum [33]. Studies by Connor et al. [34] also demonstrated that GDNF expressing Ad injected into the striatum and SN of aged rats, one week prior to 6-OHDA lesioning, allowed the production of GDNF at DA nerve terminals. However, only striatal injections of Ad-GDNF protected against the development of behavioral deficits characteristic of unilateral DA depletion. These results show that increased levels of striatal, but not solely nigral, GDNF biosynthesis prevents DA neuronal loss and protects DA terminals from oxidative damage from 6-OHDA lesioning. The development of gutless Ad from first-generation vectors was inevitable because of the shortcomings of the latter. For now, gutless Ad appears to be a promising vector platform for genetic diseases where long-term gene expression is required. With the advancement of vectorology, Ad-based delivery systems may be amenable to clinical applications in the future, but many problems remain such as immunological sensitization. Lentivirus-Based Vectors Lentiviral (LV) vectors are derived from a group of pathogenic retroviruses, which include human immunodeficiency virus (HIV). The retroviral machinery requires the conversion of the RNA genome to double-stranded DNA, mediated by the reverse transcriptase enzyme that is present in the infectious virion. The last step of the replication cycle leads to the integration of the provirus into the host genome. Once integrated, the provirus is ready to be expressed. The first retroviral vectors used for gene transfer were murine leukemia virus. Their use in the CNS was largely limited to ex vivo gene therapy as they were not able to transduce nondividing neuronal cells [11]. Lentiviral-based vectors share the properties of commonly used retroviruses with additional advantages: they can infect both dividing and terminally differentiated cells such as neurons; they have a large cloning capacity (⬎9 kb); they can be stably integrated into the genome of the target cells; and they do not encode viral proteins that can trigger an immune response. In current versions of HIV-1-based LV vectors, up to 60% of the viral genome has been eliminated and only three or four of the nine genes of HIV-1 are retained [35, 36]. Viral particles are generated by transient transfection of 293T cells with a three-plasmid system consisting of packaging, envelope, and transfer vectors [37, 38]. Splitting of the viral genome limits the formation of replication-component particles [35]. Through integration, retroviral vectors offer the opportunity of long-lasting expression, a major advantage in the treatment of genetic diseases. The level of expression in the brain can be further increased by the introduction of postregulatory elements that stabilize nascent RNA transcripts [39, 40]. In most versions of LV vectors, the particles are pseudotyped with the G envelope protein of vesicular stomatitis virus,

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which gives the vector the capacity to infect a broad range of tissues including nervous tissues, and is probably responsible for their high affinity for neurons [37, 38, 41–43]. Because retroviral vectors integrate into the host genomic DNA, there are some major biosafety issues. The first is that nonspecific integration represents a potential source of genetic mutation. Host genomic integration with lentivirus appears to be a random process and important host cellular genes may be disrupted or activated [44–46]; however, in ex vivo human gene therapy for severe combined immunodeficiency X-1, a retrovirus was found to have preferentially inserted into an oncogene sequence in 2 separate patients. Other risks may result from the vector preparation itself (i.e., toxicity of viral proteins or compounds derived from the production system). Generating replication-competent retrovirus also is a major concern; primate studies had highlighted this potential risk of [47]. To minimize the risk of such recombinants, a self-inactivating version of LV has been developed [40, 43]. The self-inactivating design results in the removal of the major part of the viral transcriptional elements prior to integration, which also minimizes the chance that genes adjacent to the vector integration site will become activated. As with other gene transfer vectors, immunogenicity of retroviral vectors needs to be studied further before widespread clinical applications are possible. LV gene transfer into the monkey nigrostriatal system has been shown to induce minor perivascular cuffing, but without an apparent inflammatory response [48]. In Fisher rats, after intraportal infusion into the liver of more than 8 ⫻ 108 transducing units of an LV, a mortality rate of 74% was observed [49], clearly unacceptable for clinical implementation. Despite these caveats, significant advances in defective LV systems have provided a new perspective on gene delivery to the brain. Use of LVs in PD animal models has permitted delivery of GDNF to the striatum or SN. For example, Deglon et al. [39] were the first to examine lenti-GDNF delivery in a rodent model of PD. Their study indicated a significant sparing of nigral neurons after unilateral injection of lenti-GDNF over the SN. Georgievska et al. [50] similarly demonstrated structural and functional protection in 6-OHDA-lesioned rats with a LV. Similar results were observed in a mouse model of PD [51] by a different group. In a MPTP monkey model of PD, Kordower et al. [52] tested LV for intracerebral GDNF delivery. In treated animals, severe motor deficits were partially corrected, loss of dopaminergic neurons in SN was partially spared, and striatal dopamine innervation was preserved up to 70–80%. Consistent with these results, striatal 18F-fluorodopa uptake [Positron Emission Tomography (PET) prior to euthanasia] was increased by 300% in the lenti-GDNF-treated striatum. This work certainly supports the eventual use of lentivirus-GDNF treatment in the gene therapy of PD, though issues of long-term efficacy and

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toxicity must first be addressed. In another in vivo approach by Azzouz et al. [53], a self-inactivating minimal LV expressing TH, AADC, and GTPCH-1 in a single transcriptional unit was designed. After stereotactic delivery into the DA-denervated striatum of the 6-OHDA-lesioned rat, sustained expression of each enzyme and production of catecholamines was detected, resulting in significant reduction of apomorphine-induced motor asymmetry during testing. Expression of each enzyme in the striatum was observed for up to 5 months after injection. These data indicate that production of three catecholaminenergic enzymes by a single LV can achieve functional improvement in 6-OHDA-lesioned rats. These results are somewhat tempered by work from other groups suggesting that multiple enzymatic delivery in 6-OHDA PD rats with gene transfer did not appear to create any additive effect beyond TH gene transfer alone [Janson, pers. commun.]. HIV-2 derived LVs have been used extensively for gene delivery to human neuronal cells. HIV-2 appears to be slightly less pathogenic than HIV-1 and because of limited sequence homology; cross-packaging of HIV-2 vectors into HIV-1 cores will minimize recombination between sequences in the transfer and packaging vectors. Gene transfer of AADC gene using the above-mentioned system was first examined in vitro by D’Costa et al. [54]. SVG cells (human neuronal cells immortalized by SV40 transformation) were transduced by both HIV-1 and HIV-2 based vectors carrying a cassette containing the AADC gene. Subsequently, gene transfer was evaluated by determining the ability of the transduced cells to convert L-dopa into DA. This conversion was measured in the intracellular compartment as well as in the secreted form in the supernatant. The results showed that both HIV-1 and HIV-2 AADC vectors successfully imparted the ability on transduced cells to efficiently convert L-dopa into DA. It was noted that the observed higher transduction for HIV-1 cross-packaged vectors was partly due to the higher titer of the latter vector. This approach provides the ability to combine gene transfer and standard drug treatment. These outcomes suggest that efficient HIV-2 vectors with a therapeutic transgene selfpackaged in HIV-2 cores, or cross-packed in HIV-1 cores, can be generated for the future treatment of PD. Adeno-Associated Virus-Based Vectors Adeno-associated viral (AAV) vectors are favorable candidates as gene delivery vehicles. They have many advantageous properties for gene therapy applications. For example, the parental virus does not cause disease; no viral genes are included in AAV recombinants and therefore, host immune response is minimized, the vectors transduce dividing or nondividing cells and a wide range of cells and tissues; expression can persist, mediating impressive longterm gene expression. One main limitation of AAV vectors is their small transgene capacity of ⬍5 kb per particle.

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Wild-type AAV is the smallest (⬃20 nm) and simplest of the DNA replication defective viruses (Parvoviruses). The nonenveloped wild-type AAV particle contains a linear single-stranded DNA genome of 4.7 kb encapsidated with a simple three-protein capsid. The conversion of the single-stranded DNA genome to a double-stranded molecule is an important event required for the efficient function of AAV as a delivery vehicle. Its rate largely depends on the physiological state of the cell and may be a limiting factor for the transduction efficiency. Wild-type AAV has been shown to stably integrate into a single specific site within chromosome 19q13.3-ter [55]. Latent persistence occurs when AAV infects cells in the absence of helper virus (Ad or HSV). When cells containing an AAV provirus are superinfected with a helper virus possessing trans-acting elements (necessary for replication and packaging), the integrated AAV genome is ‘rescued’ and replicated to yield progeny AAV particles. There is divergence in tropism among various AAV serotypes (types 1–5) [56]. For example, recombinant AAV-5 and AAV-2 preferentially transduce neurons. Viral receptors strictly define the specific tissue tropism of a particular viral serotype. The general principles of AAV vector construction are based on the substitution of the AAV coding sequence with foreign DNA (transgene) to generate a vector plasmid. Only the AAV inverted terminal repeats flanking the transgene cassette must be retained intact. Current methods to produce stocks of defective AAV often use a human cell line (typically 293 cells) that is cotransfected with an AAV vector and a helper plasmid containing the AAV coding sequences (rep and cap genes flanked by Ad). The transfected cells are subsequently superinfected with Ad plasmid, which serves as a helper virus. The result of this system is a mixture of AAV vector particles and Ad particles. The Ad can be inactivated by temperature (56oC for one hour) and separated by CsCl-density centrifugation. Other, more recent, methods for obtaining high titers of AAV with no contamination by helper Ad have been developed [57, 58]; these typically use ‘triple transfection’ with a rAAV vector and two helper plasmids that serve the replicative roles of Ad and the packaging role of the AAV wild-type sequence. Purification of AAV is critical for clinical trials. A variety of chromatographic methods (e.g., ion exchange, antibody and heparin affinity resins) have been used in both conventional chromatography and HPLC systems. Vectors derived from recombinant AAV appear to exist as episomes and have not been shown to integrate to a significant degree. After AAV particles enter the nucleus, the vectors become circularized and ligated into larger concatameric molecules. Most of such molecules appear to persist for prolonged periods, perhaps even for the lifetime of the cell in the case of nondividing cells such as neurons. This phenomenon may help to explain long-term expression of transgenes delivered to the cell by recombinant AAV. Recombinant AAV vectors are considered one of the safest viral delivery systems, with minimal induction

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of innate immune response. Nevertheless, there are reports describing generation of humoral neutralizing antibody responses to AAV capsid proteins following systemic delivery. This response may reduce the efficiency of the transduction, a consideration for systemic readministration [59, 60]. AAV vectors were first introduced into clinical trials for the treatment of cystic fibrosis [61, 62], using inhaled delivery for the treatment of the lungs. In the brain, two clinical protocols have been initiated thus far using AAV, for Canavan disease [Janson C, pers. commun.] and PD [During M, pers. commun.]. In preclinical work, long-term expression of AAV transgenes has been demonstrated in the CNS, including in the SN, globus pallidus, and striatum [63–71]. Mandel and colleagues [72, 73] examined the neuroprotective effects of intranigral AAV-GDNF injected 3 weeks before or immediately after intrastriatal 6-OHDA lesions. Significant neuroprotective effects were observed on the histological level in both versions of that experiment. However, no functional recovery was detected. More recently, Kirik et al. [67] examined the regional effects of AAV-GDNF delivery. A 6-month period of sustained expression was reported. Interestingly, GDNF expression and its protective effect were observed at both injection sites (nigra and striatum), but preservation of striatal dopaminergic fibers occurred only with striatal injection of the vector. Functional recovery also occurred only when AAV-GDNF was transduced into the striatum. It appears, therefore, that protection of dopaminergic terminals in the striatum is a critical feature in promoting functional recovery. Another approach using AAV vectors, replacement of DA biosynthetic enzymes, has been examined in various animal models of PD. Injection of an AAV vector containing the TH gene resulted in expression of TH enzyme in neurons as early as 24 h postinjection and persisted up to 7 months [66]. That study was among the first attempts to use enzyme replacement strategy with AAV as the delivery system. More recent studies have confirmed the performance of AAV both in terms of efficiency and the absence of cytotoxicity [74]. AAV-TH alone, however, was reported by one group to produce neither significantly elevated L-dopa levels nor significant behavioral improvements [63]. In addition to TH, that group found that gene transfer for other enzymes (AADC, GTPCH-1) was necessary for efficient DA production. Replacement of two or even three crucial enzymes in PD can be therapeutic. Indeed, behavioral recovery and effective dopamine production was achieved in combination with therapy with AAV-TH and AAV-AADC [75]. In turn, triple transduction with AAV-TH, AAVAADC, and AAV-GTPCH 1 showed improved rotational behavior lasting at least 12 months, and elevated DA production in rat striatum, compared with double transduction with AAV-TH and AAV-AADC [69]. This strategy extended the preclinical exploration to a primate model of PD and also showed some behavioral improvement with restoration of DA synthesis [76].

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AAV/Lac-Z

AADC-IR

AAV/AADC

AADC-IR

Fig. 1. Convection-enhanced delivery (CED) of AAV2-AADC. Efficient technique for vector delivery is required for successful gene transfer. Convection-enhanced delivery can distribute AAV-2 vector in a nontraumatic and uniform fashion within monkey striatum [71]. Immunostaining of the monkey brain section for AADC shows the extent of transduction with AAV2-AADC (right). The section from the control monkey transduced with AAV2LacZ (left). Residual immunoreactivity (IR) for AADC is seen only in nucleus accumbens which is spared in PD.

An alternative approach of combined drug and gene transfer proposed by Bankiewicz and colleagues [70, 71] is based on the premise that a reduction in AADC might contribute to the loss of L-dopa therapeutic efficacy. Therefore, gene transfer to restore the decarboxylating capacity of L-dopa may result in a therapeutic gain with continued L-dopa dosage. In a MPTP monkey model [71], AAV2-AADC injected alone in the striatum was found to confer long-term (3.5 years) expression of the AADC gene (fig. 1, 2) with robust conversion of peripheral L-dopa to DA and some behavioral improvement. Modulating intrastriatal DA levels, by combination of AADC gene delivery and oral adjustments of L-dopa dosage, may provide a treatment strategy that could prolong L-dopa efficacy and reduce side effects seen from chronic high-dose oral drug therapy. Because DA levels are difficult to regulate after single or multiple gene transduction, the AADC and L-dopa approach is inherently more safe, though longterm efficacy is still unproven.

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Fig. 2. Sustained AADC gene expression following AAV2-AADC gene delivery in PD monkey. AAV2-mediated gene expression can be long lasting (over 3.5 years) [147]. Picture was taken at 9 months post-transduction. Due to the neurotropic nature of AAV2, mostly medium spiny neurons are targeted.

An interesting approach to correct the physiological circuit affected by PD with AAV was recently proposed by Luo et al. [77]. The basis of their study design was the idea that marked improvement of the motor symptoms of PD occurs following subthalamic nucleus (STN) ablation or high-frequency stimulation. The projection axons from the STN end in excitatory synapses on target neurons in the SN pars reticulata, a major output pathway to the thalamus. Luo et al. generated AAV vectors containing two isoforms of glutamic acid decarboxylase (GAD65, GAD67), an enzyme which is responsible for conversion of glutamate to gamma amino butryic acid. Adult male rats were stereotactically injected into the STN with AAV-GAD vectors. Expression of transgenes was observed up to 5 months posttransduction. Transduced neurons, when driven by electrical stimulation, produced mixed inhibitory responses associated with the gamma amino butryic acid release. Three weeks after surgery, the ipsilateral medial forebrain bundle was lesioned with 6-OHDA, while control animals received AAV-GFP (green fluorescent protein) or PBS. These lesions led to impaired general locomotor activity and apomorphine-induced rotations contralateral to the denervated side in control animals. In the GAD65-treated rats, however, abnormal apomorphine-induced rotation was decreased by 65%. Immunohistochemical data revealed that 80% of dopaminergic neurons survived in the ventral tegmental area and 35% in the SN pars compacta. These results suggest that neurons generally considered excitatory and glutamatergic can express

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GAD transcripts. Hence, AAV-GAD gene transfer into excitatory neurons may have clinical potential for the treatment of PD or other conditions associated with excessive excitation. One major concern regarding this study has been that AAV can spread from the site of administration via axonal transport, and thus expression could adversely influence neurons beyond the targeted site. Moreover, data in a primate model has not been made public and it will be important to confirm results from the rat model in a large-animal model that more accurately reflects the human physiology. In a Phase I study that was initiated at Long Island Jewish Hospital (USA), ablative surgery in the STN is proposed in case of any adverse effects involving uncontrolled expression of the GAD transcript. Hybrid Vectors As all vector systems have certain advantages and disadvantages, researchers have tried to combine elements from different viruses to create hybrid vectors with the most advantageous features for gene delivery into the CNS. Problems with current viral vectors include toxicity of viral proteins, difficulty in regulating transgene expression, and poor efficiency of transgene delivery and stability in host cells. New generations of chimeric viral vectors will be focused on targeting of specific tissues and cell types; achieving stable and regulated transgene expression through integration into the host genome or maintenance as episomal elements; accommodating large transgenes; retaining high-transduction efficiency; and minimizing adverse cytotoxic and/or immune responses. Different versions of chimeric delivery systems have already been proposed: Ad/EBV hybrid vectors, HSV/EBV/RV hybrid amplicon vectors, Ad/RV, Ad/AAV, HSV/ AAV, and others. Costantini et al. [78] used a HSV/AAV hybrid system and showed high-transduction efficiency and stability in culture.

Nonviral Approaches for Gene Therapy of PD

All the limitations of viral gene delivery systems (mentioned above) emphasize the need for alternative therapies with high effectiveness, specificity, and minimal side effects. Therefore, nonviral vectors have become an attractive option for gene delivery. Their low immunogenicity and easy large-scale production capability are among their most important characteristics. Naked DNA It was demonstrated by Wolff and coworkers [79] that simple administration of ‘naked’ or free DNA by intramuscular injection resulted in a fairly high level of expression in muscle. Later studies confirmed naked DNA gene transfer in other tissues (e.g., lung, heart, liver, kidney). The most likely mechanism

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for cellular entry of such foreign DNA is based on receptor-mediated endocytosis [80]. While syncytial muscle fibers can readily uptake naked DNA, other tissues such as brain cells are not nearly as permissive, limiting this approach. Nevertheless, a number of routes and methods have been proposed for delivery of naked DNA into peripheral tissues, which may also apply to peripheral nerves. Examples include topical or intradermal; direct injections into deeper tissues, including intratumoral injections; and intravenous or intra-arterial. Most studies with naked DNA have focused on intratumoral injections as a possible anti-tumor strategy. For example, Coll et al. [81] showed that injection of naked DNA carrying Bax or p53 genes into a xenograft model of human lung non-small cell carcinoma could inhibit tumor growth. Naked DNA can be used as a DNA tumor vaccine; one such study showed anti-tumor immunity when naked DNA encoding the tumor antigen carcinoembryonic antigen or CEA was delivered by intrasplenic administration [82]. The transfer of naked DNA is gaining growing acceptance as a form of nonviral gene therapy; however, this technique is not sufficiently efficient in the brain. Lipid Vectors Liposomes have been used as drug carriers for many years. Several different liposomal formulations have been used in clinical trials. Cationic lipid is the most commonly used for such a purpose. To further stabilize liposomal structure, various polymers (commonly polyethyleneglycol or PEG) have been used, which may result in improved pharmacokinetics and biodistribution [83]. Cationic liposome-DNA complexes (plasmid DNA encapsulated in liposomal vesicle) are the most studied nonviral gene delivery systems in humans. After reaching the target cell, the DNA is carried across the plasma membrane, either by fusion or by endocytosis. Subsequently, DNA must be released from the endosome into the cytoplasm to avoid degradation in the lysosomes. Finally, the DNA must relocate from the cytoplasm into the nucleus to direct the expression of the gene products. All of these three steps (entry into the cell, escape into the cytoplasm, entry into the nucleus) are the main areas for chemists to design optimal formulations of lipids for gene delivery into cells. In the 6-OHDA rat model, Zhang et al. [84] demonstrated that it is possible to normalize brain TH enzyme activity by liposomal gene transfer via intravenous administration. The TH gene was encapsulated in 85-nm polyethylene glycol immunoliposomes and targeted to the brain with a monoclonal antibody to the rat transferrin receptor. In this manner, the gene was successfully delivered across the bloodbrain barrier and the plasma membrane. Three days after intravenous administration, striatal TH activity was normalized in association with a 70% reduction in apomorphine-induced rotation behavior, an approach that was repeated by others [10, 85].

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Polymer-DNA Complexes (Polyplexes) Similar to cationic lipidic vectors, polycationic polymers can interact with the negative phosphate groups of DNA. These polyplexes protect DNA from degradation and enhance DNA uptake into the cell, resulting in efficient gene transfer. Cells take up condensed particles through a number of natural processes such as endocytosis, pinocytosis, or phagocytosis. Similar to lipid vectors, a polyplex has to pass the plasma and nuclear membranes. Different strategies have been proposed to improve transfection efficiency, improve specific targeting (e.g., conjugation with different ligands), prolong gene expression (e.g., insertion of regulatory sequences), and minimize toxicity. The most common polymers for DNA delivery include poly-L-lysine, protamine, polyethylenimine, and dendrimer. Several groups have already used polyplexes in animal models for cancer, an important application of gene therapy. Polyethylenimine/DNA complexes were also found to be efficient for in vivo gene transfer into neurons after stereotactic injection into the brain [86, 87]. A study by Wang et al. [88] demonstrated that polyethylenimine/DNA complexes migrate by retrograde axonal transport to neuronal cell bodies after being internalized by nerve terminals in the muscle, and confirmed the feasibility of nonviral gene delivery to the CNS via peripheral injection sites. This approach may have a number of clinical applications including PD, but specificity remains a problem.

Regulation of Gene Expression

Many proteins of therapeutic value posses a narrow window for optimal mode of action and have side effects and toxicities when overproduced. Therefore, gene therapy systems that introduce expression of an endogenous protein ideally should be regulated in vivo to achieve sustained transgene expression. For example, in the case of PD, too much DA production as a result of excessive DA biosynthetic enzyme expression can result in unmanageable dyskinesias and other serious side effects [89–91]. Early gene delivery systems generally relied on viral promoters to drive constitutive expression [11]. Disadvantages include loss of transgene expression over time and lack of well-regulated expression. Using promoters that are specific for particular cell types and tissues is one method of gene regulation, as their presence in a physiologically specific environment prevents gene silencing or shutdown of expression. The neuron-specific enolase, enkephalin, Purkinje cell-specific L7 protein, and myelin-basic protein promoters have been used as transcriptional activators in viral vectors to express transgenes in neurons, cerebellar Purkinje cells, and oligodendrocytes [92–95]. Xu et al. [96] compared a range of different mammalian CNS expression cassettes in AAV vectors using

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different promoter sequences. The highest expression of reporter genes occurred when endogenous, nonviral promoters such as neuron-specific enolase and beta-actin were used in AAV-based vectors delivered into rat brain. The commonly used basal CMV promoter was found to be the weakest of those tested in vitro and in vivo. The choice of the proper promoter, therefore, is an important component of successful transgene expression. In a retroviral-mediated gene transfer system, Cortez et al. [97] used a glial fibrillary acidic protein promoter, whose activity is up-regulated in areas of gliosis often characteristic for PD. When astrocytes were transduced with the TH gene and implanted into the striatum of rats lesioned with 6-OHDA, a significant reduction in the turning behavior occurred for at least 4 weeks after grafting. The glial fibrillary acid protein promoter is of interest for gene therapy for neurodegenerative disorders, as it is active in the CNS throughout adult life and may serve as a disease-specific activator, since expression increases following many types of brain insults. It is important to investigate additional promoters to express transgenes in subpopulations of neurons most affected in neurodegenerative diseases such as dopaminergic neurons in PD. Gene expression can be manipulated by introducing a hybrid gene formed by linking a regulatory element upstream of the gene to be transcribed. One such strategy is to use a small-molecule drug that can cross the blood-brain barrier to act on drug-dependent promoters which directly activate or repress target gene transcription. Current drug-dependent gene-regulation systems use three general types of transcription factors: (1) drug-responsive elements (e.g., tetracycline, rapamycin); (2) nuclear hormone receptors (e.g., glucocorticoid-regulated systems), and (3) heterodimeric proteins (i.e., chemical-induced dimerization). At this time, the most commonly exploited transgene regulation systems use tetracycline as the activator or suppressor. The tetracycline-repressible system (‘tet-off’) works via negative control: the expression of the target gene is on in the absence of tetracycline and off in its presence [98]. The repressible system requires two gene sequences, the tetracycline transactivator (tTA) and the target gene that contains a promoter with tetracycline-binding sites (tetracycline operator, TetO). In the absence of an antibiotic, tTA has affinity for the TetO sites and stimulates transcription of the transgene (mode ON). When tetracycline is present, tTA protein changes its conformation and reduces its affinity for TetO sites so that the transcription is shut down [99]. The magnitude of the transgene repression in vivo can be as high as ⬃100-fold. The tetracycline-inducible system (‘tet-on’) uses positive control and works in the opposite manner [99]. The rtTA (reverse tTA) gene encodes a protein that has a very low affinity to TetO sites; however, when an antibiotic is added, the rtTA protein is converted to an active form which gains the ability to bind TetO sites and activates transgene expression. A similar principle is applied in anti-progestin or other hormone-inducible

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systems. In the presence of an inducer, transgene expression is ‘on,’ while in the absence of a hormone, the promoter is not activated and the expression is effectively blocked. An advantage of drug-controlled gene transfer systems is that genes can be delivered in a relatively dose-dependent manner with more consistent and predictable expression. As these are only the first steps in controlling expression of transgenes, it is important to understand the limitations of regulated gene therapy systems before applying them in clinical trials. More studies in animal models should address issues of safety. The ideal solution would be to develop a system that would place a transgene under the control of both a tissue-specific promoter and a disease-specific promoter. The first reports of such advanced systems have already been published [100] and may soon be used in human studies.

Targeting of Viral Vectors

Viral surface proteins that bind to the specific cell receptors work as the primary means of initiating cellular attachment. The expression of specific surface molecules produces the tissue and cellular tropism for particular viral vectors. Most viral vectors transduce a relatively broad spectrum of host cells. The main goal of targeted gene therapy is to specifically infect a single cell type or group of cells and the choice of the right vector is crucial for specific and targeted gene delivery. For example, there are eight natural AAV serotypes which have been studied for gene transfer (AAV-1–8). In the majority of studies for PD, the neurotropic AAV-2 vector was used. AAV-5 has much broader tropism and also drives efficient gene expression in astrocytes and epithelial cells [56]. Other serotypes demonstrate preference for skeletal muscle (AAV-1), neurons (AAV-3), or ependymal cells (AAV-4). It is possible to modify viral surface structure by attaching or conjugating receptor ligands or antibodies. Restricting the vector’s ability to infect unwanted tissues decreases nonspecific infectivity, which is, of course, an unwelcome result of every in vivo gene therapy strategy. Incorporation of the vesicular stomatitis viral glycoprotein has been shown to increase infectivity of retrovirus [101–103], but decreases with HSV virions [104]. It is possible to generate an Ad vector expressing a chimeric fiber protein which alters the recognition profile of the virus. In the CNS, one could design a strategy with a fiber protein conjugated to a neurotrophic factor. This would preferentially target the vector to neurons expressing the receptor for the conjugated neurotrophic factor. The enhancement of the affinity of the virus for a particular cell type by modification of the viral coat could result in lowering the number of viral vector particles to

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be used in vivo, an added advantage especially because it may have an important effect on the immune response against immunogenic vectors like Ad. Nonviral vectors (e.g., DNA-polyplexes, liposomes) have practically no selectivity at the level of their incorporation into cells; therefore, introducing specific ligands has been the major solution in designing targeted gene delivery. Attachment or incorporation of antibodies is a commonly used strategy. Receptor targeting increases transduction efficiency of disease-affected cells, while decreasing gene delivery to nontarget cells. This is perhaps of importance in PD where a very specific and isolated subset of neurons in the nigra and striatum is primarily affected. Of course, the use of selected promoters that are active only within subsets of cells or the use of cell-type-specific drug-inducible promoters, are solutions to the problem of nonspecificity in this context.

The ex vivo Approach

Ex vivo gene transfer offers the potential for persistent and regulated local and widespread delivery of therapeutic agents into the CNS. Several strategies utilizing genetically engineered cells for treating PD are currently under investigation. These strategies consist of introducing therapeutic genes to cells and grafting the modified cells into the diseased brain region. In PD models, genetically modified cells may be aimed at DA replacement in the denervated striatum, whereby the therapeutic cells are transduced with multiple genes that encode for the enzymes and cofactors involved in the biosynthesis of DA; or protecting the remaining midbrain dopaminergic cells that are still functional from degeneration. The therapeutic effect also may be aimed at rescuing DA cells that have begun the process of degeneration through the production of local trophic factors. Among growth factors that have been described to support the survival and/or regeneration of the midbrain DA neurons are brain-derived neurotrophic factor, basic fibroblastic growth factor (bFGF), insulin-derived growth factor, and glial cell line-derived neurotrophic factors (i.e., GDNF, neurturin, persephin, artemin). GDNF has the most prominent and selective effect in rescuing midbrain DA neurons, increasing DA activity and improving behavioral deficits of both rodent and primate models of PD.

Source of Cells for ex vivo Gene Therapy

There are multiple potential sources of cells for ex vivo gene therapy. The cells used as delivery vehicles must meet at least three basic criteria: (1) they should not form tumors in vivo; (2) they should graft at the site of the diseased

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brain area, and (3) they should not elicit a strong host immune response. Autologous cells isolated from patient biopsies meet these criteria and neural cells derived from the brain in particular represent an attractive source. However, this source is unpractical because of the difficulties in isolating and maintaining neuronal cells in culture and obtaining adequate numbers for clinical applications. Myoblasts, skin fibroblasts, and bone marrow stem cells all have been considered for ex vivo gene therapy for PD. While myoblasts demonstrated some limited success, bone marrow stem cells transduced with either L-dopa or TH demonstrated limited neuronal differentiation and functional integration within host tissue. Skin fibroblasts have been a popular source for autologous cells; they demonstrated good survival in a primate model of PD, with expression of transgene that lasted for several months [74]. However, long-term gene expression by grafted fibroblasts has not been shown to be successful in rat models of PD. The reason for this failure may be due to inflammatory cytokine reaction to traumatic changes in the host tissue. It is important to note that these cells do not process the cellular machinery to store and release DA; to be competent for such a function within the host striatum, fibroblasts would need to be transduced with DA transporters and other genes involved in DA storage and release mechanisms [74, 105, 106]. Indeed, Lee et al. [106] cotransduced rat fibroblasts with both vesicular monoamine transporter-2 and AADC genes and demonstrated that these cells were then capable of converting L-dopa to DA and of storing DA. Transplantation of these engineered fibroblasts into a rat model of PD resulted in efficient DA production and storage. Other cell lines have been explored as a vehicle for gene transfer in PD preclinical studies, which include immortalized fibroblasts, immortalized fetal astrocytes, schwanoma and glioma cell lines, and endothelial cells [107–111]. The cells were engineered to produce enzymes or trophic factors and then grafted into the striatum of PD model. While these cell lines survived and expressed the transgene after implantation, most of them also gave rise to tumors, initiated immunological rejection, or did not integrate and died. Neural precursor cells are capable of giving rise to neurons, astrocytes and oligodendrocytes, and migrating and integrating into the local circuitry. These cells are preferred for grafting applications, as they approximate the normal physiological activity of neural cells. One approach is to isolate purified midbrain dopaminergic neurons by using a cell type-specific live monitoring technique. To achieve this selective isolation, Sawamoto et al. [112] generated transgenic mice and rats expressing GFP under the control of a 9-kb rat TH promoter. The authors demonstrated that expression of GFP protein was specific to DA neurons in the mesencephalon. The rat fetal midbrain was dissected out and dissociated cells were sorted using the fluorescent activated cell sorting. This purification step yielded an enriched population of TH-GFP positive neurons

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(⬎60%). This sorted and enriched population of DA neurons improved the behavioral deficits of 6-OHDA lesioned rats. A similar approach was applied by the same group to isolate midbrain precursor cells [113]. The GFP reporter gene was under control of the neural-specific second intronic enhancer of the nestin gene. Nestin GFP mice showed strong fluorescence in the ventricular zone where precursors are known to reside. Neurospheres generated from the fluorescent activated cell-sorted nestin-GFP precursors were implanted into the 6-OHDA rat model of PD and 5 weeks after transplantation the rats demonstrated a significant behavioral improvement. Other studies have used nonmidbrain-derived stem cells transduced with the transcription factor Nurr1 (which promotes a dopaminergic neuronal phenotype) in order to induce stable midbrain DA lineage. For instance, to induce a dopaminergic cell lineage, Wagner et al. [114] transduced C17.2 cells [115] with Nurr1 and incubated the cultured cells with soluble factors derived from ventral mesencephalic type 1 astrocytes. This treatment resulted in the induction of dopaminergic fate in 80% of the total cells [114]. The genetic modification of stem cells with Nurr1 was also required to efficiently convert embryonic stem (ES) cells to DA neurons. Kim et al. [116] demonstrated that overexpression of Nurr1 alone promoted a 10-fold increase in the number of TH-expressing neurons. The administration of Shh (Sonic hedgehog) and FGF8 resulted in an additional 5.6-fold increase in the proportion of TH-positive neurons. The ability of these newly induced TH-positive neurons to synthesize and release DA was demonstrated using HPLC. After implantation into the 6-OHDA lesioned rats, TH-positive neurons survived and extended processes within the host parenchyma. These cells were postmitotic as demonstrated by the absence of the cell proliferation marker expression Ki-67. In other studies, Nurr1 ES cells grafted in parkinsonian rats improved their rotational behavioral and motor skills as tested in the adjusting step, cylinder, and paw-reaching tests. Fetal Mesencepahlic DA Neurons In both rodent and monkey models of PD, midbrain-derived fetal tissue implants are able to survive, extend neurites, make functional synaptic contact with host neurons, and secrete DA leading to a dramatic improvement in behavioral deficits [117–120]. Based on these studies, clinical trials of primary fetal nigral cell transplantation for medically intractable PD were initiated in the 1980s. Open-label clinical trials with mesencephalic DA neurons obtained from 6- to 9-week-old aborted human fetuses demonstrated graft survival, DA storage and release (assayed with PET), and significant and persistent improvement as measured with Unified Parkinson Disease Rating Scales (UPDRS) relative to the baseline. Histopathological demonstration of striatal DA reinnervation was obtained for a period extending up to 10 years [121–125]. These encouraging

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findings have been recently called into question, however, by the first double-blind placebo-controlled clinical trial [89]. This large, placebo-controlled study involving sham surgeries enrolled 40 patients, 20 transplant subjects and 20 controls who underwent sham surgery. The graft survival rate in transplanted patient was 85%, without the use of immunosuppression. Young patients (⬍60 years) demonstrated 28% and 34% improvement in UPDRS total and motor ‘off’ components, respectively. However, despite cell survival and DA production demonstrated with PET, clinical improvement was not as dramatic in patients in the typical age range for PD (⬎60 years). Of greatest concern were 5 patients who developed severe dyskinesias and/or dystonia in the absence of levodopa. This graft-induced dyskinesia was termed ‘runaway’ dyskinesia because it was an uncontrolled, off-medication side effect. The authors’ interpretation was that the side effects were due to graft overgrowth and an excess of DA production. Subsequently, it has been argued that this conclusion was somewhat simplistic and the negative outcome of this study is partially attributable to extended culture of donor tissue, unconventional neurosurgical procedures, and an absence of immunosuppression [126, 127]. Nevertheless, together with a study by Fahn and coworkers [89] using standardized cell transplantation procedures and assessment protocols, Hagell et al. [128] observed a pronounced ‘runaway’ dyskinesias due to fetal mesencephalic DA cell grafts. As with the trial by Fahn, these dyskinesias persisted after withdrawal of L-dopa and DA agonists [128]. In the latter study, the authors argued that the ‘runaway’ dyskinesias are not caused by overgrowth of grafted cells, but could be due to micrograft DA spillover which overstimulated supersensitive receptors outside the graft-innervated area. These authors also speculated that the development of ‘runaway’ dyskinesias could be due to the extended storage or culture of donor tissue before grafting or to transplantation-evoked changes in host striatum or nondopaminergic components of the grafts. More recently, a third report on the second double-blind, placebo-controlled trial of fetal nigral transplants [91] demonstrated a failure to induce significant motor improvement relative to placebo. Moreover, patients who received grafts also developed severe ‘runaway’ dyskinesias that tended to appear 6–12 months after transplantation. Patients with a higher dose (4 donor grafts) showed improvement at 6 and 9 months and deteriorated thereafter, coincident with the termination of cyclosporine intake, suggesting a possible immune reaction against the graft. Indeed, activated microglia immunostaining with CD45 antibody demonstrated an increased immune reaction particularly surrounding the graft deposits compared to the placebo. Significant improvement was noted in patients with milder disease at baseline, UPDRS ⱕ 49. The development of runaway dyskinesias did not depend on differences in fluorodopa uptake, nor on the dose of cells implanted.

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A study by Ma et al. [129] had suggested that higher fluorodopa uptake coincides with dyskinesia side effects. Contrary to Hagell’s [128] interpretation, the use of fresh donor tissue (less than 48 h storage) did not prevent the development of debilitating side effects [91]. In view of the new risks of transplantation that were exposed, Fahn et al. recommended against using fetal nigral cells as a cell-based therapy for PD at this time, yet suggest that patients with milder disease may benefit from such a strategy if they receive grafts with a higher number of surviving cells and a more prolonged immunosuppressant. It appears likely that overproduction and focal pulsatile delivery of DA within the dennervated striatum can lead to the development of uncontrolled dyskinesias, and future use of DA tissue grafts will need to address this issue of DA regulation and immune regulation. Stem Cells: ES and Brain-Derived The use of stem cells for ‘cell therapy’ of PD requires directing the cells toward dopaminergic lineages before transplantation or at early stages of grafting. This preconditioning of stem cells has been tested in vitro using growth factors, cytokines, and conditioned media for forebrain-derived neural precursors [130, 131] and for midbrain cells as well [132–135]. Transplantation of nonimmortalized stem cells into parkinsonian animal models has led to survival, integration, and expression of TH, the rate-limiting enzyme of DA synthesis, and to behavioral improvement of the lesioned animals [133,135–138]. Clonally derived neural stem cells [115] were shown to spontaneously differentiate into TH-expressing cells in a rat model of PD, a characteristic that is dependant on the culture confluency of the clone and the host’s microenvironment [139]. DA neurons can be derived form ES cells using a multistep protocol. In a study by Kawasaki et al. [140], ES cells were maintained in an undifferentiated state in media supplemented with serum and leukemia inhibitory factor. To direct differentiation toward the DA lineage, ES cells were cocultured with PA6 stromal cells for 8 days and then ascorbate was added to the media for 6–12 days. With this treatment, 16% of the total cell population was converted into DA neurons expressing TH- and DA lineage-specific transcription factors Nurr1 and Ptx3. After implantation into the 6-OHDA rat model of PD, the ES-derived neurons maintained the DA phenotype and did not form tumors. However, behavioral analysis of these animals was not reported. In another study by Lee et al. [141], ES cells were maintained in serum and media containing leukemia inhibitory factor. Removing leukemia inhibitory factor for 4 days and then growing the cells on adhesive substrate for 24 h induced the formation of embryoid bodies. Stem cells were subsequently expanded in serum-free media for 6–10 days before inducing DA neurons with bFGF, sonic hedgehog (Shh-N), and FGF8 for an additional 6 days. Maturation of DA neurons was established by

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culturing the cells in media supplemented with cAMP and ascorbate. Under these conditions 23% of the total cells were dopaminergic. These DA neurons expressed transcription factors characteristic of midbrain DA neurons; however, they were not assessed for their functional ability to reverse behavioral deficits in PD animal model. Naïve ES cells also have been implanted into 6-OHDA denervated rat striatum [142]. In this study, ES cells gave rise to TH-immunoreactive neurons that also expressed DA transporter and AADC. Using PET and functional MRI, the grafted ES cells demonstrated the appropriate dopaminergic neuronal properties which paralleled behavioral recovery demonstrated with the apomorhine rotational test [142]. This study also reported, however, that ES cell transplantation led to the generation of lethal teratomas in 20% of implanted animals. While these findings are encouraging, further in vitro manipulation of ES cells and long-term posttransplant survival studies are required to provide assurance that tumor formation does not occur, an unacceptable outcome in a disease with existing drug and surgical therapies. One possible technology to control both growth rate and lineage of the cells before transplantation is genetic modification, such as providing a repressible regulatory unit or suicide gene that could be induced as required.

Detection of Gene Expression

PET Neuroimaging techniques and behavioral analyses make it possible to assess in vivo the state of the DA system in patient and animal models. Functional studies provide valuable information about the structure and function of DA neurons and the effects of therapeutic approaches. These techniques permit quantitative measurement of changes in DA terminals, receptors, and release of DA in vivo. PET and the use of specific radiolabeled ligands can noninvasively quantify pre- and postsynaptic markers of the DA system. Many of these tracers bind selectively to specific transporters, such as DA transporter or the vesicular monoamine transporter 2. The type of ligand utilized will determine the information we can obtain about a particular system [143]. Such noninvasive techniques are ideal for longitudinal studies in experimental models of PD. Conceivably, they could be used to monitor the progress in effectiveness of gene therapy approaches by monitoring the transgene expression. Studies from our lab demonstrated that PET was successfully applied to monitor AADC expression introduced by AAV-based vector [71]. Another study from our laboratory in the MPTP monkey model of PD demonstrated the ability to distinguish between dopaminergic changes in the putamen and the SN

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compacta using PET scanning [144]. The previously cited study by Kordower et al. [48] with AAV-GDNF used PET to monitor therapeutic effects of gene transfer in a MPTP model. The choice of imaging ligands is especially important because the use of growth factors and other therapeutic interventions often specifically target either the terminals or cell bodies. In vivo detection of gene expression, as seen in studies after AADC gene transfer in MPTP-treated monkeys, is important because it provides quantitative assessment of gene transfer. This approach applies for both in vivo and in vitro gene transfer where duration, levels, and location of AADC gene expression can be detected. Microdialysis In vivo intracerebral microdialysis has been used in rats, nonhuman primates, and humans to monitor the extracellular level of neurotransmitters. This method can be very useful to evaluate alterations in brain metabolism. Microdialysis probes, connected to microinjection pumps, are stereotactically inserted into targeted points in the brain. Artificial cerebrospinal fluid is administered at a slow rate and dialysates are collected to microtubes for chemical analysis. The amount of neurotransmitters in each fraction is determined by HPLC. Alternatively, by directly sampling cerebrospinal fluid from the lateral ventricles, the levels of amino acids can be measured. Microdialysis was used during stereotaxic thalamic surgery for PD tremor for neurochemical characterization of the target area [145]. Studies by Fedele et al. [146] confirmed that this method might be used in PD to measure amino acid release in human basal ganglia. Using this approach one can assess the level of DA restoration via gene therapy. The very same procedure was successfully used by Pernaute et al. [70] to evaluate the functional effect of AAV-mediated gene transfer of aromatic L-amino acid decarboxylase into the striatum of 6 OHDA-lesioned rats.

Conclusions

PD is characterized primarily by the degeneration of a specific population of neurons in SN and a decline in local neurotransmitter synthesis. Replacement therapy with the DA precursor L-dopa has been a mainstay of therapy for PD. However, L-dopa addresses only the biochemical consequences of the disease and leads to long-term side effects such as dyskinesias. Prevention of further loss of dopaminergic neurons by neuroprotection, perhaps using gene transfer, is one alternative approach to treatment. Advances in cellular and genetic engineering also will permit stem cell transplants to replace neurons once they have been lost. Gene therapy for PD based on in vivo or ex vivo strategies is realistic, but will depend on the progress that is made over the years to come.

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119 Redmond DE, Sladek JR Jr, Roth RH, Collier TJ, Elsworth JD, Deutch AY, Haber S: Fetal neuronal grafts in monkeys given methylphenyltetrahydropyridine. Lancet 1986;1:1125–1127. 120 Bankiewicz KS, Plunkett RJ, Jacobowitz DM, Porrino L, di Porzio U, London WT, Kopin IJ, Oldfield EH: The effect of fetal mesencephalon implants on primate MPTP-induced parkinsonism. Histochemical and behavioral studies. J Neurosurg 1990;72:231–244. 121 Freeman TB, Olanow CW, Hauser RA, Nauert GM, Smith DA, Borlongan CV, Sanberg PR, Holt DA, Kordower JH, Vingerhoets FJ: Bilateral fetal nigral transplantation into the postcommissural putamen in Parkinson’s disease. Ann Neurol 1995;38:379–388. 122 Hauser RA, Freeman TB, Snow BJ, Nauert M, Gauger L, Kordower JH, Olanow CW: Long-term evaluation of bilateral fetal nigral transplantation in Parkinson disease. Arch Neurol 1999;56: 179–187. 123 Kordower JH, Freeman TB, Snow BJ, Vingerhoets FJ, Mufson EJ, Sanberg PR, Hauser RA, Smith DA, Nauert GM, Perl DP, Olanow CW: Neuropathological evidence of graft survival and striatal reinnervation after the transplantation of fetal mesencephalic tissue in a patient with Parkinson’s disease. N Engl J Med 1995;332:1118–1124. 124 Kordower JH, Freeman TB, Chen EY, Mufson EJ, Sanberg PR, Hauser RA, Snow B, Olanow CW: Fetal nigral grafts survive and mediate clinical benefit in a patient with Parkinson’s disease. Mov Disord 1998;13:383–393. 125 Piccini P, Brooks DJ, Bjorklund A, Gunn RN, Grasby PM, Rimoldi O, Brundin P, Hagell P, Rehncrona S, Widner H, Lindvall O: Dopamine release from nigral transplants visualized in vivo in a Parkinson’s patient. Nat Neurosci 1999;2:1137–1140. 126 Brundin P, Dunnett S, Bjorklund A, Nikkhah G: Transplanted dopaminergic neurons: More or less? Nat Med 2001;7:512–513. 127 Nikkhah G: Neural transplantation therapy for Parkinson’s disease: Potential and pitfalls. Brain Res Bull 2001;56:509. 128 Hagell P, Piccini P, Bjorklund A, Brundin P, Rehncrona S, Widner H, Crabb L, Pavese N, Oertel WH, Quinn N, Brooks DJ, Lindvall O: Dyskinesias following neural transplantation in Parkinson’s disease. Nat Neurosci 2002;5:627–628. 129 Ma Y, Feigin A, Dhawan V, Fukuda M, Shi Q, Greene P, Breeze R, Fahn S, Freed C, Eidelberg D: Dyskinesia after fetal cell transplantation for parkinsonism: A PET study. Ann Neurol 2002;52: 628–634. 130 Carpenter MK, Cui X, Hu ZY, Jackson J, Sherman S, Seiger A, Wahlberg LU: In vitro expansion of a multipotent population of human neural progenitor cells. Exp Neurol 1999;158:265–278. 131 Daadi MM, Weiss S: Generation of tyrosine hydroxylase-producing neurons from precursors of the embryonic and adult forebrain. J Neurosci 1999;19:4484–4497. 132 Ling ZD, Potter ED, Lipton JW, Carvey PM: Differentiation of mesencephalic progenitor cells into dopaminergic neurons by cytokines. Exp Neurol 1998;149:411–423. 133 Studer L, Tabar V, McKay RD: Transplantation of expanded mesencephalic precursors leads to recovery in parkinsonian rats. Nat Neurosci 1998;1:290–295. 134 Yan H, Bunge MB, Wood PM, Plant GW: Mitogenic response of adult rat olfactory ensheathing glia to four growth factors. Glia 2001;33:334–342. 135 Sanchez-Pernaute R, Studer L, Bankiewicz KS, Major EO, McKay RD: In vitro generation and transplantation of precursor-derived human dopamine neurons. J Neurosci Res 2001;65: 284–288. 136 Arenas E: Stem cells in the treatment of Parkinson’s disease. Brain Res Bull 2002;57:795–808. 137 Svendsen CN, Caldwell MA, Shen J, ter Borg MG, Rosser AE, Tyers P, Karmiol S, Dunnett SB: Long-term survival of human central nervous system progenitor cells transplanted into a rat model of Parkinson’s disease. Exp Neurol 1997;148:135–146. 138 Carvey PM, Ling ZD, Sortwell CE, Pitzer MR, McGuire SO, Storch A, Collier TJ: A clonal line of mesencephalic progenitor cells converted to dopamine neurons by hematopoietic cytokines: A source of cells for transplantation in Parkinson’s disease. Exp Neurol 2001;171:98–108. 139 Yang M, Stull ND, Berk MA, Snyder EY, Iacovitti L: Neural stem cells spontaneously express dopaminergic traits after transplantation into the intact or 6-hydroxydopamine-lesioned rat. Exp Neurol 2002;177:50–60.

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140 Kawasaki H, Mizuseki K, Nishikawa S, Kaneko S, Kuwana Y, Nakanishi S, Nishikawa SI, Sasai Y: Induction of midbrain dopaminergic neurons from ES cells by stromal cell-derived inducing activity. Neuron 2000;28:31–40. 141 Lee SH, Lumelsky N, Studer L, Auerbach JM, McKay RD: Efficient generation of midbrain and hindbrain neurons from mouse embryonic stem cells. Nat Biotechnol 2000;18:675–679. 142 Bjorklund LM, Sanchez-Pernaute R, Chung S, Andersson T, Chen IY, McNaught KS, Brownell AL, Jenkins BG, Wahlestedt C, Kim KS, Isacson O: Embryonic stem cells develop into functional dopaminergic neurons after transplantation in a Parkinson rat model. Proc Natl Acad Sci USA 2002;99:2344–2349. 143 Sanchez-Pernaute R, Brownell AL, Isacson O: Functional imaging of the dopamine system: In vivo evaluation of dopamine deficiency and restoration. Neurotoxicology 2002;23:469–478. 144 Eberling JL, Bankiewicz KS, Jordan S, VanBrocklin HF, Jagust WJ: PET studies of functional compensation in a primate model of Parkinson’s disease. Neuroreport 1997;8:2727–2733. 145 Meyerson BA, Linderoth B, Karlsson H, Ungerstedt U: Microdialysis in the human brain: Extracellular measurements in the thalamus of parkinsonian patients. Life Sci 1990;46:301–308. 146 Fedele E, Mazzone P, Stefani A, Bassi A, Ansaldo MA, Raiteri M, Altibrandi MG, Pierantozzi M, Giacomini P, Bernardi G, Stanzione P: Microdialysis in Parkinsonian patient basal ganglia: Acute apomorphine-induced clinical and electrophysiological effects not paralleled by changes in the release of neuroactive amino acids. Exp Neurol 2001;167:356–365. 147 Bankiewicz KS, Daadi MM, Pivirotto P, Bringas J, Sanchez-Pernaute R, Herscovitch P, Carson R, Eckelman W, Cunningham J, Reutter B, VanBrocklin HF, Eberling JL. Long term evaluation of AAV/AADC gene transfer in parkinsonian monkeys. 33rd Annual Meeting of Society for Neuroscience, 2003, Washington DC. Neuroscience 2003, p. Program No. 299.214.

Dr. Krystof Bankiewicz Department of Neurosurgery, University of California San Francisco MCB, 1855 Folsom Street, Room 225, San Francisco, CA 94103 (USA) Tel. ⫹1 415 502 3132, Fax ⫹1 415 514 2777, E-Mail [email protected]

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Simplifying Complex Neurodegenerative Diseases by Gene Chip Analysis Clemens R. Scherzera, Steven R. Gullansa, Roderick V. Jensenb a

Laboratory for Functional Genomics, Center for Neurologic Diseases, Harvard Medical School, Brigham and Women’s Hospital, Cambridge, Mass., and b Department of Physics, Wesleyan University, Middletown, Conn., USA

Genes for many autosomal dominant or recessive neurodegenerative diseases have been already identified. However, little is known about the complex genetics behind the vast majority of sporadic or ‘idiopathic’ neurodegenerative diseases. These diseases are likely to be caused by the combinatorial effect of several susceptibility genes acting in concert with environmental risk factors. Identifying the relevant genes, elucidating their molecular function, and defining targets for neuroprotective drugs pose great challenges and will require novel scientific methodologies. These genetic strategies will help to bring the benefits of the recent genomic revolution to the clinic and the operating room, by developing treatment strategies for neurodegenerative diseases. Traditional scientific approaches have always focused on serial studies of one gene at a time. For complex diseases that are caused by a multiplicity of susceptibility genes, high-throughput analysis of many genes in parallel is a more efficient and informative approach, though cost considerations have been a major problem in the past. Gene chips or ‘microarrays’ attach probes for transcripts of tens of thousands of genes onto a rigid support such as a glass slide and permit a comprehensive genome-wide analysis of transcript changes. This chapter will discuss how gene chip technology can be applied to the investigation of neurodegenerative diseases. We will address how gene chips can identify candidate disease-modifying genes and prioritize susceptibility genes for genotyping in complex neurodegenerative diseases. We envision that in the future, gene chip analysis will efficiently detect the molecular fingerprints associated with distinct clinical states and will define unique gene activity profiles

or ‘mRNA barcodes’ for specific clinical traits. In clinical practice, these tools may assist in the diagnosis and prognosis of neurodegenerative diseases, more accurately predict individual treatment responses, and be used as markers of disease risk in presymptomatic subjects.

Current Best Practices of Microarray Technique: Refining Modifier Candidates

Primary Screen In our experience, many investigators would like to use a combination of microarrays, bioinformatics, and simple validation experiments to define a short list of one to ten high-priority candidate genes. A stepwise filtering process is generally applied to the initial microarray datasets. We typically start with error models tailored to the specific microarray platforms, to optimize quantification of the gene expression levels. We also recommend a stringent three-step statistical analysis to minimize false positives due to biological or technical variation and to correct for multiple testing. First, a selective intensity filter is applied to exclude genes with low hybridization signal intensities, because false-positive results are particularly high for low-intensity genes. With Affymetrix gene arrays, we generally require that the gene ‘Average Difference’ or ‘Signal’ be greater that the ‘Target Intensity’ (defined as the trimmed-mean expression level on the array) for at least one sample in the study. This will focus further analysis on the 30–40% most abundant transcripts. Second, a ratio threshold (generally fold changes of >1.5–2.0) is applied to eliminate small changes in expression that are of unclear technical and biological significance. Although smaller fold-changes may be statistically significant they are very difficult to verify by other means (e.g., quantitative polymerase chain reaction with reverse transcription; RT–PCR). Finally, a t-like test statistic is used to identify genes that are expressed differentially on the basis of confidence values or P values [1]. Permutation tests (e.g., Significance Analysis of Microarrays [2]) are performed to estimate the significance of the test statistic and to correct for multiple testing. The number of false positives expected by chance alone is determined by repeatedly permuting the samples’ class labels and computing t statistics for all genes in the scrambled data. Secondary Screen To qualify each gene further after the primary microarray assessment, a secondary screen may be required to independently confirm the observed changes in gene expression. If the primary screen results in a relatively short

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list (less than fifty genes), quantitative RT–PCR can be performed (on samples) for technical validation. Investigators may further prioritize genes as candidate targets on the basis of their organismal roles; for example, hormones may be favored as potential therapeutic proteins, or receptors or enzymes that are amenable to modulation by small-molecule drugs may be chosen (for further study). For genes with unknown or unclear functions, prioritizing those of greatest physiological relevance requires further analysis such as quantitative RT–PCR or protein expression analysis. Western blot or immunohistochemistry are preferred for protein analysis, but an antibody is not always readily available. The secondary screening process may obtain a more detailed dissection of the biological process using time series, more diverse biological samples, and anatomical specificity. Shotgun Microarrays Secondary screens become labor intensive, time consuming, and expensive if a large list of genes need be confirmed. Therefore, we have begun to use multiple microarray platforms for efficient technical validation of large numbers of differentially expressed genes. Different high-density oligonucleotide platforms (e.g., Affymetrix, Amersham, Agilent) spot distinct probes for the genes interrogated and have distinct technical advantages and weaknesses. Our results suggest that for the more highly expressed transcripts, 70–80% of the >2-fold gene expression changes are concordant when the same RNA sample is run on Affymetrix and Amersham arrays. In our opinion, the current optimal secondary screen takes advantage of two independent high-density oligonucleotide platforms in a cross-validation strategy that we term ‘shotgun’ or sequential microarray analysis. Error Minimization When using microarrays to identify differentially expressed genes, it is important to recognize the inherent error caused by technical and biological variations. Reproducibility and sensitivity problems can generate both falsenegative and false-positive results. But these issues can be addressed readily through robust experimental design, rigorous statistical analysis, the use of biological and technical replicates, and independent verification by quantitative RT–PCR or other microarray platforms. Although microarrays represent a powerful tool for forming initial hypotheses, it is essential to consider the limitations of interpreting biological responses through measurements of mRNA abundance alone. Measurements of mRNA do not directly reflect protein quantities, enzyme activities, or extranuclear signal transduction. Microarray experiments also may fail to resolve true ‘modifier genes’ from homeostatic responses that attempt to restore the original state of the system. Generally, microarray

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measurements fail to resolve cause from effect. Thus, successful use of microarray technology requires that sources of error be controlled carefully in the design and execution of experiments. Biological Validation The primary microarray screen will identify a shortlist of high-priority modifier candidates. Each type of selective profiling identifies differentially expressed genes characteristic for a particular RNA source. The choice of (tissue) source and controls will modify the biases flowing into the results of the screen. Invariably, validation experiments will be indicated to distinguish microarray-derived candidates that are strong modifiers of the disease process and to overcome the limitations of each RNA source. Several approaches can be taken to validate and to prioritize candidate modifiers once a shortlist has been identified. Among the most important are gene knockout and knock-in strategies in cells and model organisms, because these can replicate more closely the actions of potential modifiers and identify phenotypic changes and mechanisms. For a high-throughput genetic validation of microarray candidates, simple model organisms such as yeast, flies, and worms are most frequently used. An elegant application of this strategy resulted in the discovery of a new modifier candidate for multiple sclerosis (MS). Microarray analysis of MS lesions yielded new modifiers of MS that were validated in autoimmune encephalomyelitis [3]. In a landmark study [3], Lawrence Steinman’s group at Stanford defined microarray-derived modifiers of human MS. By combining expression analysis and high-throughput sequencing of expressed sequence tags in a rat model of MS and human MS plaque tissue, they found an increase in osteopontin mRNA abundance in both human and rat tissues. The biological role of osteopontin in the progression of MS was then further validated in knockout mice: osteopontin-deficient mice were resistant to the progressive MS subtype and had significantly more remissions compared to wild-type mice. Using microarrays as a screening tool, osteopontin is now a promising novel drug target for blocking progressive MS in humans.

Prioritizing Candidate Suppressors or Enhancers of Neurodegeneration through Gene Chip Analysis

Selective Vulnerability Profiles When using microarrays to discover modifier genes in neurodegenerative diseases, genome-wide mRNA expression profile is determined in postmortem brain tissue from patients. The investigator applies a series of noise filters and

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significance statistics to identify candidate modifier genes that are differentially expressed in patient tissue out of the tens of thousands of genes interrogated by human genome arrays. Neurodegenerative processes are highly selective for specific neuronal populations and brain regions and are often associated with characteristic histological lesions. Each neurodegenerative disease preferentially affects distinct neuronal populations and distinct brain regions and is associated with hallmark histopathological lesions. This vulnerable neuronal population is often distributed in distinct brain regions. For example, in Parkinson’s disease (PD) dopaminergic neurons localized to the substantia nigra pars compacta are predominantly affected, while dopaminergic cells in other brain regions are less vulnerable. Regional and cellular profiling techniques have been developed that are tailored to investigate the selective regional and cellular vulnerability of neurodegenerative diseases. Expression analysis of vulnerable brain regions (regional profiling), vulnerable neuronal or glial populations (cellular profiling), or characteristic histological lesions such as MS plaques [3] (lesion profiling) has lead to intriguing results reflecting the strengths and weaknesses of each approach. Regional Profiling Nonspecific gene expression changes related to neuronal loss or reactive glial proliferation must be considered in the interpretation of gene expression in affected brain regions. Hauser et al. [in preparation] have used disease controls with dopaminergic cell loss such as progressive supranuclear paralysis to control cell loss not specific to PD pathogenesis. Alternatively, expression changes of neuronal markers such as neurofilaments or of neuronal specific subpopulations such as tyrosine hydroxylase and other dopamine biosynthesis enzymes, and glial markers such as glial fibrillary acidic protein, may be used to estimate the range of gene expression changes accounted for by unspecific cell loss and gliosis alone. Validation of regional expression changes in vulnerable neuronal populations by double-labeling immunohistochemistry or doublelabeling in situ hybridization can address this concern. Analysis of gene expression in patients ‘at risk’ or at presymptomatic disease stages could reduce some of these biases but tissue availability and diagnostic uncertainty limit this approach. Cellular Profiling Laser-capture microdissection (LCM) of vulnerable neuronal populations allows direct sampling of the neuronal population of interest under the microscope [4–6]. LCM controls for some biases associated with regional profiling such as reactive gliosis or nonspecific neuron loss. Distinct considerations

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guide the interpretation of LCM expression profiles. During interpretation of results, one must take into account whether gene expression changes observed are specific to the disease in question or whether they may be generally found in dying neurons irrespective of the specific disease process. Comparison with cellular profiles in disease controls could help to estimate this bias. In addition, a selection bias might be introduced by LCM; cellular profiling might select for neurons less affected in the disease process. This is particularly a concern if advanced disease stages are profiled. For example, in PD, an estimated 70% of nigral neurons have died prior to the onset of clinical symptoms [7]. Dopaminergic neurons that survive the disease process and thus are found in postmortem tissue might reflect a particularly resistant subpopulation rather than reflecting the transcription profile of vulnerable dopaminergic cells. The cellular gene expression profile thus might identify transcripts of genes conferring enhanced resistance within the vulnerable cell population. Extraneuronal Profiling A novel approach to avoid some of these limitations has made use of altered gene expression in peripheral tissues of patients with neurodegenerative diseases. In this paradigm, neurodegenerative diseases are approached as a systemic disease with systemic changes in the expression of disease-modifying and susceptibility genes that act in a combinatorial fashion with localizing factors unique to vulnerable neuronal populations and lead to selective neurodegeneration. Biochemical and transcriptional alterations in peripheral tissues such as platelets [8], lymphocytes [9, 10], fibroblasts [11] and muscle of neurodegenerative patients have been extensively documented in Alzheimer’s disease (AD), PD, and other neurodegenerative diseases. Indeed, most genes implicated in familial AD [8, 11–13] and familial PD [14, 15] are ubiquitously expressed. To gain insight into the molecular basis of these alterations, we [23] screened differential gene expression in lymphoblasts of controls and two independent groups of AD patients using cDNA microarrays. This genomic screen identified six differentially expressed genes. One of the six genes (LR11) is a novel neuronal ApoE receptor and thus an excellent candidate modifier. Subsequent validation experiments in the brain indicated that LR11 was enriched in vulnerable cortical and hippocampal pyramidal neurons in human control brains, and that it was concentrated in neuronal endosomal-lysosomal compartments. In striking contrast to normal tissue, LR11 was diminished in AD brains with dramatic reductions in surviving neurons. In cultured cells, LR11 overexpression markedly reduced extracellular A␤ levels, providing a mechanistic link between LR11 and A␤ clearance [Levey, unpubl. observations].

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Thus, changes in LR11 expression in AD lymphoblasts and brain, and its effects on extracellular A␤, suggested an important role for this apoE receptor in AD pathogenesis. Detection of Neuroprotective Targets: Gene Chip Analysis of Early Disease Stages Candidate Modifier Screen in Animal Models Toxic and genetic animal models of neurodegenerative diseases faithfully replicate key features of human neurodegenerative diseases. Microarray analysis of tissue from animal models, which is generally more available than human tissue samples, allows for dissection of the molecular machinery involved in progressive neurodegeneration. In extension of the ‘static’ gene expression snapshot detectable in human postmortem tissue representative of the disease endpoint, transgenic animal models allow for detection of the ‘dynamic’ range of gene expression changes during the disease progression, at any selected timepoints when the animals are sacrificed. This approach is particularly valuable in the analysis of chronic progressive neurodegenerative diseases. Pathology may begin several years prior to the onset of clinical symptoms and progresses from early disease stages associated with low morbidity and good response to medications to clinically debilitating end stages associated with the depletion of select neuronal populations. Specimens from animal models can capture these changes over the entire course of a disease, in statistically meaningful numbers. For example, in PD, tremor and bradykinesia develop only after an estimated 70% of vulnerable dopaminergic neurons in the substantia nigra have already died during the presymptomatic stage, spanning a period of years [7]. It is a fundamental goal for the neurologist to develop medications that stop or slow disease progression at presymptomatic or early disease stages. Modeling changes in presymptomatic or early symptomatic stages is especially crucial for understanding molecular pathogenesis and, perhaps even more importantly, for identifying therapeutic targets that might help to slow the disease process before it reaches the threshold for clinical symptoms. In one model of PD, Drosophila expressing human ␣-synuclein (␣S) carrying the disease-linked A30P mutation in a panneural pattern faithfully replicate age-dependent onset and chronic progression of human PD. Transgenic ␣S Drosophila develop adult-onset, progressive degeneration of dopaminergic cells, with widespread Lewy body inclusions and impaired locomotor function as monitored by progressive loss of climbing ability [16]. Loss of dopaminergic neurons and inclusion formation are first detected at 10 days of age, while at day 1 post-eclosion, the A30P-␣S Drosophila are still histologically and behaviorally normal.

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To identify gene expression changes at presymptomatic, early and advanced disease stages, our group hybridized RNA extracted from fly heads to highdensity oligonucleotide arrays spotted with probes representing the entire Drosophila genome. In presymptomatic ␣S transgenics, microarray analysis was more sensitive than conventional neuropathological techniques in elucidating disease-associated changes [17]. It was interesting that despite a ‘normal’ phenotype at this stage, in the one-day-old ␣S transgenics, transcription of thirty six genes was significantly and reproducibly dysregulated. These abnormalities presaged neuronal loss, Lewy body-like inclusion formation, and locomotor impairment at later stages. We found that the ␣S signature genes are dysregulated independent of disease stage in both presymptomatic and symptomatic animals (fig. 1). This suggests that parts of the molecular machinery dysregulated during symptomatic disease stages is already altered in presymptomatic transgenics prior to the onset of neurodegeneration (fig. 1). Thus, temporal profiling of progressive gene expression changes in neurodegenerative disease models provides unbiased starting points for defining disease mechanisms and for identifying potential targets for neuroprotective drugs at preclinical stages. Discovery of Susceptibility Genes by Converging Arraying and Mapping Genes controlling a certain clinical trait may cause variation in the trait through differential transcription due to DNA polymorphisms [18] that regulate transcription. Microarray analysis can assist traditional linkage analysis by identifying polymorphic transcription and in prioritizing candidate susceptibility genes. The correlation structure between transcript abundance and classical genetic linkage has been used to identify susceptibility loci for complex diseases such as diabetes [19] and asthma [20]. Most recently, convergence of gene expression and linkage analysis implicated a novel gene, glutathione S-transferase omega in the control of age-at-onset of AD [24]. A genetic linkage screen for age-at-onset in AD and PD has identified several chromosomal regions that may harbor novel age-at-onset genes [21]. The most interesting finding was a ⬃15 cM linkage region on chromosome 10q. This linkage peak was large, spanning over fifteen megabases and several hundred genes. Gene expression analysis probing for 22,000 human genes on RNA from 6 AD patient hippocampus and matched normal controls was performed to identify genes with polymorphic transcription in AD versus control brain. Fifty-two genes were identified that demonstrated significant differences in gene expression levels between AD and controls. Four of these fifty-two genes were physically located in the chromosome 10q linkage region. Genotyping fourteen single nucleotide polymorphisms in 1773 AD and 635 PD patients spanning these 4 candidates, and one functionally related

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a

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isoform, identified allelic association (p ⫽ 0.001) for age-at-onset with glutathione S-transferase omega, one of the 4 microarray-defined candidates. Thus, integration of the independent genetic linkage, gene expression, and allelic association evidence implicated a novel gene as a significant biological factor in the control of age-at-onset in AD. The Practice of Genomic Neurosurgery: Diagnosis, Subtype-Classification, and Treatment Personalization through Microarray-Derived Biomarkers The prospect of neuroprotective therapy has highlighted the crucial need for disease-specific biomarkers that identify patients at early stages and allow monitoring of disease progression. In addition, biomarkers for disease subtypes are needed to efficiently design clinical trials for neuroprotective drugs. In our opinion, transcription levels of susceptibility, disease-modifying, and treatment-modifying genes will result in defined gene expression ‘barcodes’ based on haplotypes or single-nucleotide polymorphisms. These gene expression barcodes will serve to diagnose patients with neurodegenerative diseases, to classify disease subtypes, and to predict treatment responses. Finally, using bioinformatics techniques such as the ‘gene ratios’ [22], a small number of genes will be extracted from the gene expression patterns that best define a clinical state. This small subset of genes will then be assayed by simple and widely available standard laboratory techniques such as quantitative real-time PCR. Fig. 1. Gene expression changes presage neurodegeneration in a Drosophila model of PD (from [17]). a 51 signature genes tightly associated with A30P-␣-synuclein expression independent of disease stage are clustered by hierarchical average-linkage analysis and visualized in a colorgram. The branches of the dendrogram comprising the cluster of four independent samples of presymptomatic 1-day-old transgenics are highlighted in pink. Expression levels higher than the mean are displayed in red, lower than the mean in blue. b–d While histology and behavior are normal in presymptomatic 1-day-old ␣S-transgenics, microarray profiles reveal a PD-specific expression signature. Graphs show the average fold change of select genes in different functional classes at day 1, 10, and 30 for ␣S transgenics (left panels) and tau transgenics (right panels). In R406W-tau transgenics, expression of the ␣S-signature genes is generally unchanged (changes not significant by SAM). Time points representing symptomatic stages of PD pathology are shaded gray. Signature expression of down-regulated lipid genes (b, and green font in a), up-regulated membrane transporters (c, orange font), and defense response genes (d, blue font) is detectable at the presymptomatic stage. e Using the ␣S-associated signature genes as classifiers, blinded hierarchical average-linkage analysis correctly distinguishes the eight ␣S samples from tau transgenics and, as expected, from normal controls. f Progressive up-regulation of a set of energy genes also begins in presymptomatics. This increase may be a compensatory response different from the energy genes uniquely down-regulated at day 1 (fig. 1a). [Reproduced with permission from Hum Mol Genet 2003;12:2457–2466, Oxford University Press.]

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Acknowledgements We thank Dr. Mel Feany at Brigham and Women’s Hospital, Harvard Medical School, for her helpful comments and critical review of the manuscript. The authors are supported by grants from the Harvard Center for Neurodegeneration and Repair, the Paul B. Beeson Career Development Award in Aging Research, the George C. Cotzias Memorial Fellowship from the American Parkinson Disease Association (to C.R.S.), and the Michael J. Fox Foundation (C.R.S, S.R.G., R.V.J).

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Scherzer CR, Jensen RV, Gullans SR, Feany MB: Gene expression changes presage neurodegeneration in a Drosophila model of Parkinson’s disease. Hum Mol Genet 2003;12:2457–2466. Schadt EE, Monks SA, Drake TA, Lusis AJ, Che N, Colinayo V, Ruff TG, Milligan SB, Lamb JR, Cavet G, et al: Genetics of gene expression surveyed in maize, mouse and man. Nature 2003; 422:297–302. Eaves IA, Wicker LS, Ghandour G, Lyons PA, Peterson LB, Todd JA, Glynne RJ: Combining mouse once genic strains and microarray gene expression analyses to study a complex trait: The NOD model of type 1 diabetes. Genome Res 2002;12:232–43. Karp CL, Grupe A, Schadt E, Ewart SL, Keane-Moore M, Cuomo PJ, Kohl J, Wahl L, Kuperman D, Germer S, et al: Identification of complement factor 5 as a susceptibility locus for experimental allergic asthma. Nat Immunol 2000;1:221–226. Li YJ, Scott WK, Hedges DJ, Zhang F, Gaskell PC, Nance MA, Watts RL, Hubble JP, Koller WC, Pahwa R, et al: Age at onset in two common neurodegenerative diseases is genetically controlled. Am J Hum Genet 2002;70:985–993. Gordon GJ, Jensen RV, Hsiao LL, Gullans SR, Blumenstock JE, Ramaswamy S, Richards WG, Sugarbaker DJ, Bueno R: Translation of microarray data into clinically relevant cancer diagnostic tests using gene expression ratios in lung cancer and mesothelioma. Cancer Res 2002;62: 4963–4967. Scherzer CR, Offe K, Gearing M, Rees HD, Fang G, Heilman CJ, Schaller C, Bujo H, Levey AI, Lah JJ: Loss of apolipoprotein E receptor LR11 in Alzheimer disease. Arch Neurol 2004;61: 1200–1205. Li YJ, Oliveira SA, Xu P, Martin ER, Stenger JE, Scherzer CR, Hauser MA, Scott WK, Small GW, Nance MA, Watts RL, Hubble JP, Koller WC, Pahwa R, Stern MB, Hiner BC, Jankovic J, Goetz CG, Mastaglia F, Middleton LT, Roses AD, Saunders AM, Schmechel DE, Gullans SR, Haines JL, Gilbert JR, Vance JM, Pericak-Vance MA: Glutathione S-transferase omega-1 modifies age-atonset of Alzheimer disease and Parkinson disease. Hum Mol Genet 2003;12:3259–3267.

Clemens R. Scherzer, MD Laboratory for Functional Genomics, Center for Neurologic Diseases Harvard Medical School, Brigham and Women’s Hospital 65 Landsdowne Street, Suite 327, Cambridge, MA 02319 (USA) Tel. ⫹1 617 768 8697, Fax ⫹1 617 768 8595, E-Mail [email protected]

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Molecular Pathology of Dementia Emerging Treatment Strategies

Gunnar K. Gouras Department of Neurology and Neuroscience, Laboratory of Alzheimer’s Disease Neurobiology, Weill Medical College of Cornell University, New York, N.Y., USA

Introduction

Dementia is defined as a progressive and abnormal decline in cognition, typically over months to years. Delirium differs from dementia in that it is an acute or subacute impairment in cognitive abilities, often caused by a reversible toxic or metabolic insult. Neurodegenerative diseases of aging that cause dementia are a growing public health concern as life expectancy increases. Alzheimer’s disease (AD), the most common cause of dementia, currently afflicts about 4 million Americans. Annual costs associated with the care of patients with AD to our society (USA) have been estimated to exceed USD 100 billion annually, and will only increase unless new therapeutic approaches are devised. Major categories in the differential diagnosis of dementia include diverse neurodegenerative diseases, toxic-metabolic encephalopathies, vascular dementia, structural lesions and dementia of depression (table 1). Currently, causes of dementia warranting surgical interventions include brain masses, subdural hematoma and hydrocephalus. At times, brain tumors, such as a frontal glioma or meningioma, can present with mainly gradual cognitive impairment. An important and neurosurgically treatable cause of dementia, presenting with gradual memory impairment in the elderly, is normal pressure hydrocephalus, which is characterized by the triad of gait impairment, dementia, and urinary incontinence. Obstructive hydrocephalus presenting with dementia may be secondary to the obstruction of CSF flow, as may be caused by a colloid cyst of the third ventricle or aqueductal stenosis. It is interesting that recent work aimed at decreasing the symptoms or progression of Alzheimer’s have looked at shunting CSF fluid as one possible approach (e.g., Eunoe, COGNIShunt System), and clinical trials at Stanford

Table 1. Major categories of dementia Neurodegenerative diseases • Alzheimer’s disease • Diffuse Lewy body disease and Parkinson’s disease • Frontotemporal dementia (Pick’s disease; corticobasal ganglionic degeneration) • Huntington’s disease • Creutzfeldt-Jakob disease (prion diseases) • Progressive supranuclear palsy • Other neurodegenerative diseases (SCAs; lipid storage diseases; demyelinating diseases) Vascular dementia Toxic-metabolic encephalopathy (endocrine, infectious, nutritional, toxins) Normal pressure hydrocephalus Structural lesions • Subdural hematoma • Brain tumor • Obstructive hydrocephalus

University propose a role of CSF purification or clearance as a potential neurosurgical treatment option for selected patients, outside of those with normal pressure hydrocephalus. Other neurosurgical approaches for dementia include the delivery of in vivo or ex vivo gene therapy in the form of recombinant enzymes or growth factors, as well as stem cell transplants to regenerate lost neurons and axons. In this chapter, we will provide an overview of neurodegenerative diseases, the most common cause of dementia in the elderly, and discuss some emerging biological treatment strategies including various molecular neurosurgical approaches. Central Role of Amyloid Beta Peptide (‘Abeta’) in AD

Over the past two decades there has been tremendous progress in better understanding of the molecular biology, pathology, and genetics of AD [8, 23]. The discovery of the peptide sequence of -amyloid (Abeta), the principle component of senile plaques [7, 16] initiated the modern era of molecular biology research into AD. Despite these advances, current treatment for AD remains strictly palliative. There is only one class of medication that is currently F.D.A. approved for the treatment of AD, the cholinesterase inhibitors, and these drugs do not appear to be as effective in mid- to late-stage AD. The use of these drugs evolved from neurochemical studies conducted in the 1970–80s which demonstrated reductions of cholinergic neurotransmission in AD brain tissue, especially in the basal forebrain.

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Of the two hallmarks of AD neuropathology, neurofibrillary tangles and senile plaques, the neurofibrillary tangles have been viewed as less specific to AD because they are found in a variety of other neurodegenerative diseases. For example, familial mutations in tau, the principle component of neurofibrillary tangles, are associated with genetic forms of fronto-temporal dementia. Nonetheless, indirect evidence suggests that tau may be essential to the loss of neurons which occurs in AD [28]. It is interesting that both Abeta and tau may be elevated following traumatic brain injury, particularly chronic brain injury, suggesting that they may constitute a response to injurious stimuli. The importance of Abeta to the AD disease process was strengthened by the discovery of mutations within the Abeta precursor protein (-APP) gene that segregate with autosomal dominant forms of early onset familial AD (FAD). Subsequently, transgenic mice harboring human APP with FAD mutations were developed that reproduce AD-like brain amyloidosis [6, 11]. The discovery of autosomal dominant FAD mutations in presenilin (PS) 1 (chromosome 14) and 2 (chromosome 1) also led to the findings that these forms of FAD invariably lead to increased generation of the longer Abeta42 form of Abeta [23]. Abeta peptides range in length up to 42 or 43 amino acids. Both the Abeta N-terminus and C-terminus reveal heterogeneity, but tend to be mainly referred to in the literature as Abeta 1–40 (Abeta40) and Abeta 1–42 (Abeta42) peptides. The slightly extended Abeta42 aggregates more readily and is the main constituent of senile plaques in AD [9]. The mechanism whereby Abeta is involved in AD pathogenesis remains controversial. Numerous investigators have demonstrated that Abeta isoforms are neurotoxic when added to cultured neurons in vitro and when injected into the brain of experimental animals in vivo [29]. Accordingly, neuritic plaques found in the extracellular space in AD brains are presumed to be toxic to surrounding neurons and their processes. Recent evidence suggests that AD, analogous to a growing number of diverse neurodegenerative diseases, is also characterized by the intracellular accumulation of its disease-linked Abeta peptide. Indeed, a recent immunoelectron microscopy study observed accumulation of Abeta within neurons, especially within distal neuronal processes of transgenic APP mice with aging prior to and with the onset of plaque pathology [30]. Currently, increases in Abeta oligomers (mainly soluble Abeta40 but also insoluble Abeta42) are viewed as important neurotoxic intermediates in the development of neuronal dysfunction [13, 23]. Increased soluble Abeta levels appear to be the best Abeta correlate of cognitive dysfunction from mild cognitive function through more severe stages of AD [18; fig. 1 for schema of AD pathogenesis]. A major focus of AD research continues to be to better

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FAD mutations (APP.PS1, PS2)

Apo E

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Fig. 1. Schema of AD pathogenesis.

understand the biochemical mechanisms by which the genetically diseaselinked APP/Abeta, PS, and apolipoprotein-E are involved in neurobiology and the AD disease process. Overall, there is growing optimism that with major advances in our understating of neurodegenerative diseases of aging, therapies for these incurable disorders are not far away [8; table 2; fig. 2]. Inhibitors of ␥- and ␤-Secretase

The increasing evidence for the importance of Abeta accumulation in AD has made reduction of Abeta a leading target for AD therapy. Strategies to accomplish Abeta reduction include inhibition of the proteases that lead to the cleavage of Abeta from its larger precursor, -APP (fig. 3). The -site APP cleaving enzyme (BACE) was discovered to be responsible for the initial cleavage of APP at the N-terminus of Abeta that first produces an APP C-terminal fragment or CTF [31]. PS is viewed as critical for the subsequent -secretase cleavage of the CTFs that generates the 40 or 42 C-terminal ends of Abeta [23]. Given that BACE knockout mice appear normal while PS1 knockout mice are embryonically lethal [25], and reports linking PS to cleavages of several other important proteins, BACE inhibition currently is the leading therapeutic target for Abeta inhibition [2]. In common with other therapies directed at the CNS, efficient delivery of such secretase inhibitors could be critical and may depend on placement of indwelling infusion devices into the CSF or brain parenchyma to bypass the blood brain barrier.

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Amyloid precursor protein (APP) metabolism -secretase (BACE)

-secretase

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Fig. 2. Amyloid precursor protein (APP) metabolism.

Table 2. Outline of therapeutic treatment strategies for AD Cholinesterase inhibitors (i.e., Aricept, Exelon, Reminyl) Anti-Abeta approaches • BACE inhibition • gamma-secretase inhibition (inhibition of PS, Aph1, PEN2 and/or Nicastrin) • anti-Abeta vaccine • cholesterol-lowering agents • anti-aggregation compounds (e.g., beta-sheet breakers) • promoters of Abeta degrading enzymes (neprilysin, IDE, neutral endopeptidase) Anti-oxidants (e.g., Vitamin E) Anti-inflammatory medications (e.g., NSAIDs) Estrogen replacement therapy → NO LONGER RECOMMENDED! Anti-tau strategies Anti-apoptosis agents Neurotrophic factors (e.g., NGF) CSF shunting Stem cells Gene therapy

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Abeta Vaccine: Theory and Practice

Other Abeta-based therapeutic approaches that are under investigation include anti-Abeta vaccines, in which Abeta is deliberately administered with an adjuvant in order to stimulate the body’s natural immune response to clear the substance from the blood and the brain. Initial excitement demonstrating reductions of cerebral Abeta plaques in transgenic FAD APP mice in response to intravenous infusion of Abeta or anti-Abeta antibodies was seriously dampened by the discontinuation of a clinical trial of Abeta infusion into AD patients secondary to the occurrence of encephalitis in ⬃5% of patients. Despite this setback for an anti-Abeta vaccine, reports indicating cognitive stabilization in some patients with high anti-Abeta antibody titers provide some encouragement [10]. Overall, an immunological approach to AD has received great interest in the field, but remains a therapeutic direction in AD that is less well understood than others. Further research on the neuroimmunology of Abeta may help elucidate the mechanism whereby anti-Abeta antibodies may benefit patients with AD. Some investigators have postulated that anti-Abeta antibodies in the systemic circulation may act as a ‘peripheral Abeta sink’ that could draw Abeta out of the brain [5]. Theoretically, evidence for the sink hypothesis could also be viewed as a mechanism whereby neurosurgical CSF shunting [27] may have therapeutic potential for AD, since it would be expected to divert Abeta out of the brain.

Other Strategies Based on Reduction of Abeta

Retrospective studies suggest that intake of cholesterol-lowering 3-hydroxy-3-methyl glutaryl coenzyme A reductase inhibitors, commonly known as statins, are associated with protection against AD. In addition, treatment of APP transgenic mice with a high fat cholesterol diet increases, while treatment with statins reduces, Abeta plaque pathology [21, 22]. There is a compelling biological mechanism by which cholesterol influences Abeta generation, since the critical -secretase cleavage of Abeta occurs within the membrane lipid bilayer and could be modulated by higher or lower cholesterol; supportive evidence comes from drops in Abeta levels in cultured cells that parallels transgenic mice studies. Prospective clinical trials to assess the efficacy of cholesterol-lowering strategies for the prevention and/or treatment of AD are ongoing, and preliminary results suggest an effect of statin treatment. In the past, retrospective studies suggested that estrogen replacement for postmenopausal women might reduce the incidence of AD. Studies in tissue culture and on APP transgenic mice support the efficacy of both estrogen and

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Therapeutic anti-A targets in Alzheimer’s disease Cholesterol modulators - and -secretase inhibitors Antioxidants

Gonadal hormones

Immunotherapy

-sheet breakers

?

Intracellular A

A plaques ?

Signal transduction modulators

Metal chelators Modulators of inflammation

Fig. 3. Sites for intervention vis-à-vis Abeta for Alzheimer’s disease: treatment strategies aimed at reducing Abeta are either directed at APP/Abeta metabolism within nerve cells or Abeta associated with extracellular plaques.

noninflammatory treatments. Unfortunately, prospective studies of patients with mild-to-moderate AD have failed to demonstrate benefits for estrogen. Indeed, patients treated with estrogen experienced more adverse side effects, especially vascular complications such as deep vein thrombosis and heart attack. In addition, estrogen intake increases the risk for the development of breast and uterine cancer. Moreover, the Women’s Health Initiative Memory Study (WHIMS) [32] showed that rather than helping as had been originally proposed, estrogen-progesterone hormone replacement therapy was harmful and actually increased the occurrence of Alzheimer’s dementia over the treatment period. The use of anti-inflammatory medications such as NSAIDs has also been proposed as a possible treatment option for incipient AD, given that experimental evidence supports the role of various inflammatory processes in AD, including the role of activated microglia and brain cytokine release. However, given a lack of clinical data to support this approach and bearing in mind the results of estrogen clinical trials which appear to contradict earlier in vitro and in vivo work, caution is warranted in recommending anti-inflammatory drugs at this time. It appears that inflammatory processes do affect the amount of Abeta produced in cell culture and mouse models of AD, yet, prospective, randomized clinical trials are required to validate this approach. Metal chelation has been proposed in the past as a treatment for AD, since certain divalent or trivalent metals appear to increase aggregation of Abeta in vitro and the antibiotic clioquinol was reported to reduce plaque pathology in transgenic APP mice [4]. However, metal accumulation is very likely to be

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an epiphenomenon of other changes and there are no convincing data to support the role of metals in the pathology of AD or the use of chelation therapy. Signal transduction modulators such as GSK3, CDK5, PI3K, or PKC modulators have been proposed as treatment strategies for AD, acting via mechanisms that may affect both Abeta and tau. Augmenting the various proteases involved in degrading Abeta in brain is another possible treatment approach. Reduced clearance and degradation of Abeta may be more important for most cases of AD than the increased generation of Abeta associated with the relatively rare early onset forms of AD. Some important enzymes in the brain that have been demonstrated to degrade Abeta in vitro or in vivo include neprilysin, insulin degrading enzyme, neutral endopeptidase, and other metalloproteinase enzymes. Protease augmentation in targeted brain areas would be particularly amenable to an in vivo or an ex vivo gene transfer approach. Excitotoxicity is a general phenomenon involved in neuronal cell damage, in which glutamatergic, excitatory transmission leads to excessive cell stimulation, pathological Ca influx, and associated processes such as apoptosis. Despite failure of other glutamatergic antagonists in the past to prevent neurodegeneration, the N-methyl-D-aspartate (NMDA) antagonist memantine was reported in a prospective clinical study to improve function in patients with moderate to severe AD [20], though the biological mechanism by which it may be beneficial for AD is not known. NMDA-mediated excitotoxicity is one potential mechanism for protective effects of NMDA antagonists, though the role of NMDA toxicity has not been precisely defined in the pathophysiology and treatment of many neurodegenerative diseases. A recent neurobiological study suggested that NMDA antagonists may provide therapeutic benefit in AD by other biological effects on Abeta and neuronal physiology [12]. Increasing evidence supports the hypothesis that oxidative stress is a mechanism whereby aging is the major risk factor for age-related neurodegenerative diseases [1]. While the biochemical pathways involved in the physiologically relevant oxidative stress associated with age-related diseases of the brain require more definition, effects on the progression of AD in a large double-blind clinical study following treatment with the anti-oxidant vitamin E suggests that this approach requires further study [24]. Because oxidative species may accentuate or accelerate the negative effects of Abeta deposition, it is possible that lowering oxidative stress may have synergistic effects on lowering Abeta. Gene Therapy for Alzheimer’s? The understanding that a significant component of AD, especially early onset forms of AD, are inherited or that almost half of the more typical late

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onset cases of AD are associated with a single genetic risk factor, the apolipoprotein E4 allele, has led to a growing awareness that gene therapy may be an important direction for future therapy. Other neurodegenerative diseases are known to have, and are increasingly being found to have, complex genetic etiologies. Since a common theme in many neurodegenerative diseases (e.g., AD, Huntington’s disease, Parkinson’s disease) is the abnormal intracellular accumulation of insoluble peptides, the most direct method to reduce the expression of a disease-linked protein is reduced transcription of the protein, increased transcription of proteases involved in the degradation of a given protein, or reduction in proteases involved in the generation of toxic proteolytic fragments (e.g., generation of Abeta from APP). In addition to standard gene transfer using viral and nonviral vectors, siRNA has emerged as a powerful new method to reduce gene transcription and is being employed in experimental strategies to reduce protein accumulation associated with neurodegenerative diseases such as AD. Effective gene therapy will require improvements in vectors to reliably transduce cells in the CNS while minimizing local inflammation and/or tissue damage. Once technical obstacles for safe and effective gene transfer to the CNS can be accomplished, manipulation of genes may one day revolutionize the treatment of neurodegenerative diseases. Stem cells are increasingly being explored as a vector to deliver ex vivo gene transfer and may be useful for dementias to replenish neurons that are destroyed as a result of a neurodegenerative disease. Neurotrophic factors, important in the development of the nervous system, continue to be produced in adulthood and have been proposed as a therapeutic option for neurodegenerative diseases such as AD, particularly in combination with stem cell-based ex vivo gene therapy. Too Much Focus on Abeta? Despite this preponderance of anti-amyloid approaches for treating AD, there is not unanimous agreement in the field whether the focus on Abeta is justified. Arguments against the primary role of Abeta in AD include the following: only a small group of AD patients have mutations in APP (1%); Abeta plaques do not correlate with cognitive dysfunction as well as synaptic loss; and significant amounts of A plaques can be found in postmortem brains of people without clear clinical evidence of dementia. It should also be pointed out that despite its apparently deleterious role in AD, Abeta40 (i.e., the majority of soluble Abeta), Abeta42 (i.e., largely insoluble Abeta fraction), and other proteolytic fragments of Alzheimer’s precursor protein such as sAPPalpha or sAPPbeta may have important physiological functions. APP knockout mice were originally reported to have normal brains, but subsequent work has demonstrated abnormalities in synaptic markers in APP knockout

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mouse brain and decreased neurite extension in cultured APP knockout mouse neurons [19]. Indeed, recent work has indicated that neuronal activity increases Abeta secretion, which in turn may provide a negative feedback to modulate further neuronal activity [12]. It was reported that transection of the perforant path, the major outflow tract from the entorhinal cortex to hippocampus, reduced plaque pathology in the hippocampus of transgenic APP mice [15, 26]. Because APP can be transported down axons by fast axonal transport [14], normal transport of APP may be required to develop plaques in the terminal fields of axonal projections. Therapeutic implications include a concern that treatments which interfere with axonal transport may reduce plaques in transgenic mouse models of beta-amyloidosis, and therefore appear to have therapeutic potential for AD while in fact they may be detrimental to neurons and not beneficial in AD. Aggregation of Insoluble Proteins in Neurodegenerative Diseases There are a number of major similarities among diverse neurodegenerative diseases of aging, which include age-related development of aberrant cellular accumulation and aggregation of insoluble proteins in vulnerable brain regions; a role for oxidative stress; and the occurrence of inflammation. While it is still debated whether intra- or extracellular aggregates are directly toxic to the cell or are an attempt by the cell to defend itself, the prevailing view is that such aggregates within aging cells of the brain are likely to be detrimental. Mutations associated with Parkinson’s disease on two different proteins, parkin, a ubiquitin ligase, and ubiquitin C-terminal hydrolase L1, have pointed to a role for the ubiquitin-proteasome degradation pathway for intracellular proteins in neurodegenerative diseases [17]. Evidence also indicates that endosomallysosomal system abnormalities occur early in the development of AD [3]. These multiple lines of evidence indicate that the reduced ability of selective neuronal populations to efficiently degrade disease-linked protein aggregates are a final common pathway in neurodegenerative diseases of aging. Future neurosurgical interventions that may be critical for the treatment of neurodegenerative diseases such as AD could include placement of infusion devices for delivery of treatments as diverse as gene therapeutic agents (i.e., viral vectors), protease inhibitors, anti-Abeta antibodies to bypass the bloodbrain barrier, stem cells, and CSF shunting to reduce levels of soluble brain Abeta in patients with AD.

Acknowledgments The author thanks Dr. Michael T. Lin for his critical reading of the chapter and helpful discussions.

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Gunnar K. Gouras, MD Assistant Professor, Department of Neurology & Neuroscience Director, Laboratory of Alzheimer’s Disease Neurobiology Weill Medical College of Cornell University, 525 East 68th Street, New York, NY 10021 (USA) Tel. 1 212 746 6598, Fax 1 212 746 8741, E-Mail [email protected]

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Expanding the Role of Deep Brain Stimulation from Movement Disorders to Other Neurological Diseases Massimo Leone, Angelo Franzini, Giovanni Broggi, Gennaro Bussone Istituto Nazionale Neurologico Carlo Besta, Milano, Italy

Introduction

Deep brain stimulation (DBS) is a relatively new treatment modality, first developed in the 1980s by Benabid and colleagues [3] in France, that until recently has been limited to the treatment of complex movement disorders such as Parkinson’s disease through stimulation of the thalamus, globus pallidus, or subthalamic nucleus. However, recent work suggests that techniques of intracranial stimulation may have much wider applications in diseases that are not well managed by any other treatments. Among the new targets for DBS are intractable epilepsy, intractable dystonia, and intractable headache [1–3]. Because the molecular effects of DBS are still largely unknown, it is possible that DBS may also have applications in neurodegenerative and psychiatric diseases; as the molecular basis of disorders such as Alzheimer’s disease or schizophrenia are elucidated, and effects of DBS on gene transcription and cellular activity are better understood, additional applications in different brain regions may become apparent. It is quite plausible that DBS will offer primary or adjunct treatment options for a variety of complex neurological disorders in the future, many of which the neurosurgeons are not accustomed to treating, but which nevertheless are prevalent and debilitating, perhaps offering new career opportunities for neurosurgeons specializing in functional and restorative neurosurgery. In the special case of hypothalamic DBS, the topic of this chapter, applications outside of intractable cluster headache (CH) may include complex neuroendocrine

Table 1. The cluster headache attack, diagnostic criteria A. At least five seperate attacks fulfilling criteria below B. Severe or very severe unilateral orbital, supraorbital, and/or temporal pain lasting 15 min to 3 h if untreated C. Headache is accompanied by at least one of the following: 1. Ipsilateral conjunctival injection and/or lacrimation 2. Ipsilateral nasal congestion and/or rhinorrhea 3. Ipsilateral eyelid edema 4. Ipsilateral forehead and facial sweating 5. Ipsilateral miosis and/or ptosis 6. Sense of restlessness or agitation

or pain disorders. The field of hypothalamic microinstrumentation is in its infancy, and these studies are a first step toward defining the roles and limitations of DBS outside of the basal ganglia. CH is an interesting clinical syndrome, the excruciating severity of which is not widely appreciated. It is a primary pain syndrome characterized by unilateral and incapacitating headache attacks and also associated with ipsilateral autonomic phenomena [4]. Its prevalence is less than one per 1000 and males are more affected than females, the ratio being about 3:1 [5, 6]. There are two main forms of the disease, episodic and the chronic. About 80% of CHs occur in the episodic form; in this case, the attacks are grouped in so-called ‘cluster periods’ usually lasting 1–2 months at a time. In the chronic form, attacks occur for more than one year without remission or with remissions lasting less than one month. The duration of single attacks ranges from 15 min to 3 h at a time, from once every other day to almost continually at eight times a day [1]. Often, attacks appear at the same hours of the day or night and (for unknown reasons) cluster periods often start in autumn and spring [7]. Circadian rhythmicity is a clinical landmark of the syndrome and for this reason it also has been nicknamed as ‘clock headache.’ The excruciating pain is orbital, supraorbital, and/or temporal in location. At least one of the following autonomic phenomena are present during the attack, ipsilateral to the pain: severe conjunctival injection and/or lacrimation, nasal congestion and/or rhinorrhea, eyelid edema, forehead and facial sweating, miosis and/or ptosis, and a sense of restlessness or agitation (table 1).

Pathophysiology of CH and Links to the Hypothalamus

The pain of CH is probably the most severe known to humans, similar in severity to subarachnoid hemorrhage. Accordingly, it has been termed the

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‘suicide headache.’ Until a few years ago, CH was still regarded as a vascular headache. In the last years much has been learned about its pathophysiology and as a consequence the vascular theory of the disease has been discarded. The observation of specific events during the headache attacks involving both cranial nerves and the brain suggests that the neural phenomena are much more relevant to the pathophysiology of CHs. Cluster pain is probably initiated by the activation of the first (ophthalmic) division of the trigeminal nerve, also associated with an increase of calcitonin-gene related peptide in the ipsilateral jugular blood during the attack. Autonomic symptoms are due to the activation of the cranial parasympathetic outflow from the VIIth cranial nerve, associated with an increase of vasoactive intestinal polypeptide in the ipsilateral jugular blood during the attack [8]. The relapsing-remitting course of cluster attacks, mainly in autumn and spring, and the clockwise regularity of single attacks initially suggested that the hypothalamus, site of circadian phenomena or the ‘biological clock,’ might play a crucial role in the pathophysiology of this form of headache [9]. A number of neuroendocrinological abnormalities have been reported in CH [10–12], lending further support to this hypothesis. The first direct evidence showing a pivotal role of the hypothalamus in CH came from positron emission tomography (PET) studies. An increased regional blood flow in the posterior inferior ipsilateral hypothalamic gray matter during the acute stage of cluster attack has been shown both in nitroglycerine-induced attacks [13] and in spontaneous cluster attacks [14]. In a voxel-based magnetic resonance imaging (MRI) study, an increased neuronal density was found in the same brain region that was known to be activated in the PET studies, again ipsilateral to the pain [15]. These structural changes were seen independent of the headache state, suggesting an inherent dysfunction of the hypothalamus in CH rather than an epiphenomenon. Although CH was previously described as a vascular headache, and vascular phenomena also appears to be involved, the striking circadian rhythmicity of this strictly unilateral pain syndrome cannot be explained by a simplistic vascular hypothesis. The case report we present in this chapter illustrates the relevance of the hypothalmus to the pathophysiology of CH. We report on a patient suffering from chronic intractable CHs on the left side, who had initially received complete surgical section of the left trigeminal sensory root to relieve the pain [16, 17]. After the operation, he was completely anesthetic over the entire left trigeminal distribution and the left corneal reflex was absent but he continued to have cluster attacks. Blink reflexes of the left supraorbital nerve produced neither ipsilateral nor contralateral blink reflex responses. With the complete section of the left trigeminal sensory root, the brain cannot perceive vasodilatation or a peripheral neural inflammatory process; as a consequence, none of these peripheral structures, be they neural or vascular, are necessary for

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an attack to happen [18]. It may be concluded that CH is generated primarily from within the brain.

Pharmacological Treatments

The pharmacological treatment of CH is aimed at aborting the ongoing attacks (i.e., acute therapy) and preventing recurrent attacks during the cluster period (i.e., prophylaxis) [19, 20]. The gold standard in the treatment of acute attacks of CH is the subcutaneous administration of the 5HT1B/D agonist sumatriptan [21], also used in migraine attacks. Alternatively inhalation of 100% oxygen can be used. Verapamil [22], lithium carbonate [23], methysergide [24] and cortisone are the most effective drugs to prevent the incidence of CH. Valproic acid, topiramate, gabapentine, naratriptan, melatonin and local application of civamide or anesthesia of the greater occipital nerve may also be of some help [25].

Surgical Treatment

Radiofrequency thermocoagulation of the trigeminal ganglion has been reported to be effective in about 75% of chronic drug-resistant CH patients [26–29]. Other procedures on the trigeminal nerve have been tempted with inconsistent results [30, 31]. Complications include recurrence of headache, in which case, a repeat procedure may be necessary [28]. Other surgical sequelae are corneal analgesia with resulting potential corneal infection or opacification, and anesthesia dolorosa.

DBS of the Hypothalamus

PET has shown the activation of the ipsilateral posterior inferior hypothalamic gray matter during CH attacks [13, 14], which is apparently specific for the condition [32], while voxel-based morphometric MR has documented alterations in the same area [15], strongly suggesting that the CH generator is located there. We reasoned that the stereotactic stimulation of this area might prevent the activation and hopefully relieve intractable forms of CH [16, 17]. The first hypothalamic implantation using DBS to relieve intractable chronic CH was done in July 2000 [16]. Due to the brilliant results, both in term of painfree state and absence of relevant adverse events, 13 new patients have been implanted so far. A summary of the first implanted patient follows.

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The First Patient Implanted with Hypothalamic DBS

At the time of treatment, this patient was a 39-year-old right-handed man who had suffered daily CH attacks for 5 years [16]. The attacks lasted from 30 min up to 4 h at a time, occurred two to eight times a day, and were associated with striking oculo-facial phenomena. The majority of the attacks were on the right side and sometimes on the left, but they were never bilateral. Cerebral MR, MR angiography, and catheter angiography were unremarkable. He became completely drug resistant. He was operated on four times on the right trigeminal nerve; after the last thermal rhizotomy, the right side headache attacks ceased, but from that moment, the left-side attacks worsened, again with striking autonomic phenomena. This new onset of CH was completely drug refractory. In addition, the patient was blind on the right as a result of vitreous humor hemorrhage and left trigeminal surgery was highly contraindicated by the risk of corneal sequelae, which could have left the patient totally blind. Stereotactic electrode implantation targeting the posterior inferior ipsilateral hypothalamic gray matter was then proposed. After informed consent the operation was performed under local anesthesia using a CRW frame on July 14, 2000. The electrode (Medtronic 3089, Minneapolis, Minn., USA) was inserted at coordinates 6 mm posterior to the anterior commissure-posterior commissure midpoint, 2 mm left of the midline, and 8 mm below the commissural plane [15, 16]. Intraoperative electrical stimulation induced no side effects. The permanent generator (Soletra, Medtronic, Minneapolis, Minn., USA), embedded in a subclavicular pocket, was connected by subcutaneous tunnelization. Therapeutic stimulation was in continuous unipolar mode. The position of the permanent electrode was verified by postoperative MR. When stimulated at 180 Hz, 3 V, 60-␮s pulse width, the attacks disappeared after 48 h [16]. Twice, unknown to the patient, the stimulator was switched off and the left side attacks reappeared within 48 h later. When the stimulator was turned on again, the attacks disappeared 48 h later [16]. More than 4 years after the operation, the patient remains pain free [17].

Patient Selection for Hypothalamic DBS

First of all, it should be kept in mind how debilitating CH can be, when it has a chronic course and does not respond to any pharmacological treatments. All patients who received hypothalamic implantation suffered daily attacks in the years before the operation, notwithstanding all kinds of prophylactic drugs, including high dosage steroids. They did not tolerate the painful condition and true to the name ‘suicide headache,’ 2 of the patients had attempted suicide. Another patient had a myocardial infarction while waiting to be operated,

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probably induced by her very high intake of daily triptans (up to 78 mg injectable sumatriptan/day); thus, she could not take triptans anymore and had to increase daily steroid consumption to control in some way the pain episodes. Three weeks after increased dosage of steroids, she died because of repeated gastrointestinal bleeding. Another patient also had myocardial infarction probably related to triptan overuse and had to increase steroid daily intake; in a few months he had developed, among other side effects, a myopathy in the legs and became unable to climb stairs or to stand from the sitting position. Steroids were tapered and strength in the leg gradually improved, but the CHs worsened. He was asked to restart steroids but the myopathy worsened (confirmed with EMG). Again, steroids were stopped, symptoms of myopathy improved, but CHs worsened. In light of these side effects of standard treatments, it is easy to understand how much chronic CHs may interfere with patients’ life. The hypothalamic DBS operation should be considered only in patients with chronic CH for at least 2 years, with daily attacks [33]. From a clinical point of view, 2 years is sufficient time to apply the full range of available CH prophylactic medications [19, 33] and to exclude that a remission period does not occur spontaneously. At present, DBS should be considered only in patients with strictly unilateral CH (no side shift), since contralateral attacks are well known to develop in CH patients after procedures on the trigeminal nerve [33]. Even though we have implanted 2 patients on both sides because they suffered from intractable bilateral chronic CH, bilateral DBS cannot be recommend at this time until more experience on unilateral DBS has been accumulated [33]. Before considering this operation, it is very important to closely monitor the patient over a period of time to verify the diagnosis and assess the attacks firsthand; hence, the patient has to be admitted in order to witness the attacks. Candidates for hypothalamic DBS must be psychologically stable, with a normal psychological profile. Neuropsychological profiling has to be tested before the operation and periodically afterwards. It is also important to exclude any intracranial abnormality that may contraindicate DBS or which could be a potential underlying cause of CH [33] by performing cranial MRI with gadolinium, craniocervical transition, MRI arterial and venous angiography, and CT of the skull base. The patient must be informed that continuous hypothalamic stimulation might have effects on his/her fertility and sexual behavior, although this aspect has not been well studied. At the present stage, the effects of hypothalamic stimulation on pregnancy are completely unknown and for this reason, we recommend that pregnant patients should not receive DBS. After electrode implantation, the stimulator is turned on only when typical spontaneous CH attack has occurred [33]. A list of primary contraindications to hypothalamic DBS also are listed in table 2. All surgical candidates are informed of the classic surgical procedures that are available for the treatment of intractable chronic CH (open microvascular

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Table 2. Criteria for electrode implants in intractable chronic cluster headache 1. CCH diagnosed according to IHS criteria; in addition both of the following: a) CCH for at least 24 months b) Attacks should normally occur on a daily basis 2. Attacks must have always been strictly unilateral 3. Patients must be hospitalized to witness attacks and document their characteristics 4. All state of the art drugs for CH prophylaxis must have been tried in sufficient dosages (unless contraindicated or have unacceptable side effects, etc.) alone and in combination, where applicable. These comprise verapamil, lithium carbonate, methysergide, valproate, topiramate, gabapentin, melatonin (where available), pizotifen, indomethacin and steroids 5. Normal psychological profile 6. No medical/neurological conditions contraindicating DBS including: a) Recent myocardial infarction b) Cardiac arrhythmia c) Cardiac malformation d) Epilepsy e) Stroke f) DBS for other reasons g) Degenerative disorder of central nervous system h) Arterial hypertension or hypotension, not controlled by drugs i) Autonomic nervous system disorder j) Endocrinological illnesses k) Major disturbance in electrolyte balance (e.g., due to renal insufficiency or hyperaldosteronism) 7. Normal neurological examination except for symptoms characteristic of CH (e.g., persistent Horner’s syndrome) 8. Normal CT scan (base of the skull window). Normal cerebral MRI including cranio-cervical transition and MRI arterial and venous angiography 9. Neurosurgical team experienced at performing stereotactic implant of electrodes 10. Patient should not be pregnant 11. Ethics Committee/Institutional Review Board approval 12. Patient informed and gives written consent

decompression/lesion of cranial nerves in the cerebellopontine angle and percutaneous radiofrequency trigeminal rhizotomy). They can choose among the various surgical options once detailed informations on the procedures are given. The Surgical Protocol

Ipsilateral Posterior Hypothalamus Electrode Implantation Affixing a stereotactic apparatus (Leksell frame, Elekta, Sweden) is performed under local anesthesia. If sedation is required, low doses of midazolam

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(0.05–0.1 mg/kg) or propofol (0.5–1 mg/kg) are used [34]. Antibiotic treatment was given to all patients during the perioperative period. A preoperative MRI (brain axial volumetric fast spin echo inversion recovery) is used to obtain highdefinition anatomical images that allow for the precise determination of the AC-PC line. MR images are fused with 2 mm thick CT slices obtained under stereotactic conditions by using an automated technique that is based on a mutual-information algorithm (Frame-link 4.0, Sofamor Danek Stealthstation, Medtronic, Memphis, Tenn., USA). The workstation also provides stereotactic coordinates of the target: 5 mm behind the mid-commissural point, 8 mm below this point and 2 mm lateral from the midline [34]. A rigid cannula is inserted through a precoronaric paramedian burr hole and positioned up to 10 mm from the target. This cannula is used both as a guide for microrecording (Lead Point Medtronic, Minneapolis, Minn., USA) and for the placement of the definitive electrode (DBS-3389, Medtronic, Minneapolis, Minn., USA). Macrostimulation (1–7 V, 60 ␮s, 180 Hz) is used to evaluate potential side effects. All patients subjected to stimulus intensities higher than 4 V have shown ocular deviation that was followed by verbal reports of extreme proportions (‘I feel near to death’; ‘I am at the edge of the end’, etc). When other side effects could be ruled out at standard parameters of stimulation, the guiding cannula was then removed and the electrode was secured to the skull with microplates. The extension was then connected to the electrode, tunneled, and brought out percutaneously for subsequent trial stimulations. On the day following surgery, an additional MR study is done for the purpose of checking the electrode position. After 3–15 days of trial stimulation, the electrodes are then connected to a pulse generator (Itrel II, Medtronic) positioned subcutaneously into the subclavicular area. The following parameters of chronic stimulation have been employed: frequency 180 Hz and a 60-␮s pulse width, with gradually increasing amplitude values [16, 17].

Clinical Results of Hypothalamic DBS

The results of this study are presented in table 3. All the patients have achieved near-complete or complete pain relief, as a result of the long-term high-frequency hypothalamic stimulation that was continued in the follow-up evaluations [35]. Eight out of the 13 implanted patients have remained pain free/almost pain free without any medication (table 3), while 5 needed low doses of methysergide or verapamil to be pain free/almost pain free. It should be noted that these same drugs had been completely ineffective prior to the operative procedure. We observed no noxious side effects from chronic high-frequency hypothalamic stimulation nor did we observe any acute complications from the

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Table 3. Chronic intractable cluster headache sufferers who received hypothalamic implantation Pt. no and gender

Age Chronic Side CH since

Implant. date

Date of improvement

Amplitude (V)

Drugs

Pt. 1 M a)

39

July 14, 2000

None

Pt. 1 M b)

40

Pt. 2 M

50

Pt. 3 F

1997

Pain free 1 month later

3.0

Right May 31, 2001

Left

Immediately pain free

0.5

1997

Left

November 17, 2000

Pain free 2 months later. Occasional attacks in the last 9 months

1.1

Methysergide 3–4.5 mg/day

63

1994

Left

May 22, 2001

Pain free 2 months later

3.0

Verapamil 80–360 mg/day

Pt. 4 M

52

1997

Right October 11, 2001

Pain free 4 months later. July 2002 electrode replacement

3.1

Methysergide 4.5 mg/day

Pt. 5 M

30

2000

Left

March 22, 2002

Pain free 2 months later

1.4

None

Pt. 6 M

46

2000

Left

May 31, 2002

Pain free 1 month later

2.8

Verapamil 360 mg/day

Pt. 7 F a)

27

2001

Left

September 12, 2002

Pain free 1 month later

2.0

None

Pt. 7 F b)

27

2002

Right January 9, 2003

Pain free 5 months later

1.3

Pt. 8 M

25

2001

Right July 10, 2003

2 brief attacks in the last 30 days

2.1

None

Pt. 9 M

43

2000

Left

2 attacks/day, very brief duration and intensity: no sumatriptan need!

2.5

None

Pt. 10 M

46

2001

Right July 30, 2003

Pain free 3 weeks after

1.5

Verapamil 360 mg/day

Pt. 11 M

50

2001

Right August 26, 2003

3 attacks per week

2.0

None

Pt. 12 M

36

2000

Left

September 25, 2003

From 10 attacks/day to 2/day (decreasing frequency and pain intensity)

2.7

None

Pt. 13 M

24

2001

Left

October 15, 2003

From 10–12 attacks/day to 3/day (decreasing frequency and pain intensity)

2.1

None

July 29, 2003

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implant procedure [35]. Patient 1 presented with some signs of mild hypersexual and hyperphagic behavior prior to the operation, which seemed to be resolved by stimulation [17]. In fact, this patient showed a 25-kg weight reduction at the 18 months follow-up.

Discussion

Less than 20% of CHs have a chronic course and about 20% of the chronic forms become drug resistant. In such cases, a surgical option has to be considered. Until a few years ago, surgical treatment of CH was not done, due to limited knowledge about the pathophysiology of the disease, and was essentially based on the interruption of the autonomic pathways [36–40] (i.e., greater superficial petrosal nerve, intermedius nerve section, sphenopalatine ganglion lesions) and/or on a partial or total trigeminal lesion [29–31, 41–46] (i.e., thermal rhizotomy, glycerolysis, direct nerve sectioning, peripheral avulsions). There appears, however, to be a direct relationship between sensory deficit and subsequent discomfort with facial numbness, keratitis, dysesthesias and sometimes anesthesia dolorosa and success rate using classical surgical approaches. In addition to these troubling side effects, the recurrence rate of CH remains high [29–31, 41–46] and even a complete trigeminal deafferentiation can be followed by the persistence of attacks of CH [18]. Microvascular decompression of the trigeminal nerve could obtain pain relief without lesioning nervous structures but unfortunately, the long-term results of these procedures is unsatisfying [26]. For many years CH was considered and treated as having a peripheral vasogenic origin. However, the striking circadian and circannual rhythmicity of the disease indicated that the hypothalamus was probably involved in the pathogenesis of CH. The recent functional PET [13, 14] and morphological, voxelbased morphometry MRI [15] studies shed light on a new pathophysiological process to explain CH in which the posterior inferior hypothalamic gray matter could be the cluster generator [13]. If a central dysfunction involving hypothalamic circuitry is linked to CH, it seems reasonable to question whether surgical strategies may be used to rebalance the unbalanced or disturbed circuits. According to the current models of basal ganglia circuitry, the akinetic and rigid symptoms of Parkinson’s disease result from the hyperactivity of the globus pallidus internus and substantia nigra pars reticulata, as a consequence of an increased glutamatergic drive from a disinhibited subthalamic nucleus. Although the precise mechanisms of high-frequency DBS remain unknown, the therapeutic effect found after long-term high-frequency DBS in Parkinson’s disease seems to be a result of the inhibitory effect of current delivery to subthalamic nucleus hyperactive neurons [47]. It is possible to suggest that a similar

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mechanism may explain DBS effectiveness in relieving chronic CH. In fact, the observed increased blood flow at the hypothalamic level observed during CH attacks may originate from an increased neuronal activity at that level. Notwithstanding the hypothesis that hypothalamic DBS rebalances the hyperactive hypothalamus (accounting for the therapeutic effect in our patients), a more generic analgesic effect coming from an activation of pain-modulating pathway, such as the one involving the release of endogenous opiates, cannot be excluded at the present stage. Future work will look at the effects of DBS on the mechanisms of pain. Over 30 years ago, other authors targeted the hypothalamus to relieve painful conditions [48–50]. Surgical procedures on the posteromedial hypothalamus were published by Sano and colleagues [49, 50] in the 1970s to treat otherwise untreatable facial pain and behavioral disorders such as violence and aggression, in the days when so-called psychosurgery was in vogue. Intraoperative high-frequency stimulation of the posteromedial hypothalamus produced analgesic effects, autonomic responses such as hypertension, tachycardia, respiratory suppression, hyperpnea, tachypnea and mydriasis as well as somatomotor responses. No such effects were observed in our series of CH patients, probably because of differences in both the targeting and the stimulation parameters that have been used [16, 17, 34, 35]. Now that the physiological basis of disorders such as CH and other pain syndromes are able to be precisely measured with new techniques such as PET, fMRI, and intraoperative microdialysis monitoring, it is hoped that functional and restorative neurosurgery will re-emerge from the discredited shadows of early 1960s psychosurgeries, much in the same manner, that basal ganglia surgery has enjoyed a renaissance, since a decadeslong hiatus through the 1960s and 1970s until the pioneering work of Laitinen and colleagues in the 1980s [51].

Conclusions and Future Directions in Hypothalamic DBS

In this chapter, we report the first large series of successfully treated chronic CH sufferers using long-term high-frequency hypothalamic stimulation. These results provide clear evidence that the hypothalamic stimulation offers a safe and effective treatment for CH without any of the troublesome side effects associated with peripheral nerve lesioning procedures. The rationale underlying hypothalamic DBS in CH is based on more advanced morpho-functional studies pointing to the hypothalamus as the ‘CH generator.’ It is hypothesized that the prolonged hypothalamic stimulation rebalances the genetic and cellular mechanisms that leading to hyperfunctioning hypothalamic neurons. It should be underscored that this is the first direct therapeutic application of neuroimaging

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functional data in a primary headache syndrome, and such that the surgical approach used is reversible (by turning off or altering current) in the event of serious complications. Other conditions in which hypothalamic DBS may provide a useful experimental model in the future include complex neuro-endocrine and pain disorders.

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Ekbom K, Monstad I, Prusinski A, Cole JA, Pilgrim AJ, Noronha D: Subcutaneous sumatriptan in the acute treatment of cluster headache: A dose comparison study. The Sumatriptan Cluster Headache Study Group. Acta Neurol Scand 1993;88:63–69. Leone M, D’Amico D, Frediani F, Moschiano F, Grazzi L, Attanasio A, Bussone G: Verapamil in the prophylaxis of episodic cluster headache: A double-blind study vs. placebo. Neurology 2000; 54:1382–1385. Bussone G, Leone M, Peccarisi C, Micieli G, Granella F, Magri M, Manzoni GC, Nappi G: Double blind comparison of lithium and verapamil in cluster headache prophylaxis. Headache 1990;30:411–417. Curran DA, Hinterburger H, Lance JW: Methysergide. Res Clin Stud Headache 1967;1:74–122. May A, Leone M: Up to date on cluster headache. Curr Opin Neurol 2003;16:333–340. Lovely TJ, Kotsiakis X, Jannetta PJ: The surgical management of chronic cluster headache. Headache 1998;38:590–594. Mathew NT, Hurt W: Percutaneous radiofrequency trigeminal gangliorhizolysis in intractable cluster headache. Headache 1988;28:328–331. Jarrar RG, Black DF, Dodick DW, Davis DH: Outcome of trigeminal nerve section in the treatment of chronic cluster headache. Neurology 2003;60:1360–1362. Taha Jm, Tew JM Jr: Long-term results of radiofrequency rhizotomy in the treatment of cluster headache. Headache 1995;35:193–196. Pieper DR, Dickerson J, Hassenbusch SJ: Percutaneous retrogasserian glycerol rhizolysis for treatment of chronic intractable cluster headaches: Long-term results. Neurosurgery 2000;46:363–368. Watson CP, Morley TP, Richardson JC, Schutz H, Tasker RR: The surgical treatment of chronic cluster headache. Headache 1983;23:289–295. May A, Bahra A, Büchel C, Frackowiak RS, Goadsby PJ: PET and MRA findings in cluster headache and MRA in experimental pain. Neurology 2000;55:1328–1335. Leone M, May A, Franzini A, Broggi G, Dodick D, Rapoport A, Goadsby PJ, Schoenen J, Bonavita V, Bussone G: Deep brain stimulation for intractable chronic cluster headache: Proposals for patient selection. Cephalalgia 2004;in press. Franzini A, Ferroli P, Leone M, Broggi G: Stimulation of the posterior hypothalamus for treatment of chronic intractable cluster headaches: First reported series. Neurosurgery 2003;52:1095–1099. Leone M, Franzini A, D’Amico D, Grazzi L, Rigamonti A, Mea E, Broggi G, Bussone G: Long-term follow-up of hypothalamic stimulation to relieve intractable chronic cluster headache. Neurology 2004;62(suppl 5):355. Gardner WJ, Stowell A, Dutlinger R: Resection of the greater superficial petrosal nerve in the treatment of unilateral headache. J Neurosurg 1947;4:105–114. Sachs E Jr: Further observations on surgery of the nervus intermedius. Headache 1969;9:159–161. Sachs E Jr: The Role of nervus intermedius in facial neuralgia: Report of four cases with observations on the pathways for taste, lacrimation and pain in the face. J Neurosurg 1968;28:54–60. Stowell A: Physiologic mechanisms and treatment of histaminic or petrosal neuralgia. Headache 1970;9:187–194. Sweet WH: Surgical treatment of chronic cluster headache. Headache 1988;28:669–670. White JC, Sweet WH: Periodic migrainous neuralgia; in Pain and the Neurosurgeon: A Forty-Year Experience. Springfield, Charles C Thomas, 1969, pp 345–434. Wilkins RH, Morgenlander JC: Results of surgical treatment of cluster headache: Initial relief followed by recurrence. Neurosurgery 1991;31:91–106. Maxwell RE: Surgical control of chronic migrainous neuralgia by trigeminal gangliorhizolysis. J Neurosurg 1982;57:459–466. Morgenlander JC, Wilkins RH: Surgical treatment of cluster headache. J Neurosurg 1990;72: 866–871. North RB, Kidd DH, Piantadosi S, Carson BS: Percutaneous retrogasserian glycerol rhizotomy: Predictors of success and failure in treatment of trigeminal neuralgia. J Neurosurg 1990;72: 851–856. O’Brien MD, MacCabe JJ: Trigeminal nerve section for unremitting migrainous neuralgia; in Rose FC, Zilkha KJ (eds): Progress in Migraine Research. London, Pitman, 1981, vol 1, pp 185–187.

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Massimo Leone, MD Istituto Nazionale Neurologico Carlo Besta Via Celoria 11, IT–20133 Milano (Italy) E-Mail [email protected]

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Molecular Mediators of Pain Priya Chaudhary, Kim Burchiel Department of Neurological Surgery, L472, Oregon Health & Science University, Portland, Oreg., USA

Introduction: What Is Pain?

Pain is an universal, subjective and unpleasant sensation. Acute and subchronic pain has a protective role as it warns of tissue damage. However, chronic and severe pain offers no survival advantage and causes suffering. Particularly in chronic pain, sensory processing from an affected region becomes abnormal and innocuous stimuli (e.g., thermal, touch/pressure) that would normally not cause pain may do so (i.e., allodynia) or noxious stimuli may elicit exaggerated perceptions of pain (i.e., hyperalgesia). In addition, sensations similar to electric tingling or shocks (i.e., paresthesias) and/or sensations having unpleasant qualities (i.e., dysesthesias) may be elicited by normal stimuli. Pain is initiated by specialized sensory nociceptors in the peripheral tissues in response to noxious stimuli [1, 2]. The dorsal root ganglion (DRG) neurons provide a site of communication between the periphery and the spinal cord. Peripheral nociceptors have the machinery for encoding noxious stimuli into action potentials. Central terminals mediate synaptic transmission as well as presynaptic modulation. At the spinal cord level, pain impulses undergo substantial modulation by local mechanisms and by projections from the supraspinal structures (inhibition and facilitation). The processed signal is transmitted to the brainstem and thalamic sites and finally to the cerebral cortex, where it elicits the sensation of pain [3, 4]. Nociceptors are sensory nerve endings which respond to stimuli, which threaten or are capable of causing tissue damage. In addition to activating centripetal discharge, the nociceptive stimuli cause primary afferent (sensory) fibers to release endogenous chemicals. Cutaneous primary afferent nociceptor fibers can be classified into three types, C, A␤ and A␦ based on their soma diameter, structure and conduction velocity of their axon. Multiple classes of

Pathological condition Nerve injury Chemical and inflammatory mediators

Transcription DNA Pre-mRNA

Splicing Alternative splicing

Translation mRNA Stabilization destabilization

Functional protein (pain mediators)

Protein

Post-translational modifications like glycosylation phosphorylation dephosphorylation

Fig. 1. Schematic demonstration of gene expression steps subjected to possible regulation during pain [redrawn from 177].

C and A␦ exist with differing sensory properties. There are two main categories of A␦ and C nociceptors: A␦ mechanical nociceptors and C-polymodal nociceptors. A␦ mechanical nociceptors are activated by mechanical stimuli that damage the tissue. C-polymodal nociceptors are capable of responding to mechanical, thermal and chemical stimuli. The other nociceptor types include A␦ mechanoheat nociceptors, A␦ and C cold nociceptors and C mechanical nociceptors. Acute pain corresponds to the activation of nociceptors with little intervention from higher modulatory mechanisms. However, injury by physical, chemical or immunological means also causes long-term alterations in the expression levels of excitatory mediators, neuropeptides, neurotransmitters, inhibitory neuromodulators, neurotrophic factors, peripheral terminal receptor functions, and signal transduction molecules (fig. 1). These substances exert a variety of actions on local tissue, vasculature, and the afferent fibers. Acute nociceptive, inflammatory and neuropathic pain to some degree, all depends on the activation of primary afferent neurons in the DRG and trigeminal ganglion. A variety of mediators are involved in the central transmission. These substances, which are capable of altering the properties of nociceptors, are broadly termed as modulators (fig. 2). In this chapter, we will describe various mediators like amines (e.g., histamine, serotonin), kinins (e.g., bradykinin; BK), prostanoids (e.g., prostaglandins; PGs), cytokines (e.g., interleukins, tumor necrosis factor; TNF), neuropeptides [e.g., substance P (SP), and calcitonin gene related peptide (CGRP)], energy sources [e.g., adenosine triphospate (ATP)],

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Inhibitory influences: Opioids (␮, ␦, ␬) ␣2-adrenoceptor (␣2c) Adenosine (A1) Cannabinoids (CB1, CB2)

GABA (GABA B) Orphanin (ORL1) Somatostatin Immune cells Mast cell

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Excitatory influences: Prostanoids (EP, IP) Bradykinin (B1, B2) Histamine (H1) Serotonin (5-HT1, 5-HT2, 5-HT3, 5-HT4) ATP (P2X3) TRPV1 Sodium channels

Adrenoceptor (␣2A) Glutamate (NMDA, AMPA, KA) Acetylcholine (N) Adenosine (A2a, A3) Tachykinins (NK1, NK2) Nerve growth factor (TrkA)

Fig. 2. Excitatory and inhibitory influences on peripheral nerve activity by mediators released by tissue injury and inflammation and by a variety of agents acting on neuroreceptors [178].

diffusible gas molecules (e.g., nitric oxide; NO), ions (e.g., H⫹, Na⫹, K⫹), and neurotrophins [NT; e.g., nerve growth factor (NGF)]. Modulators such as opioids, opioid receptors, cannabinoid receptors, and somatostatin are also described (fig. 3, table 1). Effective treatments for pain can be developed by understanding the cellular mechanisms, molecular mechanisms, and mediators that produce pain. This chapter highlights the importance of pain mediators and modulators in developing novel approaches for the treatment of pain. Since gene therapy promises to provide new, effective and innovative solutions for the treatment of pain, we describe some aspects of how gene therapy can be used in pain treatment. We have organized mediators and modulators into categories such as neuroreceptors, ion channels, excitatory receptors, inhibitory receptors, immune mediators, peptides, inflammatory mediators, growth factors and signal transduction molecules, while recognizing that they work in concert to create the sensation of pain.

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PG

ATP Histamine 5-HT

EP3

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H1 5-HT4/7 CGRP½

BK B2 (B1)

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SP NK1 A2A

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gNa⫹

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NAD SP

5-HT2A

NMDA

5-HT3

AMPA/Kainate

5-HT

P2X2/ ASIC Capsaicin VDCC P2X3 ATP Protons Vanilloids

Fig. 3. Roles of diverse receptors and intracellular signals in mediating pain at the polymodal C fiber terminal [4].

Table 1. Receptors localized on primary afferent fibers and their ligands from neuronal and non-neuronal origins [181] Receptors associated with nociceptors ATP, neurokinin-1, GABA, neuropeptide Y, acetylcholine, somatostatin, prostaglandin E, cholecystokinin, adrenergic, 5-HT, glutamate, bradyinin, noradrenaline, capsaicin, opioid, angiotensin II, adenosine (A1 and A2), cannabinoid and menthol receptors Ligands with non-neuronal sources Acetylcholine, ATP, prostaglandin E, opioids, adenosine, glutamate, bradykinin, noradrenaline, serotonin Ligands in nociceptors Substance P, opioid, ATP, adenosine, neuropeptide Y, glutamate, cholecystokinin, somatostatin

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Neuronal Pain Receptors

Vanilloid (Capsaicin) Receptors: VR1/TRPV1 TRPV1 belongs to the transient receptor potential (TRP) family of ion channels [5–7]. It is a nonspecific cation channel expressed preferentially in small sensory neurons. TRPV1-expressing neurons are divided into two groups: peptidergic, expressing SP and CGRP; and nonpeptidergic, expressing P2X3 purinoceptor. TRPV1 detects painful stimuli activated by heat and tissue acidosis, or H⫹ ions [8]. Endogenous substances released from activated immune cells during inflammation also activate TRPV1 and lead to CGRP release. BK, PGE2, and NGF are compounds released during inflammation and are also known to modulate the activity of TRPV1 [9, 10]. Certain cannabinoids, which are products of lipoxygenase pathways of arachidonic acid metabolism (e.g., 12- or 15-hydroxyperoxy-eicosatetraenoic acid) and N-arachidonyl dopamine are endogenous ligands, which activate TRPV1. TRPV1 and its ligands have an important role in mediating inflammatory pain. TRPV1 knockout mice have demonstrated that TRPV1 is important for inflammation-induced hyperalgesia [11, 12]. The natural vanilloid capsaicin, an ingredient of hot pepper, activates TRPV1 and has been most commonly used to study the properties of TRPV1 receptors [5]. Most mechanistic studies of capsaicin-induced activation of nociceptive neurons have been made using cultured sensory neurons and isolated nerves in vitro. Capsaicin causes depolarization, during which there is an increase in membrane permeability to cations (Ca2⫹, Na⫹). Vanilloids have a biphasic response consisting of an initial excitatory response and a refractory phase or desensitization [13]. Capsazepine, a competitive antagonist, and ruthenium red, a noncompetitive antagonist, both block TRPV1. ATP, protein kinase C (PKC), and protein kinase A (PKA) can also modulate the properties of the TRPV1 channels. ATP acts as an allosteric factor and enhances the effect of capsaicin on rat TRPV1 channels [14]. PKC can lead to the activation of capsaicin receptor even in the absence of ligands such as H⫹ or heat [15]. PKA sensitizes the receptor to vanilloids and anandamide [16]. Analyses of mutations in TRPV1 have been the key to understanding the function of this receptor. Site-directed mutagenesis experiments identified a glutamic acid residue (Glu600) near the putative pore which is thought to serve as a key regulatory site, setting the sensitivity to noxious stimuli in response to changes in extracellular proton concentration [17]. Jordt et al. [17] also showed that protons, vanilloids, and heat promote channel opening through separate pathways, since mutations at E648 selectively abolish protonevoked channel activation without diminishing responses to other noxious stimuli.

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The example of anandamide, a cannabinoid that plays a dual role in the body, serves to indicate the complexity of the vanilloid system. It can act as a pro- or anti-inflammatory ligand depending on whether it activates TRPV1 or cannabinoid receptor B1 (CB1). Anandamide acts as a full agonist of human TRPV1 [18] and inhibits capsaicin-induced CGRP release in skin sensory afferents and from the dorsal horn, possibly through the activation of CB1 [19, 20]. Understanding the functioning of the receptor (open and closed states) and signal transduction pathways by which TRPV1 is activated could lead to the identification of novel pain-relieving targets. Alternatively, down-regulation of TRPV1 expression or factors promoting the closed state or desensitization of the channel represents a promising therapeutic strategy for novel analgesic drugs (table 1). Cannabinoid Receptors Cannabinoid and vanilloid receptors are colocalized in the primary sensory neurons and the dorsal horn of the spinal cord [7]. CB1, CB2 are G-protein coupled receptors (GPCRs). The CB1 receptors are expressed in areas involved in modulation of nociception such as periaqueductal grey, spinal cord dorsal horn, and the DRG. CB2 receptors are expressed in nonneuronal cells such as mast cells and other immune cells. Behavioral tests indicate that cannabinoids have anti-nociceptive effects in animal models of acute pain and in persistent pain following peripheral inflammation [21] or nerve injury [22]. It is now undisputable that cannabinoid receptor modulation has therapeutic value in anti-nociception, although concomitant modulatory activity of dopaminergic systems may have adverse psychotropic effects. Anandamide (an endo-cannabinoid) is formed from the hydrolysis of a phospholipid precursor catalyzed by a phospholipase D and is inactivated via reuptake by anandamide membrane transporter (AMT) and enzymatic hydrolysis by fatty acid amide hydrolase enzyme. Anandamide activates the CB1 receptor in the brain and also acts as a full agonist of TRPV1 [18, 23]. Since anandamide and capsaicin share the same TRPV1-binding site, compounds which influence the activity of AMT may facilitate the action of anandamide at the TRPV1. Activation of AMT thus enhances the activity of anandamide at the TRPV1, and AMT inhibitors block the anandamide activity. Since the anandamide-binding site on CB1 is extracellular, AMT could play an important role in distributing anandamide between the intra- and extracellular compartments and activating TRPV1 or CB1 [24]. Cold Receptors Mammals detect temperature effects with specialized neurons in the peripheral nervous system (PNS). Cold and menthol-sensitive receptor (TRPM8)

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belongs to the TRP family of excitatory ion channels and functions as a transducer of cold stimuli [25]. About 10% of trigeminal ganglion neurons express this cold receptor, and thus it is still possible that cold excites the sensory neurons by activating the cold and menthol-sensitive receptor as well as by modulating other excitatory and inhibitory channels present on these neurons. TRPM8 was the first molecule to be identified that responds to cold temperatures and stimulation with menthol. ANKTM1 has recently been characterized as a cold-activated channel. This channel has a lower activation temperature compared to the cold and menthol receptor, TRPM8. ANKTM1 shares little amino acid similarity with TRPM8. ANKTM1 is found in a subset of nociceptive sensory neurons where it is coexpressed with TRPV1 (the capsaicin/heat receptor) but not TRPM8 [26]. Understanding the role of TRPM8 and ANKTM1 may help to identify a target for therapeutic applications using cold receptors. Cold treatment is already used as a method of relief from pain, due in part to its effect on inflammation. In some cases, hypersensitivity to cold can lead to cold allodynia in patients suffering from neuropathic pain, and this also constitutes a therapeutic rationale. Proteinase-Activated Receptors Proteinases like thrombin and trypsin not only act as degradative enzymes, but also act as signaling molecules that regulate proteinase-activated receptors (PAR) [27, 28]. Proteinases are released during inflammatory processes. Proteolytic cleavage of the extracellular amino terminus of PAR exposes a tethered ligand domain, which acts as a receptor-activating ligand. Synthetic peptides corresponding to this proteolytically revealed new N-terminal domain (PARactivating peptides) constitute selective agonists for these receptors. The PAR receptor family is known to have four members PAR1, PAR2, PAR3 and PAR4 which are all G-protein coupled [29]. PARs are expressed on endothelium, platelets, inflammatory cells, fibroblasts and nociceptive primary afferents [30]. Several studies suggest that these receptors might be mediators of neurogenic inflammation and may cause nociception. PAR agonists produce thermal and mechanical hyperalgesia, which is diminished in mice lacking the NK-1 receptor [27, 28]. In the DRG, more than half of the neurons expressing PAR2 also coexpress CGRP and SP, which play a role in vasodilatation and inflammatory responses. Thus, proteases and PARs may play a previously unknown novel role in pain. This may have a potential for developing therapeutic targets in inflammation and pain. Adrenoceptors Adrenoceptors (ARs) mediate some of the main actions of the natural catecholamines, epinephrine and norepinephrine. ARs include ␣1, ␣2, ␤1, ␤2 and ␤3. ARs are members of the much larger family of GPCRs, which include

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muscarinic cholinergic receptors, serotonin receptors, dopamine receptors, neurokinin receptors as well as the photoreceptor rhodopsin. ARs are distributed at sites, which are associated with the transfer of sensory, nociceptive information. For example ␣2 AR subtypes have been found in the DRG, superficial laminae of the spinal cord, and the thalamic nuclei. Different subtypes of ␣2 ARs play a role either in anti-nociception or have been implicated in causing hyperalgesia. Clonidine is an ␣2-adrenergic agonist that has been used as analgesic agent to control severe, acute and chronic pain conditions following epidural or spinal administration. Clonidine relieves hyperalgesia in patients with sympathetically maintained pain but has no effect on sympathetically independent pain. Clonidine not only produces significant analgesia on its own but also potentiates the analgesia produced by opiates [31]. More research is needed to understand the roles played by individual AR subtypes. Cholinergic Receptors Acetylcholine activates cholinergic receptors, both muscarinic acetylcholine receptors and nicotinic acetylcholine receptors (nAChR). At least five primary mACh receptor subtypes are known (M1–M5). They are GPCRs. M1, M3, and M5 mediate their effects through increases in intracellular calcium, whereas M2 and M4 mediate their effects through decreases in cAMP production. Nicotinic receptors on the other hand are ligand-gated channels and at least 11 nAChR subunits ␣2–␣9 and ␤2–␤4 have been identified. The activation of neuronal nAChR produces significant increases in intracellular Ca2⫹ and may play a role in cellular signaling. These receptors are known to produce spinal and supraspinal analgesia. The central anti-nociceptive effects of nicotine, a neuronal nAChR agonist, have been known for many years. Epibatidine is the most potent known agonist at several nicotinic receptor subtypes and mediates anti-nociceptive effects. However, it is too toxic for use in humans [32]. Only recently a potent nAChR agonist, ABT-594 was shown to have antinociceptive properties equal in efficacy to those of morphine [33].

Ion-Gated Channels

ATP-Gated Ion Channels Micromolar concentrations of extracellular ATP (nucleotide) activate sensory neurons via ATP-gated ion channels, cell surface receptors known as P2 receptors [34]. P2 receptors are classified into two categories: the P2X family consisting of ligand-gated cation channels and the P2Y family made up of the GPCRs [35]. Seven subtypes of P2X and eight subtypes of P2Y family have been identified [36]. P2X receptors are found as homomultimeric or heteromultimeric channels.

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P2X3 is expressed selectively by nociceptors in the DRG, predominantly on the nonpeptidergic neurons [37]. DRG neurons respond to ATP by an increase in the free intracellular calcium or by depolarization. P2X3 immunoreactive terminals have also been detected in the lamina II of the dorsal horn. P2Y receptors are expressed on large DRGs and produce action potentials by light touch. These observations suggest that P2 receptors play a role in signal transduction of pain from the periphery to the spinal cord. P2X3 receptors are down-regulated following peripheral nerve injury (e.g., sciatic nerve cut) and their expression can be regulated by glial cell-derived neurotrophic factor (GDNF) [37]. In contrast to the sciatic nerve cut example, the P2X3 receptor is up-regulated in the trigeminal ganglion after nerve injury [38]. In human embryonic kidney cells expressing the P2X2 homomer or P2X2/P2X3 heteromer, acidification (pH ⬍ 6.3) increased the ATP-induced current [39]. Since inflammation causes a decrease in the tissue pH, these ATP receptors may play an important role in inflammatory pain. Suramin and PPADS are nonspecific blockers of the P2X1, P2X2, P2X3, P2X5 receptors at micromolar concentrations and other P2X receptors at higher concentrations [40]. The more specific inhibitor Trinitrophenyl-ATP, selectively inhibits P2X1, P2X3 receptors and heteromeric channels that contain one of these receptors subunits [41]. Thus, the P2X antagonist at the sensory terminal may help in reducing pain caused due to inflammation. Adenosine Receptors Adenosine and ATP influence pain transmission at peripheral and spinal sites. Four adenosine receptor types have been cloned: the A1, A2a, A2b and A3 receptors. The A2a receptor is found in the large neuronal cells of the rat DRG. At the peripheral nerve terminals adenosine A1 receptor activation causes antinociception [42] and adenosine A2 receptor activation produces pronociception [42]. Adenosine A3 receptor activation produces pain behaviors due to release of histamine and 5-HT from mast cells and subsequent actions on the sensory nerve terminal [43]. Acid-Sensing Ion Channels Acid-sensing (proton-gated) ion channels (ASICs) use protonation for the activation of ionic current suggesting the importance of pH regulation in the normal functioning of the nervous system. Severe tissue acidosis that accompanies inflammation is painful and sensory neurons respond to acidic tissue pH with increased firing. Proton-gated cation channels in sensory nerve endings are thought to be responsible for the activation of nociceptive afferents by acid. Members of the ASIC family include ASIC1a, splice variant ASIC1b (BNC2), ASIC2a (MDEG1, BNC1), ASIC2b (MDEG2), ASIC3 (DRASIC- dorsal root

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ASIC), and ASIC4 [44]. These ion channels can form homo- or heteromultimers with other ASICs and are expressed widely in the nervous system. These channels are sensitive to amiloride at high concentrations and are selective for Na⫹. No specific blockers for these individual channels have been suggested. Only recently, psalmotoxin 1 was used to inhibit currents through ASIC1a [45]. The discovery of blockers for these channels is important to evaluate the role played by these channels in nociception. Potassium Channels K⫹ channels form the largest family of ion channels. The common feature of all K⫹ channels is the presence of a conserved motif called the P domain. The 2P domain, leak/background K⫹ channels are non-voltage-gated channels. These background channels are widely distributed in the nervous system. The 2P K⫹ channels play an essential role in setting the neuronal membrane potential and in tuning the action potential duration. They are represented by TWIK-1, TWIK-2 (weak inward rectifiers), TREK-1, TREK-2 (Twik-related K⫹ channel), TRAAK (TWIK-related arachidonic acid-stimulated K⫹; lipid-sensitive mechano-gated K⫹ channels) and TASK-1, TASK-2, TASK-3 (TWIK-related acid sensing K⫹ channel; acid-sensitive outward rectifiers), [46, 47]. The TREK-1, TREK-2 and TRAAK channel activity is elicited by increasing mechanical pressure. These channels are also reversibly opened by polyunsaturated fatty acids including arachidonic acid. TREK-1 is opened by intracellular acidosis, membrane stretch, cell swelling, arachidonic acid and heat [48, 49]. PGE2 and cAMP can close the channel by a PKA-mediated phosphorylation of Ser333. Since TREK-1 is present in sensory neurons as well in the hypothalamus, it is a good candidate as a temperature sensor [50]. TASK-1, TASK-2 and TASK-3 are sensitive to variations of extracellular pH in the physiological range. TASK-3 operates in the pathophysiological range of pH, closes at pH 6.0 and cytosolic arachidonic acid (10 ␮M), which suggests that it may play a role in inflammation [51]. The recent demonstration that TASK-1, TREK-1 and TREK-2 channels are activated by inhalational general anesthetics, and that TRAAK is activated by the neuroprotective agent riluzole, indicates that this novel class of K⫹ channels are interesting targets for new therapeutic developments [52]. Sodium Channels Na⫹ channels are important in electrogenesis within primary sensory neurons. These channels are involved in multiple functions like transduction, signal amplification, and genesis of action potentials [53]. There is growing evidence that modulation of these currents/channels is an endogenous mechanism used to control neuronal excitability [54]. Voltage-gated Na⫹ channels, which produce the inward membrane current necessary for regenerative action potential production

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have emerged as targets in the study of pathophysiology of pain and in the search for new pain therapies. Nine voltage gated Na⫹ channel ␣ subunits (Nav 1.1–1.9) and three subunits (␤1–3) have been cloned [55]. Based on the sensitivity to tetrodotoxin (TTX), these currents are divided into two types, TTX-sensitive and -resistant channels. The TTX-sensitive channel is present in all sensory neurons. TTX-resistant SNS/PN3/Nav1.8 and NaN/SNS2/Nav1.9 channels have been detected in small diameter, unmyelinated sensory afferent neurons [2]. The ratio of these two types of Na⫹ channels can have a profound effect on excitability. The slowly inactivating, rapidly repriming SNS/PN3/Nav1.8 channel is the most likely candidate for the repetitive firing of the injured peripheral nerve [56]. Several studies have demonstrated that TTX-resistant Na⫹ channels can be modulated by inflammatory molecules such as PGs and serotonin through the cAMP-PKA cascade. Down-regulation of TTX-resistant Na⫹ channels (Nav1.8 and Nav1.9) and up-regulation of TTX-sensitive Type III Na⫹ channels (Nav1.3) has been detected after nerve injury [57–60]. Local anesthetics such as lidocaine and mexiletine or anticonvulsants such as carbamazepine and phenytoin have been used in the treatment of neuropathic pain, although clinical performance has been hindered by a number of side effects. There is interest in delineating mechanisms underlying membrane excitability, action potential generation and transmission in nociceptive neurons [54, 58]. Targeting sodium channels (especially the Nav1.8 and Nav1.9) in the periphery could be a novel opportunity for producing analgesia without having major side effects in the central nervous system (CNS).

Excitatory Receptors

Glutamate Receptors Glutamate receptors play a key role in pain perception. Glutamate acts through ionotropic glutamate receptors (iGluRs, coupled to ion channels) and metabotropic glutamate receptors (mGluRs, coupled to intracellular secondary messengers). Animal studies indicate that glutamate in the periphery plays an important role in response to inflammatory agents such as intraplantar formalin [61] and causes pain-related behaviors [62]. The nociceptive-specific primary afferent fibers are a source of peripheral glutamate [63]. Ionotropic glutamate receptors include those activated by ␣-amino-3hydroxy-5-methyl-4-isoxzolepropionic acid, N-methyl-D-aspartate (NMDA) and kainate. The NMDA receptor (NMDA-R) is distinctive and unique. It acts as both ligand and voltage-gated, and is selectively permeable to Ca2⫹ ions. As a consequence, NMDA-R mediated alterations in intracellular Ca2⫹ levels regulate a variety of signaling pathways, ranging from localized, acute effects on receptor and

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channels activities to long-term effects on nuclear gene transcription. The involvement of peripheral NMDA-R in inflammatory nociception offers an attractive target for antagonists that do not cross the blood brain barrier. Such agents might be powerful anti-nociceptive agents without having the CNS side effects [64]. There are eight cloned mGluRs (mGluR1–8), divided into three groups based on sequence similarity, pharmacology and intracellular effector systems. Group I consists of mGluR1 and mGluR5, group II is made up of mGluR2 and mGluR3, and group III has mGluR4, mGluR6, mGluR7, mGluR8 [65]. The mGluRs are activated upon the release of glutamate in the dorsal horn subsequent to the activation of sensory neurons. mGluRs are also activated in the peripheral primary afferent terminals of sensory neurons in response to inflammatory stimulus and in experimental neuropathic pain elicited by ligation of L5/L6 spinal nerves [66, 67]. Group I mGluRs act through the activation of phospholipase C, which leads to the release of calcium from intracellular stores and activation of PKC. mGluRs also activate other kinases that can modulate the function of vanilloid receptors, TTX-resistant channels that have been implicated in the production of pain. The mGluRs contribute to nociceptive processes such as hyperalgesia since receptor antagonists attenuate pain. mGluR5 antagonists [SIB-1757, 2-methyl6-(phenylethynyl)-pyridine (MPEP)] and mGluR1 receptor antagonists [7-(hydroxyimino)cyclo-propa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt), 2-methyl-4-carboxyphenylglycine (LY367385)], cause reversal of pain symptoms [66–68]. The reduction of hyperalgesia by mGluR antagonists is important in designing drugs that could target the painful neuropathies or inflammatory pain conditions. Elucidation of the underlying molecular mechanisms by which the glutamate receptors enhance pain sensitivity, may lead to designing inhibitors of glutamate release, selective glutamate receptor antagonists or the inhibitors of intracellular glutamate-activated pathways [69–71].

Inhibitory Receptors

g-Amino-Butyric Acid Receptors ␥-amino-butyric acid (GABA) is a major inhibitory neurotransmitter and acts via three receptor subtypes, GABAA, GABAB, and GABAC [72]. Endogenous peripheral GABA arises from primary afferent fibers (glutamate is converted to GABA by glutamate decarboxylase). GABAA receptors, present on some unmyelinated afferent axons [73] are, therefore, involved in modulating pain signaling. The GABAmimetic agents have a broad spectrum of pharmacological actions, including analgesia. Both directly acting (GABAA and GABAB

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agonists) and indirectly acting GABAergic agents (GABA uptake inhibitors and GABA-transaminase inhibitors) produce analgesia [74]. Gabapentin, a synthetic structural analog of GABA, given systemically, is clinically effective in chronic neuropathic pain conditions [75, 76]. Pharmacological actions of gabapentin are unclear. It is known to interact with an auxiliary subunit of voltage-sensitive Ca2⫹ channel and modulation of GABA and glutamate synthesis [77].

Immune Mediators

Cytokines The role of neuroinflammation and neuroimmune activation in pain involves the infiltration of immune cells to the site of injury (CNS or PNS) and activation of endothelial cells, microglia, and astrocytes. Activation of these cells leads to the production of cytokines and chemokines [78]. Proinflammatory cytokines like interleukin (IL-1, IL-6) and TNF have been implicated in the genesis and maintenance of pain [79–83]. Proinflammatory cytokines can exaggerate pain responses by directly acting on the cytokine receptors found on neurons or by indirectly stimulating the release of other substances that could act on neurons. Cytokines can cause neuronal hyperexcitability, via alterations in ion channels. DRG neurons are known to express TNF receptor type I (TNFRI) and interleukin receptor I [84, 85]. Thus, DRG neuronal response during pain is affected by the surrounding inflammatory cytokines. In parallel, antiinflammatory cytokines such as IL-4, IL-10, IL-13 and IL-1ra are produced and reduce hyperalgesic effects of the proinflammatory cytokines that are initially produced. Inflammatory pain, therefore, is the result of interplay between hyperalgesic and analgesic mediators. Drugs such as immunosuppressants influencing this interplay may also impair endogenous hyperalgesic and analgesic mechanisms. TNF␣, IL-1␣ and IL-1␤ are the first cytokines involved in Wallerian degeneration. Indirectly, these cytokines further regulate macrophage recruitment, myelin removal, survival of PNS neurons, regeneration, and pain through the regulation of NGF production [86, 87]. Increased levels of spinal interleukins have been detected following spinal nerve transection, L5 nerve root injury [88], peripheral nerve injury, acute peripheral inflammation (formalin or zymosan subcutaneous injections in the hind paw) [89], experimental traumatic spinal cord injury in rats [90] and TNF␣ injection in the sciatic nerve [82]. The role of cytokines in neuropathic pain is further demonstrated by the ability of corticosteroids (immunosuppressants), thalidomide, and anti-inflammatory cytokine IL-10 to alleviate neuropathic pain [91, 92].

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Peptide Mediators: Opioids

Opioid Peptides Dynorphins, enkephalins and ␤-endorphins (␤-EP) are the main groups of opioid peptides. Recently, a novel group of endogenous opioid peptides have been discovered in the brain and named endomorphins, including endomorphin-1 and endomorphin-2 [93]. These opioid peptide-containing neurons have been found in the thalamus, periaqueductal grey, cortex and spinal cord, regions involved in nociceptive responses. Opioids act through opioid receptors. Opioid receptors are found on the primary afferent terminals, DRG and on immune cells. Three members of the opioid receptor family cloned in the early 1990s include ␦-opioid receptor, ␮-opioid receptor and ␬-opioid receptor. Endomorphins have been shown to induce analgesia via ␦-opioid receptors [94]. These three receptors belong to a family of seven transmembrane GPCRs and share considerable homologies. In addition to the well-established opioid receptors, an orphan opioid-like receptor 1 has been cloned. Nociceptin, a novel opioid-like heptadecapeptide, is believed to be the endogenous ligand for opioid-like receptor 1. The activation of these receptors causes reduction in excitability and decreased propagation of action potentials in the sensory neurons [95]. The role of opioids in the anti-nociceptive processes has been well documented for many centuries and opioids are arguably the earliest and most useful medicines known to man. However, using opioids for chronic and neuropathic pain remains somewhat controversial. Clinical evidence suggest that neuropathic pain is not opioid resistant but that only reduced sensitivity to systemic opioids is observed, i.e., an increase in opioid dose is needed to obtain significant analgesia. This reduced efficiency may be due to changes in spinal opioid receptors or signal transduction pathways [96]. The important problem in administering chronic opioids to control pain is the development of tolerance and dependence. Problems of tolerance are not observed, however, with peripherally applied opioids [97].

Nonopioid Peptides

SP SP is one of the most intensively studied sensory neuropeptides, an undecapeptide belonging to the tachykinin peptide family, which includes SP and neurokinin A/B. These peptides act through neurokinin receptors, NK1, NK2, and NK3. A subpopulation of DRG neurons synthesizes and transports SP to the spinal cord where it is released upon noxious stimulation. Released SP

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interacts with neurons of lamina I, which express the SP receptor (SPR or neurokinin receptor). This receptor is expressed on spinothalamic and spinobrachial neurons located in the lamina I, suggesting that these neurons play a role in nociception. Increases in NK1 levels in the superficial laminae of the dorsal horn have been detected in the sciatic nerve cut and inflammatory animal models [98, 99]. Upon binding, SP and SPR are internalized [100]. This SP induced internalization of SPR has been exploited as a means of entry into spinal cord neurons in experimental models of pain treatment. SP was conjugated to the ribosome-inactivating protein saporin. This SP-conjugated neurotoxin resulted in the death of NK1 positive neurons, which led to the inhibition of hyperalgesia [101]. The data suggest that a small population of SPR-expressing neurons are important in the maintenance of hyperalgesia; however, the role of a variety of other non-SPR receptors present on these cells should not be underestimated [102]. The novel approach of receptor internalization and introduction of therapeutic compounds may be of future use in targeting of spinal neurons involved in transmitting chronic pain. CGRP Probably the most abundant neuropeptide in small sensory neurons, activation of sensory unmyelinated neurons by noxious stimuli evokes the release CGRP from peripheral nerve endings. CGRP exerts its effects through CGRP1 and CGRP2 receptors, both of which are coupled to adenylyl cyclase. Administration of neutralizing antibody to CGRP in the spinal cord produces analgesia [103]. Neurotensin Neurotensin (NT) is a brain-gut tridecapeptide with dual functionality. It acts as a neurotransmitter/neuromodulator in the nervous system, and as a paracrine and circulating hormone in the periphery. NT acts through three receptors, NTS1, NTS2, and NTS3. NTS1 and NTS2 belong to a family of GPCRs with seven transmembrane domains, whereas NTS3 is a single transmembrane domain protein. Most of the known peripheral and central effects of NT are mediated through NTS1. NT receptors have been demonstrated on small DRG neurons. Sciatic nerve transection causes a marked decrease in the number of NT receptor mRNA-positive small neurons in DRGs, NT mRNApositive neurons in the dorsal horn, and NT-immunocreactive cell bodies and fibers in laminae I-II. Thus, axotomy causes down regulation of several NT systems at the spinal level, suggesting that the possible effects of NT on primary sensory neurons is attenuated after peripheral axotomy [104, 105]. NT administration into the CSF produces dose-related anti-nociceptive responses [106], which may represent a possible NT-mediated approach to pain relief.

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Somatostatin Somatostatin or somatotrophin-release inhibiting factor was first isolated as a 14-amino acid peptide that reduced the release of growth hormone from the pituitary. Later, it was found to act as a neuromodulator in the mammalian CNS. It is now known to have an inhibitory effect on nociceptors. Five somatostatin receptor genes have been cloned, SST1–5 [107]. These receptors are G-protein coupled with seven transmembrane domains, which interact with a wide range of downstream signaling targets [108]. Experimental and clinical data suggest that the SST2 receptor might be involved in nociceptive transmission at the central and peripheral sites. Thus, SST2-selective drugs may prove to be important analgesics [109]. Neuropeptide Y Neuropeptide Y (NPY) is a 36-amino acid peptide having diverse biological activity. NPY was originally isolated from the mammalian brain tissue and its three receptors (Y1, Y2, Y3) are relatively abundant in the brain and spinal cord [110]. The anti-nociceptive property of NYP is due to the inhibition of SP release from the primary afferent fibers [111]. Elevated levels of NPY are detected in the spinal gray matter and the DRG after sciatic nerve transection. Galanin Galanin is a 29-amino acid peptide expressed in the DRG and spinal dorsal horn interneurons and regulated by nerve injury and peripheral inflammation. Three G-protein coupled galanin receptor subtypes have been identified: GAL1–3. The role of galanin in pain processing at the spinal level appears to be quite complex. It has been known to produce both facilitatory [112, 113] and inhibitory [114] effects on nociceptive behaviors. This peptide is overexpressed in sensory neurons following peripheral nerve damage. The precise role of the peptide was unclear until the generation of a galanin-knockout mouse. Galanin is now known to act as a neuromodulator and is also important in regeneration [115]. Galanin influences pain processing at the dorsal horn level, particularly via GAL1 (inhibitory) receptors on dorsal horn neurons in response to pain arising from nerve injury (neuropathic pain). GAL1 receptor agonists could perhaps be used to treat neuropathic pain [116]. A better understanding of the role of galanin and its receptors may lead to potential therapeutic treatment options. Cholecystokinin This peptide originally was isolated from mammalian gastrointestinal tract and was later detected in brain. It is also present in primary sensory neurons.

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Cholecystokinin A and B (CCKA and CCKB) receptors are found in the CNS and PNS. CCKB receptor is predominant in the brain, spinal cord and DRG. The peptide and CCKB receptor levels increase after peripheral axotomy [117, 118].

Chemical and Inflammatory Mediators

BK Biologically active kinins, including bradykinin (BK) and kallidin, are short-lived peptide mediators predominantly generated by the enzymatic action of kallikreins on kininogen precursors [119]. Kinins are involved in neurogenic inflammation through the activation of A␦ and C fibers. A diverse spectrum of physiological and pathological actions attributed to local kinin production is a consequence of the activation of GPCRs. Kinins act through B1 and B2 receptors. B1 receptors are not normally expressed but are expressed in pathological conditions and by the proinflammatory agents such as lipopolysaccharides and cytokines. B2 receptors are expressed constitutively in the PNS and CNS. Kinins also act partly through nonreceptormediated release of histamine and 5-hydroxytryptamine (5-HT) from mast cells and sensitize primary afferents through interactions with inflammatory mediators such as cytokines and PGs leading tohyperalgesia and allodynia [120]. Various transduction pathways have been suggested for the effects of BK. BK-mediated increase in membrane excitation (depolarization) reflects an increase in cation membrane conductance due to Na⫹. This action involves the production of diacyl glycerol, increase in intracellular Ca⫹⫹ and activation of PKC. The increase in calcium levels could cause neuropeptide release, PG synthesis and stimulation of NO synthase [121]. A new pathway for BK effects is through the activation of capsaicin receptors via the production of 12-lipoxygenase metabolites [122]. Experiments have found B2 to have proalgesic actions on sensory neurons. B2 receptor antagonists are effective in their anti-nociceptive properties. However, given that B2 receptors play an important role in the control of physiological processes such as the cardiovascular system, the blockade of B2 receptors has many undesirable side effects. B1 receptors are activated under inflammatory conditions and B1 receptor antagonists are thus being developed to control inflammatory pain. Considering these facts and the widespread distribution of kinin receptors in many tissues, it is no surprise that the therapeutic potential of kinins and kinin receptor antagonists remains the focus of numerous investigations.

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PGs Prostanoids include PGD2, PGE2, PGF2␣, PGI2 and thromboxane A2. The enzyme PGH synthase (PGHS), also known as cyclooxygenase (COX) catalyzes the synthesis of PG from arachidonic acid. Arachidonic acid is kept esterified by enzymes until mobilized by phospholipases (PLA2). COX1 is the constitutively active isoform generating PG required for cellular function. COX2 generates huge amounts of PG under pathological conditions. High concentrations of PGs contribute to the excitation of neurons through the suppression of a K⫹ conductance and the increase in Na⫹ (and Ca2⫹) conductance, which leads to an increase in neuropeptide release from C fiber terminals. Prostanoids exert a variety of actions on various tissues and cells. PGs act at peripheral sensory neurons and at central sites within the spinal cord and brain to evoke hyperalgesia [123]. Of the many species of PG known, PGE2 and PGI2 are the major contributors to hyperalgesia. There are at least 8 types and subtypes of the prostanoid receptors in mouse and man: DP, EP1, EP2, EP3, EP4, FP, IP, TP [124]. The PG receptors belong to a GPCR superfamily of seven-transmembrane spanning proteins. Nonsteroidal anti-inflammatory drugs (NSAIDs; e.g., aspirin, indomethacin, ibuprofen), block PGH synthase-derived PG synthesis and are commonly used analgesics and anti-inflammatory agents [125]. Aspirin blocks substrate access and orientation at the COX active site by covalently acetylating a serine residue. The coxibs, e.g., celecoxib (Celebrex) and rofecoxib (Vioxx), are newer selective COX-2 inhibitors that have been used clinically for managing pain [126]. COX-1-derived ‘homeostatic’ PGs are not inhibited by the coxibs. Second-generation coxibs, e.g., valdecoxib and etoricoxib, are under development. Serotonin (5-HT) Serotonin or 5-HT is a neurotransmitter involved in various physiological processes. There exist at least 14 subtypes of 5-HT receptors known to be encoded by distinct genes. Splice variants of many of the subtypes have also been identified resulting in the discovery of at least thirty distinct protein products that recognize 5-HT as their physiological ligand [127]. The 5-HT receptors have been divided into seven subfamilies by convention. The 5-HT1, 5-HT2, 5-HT4, 5-HT5, 5-HT6, and 5-HT7 receptors couple to G-proteins, whereas the 5-HT3 receptors are 5-HT-gated ion channels [128]. Primary afferent fibers (C and A␦) are excited by 5-HT, which appears to involve the activation of 5-HT3 receptors directly gating ion channels permeable to Na⫹ (and K⫹). Like the B2 receptors, 5-HT3 receptors are coupled to PLC and initiate changes in the afferent fibers involving diacylglycerol-induced activation of PKC and IP3-induced increases in intracellular Ca2⫹. 5-HT3

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receptor antagonists have anti-nociceptive properties in several inflammatory pain models. The understanding of the serotonergic analgesic system will help in the development of new nonopioid, nonaddictive analgesics. Histamine Histamine, a constituent of mast cells, can activate polymodal nociceptors and release pain-related neuropeptides. Histamine generally elicits an itchy sensation rather than pain; however, higher concentrations may induce pain. Pharmacological studies have suggested that a subgroup of primary sensory neurons is responsive to histamine via the H1 receptor, which is coupled to PLC [129].

Growth Factors

NTs NTs are molecules promoting the survival, growth and maintenance of neurons. NGF, brain-derived neurotrophic factor, NT-3, NT4/5 are important NTs (i.e., growth regulators) essential for the development and maintenance of sensory neurons. NT receptor p75 (i.e., low affinity receptor capable of binding to all NTs) and a family of tyrosine kinases TrkA (i.e., binds NGF), TrkB (i.e., binds brain-derived neurotrophic factor and NT4/5) and TrkC (i.e., binds NT3) are located on adult sensory neurons. All the three Trk receptors show discrete but partly overlapping distributions to subpopulations of primary sensory neurons. Peripheral nerve injury results in apoptosis of DRG neurons and downregulation of TrkA in DRG and spinal cord [130]. Administration of exogenous NGF counteracts the degenerative changes in the NGF-responsive axotomized neurons [130, 131]. Recent evidence suggests that NGF is a peripherally produced mediator of some persistent inflammatory pain states. It has also been demonstrated that administration of NGF produces thermal and mechanical hyperalgesia [132]. GDNF GDNF, a member of the transforming growth factor-␤ → (TGF-␤→) superfamily, is a trophic factor with important effects on the primary sensory neurons [133, 134]. GDNF mediates its actions through a multicomponent receptor system composed of a glycosyl-phosphatidylinositol-linked protein (designated GDNFR-␣ or GFR␣-1), a ligand-binding domain, and the transmembrane protein tyrosine kinase Ret, which acts as the signal-transducing domain. About a third of the primary sensory neurons express Ret mRNA [135].

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GDNF can both prevent and reverse signs of neuropathic pain. It reduces ectopic discharges in damaged sensory neurons by normalization of expression of sodium channel [57].

Vasoactive Intestinal Peptide (VIP) and Pituitary Adenylyl Cyclase Activating Polypeptide (PACAP) PACAP and VIP are members of the vasoactive intestinal peptide/ secretin/ glucagon family of peptides with neurotransmitter, neuroprotective, and neurotrophic functions. PACAP is widely expressed in many central and peripheral neurons [136], in trigeminal ganglion [137], in gastrointestinal tract, and adrenal glands. It is expressed in two alternatively processed forms PACAP-27 and PACAP-38 and exerts its effects through three different receptors: PAC1 (previously called Type I PACAP receptor), VPAC1 (Type II), and VPAC2 (Type III) [138]. These receptors belong to a family of seven-transmembrane GPCRs. PAC1 receptors are coupled to both adenylate cyclase and phospholipase C [139, 140], and VPAC receptors are mostly coupled to adenylyl cyclase. PAC1 and VPAC receptors play an important role in the transmission of sensory information [141]. PACAP, vasoactive intestinal peptide, and other neuropeptides like CCK and NPY and their receptors are up-regulated after nerve injury [142, 143].

Other Mediators

Nitric Oxide (NO) NO, a free radical gas acts as a messenger molecule and plays a role in synaptic transmission both in the CNS and PNS. Immunohistochemical data suggests that NO synthase, the enzyme that synthesizes NO from L-arginine, is present in the CNS and PNS. Recent studies have suggested a role of NO in nociceptive processing [144]. NO modulates spinal and sensory neuron excitability through multiple mechanisms [145]. The activation of excitatory amino acid receptors such as NMDA receptors causes intraneuronal elevation of calcium, which stimulates NO synthase and production of NO [145, 146]. This formation of NO due to the activation of NMDA receptor indicates that NO may act as a mediator of NMDA-induced nociceptive effects. NO has also been implicated in the development of hyperexcitability resulting in hyperalgesia by increasing nociceptive transmitters at the central terminals. NO biosynthesis inhibitors like NG-nitroarginine-L-methyl ester produce anti-nociceptive effects.

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Signal Transduction

PKC Activation of PKC has been implicated in the induction and/or maintenance of neuropathic pain behaviors [147–149]. Spinal cord administration of GM1 ganglioside, an intracellular inhibitor of PKC translocation/activation, reverses both increased levels of membrane-bound PKC and painrelated behaviors [150]. Many different groups have used nonspecific PKC blockers, but these studies have not identified which of the ten isoforms of PKC are involved in maintaining hyperalgesia. Malmberg et al. [151] created a mouse lacking PKC gamma (PKC-␥). Mice that lacked PKC-␥ displayed normal responses to acute pain stimuli, but they failed to develop a neuropathic pain syndrome after partial sciatic nerve section. Thus, selective inhibitors of PKC-␥ may help to alleviate nerve injury-induced neuropathic pain states. Since acute pain responses in PKC-␥ null mice were not affected, the added advantage of using selective PKC-␥ inhibitors is that the acute pain responses, which have an important role in detecting injury, are left untouched. PKC-␧ has also been known to play a role in nociception. PKC-␧ is activated by NGF and regulates the responses to NGF including activation of extracellular signal-regulated kinases (ERK1, ERK2), isoforms of mitogenactivated protein (MAP) kinases, and neurite outgrowth [152]. Studies on PKC-␧ null mice indicate that PKC-␧ is required for the full expression of carrageenan-induced hyperalgesia [153], suggesting a role in pain due to inflammation. Inhibitors of PKC-␥ will help in reducing pain without affecting normal nociceptive responses.

Gene Therapy for Pain?

The explosion in research into understanding the neural mechanisms involved in pain has led to a search for more effective molecular treatment options, compared to traditional pharmacological approaches. Establishing and understanding various mediators and modulators involved in the pathophysiology of pain will help in designing novel therapeutic agents. Gene therapy offers a reasonable and physiological methodology to treat pain. Gene therapy can be attempted at various levels: transcription, mRNA stability, and/or translation. Gene transfer therapy to treat pain can focus on a combination of pharmacological, cellular and molecular approaches [154]. Pain therapy can be delivered in the CNS or PNS, or both sites simultaneously. Targets in the PNS offer advantages since pain information can be blocked before it reaches the spinal

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Table 2. Examples of potential targets for gene therapy [156] Target protein

Classification

NK-1 (SP) receptor Protein kinase C Vanilloid receptors PN3/Nav1.8 Cannabinoids Acetylcholine receptors NMDA subtype of glutamate receptors

G-protein coupled receptor kinase nonselective cation channels sodium channel G-protein coupled receptors G-protein coupled receptors ligand-gated ion channels

Table 3. Loss of function strategies [182]

Knock down or antisense technology Small molecule inhibitors Ribozymes Aptamers RNA interference (RNAi) Inhibitory peptides Antibodies

cord, and also because delivery in the PNS is not confounded by the problems of CNS administration [155, 156] (table 2). A molecule is a good target in the treatment of pain if it satisfies the following considerations: it should have a major role in pain sensation; interactions with other molecules (agonists and antagonists) should have been studied extensively; it should not disturb other normal physiological functions; and it should be expressed in specific neuronal types so that the shut-off of therapy is possible. Viral vectors, antisense oligonucleotide technology, and RNA interference (RNAi) might all be useful techniques in controlling pain by up-regulating anti-nociceptive and down-regulating pronociceptive targets (table 3). Viral Vectors Viral-derived vectors have a natural ability to penetrate cells and deliver a transgene into the host nucleus. The viral vector has the ability to attach, transfer its genome and the transgene into the host, but is incapable of replication (fig. 4). The most widely used viral vectors are derived from adenoviruses, adeno-associated virus, herpes simplex viruses (HSV), or retroviruses. The properties of various virus vector systems are described below (table 4). An

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36kb

Sequence

Inverted terminal repeats

Packaging sequence

Viral packaging

Function

Locus control region

Ligand response element

cDNA

Poly A

Heterologous sequence

Inverted terminal repeats

Drug Therapeutic RNA Tissue Removal of gene processing viral genes specificity regulation

Fig. 4. Features of an optimized adenovirus gene therapy vector. Schematic diagram of a gutted adenoviral vector with an adenoviral packaging sequence and terminal repeats (ITR), containing a minimum of adenoviral genome sequences [179].

Table 4. Comparison of properties of various vector systems [183] Features

Retroviral

Lentiviral

Adenoviral

AAV

Naked/lipid DNA

Maximum insert size Concentrations (viral particles per ml) Route of gene delivery Integration Duration of expression in vivo Stability Ease of preparation (scale-up)

7–7.5 kb

7–7.5 kb

⬃30 kb

3.5–4.0 kb

Unlimited size

⬎108

⬎108

⬎1011

⬎1012

No limitation

Ex vivo

Ex/in vivo

Ex/in vivo

Ex/in vivo

Ex/in vivo

Yes Short

Yes Long

No Short

Yes/No Long

Very poor Short

Good Pilot scale-up, up to 20–50 liters Few

Not tested Not known

Good Easy to scale-up

Very good Easy to scale-up

Few

Extensive

Good Difficult to purify, difficult to scale-up Not known

Immunological problems Preexisting host immunity Safety problems

None

Unlikely

Unlikely, Yes Yes No except maybe AIDS patients Insertional Insertional Inflammatory Inflammatory None mutagenesis? mutagenesis? response, response, toxicity toxicity

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ITR

⫹

LoxP LoxP

E1

ITR

ITR

⫹

Helper virus

Foreign gene

ITR

Vector Cre-recombinase expressing 293 cells

Reamplify in Cre-recombinase expressing cells ⫹ Helper virus

ITR

⫺

E1

ITR

ITR

⫹

ITR

LoxP Not packaged

Packaged

Fig. 5. The replication defective but still infective virus is dependent on the use of Crerecombinase expressing 293 cell line and a helper virus containing loxP-flanked packaging sequence. The Cre-recombinase enzyme excises any segment of DNA flanked by a loxP sequence (30 bp). Infection of the 293 cells with the helper virus with its ␺ sequence flanked by loxP site results in excision of the viral packaging sequence, rendering the helper virus DNA unpackagable. The helper virus provides all the functions for the packaging of the gutless virus [180].

ideal viral vector for gene therapy should be stable, have tissue-specific gene expression, and should not elicit a host immune response. A recently described helper-dependent gutless adenovirus is devoid of all viral genome. This vector is designed by deletion of all of the viral genome except for the inverted terminal repeat and the packaging (␺) sequence essential for viral packaging. This virus can carry up to 30 kb of transgene (fig. 5), making it a useful size for gene transfer. HSV, an enveloped double-stranded DNA virus, is another commonly used gene transfer virus. The wild-type virus is responsible for the common cold sore. The wild-type virus replicates initially in the skin or mucous membranes and is then taken up by sensory nerve terminals. It can then establish a life long latent state in the nucleus of the sensory ganglion neurons. This unique feature of the HSV can be exploited for applications directed towards conditions that affect the PNS [157]. Peripheral inoculation of HSV vectors on

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abraded skin [158] or scarified cornea [159] allows the introduction of a transgene to the sensory ganglia neurons without the need for other sophisticated methods of viral vector delivery. Three weeks after infection with HSV encoding rat preproenkephalin A via the hind footpads, strong expression of preproenkephalin A mRNA was detected in the rat lumbar DRG [158, 160]. Wilson and Yeomans and colleagues [161, 162] used a similar approach and demonstrated the anti-hyperalgesic effect of preproenkephalin overexpression in primary sensory neurons on sensitization of sensory afferents by dimethylsulfoxide or capsaicin application. Hao et al. [163] have used HSV vectormediated expression of proenkephalin in the DRG and demonstrated an anti-allodynic effect in neuropathic pain. They also showed that the enkephalin release enhances the effect of morphine, reducing ED50 of morphine 10-fold and the animals also did not develop tolerance to the continued production of vector-mediated enkephalin over a period of several weeks. Taken together, several of the above studies suggest that viral vector-mediated expression of proenkephalin may be a novel way to treat patients with neuropathic pain. Production of site-specific peptides is of great interest in the field of gene therapy, but these may require various modifications in order to facilitate secretion or activity in vivo. Addition of N-terminal signal peptide is not always sufficient to achieve this goal. To overcome this problem, addition of the preprosequence of mouse nerve growth factor to ␤-EP was tested [164]. Retrovirusmediated expression of a hybrid construct of the preprosequence of NGF and human ␤-EP in primary fibroblasts resulted in the secretion of ␤-EP. Transplantation of such ␤-EP-secreting cells into the brain or spinal cord could provide an ex vivo gene therapy approach for the treatment of chronic, opioidsensitive pain states [164]. Concerns about viral vector distribution in the CNS have limited current gene therapy efforts (table 5). The problem of CNS distribution might be overcome by transferring genes to the meninges surrounding the spinal cord. For example, a recombinant adenovirus encoding a secreted form of ␤-EP was delivered by intrathecal infusion and the resulting increase in ␤-EP secretion by the meningeal piamater cells attenuated inflammatory hyperalgesia in a carrageenan-injection model of persistent pain. This method can be adapted to treat pain in neurodegenerative disorders in which broad spatial distribution of therapeutic effect is critical [165]. Viral vectors have also been used to deliver antisense molecules to control the expression of specific genes in vivo [166, 167]. Thus, viral vector approaches can be used to treat chronic pain states in which plastic changes occur in the neuronal systems. However, in spite of attempts in carefully designing viral vectors, they have not been widely accepted for the use in the treatment of pain.

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Table 5. Comparison of viral vectors and antisense oligonucleotides [155] Advantages Viral vectors

Disadvantages

• effective delivery of large • immunogenicity prevents exogenous DNA

• longer duration of action • possibility of specific •

targeting effective in nondividing cells

• • • •

• • ODN

• specificity of gene • • • •

inhibition minimal immunogenicity effective in nondividing cells not integrated into host genome short duration of action

repeated administration of viral vector direct cellular toxicity inability to target specific subset of cells difficulty in penetrating blood brain barrier may cause insertional mutagenesis (due to integration of viral vector into host genome) possibility of viral replication possibility of creation of a new recombinant virus in vivo (for retrovirus)

• difficulty in generating • • •

and isolating an active oligomer difficulty in gaining intracellular access toxicity may be caused by nonspecific effects of the ODN difficulty in penetrating blood brain barrier

Antisense Oligonucleotide Technology Antisense oligonucleotides are complementary to a portion of target mRNA [168] and have advantages over viral vectors (table 5). Binding of the antisense molecule to target mRNA disturbs the ability of the mRNA to be read by the translational machinery, and thus blocks the synthesis of the encoded protein (fig. 6). Antisense oligonucleotides are modified to enhance entry into cells and are made to be resistant to nucleases within the cell. Three regions in a DNA sequence are considered best for standard antisense design; the 5⬘ cap region, the AUG initiation codon, and the 3⬘ untranslated region of mRNA. Most antisense molecules are 15–20 bases long. This length is sufficient to pick out an unique sequence from others. The oligonucleotides enter the cells by fluid phase pinocytosis, receptor-mediated endocytosis or both.

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Stimuli Pain

mRNA

DNA Antisense oligo

Antisense oligo

Pronociceptive protein

mRNA

DNA

Nucleus

Nucleus

Normal gene activity

Block of gene activity

Fig. 6. Antisense oligonucleotide and potential sites of action [155].

Antisense technology might be used to knock down targets involved in nociception such as NMDA receptors, PKC, neurokinin 1 receptor, and sodium channels [155] (table 6). Although this seems to be a feasible technology, there are practical difficulties in designing a perfect oligomer with greatest specificity and determining that the significant effects observed are due to the antisense oligomer, and not due to other nonantisense effects. RNAi Post-transcriptional gene silencing and RNAi involve the specific suppression of genes by complementary dsRNA [169]. RNAi provides a powerful method of gene silencing in eukaryotic cells. Specific genetic interference by double-stranded RNA in Caenorhabditis elegans was first discovered by Fire et al. [170] in 1998. Double-stranded RNA rather than single-stranded antisense RNA is introduced within cells. Once inside the cell, the double-stranded RNA molecules are cleaved by ribonuclease III into twenty-one to twenty-two nucleotide short interfering RNAs which are replicated by an RNA-dependent RNA polymerase. The short interfering RNA duplexes bind to a nuclease complex to form the RNA-induced silencing complex, which then targets the homologous endogenous mRNA sequence, thus blocking further protein synthesis [171]. However, not many experiments have been performed to determine the utility of RNAi as a method of gene knockdown in postmitotic mammalian neurons. Only recently, Krichevsky and Kosik [172] have applied the RNAi to postmitotic primary neuronal cultures. Thus, in the future one would potentially be able to specifically block target molecules in DRG neurons to control pain.

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Table 6. Effects of antisense oligonucleotide sequence in animal pain models [177] mRNA

Species

Effects

References

c-fos

Rat

↓ c-fos protein immunoreactivity

[184]

DOR

Mice

↓ DOR protein but not mRNA and anti-nociception produced by DOR-selective agonist (D-Ala2) deltorphin II

[185]

GAL

Rat

↓ axotomy-induced upregulation of Gal protein, but no change in GAL mRNA

[186]

NK-1 R

Rat

↓ behavioral response to formalin and NK-1 receptor protein immunoreactivity in spinally SP-treated rats

[187]

Nav1.8 SNS/PN3

Rat

↓ SNS/PN3 protein immunoreactivity decreased in DRG and chronic nerve or tissue injury-induced hyperalgesia and allodynia ↓ TTX-R Na⫹ current density in cultured sensory neurons and PGE2-induced hyperalgesia

[188] [189] [190]

PKC ␣

Human (in vitro)

Inhibition of phorbol ester-induced reduction of bradykinin-evoked calcium mobilization

[191]

NMDAR1

Mouse

↓ pain behavior and decreased receptor binding

[192]

Rat

↓ immunoreactive staining for NMDA-R1 and ↓ formalin-evoked behaviors

[193]

Rat

Delayed onset of mechanical and thermal hyperalgesia in chronic neuropathic pain model

[194]

PSD95/SAP90

Other Gene Therapy Methods Ribozymes are RNA molecules, which also act as enzymes. These catalytic RNA molecules can be designed to recognize and bind to a specific mRNA and cause cleavage of mRNA, thus preventing its translation into protein. While they represent an alternative to RNAi, achieving specificity and delivery of these enzymes within the living tissue is difficult. Neural stem cells are self-renewing precursors of neurons and glia with numerous potential ex vivo gene therapy applications. The advantages of using these precursors include their theoretically limitless clonal expansion in tissue culture [173]. Neural progenitor cells could be genetically modified to express exogenous genes for neurotransmitters, neurotrophic factors, or various ion

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channels. Other ex vivo transfected (iso- or xenogenic) cells can also be used to treat nervous system disorders. Several drawbacks using such methods include risk of toxicity, possible tumor formation, instability of transgenes, and lack of cell specificity. In spite of these challenges, Ishii et al. [174] have successfully used a combination of cell transplantation and gene transfer for the delivery of ␤-EP into the subarachanoid space in rats. The rats that received ␤-EP producing cells showed prominent analgesic effects for up to a month after transplantation. Another study used xenogenic tumor cells secreting ␤-EP and immunologically isolated in polymer capsules (microcapsules) to reduce pain when transplanted into the CSF of rats [175]. Synthetic DNA delivery systems like liposomes have become increasingly popular methods of gene transfer. Introduction of DNA into cells can now be safely achieved by complexing DNA with cationic lipids. These complexes are endocytosed into the cells, which involves binding, internalization, formation of endosomes, fusion with lysosomes and lysis. Finally, the DNA which survives endocytotic processing and degradation by nucleases reaches the nucleus [176]. Liposomes have been successfully used to inject DNA complexes into rodent brains, but gene expression is transient. These methods could be used in the treatment of various neurological diseases including the treatment of intractable pain, where transient expression of the transgene is needed.

Future Directions in Pain Research

Pain sensation is complex and involves integrative mechanisms at the PNS and CNS. Neuropathic pain is unresponsive to most conventional therapy. In recent years, much has been elucidated concerning neuroanatomical circuits, mediators of pain and transduction pathways involved in pain processing. This information has led to the development of new and unconventional therapeutic options for the treatment of pain. Ion channels (e.g., Nav1.8, Nav1.9), neurotransmitters, neuropeptides and their receptors present on pain-sensing neurons are important potential targets for therapy. PKC and other kinases also offer as targets for analgesic development. Among gene therapy options, the use of antisense oligonucleotides seems promising but delivery to specific cell types remains problematic. Viral vectors are attractive candidates but safety and targeting issues remain. Modulation of proteins involved in hyperalgesia by understanding the proteome will lead to more effective therapies for pain relief. Focal drug delivery via microinfusion systems may be necessary adjuncts to analgesic design. In the future, pain management will be a multidisciplinary approach that will include pharmacological intervention, minimally invasive procedures, and gene therapy targeted to

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specific cell types or specific neuron class with high efficacy and minimum side effects. It is difficult to predict which of these approaches may lead to a clinically applicable means of producing analgesia. What is certain is that each of these therapies must be precisely regulated for optimal clinical effects with optimal pharmacological specificity. However this field evolves, future analgesics depend on a growing knowledge of the nociceptive system and its aberrations.

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164 Beutler AS, Banck MS, Bach FW, Gage FH, Porreca F, Bilsky EJ, Yaksh TL: Retrovirus-mediated expression of an artificial beta-endorphin precursor in primary fibroblasts. J Neurochem 1995; 64:475–481. 165 Finegold AA, Mannes AJ, Iadarola MJ: A paracrine paradigm for in vivo gene therapy in the central nervous system: Treatment of chronic pain. Hum Gene Ther 1999;10:1251–1257. 166 Finegold AA, Perez FM, Iadarola MJ: In vivo control of NMDA receptor transcript level in motoneurons by viral transduction of a short antisense gene. Brain Res Mol Brain Res 2001; 90:17–25. 167 Collin E, Mantelet S, Frechilla D, Pohl M, Bourgoin S, Hamon M, Cesselin F: Increased in vivo release of calcitonin gene-related peptide-like material from the spinal cord in arthritic rats. Pain 1993;54:203–211. 168 Myers KJ, Dean NM: Sensible use of antisense: How to use oligonucleotides as research tools. Trends Pharmacol Sci 2000;21:19–23. 169 McManus MT, Sharp PA: Gene silencing in mammals by small interfering RNAs. Nat Rev Genet 2002;3:737–747. 170 Fire A, Xu S, Montgomery MK, Kostas SA, Driver SE, Mello CC: Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans. Nature 1998;391:806–811. 171 Hammond SM, Caudy AA, Hannon GJ: Post-transcriptional gene silencing by double-stranded RNA. Nat Rev Genet 2001;2:110–119. 172 Krichevsky AM, Kosik KS: RNAi functions in cultured mammalian neurons. Proc Natl Acad Sci USA 2002;99:11926–11929. 173 Zlokovic BV, Apuzzo ML: Cellular and molecular neurosurgery: Pathways from concept to reality. II. Vector systems and delivery methodologies for gene therapy of the central nervous system. Neurosurgery 1997;40:805–812; discussion 812–813. 174 Ishii K, Isono M, Inoue R, Hori S: Attempted gene therapy for intractable pain: Dexamethasonemediated exogenous control of beta-endorphin secretion in genetically modified cells and intrathecal transplantation. Exp Neurol 2000;166:90–98. 175 Saitoh Y, Taki T, Arita N, Ohnishi T, Hayakawa T: Analgesia induced by transplantation of encapsulated tumor cells secreting beta-endorphin. J Neurosurg 1995;82:630–634. 176 Luo D, Saltzman WM: Synthetic DNA delivery systems. Nat Biotechnol 2000;18:33–37. 177 Luo Z: Molecular dissection of pain mediators. Pain Reviews 2000;7:37–64. 178 Sawynok J: Topical and peripherally acting analgesics. Pharmacol Rev 2003;55:1–20. 179 Nabel GJ: Development of optimized vectors for gene therapy. Proc Natl Acad Sci USA 1999; 96:324–326. 180 Parks RJ, Chen L, Anton M, Sankar U, Rudnicki MA, Graham FL: A helper-dependent adenovirus vector system: Removal of helper virus by Cre-mediated excision of the viral packaging signal. Proc Natl Acad Sci USA 1996;93:13565–13570. 181 Besson JM: The neurobiology of pain. Lancet 1999;353:1610–1615. 182 Henning SW, Beste G: Loss-of-function strategies in drug target validation. Curr Drug Discov 2002;May:17–21. 183 Verma IM, Somia N: Gene therapy – Promises, problems and prospects. Nature 1997;389:239–242. 184 Huang W, Simpson RK Jr: Antisense of c-fos gene attenuates Fos expression in the spinal cord induced by unilateral constriction of the sciatic nerve in the rat. Neurosci Lett 1999;263:61–64. 185 Lee CE, Kest B, Jenab S, Inturrisi CE: Effect of supraspinal antisense oligodeoxynucleotide treatment on delta-opioid receptor mRNA levels in mice. Brain Res Mol Brain Res 1997; 48:17–22. 186 Ji RR, Zhang X, Wiesenfeld-Hallin Z, Hokfelt T: Expression of neuropeptide Y and neuropeptide Y (Y1) receptor mRNA in rat spinal cord and dorsal root ganglia following peripheral tissue inflammation. J Neurosci 1994;14:6423–6434. 187 Hua XY, Chen P, Polgar E, Nagy I, Marsala M, Phillips E, Wollaston L, Urban L, Yaksh TL, Webb M: Spinal neurokinin NK1 receptor down-regulation and antinociception: Effects of spinal NK1 receptor antisense oligonucleotides and NK1 receptor occupancy. J Neurochem 1998;70:688–698. 188 Lai J, Gold MS, Kim CS, Bian D, Ossipov MH, Hunter JC, Porreca F: Inhibition of neuropathic pain by decreased expression of the tetrodotoxin-resistant sodium channel, NaV1.8. Pain 2002; 95:143–152.

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Porreca F, Lai J, Bian D, Wegert S, Ossipov MH, Eglen RM, Kassotakis L, Novakovic S, Rabert DK, Sangameswaran L, Hunter JC: A comparison of the potential role of the tetrodotoxin-insensitive sodium channels, PN3/SNS and NaN/SNS2, in rat models of chronic pain. Proc Natl Acad Sci USA 1999;96:7640–7644. Khasar SG, Gold MS, Levine JD: A tetrodotoxin-resistant sodium current mediates inflammatory pain in the rat. Neurosci Lett 1998;256:17–20. Levesque L, Dean NM, Sasmor H, Crooke ST: Antisense oligonucleotides targeting human protein kinase C-alpha inhibit phorbol ester-induced reduction of bradykinin-evoked calcium mobilization in A549 cells. Mol Pharmacol 1997;51:209–216. Rydh-Rinder M, Berge OG, Hokfelt T: Antinociceptive effects after intrathecal administration of phosphodiester-, 2⬘-O-allyl-, and C-5-propyne-modified antisense oligodeoxynucleotides targeting the NMDAR1 subunit in mouse. Brain Res Mol Brain Res 2001;86:23–33. Garry MG, Malik S, Yu J, Davis MA, Yang J: Knock down of spinal NMDA receptors reduces NMDA and formalin evoked behaviors in rat. Neuroreport 2000;11:49–55. Tao F, Tao YX, Gonzalez JA, Fang M, Mao P, Johns RA: Knockdown of PSD-95/SAP90 delays the development of neuropathic pain in rats. Neuroreport 2001;12:3251–3255.

Kim Burchiel, MD Chairman, Department of Neurological Surgery, L 472 Oregon Health & Science University, 3181 SW Sam Jackson Park Road Portland, OR 97239 (USA) Tel. ⫹1 503 494 4173, Fax ⫹1 503 494 7161, E-Mail [email protected]

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Gene Transfer in the Treatment of Pain David Fink a, Marina Mataa, Joseph C. Gloriosob a

Department of Neurology, University of Michigan and VA Ann Arbor Healthcare System, Ann Arbor, Mich., and bDepartment of Molecular Genetics and Biochemistry, University of Pittsburgh, Pittsburgh, Pa., USA

Introduction

The Physiology of Pain Pain is an unpleasant sensory and affective experience that serves an essential biological role in alerting an organism to tissue damage. The perception of acute pain is essential for survival in a potentially hostile environment, and an elaborate set of specialized high threshold sensory transducers, nociceptors that respond to painful heat, cold, pressure, and alterations in the peripheral microenvironment are designed to detect these acute stimuli. In the setting of chronic tissue damage, changes in pH and ionic composition of the peripheral microenvironment and the release of bioactive peptides such as cytokines, growth factors, and kinins, all act to sensitize peripheral nociceptors. In addition, continued neural transmission through pain pathways leads to central changes at the level of the spinal cord and higher centers that together result in a heightened pain experience. While these changes may serve an adaptive role in preventing the use of an injured body part, thus promoting recovery and repair, the same processes lead to spontaneous or exaggerated pain that does not serve any functional biological purpose. Pain that persists beyond the course of the acute insult or pain that accompanies a chronic primary process that cannot be cured represents a major medical and social problem resulting in enormous cost to the individual and to society. Such chronic pain may result from continued peripheral injury (inflammatory or ‘nociceptive’ pain) or from damage to neural structures in the absence of peripheral tissue damage (neuropathic or central pain). Derivatives of the active agents extracted from the poppy seed (opiate drugs) and willow bark (nonsteroidal anti-inflammatory drugs) remain among our most effective and widely used analgesic drugs, but increased understanding of

the pathways involved in acute pain perception and the modifications that occur in chronic pain have now set the stage for the rational design of novel therapeutic agents to treat chronic pain. Peripheral nociceptors consisting of cells with unmyelinated (C-fibers) or thinly myelinated (A␦ fibers) axons represent a subclass of neurons whose cell bodies are located in the dorsal root ganglion (DRG). The central projection of the bipolar axons of the primary nociceptors synapse on ‘second order’ neurons in the dorsal horn of spinal cord in a regional and anatomically defined manner. Second order neurons located in the dorsal horn project rostrally to the thalamus and the parabrachial nucleus in the brainstem. Pain-related neurons in the thalamus project primarily to somatosensory cortex, conveying the discriminative aspects of the pain sensation; neurons in the parabrachial nucleus project to the hippocampus and amygdala (among other brain regions) to mediate the affective components of the pain experience. Descending pathways, integrated in the periaqueductal gray of the midbrain and relayed through the nucleus raphe magnus, project caudally to the dorsal horn of spinal cord to synapse with inhibitory interneurons. The principal neurotransmitter released from axons of the primary nociceptor at the dorsal horn is glutamate, although corelease of peptides including substance P, neuropeptide Y, dynorphin and galanin from the primary afferent serve to modulate the pain response. In the dorsal horn, intrinsic inhibitory interneurons may modulate the transmission of nociceptive information through the release of neurotransmitters such as ␥-aminobutyric acid (GABA) acting on GABAA and GABAB receptors, enkephalin acting through ␦ opioid receptors, and endomorphin 1 and 2 acting at the ␮ opioid receptor. Descending projections from brainstem nuclei control the activity of the inhibitory interneurons through the release of monoamine neurotransmitters including norepinephrine and serotonin. In states of chronic pain, there are post-translational or transcriptional changes in primary nociceptors that alter the threshold, excitability, or transmission properties of these neurons. A prolonged increase in the activity of peripheral nociceptors also results in the sensitization of second order neurons, through phosphorylation of ion channels and receptors as well as transcriptional changes in second order neurons. In addition, there may be remodeling in the dorsal horn, including sprouting of primary afferents after injury and loss of inhibitory interneurons. Imaging studies have also identified complex central changes in the activity of subcortical and cortical structures that occur in states characterized by chronic pain. The most direct approach to treating pain is to alleviate the primary inciting cause of the pain. But when the primary disease process cannot be cured, or the pain persists after the identified inciting cause has been treated, other approaches are required. Many of the neurotransmitters or neurotransmitter receptors that are the targets of pharmacological therapy are widely distributed

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through the nervous system and may be present in other organs as well. As a result, the dose of many of the drugs that may be used to treat pain are limited by side effects resulting from the action of these agents on neural pathways unrelated to pain processing, or from the action of these agents directly on non-neural tissues. Opioid receptors, for example, are present on peripheral nociceptors, on the second order projection neurons in the dorsal horn of spinal cord, and in brainstem and brain centers involved in pain processing and other cognitive and affective functions as well as in the urinary bladder and the intestine. As a result, high-dose treatment with opiate drugs may be complicated by alterations in mood and/or cognition, urinary retention, and constipation that limit the dose that may be administered. Gene transfer offers the possibility to achieve local release of analgesic substances to act at the spinal or peripheral level to maximize the analgesic effectiveness while minimizing side effects.

Cell Transplantation for Pain Relief

One method of gene transfer involves the transplantation of cells that carry and express the gene of interest. Chromaffin cells of the adrenal medulla naturally express and release a number of neuroactive substances, many of which are involved in the pain-processing pathway at the spinal level. These include serotonin, GABA, galanin, and met-enkephalin [1, 2]. Accordingly, the injection of chromaffin cells into the lumbar subarachnoid space reduces pain-related behavior in models of neuropathic pain, and in rodent models, inflammatory pain induced by subcutaneous injection of a dilute solution of formalin [3, 4]. Such grafts reduce touch-induced elevation of c-fos expression in spinal cord [5] and prevent the loss of endogenous GABA synthesis in the dorsal horn [6]. Several different mechanisms have been implicated in the analgesic effect of chromaffin cell grafts. Chromaffin cell grafts release met-enkephalin, and the level of met-enkephalin in CSF is increased following grafting [7], but the grafts also elevate CSF catecholamine levels [8]. It is possible that indirect effects, such as catecholamine stimulation by the release of inhibitory neurotransmitters from dorsal horn interneurons might account for some of the effects of the graft. In a neurophysiological study Hentall et al. [9] demonstrated that the intrathecal transplantation of chromaffin cells prevented the normal development of ‘windup’, a phenomenon of electrophysiological potentiation that is characteristic of chronic pain, in second-order wide dynamic range neurons of the dorsal horn. It appears from those studies that interference with potentiation is due to the release of molecules that persistently block the NMDA receptor (or block cellular events mediated by these receptors), separate from the possible effect of inhibitory neurotransmitters from the graft [9].

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Cells also can be modified to produce specific desired gene products. Wu et al. [10] showed that AtT-20 cells, a cell line which produces and releases ␤-endorphin, and AtT-20/hENK cells, an AtT-20 cell line transfected with the human proenkephalin gene (PE) and secreting enkephalin and ␤-endorphin, implanted into the mouse subarachnoid space produced an isoproterenolstimulated anti-nociceptive effect that was dose related and could be blocked by naloxone. Mice receiving AtT-20 cell implants developed tolerance to ␤-endorphin and the ␮-opioid agonist DAMGO, whereas mice receiving genetically modified AtT-20/hENK cell implants developed tolerance to the ␦-opioid agonist DPDPE. Genetically modified AtT-20/hENK cell implants, but not AtT-20 cell implants, reduced the development of acute morphine tolerance in the host mice [10]. Transplants of other cell lines modified to secrete substances that might act as inhibitory neurotransmitters at the spinal level to block nociceptive neurotransmission have included the demonstration that a neuronal cell line genetically modified to secrete galanin [11] or engineered to secrete GABA [12] are anti-nociceptive in models of chronic neuropathic pain. A cell line engineered to produce and release brain-derived neurotrophic factor has been shown to reduce allodynia and hyperalgesia in the chronic constriction injury model of neuropathic pain [13]. The mechanical alternative to cell transplantation is peptide delivery using an intrathecal pump. Both approaches target the pharmcological agent to the lumbar spinal cord in order to minimize the effect of these agents on the brain and on peripheral organs. A theoretical advantage of cell transplantation is the ability of the cells to deliver peptide neurotransmitter (such as enkephalin) in their natural conformation and not in a derivative state. Intrathecal pumps may be used to deliver opioid analgesic drugs such as morphine. Intrathecal delivery reduces the dose requirement and thus limits side effects, but tolerance may develop. Even intrathecal administration of the modified derivatives is limited by the very short half-life of these agents. While cell transplants produce a continuous release of the native peptide, release of additional substances (e.g., ␤-endorphin, serotonin) which may add to the effectiveness of these transplants in the animal models may complicate the practical implementation of the cells as agents for human treatment. In addition, the possibility of an immune reaction or nonspecific scar formation resulting from the injection of foreign material into the subarachnoid space is not fully known. Nonetheless, transplantation of encapsulated bovine chromaffin into the subarachnoid space has been tested in patients with severe chronic pain not satisfactorily managed with conventional therapies. The patients received no pharmacological immunosuppression. Histological analysis, immunostaining, and analysis of secretory function all confirmed survival and biochemical function of the encapsulated cells up to 6 months after

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implantation. Reductions in morphine intake and improvement in pain ratings were observed in several patients [14]. Similar results were observed in an open phase II trial of patients with intractable pain from cancer who received adrenal medullary allografts [15]. While analgesic efficacy was suggested by a reduction or stabilization in opioid use, a controlled trial has not yet been reported.

Adenoviral Gene Transfer to Meningeal Cells in the Treatment of Pain

Some of the problems attendant on injection of foreign cells into the subarachnoid space can be avoided by the direct transfer of the gene of interest into meningeal cells lining the subarachnoid space. For example, Finegold et al. [16] used a first-generation replication deficient adenoviral (Ad5) vector containing the coding sequence for human ␤-endorphin to transduce cells of the pia mater. Ad5-mediated gene transfer resulted in the release of ␤-endorphin into the CSF and attenuated inflammatory hyperalgesia, measured as the thermal withdrawal latency in the carageenen model of inflammatory pain in the rodent, without affecting basal nociceptive responses. Although the inflammatory response elicited by the first-generation adenoviral vectors employed in this study limited the duration of transgene expression, a similar approach using later-generation vectors might be appropriate for patients with severe refractory pain in the terminal stages of a disease process.

Herpes-Mediated Gene Transfer in the Treatment of Pain

An alternative but related approach uses vector-mediated gene transfer to transduce neurons, rather than meningeal cells. For this purpose, herpes simplex virus (HSV) has proven to be a useful gene transfer vector. HSV is a neurotropic virus that naturally infects skin and mucous membranes. Following the initial epithelial infection, HSV is taken up by nerve terminals in the skin and carried by retrograde axonal transport to the cell bodies of sensory neurons in the DRG where the viral DNA is inserted through a nuclear pore into the nucleus. The uptake and transport of the virion from the skin is an efficient process mediated first by interactions between specific viral envelope glycoproteins and high-affinity receptors in the sensory nerve terminals in the skin [17, 18], followed by specific interactions of capsid and tegument proteins with dynein molecules in the axoplasm to mediate the retrograde axonal transport along microtubules to the cell body [19]. The highly efficient delivery of viral DNA to the DRG neuronal nucleus from an original infection in the skin coupled

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with the natural ability of the viral genome to establish a life-long latent state as an intranuclear episomal element makes HSV a very effective gene transfer vector for the peripheral nervous system. Pohl et al. demonstrated that an HSV-based vector in which the HSV thymidine kinase sequence is replaced by the cDNA coding for human proenkephalin, injected under the skin in the paw of a rat, transduced DRG neurons to produce enkephalin [20]. Immunoreactive met-enkephalin is transported anterogradely (i.e., away from the cell body) in both directions in the bipolar axon from DRG neurons, towards the spinal cord and back towards the skin with a larger amount moving peripherally than centrally [21]. Electrically stimulated release of metenkephalin from nerve terminals can be demonstrated in an in vitro preparation [Pohl, pers. commun.]. Wilson et al. [22] then showed that subcutaneous inoculation of a similar tk-deleted HSV vector expressing PE reduces hyperalgesia measured by the sensitization of the foot withdrawal response after application of capsaicin (C fibers) or dimethyl sulfoxide (A␦ fibers). The effect of the vector persisted for at least 7 weeks after the inoculation of the vector subcutaneously into the dorsum of the foot. Baseline foot withdrawal responses to noxious radiant heat mediated by A␦ and C fibers were similar in animals infected with PE-encoding and ␤-galactosidase-encoding vectors, demonstrating that the PE-expressing vector selectively blocked hyperalgesia without disrupting the baseline sensory neurotransmission. This blockade of sensitization was reversed by the administration of the opioid antagonist naloxone, apparently acting in the spinal cord [22]. Deletion of HSV-TK impairs the ability of the virus to replicate in neurons while leaving replication characteristics in non-neuronal cells intact. HSV gene expression occurs in a rigid temporal sequence and only five of the more than eighty HSV genes that are expressed during the lytic replication cycle are characterized as ‘immediate early’ (IE) genes. The expression of IE genes begins immediately after the viral entry into the nucleus, activated by a viral protein (VP16) contained in the tegument, and does not require the de novo expression of other viral proteins. Deletion of even one essential IE gene from the HSV genome creates a recombinant that can be propagated in a complementing cell line that provides the essential IE gene product in trans. These IE gene-deleted HSV vectors are incapable of replication in noncomplementing cells [23]. Introduced into animals, such replication-incompetent vectors do not replicate, but instead traffic to DRG neurons and establish a persistent state in a fashion identical to that observed for the replication-competent recombinants. We have examined the pain-relieving properties of a replication-incompetent HSV vectorexpressing human PE in rodent models of inflammatory pain, neuropathic pain, and pain resulting from cancer in bone.

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Cumulative pain score

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Fig. 1. Time course of the anti-nociceptive effect of SHPE inoculation in inflammatory pain (formalin test). The cumulative pain score during the delayed phase of the formalin test (10 min to one hour after the inoculation of formalin) was significantly reduced in animals inoculated with the PE-expressing vector SHPE. The vector-mediated effect was maximal one week after the inoculation of the vector, and was no longer statistically significant in animals tested 4 weeks after vector inoculation. However reinoculation of the vector at 4 weeks reestablished the analgesic effect (28 ⫹ 7 day group). Intrathecal administration of the ␦ opioid receptor antagonist naltrexone in animals tested one week after vector inoculation (IT naltrexone) blocked the vector-mediated analgesic effect. From [31].

Injected under the skin of the foot, the PE-expressing HSV vector was detected in the DRG by PCR using primers specific for the human PE sequence, and the expression of PE mRNA was detected by RT-PCR using the same sequences [24]. In the formalin test of inflammatory pain, injection of the PE-expresssing HSV vector reduced spontaneous pain behavior during the delayed phase (10–60 min after the injection of formalin) without affecting the acute pain score. This effect was reversed by the intrathecal administration of naltrexone [24], suggesting in agreement with Wilson et al. that the site of action of the released transgene product is in the dorsal horn of spinal cord. The analgesic effect was limited to the injected limb; formalin testing on the limb contralateral to the injection showed no analgesic effect [Glorioso, unpubl. observations], further suggesting that release of enkephalin from primary afferent terminals in the dorsal horn was limited to the region of their projections in dorsal horn. HSV vector-mediated analgesic effects persisted for several weeks and then waned. Animals tested 4 weeks after vector inoculation showed no significant reduction in pain-related behavior during the delayed phase of the formalin

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Fig. 2. Time course of the anti-allodynic effect of SHPE inoculation in neuropathic pain (spinal nerve ligation model). Injection of SHPE (but not control vector SHZ) resulted in a sustained anti-allodynic effect in neuropathic pain that lasted for several weeks. Reinoculation of the vector 6 weeks after the initial inoculation re-established the antiallodynic effect. The vector-mediated pain-relieving effect was reversed by intraperitoneal administration of naloxone (data not shown). From [26].

test [24]. However, in animals reinoculated with the vector 4 weeks after the initial inoculation and then tested with formalin injection at 5 weeks, a substantial and significant anti-nociceptive effect was demonstrated, which was at least as great as the initial effect [24]. The time course of the vector-mediated effect is consistent with the known time course of the human cytomegalovirus immediate-early promoter that was used to drive transgene expression in these experiments. The fact that the reinjection could re-establish the initial analgesic effect suggests, but does not prove, that the animals had not developed tolerance to the vector-mediated release of enkephalin. The effectiveness of reinoculation also suggests that the exposure of animals to the HSV vector does not elicit a neutralizing immune response that would be capable of attenuating gene transfer from the vector inoculation. We have also examined the effect of transgene-mediated enkephalin release in the spinal nerve ligation model of neuropathic pain [25]. Isolated L5 spinal nerve ligation distal to the DRG results in a painful state that can be quantified by measures of mechanical and thermal hypersensitivity. Subcutaneous injection of the PE-expressing vector into the foot one week after spinal nerve ligation resulted in an anti-allodynic effect that lasted for several weeks [26]. The characteristics of this anti-allodynic effect were similar to those observed in the formalin model. Reinoculation of the vector at 6 weeks

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Sham

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Fig. 3. Transduction with SHPE blocks touch-induced elevation in c-fos expression in the dorsal horn. Animals with neuropathic pain from spinal nerve ligation stimulated with gentle rubbing of the paw show a characteristic increase in c-fos expression in dorsal horn neurons seen in vehicle-treated animals (a, top right panel). Subcutaneous inoculation of SHPE one week after spinal nerve ligation substantially blocked this induction in c-fos expression (a, bottom right panel). The quantitative data are shown in b. From [26].

re-established the anti-allodynic effect; the magnitude of the effect produced by reinoculation was at least as great as that produced by the initial injection and the effect persisted for a longer time after the reinoculation than that produced by the initial inoculation. Intraperitoneal naloxone reversed the anti-allodynic effect.

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Fig. 4. Transduction with SHPE reduces pain-related behavior in a model of pain resulting from cancer in bone. Rats with experimental osteogenic sarcoma of the distal femur (shown in right part of radiograph) demonstrate a spontaneous pain behavior that is reduced significantly in those animals that were inoculated subcutaneously in the foot with SHPE one week after tumor implantation. The analgesic effect of the vector was reversed by intrathecal naltrexone. From [28].

The effect of this vector-mediated anti-allodynic effect in neuropathic pain was continuous throughout the day. Animals tested repeatedly at different times through the day showed a similar elevation in threshold at all times tested [26]. Intraperitoneal morphine produced a greater anti-allodynic effect than the vector alone, but the inoculation of the maximum dose of morphine produced an anti-allodynic effect that persisted for only 1–2 h before waning. The effect of vector-mediated enkephalin (acting predominantly at ␦ opioid receptors) and morphine (acting predominantly at ␮ opioid receptors) was additive. The ED50 of morphine was shifted from 1.8 ␮g/kg in animals with neuropathic pain from spinal nerve ligation treated with PBS or inoculated with a control vector

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expressing lacZ, to 0.15 ␮g/kg in animals that had been injected with the PE-expressing vector one week after spinal nerve ligation. Twice daily inoculation of morphine (10 mg/kg IP) in spinal nerve ligated animals resulted in the development of tolerance by one week; beyond that timepoint, the continued twice-daily administration of morphine had no anti-allodynic effect. Animals that had been inoculated with the PE-expressing vector one week after spinal nerve ligation continued to demonstrate the anti-allodynic effect of the vector despite the induction of tolerance to morphine [26]. We also examined the effect of vector-mediated expression of PE in a rodent model of pain resulting from cancer in bone [27]. Implantation of NTCT 2,472 cells into the distal femur resulted in a significant spontaneous pain-related behavior that increased between 2 and 3 weeks after tumor injection. Animals that received a subcutaneous inoculation of the PE-expressing vector into the plantar surface of the foot one week after tumor injection showed a significant reduction in the ambulatory pain score when compared to control vector-inoculated tumor-bearing animals at both 2 and 3 weeks after tumor injection, an effect that was reversed by intrathecal naltrexone [28]. Radiographical analysis of tumor-bearing mice inoculated with SHZ or SHPE demonstrated bone loss indicative of the presence of the osteolytic tumor, and there was no evidence that transgene expression had any effect on tumor growth [28]. Similar effects have been demonstrated in adjuvant-induced polyarthritis in the rat [29]. Using a replication-competent HSV vector expressing PE, Pohl et al. showed that subcutaneous inoculation of the vector not only markedly improved the locomotion and reduced hyperalgesia, but also that the release of enkephalin from the peripheral terminals of the DRG axons resulted in a slowing of the progression of bone destruction. In that model both the slowing of joint destruction as well as reversal of the analgesic effect by a peripherally acting substituted naloxone analog suggest that the site of action of transgenemediated enkephalin released from transduced neurons is at the peripheral projection rather than in the spinal cord. However, the effect on joint destruction appears to be unique to the model of arthritis employed [Pohl, pers. commun.]. Whether this approach will be effective in the treatment of human pain should be determined quite soon. A proposal for a phase I human trial to examine the safety and tolerability of subcutaneous inoculation of an HSV vector deleted for the IE genes ICP4, ICP22, ICP27 and ICP41, and expressing human PE under the control of the human cytomegalovirus immediate-early promoter was presented to the Recombinant Advisory Committee at the NIH (for details, see RAC Protocol #0201–529 at http://www.webconferences.com/nihoba/ 20–21_june_02.htm). The study protocol describes the enrollment of 18 patients with cancer metastatic to a vertebral body resulting in pain unresponsive to maximal conventional management, who will receive an inoculation of the

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HSV vector subcutaneously in the dermatome corresponding to the radicular distribution of the pain. In this dose-escalation trial, 3 patients will be enrolled at each dose, increasing at half-log intervals. Pain treatment by transduction of DRG neurons using an HSV vector will be limited to pain syndromes that result in regional or focal pain. The noninvasive means of delivery of the vector, the focal effect of transgene expression, and the synergy with opiate drug treatment are chief advantages of this approach. However, once the transgene is introduced into the DRG neuron it cannot be removed, and using the current vectors, transgene expression is not regulated. This should not prove a problem for the use of enkephalin, and the transgene expression driven by the human cytomegalovirus promoter is transient. But in the use of other transgenes, or promoters designed to drive long term gene expression, these issues will resume further consideration and vector engineering. The fact that these patients are facing a fatal course of the disease with severe pain that is frequently not responsive even to high-dose systemic opioids strengthens the rationale for local, targeted gene transfer for pain relief.

Gene Transfer to the Brain in Models of Pain

There are other sites within the neuraxis where pain transmission may be interrupted by vector-mediated neurotransmitter expression. Injection of a replication-competent (tk-deleted) HSV vector expressing proenkepahlin into the amygdala bilaterally has been shown to reduce pain-related behavior in the delayed phase of the formalin test (animals tested 4 days after vector inoculation) [30], and injection of an HSV amplicon vector expressing glutamic acid decarboxylase to result in the release GABA in brain nuclei also reduces painrelated behaviors [Jasmin and Rabkin, pers. commun.]. While these experiments demonstrate that focal neurotransmitter effects can be achieved by gene transfer to the brain as well as the peripheral nervous system, applications to human clinical therapies are likely to take longer to develop, given the complicated neuropharmacology of CNS function.

Conclusion

Cell transplantation or vector-mediated gene transfer, by providing a means to target expression focally in the nervous system, may allow the use of short-lived macromolecules identical to the endogenous substances to enhance pain relief in specific situations. In the future, other macromolecules acting to interrupt the process responsible for the development of the pain

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(such as anti-inflammatory cytokines or specific neurotrophic factors in neuropathic pain) may be delivered in a similar fashion.

Acknowledgments This work was supported by grants from the NIH (JCG and DJF), the Department of Veterans Affairs (MM and DJF), and the Juvenile Diabetes Foundation Research International (DJF).

References 1 2 3 4 5 6

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Wilson SP, Chang KJ, Viveros OH: Opioid peptide synthesis in bovine and human adrenal chromaffin cells. Peptides 1981;2(suppl 1):83–88. Unsicker K: The trophic cocktail made by adrenal chromaffin cells. Exp Neurol 1993;123:167–173. Hama AT, Sagen J: Alleviation of neuropathic pain symptoms by xenogeneic chromaffin cell grafts in the spinal subarachnoid space. Brain Res 1994;651:183–193. Siegan JB, Sagen J: Attenuation of formalin pain responses in the rat by adrenal medullary transplants in the spinal subarachnoid space. Pain 1997;70:279–285. Sagen J, Wang H: Adrenal medullary grafts suppress c-fos induction in spinal neurons of arthritic rats. Neurosci Lett 1995;192:181–184. Ibuki T, et al: Loss of GABA-immunoreactivity in the spinal dorsal horn of rats with peripheral nerve injury and promotion of recovery by adrenal medullary grafts. Neuroscience 1997;76: 845–858. Sagen J, Kemmler JE: Increased levels of Met-enkephalin-like immunoreactivity in the spinal cord CSF of rats with adrenal medullary transplants. Brain Res 1989;502:1–10. Sagen J, Kemmler JE, Wang H: Adrenal medullary transplants increase spinal cord cerebrospinal fluid catecholamine levels and reduce pain sensitivity. J Neurochem 1991;56:623–627. Hentall ID, Noga BR, Sagen J: Spinal allografts of adrenal medulla block nociceptive facilitation in the dorsal horn. J Neurophysiol 2001;85:1788–1792. Wu HH, Wilcox GL, McLoon SC: Implantation of AtT-20 or genetically modified AtT-20/hENK cells in mouse spinal cord induced antinociception and opioid tolerance. J Neurosci 1994;14: 4806–4814. Eaton MJ, et al: Lumbar transplant of neurons genetically modified to secrete galanin reverse pain-like behaviors after partial sciatic nerve injury. J Peripher Nerv Syst 1999;4:245–257. Eaton MJ, et al: Transplants of neuronal cells bioengineered to synthesize GABA alleviate chronic neuropathic pain. Cell Transplant 1999;8:87–101. Cejas PJ, et al: Lumbar transplant of neurons genetically modified to secrete brain-derived neurotrophic factor attenuates allodynia and hyperalgesia after sciatic nerve constriction Pain. 2000; 86:195–210. Buchser E, et al: Immunoisolated xenogenic chromaffin cell therapy for chronic pain. Initial clinical experience. Anesthesiology 1996;85:1005–1012; discussion A29–A30. Lazorthes Y, et al: Human chromaffin cell graft into the CSF for cancer pain management: A prospective phase II clinical study. Pain 2000;87:19–32. Finegold AA, Mannes AJ, Iadarola MJ: A paracrine paradigm for in vivo gene therapy in the central nervous system: treatment of chronic pain. Hum Gene Ther 1999;10:1251–1257. Spear PG, Eisenberg RJ, Cohen GH: Three classes of cell surface receptors for alphaherpesvirus entry. Virology 2000;275:1–8. Shukla D, Spear PG: Herpesviruses and heparan sulfate: An intimate relationship in aid of viral entry. J Clin Invest 2001;108:503–510.

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Smith GA, Enquist, LW: BREAK INS AND BREAK OUTS: Viral interactions with the cytoskeleton of mammalian cells. Annu Rev Cell Dev Biol 2002;18:135–161. Antunes Bras JM, et al: Herpes simplex virus 1-mediated transfer of preproenkephalin A in rat dorsal root ganglia. J Neurochem 1998;70:1299–1303. Antunes Bras J, et al: Met-enkephalin is preferentially transported into the peripheral processes of primary afferent fibres in both control and HSV1-driven proenkephalin A overexpressing rats. Neuroscience 2001;103:1073–1083. Wilson SP, et al: Antihyperalgesic effects of infection with a preproenkephalin-encoding herpes virus. Proc Natl Acad Sci USA 1999;96:3211–3216. DeLuca NA, McCarthy AM, Schaffer PA: Isolation and characterization of deletion mutants of herpes simplex virus type 1 in the gene encoding immediate-early regulatory protein ICP4. J Virol 1985;56:558–570. Goss JR, et al: Antinociceptive effect of a genomic herpes simplex virus-based vector expressing human proenkephalin in rat dorsal root ganglion. Gene Ther 2001;8:551–556. Kim SH, Chung JM: An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 1992;50:355–363. Hao S, et al: Transgene-mediated enkephalin release enhances the effect of morphine and evades tolerance to produce a sustained antiallodynic effect. Pain 2003;102:135–142. Schwei MJ, et al: Neurochemical and cellular reorganization of the spinal cord in a murine model of bone cancer pain. J Neurosci 1999;19:10886–10897. Goss JR, et al: Herpes vector-mediated expression of proenkephalin reduces pain-related behavior in a model of bone cancer. pain. Ann Neurol 2002;52:662–665. Braz J, et al: Therapeutic efficacy in experimental polyarthritis of viral-driven enkephalin overproduction in sensory neurons. J Neurosci 2001;21:7881–7888. Kang W, et al: Herpes virus-mediated preproenkephalin gene transfer to the amygdala is antinociceptive. Brain Res 1998;792:133–135. Chen X, et al: Herpes simplex virus type 1 ICP0 protein does not accumulate in the nucleus of primary neurons in culture. J Virol 2000;74:10132–10141.

David Fink, MD 1914 Taubman Center/0316, 1500 E Medical Center Drive Ann Arbor, MI 48109-0316 USA Tel. ⫹1 734 936 9070, Fax ⫹1 734 763 5059, E-Mail [email protected]

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Neurovascular Disorders Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 336–376

Gene Discovery Underlying Stroke Frank C. Baronea, Simon J. Read b a

High Throughput Biology, GlaxoSmithKline, King of Prussia, Pa., USA, RIRA, Astra Zeneca, Mereside, Alderley Park, Macclesfield, Cheshire, UK

b

Introduction

As first-pass efforts to complete sequencing of the entire human genome are now concluded [1], there is an increased interest in the application of genomic approaches to aid in the discovery, development, and rationale use of drugs. As databases of differential gene expression have expanded, so has the expectation of identifying novel drug targets for disease intervention. Indeed, significant work has already been carried out to understand gene expression changes in many diseased organ systems, including the ischemic brain [2–6]. Early epidemiological studies of the 1970s provided initial evidence for a genetic influence in stroke. The Framingham study was one of the first studies to suggest that a positive parental history of stroke contributed significant risk to the offspring [7]. Thirty years later, stroke remains an area of substantial unmet medical need. The complexity of stroke undoubtedly reflects the heterogeneity of the human stroke population, the contribution of monogenic and polygenic disorders to this disease process, and the interactions of these with a multitude of environmental factors. This chapter focuses on genetics of risk and sensitivity to ischemic stroke. It will discuss how inheritance relates to the broader stroke population and provide a detailed discussion of the stroke genomics literature. It will describe how pre-clinical models of spontaneous stroke can be applied to humans to identify the chromosomal loci of risk, and how the changes in gene expression associated with stroke are associated with poststroke brain injury, resolution of brain injury, and brain recovery processes. In addition, it will provide a detailed discussion of several differential gene expression analyses techniques. This will include a detailed discussion of genes identified using different techniques and the importance of a stroke model that has been well characterized and representative of the

type of stroke most often observed in man. Issues of validation of potential stroke targets, the relevance of the expression of neuroprotective and neurodestructive genes and their specific timings, genes involved in endogenous brain protection and in brain recovery of function/plasticity, and the emerging problems with handling novel/unknown genes that may be discovered from these analyses of differential gene expression also will be addressed.

Ischemic Stroke

Stroke is the third largest cause of death in the USA, ranking only behind heart disease and cancer. It is the leading cause of disability in the USA and has the highest disease burden cost. Ischemic strokes comprise the majority of strokes, between 70–80% of all strokes. No medical treatment is approved for the treatment of acute ischemic stroke other than thrombolytic agents such as tPA, which for optimum results must be administered within 3 h after stroke onset. At most centers, only 1–2% of the stroke patients meet the criteria for treatment with this thrombolytic agent. Aspirin and anticoagulants (where embolic phenomena are documented) also are utilized as preventative therapy. Estimates indicate that there are about 775,000 new stroke cases per year in the USA, with a prevalence of about 4 million surviving, but at an increased risk of a secondary cardiovascular event. In the USA, stroke is costly, with an annual health care cost of $30–50 billion. Estimates indicate that stroke is responsible for half of all the patients hospitalized for acute neurological disease [8, 9]. Stroke risk factors include both genetic and environmental factors. Stroke risk factors that can be treated include high blood pressure, heart disease, cigarette smoking, transient ischemic attacks, and high red blood cell count. Risk factors for stroke that cannot be changed include age, gender (men have ⬃20% greater risk of stroke than women), race (African-Americans have a much higher risk of death and disability from stroke), diabetes mellitus, prior stroke, and family history of strokes. Other controllable risk factors, secondary risk factors, for stroke that also contribute to heart disease include high blood LDL-cholesterol and lipids, physical inactivity, and obesity [10].

Genetics of Increased Stroke Risk

The strongest evidence for a genetic risk to stroke comes from twin studies. Proband concordance rates have long been used to identify the heritability of a trait or disorder. The concept of concordance is that for a disorder of genetic predisposition, the rate will be higher for monozygotic twins than dizygotic twins.

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Aside from genetic influence, it is assumed that other factors, such as environmental exposure will be approximately similar for both types of twins [11]. An elevated proband-wise concordance rate for stroke risk in monozygotic twins over dizygotic twins (17.7 vs. 3.6%) has confirmed a genetic predisposition to stroke with a role of environmental factors [12]. A more recent twin study has refined cohort analysis to stroke risk by assessing individual stroke phenotypes that may be influenced by genetic factors [13]. In this study, the phenotype of white matter hyperintensity volumes using magnetic resonance imaging (MRI) was applied and genetic factors accounted for 71% of the variation in this endpoint [13]. A large number of familial studies have verified that a history of paternal or maternal stroke is associated with an occurrence of stroke in offspring, and that a positive paternal history of stroke was an independent prognostic predictor of stroke [14–16]. In a cohort of men studied since 1913, maternal history of stroke increased relative stroke risk by 3-fold [17]. Similarly, it has been reported that a positive family history of stroke in any first-degree relative was an independent predictor of stroke mortality in women aged 50–79, but not in men [18]. Moreover, a family history of stroke in men was an independent predictor of coronary heart disease aged 50–64 years, indicating that genetic risk factors for stroke may be shared with other cardiovascular disorders that have a high genetic component [18]. Indeed, studies of the relative risk of other cerebrovascular diseases with less heterogeneous phenotypes are able to document strong patterns of inheritance. Subarachnoid hemorrhage occurs with a relative risk of 6.6 in first-degree relatives compared to second-degree relatives [19]. Defining specific stroke subtypes may be the key to elucidating the exact degree of genetic contribution to any particular phenotype. From twin studies, it appears that the extent to which genetic factors may contribute to stroke risk varies with age. These factors are caveats to the identification of therapeutic targets from candidate gene strategies, and one must remember that a candidate gene approach for inheritance of risk factors may only be relevant to a highly limited stroke subpopulation.

Simple Stroke-Like Diseases: Single Gene Mutations

Identification of possible genetic determinants of stroke risk has been hampered by the lack of similar patient populations. Mendelian disorders with specific stroke-like phenotypes have been explored as genetic models of the more general population. These disorders include CADASIL (cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy) [20], MELAS (mitochondrial encephalopathy, lactic acidosis-and stroke-like

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syndrome) [21], Sneddon’s syndrome [22], familial hemiplegic migraine [23] and hereditary coagulopathies [24]. Although these subgroups contribute little to the overall prevalence of stroke, genes identified from them are hoped to highlight potential commonalities in the wider patient population. Studies on CADASIL and MELAS are examples of such approaches. CADASIL was originally described as an inherited, autosomally dominant dementia with multiple infarcts [25]. Epidemiologically, CADASIL is limited to sporadic cases in Europe [26, 27] and North America [28, 29]. The principal symptoms of the CADASIL are migraine with aura, ischemic stroke, and psychiatric symptoms including dementia [30]. In these patients, T2-weighted MRI reveals small periventricular white matter hyperintensities often involving the internal capsule [31]. The CADASIL gene, identified as Notch 3, is located at the chromosomal loci 19p13.1–13.2 [27, 32]. The Notch genes regulate the lin12/sel-12 signaling pathway that is known to be important in development, although the normal adult function of Notch genes remains unknown [33]. An interesting association of the Notch 3 gene with Alzheimer’s disease has also been discovered. Notch gene products interact with the presenilin 1 pathway as substrates for ␥-secretase. This enzyme is known to have a key pathological role in the production of A␤ peptide, although the modulatory role that Notch 3 may have in this disease process is undefined [34, 35]. The Notch 3 gene encodes a transmembrane protein composed of 2,321 amino acids, presumed to have a receptor function and located primarily on smooth muscle cells [30]. In CADASIL, approximately 90% of patients have missense mutations in extracellular domains of the protein product, whilst in about 70% of patients, the mutation is located within exons 3 and 4 [35]. All known mutations associated with CADASIL result in the removal or addition of cysteine residues and it is proposed that the expression of these mutated Notch 3 proteins results in cerebral vascular smooth muscle dysfunction [36]. Whether abnormalities in Notch signaling impact on the broader stroke population is at present unknown, although the pathogenesis of CADASIL, characterized by the progressive disruption of vascular endothelium with secondary fibrosis and thrombosis is typical of some stroke subpopulations [37]. Anticoagulant therapy has been tried in CADASIL without positive results [30]. More broadly, CADASIL also has close relationships to Alzheimer’s disease; signaling components of the presenilin pathway are shared with the Notch pathway [38]. The presenilin-1-regulated ␥-secretase cleaves both the Notch intracellular domain and ␤-amyloid precursor protein for subsequent translocation to the nucleus and binding to DNA [38]. Therefore, whilst the pathology of CADASIL may bear similarity to stroke, the cell biology also has potential connections with Alzheimer’s disease. Since vascular risk factors and/or disease can impact on both vascular dementia and Alzheimer’s disease, these relationships are intriguing [39–41].

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MELAS are characterized by migraine-like headache, nausea, seizures, and stroke-like episodes. Lesions are most commonly found in the occipital and parietal regions, with high lactate levels found within lesions using proton nuclear magnetic resonance [42]. Patients typically have mutations of mitochondrial DNA for the tRNA-leu gene at an A-G transition mutation at nucleotide position 3243 [42, 43] and at a T-C transition at position 3271 [44]. It has been speculated that as mutations accumulate, a gradual mitochondrial dysfunction develops [45]. It is unclear how widespread such mutations are in the broader stroke population. Indeed, cases of MELAS have been reported without a family history, suggesting that these point mutations may be spontaneous [46]. Pharmacological interventions have reflected a unique nature of MELAS within stroke/cardiovascular disease subpopulations. Antithrombotic therapy has been employed in MELAS patients for cardiac complications associated with left ventricular dysfunction [47]. CADASIL and MELAS demonstrate that several relatively rare ‘strokelike’ syndromes can be used to explore potential genetic determinants of stroke. Parallel strategies have been adopted with similar success in other more complex, multifactorial polygenic traits such as hypertension [48–50]. Genes such as 11␤-hydroxylase in glucocorticoid-remediable aldosteronism have been shown to mediate hereditary hypertension in these patients [51]. However, as in stroke genetics, narrowing heterogeneity and studying single gene or Mendelian disorders may have limited application to the broader patient population.

Ischemic Stroke: Complex Genetic Associations

In common with many diseases, there are individuals with complex genetic profiles which confer vulnerability to stroke, as well as poststroke gene expression, which can contribute to increased cerebral ischemic stroke effects. Candidate gene studies in heterogeneous stroke populations minimize issues of limited patient population by the choice of a functionally relevant gene and its relationship with a particular phenotype. This is termed ‘association’ and is a statistical measure of the dependence of a particular phenotype (e.g., ischemic stroke with the presence of a particular candidate gene/allele). Therefore, association can be positive (i.e., significant statistical relationship/association between the gene of choice and phenotype), or negative (i.e., absence of significant relationship/association between gene/allele and phenotype). Candidate gene polymorphisms with a positive association with stroke include: apolipoprotein E [52–54], ACE ([55–58], fibrinogen [59], Factor V [60] and Prothrombin [61]. Those gene polymorphisms with a negative association with stroke include: eNOS [62], methyl-tetrahydrofolate [61, 63], angiotensinogen [55], Factor V

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[64, 65], Factor VII [65], Factor VIII [66] and prothrombin [67]. Atrial natriuretic peptide (ANP) has a particularly strong positive association with stroke [68–70]. Candidate gene choice is frequently driven by accepted stroke risk factors (e.g., hypertension, hemostasis and abnormalities in lipid metabolism) and indeed significant positive associations of numerous markers with ischemic stroke have been identified. At present, however, it is difficult to identify candidate gene associations with ischemic stroke [4]. Reproducibility of these gene expression associations in different patient populations (e.g., different race or genetic backgrounds) also is not known. The bewildering combination of possible outcomes for candidate gene association studies is related to the genomic and phenotypic heterogeneity of the global stroke population. Studies are typically designed with case controls or by cohorts to enable close approximation of phenotype between affected and nonaffected individuals. Superimposed upon these levels of variation are issues in the timing of stroke onset, in the variability of environmental influences and penetrance (i.e., not all individuals of a given genotype will express the phenotype). Finally, although the human genome project has been completed [1], identifying functionality of gene products lags significantly behind. Currently, it is estimated that only approximately 10% of the human genome has been ascribed function [24]. Certainly more work needs to be done in this area, and issues related to stroke genomics that include risk and the expression of genes underlying brain vulnerability and ischemic sensitivity must be considered.

Preclinical Models of Spontaneous Stroke

It is with these caveats in mind that studies have focused on animal models of spontaneous stroke, where environmental and genetic variability can be controlled. Bioinformatic approaches using synteny can facilitate the matching of ‘stroke loci’ found in stroke-prone rats to candidate genes on the human chromosome. Heterogeneity of risk factors and life events in humans has made it advantageous to study rodent models. Highly homogeneous populations of stroke-prone rats have been isolated from the incompletely inbred, spontaneously hypertensive rat (SHR) and then inbred further for this phenotype. Initial studies using this stroke-prone rat indicated that the degree of functional collateral blood flow after occlusion of the middle cerebral artery (MCAo) was inherited in an autosomally recessive manner [71–73]. The authors studied luminal diameters in vascular anastomoses between middle and anterior cerebral arteries and hypothesized that a single gene not directly linked to hypertension determined the collateral flow phenotype. Further genetic comparisons between strains were hampered by heterogeneity.

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Narrowing the genotype by further crossing SHR rats with stroke-prone animals allowed cosegregation of genes defining various stroke phenotypes and for homogeneity of alleles for hypertension [74]. Two separate groups have utilized these inbred populations for identification of genes associated with manifestation of specific stroke phenotypes. A genome-wide screen was performed in an F2 cross-obtained by mating stroke-prone and SHR rats, in which latency to stroke was used as a phenotype [75]. This study identified three major quantitative trait loci (QTLs) designated, STR-1, STR-2, and STR-3. Of these, STR-2 and STR-3 conferred a protective effect against stroke in the presence of stroke-prone alleles and STR-2 colocalized with the candidate gene encoding ANP and brain natriuretic peptide (BNP). Furthermore, interactions between alleles from within STR-1 and STR-2 suggested that this phenotype was a reasonable model of the polygenicity of stroke in man. Follow-up sequencing to characterize ANP and BNP as candidates for stroke revealed point mutations in ANP and no differences in BNP. In vitro functional studies indicated lower ANP promoter activation in endothelial cells from stroke-prone rats versus SHR, with significantly lower ANP expression in the brain and no difference in BNP expression [68]. To determine the in vivo significance of the STR-2 lowered ANP promoter activation in stroke-prone animals, in comparison to stroke-resistant animals, a cosegregation analysis of stroke occurrence in SHR stroke-prone rats/SHR stroke-resistant F2 descendants and ANP expression was performed [69]. It was found that reduced expression of ANP did cosegregate with the appearance of early strokes in F2 animals [70]. Therefore, although lowered ANP expression may be part of the phenotype of the protective STR-2 QTL, it is unlikely that this is the primary protective mechanism in these animals. Parallel human studies of the role of ANP in cerebrovascular disease have confirmed that variation in the ANP gene may represent an independent risk factor for stroke in humans [4, 75] and emphasizes the utility of this cohort of animals as a model of ANP dysfunction in multiple subtypes of stroke. Two other groups have utilized a modified model of the stroke-prone animal, employing F2 hybrids derived from crossing the stroke-prone SHR with Wistar-Kyoto rats [76, 77]. One group [76] used brain weight poststroke as the phenotype for linkage analysis, after the discovery that F2 animals had higher levels of brain edema formation poststroke. This group found clear evidence of the linkage of phenotype to a gene on chromosome 4, which contributed to the severity of brain edema and was independent of blood pressure and STR3 identified by others [75]. The other group [77] designed studies to identify the genetic component responsible for large infarct volumes in the stroke-prone rat in response to a focal ischemic insult. To do this, they performed a genome scan in an F2 cross-derived from the stroke-prone rat and the normotensive Wistar-Kyoto rat [77]. Unlike others [75], they were only able to identify one

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major QTL responsible for large infarct volumes. This QTL was located on rat chromosome 5, and like STR-2, it colocalized with ANP and BNP, and was blood pressure independent. Unlike STR-2, this locus showed a much higher significance (lod 16.6) and accounted for greater (i.e., 67%) of phenotypic variance [77]. Subsequent studies identified that infarct volumes in the F1 rats were approximately identical to those of the stroke-prone animals, suggesting a dominant mode of inheritance [78]. Authors have argued over the significance of the overlap of STR-2 identified by some [55] with the QTL identified by others [77] on chromosome 5. It is unclear how the two phenotypes studied, latency to stroke (i.e., relative risk) [75] and size of infarct after occlusion (i.e., sensitivity to focal ischemia) [77], should physiologically relate to each other. However, this may only become apparent when individual genes are cosegregated with each phenotype. At present, altered ANP expression appears to play a role in the phenotype described by one group [75], but has been excluded from a role in the colony used by the other group [77, 79]. What can be concluded from each of these stroke-prone rat models? Certainly, each represents a unique and valid model of stroke for the study of inheritance, and for a role of candidate genes, in particular, stroke phenotypes. Neither colony represents a definitive model of human stroke, although linking identified candidate genes in these stroke-prone colonies to the human population has made progress [70]. One such research strategy that we have used is the analysis of genomic synteny between the rat and human genome. This bioinformatic approach seeks to align regions of homology using evolutionary conserved markers and has been applied with some success in relating animal models to human genetics in other disease paradigms such as non-insulin-dependent diabetes [80]. Relating identified loci from stroke-prone animals to the human genome offers a strategy for potential identification of candidate genes. For example, the STR-2 region of rat chromosome 5 shows well-conserved gene order and synteny with the human chromosome region 1p35–36. The high level of synteny between these regions makes this region ideal for rat-human comparative analysis. Sequence tagged sites localized to this region have been identified and mapped to human transcript clusters. As many as 132 transcripts have been identified in this region. The main candidates with some rationale for involvement in stroke are shown in table 1. Interestingly, only a few candidate genes identified at 1p35–36 have been examined in association studies. ANP has recently been assessed for association with multiple subtypes of stroke [70]. The polymorphism G664A, responsible for a valine-methionine substitution in proANP peptide was found to be positively associated with the occurrence of stroke [70]. In contrast, methylenetetrahydrofolate, another marker located at 1p35–36, was negatively associated

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Table 1. 1p36–p35 positional candidates with a biological rationale in stroke* Homology

Rationale in stroke

CD30L receptor precursor

Nerve growth factor receptor superfamily Mouse KO increases neuronal damage in response to insults Depletion of complement system improves outcome following cerebral ischemia Role in cell adhesion May be related to sustained contraction during cerebral vasospasm Critical upstream activator of the caspase cascade in vivo Role in neuronal development

Tumor necrosis factor receptor 2 Human MASP-2 serine protease protein – complement processing protease gp40 mucin – putative influenza virus receptor Human Tropomyosin-related proteinexclusively neuronal/brain expression Human protease proMch6 (Mch6)/CASPASE-9 EPHRIN RECEPTOR EphA2/Tyrosine-protein kinase receptor ECK Human PDGF-associated protein Endothelin-converting enzyme E1 WNT4 protein precursor

EPHRIN RECEPTOR EphB2/Tyrosine-protein kinase receptor ERK Stathmin – v high brain expression Corticosteriod-binding protein – yeast putative bicistronic heat shock proteins Platelet-activating factor receptor Dishevelled-1

Atrial natriuretic peptide A Brain natriuretic peptide B Complement component 1, q subcomponent, alpha polypeptide (C1QA) 5,10-Methylene-tetrahydrofolate reductase

Brain-specific angiogenesis inhibitor (BAI2) Platelet phospholipase A2, group IIA

Barone/Read

Unknown Enzyme that produces potent vasoconstriction Possible role in synaptic plasticity. Linked to JNK signaling and indirectly to Notch (CADASIL) Role in neuronal development Phosphorylated by CAM kinase II Stress response Unknown Possible role in synaptic plasticity. Linked to JNK signaling and indirectly to Notch Localized with LOD peak hypertension, see text Localized with LOD peak hypertension, see text Depletion of complement system improves outcome following cerebral ischemia Heterozygous mutations are significant cause of stroke in general population, see text Regulator of angiogenesis Antiplatelet agents modify stroke risk

344

Table 1 (continued) Homology

Rationale in stroke

Sodium hydrogen exchanger-1

pH regulator of acidity associated with postischemic damage PAF involved in arterial thrombosis; Antiplatelet agents modify stroke risk

Platelet-activating factor receptor (PTAFR)

*This table documents the identification of human candidate genes that are syntenic to the STR2 region of rat chromosome 5 identified in a cohort of SHR-stroke prone animals. STR2 shows well-conserved gene order and synteny with the human chromosome region 1p35–36 (between D1S503–D1S2667). The high level of synteny between these regions makes this region ideal for rat-human comparative analysis. Sequence tagged sites localized to this region have been identified and mapped to human transcript clusters. Many transcripts, specifically 132 of them, have been identified in this region, with the main candidates listed above.

with occurrence of stroke [79]. Further studies may elucidate the predictability of markers of 1p35–36 and association with stroke for those genes listed in table 1. In contrast, rat-human synteny in the regions of the rat STR-1 and STR-3 loci are not well conserved, as several disruptions of synteny appear to have been introduced during evolution. It may be difficult to determine the exact regions of synteny between these rat loci and human chromosomal loci, and thus to extrapolate the candidate genes from rat to human. Human chromosomal regions syntenic with STR-1 span regions of two human chromosomes, around 16p11 and 19q13. Human synteny with the STR-3 region also appears to be disrupted, with regions of synteny mapping telomerically to opposite arms of chromosome 7 (7p21 and 7q35). Of course, this is a problem of animal modeling of human diseases in general and is not restricted only to ischemic stroke.

Stroke-Associated Gene Expression in the Evolution of Brain Injury

Cerebral ischemia is a powerful stimulus for the de novo expression and up-regulation of numerous genes [2, 4, 5, 81, 82]. In terms of isolation of gene candidates for a neuroprotective strategy, interpretation of expression changes has proven difficult. The multitude of animal models of ischemia with varying

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genetic heterogeneity and infarct pathophysiology, is also complicated by spatial and temporal variations that have largely confounded interpretation. Furthermore, assays of differential expression have varying sensitivity to the relative fold-increase or -decrease in mRNA expression. As a result, ‘fishing’ exercises (i.e., differential expression detection studies) will often result in ‘catches’ (i.e., hits) of differential gene expression that vary depending on the assay employed. Bearing in mind this bewildering array of complexity, the next section addresses animal model(s) that might be utilized with differential gene expression analysis, target confirmation methodology that is necessary following the identification and confirmation of a differentially expressed gene (i.e., a ‘hit’), and the functional assessment of these genes in the disease process. A hierarchical critical path that depicts the path from target identification to target confirmation/validation is depicted schematically in figure 1.

Clinical Relevance of Ischemic Stroke Models

The failure of several putative neuroprotective agents in recent large multicentered clinical trials [83, 84] has led to critical re-examination of preclinical models of ischemia [85]. Heterogeneity in the human stroke population and the multitude of well-defined animal models of ischemia have led to attempts to refine model choice as related to patient subgroups [86, 87]. In an effort to stratify patient groups that can be predicted using specific animal models, authors have focused on the use of MRI signatures, particularly perfusionweighted or diffusion-weighted imaging (PWI, DWI) mismatches. Two main groups of acute stroke patients are identifiable: those with evolving infarcts in which lesion PWI ⬎ DWI, or those with a stabilized infarct where PWI ⱕ DWI [88, 89]. Such PWI/DWI assessments have been proposed to correlate to the extent of salvageable tissue, with approximately 70% of patients exhibiting PWI lesions ⬎ DWI at 6 h poststroke, and about 50% of patients exhibiting this mismatch at 24 h poststroke [89]. Applying the same imaging paradigms to animal models of focal ischemia should enable translation of preclinical pathophysiology into predictive outcomes in the appropriate patient population. However, detailed comparisons of the development of PWI/DWI signatures between animal models of ischemia are difficult to establish due to the use of various rat strains, anesthetics, and modes of ischemia induction. However, broad comparisons are possible by exploring the development of DWI signal as a marker of lesion volume with respect to time. Data in certain animal models of focal stroke can show a delay in the development of DWI hyperintensity (i.e., brain lesion size) that lags

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Critical path from expression to target validation Animal model

mRNA differential expression

RDA, Microarrays, SAGE, SSH, DD

Reproducibility/commonality of ‘hits’?

Expression studies

Confirm differential expression Expression analysis

Northern analysis Quantitative RT-PCR In situ hybridization (ISH)

mRNA localization

Confirmation and localization of protein expression

ELISA, Western analysis, Immunohistochemistry (IHC)

Functional studies

Genetic/transcriptional modification

In-Vivo pharmacological studies

Targeted gene KO Antisense Adenoviral transfection Appropriate PK tool compound treatment

Fig. 1. Critical pathway for target identification and confirmation. Following selection of an animal model appropriate to clinical subpopulation, broad mRNA differential expression strategies are adopted employing differential expression assays such as RDA, microarrays, subtractive hybridization (SSH), serial analysis of gene expression (SAGE) and/or differential display (DD). Across these assays, reproducibility in identified hits are explored as a technique of prioritizing subsequent studies to confirm differential expression. Comprehensive expression analysis using RT-PCR or Northern analysis allows confirmation of identified hits and fully quantified temporal profiling. This can also include RNA localization using in-situ hybridization (ISH). Protein confirmation by ELISA, Western analysis and immunohistochemistry (IHC) follows mRNA profiling, to confirm translation. In an ischemic brain, pooling of mRNA and uncoupling of translation can occur as indicated in the text. Finally, functional studies encompassing target gene knockout, adenoviral transfection and in vivo pharmacology complete validation of a potential target gene. Appropriate chemistry directed against the verified biological target could result in the eventual discovery of a drug for stroke.

behind a perfusion deficit (i.e., PWI changes associated with stroke or focal ischemia). This delay is attributable, in part, to the relative contribution of insufficient collateral flow and the peri-infarct depolarization that injures the poorly perfused, ischemic brain during infarct evolution [87, 90].

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Direct MCAo by proximal electrocoagulation of the middle cerebral artery produces an expanding DWI lesion, with an initial marked expansion at 4 h followed by a small increase from 4 to 24 h [91]. Electrocoagulation occlusion of the distal MCA produces a much more rapidly evolving infarct with a near maximal DWI lesion observed by 1–2 h [92, 93]. Available embolic models of focal stroke using intra-arterial injection of thrombin [94] or aged [95] or fibrin-rich [96] clots have reported similar expansion of DWI hyperintensity. For example, following thrombin injection, DWI hyperintensity is apparent at 80 min postadministration, with the volume gradually expanding up to 24 h [94]. Intraluminal suture occlusion produces a range of DWI lesion progression dependent on whether the filament is introduced via the common carotid artery [97] or the external carotid artery [98]. Permanent MCAo via common carotid artery suture produces a rapid evolution of DWI hyperintensity within minutes, followed by maximal expansion by 2 h [99, 100]. In comparison, permanent MCAo without occluding the common carotid artery (i.e., via the external common carotid artery) [98], evokes an initial rapid expansion of DWI hyperintensity over the first 30 min followed by final infarct volume reached at 7 h [101–103]. A close inspection of this model identifies it as exhibiting a flow mismatch similar to that in man, i.e., it represents the type of evolving infarct that should provide information relevant to human stroke [87]. For this reason a number of groups [6, 104, 105] have decided to use this model for differential gene expression studies.

Differential Gene Expression Methodologies

The detection of genes that are differentially expressed due to stroke can be identified using simpler techniques such as Northern blotting, RT-PCR, or in situ hybridization. These techniques involve the selection and study of a specific gene of interest based on previous data that provides a biological rationale for study in stroke or another specific disease. However, more sophisticated screening techniques are now available that can identify groups of differentially expressed genes, both known and unknown. These screening techniques include subtractive hybridization, differential hybridization, representational differential analysis (RDA), serial analysis of gene expression (SAGE), and differential display [106]. The identification of differentially expressed genes in stroke has employed the simpler techniques as well as these newer techniques. Variations in assay and threshold of detection can result in the isolation of gene sets that differ according to assay selection. Therefore, to ensure maximum confidence in the detection of adaptive up-regulation of gene expression,

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reproducibility of genes and pathways should be verified across several differential expression techniques and independent cross-validation of a gene’s up-regulation should be emphasized. A summary of stroke-associated gene expression discovered by all the techniques is listed in table 2. Only those gene/message expression changes in the rat following MCAo were confirmed using more than one expression detection technique. A brief summary of the more complex techniques is provided below. Differential Display Differential display is a means of comparing all poly-A mRNA between experimental and control populations. In this technique, mRNA is converted into first strand cDNA with reverse transcription followed by polymerase chain reaction (PCR) with multiple sets of primers. The PCR products are then displayed with control and experimental samples side-by-side on high-resolution denaturing gel. In this way, differential gene expression is apparent. This technique has been applied with success to isolate differentially expressed products following experimental MCAo. For example, differential display was used after rat MCAo to discover a gene that encodes adrenomedullin, a member of the calcitonin gene-related peptide family [113]. This analysis was followed by temporal studies using Northern analysis, which confirmed that expression of mRNA levels increased in the ischemic cortex at 3 and 6 h after MCAo and levels remained elevated for up to 15 days. Immunohistochemical studies to confirm protein expression then localized adrenomedullin to ischemic neuronal processes. In functional studies, synthetic adrenomedullin microinjected into the preconstricted rat pial arteries produced dose-dependent relaxation of the vessels. In addition, intracerebroventricular administration of adrenomedullin, prior to and after MCAo, increased the degree of focal ischemic injury. Other groups have also used this technique in ischemia to identify differentially expressed mRNAs such as a zinc transporter gene [144] and an ADP-ribosylation factor like gene [145]. Other examples include the transcription factor SEF-2 [146], proteosome p112 [146], and ST-38 chemokine [147] following rat MCAo. Differential display is useful but very labor intensive. It is most useful for examining several RNA samples simultaneously and has been used extensively for temporal, doseresponse, and multiple treatment studies. Although differential display is ‘semiquantitative,’ only relatively a small amount of total RNA (approximately 15 ␮g) are required. Some problems include high false positive rates that cannot be confirmed by RT-PCR or Northern blotting. Modifications such as subtracted differential display, which removes unregulated cDNA by mRNA subtraction prior to differential display [148], represent improvements. Confidence in an isolated candidate gene can be improved by using independent follow-up assays of gene expression in parallel.

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Table 2. Summary of differential gene expression changes determined by techniques that measure transcription differences following focal ischemia stroke in the rat* Barone/Read

Gene

350

Stroke pMCAo ⫽ p tMCAo ⫽ t

Early RNA expression ⱕ24 h

Later RNA expression ⱖ24 h

RNA/Protein cross-validation techniques

Protein demonstrated/ verified

Reference(s)

Immediate early genes NGFI-A NGFI-B NGFI-C NGFI-C Nurr-1 erg-2 erg-3 Zif 268, c-fos NF-␬B

p p p p p p p p t

Yes Yes Yes Yes Yes Yes Yes Yes Yes

No No No No No No No No No

No No No No No No No No Yes

107 107 107 3 107 107 107 108 109

NF-␬B

t

Yes (subunit specific)

Yes (subunit specific)

Yes

110

Activating transcription factor c-fos, c-jun, zif 268 ATF-3

t

No

Yes

111

p p, t

Yes (decrease) Yes Yes

No ND

ISH ISH ISH ISH ISH ISH ISH Northern analysis IHC Gel shift analysis Western blotting IHC Gel shift analysis IHC Western analysis Northern analysis RT-PCR, ISH, IHC

No No

108 105

Cytokines IL-1 receptor

p

No

RT-PCR

No

113

IL-1RA

p

Yes

RT-PCR

No

113

Yes (subunit specific) Yes

Gene Expression in Stroke

IL-1RA IL-1B IL-1B IL-1␤ IL-1␤ IL-2 IL6, Zif 268, c-fos CINC/IL-2 IL-10 TNF-␣ TNF-␣ TNF-␣ TNF-␣

p p p p t p p p p p t p p

Yes Yes Yes Yes Yes No change Yes Yes Yes (6 h) Yes Yes Yes Yes

ND Yes No No Yes No change No ND No Yes Yes No Yes

LIF

t

Yes

Yes

LIF SOCS-3 IL-1␤ TNF␣

p p p, t p, t

Yes Yes Yes Yes

Yes Yes No No

Inflammation COX-1 COX-2 COX-2 MCP-1 MCP-1

t t p t p

No change Yes

No change Yes Yes Yes Yes

Yes Yes

RT-PCR Northern analysis ISH RT-PCR Northern analysis RT-PCR Northern analysis ISH RT-PCR ISH, IHC, RT-PCR Northern analysis RT-PCR Northern analysis IHC RT-PCR Western blot IHC RT-PCR RT-PCR RT-PCR, ISH RT-PCR, ISH RT-PCR RT-PCR, IHC RT-PCR ELISA Nothern analysis RT-PCR, IHC

No No No No No No No No No Yes No No Yes

114 115 116 117 118 117 108 119 117 120 108 117 121

Yes

122

No No No No

104 104 105 105

No Yes No Yes Yes

123 123 104 124 125

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Table 2 (continued) Barone/Read 352

Gene

Stroke pMCAo ⫽ p tMCAo ⫽ t

Early RNA expression ⱕ24 h

Later RNA expression ⱖ24 h

RNA/Protein cross-validation techniques

Protein demonstrated/ verified

Reference(s)

MCP-1 MCP-1 iNOS iNOS MCP-3 IP10 CXCR3 Heme oxygenase LPS-binding protein

p p t t p, t p p p, t p

No Yes Yes Yes Yes Yes Yes Yes No

Yes Yes No Yes Yes Yes Yes ND Yes

Northern analysis RT-PCR RT-PCR RT-PCR, IHC Northern analysis Northern analysis Northern analysis RT-PCR, ISH RT-PCR

No No No Yes No No No ND ND

126 104 123 127 128 129 130 105 6

Apoptosis Bax Caspase 1,6,7,8,11 Caspase 2,9 Caspase 3 Caspase-3 Fas, Fas-L TR3-death receptor p75 NGF-R Arc BIS Arc

t p p t p p p p p p, t p

Yes Yes No change No Yes Yes Yes Yes Yes Yes

No ND ND Yes No ND ND ND No ND Yes

ISH RT-PCR RT-PCR ISH RT-PCR RT-PCR RT-PCR RT-PCR ISH RT-PCR, ISH RT-PCR

No No No No No No No No Yes No No

131 132 133 131 133 133 133 133 3 105 104

Growth factors VEGF

p

Yes

No

ISH, IHC

Yes

134

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VEGF

t

Yes

Yes

VEGF receptor VGF BDNF TGF␤-1 TGF-␤1 Narp Cathepsin C Cystatin B Agrin JAK-2 cpg-21 Narp

p p t p p p, t p p p p p p

No Yes Yes No ND Yes No No Yes Yes Yes Yes

Yes Yes No Yes Yes ND Yes Yes No No No No

Other genes Adrenomedullin

p

Yes

Yes

HIF-1 Hsp-27 Hsp-70 Hsp-70

p p p t

No ND ND Yes

Yes Yes Yes No

GADD 45

t

Yes

No

MIP-1␣

p

Yes

Yes

MIP-1␣ MIP-3␣ CRH

p, t p p

Yes

No Yes No

Yes

Northern analysis Western blotting ISH, IHC RT-PCR ISH Northern analysis RT-PCR RT-PCR, ISH RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR Northern analysis IHC ISH, IHC RT-PCR RT-PCR In situ autoradiography In situ autoradiography Northern analysis RT-PCR, IHC In situ RT-PCR ISH

Yes

135

Yes No No No No No No No No No No No

134 104 136 143 104 105 6 6 6 6 6 6

Yes

112

Yes No No No

134 104 104 137

No

137

Yes

125

No No No

138 104 139

Table 2 (continued) Barone/Read 354

Gene

Stroke pMCAo ⫽ p tMCAo ⫽ t

Early RNA expression ⱕ24 h

Later RNA expression ⱖ24 h

RNA/Protein cross-validation techniques

Protein demonstrated/ verified

Reference(s)

NT-3 Trk-B Osteopontin Osteopontin

t t p p

Yes (decrease) Yes Yes No

No No Yes Yes

No No Yes Yes

136 136 140 141

Osteoactivin TIMP-1 TIMP-1

p p p

ND Yes Yes

Yes No Yes

No No No

104 142 142

TIMP-1 CD14 CD44 GADD45␥ Xin Hsp-70 Cyr61 Lox-1 Rad G33A HYCP2 Mim-3 CELF

p p p p p p, t p, t p, t p p p p p

ND ND ND ND

Yes Yes Yes Yes Yes ND ND ND No No No No No

ISH ISH ISH, IHC Northern analysis IHC RT-PCR Northern analysis Subtractive cDNA libraries Southern analysis RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR, ISH RT-PCR, ISH RT-PCR, ISH RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR

No No No No No No No No No No No No No

104 104 104 104 104 105 105 105 6 6 6 6 6

Yes Yes Yes Yes Yes Yes Yes Yes

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Tenascin DAF Cip-26 PHAS-I TBFII Spr Glycerol-3 phosphate dehydrogenase PRG1

p p p p p p p

Yes Yes No No No No No

p

No

Yes Yes

RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR RT-PCR

No No No No No No No

6 6 6 6 6 6 6

Yes

RT-PCR

No

6

No No Yes Yes

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*This table only lists increased message expression that has been validated within or between labs independently. The importance of this validation in addition to the verification of translated protein for candidate genes is emphasized in the text. Permanent MCAo ⫽ pMCAo; transient MCAO ⫽ tMCAo; ND ⫽ not determined; ISH ⫽ in situ hybridization; IHC ⫽ immunohistochemistry.

Subtractive Hybridization Subtractive hybridization compares qualitative differences in gene expression between two experimental paradigms. This is usually achieved by hybridization of biotinylated ‘driver’ cDNAs to the mRNA pool from the target tissue. Duplexes of driver cDNAs and target mRNAs are then removed, resulting in a pool of target mRNAs expressed only by the target tissue [149]. Down-regulated mRNAs are determined by carrying out the reaction in reverse. Modifications to the assay include suppression subtractive hybridization and RDA, where the polymerase chain reaction replaces physical subtraction methods to enrich for differentially expressed transcripts. Such modifications emphasize differential mRNA of both low and high abundance, rather than biasing selection of only highly expressed genes as is the case with the more basic subtractive hybridization methodology. Suppression subtractive hybridization has been used to identify candidate genes with putative roles in experimental cerebral ischemia. For example, the induced expression of a rat homolog to human monocyte chemotactic protein-3 (MCP-3) was identified in the ischemic brain [128]. Independent Northern analysis identified increases in MCP-3 mRNA observed at 12 h postischemia, with 49-fold and 17-fold increases over control in permanent and temporary MCAo, respectively. Significant induction of MCP-3 in the ischemic cortex was sustained up to 5 days after ischemic injury. In other models, subtractive hybridization has been less widely used to identify candidate genes, perhaps due to the technique demands of the subtraction approach, although false positives are less frequent. The subtractive hybridization approach also has been used to successfully identify a novel cDNA clone (pGSH3), expressed only after ischemia in the gerbil cortex [149], which turned out to be a homolog of a 72-kilodalton human heat-shock protein (hsp70) gene. Basal cortical levels were found to be low, but 8 h after a 10-min transient forebrain ischemia, gene expression became prominent in the cerebral cortex. RDA RDA is a relatively novel PCR-coupled, genome subtractive process [150] that until recently [104, 105] had not been used to assay differential expression in models of cerebral ischemia. RDA is conceptually similar to subtractive hybridization, but the unavailability of a commercially produced kit for RDA has meant that it has been less broadly exploited. RDA was originally established to monitor differences in genomic DNA content between individuals, it was later modified to identify differences in gene expression [150]. The robust gene expression changes that characterize the MCAo model are detectable with RDA, as we have recently been able to show [104]. Subtracting ischemic cortex from rats 24 h following permanent MCAo from similarly treated tissue

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from sham-operated animals, we identified candidate ischemia-regulated transcripts. Primary confirmation of the accumulation of these gene products in the ischemic cortex was confirmed using SYBR Green RT-PCR, followed by the more comprehensive time course analysis using TaqMan RT-PCR in selected cases. Several genes identified through this approach had previously been reported to increase following MCAo, such as heat shock proteins (hsp 27 and hsp 70) and others (MCP-1, MIP3␣, COX-2, TGF-␤1, tissue inhibitor of matrix metalloproteinase (TIMP-1) and Arc), but some were newly identified as MCAo-induced genes in this study (LIF, SOCS-3, VGF, CD44, CD14, CD81, osteoactivin, GADD45␥ and Xin) [104]. In another study [105] using this technique, 128 unique gene fragments were isolated and 13 were selected for RT-PCR analysis. Many transcripts were verified to be differentially expressed by RT-PCR, including four genes not previously implicated in stroke: neuronal activity-regulated pentraxin (Narp), cysteine rich protein 61 (cyr61), Bcl2binding protein Bis (Bcl-2-interacting death suppressor), and lectin-like ox-LDL receptor (Lox-1). Microarray Analysis All the above strategies identify relatively small numbers of differentially expressed genes. Large numbers of DNA fragments (110–450 bp) are produced in the process, which need to be confirmed and frequently extended to full lengths to obtain gene identity. Although all of these technologies are useful for isolating candidate genes, they are of limited utility in providing a broad characterization of the expression of large number of genes within a particular model. Array-based technology, on the other hand, allows large-scale and prospective analysis of gene expression as well as time-response profiling and drug treatment analysis (pharmacogenomics). Whether using arrays of oligonucleotides [151, 152] or gene fragments [153], the array technology allows parallel expression monitoring of numerous genes at the same time [154]. The limitations and biases of the technique are obviously in the selection of genes to study on the array. The power and quality of microarrays has continued to improve significantly, and now thousands of genes can be evaluated at a time. The pioneer study [3] using this technique in the context of stroke was applied to studying gene expression in a proximal MCAo electrocautery model [155]. Oligonucleotide probe arrays were employed with 750 predetermined genes optimized for gene expression in rat bone and cartilage. The gene chip (Roche, ROEZ002) was used to monitor gene expression after 3 h of permanent focal ischemia. To determine genes differentially expressed as a consequence of ischemia, the authors took tissue from the ipsilateral frontal and parietal cortices and compared their expression to corresponding regions on the contralateral side. A significant change in transcription was defined as a 2-fold or greater

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increase or decrease in expression compared to the contralateral hemisphere. The authors described a significant up-regulation of 24 genes in the parietal and frontal cortices and striatum, with particularly robust changes in c-fos, NGFI-A, NGFI-B, NGFI-C, Krox20, Nor-1, cyclooxygenase-2, and Arc. This study clearly demonstrated the utility of array technology in the analysis of gene regulation following experimental cerebral ischemia. The use of arrays optimized for bone and cartilage genes unfortunately limited the usefulness to 15% of the total gene representation on the array. Nevertheless, key gene families such as the phosphatases (MKP-1 and MKP-3) and the chemokines (MCP-1 and MIP-1␣) were represented and expression profiles agreed with previous findings [156, 157]. No change in ‘housekeeping’ genes GAPDH and ␤-actin were found (ipsilateral vs. contralateral). Another outstanding study [6] used the rat Affymetrix U34A oligonucleotide array to assess 8740 transcripts in the peri-infarction rat cortex 24 h after thread MCAo, a model representing slow stroke evolution, as in man [98]. Using strict analysis criteria, less than 4% of transcripts were regulated (e.g., 264 were up-regulated and 64 were down-regulated), of which 163 had not been reported to be modified in stroke previously. In terms of functional groups, G-protein-related genes were least variable, while cytokines, chemokines, stress proteins and cell adhesion and immune molecules were most modulated. Quantitative RT-PCR of selected genes identified early up-regulated genes including Narp, Rad, G33A, HYCP2, Pim-3, Cpg21, Jak2, CELF, Tenascin, and DAF. Late up-regulated genes (⬎24 h) included cathepsin C, Cip-26, cystatin B, PHAS-I, TBFII, Spr, PRG1, and LPS-binding protein [6]. Implications of up-regulated glycerol 3-phosphate dehydrogenase, plasticity-related transcripts, gene regulation related to cell survival, death and tissue repair and functional recovery, and biochemical pathways related to gene changes were evaluated [6]. SAGE SAGE yields information about absolute transcript numbers of many, if not all, genes expressed in a given tissue and therefore allows for the identification of differentially expressed genes when applied to tissues in different conditions [158–160]. The technique is based on the reduction of each expressed transcript sequence to short (14–15 bp), yet representative, sequences (tags) at a defined position, which are concatenated into long molecules. Sequencing these molecules reveals the identity of multiple transcripts simultaneously. The number of times a particular tag is detected in a SAGE library, therefore, provides a quantitative and digital measure of gene expression [158–160]. In a recent and an elegant study, differentially expressed genes in mouse brain 14 h after the induction of focal cerebral ischemia were determined using SAGE [160]. From the estimated 30,000 genes of the mouse genome, at least 24,590 genes were detected

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by SAGE. Analysis of ⬎60,000 transcripts revealed 83 up-regulated and 94 down-regulated transcripts, defined as greater than or equal to 8-fold difference from baseline. Up-regulated genes were classified as transport and secretion (4%), ribosomal proteins (4%), DNA/RNA metabolism (4%), cytoskeletal (6%), protein folding and degradation (12%), intermediary metabolism (16%), signal transduction (22%), or unclassified (32%). Metallothionein-II (MT-II) was found to be the most significantly up-regulated transcript in the ischemic hemisphere. MT-I and MT-II both appear to be induced by metals, glucocorticoids, and inflammatory signals in a coordinated manner, yet their function remains unknown. Up-regulation of both MT-I and MT-II was confirmed by Northern blotting. MT-I and MT-II mRNA expression increased immediately after 2 h of transient ischemia, with a maximum after 16 h. Western blotting and immunohistochemistry revealed MT-I/-II up-regulation in the ischemic hemisphere, whereas double-labeling demonstrated colocalization of MT with markers for astrocytes as well as for monocytes/macrophages. The completeness of this study (fig. 1) was demonstrated by the use of MT-I- and MT-II-deficient mice, which developed an approximately 3-fold larger infarcts than wild-type mice and a significantly worse neurological outcome [160].

Assay Variation and Confidence in Identified Gene Expression

In the preceding sections we discussed many of the techniques available for the detection of differential gene expression and some of the ‘within assay’ issues associated with each technique. Next, we will discuss in more detail model-to-model differences, the importance of the poststroke timings of RNA sampling, and different experimental paradigms in stroke that might help us discover genes that have roles in brain protection or tolerance, and studies that can be used to look for genes that might contribute to the recovery/plasticity of the brain postinjury. There are several issues which warrant discussion including the significant variability between techniques, and the identification of false positive and false negative results. Assays of differential expression have an inherent variability dependent on assay methodology, sensitivity, and reaction efficiency. When exploring disease paradigms which are powerful stimulators of gene expression such as cerebral ischemia, the usual tendency is to highlight gene sets or functional groups that are up-regulated and differentially expressed. Given the large numbers of genes identified, it is difficult to confirm all the differentially expressed genes and false positive and false negative differential gene expression becomes an issue. False positives can broadly be defined as genes whose differential expression is not subsequently confirmed by an independent study

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(i.e., RT-PCR, Northern analysis, in situ hybridization, etc.). False negatives, in contrast, are genes that are, in fact, differentially expressed, but not detected as such by the complex assay employed (e.g., subtractive hybridization, RDA, DNA microarray). To manage these issues, we have employed the strategy of using multiple assays of differential expression on the same RNA pool and then cross-validating all of the numerous differentially expressed products between assays. Identifying commonalities in expression across assays increases confidence in particular results, and genes identified across two or more assays receive a higher priority for confirmatory studies. A table of descending confidence in ‘hits’ can then be constructed. This technique for handling large numbers of ‘hits’ avoids issues of biasing the identification of differential expression to a single assay and also interassay variability. Subsequent analysis by Taqman RT-PCR has confirmed that robust differential gene expression identified across all assays had particularly high-fold increases in differential expression. This strategy is also useful for identifying false negatives (i.e., products differentially expressed but not detected as such in assays) [4]. The ‘complimentary-techniques’ approach at target validation maximizes the coverage of differential gene expression by minimizing the losses due to the technical vagaries of any single technique.

Confirmation: An Integral Part of Differential Gene Expression

The techniques cited above for the identification of differentially expressed mRNAs represent starting points for the study of gene expression following stroke. All data derived by these methods require confirmation from an independent study to remove false positives, and this usually forms part of a broader analysis of expression of the gene that has been identified [3–6, 104–106, 160]. Traditional methods for analyzing gene expression include techniques such as Northern blotting, RNAse protection, in situ hybridization, and semi-quantitative RT-PCR. All of these methodologies have been used to study the expression of individual genes or small groups of genes in stroke models. Differential screening methodologies ideally generate large numbers of ‘hits’ which require rapid confirmation in a high throughput system. Recently, real-time quantitative RT-PCR techniques, such as ‘Taqman’ probes or SYBR green [161] to monitor an accumulating PCR product in real time, allow an accurate comparison of initial PCR template numbers. These assays can be carried out in 96 or 384 well formats and can utilize robots, reducing operator time and error. With these techniques, it is possible to carry out rapid confirmation of many differentially expressed genes simultaneously

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or to undertake a more detailed expression analysis of a single ‘hit’ [142]. For example, Taqman RT-PCR has been extensively applied to temporal profiling of caspase expression following MCAo in rats [132, 133] and SYBR green has been used to confirm differentially expressed genes identified by RDA [104]. Taqman is a PCR-based technique that is more sensitive than other confirmatory technologies such as Northern blotting. Typically, Taqman RT-PCR uses approximately 50 ng of total RNA per gene, while Northern blotting uses 10–20 ␮g. Additionally, Taqman RT-PCR is as sensitive as in situ hybridization, with the added advantage of higher throughput. Perhaps most importantly for paradigms such as MCAo, where gene expression can exceed 600-fold over that observed in naïve animals, Taqman PCR can quantitate gene expression over five to six orders of magnitude without multiple dilution series, as necessitated by other assays [115]. Clearly, PCR-based technologies such as Taqman RT-PCR and SYBR Green RT-PCR, whilst in their infancy in application to the study of cerebral ischemia [104, 132, 133], offer advantages for confirmation and expansion of data on differential gene expression. These techniques are also of value in testing hypotheses about genes already known to be regulated in stroke models, where differential expression is suggested by other biological evidence/data. The sensitivity of PCR-based methodologies suggests that sufficient RNA can be isolated from a single animal to allow the simultaneous assessment of several hundred genes. A large body of data can be amassed and the expression of many different genes compared in a single study, without drawbacks such as variation between studies, operators and cohorts of animals. The major drawback of high-throughput quantitative RT-PCR is that while it allows for the rapid assessment of changes in gene expression at the level of mRNA, it is not able to provide information on the precise cellular localization of such changes. A detailed understanding of stroke models utilized for differential gene expression is essential (i.e., the models have to be adequately characterized over time for cellular changes). The cell-type and intracellular locations of changes in gene expression are important. For example, neurons and oligodendrocytes die within the ischemic infarct [162, 163] particularly after 12 h of ischemia. Astrocytes and microglia are decreased in number in the core region of the lesion and proliferation of both of these cell types occurs in the marginal areas [164]. Polymorphonuclear leukocytes, and later macrophages, invade the lesion after around 12 h and for days after [163–165]. Changes in gene expression have to be understood in the context of these evolving cell types present at any given time after stroke. Ultimately, expression profiling must involve techniques such as in situ hybridization and immunohistochemistry, which allow the localization of expression to be viewed in relation to the structure of the evolving lesion, and the identification of the types of cells in which expression is occurring.

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Confirmation of expression and time course of protein translation is essential for verification that up-regulated gene transcription has proceeded to the formation of protein. This point is especially pertinent given the severe energy perturbations in the ischemic brain. During this state, transcription and translation can become uncoupled due to energetic demands of assembling protein. This effect is temporally and spatially dependent and has been extensively reviewed [5, 81], including the uncoupling of mRNA transcription and protein translation following MCAo [4]. Issues associated with the sensitivity of protein-detection assays must be considered for some proteins. Ultimately, it is proteins which are pivotal in cellular function, and thus proteomic analysis in addition to mRNA analysis will point the way ahead.

Functional Studies,Transgenic Studies, and in vivo Pharmacology

There are already many examples from the literature where transgenic animal studies and/or pharmacological studies have coincided with gene expression studies to demonstrate the involvement of specific gene expression in focal stroke injury or protection. For example, cyclooxygenase-2-deficient mice are known to exhibit reduced susceptibility to brain injury [166]. IL-1ra was shown to be neuroprotective in brain injury [167] well before the altered expression of the IL-1 system in stroke was demonstrated [114]. IL-6 also has been shown to be neuroprotective in stroke [168, 169]. In addition, it has been shown that blocking thyrotropin-releasing hormone provides a significant protection against ischemic brain damage and associated neurological deficits [170, 171]. Following stroke, treatment with BDNF reduces brain injury in the MCAo model [172]. Genes for all these proteins have been shown to be up-regulated in stroke models. One recent example is Metallothionene-II as a major neuroprotective gene in mouse focal cerebral ischemia [160]. In this study, changes in the metallothionene-II gene were verified using multiple methods including immunohistochemistry, in situ hybridization and Western blot. In addition, stroke in the metallothionene knockout mouse resulted in stroke three times larger than in wild-type mice [160].

Additional Models for Discovering Gene Targets in Brain Injury

Neuroprotective or Neurodestructive Gene Expression As pointed out previously [2], focal ischemia stimulates multiple gene expression changes. Focal ischemia is a very powerful reformatting and reprogramming stimulus for the brain. There are broad and robust gene expression

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responses that occur following the focal stroke that are exhibited as temporal episodes or ‘waves’ of expression of different groups of genes [2]. These waves are largely comprised of increased expression of inflammatory cytokines including IL-6 and IL-1ra. In addition, growth factors (e.g., BDNF) that might be expected to play a neuroprotective role following stroke also increase in this time frame. The increased cytokine gene expression appears to drive leukocyte infiltration, a poststroke brain response to injury, and is associated with secondary brain injury and repair processes following stroke. Later waves of new gene expression include mediators, which appears to be important in tissue remodeling (i.e., resolution of ischemic tissue injury) and perhaps recovery of function. These issues are important in relation to the models suggested below that may provide new directions in future differential gene expression analysis. Preconditioning Stress in Brain Tolerance Strategies Certain stimuli that can cause injury will protect the brain against subsequent, severely injuring stimuli if applied at a low intensity (i.e., subthreshold for injury) prior to that severe injury. This phenomenon involves complex processes involved in endogenous organ protection. For ischemic stimuli, this phenomenon has been termed ischemic preconditioning (PC) or ischemic tolerance (IT). PC is a reaction to a potentially noxious stimulus such as hypoxia, ischemia, or inflammation. A short ischemic preconditioning event can result in a resistance to severe ischemic tissue injury. This phenomenon has been described in brain and heart, and may represent a fundamental cell response to certain types or levels of injury [173–176]. PC or IT in the brain is associated with a protected state that develops over hours, persists for days or longer and involves de novo protein synthesis [177, 178]. MRI can demonstrate the evolution of infarction and its reduction by tolerance induction [179–181]. Functional or motor effects are protected by PC [177]. It is interesting that the stroke-prone rat (discussed earlier in the chapter as a model of spontaneous stroke and used as an experimental model for gene associations) are significantly more sensitive to cerebral ischemia [92] and exhibit greater brain injury to cerebral ischemia and also exhibit a significantly reduced degree of IT to PC [179–181]. The brain changes associated with brain ischemia involve a progression of both injurious and protective processes as brain injury evolves and is then repaired following an insult such as a stroke. Focal cerebral ischemia induces a complex series of mechanisms [2, 173, 176] that result in infarcted tissue, a situation in which neurodestruction has overwhelmed neuroprotection. The major pathophysiological mechanisms of tissue destruction in stroke involve acute mechanisms of excitotoxicity and delayed mechanisms of inflammation and

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apoptosis. The corresponding protective tissue responses include up-regulation of neurotrophic factors and protective or ‘stress’ genes/proteins. Later regenerative or restorative processes may allow recovery of some brain function poststroke [2], but generally are not sufficient to correct the initial damage. Because IT paradigms also provide insight into the mechanisms of endogenous brain neuroprotection in the absence of any destructive processes, it is believed that understanding the signaling and mechanisms involved in precondition-induced IT can discover new targets and approaches to protect the brain and other end organs from the injury/disease. IT has been demonstrated in the human brain [182, 183], and so differential gene expression strategies in IT experimental models have physiological relevance for studying apoptosis and other poststroke complications [184]. This endogenous brain protection phenomenon appears to represent a fundamental protective response to injury following prior stress [173, 176, 185, 186]. Models of brain protection provide an opportunity to identify novel protective gene expression associated with the development of brain tolerance. Data from differential gene expression suggests that neurotrophic factors, stress proteins, and cytokines contribute to the tolerance response to ischemia and other forms of stress in the brain [173, 174, 176–178, 187–189]. For example, IT is associated with an increased expression of the neuroprotective protein IL-1ra [177] and heat shock proteins [178] and a reduced postischemic expression of the early response genes, c-fos and zif268 [177]. A number of techniques including suppressive subtractive hybridization methodology have been applied to discover genes responsible for IT following PC [190]. With suppressive subtractive hybridization, tissue inhibitor of matrix metalloproteinase (TIMP-1) was identified as one candidate molecule in the stroke response and IT. Northern analysis confirmed that TIMP-1 mRNA was significantly elevated at 24 h and 2 days after PC, which corresponded well to the onset of IT [190]. Strategies in Brain Recovery, Plasticity and Recovery of Function Gene changes occurring one or more days after stroke might provide insight into reparative and recovery processes. While neurological functional deficits occur following stroke, there may be a recovery of brain function that occurs spontaneously or improves with training following stroke [2, 191–193]. Sampling tissue during functional brain recovery in animal models (i.e., at later time periods poststroke) might be expected to provide an opportunity to identify novel genes important for long-term brain regeneration or plasticity. These studies would be amenable to differential gene expression analyses, but would be profiled at later poststroke timepoints or under treatment conditions shown to facilitate such brain regeneration/recovery, for example after the introduction

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of putative neuroprotective or neuroregenerative agents. It is again interesting that stroke-prone rats that are more sensitive to and exhibit greater brain injury to cerebral ischemia also exhibit a greater degree of neurological deficits with significantly less spontaneous recovery of neurological deficits poststroke [92, 180, 181]. Recent data indicating complete recovery of neurological function in the SHR, and by comparison the lack of improvement/recovery of neurological function in the stroke-prone rat (i.e., with similar degrees of absolute brain injury) suggests that the stroke-prone rat might be a valuable model for the evaluation of neurodegenerative drugs poststroke [181]. From the same point of view, differential gene expression studies could be used in the future to compare SHR-SP and SHR in order to elucidate mechanisms and to discover new targets that facilitate neurobehavioral recovery of the injured brain. Gene Therapy and Gene Target Validation In recent years, clinical progress in gene therapy has proceeded in parallel with in vivo gene transfer for physiological studies and gene validation. Therapeutic neovascularization for ischemic diseases has been one particularly encouraging area of study. For example, animal models involving intramuscular injection of naked plasmid or adenoviral carried DNA encoding vascular endothelial growth factor (VEGF) have shown promotion of angiogenesis in ischemic limbs [194, 195]. Similarly, in clinical trials, VEGF gene transfer augments the population of circulating endothelial progenitor cells and transiently increases plasma levels of VEGF [196]. Furthermore, myocardial transfer of naked plasmid DNA phVEGF(165) has been found to augment perfusion of ischemic myocardium and reduces the size of defects documented at rest by single-photon emission CT-imaging [197]. Ex vivo gene transfer, employing the modification of cultured cells and subsequent implantation into a host organism, is a proven strategy for recovery from CNS injury and could incorporate cells expressing genes that confer protection from stroke. An analogous area of research has been in recovery from long-term rodent hemiparkinsonism by implantation of cells following 6-OHDA lesioning, which has been found to improve behavioral deficit for up to 13 months [198]. In vivo gene transfer, the delivery of a gene directly to recipient somatic cells, has also been explored for neuroprotection and recovery from CNS injury. The delivery of the proto-oncogene bcl-2 has been examined in gerbil models using adeno-associated virus vectors. Transduction of both pre- and postforebrain ischemia was found to prevent DNA fragmentation in hippocampal CA1 neurons, commonly associated with cell death induced by ischemia [199]. Adenoviral transfection of the endogenous cytokine antagonist IL-1ra also has demonstrated neuroprotection in transient focal cerebral ischemia and

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reperfusion models in the mouse [200, 201]. In addition, the neuroprotective potential of heat shock protein-70 on brain injury, including that produced by brain ischemia, has been tested with viral vectors. Use of transgenic animals and gene transfer technology to overexpress heat shock protein-70 clearly protects the brain [202, 203] in the context of stroke.

The Elusiveness of Novel Gene Targets and the Future

Initially, the movement to discover differential gene expression in brain destruction or protection was driven by the hope of discovering novel genes that would provide pathways to therapeutics. The complexity of understanding and applying resources to gene fragments in the hopes of ultimately reaching this ‘therapeutic nirvana’ has not yet occurred. We have identified several genes that have been differentially expressed in ischemic or tolerant brain tissue, and which appear to be important, but there still exists a therapeutic void. In pharmaceutical development, the odds of developing a successful drug are generally better for the pursuit of known genes as therapeutic targets, although the new science of pharmacogenetics as a lead optimization tool may change this manner of operation. Many other factors can make investing resources into work on unknown genes costly, risky, and difficult to pursue. Some of these include the absence of any understanding of an identifiable function for the unknown protein, and if it is in fact associated with a novel gene, and lack of any concrete, supporting information that the novel gene/protein has any involvement in the pathophysiology of stroke rather than being an epiphenomenon. In spite of all this, it is clear that the therapeutic targets of the future exist in the complex patterns of gene expression underlying stroke. As such, unknown or novel genes represent a potential source of collaboration between academic and industrial laboratories. Potential novel gene products could be evaluated for tissue distribution, function and relevance in tissue injury and protection, in a collaborative and productive setting. Clearly, the methodology is available to identify differential gene expression in stroke in addition to other conditions of brain disease. However, the methodology needs to be developed with the caveats discussed above. If one operates as we have suggested, using cross-validating technologies in animal models and human tissue to validate ‘hits,’ there is potential for many significant opportunities for the biological discovery, not only related to stroke, but in many other conditions such as end organ failure in various cardiovascular diseases, in areas such as oncology, and perhaps extending further to other very complex problems such as substance abuse, tolerance, addiction and drug dependency.

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Acknowledgements The authors would like to thank their collaborators in this research, especially Dr. Andy Parsons, Dr. David Harrison, Dr. Karen Philopia, Dr. Karen Kabnick, Dr. Shawn O’Bien, Dr. Steve Clark, Dr. Mary Brawner, Dr. Giora Feuerstein, Dr. Xinkang Wang, Dr. Ray White, Dr. Stewart Bates, Dr. Jeff Legos and Dr. Israel Gloger. It has been a pleasure working with all of them in this exciting area.

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187 Chen J, Simon RP: Ischemic tolerance in the brain. Neurology 1997;48:306–311. 188 Wang XK, Li X, Currie RW, Willette RN, Barone FC, Feuerstein GZ: Application of real-time polymerase chain reaction to quantitate induced expression of interleukin-1beta mRNA in ischemic brain tolerance. J Neurosci Res 2000;59:238–246. 189 Wang XK, Li X, Erhardt JA, Barone FC, Feuerstein GZ: Detection of tumor necrosis factor-alpha mRNA induction in ischemic brain tolerance by means of real-time polymerase chain reaction. J Cereb Blood Flow Metab 2000;20:15–20. 190 Wang XK, Yaish-Ohad S, Li X, Barone FC, Feuerstein GZ: Use of suppression subtractive hybridization strategy for discovery of increased tissue inhibitor of matrix metalloproteinase-1 gene expression in brain ischemic tolerance. J Cereb Blood Flow Metab 1998;18:1173–1177. 191 Rossini PM, Pauri F, Cicinelli P, Pasqualetti P, Traversa R, Tecchio F: Neuromagnetic recordings and magnetic brain stimulation in the evaluation of sensorimotor hand area interhemispheric differences: Normative, experimental and patients’ data. Electroencephal Clin Neurophysiol 1999; 50(suppl):210–220. 192 Dobkin BH: Functional rewiring of brain and spinal cord after injury: The three Rs of neural repair and neurological rehabilitation. Curr Opin Neurol 2000;13:655–659. 193 Hunter AJ, Hatcher J, Virley D, Nelson P, Irving E, Hadingham SJ, Parsons AA: Functional assessments in mice and rats after focal stroke. Neuropharmacology 2000;39:806–816. 194 Lee LY, Patel SR, Hackett NR, Mack CA, Polce DR, El-Sawy T, Hachamovitch R, Zanzonico P, Sanborn TA, Parikh M, Isom OW, Crystal RG, Rosengart TK: Focal angiogen therapy using intramyocardial delivery of an adenovirus vector coding for vascular endothelial growth factor 121. Ann Thorac Surg 2000;69:14–24. 195 Gowdak LH, Poliakova L, Wang X, Kovesdi I, Fishbein KW, Zacheo A, Palumbo R, Straino S, Emanueli C, Marrocco-Trischitta M, Lakatta EG, Anversa P, Spencer RG, Talan M, Capogrossi MC: Adenovirus-mediated VEGF(121) gene transfer stimulates angiogenesis in normoperfused skeletal muscle and preserves tissue perfusion after induction of ischemia. Circulation 2000;102:565–571. 196 Kalka C, Masuda H, Takahashi T, Gordon R, Tepper O, Gravereaux E, Pieczek A, Iwaguro H, Hayashi SI, Isner JM, Asahara T: Vascular endothelial growth factor(165) gene transfer augments circulating endothelial progenitor cells in human subjects. Circ Res 2000;86:1198–1202. 197 Vale PR, Losordo DW, Milliken CE, Maysky M, Esakof DD, Symes JF, Isner JM: Left ventricular electromechanical mapping to assess efficacy of phVEGF(165) gene transfer for therapeutic angiogenesis in chronic myocardial ischemia. Circulation 2000;102:965–974. 198 Cao L, Zhao YC, Jiang ZH, Xu DH, Liu ZG, Chen SD, Liu XY, Zheng ZC: Long-term phenotypic correction of rodent hemiparkinsonism by gene therapy using genetically modified myoblasts. Gene Ther 2000;7:445–449. 199 Shimazaki K, Urabe M, Monahan J, Ozawa K, Kawai N: Adeno-associated virus vector-mediated bcl-2 gene transfer into post-ischemic gerbil brain in vivo: Prospects for gene therapy of ischemiainduced neuronal death. Gene Ther 2000;7:1244–1249. 200 Yang GY, Zhao YJ, Davidson BL, Betz AL: Overexpression of interleukin-1 receptor antagonist in the mouse brain reduces ischemic brain injury. Brain Res 1997;751:181–188. 201 Yang GY, Mao Y, Zhou LF, Ye W, Liu XH, Gong C, Lorris BA: Attenuation of temporary focal cerebral ischemic injury in the mouse following transfection with interleukin-1 receptor antagonist. Brain Res Mol Brain Res 1999;72:129–137. 202 Yenari MA, Fink SL, Sun GH, Chang LK, Patel MK, Kunis DM, Onley D, Ho DY, Sapolsky RM, Steinberg GK: Gene therapy with HSP 72 is neuroprotective in rat models of stroke and epilepsy. Ann Neurol 1998;44:584–591. 203 Yenari MA, Giffard RG, Sapolsky RM, Steinberg GK: The neuroprotective potential of heat shock protein 70 (HSP 70). Mol Med Today 1999;5:525–531.

Frank C. Barone, PhD High Throughput Biology, UW2108, GlaxoSmithKline 709 Swedeland Road, King of Prussia, PA 19406 (USA) Tel. ⫹1 610 270 6824, Fax ⫹1 610 270 6505, E-Mail [email protected]

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Molecular Mediators of Hemorrhagic Stroke R. Loch Macdonald Department of Neurosurgery, University of Chicago Medical Center, Chicago, Ill., USA

Introduction

This chapter will discuss events mediating hemorrhagic stroke at a molecular level and underlying diseases that increase the risk of developing such stroke. Hemorrhagic stroke encompasses intracerebral hemorrhage (ICH) that may be secondary to hypertension; congenital and acquired disorders of blood coagulation; intracranial vascular diseases such as vascular malformations, aneurysms, and amyloid angiopathies; and subarachnoid hemorrhage (SAH). Information on the etiological and pathophysiological mechanisms involved in underlying causes of ICH or SAH (e.g., hypertension) will be presented. The treatment of spontaneous ICH is currently limited to surgical evacuation of the hematoma, ventricular drainage, medical measures to reduce intracranial pressure, and supportive care; biologically based therapies that have been tested in experimental settings will be discussed. Etiology of ICH

Hypertension Most large series have found that the most common disease associated with ICH is hypertension [163]. Additional risk factors for ICH include age, race, and severe hypocholesterolemia. Some cases of ICH may be related to various congenital coagulopathies, including more specifically for the brain mutations in the genes for the A subunit of factor 13, which is involved in crosslinking fibrin [18]. The mechanism of hemorrhage is unclear, but studies demonstrate an association between ICH associated with hypertension (most

commonly in the putamen, thalamus, pons, and deep cerebellar nuclei) and lipohyalinosis of small penetrating arteries and arterioles. A population-based study [209] of 188 cases of ICH in the Cincinnati area found that 121 (64%) of hemorrhages were nonlobar and that 54% of the nonlobar hemorrhages were attributable to hypertension. Hypertension causes numerous detrimental effects on the circulation including decreasing endothelium-dependent relaxations via effects on the endothelium and accelerating atherosclerosis. Charcot-Bouchard aneurysms [21] of the penetrating arterioles (i.e., chronically dilated arterioles secondary to hypertension) were traditionally thought to be the source of hypertensive ICH. This assumption has been questioned by autopsy studies, which found these aneurysms difficult to identify [19]. Alternative explanations are that hypertension causes lipohyalinosis of penetrating arterioles, which damages the arteriolar wall leading to dissection and hemorrhage. It is also possible that lipohyalinosis occludes arterioles and there is hemorrhage into the area of brain infarcted by the occlusion or from the occluded arteriole due to an increased upstream pressure. The molecular basis of hypertension is complex. Adoption and twin studies suggest that more than half the variability in blood pressure between individuals in Western populations is genetically determined [195]. Blood pressure is not determined by a single gene, and animal models suggest the mode of inheritance is governed by at least 6–10 genes. In addition, genes that encode proteins in the second messenger pathways for the primary regulatory proteins may be involved, enhancing the complexity of the task involved in identifying the genes. Hypertension is a heterogeneous condition in which underlying genotypes may give rise to different diseases associated with hypertension. Polygenic inheritance, variable penetrance, gene-environment interactions, genetic and pathological heterogeneity, and late age of onset make the molecular genetics of diseases like hypertension and stroke difficult to study, especially using traditional linkage-based genetic analysis [195]. Candidate genes involved in the renin-angiotensin-aldosterone system and in the regulation of intracellular ion homeostasis have been most extensively investigated, but many other genes are likely to be involved, including those regulating vascular tone and cardiovascular function. There have been reports of associations between angiotensin-converting enzyme alleles and hypertension [221] and the sodium-potassium ATPase and hypertension [184]. Amyloid Angiopathies Amyloid angiopathy (i.e., cerebrovascular amyloidosis) is an important cause of lobar ICH [202]. In amyloid angiopathy there is deposition of eosinophilic material in the walls of the cerebral and cerebellar arteries, including leptomeningeal arteries, arterioles, venules and capillaries. These patients

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develop lobar ICH late in life, usually after the age of 70. There may be multiple hemorrhages over time and rarely simultaneous hemorrhages in multiple areas of the brain. The coexistence of brain amyloid deposition that may be associated with Alzheimer’s disease (AD) has been noted pathologically and correlates with the clinical evidence of dementia in approximately 30% of patients with amyloid-related ICH [202]. The exact relationship between sporadic amyloid angiopathy and AD pathogenesis is unclear. Amyloid angiopathy can occur in AD along with amyloid plaques, neurofibrillary tangles, and granulovacuolar degeneration, and is found in 75–100% of AD cases, whereas amyloid angiopathy can occur in the absence of dementia and the other neuropathological changes of AD. This pattern suggests the underlying genetic and etiological variability in the disease. Individuals with the apolipoprotein-E4 allele have been found to be at increased risk of developing late-onset AD [27]. This observation led to interest in whether patients with amyloid angiopathyrelated ICH were more likely to have this allele. Woo et al. [209] reported that there is an association between the ␧2 allele (apoE2) or ␧4 allele (apoE4) and ICH from amyloid angiopathy, consistent with other findings [46, 126]. Polymorphisms in the endoglin gene may also be associated with spontaneous ICH [4]. Endoglin is a membrane glycoprotein primarily associated with human vascular endothelium; a component of the transforming growth factor-␤ (TGF-␤) receptor complex, it binds TGF-␤1 with high affinity and mutations are associated with hereditary hemorrhagic telangiectasia (HHT1). A number of inherited forms of amyloid angiopathy have been described. Dutch families living in a small area of the Netherlands were described with hereditary cerebral hemorrhage with amyloidosis, known as ‘Dutch-type’ amyloidosis [13]. These patients develop ICH due to amyloid angiopathy of meningeal and cortical penetrating arteries and arterioles. Some patients develop dementia that is thought to be secondary to multiple ICH; typical AD does not develop and the neuropathological characteristics of the changes in the brain appear different (e.g., neurofibrillary tangles are absent). The inheritance of ‘Dutch-type’ cerebral hemorrhage with amyloidosis is autosomal dominant and is due to a mutation of the amyloid precursor protein. Hereditary cerebral hemorrhage with amyloidosis of the Icelandic type is an autosomal dominant disorder causing ICH [140]. This amyloidosis is due to a mutation in the gene encoding the cysteine protease inhibitor cystatin C (gamma-trace) found on chromosome 20. The mutation causes the protein to form amyloid deposits in cerebral vessels. This cause of ICH is quite rare; screening of patients with sporadic ICH for mutations in specific exons of cystatin C and the amyloid precursor protein did not reveal mutations [45]. Gerstmann-Sträussler-Scheinker disease is a spongiform encephalopathy that results from a mutation in the prion protein. Brain deposition of amyloid

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occurs in this disease as well as other spongiform encephalopathies, although ICH is not an usual feature. A prion protein cerebral amyloid angiopathy, however, has been described in which a mutation in the prion protein is associated with its deposition as amyloid in the brain and cerebral blood vessels [44]. Again, ICH is not common in this disease. However, patients with oculoleptomeningeal amyloidosis can present with ICH [165]. This condition is associated with amyloid deposited in the cerebral vasculature that is composed of transthyretin or prealbumin. Additional mutations in transthryetin leading to amyloid angiopathy are described in type 1 familial amyloidotic polyneuropathy [2]. Understanding why some forms of amyloid angiopathy such as ‘Dutchtype’ amyloidosis lead to ICH, whereas others in which another protein is deposited do not cause ICH will be important in determining the pathogenesis of amyloid-related ICH. Arteriovenous Malformations and Other Vascular Malformations Intracranial vascular malformations are classified as arteriovenous (AVM), cavernous, venous, capillary, or mixed. The first three types are the most important clinically. Most AVMs are sporadic and there are no underlying genetic factors predisposing to their occurrence. They may be associated with the Osler-Weber-Rendu and Wyburn-Mason syndromes. Familial occurrence of AVMs is documented, but these cases make up less than 1% of sporadic single AVMs [35, 217]. Some of these patients have multiple AVMs and inheritance may be related to Osler-Weber-Rendu syndrome. Roger Wyburn-Mason [210] described the association of retinal and midbrain AVMs and occasionally facial nevi in a 1943 monograph. The defining features of this disorder and its genetic basis are still uncertain. Additional rare familial disorders with cerebrovascular manifestations include multiple systemic hemangiomatosis which is characterized by cavernous malformations of the skin, viscera, central nervous system and peripheral nerves [35]; blue rubber bleb nevus syndrome which consists of bluish cavernous malformations of the skin and various cerebrovascular malformations; cutaneomeningospinal angiomatosis (Cobb’s disease or dermatospinal angioma) which is characterized by skin port-wine stain associated with spinal AVM; and hereditary neurocutaneous angiomatosis, which is characterized by multiple skin cavernous malformations or AVMs along with brain AVMs and venous anomalies. Osler-Weber-Rendu syndrome (hereditary hemorrhagic telangiectasia) is inherited in an autosomal dominant fashion and is characterized by capillary telangiectases of the skin, mucosa, and viscera [50]. Penetrance is high but the phenotype is variable. Several mutations have been identified. HHT1 is defined by a mutation in the gene for endoglin on chromosome 9q, a transforming growth factor-␤ binding protein on endothelial cells. A second mutation,

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termed HHT2, was identified on chromosome 12q and encodes activin-1 like receptor, which is also a cell-surface receptor for the TGF-␤ family of growth factors. The phenotype varies, but there may be multiple systemic telangiectasias as well as AVMs in the brain. These patients also tend to have pulmonary AVMs. The incidence ranges from one in 2351 among the French population of Ain to one in 40,000 in England. The most common clinical manifestation is epistaxis. Cavernous malformations are known to have a genetic basis. Kufs [91] may have been the first to suggest that cavernous malformations might be inherited. A large Mexican-American family was reported in 1982, in whom a pattern of autosomal dominant inheritance with variable penetrance seemed present [60]. Rigamonti et al. [120, 166] described 24 patients with cavernous malformations, of which 13 were members of 6 unrelated Mexican-American families. The familial cavernous malformations differed from sporadic cases in that 75% of familial lesions were multiple, whereas only 10–25% of sporadic ones were. They estimated that 30–50% of cavernous malformations were familial although more conservative estimates are as low as 6% [70]. The location of genes for familial cavernous malformations were mapped to chromosomes 7q (CCM1) [31, 48, 104], 7p (CCM2) and 3q (CCM3) [29]. One of the genes identified as mutated in CCM1 in French and MexicanAmerican families is KRIT1 [94, 169]. The function of this protein and how mutations in it lead to the formation of cavernous malformations are unknown. Denier et al. [30] noted that KRIT1 messenger ribonucleic acid (mRNA) was expressed ubiquitously during mouse development from E7.5 to E9.5, after which expression became restricted to neurons and epithelia. Vascular expression was restricted to large embryonic blood vessels. KRIT1 protein colocalized with microtubules in endothelial cells and has been reported to interact with Krev1 and integrin cytoplasmic domain-associated protein-1 [49]. Based on these findings, it was hypothesized that KRIT1 is involved in the determination of endothelial cell shape and function and disruption of KRIT1 leads to abnormal endothelial tube formation and cavernous malformations. Developmental venous anomalies were previously called venous malformations, but now are believed to be congenital variations in venous drainage. Vikkula et al. [201] described a mutation in the receptor tyrosine kinase TIE2 gene on chromosome 9p21 in two families with multiple venous malformations. This mutation was inherited in an autosomal dominant manner. The mutation results in an increased activity of TIE2, an endothelial cell-specific receptor tyrosine kinase expressed in developing endothelial cells and important in blood vessel development. The venous malformations in these families were in the skin and mucosa but not the brain.

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Intracranial Aneurysms The common saccular intracranial aneurysm is an acquired lesion that usually occurs at an arterial bifurcation and is secondary to hemodynamic stressmediated degeneration of the internal elastic lamina. Factors predisposing to the development and the rupture of intracranial aneurysms include age, gender, cigarette smoking, hypertension and familial predisposition. There is some geographical variation in the incidence of SAH with higher rates reported in Japan and Finland [6, 73, 206]. The risk of SAH in African-Americans in the Greater Cincinnati area was twice that of Caucasians [14]. Such geographical and racial differences could reflect genetically based population differences that, if identified, could lead to insight into the etiology and pathogenesis of intracranial aneurysms. On the other hand, they could result from variations in the age distribution, availability of medical care and prevalence of smoking, alcohol intake, atherosclerosis and hypertension in the populations studied. Of course, some of these risk factors themselves may have underlying genetic bases. Some intracranial aneurysms are ‘familial’ in that they appear to be inherited by an autosomal dominant or multifactorial mechanism. It is difficult to exclude the presence of shared environmental factors in these cases. Studies of identical twins reared apart have been used to examine the genetic basis of diseases and stroke [5], but such studies have not been conducted for intracranial aneurysms. Familial aneurysms are usually defined by aneurysms occurring in two or more first- to third-degree relatives. First-degree relatives of patients with SAH had a 3-fold elevated risk of developing SAH compared to the general population of Denmark [41]. In a series of 485 patients with SAH, 16 of 237 (7%) of respondents to a questionnaire reported having blood relatives with the same condition [133]. The genetic basis for familial aneurysms is unknown at this time. Several investigators have searched for genes associated with sporadic intracranial aneurysms. Alleles of the ␣1 antitrypsin gene that are associated with reduced enzymatic activity were found to be more common in patients with aneurysms than controls in one study [175]. Further work is needed to confirm this relationship. A study in a Japanese population identified a genetic locus associated with intracranial aneurysms on chromosome 7q11 [142], and later refined that locus to the gene for collagen type I ␣-2 (COL1A2) at 7q22.1. Experimental studies of intracranial aneurysms in animals have focused on induced hypertension, inhibition of collagen cross-linking with ␤-aminopropionitrile, a toxin which causes lathryism, and carotid artery occlusion to induce flow changes in the cerebral circulation. To examine the molecular basis for aneurysm formation, Peters et al. [152] used global gene expression analysis (SAGE-Lite) to examine gene expression in an aneurysm from a 3-year-old girl. There was significant overexpression of genes encoding extracellular

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matrix components including various collagen isoforms and elastin, and genes involved in extracellular matrix turnover (TIMP-3, OSF-2), cell adhesion and antiadhesion (SPARC, hevin), cytokinesis (PNUTL2), and cell migration (tetraspanin-5). Their interpretation from this single gene expression analysis was that the aneurysm was characterized by typical wound healing and tissue remodeling responses, but the underlying basis for its formation could not be established. Inducible nitric oxide synthase (iNOS) has been detected at the orifices of human and rat aneurysms by immunohistochemical staining, whereas it is normally absent from cerebral arteries [39]. Aminoguanidine, an inhibitor of NOS, decreased both early aneurysmal changes and the incidence of aneurysms in rats. The defibrinogenic agent, batroxobin, which may decrease shear stress by reducing blood viscosity, prevented the induction of iNOS as well as early aneurysmal changes in their model. These data suggest that NO formed by iNOS may contribute to the formation of cerebral aneurysms. Other diseases that may be associated with intracranial aneurysms include fibromuscular dysplasia, coarctation of the aorta, autosomal dominant polycystic kidney disease (PKD), and Ehlers-Danlos (ED) syndrome [174]. There are other genetic syndromes in the literature in which aneurysms have occurred but the relationship in these cases is more speculative. The most common disease in which cerebral aneurysms occur may be fibromuscular dysplasia or FMD [117]. FMD was reported in the renal arteries of 1.1% of 819 autopsies [61]. In a review of five series of patients undergoing angiography (approximately 22,000 patients), FMD was noted in about 0.5% of cases (0.25–0.61%). About 85% of FMD cases are in women. Mettinger et al. [117] has reported that 21% of 284 patients with FMD had intracranial aneurysms. Hypertension from renal artery involvement was not necessarily present. The incidence of FMD in patients presenting with intracranial aneurysms and SAH is uncertain. Clinical presentation may be with cerebral infarction, intracranial aneurysm, or as an incidental finding. Pathologically, FMD is characterized by the intimal proliferation of smooth muscle cells that have a phenotype altered to resemble myofibroblasts. There is an associated proliferation of extracellular matrix and collagen. Genetic factors may play a role in some cases, although well-documented familial cases are rare. The inheritance pattern appears to be autosomal dominant with reduced penetrance in males. There is no conclusive genetic basis, but Bofinger et al. [12] studied polymorphisms of angiotensin converting enzyme and found that one allele was associated with FMD. Autosomal dominant PKD is also associated with intracranial aneurysms. The relationship between these diseases has been studied in 88 patients selected from 378 known cases of PKD [20]. Cranial CT was performed in 60 patients, cerebral angiography in 21, and both procedures in 11. Four patients had aneurysms (4% incidence, 95% confidence interval

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0.1–9%). These results suggest an increased risk of aneurysms in patients with PKD. Other reports have detected intracranial aneurysms in upto 40% of patients with PKD. At least two genetic defects are identified in PKD [3]. About 85% of cases (PKD1) are due to mutations in the gene for polycystin located on chromosome 16p, which is a large glycoprotein that is postulated to be important in cell-cell and cell-extracellular matrix interactions. PKD2 is due to mutations in a gene on chromosome 4, which encodes a protein that is postulated to be a membrane spanning protein, perhaps an ion channel. PKD is also associated with hypertension secondary to kidney failure, and it has been suggested that the latter leads to aneurysms in these cases, although there are cases reported without well-documented hypertension. ED syndrome is another collagen disease that is characterized by hyperelasticity of the skin, joint hypermobility, bruising, and abnormal scarring. The skin may be relatively normal and the joints not excessively mobile in ED Type 4, which is of interest in stroke. These patients develop spontaneous dissections of large extracranial arteries as well as intestinal ruptures. Carotid cavernous fistulas are the most common cerebrovascular lesion although aneurysms also are reported [3, 173]. The molecular basis of ED syndrome is mutations in the gene for type III procollagen. Type III collagen is a major extracellular structural collagen in the walls of blood vessels and is composed of three ␣-helix polypeptide chains that form a triple helix. The type III collagen gene is located on chromosome 2 and numerous mutations causing ED Type 4 have been reported. Alberts [2, 3] found only one well-documented case of a patient with ED Type 4 and a true saccular intracranial aneurysm, suggesting that simple defects in type III collagen are not an usual cause of these aneurysms. Studies of humans with familial and sporadic intracranial aneurysms have not found mutations in type III collagen to be responsible [92]. Marfan’s syndrome may be associated with saccular, fusiform, or dissecting aneurysms [3]. This disease results from mutations in fibrillin, a gene on chromosome 15q. Fibrillin is a glycoprotein that is a part of elastic and nonelastic microfibrils. Most aneurysms described are extracranial or in the cavernous internal carotid artery. Few true saccular aneurysms have been identified. These patients are prone to dissections of the large elastic and muscular arteries and dissections could underlie some of the reported cases of aneurysmal disease. Patients with coarctation of the aorta were suggested to be at increased risk of having intracranial aneurysms and of SAH at a young age. In early reports, many of the patients had hypertension which may have increased the incidence of aneurysmal disease. Early diagnosis and treatment of the coarctation was reported in 182 patients who were followed for a total of 3288 patient-years. There were no deaths from intracranial hemorrhage, suggesting

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that early repair and prevention of chronic hypertension in these patients reduces or eliminates the risk of intracranial aneurysm [11]. More study is needed to ascertain whether aneurysms in these patients are due to arterial hypertension or a common vascular defect.

Pathophysiology of ICH

Direct and Indirect Molecular Effects Pathophysiological mechanisms of brain damage from ICH are hypothesized to include direct damage to the brain due to physical disruption by the hematoma and secondary injury due to the enlargement of the hematoma, increased intracranial pressure, brain edema, toxic effects of substances released from the blood clot, and possibly ischemia around the clot. Two studies in which computed tomography was performed sequentially after ICH documented that enlargement occurs in about one quarter of patients if the first CT scan is obtained within one hour of the ictus [15, 37]. The risk of the enlargement decreases with time. Microscopically, there is extracellular edema appearing around ICH in humans within a time course of hours. Polymorphonuclear lymphocytes appear around the ICH by 2 days and peak at 4 days. Microglia are seen at this time and ingest ICH and damaged brain tissue. Gliosis, characterized by gemistocytic astrocytes appears after days and may persist for months to years. The pathogenesis of brain injury secondary to SAH is multifactorial and has not been extensively investigated. Processes involved include global and focal ischemia from increased intracranial pressure and decreased cerebral perfusion pressure, cerebral herniation, ICH, subdural hemorrhage, hydrocephalus, and aneurysm rebleeding. It is assumed that most of the initial damage in patients who are neurologically impaired after SAH is due to global ischemia since intracranial pressure rises with bleeding and reduces cerebral perfusion pressure. Whether or not there are additional direct effects of the SAH itself on the brain is open to question, but the possibility has been raised based on experimental and clinical studies demonstrating that cerebral blood flow (CBF) is reduced after SAH in proportion to the clinical grade and that this often is accompanied by reduced cerebral metabolic demand. Mitochondrial respiration, sodium-potassium ATPase activity, extracellular potassium and calcium are altered in brain tissue of experimental animals exposed to subarachnoid blood, although the relationship of these changes to the alterations in CBF and metabolism has not been studied [36, 72, 105, 218]. Molecular changes may occur in the brain after SAH as a result of the SAH itself or due to secondary processes listed above that often result in ischemia.

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SAH increases c-fos, c-jun and HSP70 mRNA in a number of brain regions in rats [55, 135]. Neonatal capsaicin treatment which destroys unmyelinated c fibers, or sectioning of meningeal afferents, results in reduced c-fos in the trigeminal nuclear complex, parabrachial nucleus, and medullary lateral reticular nucleus, but not in nucleus of tractus solitarius, area postrema, ependyma, piamater and arachnoid. This pattern suggests that SAH directly induces c-fos protein expression in the nucleus of the tractus solitarius, area postrema, ependyma, pia mater and arachnoid and that in other areas (trigeminal nuclear complex, parabrachial nucleus, and medullary lateral reticular nucleus) induction may be secondary to trigeminovascular nerve fibers. Creation of SAH in rats by endovascular perforation of the intracranial anterior cerebral arteries causes an SAH that may resemble that occurring in man since intracranial pressure is elevated for some time [9, 200]. This pattern differs from other models of SAH, where blood is injected slowly into the cerebrospinal fluid cisterns or a blood clot is placed surgically around the arteries. SAH caused by endovascular perforation increases HSP70 protein in multiple brain areas bilaterally for up to 5 days, whereas cisternal blood injections do not [115]. HSP70 is known to be induced by ischemia, which was postulated to be the cause in these experiments although CBF was not measured. Other experiments were conducted in which HSP70 induction in the hippocampus occurred when SAH in rats was induced by blood injections that increased intracranial pressure, whereas saline injections produced only transient elevations in intracranial pressure and transient HSP70 expression. These data are consistent with a role for ischemia in these models and in acute changes after SAH. Acute vasoconstriction with reduction in CBF has been reported to occur in this model and it has been reported to be reversible with NO donors, suggesting that decreased NO availability contributes to the reduced blood flow [10, 178]. Erythrocyte lysis and hemoglobin are probably involved in the delayed cerebral vasospasm that is discussed below. Interest in their role earlier after SAH also has been raised based on animal experiments performed by Matz et al. [111] Hemoglobin metabolism is catalyzed initially by heme oxygenase [HO], an enzyme that exists in three different forms. The inducible form, HO-1, was induced diffusely in microglia throughout the brain of rats after cisternal injection of whole blood, erythrocyte hemolysate, or oxyhemoglobin. Focal areas of expression of HO-1 and HSP70 and of DNA fragmentation were also noted after the injection of hemolysate [111, 112]. Since HSP70 is induced in areas of ischemia, two conclusions can be drawn. The lack of widespread increase in HSP70 suggests that there was no overall stress response or ischemia in the brain in this model, whereas focal expression is probably due to ischemia secondary to acute vasospasm or direct toxic effects of the blood products. The focal areas were blocked by the pretreatment of rats with

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tirilazad-like anti-oxidants, U101033E and U74389G [197]. Induction of HO-1 expression in the brain after SAH in rats occurred after injection of erythrocyte hemolysate, but injection of the vasoconstrictor endothelin did not [93]. Since the degree of vasospasm of the basilar artery was similar in both cases, induction of HO-1 may be due to effects of blood and not vasospasm. Evidence for direct effects of SAH on the brain was presented by Matz et al. [114]. Injection of erythrocyte hemolysate into the cortical subarachnoid space of mice produced DNA fragmentation and laddering and terminal deoxyuridine nick endlabeling of cells, consistent with apoptotic cell death in cortex after SAH. In this model, transgenic mice overexpressing copper-zinc superoxide dismutase, an antioxidant enzyme, exhibited less cell death [113]. iNOS protein was increased in microglia, astrocytes and neurons of the brains of rats 7 days after SAH and vasospasm induced by two injections of blood into the cisterna magna 2 days apart [208]. Brain Edema Most research has focused on brain edema and ischemia around intracerebral hematomas. Cerebral edema is defined as an increase in the brain water content. The most common classifications are vasogenic, cytotoxic, or interstitial. Edema develops within hours of hemorrhage and peaks at 4–10 days in humans before resolving [194]. Edema after ICH is probably a combination of vasogenic edema due to blood brain barrier breakdown and cytotoxic edema due to cellular injury. The time course is shorter in animal models, with a peak at 3–4 days. Animal models of ICH suggest that different processes contribute to edema formation at different times. Pathophysiological mechanisms involved in edema may include hydrostatic pressure from clot formation, clot retraction, thrombin release during coagulation, erythrocyte lysis with hemoglobin release, complement activation, disruption of the blood brain barrier, and ischemia reperfusion [211]. Edema developing within the first hours probably is secondary to hydrostatic pressure changes and clot retraction. This conclusion is based on studies demonstrating that the blood brain barrier is intact within 8 h of ICH in pigs [204] and blood clotting extrudes serum from the clot, which then appears on CT scan as a hypodense rim around the ICH. A series of experiments using a rat model of ICH have provided important information about the pathogenesis of edema developing after this initial phase of clot retraction. Injection of pure thrombin into the rat brain caused cerebral edema 24 h later that was prevented by the selective thrombin inhibitors hirudin and ␣-NAPAP [95]. Injection of plasma with prothrombinase to activate the coagulation cascade led to edema formation after 24 h, whereas injection of pure serum, plasma or washed, intact erythrocytes alone

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did not cause edema [96]. Edema 24 h after ICH was also reduced by hirudin, further supporting a role for thrombin generated by coagulation in the generation of edema at this time after ICH in rats. Thrombin-induced edema was suggested to be secondary to the disruption of the blood brain barrier and to cytotoxic effects of thrombin on glia based on studies showing normal CBF but disruption of the blood brain barrier at the time of thrombin-induced edema as well as studies in vitro documenting toxic effects of thrombin on C6 glioma cells [97]. Additional evidence supporting a role for blood coagulation in generating brain edema was that injection of heparinized whole blood into rats produced less edema than injection of unheparinized blood that subsequently clotted [212]. These findings are consistent with human studies reporting that edema around thrombolysis-related ICH is less than that around ICH occurring in patients with normal clotting [42]. In patients who are anticoagulated or have had intravascular thrombolytics, there would be expected to be less clot retraction and thrombin generation. Activation of complement also contributes to edema formation at both 24 and 72 h after ICH in rats [71]. Possible mechanisms are the formation of membrane attack complexes by complement that facilitate erythrocyte lysis, which is known to release hemoglobin and possibly other substances from the erythrocyte that can produce edema. The membrane attack complexes also may damage cells in the brain itself, leading to neuronal injury and edema as well as leading to further breakdown of the blood brain barrier. When various blood fractions were injected into the brain of rats, edema occurred after exposure to lysed erythrocytes and hemoglobin but not immediately after the injection of intact erythrocytes [212]. Hemoglobin, its breakdown products and/or reactions induced by them are numerous and may be toxic to a variety of cell types including neurons [32, 116]. Hemoglobin, hemin, bilirubin, or FeCl2 injections into the basal ganglia of rats all increased brain water content 24 h after injection. Hemoglobin is metabolized by HO enzymes, the inducible form of which is called HO-1. This enzyme was up-regulated after hemoglobin injection. Interestingly, inhibition of HO with tin protoporphyrin-9 or scavenging of iron by deferoxamine reduced hemoglobin-mediated edema. These animal studies suggest that within the first hours of ICH, clot retraction and plasma proteins derived from the hematoma initiate the development of cerebral edema. Initially, the blood brain barrier is intact but it breaks down to some extent, possibly secondary to thrombin generated by clotting in the hematoma and by cytotoxic effects of thrombin, resulting in vasogenic edema. There may also be leakage of additional thrombin, clotting factors, and complement proteins that produce further edema by mechanisms discussed above. These processes are believed to be important in edema formation within 24–72 h of ICH. With time there is lysis of erythrocytes and edema formation

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secondary to effects of the released erythrocyte contents and this contributes to edema at longer times after ICH. Ischemia Numerous studies have measured regional CBF around ICH in animal models and in humans [164]. Animal experiments have produced conflicting results as to whether ICH reduces CBF and for how long. This ambiguity is because the rate of development of the ICH, as well as its location, final volume, and possibly the animal species studied affect changes in CBF that may be observed. Many early studies modeled ICH by inflating a balloon in the brain, which may produce more marked changes in the CBF and less edema than blood. Recent studies of ICH in dogs and pigs found no reduction in CBF and no evidence of ischemia around the ICH [164, 204]. Qureshi et al. [162] have reviewed studies of CBF after ICH in humans, with similar findings as in recent animal studies. Within 48 h of ICH there may be some reduction in CBF around the ICH, secondary to the reduced cerebral metabolic demand. Positron emission tomography studies suggest that regional oxygen extraction is normal or reduced. CBF subsequently may normalize, increase above normal, or remain depressed before returning to normal after 14 days. Overall, it appears that the pathogenesis of edema and brain injury around ICH is multifactorial but is related to factors other than mass effect from the hematoma and that ischemia and reperfusion play a relatively minor role in most cases [211]. Apoptosis and Inflammation Additional processes documented to occur around experimental ICH include apoptosis and inflammation. Neurons and astrocytes around ICH in rats may demonstrate DNA fragmentation and changes characteristic of apoptosis [63, 110]. Histological studies demonstrate areas of brain necrosis in these models, but these studies suggest that brain damage occurs by active processes such as apoptosis as well. Inflammation is observed around experimental ICH and its contribution to the formation of edema is suggested by studies demonstrating that the depletion of leukocytes and platelets by whole-body irradiation reduces edema around ICH in rats [77]. Early attempts to inhibit inflammation may demonstrate some benefit, but inflammation probably evolved to maintain the life of the organism in the face of injury and may have beneficial effects as well. Whether selection pressures or whatever mechanisms of evolution selected for favorable responses specifically to ICH is open to question, but it is reasonable to assume that there are favorable aspects to inflammation and inhibition is of unproven clinical benefit. The detailed nature of the inflammatory response to ICH is unknown but as more selective pharmacological and

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biological methods to manipulate inflammation become available, this would seem to be an area worthy of further investigation. Microvascular Vasospasm Most of the delayed deterioration that can complicate SAH has been attributed to the delayed cerebral vasospasm in large arteries, discussed below. Whether or not the arteries and arterioles are affected after they enter the brain substance has been studied in several experimental models and using positron emission tomography in humans. Experimental models have generally not found evidence that SAH constricts the penetrating arteries [128] or that they are abnormal pharmacologically [80]. Ohkuma et al. [136] reported that corrosion casts of cerebral arteries after SAH in dogs did demonstrate microvascular spasm [139]. Yet the potential for fixation artifacts and postmortem changes renders these studies virtually impossible to interpret in the opinion of this author. Clinical studies have usually been consistent with dilation of small arteries during vasospasm [47, 64] although recent data have been interpreted as suggesting that there is small artery spasm after SAH [137, 219]. These studies did not measure the arterial diameter however, so the hypothesis of microvessel spasm remains unproven. Cerebral Vasospasm Most work on SAH has focused on cerebral vasospasm. Vasospasm is associated with changes in the endothelial, smooth muscle, and adventitial fibroblasts of the affected major subarachnoid cerebral arteries. These changes can be observed grossly, histologically, ultrastructurally, or as changes in expression of mRNA and protein or function of the artery, measured at the single cell, excised artery or in vivo level. Global Changes in Gene Expression in Vasospasm Differential display, cDNA arrays and serial analysis of gene expression can be used to ascertain differences in mRNA expression levels between two or more sets of samples. A large number of differences may be produced that can be used to generate hypotheses about disease pathogenesis. Several investigators have reported the analysis of vasospastic arteries using high-throughput molecular techniques. For example, fluorescent differential display was used to screen for changes in gene expression after SAH in rats [192]. Onda et al. [141] reported that eleven known genes were up-regulated after SAH in dogs, which included growth factor genes (VEGF, BiP protein; glucose-regulated protein 78), growth-arrest and DNA damage-inducible protein (gadd45), neuromodulin, protein disulfide isomerase-related protein P5, acid sphingomyelinase-like phosphodiesterase) and inflammation genes (monocyte chemotactic protein 1,

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cystatin B, inter-␣-trypsin inhibitor family heavy chain-related protein, serum amyloid A protein, glycoprotein 130). They found that Frizzled-6, the gene for a human WNT receptor (involved in development and carcinogenesis), was down-regulated. The middle cerebral arteries of monkeys with or without vasospasm after SAH were analyzed using a cDNA array containing oligonucleotides for 5184 genes [102]. Five hundred and thirty seven of the genes (10%) were expressed in the arteries. One hundred and sixty four did not change significantly (31%) and 373 genes (69%) were differentially expressed at 3, 7 or 14 days after SAH. Many showed increasing expression with time. Functions of differentially-expressed genes included regulation of gene expression, cell proliferation, inflammation, membrane proteins and receptors, and kinases and phosphatases. Smooth Muscle Contraction Smooth muscle contracts in response to electrical, chemical, and mechanical stimulation. These stimuli cause an increase in intracellular free Ca2⫹. The source of Ca2⫹ may be influx from the extracellular space, release from intracellular stores, or a combination. Ca2⫹ influx may occur through voltage-gated or possibly receptor-operated Ca2⫹ channels. Release from intracellular stores is via the inositol triphosphate pathway or Ca2⫹-induced Ca2⫹ release. The increased Ca2⫹ binds calmodulin and activates myosin light chain kinase, which phosphorylates myosin light chain. This increases actin-activated myosin ATPase activity, cross-bridge cycling, and contracts the muscle. There is a variable relationship between intracellular Ca2⫹ concentration, contraction velocity, force maintenance and the level of phosphorylation of myosin light chain. Contraction results in all cases from an interaction between actin and myosin. After the initial phase, the contraction or force development may persist with lower rates of cross-bridge cycling. This usually is associated with the return of intracellular Ca2⫹ to basal or near basal levels, reduced myosin light chain phosphorylation and decreased cross-bridge cycling. The persistence of contraction in the absence of the processes that mediate it initially involves various secondary processes that may include a latch bridge state, thin-filament (actin) regulatory processes and/or changes in Ca2⫹ sensitivity of the contractile apparatus. Second messenger systems that involve protein kinase C, tyrosine kinases, and mitogen-activated protein (MAP) kinases also regulate smooth muscle contraction and have been the subject of some study in vasospasm. Kinases are enzymes that phosphorylate proteins. The regulation of smooth muscle contraction is complex and is reviewed elsewhere [7, 99]. Several investigators have measured the levels of contractile proteins in vasospastic arteries [56, 119]. Most have reported reductions in the actin and myosin proteins during vasospasm and this was postulated to be due to proteolytic

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degradation secondary to calpains that are activated by increased intracellular Ca2⫹ in vasospastic smooth muscle cells. A role for calpains in vasospasm was supported by the evidence of calpain activation in cerebral arteries for days after SAH in dogs and rabbits [98, 215] and the fact that topical application of calpain inhibitors to the dog basilar artery reduced the degree of vasospasm [118]. The results of assays of myosin light chain phosphorylation and of amounts of caldesmon and calponin in vasospastic arteries, however, have been inconsistent [16, 38, 56, 139, 190]. In addition, detailed studies in a nonhuman primate model of vasospasm demonstrated that vasospasm could be maintained for up to 2 weeks by repeated stimulation with blood clot and relaxation of the vasospastic arteries became progressively slower with increasing duration of exposure to perivascular blood clot [189, 222]. These authors concluded that contractile processes were relatively preserved after SAH, whereas there was a reduction in the ability of arteries to relax after SAH. Reductions, if any, in contractile proteins, did not impair the ability of the artery to remain contracted. Numerous intracellular signal transduction mechanisms mediating smooth muscle contraction have been investigated in vasospasm [99]. The complexity of these pathways consisting of numerous enzyme isoforms, interactions between parallel cascades as well as differences between species and in the biochemistry of the cerebral arteries at different times after SAH, and use of nonspecific pharmacological agents to draw conclusions about relative roles of the various pathways, all have complicated interpretation of these studies. The protein kinase C pathway is perhaps the most extensively investigated. This family of kinases consists of at least eleven isoforms with differing requirements for Ca2⫹, phospholipids, and diacylglycerol for activation [83]. Inhibitors of protein kinase C such as H-7 were reported to reverse established vasospasm in dogs [108]. In addition, vasospastic dog basilar artery contains increased levels of diacylglycerol, an endogenous activator of protein kinase C and demonstrates increased protein kinase C activity [99, 109]. Peterson and colleagues [216] reported, however, that diacylglycerol concentrations were not elevated after SAH in dogs and that protein kinase C activity was not significantly elevated. This observation can be reconciled with their studies in vitro, which showed that minor changes observed in protein kinase C activity and membrane translocation could significantly alter smooth muscle contraction [81]. A major limitation of those studies is the lack of specificity of the pharmacological drug protein kinase inhibitors that were used. H-7, staurosporine and similar drugs such as fasudil (AT877, HA1077) are kinase inhibitors with varying degrees of specificity for multiple kinases involved in smooth muscle contraction. In the absence of assays for each kinase in treated tissue and/or of the concentration of drug achieved in the tissue, it is difficult to be certain as to the mechanism of the effect of these drugs.

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Nishizawa et al. [131, 132] have conducted a series of studies to test their hypothesis that NO, which is tonically released from cerebral arteries, inhibits or reduces the activation of protein kinase C. Therefore, a reduction in NO would increase protein kinase C activity and contribute to vasospasm. A correlation was found between vasospasm, reductions in arterial cyclic guanosine monophosphate which is increased by NO, and increased protein kinase C activity. Vasospastic arteries also showed an increase in protein kinase C-dependent tone when studied under isometric tension in vitro [130]. Protein kinase C may produce vasospasm by other mechanisms. Fujikawa et al. [38] noted that the pattern of myosin light chain phosphorylation in vasospastic dog arteries was consistent with phosphorylation by myosin light chain kinase but not protein kinase C. Phosphorylation of calponin was detected in these arteries, which was suggested to contribute to vasospasm indirectly through PKC. Tyrosine kinases are another class of kinases that are involved in smooth muscle contraction and that have been postulated to be involved in vasospasm. They are classified as nonreceptor, membrane-bound (such as pp60c-src), membrane-spanning receptor (typically growth factor receptors such as insulin, platelet-derived growth factor and epidermal growth factor receptors), and cytosolic tyrosine kinases (e.g., c-abl, c-fes). The receptor tyrosine kinases activated by growth factors in some cases act through signal transduction pathways such as the ERK/MAP kinase pathway, discussed below. Several in vitro studies where solutions of erythrocyte hemolysate were added to cerebral arteries suspended under isometric tension were purported to support a role for tyrosine kinase activation in the contractions induced by hemolysate, since tyrosine kinase inhibitors reduced the contractions [85]. These studies are supportive but provide no direct evidence for a role of tyrosine kinase activation in vasospasm. More direct evidence was the observation that vasospasm after SAH in dogs was associated with phosphorylation of known intracellular substrates of tyrosine kinases such as Shc, Raf1, and MAP kinases [38]. Furthermore, topical application of the tyrosine kinase inhibitor, genistein, reversed vasospasm. Vascular smooth muscle contains several types of MAP kinases [1]. MAP kinases were identified and named based on their activation in response to stimulation of cells by mitogens. Growth factors (epidermal, platelet-derived, fibroblast, nerve, insulin and insulin-like growth factors) and other agonists, some of which contract smooth muscle, including vasopressin, angiotensin II, platelet-activating factor, endothelins(ETs) and muscarinic agonists, all act on various cell membrane receptors and result in the activation of intracellular MAP kinases. There are at least three interacting MAP kinase pathways, each of which consists of a kinase that activates the MAP kinase and a kinase that activates MEK (MEK kinase). One postulated function of MAP kinases in smooth muscle is to phosphorylate caldesmon, which would remove the

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inhibitory effect of caldesmon on contraction. Zubkov et al. [224] reported that PD-98059, an inhibitor of the MAP kinase pathway, reduced contractions and prevented MAP kinase immunoreactivity increase in rabbit basilar artery exposed to erythrocyte hemolysate. In vivo investigations of the MAP kinase pathway in vasospasm are limited to one study showing that the ERK pathway was activated during vasospasm after SAH in dogs [38]. The most recent interest in kinase involvement in vasospasm has centered on the role of Rho kinase. Rho proteins are low-molecular-weight G proteins belonging to the Ras superfamily of 20 to 30 kDa GTP-binding proteins [199]. This class of G proteins is distinct from the heterotrimeric G proteins [177]. The Rho G proteins are involved in cytoskeletal organization, membrane trafficking, regulation of transcription, control of cell growth and development and smooth muscle contraction. They function in a complex pathway that includes numerous other regulatory proteins, but the main effect of interest is the activation of the Rho kinases such as p160ROCK (ROCKII). Rho kinase phosphorylates the myosin-binding subunit of myosin light chain phosphatase. This reduces the phosphatase activity and maintains myosin light chain phosphorylation and smooth muscle contraction. Other actions may occur as well. Interestingly, fasudil (HA1077 or AT877), which was reported to decrease vasospasm and improve outcome in patients with aneurysmal SAH [182], is a protein kinase inhibitor with order of potency p160ROCK ⬎ cAMP-dependent protein kinase ⬎ protein kinase C ⬎ myosin light chain kinase. Biochemical studies of vascular smooth muscle during vasospasm suggest that the activation of Rho kinase and the resulting Ca2⫹ sensitization may contribute to vasospasm [170]. Most of the actions of fasudil that inhibit kinase activity should reduce vasospasm, making it a candidate drug for the treatment of vasospasm. The relatively modest effects observed clinically would need to be reconciled with this, perhaps being related to the inability to administer adequate doses without producing hypotension or other undesirable cardiovascular effects. Smooth Muscle Relaxation Multiple mechanisms can produce smooth muscle relaxation including removal of the contractile stimulus which reduces intracellular Ca2⫹. There may be dephosphorylation of myosin light chain by myosin light chain phosphatase mediated by cAMP-protein kinase A, reduction in intracellular Ca2⫹ by activation of K⫹ channels and relaxation mediated by the guanylate cyclase-cGMPprotein kinase G system. Vasodilatory pathways may be selectively impaired during vasospasm [186, 222]. There is little evidence for impairment in the cAMP-protein kinase A relaxation pathway after SAH [147, 153]. More evidence points to abnormalities in K⫹ channels and the cGMP pathway in vasospasm. Vascular smooth muscle is

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depolarized during vasospasm in dogs and nicroandil, a K⫹ channel opener that also may activate soluble guanylate cyclase, reversed vasospasm in this model [57]. Vasodilation of the rat basilar artery after SAH was impaired in response to agents that produce relaxation by the cGMP pathway such as acetylcholine and sodium nitroprusside [186]. Relaxation to papaverine, 8-bromoguanosine 3⬘,5⬘-cyclic monophosphate and brain natriuretic peptide (an activator of particulate guanylate cyclase) were not affected and relaxation to aprikalim and calcitonin-gene related peptide, which activate ATP-sensitive K⫹ channels, were augmented. Endothelial Dysfunction and Vasospasm Endothelial dysfunction is widely believed to be important in the genesis of vasospasm. Questions remaining include the mechanism by which perivascular blood clot injures the endothelium and whether or not such changes are a cause or consequence of vasospasm. Cerebrovascular endothelium synthesizes vasoconstrictors such as ETs and prostaglandin F2␣ and vasodilators such as NO, prostacyclin, superoxide anion radical, and endothelium-derived hyperpolarizing factor [33]. Perivascular nerves and astrocytes also may synthesize some of these compounds. Any reduction in vasodilators and increase in vasoconstrictors could contribute to vasospasm. As mentioned above, prostacyclin and the cAMP system do not seem to be substantially altered after SAH. On the other hand, much interest has focused on NO which is synthesized by NOS. Endothelial NOS is localized primarily to endothelial cells and neuronal NOS to perivascular nerves [220]. There is a basal release of NO from these sources that reduces the tone of cerebral arteries and the removal or impairment of response could contribute to vasospasm. Endothelium-dependent relaxation is impaired after SAH and different pharmacological studies have demonstrated impairment at all of the possible sites in the pathway including impaired synthesis and release of NO [68], scavenging and destruction of NO before it reaches the smooth muscle [76, 214], and impaired ability of smooth muscle to generate cGMP and relax in response to NO [34, 86]. These studies are based mainly on the pharmacological study of excised arteries and on biochemical measurements of substances in the arteries. No comprehensive study has been carried out. The level of soluble guanylate cyclase protein was reduced in vasospastic dog basilar artery although there was no significant reduction in endothelial NOS [79]. In a monkey model of SAH, the reverse was found in which there was a significant reduction in mRNA coding for endothelial NOS, whereas levels of neuronal NOS and soluble guanylate cyclase were not reduced [66]. In the same model, another group reported that the immunoreactivity for neuronal NOS was reduced [158].

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iNOS usually is not detectable in unstimulated cells, but it may be induced by inflammatory mediators such as lipopolysaccharide, cytokines (interferongamma, IL-1␤, TNF-␣) and cerebral ischemia and decreased by IL-4, IL-10, TGF-␤, basic FGF, aldosterone, HSP70, insulin-like growth factor, dexamethasone and NO. Increased iNOS expression has been documented to occur in arteries in response to injuries such as endothelial removal and balloon angioplasty. iNOS produces larger amounts of NO. These may be protective under these circumstances by reducing platelet and leukocyte adherence, decreasing smooth muscle proliferation and acting as a compensatory mechanism in response to endothelial dysfunction or loss [84]. On the other hand, the production of NO by iNOS in inflammatory cells probably has physiologically important antimicrobial and antitumor effect. NO may combine under specific conditions with superoxide anion radical to produce peroxynitrite (ONOO⫺) which can damage proteins and other biological molecules. A series of studies by Sayama et al. [171, 172] reported that SAH in rats is associated with an increase in iNOS mRNA in basal portions of the brain and that immunoreactivity to iNOS was present in inflammatory cells in the subarachnoid space. Vasospasm was reduced by the administration of the relatively selective iNOS inhibitor, aminoguanidine. Another pathway of smooth muscle relaxation involves membrane hyperpolarization. There are many mechanisms involved, but one is mediated by endothelial cells that may produce a diffusable substance, endotheliumderived hyperpolarizing factor that acts on smooth muscle cells to hyperpolarize them and thereby cause vascular relaxation [34]. Since the membrane potential in smooth muscle cells is controlled in large part by K⫹ conductance, this substance may activate K⫹ channels, leading to K⫹ efflux, membrane hyperpolarization, closing of voltage-gated Ca2⫹ channels, reduction in intracellular Ca2⫹ and thereby smooth muscle relaxation [58]. The precise nature of the endothelium-derived hyperpolarizing factor is unclear, although in the coronary arteries they may be cytochrome P450 metabolites produced from metabolism of arachidonic acid such as epoxides (e.g., epoxy-eicosatrienoic acid) formed by the epoxygenase pathway. There has been little specific investigation of endothelium-derived hyperpolarization in vasospasm, but it has been suggested that of the naturally occurring vascular relaxation mechanisms, those mediated by K⫹ channels may be the least affected by SAH and may thus be a useful therapeutic target [147, 187]. Of the contractile substances released by endothelial cells, the ET system is most widely studied. There are three 21-amino acid long ET peptides, ET-1,2,3 [34]. They show sequence homology to sarafotoxin S6c, the venom of the snake Atraclaspis engadensis. ETs are synthesized as preproETs of approximately 200 amino acids and then cleaved by endopeptidases to big ETs and then to ETs by the ET converting

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enzymes [34]. ET acts on ETA receptors found predominantly on smooth muscle, leading to contraction, on ETB receptors located on smooth muscle where they may mediate contraction (ETB1) and on endothelial cells (ETB2) where they mediate relaxation by release of NO and/or prostacyclin. The ETs have been suggested to be important in the pathogenesis of vasospasm based on studies reporting that ET concentrations are elevated in cerebrospinal fluid after SAH in humans and experimental animals [75, 106, 179, 193] and in arteries of animals with SAH [183], and that antagonists of ET receptors prevent or reduce vasospasm in animals [17, 129, 138, 205, 225]. Contradictory reports have appeared on ET antagonist effects [28, 40, 51, 89, 223] and most are consistent with the lack of demonstrated effect of TAK-044, an ET receptor antagonist, on vasospasm in humans [181]. In data from experimental models, expression of ETA receptor mRNA was increased after SAH in dogs [74]. Vasospasm was prevented in this model by intrathecal administration of the ETA receptor antagonist, BQ-123. In a monkey model of SAH, mRNA levels of preproET-1, preproET-3, and ETA receptors were unchanged in vasospastic arteries 7 days after SAH, whereas there was a significant increase in the ETB [65]. These findings are consistent with other studies in rabbits and dogs showing that SAH is associated with a shift from ETA to ETB receptor expression in the vasospastic arteries [167]. The ET system has been used as a target for the antisense approach to reducing expression of genes. Antisense oligonucleotides are short DNA sequences that are taken up by cells and contain a sequence that is complementary to the gene of interest. There are several possible mechanisms of action which results in blocking transcription and/or translation of the gene. Antisense oligoncleotides directed at preproET-1 were incorporated into all layers of the basilar artery after intracisternal injection into rats, and were shown to reduce contractions of the basilar artery exposed in situ to whole blood hemolysate for up to 72 h after treatment [145]. In dogs, intracisternal antisense oligoDNAs to preproET-1 did not prevent vasospasm although they did when administered in combination with tissue plasminogen activator [137]. The role of ETs in vasospasm remains incompletely investigated. Interpretation of the results of studies are complicated by the observation that cerebral ischemia may be associated with an increase in ET-1 in brain, arteries, and cerebrospinal fluid [157], that contractions of arteries to various substances can be potentiated by subthreshold concentrations of ET-1, and that receptor sensitivity may also be altered. Gene Expression in Vasospasm One of the central processes in vasospasm is the prolonged hemolysis of erythrocytes in the subarachnoid space after SAH, leading to the release of hemoglobin and its breakdown products. Physiological metabolism of hemoglobin is

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mediated by the initial release of heme from the globin chain that is facilitated by the oxidation of ferrous (Fe2⫹) iron in oxy- or deoxyhemoglobin to ferric (Fe3⫹) iron in methemoglobin. The heme groups are enyzmatically broken down by HO into biliverdin, free iron and carbon monoxide. HO-2 is a constitutively expressed form that is found in many tissues, whereas HO-1 is an inducible enzyme that is increased in response to heavy metals, oxidative stress, ultraviolet light, heme, and numerous other stimuli. There is increased hemoglobin and oxidative stress in the subarachnoid space after SAH and it is therefore perhaps not surprising that HO-1 mRNA was found to be increased in the basilar artery of rats with SAH [190]. More interesting was the finding that preventing the increase in HO-1 with antisense oligodeoxynucleotides aggravated vasospasm. Ono et al. [144] reported that HO-1 protein was increased in the cerebral arteries after SAH in monkeys. In addition, the iron released by the HO metabolism of heme would be expected to increase ferritin protein, as ferritin is an iron-sequestering protein. Indeed, an increase in ferritin protein was found in this model. Effects of modulating these responses were not reported. Inf lammation Inflammation is the response of a living tissue to injury. From an evolutionary perspective, one would expect it to mediate reactions that would favor survival of the organism. To some extent, however, detrimental effects have been emphasized in the medical literature. Evidence suggests that inflammation may have beneficial and detrimental effects. For example, administration of proinflammatory cytokines early after spinal cord injury in mice exacerbated injury, whereas administration later appeared to reduce injury [87]. Moreover, activated macrophages and microglia have been shown to promote axonal regrowth after spinal cord injury in rats [161]. The hypothesis that vasospasm is an inflammatory disease or that inflammation in general worsens vasospasm is simplistic in that it considers inflammation to be a single process. Few studies, however, have attempted to assess roles of specific aspects of the inflammatory reaction in vasospasm. A classic study by Weir et al. [207] involved repeated blood injections given one week apart into monkeys and suggests that a delayed-type hypersensitivity (Type IV) reaction does not contribute to vasospasm since this did not produce worsening vasospasm. Kubota et al. [90], however, studied the kinetics of lymphocyte subsets in the cerebrospinal fluid of rats after SAH and found a pattern that resembled such a reaction. It remains unknown as to whether this immune reaction is an epiphenomenon since the former study did not perform immunological studies, and the latter did not modify the reaction to assess the effect on vasospasm. Support for inflammatory mechanisms in vasospasm is provided by studies demonstrating immunoglobulin and complement components in the walls of

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vasospastic arteries of animals [53] and man [69] and of elevated levels of circulating immune complexes and activated complement in humans with SAH [82, 149, 151, 196]. Outcome of patients after SAH is worse if there are elevated levels of the proinflammatory cytokines IL-1 receptor antagonist and TNF-␣ in the cerebrospinal fluid [107]. Anti-inflammatory drugs such as corticosteroids improved outcome after SAH in Phase 2 [25, 168] and Phase 3 trials in humans [59] and reduced vasospasm in animal models [24, 26, 213]. Experimental evidence suggesting that cyclosporine A reduces vasospasm after SAH in dogs [155] and monkeys [52] is not supported by subsequent studies in humans [103], nor did a similar drug, FK506, prevent experimental vasospasm [124, 129] in animal models. One aspect of inflammation that seems to be important is the role of complement in accelerating erythrocyte hemolysis in the subarachnoid space after SAH [154]. Inhibition of complement-mediated reactions in rabbits reduces vasospasm [43]. Another role of complement may be to form membrane attack complexes [150]. Experiments using freshly isolated rat cerebral artery vascular smooth muscle cells in vitro showed that these complexes caused large increases in membrane conductance. These could be formed in the subarachnoid space after SAH and could lead to smooth muscle cell damage and perhaps contraction. One of the initial reactions in inflammation is the up-regulation of adhesion molecules on the surface of endothelial cells. Several groups have reported an increased immunoreactivity to intercellular adhesion molecule-1 or during vasospasm after SAH in rats and after perivascular blood placement around the femoral artery in rats [54, 185]. Humans with aneurysmal SAH also have elevated levels of immunoreactivity to intercellular adhesion molecule-1 as well as other adhesion molecules (vascular cell adhesion molecule-1, L-selectin) in cerebrospinal fluid [160]. Blockade of intercellular adhesion molecule-1 with monoclonal antibodies reduced perivascular inflammation and vasospasm in several vasospasm models [8, 148]. Ono et al. [143] administered antisense oligodeoxynucleotides to NF-␬B intrathecally to rabbits with SAH. Vasospasm was reduced and importantly, unlike many other studies, evidence that the oligonucleotides had the desired effect was provided by showing reduced activity of NF-␬B as measured by gel-shift assay. Remodeling, Fibrosis, Proliferation, and Phenotype Changes Vasospastic arteries have reduced contractility and compliance compared to normal arteries, and they develop histopathological changes within all layers of the arterial wall [99, 101, 203]. The mechanism of these changes and whether they cause or contribute to vasospasm or are only an epiphenomenon is unknown. The characteristic response of an artery to injury is intimal proliferation [176]. Interestingly, this can occur after injury to the tunica intima, but

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also to perivascular processes such as the placement of a cuff around an artery [121]. There is an intimal proliferation in arteries that develop vasospasm after SAH but this usually occurs after angiographic vasospasm and is rare enough a cause of symptoms so as to be a subject of case reports [88, 159]. That it does develop, however, proves that there has been some type of vascular injury. Decreased arterial compliance in vasospastic arteries represents an increased stiffness of the arterial wall and could be due to an increase in or an alteration in the extracellular matrix or in the smooth muscle cells themselves [78, 123, 125]. Increased elastin and collagen have been detected in vasospastic arteries [123, 125]. Procollagen I and III mRNA were increased 7 and 14 days after clot placement around the rat femoral arteries, whereas TGF-␤ mRNA was increased at 3 days [78]. It was suggested that the growth factor increase mediated the subsequent increase in collagen expression. Some investigators were unable to document an increase in collagen in arteries during vasospasm [100]. A causative role of collagen synthesis in vasospasm was demonstrated by Onoda et al. [146] who had reported the ability to reduce femoral artery vasospasm in rats by the administration of antisense oligodeoxynucleotides to procollagen type I [146]. Molecular Therapy for Vasospasm Some gene therapy approaches for vasospasm have been mentioned above where appropriate and the subject has been reviewed by other authors in this volume [62]. As suggested by the plethora of pathways involved, many approaches might be applicable to treating vasospasm. Other phenomena such as cerebral ischemia also could be targeted and this is a situation where the time taken for gene expression to be increased or decreased could be less of an issue since vasospasm onset is delayed for days after SAH. Studies using marker genes with adenovirus as a vector demonstrate that widespread transfection of cells in the leptomeninges and the adventitia of large vessels can be achieved after intrathecal injection into the normal subarachnoid space [23]. SAH did not seem to affect gene transfer in vivo [122] in dogs, but SAH in humans might obstruct cerebrospinal fluid circulation more substantially than in these animal models. Injection of adenovirus expressing NOS into the cisterna magna of dogs with SAH did not prevent vasospasm [22, 188]. Whether expression of other genes might work awaits further study.

Biological Therapies for ICH

At present, few biological therapies have been tried for experimental ICH and most therapy is strictly supportive. Some current treatments are of

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unproven efficacy; the author considers it as important to consider that dexamethasone may be neurotoxic and phenytoin also interacts by inducing liver enzymes and may reduce the neuroprotective effects of folic acid [180, 191, 198]. Targets for such therapy might be reducing brain edema by inhibiting some of the pathways discussed above. Identification of apoptosis in cells around experimental ICH raises the possibility of neuroprotective strategies aimed at preventing apoptosis. Because hypertension is an important risk factor for ICH and SAH, treatment of primary hypertension is important, and gene therapy for experimental hypertension has met with some success [156]. These treatments have not been tried clinically, but clearly there would be great advantages to a gene therapy for hypertension that would eliminate the need for daily administration of medications, often at substantial cost, and if properly modulated, with fewer side effects. Some approaches taken have been either to increase expression of genes mediating vasodilation or to decrease genes for vasoconstriction. Overexpression of genes for proteins such as kallikrein, adrenomedullin, atrial natriuretic peptide, and NOS have successfully reduced blood pressure in rodent models using viral and nonviral methods. Reducing vasoconstrictor gene expression utilizes an antisense approach and has led to successful blood pressure reductions in rodent models with the inhibition of angiotensinogen, angiotensin receptors, and adrenergic receptor subtypes. Challenges remain, including maintaining duration of expression and achieving physiological levels of blood pressure that are regulated in a normal manner. Other approaches may target underlying causes of SAH such as aneurysms, using molecular techniques; for example, fibrosis within experimental aneurysms can be increased by the placement of gelatin-containing basic fibroblast growth factor in the aneurysm [67], leading to the obliteration of the aneurysm sac. This or other gene therapies to increase proliferation of fibroblasts and/or arterial wall cells within the aneurysm sac could be used as adjuncts to endovascular treatment of aneurysms. Similar strategies can be envisioned for the treatment of AVM.

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R. Loch Macdonald, MD Professor, Section of Neurosurgery University of Chicago Medical Center 5841 South Maryland Avenue, Chicago, IL 60637 (USA) Tel. ⫹1 773 702 2123, Fax ⫹1 773 702 3518, E-Mail [email protected]

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Advances towards Cerebrovascular Gene Therapy Yoshimasa Watanabe, Donald D. Heistad Departments of Internal Medicine and Pharmacology, University of Iowa College of Medicine, Cardiovascular Center, and Veterans Affairs Medical Center, Iowa City, Iowa, USA

Introduction

More than a decade has passed, since in vivo gene transfer to blood vessels was first reported [1]. At present, results from clinical trials indicate that gene therapy holds promise for vascular diseases, including coronary artery disease and limb ischemia [2–6]. Gene therapy has several potential advantages over pharmacological therapies currently in use. First, disease-specific effects are expected by introduction or manipulation of selected genes. Second, overexpression of therapeutic genes can provide prolonged production of therapeutic molecules. Third, by local gene transfer, therapeutic molecules can be delivered to a target tissue selectively, while minimizing systemic or nonspecific effects. Previously, we suggested several strategies for treatment of cerebrovascular diseases, which might be achieved by gene therapy [7]. These strategies include the prevention of vasospasm after subarachnoid hemorrhage (SAH); stimulation of collateral blood flow to ischemic regions of the brain by angiogenesis; and treatment of atherosclerotic lesions of the extracranial cerebral arteries by inhibiting thrombosis or proliferation of lesions, stabilizing plaques, and preventing restenosis after angioplasty. Recent progress in studies using animal models demonstrates the feasibility of gene therapy for several cerebrovascular diseases, although some obstacles must be solved before clinical use is possible. Here, we summarize progress in gene therapy targeted to cerebral arterial pathophysiology under experimental settings, and discuss future directions towards cerebrovascular gene therapy in humans. Basic strategies for gene therapy may consist of (1) expression of deficient genes or ‘reconstitution’; (2) overexpression of therapeutic genes;

(3) down-regulation of pathogenic gene expression. Currently, these three alternative strategies are achieved by the delivery of genes or nucleotides with therapeutic potency into the target tissue or cells. In general, maintaining long-term effects is the primary limitation of current gene therapy techniques, especially for the treatment of chronic diseases. Nevertheless, a transient therapeutic effect may be suitable for the treatment of several cardiovascular disorders, such as prevention of restenosis after angioplasty and therapeutic angiogenesis for ischemic tissue [8].

Basic Methods for Experimental Cerebrovascular Gene Therapy

Choice of Gene Delivery System Direct delivery of ‘naked’ plasmid DNA is effective in skeletal muscle [9]. However, because of the low efficiency of naked DNA-mediated gene transfer into the vascular wall, viral or nonviral vectors have been used for vascular gene transfer in most studies [10]. Replication-deficient recombinant adenoviruses are widely used for the delivery of genes both in experimental gene transfer to cerebral blood vessels and in some clinical trials of cardiovascular gene therapy [8, 10]. Unlike retroviral vectors, both dividing and nondividing cells are efficiently transfected with adenovirus Ad vectors, which is an advantage of Ad vectors in transfecting quiescent vascular cells [11]. The vectors have a relatively large capacity for cDNA inserts and are easily prepared in high viral titers [11]. Stimulation of immune and inflammatory responses, however, is a serious limitation of Ad vectors. For example, Ad-mediated gene transfer into the lumen of arteries provokes infiltration of T cells and up-regulation of adhesion molecules in the vascular wall, intimal hyperplasia, and increased turnover of endothelial cells, resulting in the early loss of transgene expression [12–14]. To minimize adverse effects of Ad vectors, low titers of the virus could be used if enhanced efficiency of transfection allows adequate expression of a transgene [15]. Concomitant use of cationic polymer or lipids or calcium phosphate with Ad vectors enhances transgene expression in cerebral arteries in vivo and ex vivo [16–18]. Ad vectors have been altered to reduce immune responses in vascular tissue, although they still have substantial immunogenicity [19–21]. A recent technology for constructing a new generation of Ad vectors may help to circumvent this problem [22]. Helper-dependent, or ‘gutless’ Ad vectors, which do not contain most genes encoding viral proteins, are less toxic but maintain efficient production of transgene expression in animals. However, difficulty in purification (i.e., contamination by helper virus, which is required for the

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production of the helper-dependent vector) and great difficulty in large-scale production are major limitations of this vector. Adeno-associated viruses may be an alternative for Ad vectors. Adenoassociated viral vectors have been shown to be useful in transducing vascular cells in the carotid artery with reporter genes in vivo [23–25]. Adeno-associated viruses represent useful vectors with low toxicity, but have limited transgene capacity [22]. Cationic liposomes form a complex with DNA molecules and facilitate uptake of DNA by cells. Nonviral methods have been used for gene transfer to vascular tissue in vivo [10]; however, efficiency of gene transfer is a major limitation of this method [26, 27]. Nevertheless, cationic liposome-mediated gene transfer is attractive because of its potential for delivering large DNA molecules without substantial toxicity [28, 29]. The oligo-deoxyribonucleotide (ODN)-based strategy is an alternative to gene therapy based on gene transfer techniques [30]. This strategy includes techniques utilizing specific interactions of antisense, ribozyme, or ‘decoy’ ODNs with gene expression machinery. Expression of a target gene is inhibited by inactivation of the mRNA (antisense or ribozyme ODNs) or the transcription factor (decoy ODNs). In addition, recent findings in cellular mechanisms of gene silencing have been applied to the development of a new gene targeting technology. Transfection of cells with a small interfering RNA (siRNA), an RNA fragment, specifically recognizes and then degrades a target mRNA [31]. The siRNA technique is available for in vivo use by Ad vector-mediated delivery, though it is not clear whether this method is applicable to gene therapy for vascular diseases [32]. In vivo Gene Transfer to the Cerebral Circulation The first successful in vivo gene transfer to blood vessels was achieved by the intraluminal administration of a retroviral vector containing a reporter gene into segments of the iliofemoral artery of pigs [1]. In rat carotid arteries, efficient transgene expression was produced by this approach using Ad vectors [26, 33]. In rabbit carotid arteries, functional overexpression of nitric oxide synthase (NOS) in endothelium was also demonstrated [34, 35]. To date, the intraluminal approach is the most common technique for in vivo gene transfer to the carotid artery of experimental animals. However, in order to achieve effective transfection of cells in the vessel wall by this approach, transient interruption of blood flow is usually required for 10–30 min, which would produce unacceptable cerebral ischemia in patients. In addition, delivery of genes or nucleotides is limited to a short segment of the vessel, and medial smooth muscle cells are not transfected unless endothelial integrity is disrupted [36, 37].

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Fig. 1. Adenovirus-mediated gene transfer of bacterial ␤-galactosidase (␤Gal) to cerebral arteries. Rat brain was examined histochemically after intracisternal injection of adenovirus encoding ␤Gal. Expression of ␤Gal (blue staining) was seen on the ventral surface of the brain, especially along major cerebral arteries. (Reprinted with permission from [38].)

To circumvent these problems, methods for perivascular delivery of transgenes have been developed. For the intracranial circulation, transfection of vascular and perivascular cells is achieved by the injection of vectors into cerebrospinal fluid (CSF). Ooboshi et al. [38] injected an Ad vector containing cDNA for reporter gene ␤-galactosidase (␤Gal), into the cisterna magna of rats. One and 3 days after the injection of Ad, expression of ␤Gal was observed in adventitial cells of large cerebral arteries and cells in perivascular tissue (fig. 1). The same technique has been applied to Ad-mediated gene transfer in other species including primates [39–42]. In several related studies, ODNs were administered via the cisterna magna and distributed in all layers of intracranial cerebral arteries [43, 44].

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Fig. 2. Perivascular gene transfer to the carotid artery. Ad␤Gal was injected within the sheath of the common carotid artery of atherosclerotic monkeys. Adventitial cells expressing ␤Gal were stained blue. (Reprinted with permission from [45].)

Functional expression of transgene products has been demonstrated by in vivo gene transfer of vasoactive molecules using the perivascular approach for intracranial arteries. After injection of Ad containing cDNA for endothelial NOS (eNOS) into CSF, eNOS is overexpressed in adventitia of the basilar artery of dogs [39]. Overexpressed eNOS in adventitia is functionally linked to nitric oxide-dependent relaxation induced by bradykinin, which is normally endothelium-dependent, in vessel rings denuded with endothelium. Gene transfer of CGRP increases the level of cyclic adenosine monophosphate in the basilar artery and attenuates contraction induced by histamine and serotonin in vitro [42]. For the extracranial carotid artery, injection of vectors into the periarterial sheath is a practical way of perivascular gene delivery [45] (fig. 2). In the rabbit carotid artery, functional overexpression of eNOS can be achieved using this approach [46]. After Ad-mediated gene transfer to the periarterial sheath, neointimal formation, which is commonly seen after intraluminal approach is not induced, and inflammatory responses are confined to the adventitia [47]. As a less invasive method, local gene transfer of a secretory peptide to a remote site may be an alternative strategy. For example, a vector can be injected into skeletal muscle, and a therapeutic transgene product can be locally overexpressed in the skeletal muscle and secreted into the systemic circulation

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[48, 49]. In addition to local gene transfer, intravenous administration of vectors is another possible method. The liver is a major site of transgene overexpression after intravenous gene transfer, and therapeutic gene products such as factor VIII for hemophilia A are secreted into the systemic circulation [50]. Vascular effects of circulating transgene products may be produced by binding to the vessel wall. For example, extracellular superoxide dismutase (ECSOD), an isoform of superoxide dismutase (SOD) that is released into the extracellular space [51], is released from liver into the blood and binds to the vessel wall after intravenous gene transfer [52]. Binding of ECSOD to vessel wall is mediated by the interaction between heparan sulfate proteoglycans in vascular tissue and the heparin-binding domain of the enzyme. Heparin-binding ECSOD, but not a truncated form of the enzyme lacking the heparin-binding domain, reduces arterial pressure and improves endothelial dysfunction after intravenous gene transfer in hypertensive rats [52]. Thus, intravenous gene transfer of secretory proteins can take advantage of endogenous extracellular binding sites to localize effects in blood vessels.

Experimental Gene Therapy for Cerebrovascular Diseases

Cerebral Vasospasm after SAH Vasospasm after SAH may be a good target for gene therapy because of the unique characteristics of the syndrome. Delayed onset of vasospasm allows a period of time for transgene expression or down-regulation of expression of a specific gene, and the transiency of the risk of the syndrome will not require prolonged effects of gene therapy. In addition, a perivascular approach via CSF will allow widespread exposure of cerebral vessels to vectors at the base of the brain, which are covered by subarachnoid hematoma after SAH. Existence of a hematoma at the site of SAH does not attenuate transgene expression in cerebral vessels mediated by an Ad vector [53, 54]. Furthermore, increase in the efficiency of transfection or expression after SAH, by up-regulation of Ad receptor expression or increased activity of the promotor encoded in an Ad vector, may enhance transgene expression in vascular adventitia [54]. Vasospasm after SAH is probably caused by multiple, complex mechanisms [55, 56]. Several strategies, including overexpression of molecules involved in vasorelaxation or cytoprotective mechanisms and down-regulation of putatively pathological genes, have been tested for experimental gene therapy for vasospasm (table 1). Several studies have examined effects of local overexpression of nitric oxide (NO) synthase, because there is substantial evidence that indicates impairment of endothelium-dependent, NO-induced vasorelaxation after SAH [57]. In rings of canine basilar arteries excised after experimental SAH, gene transfer of

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Table 1. Gene therapy for vasospasm after experimental subarachnoid hemorrhage Target genes

Strategy

Delivery method

Treatment protocol

Reduction of spasm

Animals

Refs

eNOS eNOS ppCGRP HO-1 ECSOD ppET-1 MAPK NF-␬B

GT GT GT GT GT AS AS Decoy

Ad Ad Ad Ad Ad ODN ODN ODN

At SAH 24-hr pretreatment 30-min posttreatment At SAH 30-min posttreatment 30-min posttreatment At SAH At SAH

No Yes Yes Yes Yes Yes* Yes Yes

D D Rbt, D R Rbt D R Rbt

58 59 69, 70 75 81 84 88 44

eNOS, endothelial nitric oxide synthase; ppCGRP, preprocalcitonin gene-related peptide; HO-1, hemeoxygenase-1; ECSOD, extracellular superoxide dismutase; ppET-1, preproendothelin-1; MAPK, mitogen-activated protein kinase, NF-␬B, nuclear factor-␬B; GT, gene transfer; AS, antisense; Decoy, transcription factor decoy; Ad, adenovirus; ODN, oligodeoxynucleotide; SAH, subarachnoid hemorrhage; D, dog; Rbt, rabbit; R, rat. *In combination with recombinant tissue plasminogen activator.

eNOS partially restores bradykinin-induced, NO-dependent relaxation [54]. However, gene transfer of eNOS after SAH in dogs failed to protect against the constriction of the basilar artery or decreased cerebral blood flow, despite the elevation of local NOS activity and NO production [58]. Another study reported the reduction of vasospasm by gene transfer of eNOS prior to induction of SAH, although the study design is not clinically relevant [59]. Failure to prevent vasospasm by eNOS gene transfer after SAH may be due in part to inactivation of NO by oxyhemoglobin [58, 60]. In addition, imbalance between the activities of soluble guanylate cyclase and phosphodiesterase, resulting in decreased levels of cyclic guanosine monophosphate (cGMP), may contribute to impaired responses of arterial smooth muscle to NO [61, 62]. Instead of altering NO-cGMP mediated mechanisms, we have employed the vasodilator effects of calcitonin gene-related peptide (CGRP) for the prevention of experimental cerebral vasospasm by gene transfer. In cerebral arteries after experimental SAH, the smooth muscle cell membrane is depolarized, and relaxation in response to ATP-sensitive K⫹ channel (KATP channels) activators is preserved or enhanced [61, 63–65]. CGRP, a neuropeptide, which is contained in cerebral sensory nerves, hyperpolarizes the membrane of arterial smooth muscle cells and relaxes cerebral arteries at least in part via opening of K⫹ channels, including KATP channels [66–68]. We constructed a recombinant Ad containing prepro-CGRP gene, and demonstrated the prevention of vasospasm by intracisternal gene transfer of CGRP after experimental SAH in rabbits [69] (fig. 3).

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a

c

b

⌬Diameter (%)

0

e

d

Control

AdCGRP

⫺10

⫺20

⫺30

Fig. 3. Effects of gene transfer of preproCGRP on vasospasm following experimental subarachnoid hemorrhage (SAH) in rabbits. Adenovirus-encoding preproCGRP (AdCGRP), control virus (Ad␤Gal), or vehicle was injected into the cisterna magna 30 min after intracisternal injection of autologous blood. a–d Angiograms of cerebral arteries of rabbits treated with Ad␤Gal (a and b) or AdCGRP (c and d). Vertebral arteriography was performed before (a and c), and 2 days after (b and d) SAH. e The percentage changes in the diameter (⌬diameter) of the basilar artery 2 days after SAH. Values are mean ⫾ SEM.

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This was, to our knowledge, the first successful transfer of vasoactive genes in vivo for the prevention of vasospasm after SAH. Vasospasm is also reduced by the same treatment in dogs after SAH [70]. Oxidative stress in cerebral arteries has been implicated in the pathogenesis of vasospasm after SAH, and several compounds that scavenge reactive oxygen species are under clinical trials [71]. Heme oxygenase-1 (HO-1), an inducible isoform of key enzymes for the catabolism of heme, appears to have an important role in cellular protection against oxidative stress [72]. Transient expression of HO-1 in cerebral arteries occurs at an early stage after experimental SAH [73, 74], and suppression of induction of HO-1 by an antisense ODN for HO-1 exacerbates vasospasm after experimental SAH [73]. Vasospasm and reduction of cerebral blood flow induced by hemoglobin was prevented, and vasospasm after experimental SAH was ameliorated after intracisternal gene transfer of HO-1 in rats [75]. As a defense mechanism against oxidative stress, we have examined the effect of local overexpression of ECSOD on experimental vasospasm. We speculated that increased extracellular superoxide (O2⭈⫺) plays a role in the pathophysiology of vasospasm. Several previous studies in experimental animals suggested the pathophysiological importance of superoxide O2⭈⫺ in cerebral vasospasm after SAH [76–79]. We found that intracisternal gene transfer of ECSOD increased local expression and activity of ECSOD after SAH in rabbits and reduced vasospasm [80, 81]. Interestingly, gene transfer of a variant ECSOD lacking tissue-binding ability produced high levels of ECSOD in CSF, but had no effect on vasospasm, suggesting that binding of ECSOD to the vessel wall is important for the reduction of vasospasm [81]. In addition to overexpression of therapeutic genes, ODN-based targeting of genes putatively involved in pathophysiology of vasospasm has been tested in several studies. The target genes of this strategy include preproendothelin-1 (ppET-1), mitogen-activated protein kinase (MAPK), and nuclear factor-␬B (NF-␬B). Evidence from several studies implicates endothelin-1 (ET-1) in the pathophysiology of cerebral vasospasm after SAH [55, 57, 82, 83]. Pretreatment with an intracisternal injection of an antisense ODN against ppET-1, a precursor of ET-1, inhibits expression of mRNA of ppET-1 and attenuates hemolysate-induced cerebral vasospasm in rats [43]. In dogs with SAH, however, antisense ODN against ppET-1 was found to have little effect, perhaps because a physical barrier of subarachnoid clot prevented ODNs from entering into the basilar artery [84]. MAPK, a key molecule involved in the ras p21 signaling pathway in cells, regulates the contraction of smooth muscle in some vascular tissues and may be involved in abnormal contraction of cerebral arteries after SAH [85–87]. In rats, intracisternal injection of an antisense ODN against MAPK inhibits MAPK expression in the basilar artery and reduces vasospasm after SAH [88].

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Increased expression of inflammatory cytokines and cell adhesion molecules in cerebral arteries may be involved in the pathophysiology of vasospasm after SAH [89–91]. Thus, NF-␬B, a key transcription factor in the regulation of expression of inflammatory cytokines and cell adhesion molecules [92], is a possible target for the treatment of vasospasm. NF-␬B is activated in the basilar artery of rabbits after SAH, and pretreatment with ‘decoy’ ODN for NF-␬B reduces vasospasm [44]. Carotid Artery Stenosis Restenosis of arteries is a major problem after coronary angioplasty and stenting and is a potential target for gene therapy [93]. Carotid endarterectomy and angioplasty with stenting are now in wide use for the treatment of stenotic lesions of carotid arteries. Carotid restenosis is also a target for gene therapy, although the incidence of restenosis in the carotid artery may not be as high as that in coronary and iliofemoral arteries [94–96]. There are reports of gene therapy for restenosis after balloon injury of the carotid artery in small animals (table 2). Inhibition of smooth muscle proliferation by blocking progression of the cell cycle has been the most common approach to experimental gene therapy for restenosis of the carotid artery. The first demonstration of an inhibitory effect of antisense ODN examined the role of a proto-oncogene c-myb in neointima formation in balloon-injured carotid artery of rats [97], but results were not supported by later studies [98, 99]. Local delivery of antisense ODN against c-myc, another proto-oncogene, appears to be effective after carotid artery balloon injury in rats [98, 100], and was tested in a clinical trial for the prevention of restenosis after coronary angioplasty and stenting [101]. Antisense ODN-based gene targeting of essential components of the cell cycle, including cyclin-dependent kinases (cdc2 and cdk2 kinases), proliferating-cell nuclear antigen, and cyclins B and G1, has been shown to reduce the formation of neointima in rat carotid artery [102–107]. Neointimal formation is inhibited also by transfecting smooth muscle cells in injured vessel with cell cycle regulatory genes. This strategy includes overexpression of cyclin-dependent kinase inhibitors (p21CIP1, p27KIP1 and a fusion protein of p27KIP1 and p16INK4b), wild-type p53 (an activator of p21CIP), cell cycle-associated transcription factors (GAX and GATA-6), and retinoblastoma proteins, which inhibit a key transcription factor E2F [108–119]. Gene transfer of a fusion protein that consists of fragments of Rb protein and E2F induces cell-cycle arrest in smooth muscle cells and reduces the neointimal development after balloon injury [120]. Decoy administration of E2F is an unique strategy to inhibit smooth muscle proliferation in injured carotid arteries of rats [121]. This technique is applied to the prevention of vein-graft occlusion after coronary bypass surgery in ongoing clinical trials [5, 122].

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Table 2. Gene therapy for carotid neointimal formation after arterial injury Target genes

Strategy

Delivery method

Approach

Animals

Refs

c-myb, c-myc Cdc2 kinase, Cdk2 kinase PCNA Cyclin B1, Cyclin G1

AS AS AS AS

PV, IV/US PV, IL PV IL

R, P R R R

97–99, 100, 177 102–105 102, 106 105, 107

p21CIP1, p27KIP1, p16INK4b p53 GAX, GATA-6 Rb proteins E2F HSVtk (w/ganciclovir i.v.) Fas-ligand Bcl-xL Hemeoxygenase-1 bFGF PDGFR (␤-chain) PDGFR (extracellular domain) ␤-ARK (G␤␥-binding domain) H-ras, MAPKK Activator protein-1 BMP-2 Fortilin Hirudin, TFPI TIMP-1, TIMP-2 TGF-␤1 MCP-1 NF-␬B (p65) NOS isoforms Protein kinase G (catalytic domain) CNP Tissue kallikrein PGI2 synthase

GT GT GT GT Decoy GT GT AS GT GT (DN) AS GT

ODN ODN, HVJ-L ODN HVJ-L, Retrovirus Ad Pl, Ad, HVJ-L Ad Ad HVJ-L Ad Ad ODN Ad Ad ODN Ad

IL IL, IV/US IL IL IL IL IL IL IL IL PV IL

R, Rbt R, Rbt R R R R R Rbt R R R R

108–112 113, 114, 178 115, 116 117–120 121 123 110, 124 125 126 127 128 129

GT

Ad

IL

R

130

GT (DN) Decoy GT GT GT GT AS GT (DN) AS GT GT

Pl, Ad ODN Ad Ad Ad Ad Pl Pl ODN Ad, HVJ-L Ad

PV, IL IL IL IL IL IL PV Intramuscular PV IL IL

R Rbt R R R, Rbt R Rbt R, Rbt, M R R, Rbt R

131–133 135 136 137 138, 139 141–143 144 48, 147 148 152–154, 159 155

GT GT GT

Ad Ad Pl, HVJ-L

IL IL IL

R, Rbt R R

156, 157 158, 159 161–163

PCNA, proliferating cell nuclear antigen; HSVtk, herpes simplex virus thymidine kinase; bFGF, basic fibroblast growth factor; PDGFR, platelet-derived growth factor receptor; ␤-ARK, ␤-adrenergic receptor kinase; MAPKK, mitogen-activated protein kinase kinase; BMP, bone morphogenetic protein; TFPI, tissue factor pathway inhibitor; TIMP, tissue inhibitor of matrix metalloproteinase; TGF, transforming growth factor; MCP, monocyte chemoattractant protein; NF-␬B, nuclear factor-␬B; NOS, nitric oxide synthase; PGI2, prostaglandin I2; AS, antisense; GT, gene transfer; Decoy, transcription factor decoy; DN, dominant negative; ODN, oligodeoxynucleotide; HVJ-L, hemagglutinating virus of Japan-liposome complex; Ad, adenovirus; Pl, plasmid; PV, perivascular; IV/US, ultrasound-supported gene/nucleotide delivery; IL, intraluminal; R, rat; P, pig; Rbt, rabbit; M, monkey.

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Besides the cytostatic strategy achieved by stasis of the cell cycle, another strategy is the induction of cell death in proliferating smooth muscle cells. Gene transfer of herpes simplex virus thymidine kinase followed by systemic administration of ganciclovir, which is a cytotoxic combination for neoplastic cells, is effective in reducing development of neointima [123]. Local overexpression of Fas ligand, an activator of the extrinsic pathway of apoptosis, and down-regulation of Bcl-xL, an anti-apoptotic regulator protein, facilitate apoptosis of neointimal cells and inhibit neointimal formation [110, 124, 125]. In addition, overexpression of Bcl-xL induces regression of established neointimal lesion [125]. Fas-ligand overexpression may be advantageous in the context of Ad-mediated gene transfer, because it inhibits infiltration of Fas-expressing T cells into the arterial wall in response to the Ad vector treatment [110, 124]. After gene transfer of HO-1, increase in the number of apoptotic cells and decrease in the number of proliferative cells were observed in the medial wall of injured carotid arteries, although the mechanisms of these effects are not clear [126]. Mitogenic signal transduction in smooth muscle cells may also be a target for inhibition of restenosis by gene therapy. In balloon-injured carotid arteries, neointimal formation is inhibited by treatment with antisense ODNs for basic fibroblast growth factor, or platelet-derived growth factor (PDGF) receptors [127, 128]. Local gene transfer of the extracellular domain of PDGF receptors, which binds PDGF-B chain and antagonizes the effect of PDGF, also inhibits the formation of neointima [129]. Inhibition of intracellular signaling molecules of mitogenesis also reduces neointimal hyperplasia after injury. This strategy includes overexpression of an inhibitory peptide against the ␤␥ subunit of heterotrimeric G proteins, dominantnegative mutant of ras p21, or MAPK kinase [130–133]. Decoy administration against activator protein-1, a transcription factor activated through a signaling pathway mediated by MAPKs in injured arterial wall [134], inhibits the formation of neointima [135]. Bone morphogenetic protein-2, a member of the transforming growth factor superfamily with bone-inducing activity, is discussed elsewhere in this volume in the context of increasing bony fusion rates in spinal surgery. In the context of cerebrovascular disease, bone morphogenetic protein-2 has growth-inhibitory effects on smooth muscle cells, and limits the development of neointima after local gene transfer to the injured rat carotid artery [136]. Thus bone morphogenetic protein might be useful as a gene transfer strategy to prevent restenosis. A recent study also demonstrated that gene transfer of fortilin, a novel anti-apoptotic factor, inhibited cellular proliferation in the vascular wall and reduced intimal thickening after balloon injury [137]. Overexpression of hirudin or tissue factor pathway inhibitor in balloon-injured carotid artery inhibits the activation of thrombin, a potent mitogen for vascular smooth muscle cells, and reduces neointima formation [138,

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139]. This strategy may be advantageous, because the prevention and reduction of thrombosis is also important for the treatment of arteriosclerotic lesions. Both proliferation and migration of smooth muscle cells are major features of neointima formation after arterial injury [140]. Effects of inhibition of cell migration by tissue inhibitors of metalloproteinase, which inhibit matrix metalloproteinases, have been tested in injured carotid arteries. Matrix metalloproteinases contribute to the digestion of extracellular matrix prior to the migration of smooth muscle cells. Local or systemic gene transfer of tissue inhibitors of metalloproteinase inhibits (or delays) progression of intimal hyperplasia [141–143]. Inhibition of the deposition of extracellular matrix may also be effective in reduction of neointimal formation after balloon injury. Antisense ODN against transforming growth factor-␤1 inhibits the production of proteoglycans in injured vascular wall and reduces the development of neointima in the rabbit carotid artery [144]. Recent studies suggest that inflammatory responses and oxidative stress in the vascular wall contribute to neointima formation after arterial injury [145, 146]. Thus, suppression of inflammation and oxidative stress are possible strategies for the treatment of restenosis. Gene transfer of a dominant negative mutant of monocyte chemoattractant protein-1 or antisense ODN-mediated down-regulation of transcription factor NF-␬B suppresses inflammatory changes in the vascular wall and reduces intimal hyperplasia after arterial injury [48, 147, 148]. Enhancement of cellular protection against oxidative stress by gene transfer of ECSOD inhibits neointima formation after balloon injury of the rabbit aorta [149]. Gene transfer of NOS isoforms is a diverse strategy for anti-restenotic therapy, because NO has anti-proliferative as well as anti-inflammatory and anti-thrombotic properties, and is diffusible after release from its site of production [150]. For example, von der Leyen et al. [151] demonstrated the inhibition of neointima formation in balloon-injured rat carotid artery after gene transfer of eNOS. Local overexpression of constitutive NOS isoforms inhibits the proliferation of smooth muscle cells in balloon-injured carotid artery of rats, and inflammatory response in the carotid artery of hyperlipidemic rabbits [152, 153]. Reduced neointimal formation has been shown in rat carotid balloon injury model after gene transfer of inducible NOS, at a relatively lower titer of the Ad vector than after eNOS gene transfer [154]. In accordance with these studies, overexpression of a constitutively active mutant of protein kinase G, an intracellular effector of the NO-cGMP system, is also effective [155]. Locally overexpressed C-type natriuretic peptide, a secreted peptide activator of particulate guanylate cyclase, reduces thrombus formation, inflammatory response, and intimal thickening in injured carotid arteries [156, 157]. Interestingly, these effects of C-type natriuretic peptide are likely to depend on increased NO production through enhanced induction of NOS in the vessel wall after injury [157].

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Gene transfer of human tissue kallikrein, which converts kininogen into vasoactive kinin, inhibits neointima formation probably through activating NO-cGMP system [158–160]. Increased tissue levels of cyclic adenosine monophosphate may also contribute to this anti-proliferative effect of overexpressed kallikrein [158, 160]. Increased production of prostacyclin (PGI2), which increases cellular cyclic adenosine monophosphate level in injured carotid artery after gene transfer of PGI2 synthase results in reduced intimal hyperplasia [161–163]. Because endothelium is important for maintaining physiological homeostasis of the arterial wall by mechanisms including the production of NO and PGI2, facilitation of re-endothelialization is a reasonable strategy for the treatment of injured arteries. Gene transfer of vascular endothelial growth factor (VEGF), a stimulator of endothelial proliferation, accelerates re-endothelialization, suppresses intimal hyperplasia, and reduces the frequency of thrombotic occlusion after balloon injury of the femoral artery of rabbits [164]. VEGF gene transfer has been tested for the prevention of coronary restenosis in patients undergoing angioplasty [4]. Local overexpression of PGI2 has also been reported to accelerate endothelial regeneration after balloon injury [161]. Ischemia Gene transfer of angiogenic growth factors is a promising strategy for the development of collateral circulation in ischemic tissue. Therapeutic angiogenesis by gene transfer of angiogenic growth factors, including VEGF, hepatocyte growth factor, and basic fibroblast growth factor, has been tried for the treatment of myocardial and limb ischemia in clinical trials [2, 3, 6, 30]. In recent studies using a cerebral hypoperfusion model in rats, intrathecal gene transfer of hepatocyte growth factor or VEGF stimulated angiogenesis in the brain and increased the cerebral blood flow [165]. Gene transfer of basic fibroblast growth factor is also effective in inducing neovascularization in the brain of normal rats [166]. Thus, this strategy may be applicable to the development of collateral vessels in the brain for the prevention and treatment of cerebral ischemia. Other gene transfer strategies may be useful for treating the effects of ischemic or repairing ischemia-damaged tissue, but these fall outside the scope of this chapter.

Current Problems and Future Directions

Cerebral Vasospasm Prevention of vasospasm after SAH is a promising target for the clinical use of gene therapy techniques. Most studies, however, have been performed in SAH models of small animals. Because the severity and temporal profile of vasospasm in small animals are different from those in humans, findings must

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be confirmed in larger animals, such as dogs and primates. Mechanisms of development, maintenance, and resolution of cerebral vasospasm are not fully understood, so there is no clear target for the treatment of vasospasm. Progress in basic research addressing pathogenesis of vasospasm is important for the establishment of effective gene therapy. One of the major obstacles to the use of Ad-mediated gene transfer for treatment of vasospasm after SAH is that Ad vectors induce inflammatory responses in cerebral arteries [167]. Inflammation is also an important pathophysiological change in cerebral vessels after SAH [90, 168, 169]. Another limitation of Ad gene transfer is that transgene expression is achieved only in adventitial cells of cerebral arteries by the perivascular approach [38, 40, 53]. Thus, it is difficult to alter gene expression of smooth muscle cells and endothelial cells, where important pathology occurs. ODNs produce little inflammation and diffuse deep into all vessel layers [43, 44]. However, uptake of ODNs by cells in the vessel wall may be insufficient to prevent vasospasm because subarachnoid blood can prevent ODNs from reaching the arterial wall [84]. Efficacy of ODNs in prevention of vasospasm, as summarized earlier, is unclear. Carotid Stenosis Before the clinical use of gene therapy for restenosis of the carotid artery, a better delivery system for vectors or plasmids must be developed. Methods used in most studies are not relevant to clinical settings, because vectors or plasmids are placed in the lumen of the carotid artery with interruption of blood flow for more than 10 min. Several studies suggest the feasibility of a perivascular approach for prevention of neointimal formation in injured carotid arteries [97–100, 106, 128, 133, 170]. Gene transfer into skeletal muscle, with systemic release of a secretory peptide, is an alternative approach [48]. Recent developments in catheter- or stent-based vector delivery systems may enable safe and effective intraluminal approaches for the carotid artery [171–175]. In addition, a new method using intravenous ultrasound contrast agents may be useful for intraluminal vascular gene delivery [176]. An ODN that is bound to microbubbles prepared as a contrast agent is effectively transduced into the wall of the extracranial carotid artery within the area of insonation [177, 178]. There is considerable concern, however, that the approach may produce additional endothelial damage. In patients with carotid atherosclerotic lesions, prevention of thrombotic events (both acute occlusion and distal embolism) and development of stenosis is an important target for gene therapy. Established atherosclerotic lesions are stabilized after gene transfer of a dominant negative mutant of monocyte chemotactic protein-1 [49]. Progression of atherosclerotic lesions is also reduced after gene transfer of the mutant monocyte chemotactic protein-1 [49] in the

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aorta of mice with deficiency of apolipoprotein E, which otherwise develop spontaneous atherosclerosis. In an arterial thrombosis model in the femoral artery of rabbits, local gene transfer of tissue plasminogen activator inhibited the formation of thrombus in the vessel [179]. The concepts of these studies may be useful for developing gene therapy for carotid atherosclerosis. Ischemia and Collateral Circulation It is not yet clear whether therapeutic gene transfer for angiogenesis is useful for focal angiogenesis to regions of the brain that are at risk for ischemia. A recent study has demonstrated that neovascularization in the ischemic limb of mice is effectively achieved by intravenous administration of bone marrow-derived endothelial progenitor cells transfected ex vivo with the VEGF gene [180]. This endothelial progenitor cell-mediated gene therapy may be applicable to focal cerebral ischemia, because endothelial progenitor cells incorporate into the vessels in the ischemic area of the brain after occlusion of middle cerebral artery in mice [181]. Several issues have been raised regarding the safety of therapeutic gene transfer for angiogenesis after accumulation of experience from clinical trials [8]. For example, it is speculated that vascular malformations could develop at the site of neovascularization and predispose to brain hemorrhage.

Conclusion

In general, safety is the paramount issue and obstacle for the establishment of cerebrovascular gene therapy in the clinic. Development of safe and efficient vectors for gene/nucleotide delivery in humans is urgently required. In addition, tissue-specific targeting of vectors or transgene expression will be useful for reducing the toxicity of the vectors or transgene products [120, 182, 183]. Data will also be needed regarding pharmacokinetics and toxicity of vectors and therapeutic genes or nucleotides [184]. It is not likely that gene therapy will be clinically useful for cerebrovascular diseases in the near future, but we are optimistic that the improvement of methods for gene therapy will ultimately result in its clinical use.

Acknowledgments We thank Drs. Yi Chu, Frank Faraci, and Carol Gunnett for their critical evaluation of this manuscript. Original studies by authors were supported by funds from the Veterans Administrations, NIH Grants HL16066, HL62984, NS24621, HL14388, DK54759, the Carver Research Program of Excellence, the Wendy Hamilton Trust, and an award from the American Heart Association 0120641Z.

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159 Murakami H, Miao RQ, Chao L, Chao J: Adenovirus-mediated kallikrein gene transfer inhibits neointima formation via production of nitric oxide in rat artery. Immunopharmacology 1999;44: 137–143. 160 Emanueli C, Salis MB, Chao J, Chao L, Agata J, Lin K-F, Munaò A, Straino S, Minasi A, Capogrossi MC, Madeddu P: Adenovirus-mediated human tissue kallikrein gene delivery inhibits neointima formation induced by interruption of blood flow in mice. Arterioscler Thromb Vasc Biol 2000;20:1459–1466. 161 Numaguchi Y, Naruse K, Harada M, Osanai H, Mokuno S, Murase K, Matsui H, Toki Y, Ito T, Okumura K, Hayakawa T: Prostacyclin synthase gene transfer accelerates reendothelialization and inhibits neointimal formation in rat carotid arteries after balloon injury. Arterioscler Thromb Vasc Biol 1999;19:727–733. 162 Todaka T, Yokoyama C, Yanamoto H, Hashimoto N, Nagata I, Tsukahara T, Hara S, Hatae T, Morishita R, Aoki M, Ogihara T, Kaneda Y, Tanabe T: Gene transfer of human prostacyclin synthase prevents neointimal formation after carotid balloon injury in rats. Stroke 1999;30: 419–426. 163 Yamada M, Numaguchi Y, Okumura K, Harada M, Naruse K, Matsui H, Ito T, Hayakawa T: Prostacyclin synthase gene transfer modulates cyclooxygenase-2-derived prostanoid synthesis and inhibits neointimal formation in rat balloon-injured arteries. Arterioscler Thromb Vasc Biol 2002; 22:256–262. 164 Asahara T, Chen D, Tsurumi Y, Kearney M, Rossow S, Passeri J, Symes JF, Isner JM: Accelerated restitution of endothelial integrity and endothelium-dependent function after phVEGF165 gene transfer. Circulation 1996;94:3291–3302. 165 Yoshimura S, Morishita R, Hayashi K, Kokuzawa J, Aoki M, Matsumoto K, Nakamura T, Ogihara T, Sakai N, Kaneda Y: Gene transfer of hepatocyte growth factor to subarachnoid space in cerebral hypoperfusion model. Hypertension 2002;39:1028–1034. 166 Yukawa H, Takahashi JC, Miyatake S-I, Saiki M, Matsuoka N, Akimoto M, Yanamoto H, Nagata I, Kikuchi H, Hashimoto N: Adenoviral gene transfer of basic fibroblast growth factor promotes angiogenesis in rat brain. Gene Ther 2000;7:942–949. 167 Lüders JC, Weihl CC, Lin G, Ghadge G, Stoodley M, Roos RP, Macdonald RL: Adenoviral gene transfer of nitric oxide synthase increases cerebral blood flow in rats. Neurosurgery 2000;47: 1206–1215. 168 Peterson JW, Kwun BD, Hackett JD, Zervas NT: The role of inflammation in experimental cerebral vasospasm. J Neurosurg 1990;72:767–774. 169 Handa Y, Kabuto M, Kobayashi H, Kawano H, Takeuchi H, Hayashi M: The correlation between immunological reaction in the arterial wall and the time course of the development of cerebral vasospasm in a primate model. Neurosurgery 1991;28:542–549. 170 Laitinen M, Zachary I, Breier G, Pakkanen T, Hakkinen T, Luoma J, Abedi H, Risau W, Soma M, Laakso M, Martin JF, Yla-Herttuala S: VEGF gene transfer reduces intimal thickening via increased production of nitric oxide in carotid arteries. Hum Gene Ther 1997;8:1737–1744. 171 Varenne O, Pislaru S, Gillijns H, Van Pelt N, Gerard RD, Zoldhelyi P, Van de Werf F, Collen D, Janssens P: Local adenovirus-mediated transfer of human endothelial nitric oxide synthase reduces luminal narrowing after coronary angioplasty in pigs. Circulation 1998;98:919–926. 172 Marshall DJ, Palasis M, Lepore JJ, Leiden JM: Biocompatibility of cardiovascular gene delivery catheters with adenovirus vectors: An important determinant of the efficiency of cardiovascular gene transfer. Mol Ther 2000;1:423–429. 173 Nakamura T, Morishita R, Asai T, Tsuboniwa N, Aoki M, Sakonjo H, Yamasaki K, Hashiya N, Kaneda Y, Ogihara T: Molecular strategy using cis-element ‘decoy’ of E2F binding site inhibits neointimal formation in porcine balloon-injured coronary artery model. Gene Ther 2002;9: 488–494. 174 Ye Y-W, Landau C, Willard JE, Rajasubramanian G, Moskowitz A, Aziz S, Meidell RS, Eberhart RC: Bioresorbable microporous stents deliver recombinant adenovirus gene transfer vectors to the arterial wall. Ann Biomed Eng 1998;26:398–408. 175 Klugherz BD, Song C, Defelice S, Cui X, Lu Z, Connolly J, Hinson JT, Wilensky RL, Levy RJ: Gene delivery to pig coronary arteries from stents carrying antibody-tethered adenovirus. Hum Gene Ther 2002;13:443–454.

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176 Porter TR, Xie F: Therapeutic ultrasound for gene delivery. Echocardiography 2001;18:349–353. 177 Porter TR, Hiser WL, Kricsfeld D, Deligonul U, Xie F, Iversen P, Radio S: Inhibition of carotid artery neointimal formation with intravenous microbubbles. Ultrasound Med Biol 2001;27: 259–265. 178 Taniyama Y, Tachibana K, Hiraoka K, Namba T, Yamasaki K, Hashiya N, Aoki M, Ogihara T, Yasufumi K, Morishita R: Local delivery of plasmid DNA into carotid artery using ultrasound. Circulation 2002;105:1233–1239. 179 Waugh JM, Kattash M, Li J, Yuksel E, Kuo MD, Lussier KM, Weinfeld AB, Saxena R, Rabinovsky ED, Thung S, Woo SLC, Shenaq SM: Gene therapy to promote thromboresistance: Local overexpression of tissue plasminogen activator to prevent arterial thrombosis in an in vivo rabbit model. Proc Natl Acad Sci USA 1999;96:1065–1070. 180 Iwaguro H, Yamaguchi J, Kalka C, Murasawa S, Masuda H, Hayashi S, Silver M, Li T, Isner JM, Asahara T: Endothelial progenitor cell vascular endothelial growth factor gene transfer for vascular regeneration. Circulation 2002;105:732–738. 181 Hess DC, Hill WD, Martin-Studdard A, Carroll J, Brailer J, Carothers J: Bone marrow as a source of endothelial cells and NeuN-expressing cells after stroke. Stroke 2002;33:1362–1368. 182 Peng K-W, Russell SJ: Viral vector targeting. Curr Opin Biotechnol 1999;10:454–457. 183 Wickham TJ: Targeting adenovirus. Gene Ther 2000;7:110–114. 184 Pislaru S, Janssens SP, Gersh BJ, Simari RD: Defining gene transfer before expecting gene therapy. Putting the horse before the cart. Circulation 2002;106:631–636.

Donald D. Heistad, MD Department of Internal Medicine, University of Iowa College of Medicine 200 Hawkins Dr., Iowa City, IA 52242 (USA) Tel. ⫹1 319 356 2706, Fax ⫹1 319 353 6343, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 439–457

Ex vivo Gene Therapy and Cell Therapy for Stroke Douglas Kondziolka a, Jason Sheehanb, Ajay Niranjana a

Department of Neurological Surgery, University of Pittsburgh, Pittsburgh, Pa., Department of Neurological Surgery, University of Virginia, Charlottesville, Va., USA b

Introduction

The field of neural transplantation for treatment of neurological diseases began decades ago when Bjorklund and Stenevi [1] demonstrated that transplantation of dopamine-secreting neurons into the rat striatum improved functionality in previously damaged nigrostriatal pathways. These results were later expanded on by other groups [2]. This initial work in cell transplantation spawned additional research to understand the pathophysiology of neurodegenerative diseases such as Parkinson’s (PD) and Huntington’s (HD) diseases as well as injury-related neurological disorders such as ischemia or trauma. Preclinical and clinical studies have demonstrated proof-of-principle for restorative neurosurgical procedures that replace missing neurotransmitters and rebuild damaged neuronal circuitry. Recent advances in molecular biology, cell engineering, and stem cell technology may afford even more novel approaches for neural transplantation. Cell and Tissue Transplantation in the Brain

Schmidt et al. [3] first proposed the concept of using dissociated tissue from the central nervous system for intraparenchymal implantation. In a series of reports, they demonstrated that cellular suspensions of mesencephalic, dopaminergic cells could be effectively implanted into rat parenchyma, survive, and promote regeneration [4–8]. PD, which is a complex neurological disorder characterized in part by degeneration of dopaminergic neurons in the substantia nigra par compacta, became the first widely applied model for

neural transplantation. The successes and failures of neural transplantation with regard to PD have yielded valuable therapeutic strategies for other neurological diseases. For instance, neurotransplantation research has been extended to other neurodegenerative diseases such as HD and to spinal cord regeneration and posthypoxic injury. All of these neurological conditions share common challenges related to neurotransplantation: (1) selection of an appropriate and readily available source of tissue; (2) refinement of tissue preparation protocols; (3) development of safe and effective techniques for surgical implantation of cells and tissue, and (4) creation of a suitable environment in vivo, which fosters survival of transplanted neuronal tissue and restoration of damaged neuronal connections.

Tissue Sources and Preparation for Neuronal Implantation

Finding a suitable source for neuronal implants poses both scientific and ethical challenges. Prior to 1988, researchers were free to conduct federally financed research using fetal tissue sources. However, research on human fetal tissue transplants using material derived from elective abortions was banned from National Institutes of Health (NIH) funding during the administration of President Ronald Reagan due to the pressure from conservative religious groups, which influenced scientific policy. Despite the ban on federal funding in human subjects, research continued in animal models, and private funding also ensured that transplantations continued. In 1993, with groups from around the world reporting promising results with fetal cell implantation and a change to a Democratic administration, which lifted the NIH funding ban, fetal neurotransplantation research in human subjects was allowed to proceed in the USA. Much has been written about ethical issues surrounding fetal tissue research; the facts remain that fetal tissue research has a long track record of success and is legal [9–13]. One major problem of donated tissue is that it is often not suitable for in vivo use. In a recent examination of approximately 1,500 embryonic donors in five NIH-funded tissue banks, only seven samples were believed to be suitable for transplantation in PD patients [14]. Other studies have indicated that multiple donors or first-trimester embryonic tissues are not absolute criteria for successful human grafting [15–17]. The cryopreservation of tissue is believed to be less favorable for long-term neuronal survival and maturation [18]. Fetal cell aggregates in long-term suspension cultures are another viable tissue culturing protocol that has been explored. Logistically and economically, cryopreserved cell lines are the most favorable approach and have been shown to be effective in a stroke transplantation trial at the University of Pittsburgh [19, 20]. Despite the limited number of suitable embryonic donor tissue in NIH-funded banks,

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a degree of optimism is warranted with the stem cell and neuronal precursor line research that may soon replace primary human brain tissue as the major sources of donor cells, unless further legislation impairs access to that material. Prior to implantation of cells, in vitro processing is beneficial. Cell culturing techniques permit the selection of a viable and well characterized subset of neuronal cells for implantation. Glial-neuronal interactions and neurotrophin exposure in vitro can help to protect grafted cells from detrimental host response and promote long-term survival in vivo [21, 22]. Some grafted cells have been shown to undergo apoptosis or programmed cell death mediated by caspase activity within the first 15 days postimplantation [23, 24]. Therefore, in vitro inhibition of caspase activity prior to implantation or other ‘conditioning’ could lead to decreased apoptosis and, in turn, improved overall viability of grafts [25].

Immunosuppression and Overall Success of the Graft

The need for either short- or long-term immunosuppression has been the subject of much debate in the neural transplantation field. The necessity and duration of immunosuppression are generally a function of the degree of graftversus-host response induced by the implanted tissue. Most agree that xenografts require long-term immunosuppression. However, in 1991, Henderson et al. [18] demonstrated that long-term clinical benefits were afforded in 12 advanced PD patients who underwent graft implantation despite the absence of immunosuppression. Although human and primate studies of allogeneic transplants do not induce immunity, definitive rejection has not been demonstrated [26–30]. Others argue that immunosuppression may affect symptoms associated with PD and other neurological conditions [30]. The annual cost (approximately USD 10,000) and 1–10% per year complication risk associated with immunosuppression must be factored into the equation [31]. Despite the cost, risks, and equivocal research results, most researchers still favor a short-term course of immunosuppression with agents such as cyclosporine (5–10 mg/kg) and corticosteroids [32]. Little is known about the utility of other immunosuppression agents such as tacrolimus for brain transplantation [33]. The reaction of the host’s cells to the graft is critical in terms of the graft’s survival, integration, and functionality. Research in murine transplant models has suggested that a portion of the functional benefit of fetal transplantation may be due to the host’s reaction alone or the surgical procedure itself. Release of neurotrophic factors by the host parenchyma may enhance the surrounding neuropil. Host sprouting adjacent to a dopaminergic graft has been demonstrated to be quite substantial [34, 35]. Bankiewicz et al. [36] have demonstrated clinical improvement in hemiparkinsonian monkeys after a surgical procedure that

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resulted in cavitation alone. As such, the regenerative capacity of the host parenchyma may play a considerable role in the degree of functional recovery achieved following neuronal grafting. The microglial response also plays a role in the overall success or failure of the graft. Microglia contribute to the immune-mediated response of the host by functioning as antigen-presenting cells [37]. The role of microglia is important not just following the brain injury or development of intracranial pathology but also for the functioning of the brain under normal conditions [38]. Microglia have been shown to produce growth factors and trigger a regenerative cascade following central nervous system injury [39–43]. In fact, through the secretion of neurotrophins, microglia can promote the survival and development of TH-positive neurons in vitro and nigrostriatal dopaminergic neurons in vivo [44–46]. Thus, the host’s neuroglial response plays a pivotal role in the graft’s survival, differentiation, and function.

Cerebral Infarction as a Model for Cell Transplantation

Stroke is the third leading cause of death and the most common cause of lasting disability in the USA [47]. The incidence of strokes in the USA is approximately 750,000 cases per year [47]. One third of the strokes are fatal and the majority of survivors demonstrate significant residual impairments [48, 49]. As the population shifts to an older median age and people live longer as a result of improved therapies for other diseases, the prevalence of stroke-related morbidity and mortality will increase. Recent research and clinical efforts have focused on developing and refining treatments for acute stroke with a typically narrow peri-ischemic window for the administration of therapeutic agents. At present, no effective treatment for chronic stroke patients with fixed neurological deficits is available. Transplantation of neural cells into the stroke penumbra has been evaluated by a number of investigators as an approach for ameliorating functional deficits [50]. The two most common models of brain ischemia are the murine hippocampal stroke model, which produces well-defined lesions particularly in the CA1 region, and the middle cerebral artery occlusion model in the rat [51–55]. A number of reports using both models have demonstrated survival and integration of cortical transplants within infarcted regions [52, 56–59]. Moreover, stroke models have shown improved function following neuronal transplantation [60–66]. Treatment of human cortical stroke by transplant has been discouraging because of the heterogeneous make-up and the large volume of neuronal tissue affected [67]. However, as a result of the successes with transplantation for

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neurodegenerative diseases (e.g., PD and HD), some consensus exists that transplantation has potential for treatment of striatal stroke [67].

Use of a Human Neuronal Cell Line for Neurotransplantation

The logistical difficulties inherent in obtaining large quantities of human fetal neurons stimulated a search for alternative sources of tissue for transplantation. One such source of tissue is the human teratocarcinoma-initiated neuronal cell line (NT2). Andrews et al. [68] initially established this nontumorigenic, embryonic cell line from a human teratocarcinoma. Treatment of NT2 cells with retinoic acid causes differentiation into a mature neuronal phenotype, similar in morphology and behavior to terminally differentiated, postmitotic neurons [69, 70]. Following transplantation, NT2 cells demonstrate survival, formation of synaptic processes, expression of neurotransmitters, and integration into the host’s parenchyma [70–72]. In particular, the cells express three neurofilament proteins (NFL, NFM, and NFH); microtubule-associated protein 2; glial-derived neurotrophic factor (GDNF); and possibly the axonal protein tau [Borlongan, pers. commun.]. As a result of the neuronal phenotype and virtually unlimited supply, NT2 cells are a useful source for neurotransplantation in animal models. NT2 cells have the following advantages: (1) they do not harbor known pathogens or infectious agents potentially present in allografts or xenografts; (2) they have been extensively characterized in vitro; (3) they are amenable to genetic engineering, and (4) they have been utilized in animal models for PD and trauma [73–76]. The notion of restoring function postischemic injury by transplanting human neuronal cells has attracted a number of proponents [67]. There is evidence for the feasibility of this approach; for example, fetal tissue transplantation in a rat model of transient focal cerebral ischemia demonstrated a restoration of behavioral and motor functions [77]. Borlongan et al. [50, 67] reported that transplantation of neuronally differentiated NT2 cells could ameliorate the deficits following stroke. These preclinical studies were conducted in a transient, focal ischemic model rather than a global one, so as to maximize the chances of functional recovery. Animals treated with NT2 neurons and cyclosporine demonstrated improvement in behavioral deficits during a 6-month window of observation and evaluation. In particular, animals treated with NT2 neurons exhibited recovery in the passive avoidance test (i.e., behavioral assessment) and elevated body swing test (i.e., motor assessment) [78, 79]. Control groups receiving fetal rat cerebellar cells, cell culture medium alone, or cyclosporine, failed to demonstrate significant behavioral improvement. A similarly favorable behavioral result following NT2 neuronal transplantation was seen in a middle cerebral artery occlusion (MCAO) rat model [80].

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In view of these results, the initial objectives in a 12-patient Phase I clinical trial performed at the University of Pittsburgh were to demonstrate the safety of neuronal-cell transplantation in immunosuppressed striatal stroke patients [81, 82]. In a 24-month follow-up period, no adverse events related to the implantation occurred. A postmortem examination of one patient from this study who died of unrelated causes clearly demonstrated NT2 neuron survival 27 months after surgery and the absence of any adverse brain parenchymal effects [20]. The Phase I clinical stroke study also provided some evidence of possible functional recovery from stroke-related neurological deficits following neuronal cell transplantation. The mean National Institutes of Health Stroke Scale score decreased and the mean European Stroke Scale total scores increased, suggesting improvement in transplanted groups. Furthermore, total European Stroke Scale scores and composite motor subscale scores of the European Stroke Scale increased to a larger extent in the 4 patients who received 6 million cell implantation as compared to those receiving only 2 million cells. The Barthel Index and SF-36 scores decreased in the group receiving 2 million cells and increased in the group receiving 6 million cells. Overall, in the group receiving 6 million cells, outcome measurements demonstrated some improvement in stroke-related deficits [82]. More recently, serial [18F] fluorodeoxyglucose positron emission tomography after human neuronal implantation for stroke has been conducted. Fluorodeoxyglucose positron emission tomography can be utilized to map metabolic brain response at the site of cell implantation. In that same group of 12 patients who underwent implantation of human neuronal cells, fluorodeoxyglucose positron emission tomography imaging was performed 1 week, 6 months, and 12 months postoperatively [19, 82]. Increased glucose metabolic activity in the stereotactic target and surrounding tissue was noted at 6 and 12 month timepoints; this increase correlated with improved motor performance scores [19]. Thus, it would appear that enhanced cellular function at the implantation site occurred in some patients and correlated with improved neurological function. Although the Phase I clinical stroke trial at the University of Pittsburgh demonstrated encouraging preliminary results, many questions remain unanswered and new questions have arisen. Underlying neuronal mechanisms that led to improved neurological function have yet to be established, and could be nonspecifically related to cell grafting. For example, instrumentation of the striatum or use of immunosuppressive drugs may have contributed to the observed effects. Some possible cellular mechanisms for neurological improvement include the following: neurotrophin secretion by grafted cells; increased neurotransmitter production by resident cells; re-establishment of local interneuronal connections in response to grafting; improvement in regional oxygen tension in response to grafting; or reduction in glial reactivity in response to grafting.

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A two-center, Phase 2 (dose response) trial is underway to evaluate the utility of neurotransplantation for patients who have suffered basal ganglia infarcts. Eighteen patients have received either 5 or 10 million cells delivered into twentyfive implants along five trajectories. Four additional patients were assigned to an observational control group. All patients received a focused stroke rehabilitation program. Further studies are planned to examine the role of immunosuppression, patient age, severity of neurological deficit, number of implants and implanted cells, choice of outcome measures, and randomization schemes.

Cellular Support for Graft Survival and Axonal Reconnection

One of the most important factors in neuronal survival and integration of the graft is the creation of a receptive milieu in the host. In 1951, LeviMontalcini [83] first presented her work on growth factors at the New York Academy of Sciences. While working for Viktor Hamburger, Levi-Montalcini had grafted a malignant mouse tumor into a developing embryo. She noted that nerve fibers had emerged not only from an adjacent sensory ganglia but even more from the sympathetic ones. These nerve fibers had extended chaotically and thereby completely invaded both the neoplastic tumor tissue and practically all of the embryo’s organs [84]. The humoral factor secreted by the tumor was eventually characterized as nerve growth factor (NGF) [85]. NGF was the first member of a family of endogenous growth factors. Many of these growth factors or neurotrophins play a role in the central and peripheral nervous system under normal and pathological conditions. Neurotrophins interact with cells through high affinity, tyrosine kinase receptors (trk A, trk B) or lower affinity p75 receptor [86]. The broad extent of neurotrophin functional roles and their complex cell signaling pathways make them a continued focus of research efforts. Depending upon the cell type and conditions, a particular neurotrophin may have either pro- or anti-apoptotic cellular responses [87]. Neurotrophins have been the subject of studies to evaluate their therapeutic role in neurodegenerative diseases [88–90]. Growth factors are crucial for the development and survival of neural progenitor cells in both the central and peripheral nervous system [91]. Even factors that were once thought to be neurotoxic (e.g., tumor necrosis factor) have been shown to have neuroprotective effects under certain circumstances [92]. Neurotrophins have been used as neuroprotectants to slow, halt, or reverse the progression of neuronal degeneration [90]. They also have been utilized to enhance survival of neuronal and non-neuronal grafts [90]. Unfortunately, neurotrophins themselves do not pass the blood-brain barrier in significant amounts. Several novel techniques have been utilized to circumvent this problem and include grafting of cells which

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secrete selective neurotrophins; stereotactic intraparenchymal or intraventricular delivery of neurotrophins; conjugation of neurotrophins to antibodies and delivery via receptor-mediated transport; peripheral administration of bioactive neurotrophin subunits [93–95]. In brain transplantation, neurotrophins have favorably influenced the survival, integration, and neurotransmitter expression of grafted and host cells. Treatment of nigral dopaminergic neurons or fetal cerebral cortex with brain-derived neurotrophic factor (BDNF) was found to improve the functional performance and neuronal graft survival in a rat Parkinsonian model [96]. Interestingly, BDNF produced by dorsal interneurons was found to stimulate proliferation and differentiation of motor neuron precursors after anterograde transport [97]. Neurotrophin-3 enhanced survival of dopaminergic neurons in striatal grafting of fetal mesencephalic cells [98]. Glial cell line-derived neurotrophic factor (GDNF) not only promoted survival, but also the sprouting of fetal transplants in PD models [98–101]. Unfortunately, results of the intraventricular delivery of GDNF in the treatment of PD has been discouraging [102]. NGF-supplemented cellular grafts can prevent degeneration of basal forebrain cholinergic neurons, increase the number of neurons, and stimulate the neuronal maturation [103–105]. The trophic effects of NGF-secreting stem cells prevented degeneration of vulnerable cholinergic striatal neurons in a rodent model of HD [106]. Neurotrophins could also be delivered following a stroke in order to compensate for the death or impairment of ischemic cells. Using a transient middle cerebral artery rat stroke model, Li et al. [107] demonstrated that neurological improvement resulting from human marrow stromal intravenous therapy was most likely derived from the increased production of NGF and BDNF. In a similar animal model of stroke, overexpression of NGF, BDNF, GDNF, and ciliary neurotrophic factor through recombinant adeno-associated viral gene transfer was found to reduce neuronal death and lead to functional sparing [108, 109]. As noted previously, the neuron-like cells utilized in the stroke trial at the University of Pittsburgh secrete GDNF, which may have contributed to the favorable results observed thus far in terms of both graft survival and improved functional outcome. Delivery of neurotrophins themselves appears to be neuroprotective following infarction in other animal models [110–113]. Fibroblast growth factor gene expression was observed to be upregulated within 3 days of a focal cerebral infarction in rats [114]. Moreover, exogenous administration of fibroblast growth factor after infarction reduced infarct size and enhanced functional recovery [114]. Apoptosis or programmed cell death, which plays a major role in neuronal development, may also occur in neurodegenerative diseases such as Alzheimer’s and PD [115, 116]. In animal models of stroke, apoptosis has been implicated in neuronal loss following ischemic injury [117, 118]. Neurotrophins appear to

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attenuate the apoptotic signaling pathways, and may have value in this context. Anti-apoptotic signaling activates transcription factors (e.g., nuclear factor-␬B) and induces expression of stress proteins, antioxidant enzymes, calcium regulatory agents, ion channel modulators, and mitochondrial stabilizing proteins (e.g., Bcl-2) [119]. Neurotrophins may have value in the treatment of neuronal injury or loss from ischemic or degenerative conditions.

Gene Therapy for Ischemia with Genes for Neurotrophic Factors

Gene delivery systems using nonviral and viral vectors have been reported for the treatment of genetic diseases, cancers, and other conditions [120, 129, 130]. Replication-defective herpes simplex virus (HSV) type 1 was among the first vectors to be used in experimental gene transfer into the brain [121–123]. Retroviral vectors also were developed and tested for this purpose [124, 125]. However, many retroviral (nonlentiviral) vectors do not infect nondividing cells such as neurons, and may be associated with risks of mutagenesis and tumorogenesis. [126, 127]. Replication-defective lentivirus can transduce a gene to nondividing cells and affords long-term expression, but retains the potential to cause insertional mutagenesis [128]. To deliver neurotrophic factor protein in ischemic brain, Kitagawa et al. [131] chose an adenovirus vector containing the GDNF gene (Ad-GDNF). The protective effect of the Ad-GDNF transfer was examined after transient MCAO in the rat. That study demonstrated reduction in infarct volume in the animals that were pretreated with Ad-GDNF 24 h before a subsequent 90 min of transient MCAO. The reduction in the infarct size was not associated with changes in regional cerebral blood flow, as compared with the vehicle or Ad-LacZ animal groups. Immunohistological analyses showed that treatment with Ad-GDNF greatly reduced the number of TUNEL, caspase-3, and cytochrome c-positive cells. No leukocyte infiltration was detected in Ad-GDNF-treated rats. Inhibition of the cytosolic release of cytochrome c by Ad-GDNF suggests a target of protection in an apoptotic pathway through cytochrome c and caspase-3 in the penumbra [132]. In another study Casper et al. [133] performed ex vivo transduction of neural cell aggregates with replication defective HSV containing cDNA of human vascular endothelial growth factor (HSVhvegf) and transplanted the cells into the rat brain. They demonstrated a dose-dependent increase in blood vessel density within transplants transduced with HSVvegf. The transplants were vascularized at a faster rate for up to 4 weeks and the size of HSVvegf transplant was twice that of control at 8 weeks. The rate of vascularization may be a major factor in survival of transplanted cells into the brain and can be improved by ex vivo transduction with HSVvegf.

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Several other gene transfer studies for cerebral ischemia have been performed in animal models; for example, studies of the protective effects of adenovirus-mediated neuronal apoptosis inhibitory protein and an interleukin-1 receptor antagonist against ischemic brain injury [134, 135]. HSV-mediated HSP72 or Bcl-2 gene transfer also showed an improvement in focal ischemiainduced brain damage [135–139]. These viral vectors have certain disadvantages such as toxicity and low efficacy of gene expression; development of other vectors is necessary for in vivo gene therapy approaches in patients.

Potential of Neural Stem Cell Transplantation to the Ischemic Brain

Several studies have been performed to transplant neural cells into the ischemic brain. For example, fetal rat hippocampal neurons were stereotaxically transplanted into 5-day-old ischemic hippocampal CA1 lesions of the adult rat [52]. In the recipient brain, the transplanted cells survived well and formed clusters in the host CA1 subfield at 14 or 100 days after the transplantation. Fetal CA1 grafts also promoted recovery from cognitive deficits in marmosets induced by excitotoxic damage to the CA1 field [140]. Li et al. [141] used hematopoietic myeloid progenitor cells (1 ⫻ 105 per ␮l) cultured from adult mouse bone marrow to implant into the penumbra of the ischemic striatum at 4 or 24 h after 3 h of transient MCAO in mice. In another experiment, fresh bone marrow cells (1 ⫻ 106 per 10 ␮l), including the supportive stromal tissue obtained directly from adult rats, was grafted into the penumbra of the ischemic striatum at 4 or 24 h after 2 h of transient MCAO in rats. The adult hematopoietic bone marrow cells survived in the adult mouse and rat brains after ischemia. In other experiments, neuroepithelial stem cell lines (MHP36) were genetically engineered with a temperature-sensitive gene so that the cells would replicate at 33⬚C, but cease replication and differentiate into neurons or glia at the body temperature of 37⬚C [142]. With this cell line (MHP36), Hodges et al. [143], Virley et al. [144] showed functional recovery in adult rats after 15 min of global ischemia, and also found an extensive repopulation of the grafted cells into neuronal and glial phenotypes in the hippocampus. In further studies MHP36 stem cell restored cognitive function in marmosets after NMDAinduced excitotoxic hippocampal CA1 lesions as effectively as fetal homografts. Marrow stromal cells (MSCs) have also been used in experimental stroke models. MSCs also have the capacity to pass through the blood-brain barrier and migrate throughout the forebrain and cerebellum, and they differentiate into astrocytes and neurons after injection into neonatal mouse brain [145].

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Transplantation of adult MSCs directly into the adult rat brain and spinal cord has resulted in the reduction of functional deficits associated with stroke [146], traumatic brain injury, and spinal cord injury [147]. MSCs can form neural phenotypes and migrate when placed in damaged brain [146, 147]. The route of administration is an important aspect of genetic and cellular therapies for stroke. Direct injection of vectors into the brain parenchyma or ventricle is one option that has been tested extensively, but less invasive procedures would be preferable if possible. To test whether the intravenous route could be used to send MSCs into the brain to reduce neurological functional deficits after stroke in rats, Chen et al. [148] performed an intravenous infusion of bone marrow derived-MSCs. These investigators subjected rats to 2 h of MCAO and treated the animals with infusion of MSCs. They demonstrated significant recovery of somatosensory behavior and Neurological Severity Score (p ⬍ 0.05) in animals infused with 3 ⫻ 106 MSCs at day 1 or 7 days after MCAO compared with control animals. Morphological analysis of the tissue indicated that BrdU-labeled MSCs were more likely to enter into damaged brain than into contralateral nonischemic brain. Many MSCs survived, and a few cells expressed protein markers for parenchymal cells. Neural stem cells have wide appeal as vectors for gene transfer, and may represent the next logical step in clinical trials for stroke treatment. The stem cell possesses the potential of self-renewal as well as multidirectional differentiation. Stem cells are influenced by neurotrophic factors to differentiate into a given neural lineage. It is expected that neural stem cells have the potential to compensate and recover neural functions that were lost because of ischemic stroke. Neural stem cells are believed to be present even in mature adult mammalian brain [149, 150], and could be directed toward expansion and self-repair if the proper stimuli could be applied. Immortalized multipotent neural stem and progenitor cells are another preferred source of tissue for gene manipulation and ex vivo gene transfer to the brain [151]. These cells can be genetically transduced ex vivo (e.g., with growth factors) and maintained as cell lines in culture. Targeted intracerebral delivery of neurotrophic factors has emerged as an interesting therapeutic strategy to repopulate an ischemic area of the brain. The concept behind immortalization is to halt the program of development by inducing the cell in a mode of continuous cell cycle. The most common method of immortalizing cells is to introduce an oncogene such as large T antigen. tsA58 is a temperature-sensitive mutated allele of the large T antigen that is stable at 33⬚C in cultured cells but degrades at body temperature (37–39⬚C). The resultant conditionally immortalized neural progenitors have the ability to differentiate and integrate after implantation into adult brain. Martinez-Serrano and Bjorklund [151] studied protection of ischemia-induced striatal cell death using transplants of conditionally

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immortalized NGF-secreting H1B5 cells. They demonstrated that NGF-secreting H1B5 cell implantation resulted in the reduction of the effect of subsequent MCAO. The results obtained with the NGF-secreting H1B5 cell line have stimulated the development of procedures for ex vivo gene transfer of other CNSactive neurotrophic factors, such as BDNF, neurotrophin-3, NT-4/5, ciliary neurotropic factor and GDNF.

Conclusions

Tremendous accomplishments in neuroscience, molecular biology, and genetics provide a solid foundation for neurotransplantation. Restorative neurosurgical procedures will continue to evolve as advances are made in these fields. Selection of a suitable graft material, safe and effective implantation techniques, and establishment of a hospitable milieu for graft survival and integration will be necessary to achieve optimal patient outcomes. Cerebral infarction and neurodegenerative disorders are appropriate initial candidates for neurotransplantation research. In future, it may be possible to employ multiple gene therapies strategy targeted to specific areas in the ischemic brain. Neurons may be protected by virus-mediated gene expression of neurotrophic factors, and angiogenic genes such as VEGF or bFGF may be used to generate new vessels and improve collateral circulation. Ex vivo-manipulated stem cells could be transplanted into the ischemic lesion for integration and differentiation into neurons, to assist with restoring neuronal density to the ischemic or infarcted brain.

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93 Granholm AC, Albeck D, Backman C, Curtis M, Ebendal T, Frieden P, Henry M, Hoffer B, Kordower J, Rose GM, Soderstrom S, Bartus RT: A non-invasive system for delivering neural growth factors across the blood-brain barrier: A review. Rev Neurosci 1998;9:31–55. 94 Pan W, Banks WA, Kastin AJ: Permeability of the blood-brain barrier to neurotrophins. Brain Res 1998;788:87–94. 95 Friden PM, Walus LR, Watson P, Doctrow SR, Kozarich JW, Backman C, Bergman H, Hoffer B, Bloom F, Granholm AC: Blood-brain barrier penetration and in vivo activity of an NGF conjugate. Science 1993;259:373–377. 96 Zhou J, Bradford HF, Stern GM: Influence of BDNF on the expression of the dopaminergic phenotype of tissue used for brain transplants. Brain Res Dev Brain Res 1997;100:43–51. 97 Kawaja MD, Rosenberg MB, Yoshida K, Gage FH: Somatic gene transfer of nerve growth factor promotes the survival of axotomized septal neurons and the regeneration of their axons in adult rats. J Neurosci 1992;12:2849–2864. 98 Espejo M, Cutillas B, Arena TE, Ambrosio S: Increased survival of dopaminergic neurons in striatal grafts of fetal ventral mesencephalic cells exposed to neurotrophin-3 or glial cell line-derived neurotrophic factor. Cell Transplant 2000;9:45–53. 99 Kordower JH, Emborg ME, Bloch J, Ma SY, Chu Y, Leventhal L, McBride J, Chen EY, Palfi S, Roitberg BZ, Brown WD, Holden JE, Pyzalski R, Taylor MD, Carvey P, Ling Z, Trono D, Hantraye P, Deglon N, Aebischer P: Neurodegeneration prevented by lentiviral vector delivery of GDNF in primate models of Parkinson’s disease. Science 2000;290:767–773. 100 Granholm AC, Mott JL, Bowenkamp K, Eken S, Henry S, Hoffer BJ, Lapchak PA, Palmer MR, van Horne C, Gerhardt GA: Glial cell line-derived neurotrophic factor improves survival of ventral mesencephalic grafts to the 6-hydroxydopamine lesioned striatum. Exp Brain Res 1997;116: 29–38. 101 Stromberg I, Bjorklund L, Johansson M, Tomac A, Collins F, Olson L, Hoffer B, Humpel C: Glial cell line-derived neurotrophic factor is expressed in the developing but not adult striatum and stimulates developing dopamine neurons in vivo. Exp Neurol 1993;124:401–412. 102 Kordower JH, Palfi S, Chen EY, Ma SY, Sendera T, Cochran EJ, Mufson EJ, Penn R, Goetz CG, Comella CD: Clinicopathological findings following intraventricular glial-derived neurotrophic factor treatment in a patient with Parkinson’s disease. Ann Neurol 1999;46:419–424. 103 Tuszynski MH, Roberts J, Senut MC, U HS Gage FH: Gene therapy in the adult primate brain: Intraparechymal grafts of cells genetically modified to produce nerve growth factor prevent cholinergic neuronal degeneration. Gene Ther 1996;3:305–314. 104 Diaz-Cintra S, Rivas P, Cintra L, Aguilar A, Guiterrez G, Perez E, Excobar M, Bermudez-Rattoni F: Morphometric study of fetal brain transplants in the insular cortex and NGF effects on neuronal and glial development. Cell Transplant 1995;4:505–513. 105 Westlund KN, Lu Y, Kadekaro M, Harmann P, Terrell ML, Pizzo DP, Hulsebosch CE, Eisenberg HM, Perez-Polo JR: NGF-producing transfected 3T3 cells: Behavioral and histological assessment of transplants in nigral lesioned rats. J Neurosci Res 1995;41:367–373. 106 Kordower JH, Chen EY, Winkler C, Fricker R, Charles V, Messing A, Mufson EJ, Wong SC, Rosenstein JM, Bjorklund A, Emerich DF, Hammang J, Carpenter MK: Grafts of EGF-responsive neural stem cells derived from GFAP-hNGF transgenic mice: Trophic and tropic effects in a rodent model of Huntington’s disease. J Comp Neurol 1997;387:96–113. 107 Li Y, Chen J, Chen XG, Wang L, Gautam SC, Xu YX, Katakowski M, Zhang LJ, Lu M, Janakiraman N, Chopp M: Human marrow stromal cell therapy for stroke in rat: Neurotrophins and functional recovery. Neurology 2002;59:514–523. 108 Andsberg G, Kokaia Z, Klein RL, Muzyczka N, Lindvall O, Mandel RJ: Neuropathological and behavioral consequences of adeno-associated viral vector-mediated continuous intrastriatal neurotrophin delivery in a focal ischemia model in rats. Neurobiol Dis 2002;9:187–204. 109 Hermann DM, Kilic E, Kugler S, Isenmann S, Bahr M: Adenovirus-mediated GDNF and CNTF pretreatment protects against striatal injury following transient middle cerebral artery occlusion in mice. Neurobiol Dis 2001;8:655–666. 110 Zhao LR, Risedal A, Wojcik A, Hejzlar J, Johansson BB, Kokaia Z: Enriched environment influences brain-derived neurotrophic factor levels in rat forebrain after focal stroke. Neurosci Lett 2001;305:169–172.

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111 Zhang Y, Pardridge WM: Neuroprotection in transient focal brain ischemia after delayed intravenous administration of brain-derived neurotrophic factor conjugated to a blood-brain barrier drug targeting system. Stroke 2001;32:1378–1384. 112 Ferrer I, Krupinski J, Goutan E, Marti E, Ambrosio S, Arenas E: Brain-derived neurotrophic factor reduces cortical cell death by ischemia after middle cerebral artery occlusion in the rat. Acta Neuropathol (Berl) 2001;101:229–238. 113 Hortobagyi T, Harkany T, Reisch R, Urbanics R, Kalman M, Nyakas C, Nagy Z: Neurotrophinmediated neuroprotection by solid fetal telencephalic graft in middle cerebral artery occlusion: A preventive approach. Brain Res Bull 1998;47:185–191. 114 Kawamata T, Speliotes EK, Finklestein SP: The role of polypeptide growth factors in recovery from stroke. Adv Neurol 1997;73:377–382. 115 Kanazawa I: How do neurons die in neurodegenerative diseases? Trends Mol Med 2001;7:339–344. 116 Jellinger KA: Cell death mechanisms in Parkinson’s disease. J Neural Transm 2000;107:1–29. 117 Morita-Fujimura Y, Fujimura M, Yoshimoto T, Chan PH: Superoxide during reperfusion contributes to caspase-8 expression and apoptosis after transient focal stroke. Stroke 2001;32:2356–2361. 118 Benchoua A, Guegan C, Couriaud C, Hosseini H, Sampaio N, Morin D, Onteniente B: Specific caspase pathways are activated in the two stages of cerebral infarction. J Neurosci 2001;21: 7127–7134. 119 Mattson MP, Culmsee C, Yu ZF: Apoptotic and antiapoptotic mechanisms in stroke. Cell Tissue Res 2000;301:173–187. 120 Breakfield XO: Gene delivery into the brain using virus vectors. Nat Genet 1993;3:187–189. 121 Pallela TD, Hidaka Y, Silverman LJ, Levine M, Glorioso J, Kelley WN: Expression of human HPRT mRNA in brains of mice infected with a recombinant herpes simplex virus-1 vector. Gene 1989;80:137–144. 122 Fink DJ, Sternberg LR, Weber PC, Mata M, Goins WF, Glorioso JC: In vivo expression of [beta]galactosidase in hippocampal neurons by HSV-mediated gene transfer. Hum Gene Ther 1992;3: 11–19. 123 Wolfe JH, Deshmann SL, Fraser NW: Herpes virus vector gene transfer and expression of [beta]glucuronidase in the central nervous system of MPS VII mice. Nat Genet 1992;1:379–384. 124 Price J, Turner D, Cepko C: Lineage analysis in the vertebrate nervous system by retrovirusmediated gene transfer. Proc Natl Acad Sci USA 1987;84:156–160. 125 Walsh C, Cepko CL: Clonally related cortical cells show several migration patterns. Science 1988; 241:1342–1345. 126 Mulligan RC: The basic science of gene therapy. Science1993;260:926–932. 127 Karpati G, Lochmuller H, Nalbantoglu J, Durham H: The principles of gene therapy for the nervous system. Trends Neurosci 1996;19:49–54. 128 Kafri T, Blomer U, Peterson DA, Gage FH, Verma IM: Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors. Nat Genet 1997;17:314–317. 129 Ono T, Fujino Y, Tsuchiya T, Tsuda M: Plasmid DNAs directly injected into mouse brain with lipofectin can be incorporated and expressed by brain cells. Neurosci Lett 1990;117:259–263. 130 Danko I, Fritz JD, Latendresse JS, Herweijer H, Schltz E, Wolf JA: Dystrophin expression improves myofiber survival in mdx muscle following intramuscular plasmid DNA injection. Hum Mol Genet 1993;2:2055–2061. 131 Kitagawa H, Sasaki C, Sakai K, Mori A, Mitsumoto Y, Mori T, Fukuchi Y, Setoguchi Y, Abe K: Adenovirus-mediated gene transfer of GDNF prevents ischemic brain injury after transient MCA occulusion in rats. J Cereb Blood Flow Metab 1999;19:1336–1344. 132 Fujimura M, Morita-Fujimura Y, Murakami K, Kawase M, Chan PH: Cytosolic redistribution of cytochrome c after transient focal cerebral ischemia in rats. J Cereb Blood Flow Metab 1998;18: 1239–1247. 133 Casper D, Engstrom SJ, Mirchandani GR, Pidel A, Palencia D, Cho PH, Brownlee M, Edelstein D, Federoff HJ, Sonstein WJ: Enhanced vascularization and survival of neural transplants with ex vivo angiogenic gene transfer. Cell Transplantat 2002;11:331–349. 134 Betz AL, Yang GY, Davidson BL: Attenuation of stroke size in rats using an adenoviral vector to induce overexpression of interleukin-1 receptor antagonist in brain. J Cereb Blood Flow Metab 1995;15:547–551.

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135 Xu DG, Crocker SJ, Doucet J-P, St-Jean M, Tamai K, Hakim AM, Ikeda J-E, Liston P, Thompson CS, Korneluk RG, MacKenzie A, Robertson GS: Elevation of neuronal expression of NAIP reduces ischemic damage in the rat hippocampus. Nat Med 1997;3:997–1004. 136 Linnik MD, Zahos P, Geschwind MD, Federoff HJ: Expression of bcl-2 from a defective herpes simplex virus-1 vector limits neuronal death in focal cerebral ischemia. Stroke 1995;26:1670–1674. 137 Lawrence MS, McLaughlin JR, Sun G-H, Ho DY, McIntosh L, Kunis DM, Sapolsky RM, Steinberg GK: Herpes simplex viral vectors expressing bcl-2 are neuroprotective when delivered after a stroke. J Cereb Blood Flow Metab 1997;17:740–744. 138 Yang GY, Zhao YJ, Davidson BL, Betz AL: Overexpression of interleukin-1 receptor antagonist in the mouse brain reduces ischemic brain injury. Brain Res 1997;751:181–188. 139 Yenari MA, Fink SL, Sun GH, Chang LK, Patel MK, Kunis DM, Onley D, Ho DY, Sapolsky RM, Steinberg GK: Gene therapy with HSP72 is neuroprotective in rat models of stroke and epilepsy. Ann Neurol 1998;44:584–591. 140 Hodges H, Virley D, Williams C, Ridley RM, Sinden JD, Kershaw TR, Harland S, Gray JA, Lantos PL: Fetal CA1 grafts promote recovery from cognitive deficits induced by excitotoxic damage to the CA1 field in marmosets. J Cereb Blood Flow Metab 1997;17(suppl 1):S451. 141 Li Y, Chopp M, Chen J, Gautam SC, Dou D, Zhang L, Wang L, Xu Y, Powers C, Zhang X, Jiang F: Intracerebral grafting of adult bone marrow cells after stroke in adult mice and rats. J Cereb Blood Flow Metab 1999;19(suppl 1):S615. 142 Sinden JD, Rashid-Doubell F, Kershaw TR, Nelson A, Chadwick A, Jat PS, Noble MD, Hodges H, Gray JA: Recovery of spatial learning by grafts of a conditionally immortalized hippocampal neuroepithelial cell line into the ischemia-lesioned hippocampus. Neuroscience 1997;81:599–608. 143 Hodges H, Virley D, Ridley RM, Sinden JD, Kershaw TR, French S, Harland S, Gray JA, Lantos PL: Conditionally immoral MHP36 cells restore cognitive function in marmosets after excitotoxic hippocampal CA1. J Cereb Blood Flow Metab 1999;19(suppl 1):S617. 144 Virley D, Ridley RM, Sinden JD, Kershaw TR, Harland S, Rashid T, French S, Sowinski P, Gray JA, Lantos PL, Hodges H: Primary CA1 and conditionally immortal MHP36 cell grafts restore conditional discrimination learning and recall in marmosets after excitotoxic lesions of the hippocampal CA1 field. Brain 1999;22:2321–2335. 145 Kopen GC, Prockop DJ, Phinney DG: Marrow stromal cells migrate throughout forebrain and cerebellum, and they differentiate into astrocytes after injection into neonatal mouse brains. Proc Natl Acad Sci USA 1999;96:10711–10716. 146 Li Y, Chopp M, Chen J, Wang L, Gautam SC, Xu Y, Zhang ZG: Intrastriatal transplantation of bone marrow stromal cells (MSCs) improves functional recovery after stroke in adult mice. J Cereb Blood Flow Metab 2000;20:1311–1319. 147 Chopp M, Zhang XH, Li Y, Wang L, Chen J, Lu D, Lu M, Rosenblum M: Spinal cord injury in rat: Treatment with bone marrow stromal cell transplantation. Neuroreport 2000;11:3001–3005. 148 Chen J, Li Yi, Wang L, Zhang Z, Lu D, Lu M, Chopp M: Therapeutic benefit of intravenous administration of bone marrow stromal cells after cerebral ischemia in rats. J Neurol Sci 2001; 189:49–57. 149 Eriksson PS, Perfilieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, Gage FH: Neurogenesis in the adult human hippocampus. Nat Med 1998;4:1313–1317. 150 Gould E, Reeves AJ, Graziano MS, Gross CG: Neurogenesis in the neocortex of adult primates. Science 1999;286:548–552. 151 Martinez-Serrano A, Bjorklund A: Ex vivo nerve growth factor gene transfer to the basal forebrain in presymptomatic middle-aged rats prevents the development of cholinergic neuron atrophy and cognitive impairment during aging. Proc Natl Acad Sci USA 1998;17;95:1858–1863.

Douglas Kondziolka, MD Department of Neurological Surgery Suite B-400, UPMC, 200 Lothrop Street, Pittsburgh, PA 15213 (USA) Tel. ⫹1 412 647 6782, Fax ⫹1 412 647 0989, E-Mail [email protected]

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Neuro-Oncology Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 458–498

Neurosurgical Applications for Polymeric Drug Delivery Systems Paul P. Wang a, Henry Brema,b Departments of aNeurological Surgery and bOncology, The Johns Hopkins Hospital, Baltimore, Md., USA

Introduction

Malignant brain tumors represent one of the most devastating forms of human illness. Each year, approximately 16,800 Americans are diagnosed with primary brain tumors and 13,100 die from the disease [1]. Half of all primary brain tumors originate from glial cells and are thus classified as gliomas. Astrocytomas, which represent 3 out of 4 gliomas, include a heterogeneous group of tumors ranging from low-grade lesions and high-grade anaplastic astrocytomas, to its most common and aggressive variant, the glioblastoma multiforme (GBM). Historically, the standard of care for high-grade malignant gliomas includes surgical biopsy for pathological diagnosis [2], surgical debulking of accessible tumor, and adjuvant radiation therapy and chemotherapy [3–7]. Unfortunately, despite recent advances in neuroradiology and neurosurgical techniques, longterm patient survival remains dismal. Median survival after surgical resection alone is 6 months, and only 7.5% of patients survive 2 years. Adjuvant radiation therapy extends median survival to 9 months, and adjuvant systemic chemotherapy has been minimally effective [8, 9]. Due to the highly invasive nature of malignant gliomas, a gross total resection of the tumor almost always leaves behind remnants at the microscopic level. Broader surgical resection results in an increased risk of damaging functional brain tissue, resulting in immediate morbidity and neurological deficit. Similarly, increasing the dose or field of a radiation protocol introduces unacceptable side effects such as tissue edema or necrosis. Therefore, efforts have been made to improve adjuvant chemotherapy modalities for treating malignant brain tumors, both in the development of more effective agents and in the advancement of drug

delivery methods. The introduction of biodegradable controlled-release localdelivery polymers to treat malignant brain tumors has been an important advancement in improving chemotherapy delivery methods. While the benefits of local-delivery polymers are still debatable, local administration has a strong rationale, which stands to improve patient outcomes as the currently available chemotherapeutic drugs improve. This chapter chronicles the history of local delivery techniques for treating gliomas, their clinical applications, and future technologies that may advance the use of local-delivery systems in neurosurgery.

Unique Issues of Drug Delivery into the Central Nervous System

The unique environment of the central nervous system (CNS) presents significant challenges for delivering chemotherapy to brain tumors. The CNS is physiologically isolated by the blood-brain barrier (BBB), composed of tight junctions between endothelial cells of capillaries supplying the CNS [10], as well as anchoring astrocytic foot processes. The BBB forms a pharmacological barrier that under most conditions can prevent the influx of large molecules into the brain. Anti-neoplastic agents that are large, ionically charged, or hydrophilic do not readily penetrate the BBB [11] (fig. 1), and intolerably high systemic drug levels may be necessary to achieve therapeutic doses within the CNS [12]. The approaches to improving drug delivery to brain tumor patients fall into three categories: (1) tailoring existing agents to maximally utilize the natural permeability properties of the BBB; (2) disrupting the BBB before systemic delivery of agents, and (3) circumventing the BBB with direct delivery of agents to the CNS. Using the first approach, existing drugs can be manipulated pharmacologically to create smaller, more lipophilic, or ligand-adapted agents, which traverse the BBB more readily. For example, both lumustine (CCNU) and semustine (methyl-CCNU) are lipophilic variants of carmustine (BCNU) [13], a previously existing chemotherapeutic agent with modest efficacy in the treatment of malignant brain tumors. Clinical trials have not shown any significant efficacy of either agent when given systemically [5]. Anti-neoplastic agents also can be linked with a carrier or ligand capable of penetrating the BBB. For example, dihydropyridine is a lipophilic carrier that readily crosses the BBB, and has been shown to deliver a variety of drugs intracranially, including chemotherapy agents, antibiotics, and neurotransmitters [14]. Receptor-specific monoclonal antibodies also have been used to deliver drugs across the BBB [15]. The second approach to delivering drugs across the BBB involves two steps, disruption of the BBB and systemic delivery of drugs across the compromised BBB. For example, the diuretic mannitol can cause acute dehydration of endothelial cells, which in turn shrinks the cells and widens the tight junctions

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Fig. 1. Cerebrovascular permeability versus octanol-water partition coefficient of selected chemicals and drugs. (Reprinted with permission from Elsevier Science [12].)

of the BBB, when infused intra-arterially in hyperosmolar concentrations. In a study investigating the efficacy of premedicating brain tumor patients with mannitol before the administration of carboplatin and etoposide, Williams et al. [16] demonstrated that 4 out of 4 patients with primitive neuroectodermal tumors and 2 out of 4 patients with CNS lymphomas had a clinical response following mannitol and drug administration. However, mannitol as an adjuvant to chemotherapy has showed no benefit to patients with GBM, oligodendrogliomas, or metastatic carcinomas, probably because it does not improve the delivery of drug to the tumor site despite its ability to increase penetrance of agents across the BBB [17]. Another drug that directly disrupts the BBB is the bradykinin agonist RMP-7, which has been shown in experimental brain tumor models to increase the uptake of carboplatin [18]. However, in vivo efficacy is limited because like mannitol, it does not necessarily improve the delivery of the antineoplastic agents to the tumor site after it has crossed the BBB [19]. The third approach to overcoming the BBB is to completely bypass it by local delivery of the drug. The three main strategies within this approach

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are: (1) delivery via cerebrospinal fluid (CSF) exposure; (2) drug administration via catheters, and (3) delivery via sustained-release local-delivery polymers. The local-delivery approach theoretically provides for the maintenance of sustained significant local levels of the drug at the tumor site while avoiding dangerously high systemic exposure. Such a strategy seems particularly appropriate for the management of malignant gliomas, since 80–90% recur after conventional treatment within 2 cm of the original resection bed [20]. CSF infusion requires the introduction of the drug via either an intraventricular catheter or lumbar puncture. The BBB does not extend to the ependymal lining of the ventricular system and delivery via CSF infusion bypasses the BBB. Initial results have been disappointing mainly due to poor penetration of the drug into the brain parenchyma [21]. Delivery of anti-neoplastic agents through catheter systems requires accurately placing a catheter’s tip within the tumor site and infusing from a reservoir of drug, through the catheter, and directly to the tumor. One of the earliest such systems is the Ommaya reservoir [22], which can deliver intermittent bolus injections of chemotherapy to the tumor. The introduction of implantable pumps such as Infusaid (Norwood, Mass., USA) [23], MiniMed PIMS (Sylmar, Calif., USA) [24], and Medtronic SynchroMed (Minneapolis, Minn., USA) [25] allows for the constant infusion of drug over extended periods of time. These devices may be limited by mechanical failure, obstruction by clot or tissue debris, and infection. Biocompatible sustained-release polymers are impregnated with chemotherapeutic agents and surgically implanted in a tumor cavity after surgical resection and debulking of malignant gliomas. The polymers are designed to slowly release the drug over time. They fall into two categories, biodegradable and nonbiodegradable. The former release drug as the polymer matrix breaks down over time, while the latter leaves behind an intact polymer matrix after all of the therapeutic agents have been released [26].

Development of Biocompatible Polymers

Historical Perspective In 1976, Langer and Folkman [27] reported the sustained and predictable release of macromolecules from the ethylene vinyl acetate (EVAc) copolymer, a nonbiodegradable polymer initially developed for possible applications within the clothing industry. Drug incorporated into an EVAc polymer matrix diffuses through the micropores of the matrix at a rate dependent on multiple chemical properties of the drug, including molecular weight, charge, and water solubility. The polymer was deemed safe after rabbit cornea studies demonstrated its biocompatibility [28] and rat brain studies demonstrated its inertness [29–33].

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Bulk erosion

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Fig. 2. a Bulk erosion of biodegradable controlled-release polymer implants leads to unpredictable release profiles. b Polymers exhibiting surface erosion release drug at nearly constant rate (zero-order kinetics) as they dissolve in water. (Reprinted with permission [56].)

Since its introduction, EVAc polymers have been incorporated into numerous clinical applications, including glaucoma treatment, dental care, contraception, insulin therapy, asthma treatment, and chemotherapy. Recently, EVAc polymers have been successfully used to deliver neurotrophic growth factors to treat peripheral nerve injuries in a rat brachial plexus injury model [34]. However, EVAc polymers have not been approved by the Food and Drug Administration (FDA) for intracranial use [35]. In addition to the disadvantage of a permanent foreign body left behind after drug delivery, EVAc polymers demonstrate firstorder release kinetics, with the release rate decreasing over time [36]. To avoid the disadvantages of a residual indwelling matrix, a series of biodegradable polymer systems were developed. By releasing drugs with a combination of diffusion and polymer degradation, sustained release of drugs could be accomplished without leaving behind a permanent foreign body (fig. 2, 3). The first such biodegradable polymer included the family of lactide/glycolide polyesters, where the lactic acid and glycolic acid monomers are polymerized with ester bonds. The polylacticcoglycolic acid (PLGA) polymers exhibited several advantages over the nonbiodegradable EVAc polymers. In addition to biodegradability, PLGA polymers could be tailored to provide a range of drug delivery rates by varying the ratio of lactic acid to glycolic acid monomers [37]. Biocompatibility was demonstrated in the rat brain [38–40], as well as by polymers fashioned into sutures, which are now in wide clinical use [41, 42]. Often shaped into injectable microspheres [43], PLGA polymers have been successfully used to deliver a variety of drugs, including steroids, anti-inflammatory agents, antibiotics, anesthetics, narcotics, and chemotherapeutic agents [37, 44–53]. A microsphere variant also has been designed specifically for stereotactic implantation into the brain [54]. Another variant, manufactured by covalently linking the polymer matrix to a polyethylene glycol coating, has been

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Diffusion

Degradation

Fig. 3. Polymer implants releasing drug by degradation provide more predictable release profiles compared to diffusion. (Reprinted with permission [56].)

shown to reduce opsonization and elimination in the immune system [55]. A disadvantage to PLGA polymers is the drug release characteristic of bulk erosion, similar to the dissolution of a sugar cube in water (fig. 2a), which can lead to sporadic dumping of the drug, suboptimal tissue exposure, and unexpected acute toxicity [56]. In 1985, Leong et al. [62] formulated the polyanhydride poly[bis(pcarboxyphenoxyl)] propane-sebacic acid (pCPP:SA) matrix. Like the PLGA polymer, pCPP:SA is biodegradable by spontaneously breaking down to dicarboxylic acids in water. Unlike PLGA polymer, the pCPP:SA matrix offers several important properties. First, because of its extreme hydrophobicity, the matrix shields the incorporated drug from aqueous media, an important feature for compounds with short biological half-lives. Second, in contrast to the PLGA breakdown by bulk erosion (i.e., like a sugar cube), pCPP:SA breakdown is limited to its surface, a

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property called surface erosion (i.e., like peeling an onion) (fig. 2b). This property confers a theoretical advantage of constant-rate or zero-order drug delivery. Like the previously introduced polymer systems, the pCPP:SA matrix can also be manufactured into an almost endless variety of shapes, including microspheres, sheets, rods, and wafers [57–63]. Also like the PLGA polymer, altering the ratio osf the two monomers CPP and SA can vary the rate of breakdown of pCPP:SA, and thus the rate of drug delivery. For example, a 1 mm thick polymer composed of pure CPP would require 3 years to degrade, but a mixture of 20% CPP and 80% SA or pCPP:SA (20:80) would only require 3 weeks [60]. Multiple studies have demonstrated the biocompatibility of pCPP:SA. The dicarboxylic acid breakdown products are neither mutagenic, teratogenic, nor cytotoxic in standard assays [64]. In vitro proliferation assays with endothelial cells and smooth muscle cells showed no inhibition properties in the pCPP:SA matrix [64]. There was no identifiable inflammation in the rabbit cornea assay [28, 62]. Biocompatibility with neural tissue was demonstrated in the brains of rats [65], rabbits [66], and monkeys [67]. Due to its favorable drug release characteristics and biocompatibility, pCPP:SA has been used extensively in the clinical setting, and its current applications in neuro-oncology are discussed in the next section. A variety of other local-delivery sustained-release systems have been developed for intracranial use. The fatty acid dimmer-sebacic acid (FAD:SA) polymers [68–70] are well suited for releasing hydrophobic chemotherapeutic agents [71–73]. Polyethylene glycol-coated liposomes and gelatin chondroitin sulfate-coated microspheres have been developed to successfully release anthracyclines and cytokines, respectively [74, 75]. Commonly used surgical materials, such as fibrin glue [76], gelatin sponges [77], Surgicel (oxidized regenerated cellulose) [77], polymethylmethacrylate [78], and silastic tubing [79], have all been used for intracranial local delivery of drugs.

Clinical Applications of Polyanhydride Polymers for Drug Delivery BCNU (Gliadel)

Development and Clinical Use The initial drug chosen for the development of sustained local delivery of intracranial chemotherapy was carmustine (BCNU) due to its widespread use as a systemic agent for treating malignant brain tumors and its established mechanism of action against glioma cells. Belonging to the family of nitrosureas, BCNU is a DNA alkylating agent (i.e., chloroethylation of guanine at the O6 position) and causes DNA chain termination, impairing the mitotic activity of cancer cells. Its relatively low molecular weight and lipid solubility allows it to cross the BBB

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and accumulate in the CNS [13]. However, its effectiveness as a systemic agent for brain tumors is limited by its relatively short half-life (⬍15 min) and numerous serious side effects, including bone marrow suppression and pulmonary fibrosis. Clinical trials investigating its efficacy with systemic delivery demonstrated only modest improvements in survival [4, 6]. However, by incorporating BCNU into local-delivery polymers, it was hoped that the efficacy would be significantly increased and dose-limiting side effects could be avoided. Preclinical Trials Preclinical studies investigating BCNU-loaded polymer preparations first studied the in vivo release kinetics of BCNU-loaded polymers. The initial study used an EVAc copolymer loaded with BCNU and implanted into the rat brain [33]. BCNU concentrations were measured with the Bratton-Marshall assay in both cerebral hemispheres, as well as in serum samples collected at various timepoints after polymer placement. In the hemisphere ipsilateral to the polymer, BCNU levels peaked at 4 h and remained elevated through day 7. Levels in the contralateral hemisphere and in the serum were lower by at least an order of magnitude throughout the time course of the experiment. This study demonstrated the proof of principle of the ability of polymer technology to provide both local delivery and sustained release of chemotherapy within the CNS. A second experiment, again using a rat intracranial model, compared kinetics and biodistribution of a BCNU and pCPP:SA (80:20) copolymer with direct stereotactic injection of BCNU [80]. Using tritiated BCNU, the distribution of drug was assessed by quantitative autoradiography in brain sections collected at various timepoints following implantation/injection. Animals with BCNU polymers had approximately 50% of the ipsilateral hemisphere exposed to BCNU at day 3, and 10% exposed at day 14. A polymer containing only 600 ␮g of BCNU provided tissue concentrations of up to 6 mM at 10 mm from the implantation site on both days 3 and 7. In contrast, animals directly injected with BCNU showed an initial broadly distributed BCNU spike from 1 to 3 h postinjection, which then rapidly disappeared. A third experiment implanted 20% (w/w) BCNU-loaded pCPP:SA polymers in a primate intracranial model; tumoricidal concentrations of BCNU was demonstrated up to 4 cm from the polymer site at 24 h after surgery [81]. At 30 days, active drug was still present up to 1 cm from the polymer. This set of experiments confirmed that polymer technology can provide local, sustained, and clinically significant in vivo delivery of chemotherapy agents within the CNS. A second phase of preclinical experiments investigated the efficacy of BCNU-loaded polymers in animal models of brain cancer. In 1993, Tamargo et al. [29] compared the efficacy of polymer-delivered BCNU with systemic BCNU using both the rat flank and intracranial 9L gliosarcoma models. In the flank model using EVAc polymers, tumor growth delay was significantly

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longer in polymer-delivered BCNU compared to systemic delivery (16.3 vs. 11.2 days, p ⬍ 0.05). In the intracranial model, a 10 mg polymer with 20% (w/w) BCNU polymers improved survival in animals with established 9L gliosarcoma. Compared to controls, the EVAc and pCPP:SA polymers increased median survival times 7.3-fold and 5.4-fold, respectively. In contrast, systemic BCNU increased survival only 2.4-fold. A second study using the same established rat intracranial 9L gliosarcoma model compared the efficacy of 20% (w/w) BCNU-loaded pCPP:SA polymers with direct stereotactic injection of an equivalent dose of BCNU [82]. Median survival improved by 271% compared to that of control animals in the polymer group. In contrast, stereotactic injection improved median survival only by 36%. There were also twice as many long-term survivors in the polymer group. A third set of studies established the optimal pCPP:SA monomer ratio and dosing percentage of BCNU [83]. First, in vitro release kinetics were compared between 50:50 and 20:80 pCPP:SA formulations with ranges of drug concentration between 4% and 32% BCNU-loaded polymers. In theory, lowering the proportion of CPP should slow polymer degradation, and thus increase sustained release. In fact, the release kinetics revealed minimal differences between the two formulations loaded at 4% BCNU, but comparisons between the 32% polymers revealed a 150% increase in release duration (18 vs. 7 days) for the 50:50 formulation. Next, the efficacy of polymer loads of 0, 4, 8, 12, 20, and 32% (w/w) were compared in both 50:50 and 20:80 pCPP:SA polymer formulation. When tested against the established rat intracranial 9L gliosarcoma model, survival was maximized in the 20% loaded pCPP:SA (20:80) formulation, which provided a 63% survival rate at 200 days compared to a median survival of ⬍20 days in controls. An additional toxicity study using the cynomolgus monkey intracranial model demonstrated that 20% BCNU-loaded pCPP:SA (20:80) polymers had no systemic or local morbidities, and MRI images obtained 150 days after implantation showed no evidence of edema or mass effect [83]. A final set of preclinical experiments investigated the efficacy of local polymer delivery of various anti-neoplastic agents, including BCNU, to combat brain metastases [84]. First, maximum nontoxic doses of each agent in pCPP:SA polymer were established in the mouse intracranial model. Next, efficacy was tested with and without concurrent radiation therapy in several metastatic tumor lines, including the B16 melanoma, RENCA renal cell carcinoma, CT26 colon cancer, and Lewis lung carcinoma. While both BCNU-loaded polymers and radiation treatments were effective alone, they were much more effective in combination against B16 melanoma (median survival 35 vs. 21.5 days for controls; p ⫽ 0.0005), RENCA renal cell carcinoma (38.5 vs. 12 days; p ⬍ 0.007), and Lewis lung carcinoma (23 vs. 21 days; p ⫽ 0.001). A later study showed

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intracranial BCNU polymers to be effective with (41 vs. 17 days; p ⫽ 0.02) and without irradiation (⬎200 vs. 17 days; p ⬍ 0.0001) against the EMT-6 breast cancer in mice [85]. These encouraging findings have led to clinical trials of BCNU-loaded pCPP:SA treatment for metastatic brain tumors. Clinical Experience with Recurrent Gliomas Based on the preclinical findings described above, approval was obtained for a multicenter phase I-II human trial using pCPP:SA for the treatment of malignant gliomas [86]. Enrollment criteria limited patients to those with recurrent malignant gliomas who had previously undergone a craniotomy for debulking and in whom standard therapy had failed. Other eligibility requirements included an indication for reoperation, a single tumor focus with ⱖ1 cm3 of enhancing volume on radiographic imaging, completion of external beam radiotherapy, a Karnofsky Performance Scale (KPS) score of ⱖ60, and no exposure to nitrosureas during the 6 weeks prior to polymer implantation. Twenty-one patients were enrolled, and three different polymer loads were tested: 1.93, 3.85, and 6.35% (w/w). Each polymer weighed 200 mg, and most patients received a maximum eight wafers within the tumor cavity following debulking (fig. 4). Tumor volumes were similar in all groups. Following treatment with polymeric BCNU as described above, there was no evidence of systemic toxicity, measured by frequent blood chemistry, hematological, and urinalysis exams. Although KPS scores fell slightly during the immediate postoperative period, they returned to baseline and remained stable for at least 7 weeks, indicating quality of life preservation during the chemotherapeutic period. The implanted polymers were detectable on postoperative CT and MRI. They appeared as areas of decreased signals on T1-weighted MRIs, and some were visible on CT as long as 49 days after surgery. In 13 of 21 patients, routine protocol scans revealed some areas of enhancement around the implant sites, which generally resolved spontaneously and did not correlate with any neurological decline or other toxic sequelae [87]. Over the course of study, 10 patients required reoperation for declining neurological status with increasing enhancement on MRI or CT. The most notable intraoperative finding was a rim of necrotic tissue up to 1 cm thick around the wafer(s), similar to that described in interstitial brachytherapy. The KPS scores were generally improved following the removal of this tissue. The overall median survival times were 46 weeks after implant and 87 weeks after initial diagnosis, with 86% of patients alive after more than one year of diagnosis. On the basis of this work, the 3.85% BCNU-loaded polymer was chosen for further study. The next clinical trial was a rigorous multicenter, prospective, randomized, double-blinded, and placebo controlled phase III study [88] investigating the efficacy of 3.85% BCNU (w/w) pCPP:SA polymers in treating 222 patients

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Fig. 4. After surgical debulking, the tumor resection cavity is lined with up to eight 200 mg 3.8% (w/w) BCNU pCPP:SA (20:80) (Gliadel®) polymer, where the loaded drug is gradually released as they dissolve.

with recurrent malignant gliomas at 27 medical centers in the North America. Patients were randomized to receive either a BCNU treatment polymer or a blank placebo. Selection criteria was the same as for the preceding study, except that no chemotherapy was allowed for 4 weeks preceding surgery, and systemic chemotherapy was allowed as early as 2 weeks after surgery. Individual patients and their surgeons decided upon additional operations for tumor recurrence during the study period. Mean age was 47.8 years, and randomization rendered the treatment and placebo groups well matched for age, tumor type, and preoperative KPS scores. All patients previously underwent external beam radiotherapy, and 52.7% of the treatment group and 48.2% of the control group had previously undergone chemotherapy.

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The median postoperative survival of the patients implanted with treatment polymer was 34 weeks compared to 23 weeks in the placebo group (hazard ratio 0.67, p ⫽ 0.006) (fig. 5). The 6-month survival rate was 60% in the treatment group and 47% in the placebo group. Remarkably, among GBM patients (n ⫽ 145), there was a 50% increase in 6-month survival within the treatment population compared to control population (p ⫽ 0.02). The BCNU-loaded polymer was again shown to be safe and well tolerated, with no evidence of bone marrow suppression or systemic toxicity. Within 6 months of polymer placement, 11.8% of treatment and 11.6% of control patients underwent reoperation. Intracranial infection was more common in the treatment group than the placebo group (4/110 or 3.6 vs. 1/112 or 0.9%), but this adverse event did not reach statistical significance. Of the eleven brains from the treatment group that were examined postmortem, there were mild inflammatory reactions without marked necrosis. These studies established that BCNU-polymers are safe and relatively effective in the treatment of recurrent malignant gliomas. Based on the mean survival rates and safety data, the FDA in 1996 approved 3.85% BCNU-loaded PCPP:SA polymer (Gliadel®) for the treatment of recurrent GBM. This was the first time in 23 years that the FDA approved a new treatment for malignant gliomas.

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Clinical Experience with Gliadel Used as Initial Therapy In general, any oncology treatment found effective at recurrence has been subsequently shown to be even more effective as initial therapy. After the establishment of Gliadel’s role in treating recurrent malignant gliomas, attention naturally turned to examining its role during initial disease presentation. A phase I-II trial with 22 enrolled patients in three medical centers was designed to determine the safety of Gliadel polymers at the time of initial surgery [89]. Like the previous studies, the polymer wafers weighed 200 mg, and most patients received a maximum of eight wafers. Inclusion criteria required a single enhancing tumor focus ⱖ1 cm3, age ⬎18 years, and a KPS score of ⱖ60. The mean age was 60, and all the patients received postoperative external beam radiation therapy averaging 5000 rad. No patients received additional chemotherapy during the first 6 months after surgery. There was no perioperative mortality, nor was there evidence of systemic or local toxicity attributable to the polymer. Twenty-one of 22 patients received a pathological diagnosis of GBM. Median survival was 44 weeks from implantation with 4 patients surviving ⬎18 months. This phase I-II study demonstrated that Gliadel was a safe treatment option for the patients newly diagnosed with malignant gliomas. Based on these encouraging findings, a phase III, multicenter, randomized, double-blinded, placebo-controlled study of the efficacy of locally implanted Gliadel against newly diagnosed malignant gliomas was begun [90]. Admission criteria included the presence of a single tumor focus with ⱖ1 cm3 of enhancement, age between 18 and 65 years, a KPS score ⱖ60, and a histopathological diagnosis of malignant glioma on intraoperative frozen section. Again, 200 mg wafers were used. Originally intended for 100 patients, the study was terminated prematurely at 32 patients due to the unavailability of the drug. There were 16 patients in each arm of the study. The median age was 55.5 years for the BCNU group and 53 years for placebo. The median KPS score was 75 for the treatment group and 90 in the control group. There was a discrepancy in tumor pathology; all placebo patients harbored GBMs, but only 11 of 16 BCNU patients had the same diagnosis. With all 32 patients included in the analysis, the median survival of the treatment group was 58.1 weeks compared to 39.9 weeks for the control group (p ⫽ 0.012) (fig. 6). When limited to the subgroup of GBM patients, the treatment group had a median survival of 53.3 weeks versus 39.9 weeks for the placebo group (p ⫽ 0.008). Perhaps more striking was the fact that 3 out of 4 enrolled patients with GBM alive 3 years after the termination of the study were in the BCNU-loaded polymer group. Age and preoperative KPS score significantly impacted survival, while tumor type impacted survival but not in a

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statistically significant way. Again, no local or systemic toxicity attributable to the polymers was noted. A larger phase IV, multicenter, randomized, double-blinded, placebocontrolled study was recently carried out involving 240 patients to definitively assess the role of Gliadel in initial therapy [91]. This study demonstrated that treatment with Gliadel increased median survival (13.9 vs. 11.6 months, p ⫽ 0.003) compared to controls treated with blank polymers. Based partially on these results as well as the results of previous studies, in 2003 the FDA approved Gliadel for the treatment of initially diagnosed malignant glioma. This step represents another major milestone in the evolution of local-delivery polymer chemotherapy for the treatment of brain tumors. Clinical studies investigating the efficacy of BCNU-polymer treatment has by no means concluded. A phase I-II dose-escalation study was recently completed to establish the maximal nontoxic BCNU loading in 20:80 PCPP:SA polymers. Similar to the results in animal models [83], the maximal tolerated dose was determined to be 20% BCNU (w/w) [92]. At this time, Gliadel is still loaded only at 3.85%. Future clinical trials include a phase III trial to evaluate a 20% BCNU-loaded wafer, as well as trials investigating the

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use of Gliadel for metastatic tumors and in combination with other drugs, and the use of chemotherapy resistance modification in combination with Gliadel. Other Neuro-Oncology Applications The successful development of BCNU-loaded sustained-release polymers from the laboratory to standard clinical use has served as a model for future investigation. With local delivery, agents that have never been seriously considered for neuro-oncology due to systemic toxicity can now be examined. The following agents have been either successfully incorporated into polymers or successfully used as adjuvants for polymer-based treatment. O6-Benzylguanine A limitation of Gliadel is the acquired resistance of many brain tumors to BCNU through the expression of O6-alkylguanine-DNA alkyltransferase (AGT), a DNA-repair protein found in the majority of brain tumors treated with BCNU [93]. BCNU exerts its anti-neoplastic effects by chloroethylation of DNA at the O6 position of guanine. By removing adducts at this position, AGT is able to protect tumor cells from BCNU. O6-Benzylguanine (O6-BG) is a substrate analog that can irreversibly inactivate AGT by transferring a benzyl group to a cysteine residue at the active site of AGT [94]. The O6-BG-mediated AGT inhibition increases tumor sensitivity to BCNU, but when given in combination with systemic BCNU, maximal tolerated doses of BCNU were reduced up to 6-fold in animal models due to bone marrow toxicity [95]. Presumably, the same increased BCNU sensitivity O6-BG imparted to tumor cells also affected cells vulnerable to BCNU-related toxicity. Rhines et al. [96] postulated that by combining systemic O6-BG with intracranial local-delivered BCNU polymers instead of systemic BCNU, the side effects of combined treatment could be significantly reduced. Using the established rat intracranial F98 glioma model (a tumor line with high AGT activity), they showed that by giving systemic O6-BG with BCNU-loaded pCPP:SA polymers, median survival was improved in the combination therapy over animals receiving O6-BG alone (34 vs. 22 days, p ⫽ 0.0002) or BCNU polymer alone (34 vs. 25 days, p ⫽ 0.0001). They also found that it was not necessary to reduce BCNU polymer load when systemic O6-BG was introduced. The animals did not exhibit bone marrow or gastrointestinal toxicity. These results suggest that the concurrent use of O6-BG and BCNU polymers may be an important addition to the treatment of malignant brain tumors. In fact, preliminary phase I clinical trials investigating O6-BG as an adjuvant to BCNU treatment have been completed [97]. Current ongoing efforts include incorporating O6-BG within polymers for local delivery, and dose-escalation studies for systemic and local-delivered O6-BG.

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Paclitaxel Paclitaxel (Taxol) is a microtubule-binding agent, which has been shown to have tumoricidal activity against non-small cell lung cancer, breast cancer, and ovarian cancer. In vitro studies have shown paclitaxel to be potent against rat and human glioma cell lines [98], but not permeable across the BBB [99], thus making it an excellent candidate for local polymeric delivery. In vitro kinetics studies demonstrated that 20–40% (w/w) paclitaxelloaded pCPP:SA (20:80) polymers released for up to 1000 h. Biodistribution studies revealed tumoricidal concentrations of paclitaxel in the rat brain for ⬎30 days after implantation. In the established rat intracranial 9L gliosarcoma model, median survival was improved from 19.5 days in rats treated with blank polymers to 61.5 days with 20% paclitaxel-loaded polymers (p ⬍ 0.001) [100]. Clinical trials utilizing a paclitaxel-loaded PACLIMER [101] microsphere delivery system are currently in progress for ovarian cancer. Toxicity trials on dogs for the treatment of intracranial tumors are ongoing; the initial results show no early mortality or significant morbidity that can be attributed to the paclitaxel polymer [102]. Phase I-II clinical trials are being planned for implementation as soon as these canine toxicity studies are completed and can demonstrate safety. Camptothecin The camptothecins are a family of inhibitors of DNA-replicating enzyme topoisomerase I [103]. While in vitro and in vivo studies showed great promise, clinical trials demonstrated unexpected toxicities with systemic administration [104]. This prevented its use as a systemic agent for gliomas, but made it an excellent candidate for local polymer delivery [32]. Among the family of camptothecins, sodium camptothecin was selected due to its chemical properties which make it easy to load onto polymers. Initially incorporated onto the EVAc polymers, where in vitro kinetics experiments demonstrated its sustained release, local-delivery sodium camptothecin demonstrated dramatically increased efficacy in the established rat intracranial 9L gliosarcoma model, where 50% (w/w) polymers extended median survival from 19 days in control animals to ⬎120 days (p ⬍ 0.001). In addition, while none of the control rats survived beyond 32 days, 59% of treatment animals survived ⬎120 days. In contrast, systematic camptothecin had no impact on survival. No local or systemic toxicity was observed in the polymer-implanted animals. Sodium camptothecin loaded onto pCPP:SA polymers was also tested, both in the previously described mouse metastatic tumor study [84], and, most recently, in the established rat intracranial 9L gliosarcoma model [105]. The metastatic study showed that the camptothecin-loaded pCPP:SA polymer was effective only in combination with radiation therapy and only against the

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B16 melanoma (median survival 27.5 vs. 19 days; p ⫽ 0.043). In the 9L gliosarcoma model, median survival was extended with 50% (w/w) camptothecin-loaded polymers from 17 days in control animals to 69 days (p ⬍ 0.001). No local or systemic toxicity was noted. The polymers were also shown to release active camptothecin for up to 1000 h. Current ongoing efforts include preclinical studies examining the efficacy of various camptothecin analogs [106]. Quinacrine Quinacrine was an early anti-malarial drug and is rarely used anymore in the clinical setting. While its exact mechanism of action is unknown, it demonstrates a wide variety of intracellular actions, including the ability to reduce mutagenicity in leukemia and glial cell lines [107, 108]. In the rat flank C6 glioma model, quinacrine has been shown to reduce tumor growth when delivered orally at its maximally tolerated dose (20 mg/kg) in conjunction with systemic BCNU therapy, compared to systemic BCNU therapy alone [108]. However, preliminary work by us has shown that in the established rat intracranial 9L gliosarcoma model, oral quinacrine exhibits no effect when given with intracranial BCNU-loaded 3.85% (w/w) pCPP:SA polymers (fig. 7a). It is likely that the CNS delivery of quinacrine to the tumor site is inadequate, since quinacrine exhibits a 10% penetrance into the CNS. However, the coimplantation of 15% (w/w) quinacrine-loaded pCPP:SA polymers with BCNU-loaded polymers does result in a significant increase in survival (fig. 7b). To exhibit this synergistic effect, it is necessary that the quinacrine polymer be implanted 2 days before the BCNU polymer; placing both polymers in the tumor bed concurrently results in no benefit whatsoever (fig. 7c). Current ongoing work includes investigations into the molecular basis behind this synergistic effect. Possible mechanisms include quinacrine’s documented anti-mutagenicity, interference with the outward transport of BCNU out of the glial cell, enhancement of the BCNU-induced apoptosis, and anti-AGT activity. Mitoxantrone Mitoxantrone is a dihydroxyanthracenedione derivative used in the treatment of advanced breast cancer, non-Hodgkin’s lymphoma, acute nonlymphoblastic leukemia, and chronic myelogenous leukemia in blast crisis, and has been FDA approved for the treatment of hepatic and ovarian cancer. Its use for CNS malignancies has been limited by its poor CNS penetrance and dose-limiting myelosuppression. Recently, DiMeco et al. [109] demonstrated that with up to 10% (w/w) mitoxantrone-loaded pCPP:SA wafers median survival was significantly improved compared to controls (50 vs. 19 days, p ⬍ 0.0001). Further animal studies in combination with other chemotherapy agents are ongoing.

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Immunotherapy The role of local delivery of immunotherapy to combat malignant gliomas has mainly involved the use of cytokines, such as interleukins (IL), interferons, and colony-stimulating factors, which are produced by cells of the immune system to generate and maintain an immune response. Because of genetic mutations, tumor cells express proteins foreign to the host, thereby rendering them vulnerable to an immune response. Effort has been made in using local delivery to release cytokines into brain tumors because cytokines are impermeable to the BBB and carry with them systemic toxicity. Two general strategies have been made in providing for the local delivery of cytokines. The first is the sustained release of cytokine at the tumor site using irradiated tumor cells transduced to secrete the cytokine in a paracrine fashion. Thompson et al. [110] demonstrated with the C57BL/6 mouse intracranial melanoma model that IL-2-transduced tumor cells could generate an immune response to wild-type tumor via direct injection to the tumor site, but not when injected in the flank. In contrast, GM-CSFtransduced cells generated an immune response via flank injection, but not when injected intracranially. Synergy was noted when intracranial IL-2transduced cells and subcutaneously flank GM-CSF-transduced cells were injected simultaneously. A study using the same animal model demonstrated several interesting findings [111]. First, rats with intracranially implanted IL-2-transduced cells demonstrated increased survival compared to controls when challenged with tumor both intracranially and in sites distal to the brain. Second, after successful rejection of an initial challenge, these animals also exhibited immunological memory by mounting an immune response and increasing survival in the face of a second tumor challenge, both intracranial and in sites distal to the brain. In contrast, identical or 10-fold increased doses of subcutaneously injected IL-2transduced cells failed to elicit such memory responses. Finally, the study demonstrated with gene knockout mice that natural killer cells but not CD4⫹ T cells were most responsible for the anti-tumor immune response. Because flank-injected IL-2 paracrine cells did not elicit a similar response, the study postulated that immune cells within the CNS had different cytokine requirements than their counterparts in the periphery. A third study identified IL-12 as a potential candidate for local paracrine delivery [112]. In addition to its immune regulatory effects, IL-12 also exhibits anti-angiogenesis properties. By challenging the established rat intracranial 9L gliosarcoma model, tumor cells transduced with IL-12 were implanted intracranially. Expression of IL-12 was confirmed by reverse transcriptasepolymerase chain reaction. Furthermore, in addition to prolonging survival, local paracrine delivery of IL-12 also induced immunological memory to the

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animals. A second injection of wild-type 9L gliosarcoma tumor cells also elicited an immune response. Several experiments have recently examined the interaction between local paracrine immunotherapy and local polymer-delivered chemotherapy. Using the mouse intracranial F16-B10 melanoma model, an experiment demonstrated synergy between either 10% (w/w) BCNU-loaded or 1% (w/w) carboplatin pCPP:SA polymers with local paracrine IL-2-transduced cells [113]. When combining BCNU-loaded polymers with immunotherapy, 70% of the animals receiving combination therapy survived ⬎72 days, compared to none (with a median survival of 15.8 days) in controls (p ⫽ 0.0023). When combining carboplatin-loaded polymers with immunotherapy, 80% of the animals survived ⬎72 days, compared to none (with a median survival of 20.6 days) in controls (p ⫽ 0.0001). Histological examination of animals receiving combination therapy revealed rare degenerating tumors cells with a marked mixed inflammatory reaction on postimplantation day 14, and no tumor cells and resolution of the inflammatory reaction on day 72. The second general strategy for sustained delivery of cytokines involves loading them directly into polymers. In 1998, Wiranowska et al. [114] demonstrated proof of principle that polymers could deliver sustained and biologically active cytokines. By loading murine interferon-␣/␤ onto EVAc polymers, they demonstrated with both in vitro and in vivo experiments that the released interferous were biologically active. In vitro assays determined most of the activity was released within the first 4 days. In vivo trials demonstrated most of the activity was released within the first 24 h and gradually decreased over the next 3 days. In 1993, Golumbek et al. [75] introduced the gelatin chondroitin sulfate microsphere system for local delivery of drugs in an injectable mixture. In 2001, Hanes et al. [115] encapsulated IL-2 into gelatin chondroitin sulfate and confirmed that the mixture maintained a sustained release of activity over 2 weeks in vitro and up to 3 weeks in vivo. Using the rat intracranial 9L gliosarcoma model, the mouse intracranial B16-F10 melanoma model, and the mouse liver CT26 carcinoma model, they then demonstrated statistically increased effectiveness in generating a protective immune response when injecting the gelatin chondroitin sulfate-IL-2 mixture into tumor compared to controls or local paracrine delivery. In B16-F10 model, 42% of animals exhibited protection on a second tumor challenge. Recently, Rhines et al. [116] demonstrated that the combination of IL-2-loaded microspheres and BCNU-loaded pCPP:SA polymers showed a statistically significant increase of median survival when compared to treatment quinacrine pCPP:SA (20:80) polymers placed day 3 after tumor implantation. c KaplanMeier survival curve for animals treated with intracranial BCNU polymers placed day 5 after 9L gliosarcoma implantation with or without intracranial 10 mg 15% (w/w) quinacrine pCPP:SA (20:80) polymers concurrently day 3 after tumor implantation.

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with either modality alone. Therefore, brain tumor vaccines using polymer delivery of cytokines is a promising avenue of investigation. Angiogenesis Inhibitors Angiogenesis is the process where new blood vessels form, and is essential for the tumor growth [117]. Without angiogenesis, the source of nutrients is limited to a few millimeters by diffusion from the tumor periphery, and tumor size is arrested in equilibrium between peripheral cell proliferation and central cell death. With angiogenesis, nutrient delivery can reach the central cells, and tumor growth becomes exponential with the potential for metastatic spread [118]. Since GBM is one of the most angiogenic of all neoplasms, the use of angiogenesis inhibitors for treating brain tumors has generated much interest. One of the earliest attempts at local polymer-delivered anti-angiogenesis agents used heparin and cortisone [119], which exhibits angiogenesis inhibition properties [120] when used in combination. Loading both drugs onto an EVAc copolymer and testing against the rabbit cornea VX2 carcinoma model, angiogenesis activity was reduced by 60% at 21 days after implantation (p ⬍ 0.05). In the same study, the drug combination loaded in pCPP:SA polymers inhibited growth in the rat flank 9L gliosarcoma model by 78% (p ⬍ 0.05). Squalamine, an aminosterol isolated from the dogfish shark, has been shown to exhibit anti-angiogenesis properties by inhibiting tumor mitogen-induced endothelial cell proliferation [121]. In the rabbit cornea VX2 carcinoma model, EVAc copolymers loaded with 20% (w/w) squalamine have been shown to inhibit vascular ingrowth. Currently, squalamine is being evaluated in clinical trials for a variety of advanced cancers [122]. Further work examining its efficacy in local polymer delivery is ongoing. Another anti-angiogenesis agent is minocycline, a broad-spectrum antibiotic with anti-collagenase properties [123]. When loaded onto the EVAc polymer and tested against the rabbit cornea VX2 carcinoma model, it inhibited neovascularization by a factor of 4.5, 4.4, and 2.9 on days 7, 14, and 21, respectively (p ⬍ 0.05 at all timepoints) [124]. In a follow-up study, the polymers were found to deliver minocycline in a sustained fashion with 55% of the drug released at 90 days [31]. With 50% (w/w) minocycline-loaded EVAc polymers challenging the rat intracranial 9L gliosarcoma model, median survival increased from 13 to 69 days (p ⬍ 0.001) when polymers were implanted simultaneously with tumor injection. When treated in the established rat intracranial 9L gliosarcoma model, minocycline-loaded polymers alone did not impact survival, but they did extend median survival by 43% when used in conjunction with surgical resection (p ⬍ 0.002), and by 90% with surgical resection and systemic BCNU. Recent work with 40–50% (w/w) minocycline-loaded PCPP:SA polymers suggest that their efficacy is significantly better than that of the EVAc polymers [125].

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Cyclophosphamide and 4-Hydroxyperoxycyclophosphamide Cyclophosphamide (Cytoxan) is an alkylating agent, widely used for the treatment of a variety of malignancies. Its active metabolite, hydroxycyclophosphamide, poorly crosses the BBB. Therefore, the drug has not been widely used for the treatment of malignant gliomas. Because cyclophosphamide requires enzymatic activation by the hepatic p450 cytochrome oxidase system, its potential for effective utilization of local-delivery polymer systems is low [126]. 4-hydroxyperoxycyclophosphamide (4-HC) is a derivative of cyclophosphamide, which spontaneously converts in vivo into the active metabolite [127], thus making it a candidate for polymeric local delivery. Because of 4-HC’s hydrophilic properties, the FAD:SA system was chosen for delivery. Pharmacokinetic and biodistribution studies demonstrated favorable release kinetics and intracranial distribution [128]. In the rat model, cerebral drug levels peaked between 5 and 20 days after implantation, in contrast to a rapid fall in levels 48 h after systemic dosing. After toxicity studies demonstrated the maximum tolerable polymer dose of 20% (w/w), efficacy was measured using the established rat intracranial 9L gliosarcoma and F98 glioma model [72]. Animals treated with the blank polymer had a median survival of 14 days with no long-term survivors, while animals treated with 4-HC loaded polymers had a median survival of 77 days with 40% surviving beyond 80 days (p ⫽ 0.004). Recently, it was shown that L-buthionine sulfoximine (BSO) potentiates the anti-tumor effects of 4-HC in the rat 9L gliosarcoma model [129] by inhibiting the glutathione S-transferase enzyme pathway, which appears to play an important role in the inactivation of alkylating agents [130]. In the established intracranial 9L gliosarcoma model, corelease of both 4-HC and BSO from the FAD:SA polymer boosted median survival 4.6 times greater than rats treated with empty polymer (61.5 vs. 13 days, p ⬍ 0.001), while release of 4-HC alone improved median survival only 2.3 times (p ⬍ 0.001). Systemic delivery of BSO in conjunction with polymer-delivered 4-HC did not impact survival. In addition, a separate experiment in this study showed that local delivery of BSO may be safer than systemic administration. BSO-loaded EVAc polymer implanted in a rat brain depleted intracranial glutathione levels without impacting hepatic levels, while systemic BSO delivery was shown to reduce hepatic glutathione levels without impacting intracranial levels. Adriamycin Adriamycin, an anthracycline antibiotic that intercalates with DNA, causing strand scission and double stranded cross breaks, exhibits tumoricidal activity on breast cancer, acute leukemia, lymphoma, and other cancers [131]. In 1983, following surgical debulking for malignant glioma, Nakazawa et al. [132]

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treated 20 patients with adriamycin injections to the tumor bed via an Ommaya reservoir. Total doses of 5.0–10.0 mg in daily 0.5-mg aliquots were injected into the reservoir. In addition, all patients received cobalt-60 irradiation and immunotherapy. This regimen achieved a one-year survival rate of 55%. Compared to systemic delivery, local infusion with Ommaya reservoirs increased the local concentration of adriamycin up to 38 times, and the drug was noted to penetrate up to 3 cm into the brain parenchyma. Subsequently, adriamycin was successfully incorporated into EVAc polymers in the shape of needles [133]. Pharmacokinetic studies demonstrated zero-order kinetics release, and further studies demonstrated that the needles significantly inhibited the growth of brain tumor xenografts in nude mice. Adriamycin has also been successfully incorporated into biodegradable pCPP:SA polymers [134]. In vitro assays demonstrated sustained release, and in vivo studies demonstrate improved median survival (33 vs. 13 days in controls, p ⬍ 0.0006) in the established rat intracranial 9L glioma model. Further animal studies of the adriamycin-loaded pCPP:SA polymers, including investigations with concurrent immunotherapy, are ongoing. 5-Fluorouracil 5-Fluorouracil (5-FU) is a thymidine analog that blocks the conversion of deoxyuridylic acid to thymidylic acid, thus depriving the cell of an essential precursor for DNA synthesis. While effective in treating various nonintracranial malignancies, its efficacy for brain tumors is limited by its systemic toxicities, which include myelosuppression and gastrointestinal mucosal injury [131]. In fact, early efficacy trials testing 5-FU’s role in treating brain tumors were disappointing [135–137]. There is a long history in the investigation of 5-FU local delivery. The earliest attempt was a 1968 study in which 5-FU was loaded into gelatin sponges and Surgicel [77]. While no evidence of toxicity was noted, no therapeutic effect was demonstrated. In 1979, pharmacokinetic studies of silastic tubes loaded with 5-FU and urokinase demonstrated continual release of both drugs for over 5 weeks [79]. When tested against the rat flank ethylnitrosourea induced gliomas, this drug-loaded silastic tube was found to be capable of inhibiting tumor growth. This study led to a clinical trial of 14 patients with malignant gliomas or metastatic disease. Thirteen patients lived for more than 8 months following implantation. Additional clinical trials using silastic tubes loaded with a ‘chemotherapy cocktail’ of 250 mg 5-FU, 6,000 IU urokinase, 1.5 mg mitomycin C, and 250 mg bromodeoxyuridine showed a median survival of 18 months for patients with malignant gliomas, with a 3-year survival rate of 16% [138, 139]. Clinically significant levels of 5-FU were measured as long as 2 years after implantation.

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In 1986, the first attempt to deliver 5-FU with polymers was made when several anti-neoplastic agents, including 5-FU, adriamycin, and mitomycin C, were loaded onto a matrix consisting of ‘glassified monomers’ with 10% polymetacrylic methyl acid and tested on 55 patients [140]. The one-year survival rate for malignant gliomas was 47%. 5-FU was then successfully loaded onto PLGA microspheres [141]. When tested against the established rat intracranial C6 glioma model, treatment with 5-FU-loaded microspheres significantly decreased mortality (p ⫽ 0.017), while treatment with placebo and bolus 5-FU injections had no effect [142]. There were no observed toxicities, and histological examination showed only mild tissue reaction. Menei et al. [143] reported treating 8 newly diagnosed GBM patients with surgical debulking and implantation of 5-FU-loaded PGLA microspheres. The patients also underwent postoperative adjuvant external beam radiation therapy. Clinically significant concentrations of 5-FU were measured in the CSF up to one month after surgery, while 5-FU levels in the blood were low and transitory. Median survival time was 98 weeks for the 8 patients, with 2 patients exhibiting disease remission at 139 and 153 weeks. Fluorodeoxyuridine, a compound related to 5-FU, has been successfully delivered from FAD-SA polymers in vitro and in vivo [144]. This study was based on the earlier work in which fluorodeoxyuridine was continuously infused with a Medtronic SynchroMed pump to treat a single patient with intracranial metastatic renal cell carcinoma [145]. A complete response was achieved in 3 months and maintained for 22 months. Methotrexate Methotrexate (MTX) is a folate antagonist widely used against a number of malignancies. Its effectiveness against brain tumors is limited, again due to impermeability across the BBB and systemic side effects, which include myelosuppression and gastrointestinal necrosis [131]. Once introduced to the brain parenchyma via direct injection, MTX disseminates widely in the brain [146]; its dissemination is comparatively poor with cisternal or intraventricular infusion [21, 147]. The first large-scale clinical trial of intracranial MTX was reported in 1987, where 269 patients were divided into five treatment groups, each receiving a different postoperative strategy following surgical resection [148]. One of the groups received ‘local chemotherapy,’ which was implantation of a Spongistan matrix soaked with 50 mg MTX. No side effects or complications were observed. While the local MTX had no statistically significant effect on overall survival, there were more long-term survivors in the group receiving local MTX than in the other groups. In an effort to improve stability and biodistribution, MTX has also been modified by covalent linking to dextran [149]. In vitro studies against the

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human H80 glioma line demonstrated that the presence of the conjugated dextran did not adversely affect tumoricidal properties. On a three-dimensional collagen lattice designed to simulate extracellular matrix, the MTX-dextran conjugate showed superior penetrance when compared to unmodified MTX. In the established rat intracranial 9L gliosarcoma model, FAD:SA polymers loaded with the MTX-dextran conjugate offered modest, but significant, improvement over controls. Other attempts to produce local delivery include loading MTX onto polymethylmethacrolate pellets [78] and PLGA copolymer matrices [150]. Despite release kinetics demonstrating release of 96–99% of the drug within only 2 days, the MTX-loaded pellets significantly improved median survival by 69% compared to controls in the rat intracranial ethylnitrosourea-induced tumor model. The MTX-loaded PLGA polymer inhibited glioma growth in the rat flank. A fibrin glue-based MTX system has also been introduced that inhibited glioma growth in the rat flank [76]. Although early clinical trials [148, 151] showed minimal toxicity of intratumoral MTX, other case reports have documented neurological side effects. For example, one patient with meningeal carcinoma developed an abulic-hypokinetic syndrome and left hemiparesis after receiving intraventricular MTX [152]. Two patients with MTX administered via an Ommaya reservoir developed large cysts at the catheter tip [153]. Platinum Drugs Carboplatin is a second generation platinum analog that causes myelosuppression when administered systemically, but is less neurotoxic than its parent compound, cisplatin [154]. Because of its solubility, carboplatin is optimally released by the FAD:SA polymer. Using the rat intracranial F98 glioma model, Olivi et al. [71] determined a maximum nontoxic dose of 5% (w/w) and then tested for efficacy. Carboplatin-loaded polymers increased median survival from 16 days in control animals (with all controls being dead by day 19) to 52 days. In a separate study, carboplatin polymers were assessed against various mouse metastatic brain tumors [84]. In combination with radiation therapy, carboplatin-loaded polymers prolonged survival against the CT26 colon carcinoma (median survival 33 vs. 20.5 days for controls, p ⫽ 0.013) and RENCA renal cell carcinoma (15 vs. 12 days, p ⬍ 0.01). The carboplatin-loaded polymers alone demonstrated efficacy against the B16 melanoma (27 vs. 16.5 days, p ⫽ 0.043), while combination with radiation was not effective. In an attempt similar to combining 4-HC and BSO, carboplatin has also been coupled with ␣-cyclodextrin to delay the decomposition and increase bioavailability [155]. Both agents were incorporated into ethylcellulose microcapsules at a 2.2% (w/w) loading, and pharmacokinetic assays demonstrated

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56% release of carboplatin over 110 days. When tested against the established rat intracranial F98 glioma, animals implanted with microcapsules loaded with only ␣-cyclodextrin exhibited a median survival of 20 days, compared to 34 days for microcapsules loaded with only carboplatin (p ⬍ 0.001), and 51 days for microcapsules loaded with both (p ⬍ 0.01 vs. carboplatin alone). Despite its neurotoxicity, there has been significant effort investigating the role of local delivery for carboplatin’s parent compound cisplatin. When loaded onto biodegradable polylactic acid polymers and tested with the established rat intracranial 9L gliosarcoma model, median survival was increased from 24 days in the control group to 32 days for animals treated with systemic cisplatin, 39 days for animals treated with local bolus infusions of cisplatin, and more than 60 days for the polymer delivery group (p ⬍ 0.00006 vs. systemic group; p ⬍ 0.001 vs. bolus group). Furthermore, histopathological ‘cure’ was seen in 8 of 12 cisplatin-loaded polymer animals versus 3 of 13 local bolus infusion animals (p ⬍ 0.01). No cures were seen in the other groups. Biocompatibility was confirmed in a follow-up study [38]. Dexamethasone Vasogenic edema is a major source of morbidity in brain tumors and is induced by malignant gliomas secondary to breakdown of the BBB [131]. High dose corticosteroid therapy can significantly alleviate the edema [156], but systemic sustained exposure leads to significant side effects, including diabetes mellitus, skin atrophy, Cushing’s syndrome, weight gain, hemorrhagic gastrointestinal ulcers, myopathies, osteoporosis, and pathological fractures [157]. When 35% dexamethasone (w/w) was loaded onto the EVAc copolymer, clinically significant delivery was demonstrated in the rat brain for up to 21 days [158]. Concurrent plasma levels were noted to be low. To assess efficacy, both systemic dexamethasone and drug-loaded EVAc polymers were tested in the rat intracranial 9L gliosarcoma-induced model [30]. Measuring edema as percentage water weight, it was demonstrated that both intracranial dexamethasoneloaded polymer (79.15%; p ⬍ 0.05) and intraperitoneal dexamethasone injections (79.16%; p ⬍ 0.05) were more efficacious compared to controls (79.45%) and intraperitoneal polymer implantation (79.39%). Bleomycin Bleomycin is a tumoricidal antibiotic used to treat testicular cancer, squamous cell carcinoma, and other malignancies. Dose-limiting systemic side effects include gastrointestinal and pulmonary toxicities. Intracranial polymeric delivery of bleomycin has been limited to two reports, where the drug was loaded into a compressed tablet form of lactose and encapsulated with ethylcellulose [159, 160]. In the canine intracranial model, the system demonstrated a release

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half life of 11 days, with CSF bleomycin levels detected for up to 20 days. In a Wistar rat intracranial glioma model, the tablet demonstrated statistically significant decreased tumor growth compared to systemically delivered bleomycin. The preparation was then placed into 6 patients undergoing craniopharyngioma resection and recurrence was prolonged in one patient. Other Neuro-Oncology-Related Agents Radiosensitizers, such as 5-iodo-2⬘-deoxyuridine (IudR), which act by replacing thymidine in replicating DNA, have been incorporated into pCPP:SA polymers and tested in animal models as adjuvants to radiation therapy [161, 162]. Anticonvulsants, such as phenytoin, have been successfully incorporated into the EVAc copolymer, and decreased cobalt-induced seizures in SpragueDawley rats [163]. Neither class of drug has seen clinical use in conjunction with local polymer drug delivery.

Other Neurosurgery Applications of Polymeric Drug Delivery

The neurosurgical applications for polymer-based drug delivery systems are not limited to treating the malignancies. A growing number of neurosurgical research endeavors to utilize this technology, including clinical applications for vasospasm, peripheral nerve injuries, and spinal fusion. Vasospasm Delayed vasospasm occurs in approximately 30% of patients 4–14 days following aneurysmal subarachnoid hemorrhage. Despite early surgical intervention and advances in microsurgical techniques and neuroimaging, it remains the leading cause of delayed morbidity and mortality in subarachnoid hemorrhage. The etiology of delayed vasospasm is unclear. However, there has been an increasing recent evidence suggesting that both inflammation and depleted levels of nitric oxide (NO) play a important role in its presentation [164, 165]. Ibuprofen, an anti-inflammatory agent, and (Z)-1-[2-(2-aminoethyl)N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA/NO), an NO donor molecule, have both been studied as potential drugs that can be loaded onto polymers to treat vasospasm. The anti-inflammatory properties of ibuprofen include its ability to inhibit the expression of certain cell adhesion molecules and therefore disrupt leukocyte endothelial cell interactions. However, dose-limiting side effects prevent its systemic administration in obtaining clinically significant levels in the CNS. In 1999, Thai et al. [166] loaded 50% ibuprofen (w/w) onto EVAc

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polymers and demonstrated the complete release of all drugs within a 12-day period. In the rat femoral artery model, ibuprofen-loaded polymers demonstrated statistically significant reduction in vasospasm compared to blank polymers, when the treatment was initiated within 6 h (98.0% vs. 69.2% vessel lumen patency, p ⬍ 0.002). No adverse reactions were observed in the animals. DETA/NO is a water soluble NO donor whose application in vasospasm treatment is under active investigation. A recent study performed by Aihara et al. [167] using a monkey intracranial subarachnoid hemorrhage model treated animals with continuous intercisternal infusion of DETA/NO via pumps for up to 7 days. No statistically significant reduction in vasospasm was encountered. However, it should be noted that in their treatment regimen, the reservoirs of the pumps were filled every 24 h with freshly prepared DETA in sterile water. Without a buffered solution, it is possible that the DETA/NO decomposed immediately on contact with aqueous carrier. On the other hand, by loading DETA/NO in EVAc polymers, this problem is avoided since the loaded drug is sequestered away from the aqueous environment due to the anhydrous nature of the EVAc lattice and will not decompose until it is released. In fact, Tamargo and colleagues [168] have shown that local 20% (w/w) DETA/NO-loaded polymers significantly reduces vasospasm in multiple animal models, including the rat femoral artery model, rabbit basilar artery model [169], and monkey intracranial model [170]. The 20% DETA/NOloaded polymers exhibited no apparent toxic side effects in any of the animal models, and further efficacy and toxicity studies are ongoing. Peripheral Nerve Injuries The incidence of obstetric brachial plexus injuries in the USA has been estimated to be as high as 2.5 per 1,000 live births [171]. Recently, using EVAc polymers loaded with neurotrophic factors glial cell-line-derived neurotrophic factor and brain-derived neurotrophic factor, Aszmann et al. [34] demonstrated with a rat neonate brachial plexus crush injury model that the combination of glial cell-line-derived neurotrophic factor/brain derived neurotrophic factor polymers resulted in dramatic motoneuron rescue effect as well as greater post-treatment strength and voluntary functionality. This finding suggests that the exogenous trophic support of motoneurons using polymer delivery systems may have a significant role in the treatment of all types of severe neonatal plexopathies. Spinal Fusion Bone morphogenic proteins (BMPs) represent a family of bone growth factors whose existence was first postulated as early as 1965 by Urist [172]. There

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has been much excitement over its possible clinical applications following its isolation, cloning, and sequencing. Active areas of research include its use in orthopedic surgery, plastic surgery and reconstruction, dentistry, and maxillofacial surgery. The most obvious clinical neurosurgical application of BMP is its role in spinal fusion (with or without instrumentation). In fact, investigations have recently culminated in the presentation of two clinical trials. Boden et al. [173] demonstrated significantly higher fusion rates in Grade I spondylolisthesis when recombinant BMP with biphasic calcium phosphate granules were introduced in posterolateral fusion procedures with or without fixation. (Also see chapter by Kang et al. in this volume.) However, Johnsson et al. [174] discovered no significant effect with an implant consisting of recombinant BMP reconstituted in a Type I bone collagen carrier for noninstrumented posterolateral fusion of Grade I-II L5-S1 spondylolisthesis. With differences in results between these studies, it is clear that the clinical role of BMP in spinal fusion will remain an active area of interest in the near future. There have been multiple trials investigating the use of polymer-based systems to deliver BMP in a spinal fusion setting (e.g., [175, 176]). Most use some form of PLGA polymer system; animal models include sheep, canine, and rabbit. To date, the research has not reached the level of human clinical trials.

Future Directions

Polymer-based delivery systems represent a proof of principle that controlled drug delivery can play a significant role in the treatment of various neurosurgical pathologies, including malignancies. Active investigation is ongoing in several other exciting approaches to sustained drug delivery in the brain. Convection-Enhanced Delivery Systems In tissue, compounds travel by diffusion, which is dependent on both the free concentration gradient and the diffusivity of the compound in the tissue. Convection, which can be used to supplement diffusion, relies on a simple pressure gradient, and is independent of molecular weight. When a drug is infused into the cerebral white matter, a pressure gradient is created, and can be used to introduce high concentrations of drug throughout the brain without structural or functional side effects [177–179]. Primate trials investigating the treatment of Parkinson’s disease symptoms have been conducted using convection-enhanced drug delivery (CEDD) [180]. (Also see chapter by Bankiewicz in this volume.) A recent study using CEDD to deliver the monoclonal antibody trastuzumab to treat metastatic breast cancer in a rat model demonstrated an increase of median

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survival of 52 days as compared to 26.5 days for intraperitoneal injection of trastuzumab (p ⫽ 0.009) and 16 days for controls [181]. Recently, CEDD was used in a study of taxol to treat 3 brain tumor patients [182]. The investigation focused on the use of diffusion-weighted MRI to monitor the effects of drug delivery. Microchip Drug Delivery A novel method of drug delivery with clinical potential is the use of newly developed microchips to control drug delivery (fig. 8a) [183]. Using solid-state silicon technology, microchip systems provide controlled release of multiple microreservoirs, thus providing for single or multiple agent delivery. Drugs, in the form of solids, liquids, or gels are released by electrochemical dissolution of a thin anode membrane covering each microreservoir. The delivery time of each reservoir can be programmed independently, thus providing for a seemingly endless permutation of release profiles and therapy combinations. The device is an integrated circuit, capable of providing its own microbattery, memory, and processing. It can be implanted surgically, mounted on the tip of a small probe, or even swallowed. Current research in this technology includes efficacy and biocompatibility studies. Alternative biodegradable ‘passive chips’ (fig. 8b) are also being developed based solely on biodegradable polymer technology [184]. Instead of an integrated silicon based circuit, the release mechanism of each microreservoir is controlled by slow degradation of a thin polymeric membrane covering each reservoir. Like the active microchip described above, current research includes efficacy and biocompatibility studies. Both ‘pharmacy-on-a-chip’ systems may potentially be used to deliver up to 1,000 different drugs on demand.

Conclusions and Future Directions in Solid Phase Drug Delivery

Tumors of the CNS represent a significant pharmacological and clinical challenge. The physiological barriers that isolate the CNS make difficult the delivery of high tumoricidal CNS concentrations of anti-neoplastic agents without causing unacceptably toxic systemic levels of drug. Biodegradable polymer technology has allowed a new approach to treating brain tumors. Gliadel, the 3.85% (w/w) BCNU-loaded pCPP:SA (20:80) polymer, represents the first successful delivery system developed from this technology, and the first new treatment for malignant gliomas approved by the FDA in two decades. Many other drug-polymer combinations are now undergoing active investigation. These studies are focusing on combinations of local-delivery approaches, such as the promising mix of immunotherapy and chemotherapy. Still newer technologies,

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Anode

Silicon nitride or dioxide

Silicon

Cathode Active substance Small reservoir opening (usually covered by gold membrane) Silicon side wall

b

a

Large reservoir opening (for reservoir filling)

Fig. 8. a Microchip. Front (left) and back views of a new microchip for controlled local release of chemicals. Dots between the three large bars (cathodes) on the front are the caps (anodes) covering the reservoirs holding the chemicals. Electrical voltage applied between the cap and cathode causes a reaction that dissolves the cap, releasing the reservoir’s contents. The back view shows larger openings through which the contents of the reservoirs are deposited (these openings are sealed after filling.) Photo by Paul Horwitz, Atlantic Photo Service, Inc. b Schematic of passive microchip. Initial models use PLGA and other existing polymer matrices for the substrate and the entire chip will be biodegradable.

such as the microchip and CEDD, may hopefully enhance the contributions that polymer drug delivery systems have made. The future of neurosurgical oncology is an exciting one. With the introduction of new technology, perhaps when a patient undergoes an operative resection/ debulking of a malignant tumor, his treatment may soon include a microchip programmed and loaded with a combination of chemotherapy agents tailored to intraoperative pathology and molecular biological diagnosis of the tumor. Drugs such as dexamethasone and phenytoin could be loaded onto several microresevoirs to treat cerebral edema and prevent postoperative seizures. Cytokineloaded microspheres and irradiated tumor cells from resected specimens could be placed directly onto the tumor site, or loaded onto other wells of the chip. The microchip(s) could then be implanted into the tumor cavity intraoperatively. Should there be a recurrence, stereotactic biopsy for diagnosis and biological properties could be followed by the implantation of microspheres loaded

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with angiogenesis inhibitors and additional chemotherapy agents. Such exciting possibilities for both the patient and neurosurgeon have been made possible by the development of local and controlled drug delivery to the CNS. Acknowledgements Research presented in this work has been supported by the National Cooperative Drug Discovery Group (UO1-CA52857 and AI 47739) of the National Cancer Institute (NCI-NIH), Bethesda, MD. Dr. Wang is a neurosurgery resident and neuro-oncology surgery fellow whose research is supported by a grant (T32 CA-09574) from the National Institute of Health/The Johns Hopkins University, Bethesda/Baltimore, Md., USA. Under a licensing agreement between Guilford Pharmaceuticals and the Johns Hopkins University (JHU), Dr. Brem is entitled to a share of royalty received by the University on sales of products described in this work. Dr. Brem and JHU own Guilford Pharmaceuticals stock, which is subject to certain restrictions under University policy. Dr. Brem is also a paid consultant to Guilford Pharmaceuticals. The terms of this arrangement are being managed by JHU in accordance with conflict of interest policies.

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35 36 37

38 39

40

41 42

43

44

45

46 47

48

49

50 51 52

53

54

55

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Polymeric Drug Delivery Systems

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56 57 58 59 60

61

62 63 64 65 66 67

68 69

70 71

72

73 74 75

76

77 78

79

Brem H, Langer R: Polymer-based drug delivery to the brain. Sci Med 1996;July/August:52–61. Mathiowitz E, Saltzman M, Domb A: Polyanhydride microspheres as drug carriers. II. Microencapsulation by solvent removal. J Appl Polym Sci 1988;35:755–774. Mathiowitz E, Langer R: Polyanhydride microspheres as drug carriers. I. Hot-melt microencapsulation. J Control Release 1987;5:13–22. Bindschaedler C, Leong K, Mathiowitz E, Langer R: Polyanhydride microsphere formulation by solvent extraction. J Pharm Sci 1988;77:696–698. Chasin M, Domb A, Ron E: Polyanhydrides as drug delivery systems; in Chasin M, Langer R (eds): Biodegradable Polymers as Drug Delivery Systems. New York, Marcel Dekker, 1990, pp 43–70. Howard MA III, Gross A, Grady MS, Langer RS, Mathiowitz E, Winn HR, Mayberg MR: Intracerebral drug delivery in rats with lesion-induced memory deficits. J Neurosurg 1989;71: 105–112. Leong KW, Kost J, Mathiowitz E, Langer R: Polyanhydrides for controlled release of bioactive agents. Biomaterials 1986;7:364–371. Mathiowitz E, Kline D, Langer R: Morphology of polyanhydride microsphere delivery systems. Scanning Microsc 1990;4:329–340. Leong KW, D’Amore PD, Marletta M, Langer R: Bioerodible polyanhydrides as drug-carrier matrices. II. Biocompatibility and chemical reactivity. J Biomed Mater Res 1986;20:51–64. Tamargo RJ, Epstein JI, Reinhard CS, Chasin M, Brem H: Brain biocompatibility of a biodegradable, controlled-release polymer in rats. J Biomed Mater Res 1989;23:253–266. Brem H, Kader A, Epstein JI, Tamargo RJ, Domb A, Langer R, Leong KW: Biocompatibility of a biodegradable, controlled-release polymer in the rabbit brain. Sel Cancer Ther 1989;5:55–65. Brem H, Tamargo RJ, Olivi A, Pinn M, Weingart JD, Wharam M, Epstein JI: Biodegradable polymers for controlled delivery of chemotherapy with and without radiation therapy in the monkey brain. J Neurosurg 1994;80:283–290. Dang W, Saltzman WM: Dextran retention in the rat brain following release from a polymer implant. Biotechnol Prog 1992;8:527–532. Domb A, Bogdansky S, Olivi A, Judy K, Dureza C, Lenartz D, Pinn MI, Colvin M, Brem H: Controlled delivery of water soluble and hydrolytically unstable anti-cancer drugs for polymeric implants. Polymer Prepri 1991;32:219–220. Shieh L, Tamada J, Chen I, Pang J, Domb A, Langer R: Erosion of a new family of biodegradable polyanhydrides. J Biomed Mater Res 1994;28:1465–1475. Olivi A, Ewend MG, Utsuki T, Tyler B, Domb AJ, Brat DJ, Brem H: Interstitial delivery of carboplatin via biodegradable polymers is effective against experimental glioma in the rat. Cancer Chemother Pharmacol 1996;39:90–96. Judy KD, Olivi A, Buahin KG, Domb A, Epstein JI, Colvin OM, Brem H: Effectiveness of controlled release of a cyclophosphamide derivative with polymers against rat gliomas. J Neurosurg 1995;82:481–486. Shikani AH, Eisele DW, Domb AJ: Polymer delivery of chemotherapy for squamous cell carcinoma of the head and neck. Arch Otolaryngol Head Neck Surg 1994;120:1242–1247. Gabizon AA: Liposomal anthracyclines. Hematol Oncol Clin North Am 1994;8:431–450. Golumbek PT, Azhari R, Jaffee EM, Levitsky HI, Lazenby A, Leong K, Pardoll DM: Controlled release, biodegradable cytokine depots: A new approach in cancer vaccine design. Cancer Res 1993; 53:5841–5844. Hirakawa W, Kadota K, Asakura T, Niiro M, Yokoyama S, Hirano H, Yatsushiro K, Kubota Y, Shimodozono Y: Local chemotherapy for malignant brain tumors using methotrexate-containing fibrin glue. Gan To Kagaku Ryoho 1995;22:805–809. Ringkjob R: Treatment of intracranial gliomas and metastatic carcinomas by local application of cytostatic agents. Acta Neurol Scand 1968;44:318–322. Rama B, Mandel T, Jansen J, Dingeldein E, Mennel HD: The intraneoplastic chemotherapy in a rat brain tumour model utilizing methotrexate-polymethylmethacrylate-pellets. Acta Neurochir 1987;87:70–75. Oda Y, Tokuriki Y, Tsuda E, Handa H, Kieler J: Trial of anticancer pellet in malignant brain tumours: 5 FU and urokinase embedded in silastic. Acta Neurochir Suppl 1979;28:489–490.

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80

81

82 83

84

85 86

87

88

89

90

91

92

93

94

95

96

97

98

Grossman SA, Reinhard C, Colvin OM, Chasin M, Brundrett R, Tamargo RJ, Brem H: The intracerebral distribution of BCNU delivered by surgically implanted biodegradable polymers. J Neurosurg 1992;76:640–647. Fung LK, Ewend MG, Sills A, Sipos EP, Thompson R, Watts M, Colvin OM, Brem H, Saltzman WM: Pharmacokinetics of interstitial delivery of carmustine, 4-hydroperoxycyclophosphamide, and paclitaxel from a biodegradable polymer implant in the monkey brain. Cancer Res 1998;58: 672–684. Buahin KG, Brem H: Interstitial chemotherapy of experimental brain tumors: Comparison of intratumoral injection versus polymeric controlled release. J Neurooncol 1995;26:103–110. Sipos EP, Tyler B, Piantadosi S, Burger PC, Brem H: Optimizing interstitial delivery of BCNU from controlled release polymers for the treatment of brain tumors. Cancer Chemother Pharmacol 1997;39:383–389. Ewend MG, Williams JA, Tabassi K, Tyler BM, Babel KM, Anderson RC, Pinn ML, Brat DJ, Brem H: Local delivery of chemotherapy and concurrent external beam radiotherapy prolongs survival in metastatic brain tumor models. Cancer Res 1996;56:5217–5223. Ewend MG, Sampath P, Williams JA, Tyler BM, Brem H: Local delivery of chemotherapy prolongs survival in experimental brain metastases from breast carcinoma. Neurosurgery 1998; 43:1185–1193. Brem H, Mahaley MS Jr, Vick NA, Black KL, Schold SC Jr, Burger PC, Friedman AH, Ciric IS, Eller TW, Cozzens JW, Kenealy JN: Interstitial chemotherapy with drug polymer implants for the treatment of recurrent gliomas. J Neurosurg 1991;74:441–446. Hammoud DA, Belden CJ, Ho AC, Dal Pan GJ, Herskovits EH, Hilt DC, Brem H, Pomper MG: The surgical bed after BCNU polymer wafer placement for recurrent glioma: Serial assessment on CT and MR imaging. AJR Am J Roentgenol 2003;180:1469–1475. Brem H, Piantadosi S, Burger PC, Walker M, Selker R, Vick NA, Black K, Sisti M, Brem S, Mohr G, Muller P, Morawetz R, Schold SC: Placebo-controlled trial of safety and efficacy of intraoperative controlled delivery by biodegradable polymers of chemotherapy for recurrent gliomas. The Polymer-brain Tumor Treatment Group. Lancet 1995;345:1008–1012. Brem H, Ewend MG, Piantadosi S, Greenhoot J, Burger PC, Sisti M: The safety of interstitial chemotherapy with BCNU-loaded polymer followed by radiation therapy in the treatment of newly diagnosed malignant gliomas: Phase I trial. J Neurooncol 1995;26:111–123. Valtonen S, Timonen U, Toivanen P, Kalimo H, Kivipelto L, Heiskanen O, Unsgaard G, Kuurne T: Interstitial chemotherapy with carmustine-loaded polymers for high-grade gliomas: A randomized double-blind study. Neurosurgery 1997;41:44–48. Westphal M, Hilt DC, Bortey E, Delavault P, Olivares R, Warnke PC, Whittle IR, Jääskeläinen J, Ram Z: A phase 3 trial of local chemotherapy with biodegradable carmustine (BCNU) wafers (Gliadel wafers) in patients with primary malignant glioma. J Neurooncol 2003;5. Olivi A, Grossman SA, Tatter S, Barker FG 2nd, Judy K, Olson J, Hilt D, Fisher JD, Piantadosi S: Results of a phase I dose escalation study using BCNU impregnated polymers in patients with recurrent malignant gliomas. J Clin Oncol 2003;in press. Silber JR, Bobola MS, Ghatan S, Blank A, Kolstoe DD, Berger MS: O6-methylguanine-DNA methyltransferase activity in adult gliomas: Relation to patient and tumor characteristics. Cancer Res 1998;58:1068–1073. Pegg AE, Boosalis M, Samson L, Moschel RC, Byers TL, Swenn K, Dolan ME: Mechanism of inactivation of human O6-alkylguanine-DNA alkyltransferase by O6-benzylguanine. Biochemistry 1993;32:11998–12006. Page JG, Giles HD, Phillips W, Gerson SL, Smith AC, Tomaszewski JE: Preclinical toxicology study of O6-benzylguanine (NSC-637037) and BCNU (carmustine, NSC-409962) in male and female beagle dogs. Presented at Proc Am Assoc Cancer Res, 1993. Rhines LD, Sampath P, Dolan ME, Tyler BM, Brem H, Weingart J: O6-benzylguanine potentiates the antitumor effect of locally delivered carmustine against an intracranial rat glioma. Cancer Res 2000;60:6307–6310. Rosenblum M, Weingart J, Dolan E, Tatter S, Judy K, Olson J, Bohan E, Fisher J: Phase I study of Gliadel combined with a continuous intravenous infusion of O6-benzylguanine in patients with recurrent malignant glioma. Presented at 38th ASCO Annual Meeting, Orlando, FL, 2002. Cahan MA, Walter KA, Colvin OM, Brem H: Cytotoxicity of taxol in vitro against human and rat malignant brain tumors. Cancer Chemother Pharmacol 1994;33:441–444.

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99 Klecker R, Jamis-Dow C, Egorin M: Distribution and metabolism of 3-H Taxol in the rat (abstract). Proc Am Assoc Cancer Res 1992;34:381. 100 Walter KA, Cahan MA, Gur A, Tyler B, Hilton J, Colvin OM, Burger PC, Domb A, Brem H: Interstitial taxol delivered from a biodegradable polymer implant against experimental malignant glioma. Cancer Res 1994;54:2207–2212. 101 Harper EWD, Lapidus RG, Garver RI Jr: Enhanced efficacy of a novel controlled release paclitaxel formulation (PACLIMER delivery system) for local-regional therapy of lung cancer tumor nodules in mice. Clin Cancer Res 1999;5:4242–4248. 102 Gabikian P, Li KW, Magee C, Morrell C, Tyler BM, Foster F, Dang W, Brem H, Walter KA: Safety of intracranial paclitaxel: Polilactofate microspheres in dogs (poster). Presented at American Association of Neurological Surgeons Annual Meeting, Chicago, IL, 2002. 103 Hsiang YH, Liu LF: Identification of mammalian DNA topoisomerase I as an intracellular target of the anticancer drug camptothecin. Cancer Res 1988;48:1722–1726. 104 Slichenmyer WJ, Rowinsky EK, Donehower RC, Kaufmann SH: The current status of camptothecin analogues as antitumor agents. J Natl Cancer Inst 1993;85:271–291. 105 Storm PB, Moriarity JL, Tyler B, Burger PC, Brem H, Weingart JD: Polymer delivery of camptothecin against 9L gliosarcoma: Release, distribution, and efficacy. J Neurooncol 2002;in press. 106 Sampath P, Amundson E, Wall ME, Tyler BM, Wani MC, Alderson LM, Colvin M, Brem H, Weingart JD: Camptothecin analogs in malignant gliomas: Comparative analysis and characterization. J Neurosurg 2003;98:570–577. 107 Giampietri A, Fioretti MC, Goldin A, Bonmassar E: Drug-mediated antigenic changes in murine leukemia cells: Antagonistic effects of quinacrine, an antimutagenic compound. J Natl Cancer Inst 1980;64:279–301. 108 Reyes S, Herrera LA, Ostrosky P, Sotelo J: Quinacrine enhances carmustine therapy of experimental rat glioma. Neurosurgery 2001;49. 109 DiMeco F, Li KW, Tyler BM, Wolf AS, Brem H, Olivi A: Local delivery of mitoxantrone for the treatment of malignant brain tumors in rats. J Neurosurg 2002;97:1173–1178. 110 Thompson RC, Pardoll DM, Jaffee EM, Ewend MG, Thomas MC, Tyler BM, Brem H: Systemic and local paracrine cytokine therapies using transduced tumor cells are synergistic in treating intracranial tumors. J Immunother Emphasis Tumor Immunol 1996;19:405–413. 111 Ewend MG, Thompson RC, Anderson R, Sills AK, Staveley-O’Carroll K, Tyler BM, Hanes J, Brat D, Thomas M, Jaffee EM, Pardoll DM, Brem H: Intracranial paracrine interleukin-2 therapy stimulates prolonged antitumor immunity that extends outside the central nervous system. J Immunother 2000; 23:438–448. 112 DiMeco F, Rhines LD, Hanes J, Tyler BM, Brat D, Torchiana E, Guarnieri M, Colombo MP, Pardoll DM, Finocchiaro G, Brem H, Olivi A: Paracrine delivery of IL-12 against intracranial 9L gliosarcoma in rats. J Neurosurg 2000;92:419–427. 113 Sampath P, Hanes J, DiMeco F, Tyler BM, Brat D, Pardoll DM, Brem H: Paracrine immunotherapy with interleukin-2 and local chemotherapy is synergistic in the treatment of experimental brain tumors. Cancer Res 1999;59:2107–2114. 114 Wiranowska M, Ransohoff J, Weingart JD, Phelps C, Phuphanich S, Brem H: Interferon-containing controlled-release polymers for localized cerebral immunotherapy. J Interferon Cytokine Res 1998;18:377–385. 115 Hanes J, Sills A, Zhao Z, Suh KW, Tyler B, DiMeco F, Brat DJ, Choti MA, Leong KW, Pardoll DM, Brem H: Controlled local delivery of interleukin-2 by biodegradable polymers protects animals from experimental brain tumors and liver tumors. Pharm Res 2001;18:899–906. 116 Rhines LD, DiMeco F, Lawson HC, Tyler BM, Hanes J, Olivi A, Brem H: Local immunotherapy with interleukin-2 delivered from biodegradable polymer microspheres combined with interstitial chemotherapy: A novel treatment for experimental malignant glioma. Neurosurgery 2003; 52:1–8. 117 Folkman J: Tumor angiogenesis: Therapeutic implications. N Engl J Med 1971;285:1182–1186. 118 Gimbrone MA Jr, Leapman SB, Cotran RS, Folkman J: Tumor dormancy in vivo by prevention of neovascularization. J Exp Med 1972;136:261–276. 119 Tamargo RJ, Leong KW, Brem H: Growth inhibition of the 9L glioma using polymers to release heparin and cortisone acetate. J Neurooncol 1990;9:131–138.

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120 Folkman J, Langer R, Linhardt RJ, Haudenschild C, Taylor S: Angiogenesis inhibition and tumor regression caused by heparin or a heparin fragment in the presence of cortisone. Science 1983; 221:719–725. 121 Sills AK Jr, Williams JI, Tyler BM, Epstein DS, Sipos EP, Davis JD, McLane MP, Pitchford S, Cheshire K, Gannon FH, Kinney WA, Chao TL, Donowitz M, Laterra J, Zasloff M, Brem H: Squalamine inhibits angiogenesis and solid tumor growth in vivo and perturbs embryonic vasculature. Cancer Res 1998;58:2784–2792. 122 Bhargava P, Marshall JL, Dahut W, Rizvi N, Trocky N, Williams JI, Hait H, Song S, Holroyd KJ, Hawkins MJ: A phase I and pharmacokinetic study of squalamine, a novel antiangiogenic agent, in patients with advanced cancers. Clin Cancer Res 2001;7:3912–3919. 123 Golub LM, Wolff M, Lee HM, McNamara TF, Ramamurthy NS, Zambon J, Ciancio S: Further evidence that tetracyclines inhibit collagenase activity in human cervicular fluid and from other mammalian sources. J Periodontal Res 1985;20:12–23. 124 Tamargo RJ, Bok RA, Brem H: Angiogenesis inhibition by minocycline. Cancer Res 1991;51: 672–675. 125 Frazier JL, Wang PP, Case D, Tyler BM, Pradilla G, Weingart JD, Brem H: Local delivery of minocycline and systemic BCNU have synergistic activity in the treatment of intracranial glioma. J Neurooncol 2003;in press. 126 Chabner BA, Collins JM (eds): Cancer Chemotherapy: Principles and Practice. Philadelphia, Lippencott, 1990. 127 Colvin M, Hilton J: Pharmacology of cyclophosphamide and metabolites. Cancer Treat Rep 1981; 65(suppl 3):89–95. 128 Buahin KG, Judy KD, Hartke C, Domb A, Maniar M, Colvin O, Brem H: Controlled release of 4-hydroperoxycyclophosphamide from the fatty acid dimer-sebacic acid copolymer. Polym Adv Technol 1992;3:311. 129 Sipos EP, Witham TF, Ratan R, Burger PC, Baraban J, Li KW, Piantadosi S, Brem H: L-buthionine sulfoximine potentiates the antitumor effect of 4-hydroperoxycyclophosphamide when administered locally in a rat glioma model. Neurosurgery 2001;48:392–400. 130 Colvin OM, Friedman HS, Gamcsik MP, Fenselau C, Hilton J: Role of glutathione in cellular resistance to alkylating agents. Adv Enzyme Regul 1993;33:19–26. 131 Goodman LS, Hardman JG, Limbird LE, Gilman AG: Goodman and Gilman’s: The pharmacological basis of therapeutics. New York, McGraw-Hill, 2001. 132 Nakazawa S, Itoh Y, Shimura T, Matsumoto M, Yajima K: New management of brain neoplasms. II. Local injection of adriamycin. No Shinkei Geka 1983;11:821–827. 133 Lin SY, L.F. C, Lui WY, Chen CF, Han SH: Tumoricidal effect of controlled-release polymeric needle devices containing adriamycin HCl in tumor-bearing mice. Biomater Artif Cells Artif Organs 1989;17:189–203. 134 Watts MC, Lesniak MS, Burke M, Samdani AF, Tyler BM, Brem H: Controlled release of adriamycin in the treatment of malignant glioma (poster). Presented at American Association of Neurological Surgeons Annual Meeting, Denver, CO, 1997. 135 Levin VA, Edwards MS, Wara WM, Allen J, Ortega J, Vestnys P: 5-Fluorouracil and 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea (CCNU) followed by hydroxyurea, misonidazole, and irradiation for brain stem gliomas: A pilot study of the Brain Tumor Research Center and the Childrens Cancer Group. Neurosurgery 1984;14:679–681. 136 Shapiro WR: Studies on the chemotherapy of experimental brain tumors: Evaluation of 1-(2-chloroethyl)-3-cyclohexyl-1-nitrosourea, vincristine, and 5-fluorouracil. J Natl Cancer Inst 1971;46:359–368. 137 Shapiro WR, Green SB, Burger PC, Selker RG, VanGilder JC, Robertson JT, Mealey J Jr, Ransohff J, Mahaley MS Jr: A randomized comparison of intra-arterial versus intravenous BCNU, with or without intravenous 5-fluorouracil, for newly diagnosed patients with malignant glioma. J Neurosurg 1992;76:772–781. 138 Oda Y, Uchida Y, Murata T, Mori K, Tokuriki Y, Handa H, Kobayashi A, Hashi K, Kieler J: Treatment of brain tumors with anticancer pellet – Experimental and clinical study. No Shinkei Geka 1982;10: 375–381.

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139 Oda Y, Kamijyo Y, Okumura T, Tokuriki Y, Yamashita J, Handa H, Aoyama I, Hashi K, Mori K: Clinical application of a sustained release anticancer pellet. No Shinkei Geka 1985;13: 1305–1311. 140 Kubo O, Himuro H, Inoue N, Tajika Y, Tajika T, Tohyama T, Sakairi M, Yoshida M, Kaetsu I, Kitamura K: Treatment of malignant brain tumors with slowly releasing anticancer drug-polymer composites. No Shinkei Geka 1986;14:1189–1195. 141 Boisdron-Celle M, Menei P, Benoit JP: Preparation and characterization of 5-fluorouracilloaded microparticles as biodegradable anticancer drug carriers. J Pharm Pharmacol 1995;47: 108–114. 142 Menei P, Boisdron-Celle M, Croue A, Guy G, Benoit JP: Effect of stereotactic implantation of biodegradable 5-fluorouracil-loaded microspheres in healthy and C6 glioma-bearing rats. Neurosurgery 1996;39:117–124. 143 Menei P, Venier MC, Gamelin E, Saint-Andre JP, Hayek G, Jadaud E, Fournier D, Mercier P, Guy G, Benoit JP: Local and sustained delivery of 5-fluorouracil from biodegradable microspheres for the radiosensitization of glioblastoma: A pilot study. Cancer 1999;86:325–330. 144 Choti MA, Saenz J, Yang X, Brem H: Intrahepatic FUdR delivered from biodegradable polymer in experimental liver metastases from colorectal carcinoma. Presented at Proc of AACR, 1995. 145 Damascelli B, Marchiano A, Frigerio LF, Salvetti M, Spreafico C, Garbagnati F, Zanoni F, Radice F: Flexibility and efficacy of automatic continuous fluorodeoxyuridine infusion in metastases from a renal cell carcinoma. Cancer 1991;68:995–998. 146 Sendelbeck SL, Urquhart J: Spatial distribution of dopamine, methotrexate and antipyrine during continuous intracerebral microperfusion. Brain Res 1985;328:251–258. 147 Blasberg RG, Patlak CS, Shapiro WR: Distribution of methotrexate in the cerebrospinal fluid and brain after intraventricular administration. Cancer Treat Rep 1977;61:633–641. 148 Diemath HE: Local use of cytostatic drugs following removal of glioblastomas. Wien Klin Wochenschr 1987;99:674–676. 149 Dang W, Colvin OM, Brem H, Saltzman WM: Covalent coupling of methotrexate to dextran enhances the penetration of cytotoxicity into a tissue-like matrix. Cancer Res 1994;54:1729–1735. 150 Zeller WJ, Bauer S, Remmele T, Wowra B, Sturm V, Stricker H: Interstitial chemotherapy of experimental gliomas. Cancer Treat Rep 1990;7:183–189. 151 Weiss SR, Raskind R: Treatment of malignant brain tumors by local methotrexate. A preliminary report. Int Surg 1969;51:149–155. 152 Uldry PA, Teta D, Regli L: Focal cerebral necrosis caused by intraventricular chemotherapy with methotrexate. Neurochirurgia 1991;37:72–74. 153 Shimura T, Nakazawa S, Ikeda Y, Node Y: Cyst formation following local chemotherapy of malignant brain tumor: A clinicopathological study of two cases. No Shinkei Geka 1992;20: 1179–1183. 154 Olivi A, Gilbert M, Duncan KL, Corden B, Lenartz D, Brem H: Direct delivery of platinum-based antineoplastics to the central nervous system: A toxicity and ultrastructural study. Cancer Chemother Pharmacol 1993;31:449–454. 155 Utsuki T, Brem H, Pitha J, Loftsson T, Kristmundsdottir T, Tyler BT, Olivi A: Potentiation of anticancer effects of microencapsulated carboplatin by hydroxypropyl ␣-cyclodextrin. J Control Release 1996;40:251–260. 156 Maxell RE, Long DM, French LA: The clinical effects of a synthetic gluco-corticoid used for brain edema in the practice of neurosurgery; in Reulen HJ, Schurmann K (eds): Steroids and Brain Edema. Berlin, Springer-Verlag, 1972, pp 219–232. 157 Melby JC: Drug spotlight program: Systemic corticosteroid therapy: Pharmacology and endocrinologic considerations. Ann Intern Med 1974;81:505–512. 158 Reinhard CS, Radomsky ML, Saltzman WM, Brem H: Polymeric controlled release of dexamethasone in normal rat brain. J Control Rel 1991;16:331–340. 159 Katakura R, Mori T, Mineura K, Suzuki J: A device for prolonged releasing of anticancer drug–bleomycin. No Shinkei Geka 1980;8:1057–1062. 160 Katakura R, Mori T, Suzuki J: A device for prolonged releasing of anticancer drug–bleomycin: Second report. No Shinkei Geka 1982;10:941–944.

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161 Williams JA, Dillehay LE, Tabassi K, Sipos E, Fahlman C, Brem H: Implantable biodegradable polymers for IUdR radiosensitization of experimental human malignant glioma. J Neurooncol 1997;32:181–192. 162 Williams JA, Yuan X, Dillehay LE, Shastri VR, Brem H, Williams JR: Synthetic, implantable polymers for local delivery of IUdR to experimental human malignant glioma. Int J Radiat Oncol Biol Phys 1998;42:631–639. 163 Tamargo RJ, Rossell LA, Tyler BM, Aryanpur JJ: Interstitial delivery of diphenylhydantoin in the brain for the treatment of seizures in the rat model (poster). Presented at American Association of Neurological Surgeons Annual Meeting, San Diego, CA, 1994. 164 Polin RS, Bavbek M, Shaffrey ME, Billups K, Bogaev CA, Kassell NF, Lee KS: Detection of soluble E-selectin, ICAM-1, VCAM-1, and L-selectin in the cerebrospinal fluid of patients after subarachnoid hemorrhage. J Neurosurg 1998;89:559–567. 165 Pluta RM, Thompson BG, Dawson TM, Snyder SH, Boock RJ, Oldfield EH: Loss of nitric oxide synthase immunoreactivity in cerebral vasospasm. J Neurosurg 1996;84:648–654. 166 Thai QA, Oshiro EM, Tamargo RJ: Inhibition of experimental vasospasm in rats with the periadventitial administration of ibuprofen using controlled-release polymers. Stroke 1999;30: 140–147. 167 Aihara Y, Jahromi BS, Yassari R, Sayama T, Macdonald RL: Effects of a nitric oxide donor on and correlation of changes in cyclic nucleotide levels with experimental vasospasm. Neurosurgery 2003;52:661–667. 168 Tierney TS, Clatterbuck RE, Lawson C, Thai QA, Rhines LD, Tamargo RJ: Prevention and reversal of experimental posthemorrhagic vasospasm by the periadventitial administration of nitric oxide from a controlled-release polymer. Neurosurgery 2001;49:945–951; discussion 943–951. 169 Gabikian P, Clatterbuck RE, Eberhart CG, Tyler BM, Tierney TS, Tamargo RJ: Prevention of experimental cerebral vasospasm by intracranial delivery of a nitric oxide donor from a controlled-release polymer: Toxicity and efficacy studies in rabbits and rats. Stroke 2002;33: 2681–2686. 170 Clatterbuck RE, Gailloud P, Tierney TS, Murphy K, Tamargo RJ: Controlled release of nitric oxide donor DETA-NO prevents cerebral vasospasm following experimental subarachnoid hemorrhage in monkeys. Neurosurgery 2002;51:545–546. 171 Jackson ST, Hoffer MM, Parrish N: Brachial-plexus palsy in the newborn. J Bone Joint Surg Am 1988;70:1217–1220. 172 Urist MR: Bone: Formation by autoinduction. Science 1965;150:893–899. 173 Boden SD, Kang J, Sandhu H, Heller JG: Use of recombinant human bone morphogenetic protein-2 to achieve posterolateral lumbar spine fusion in humans: A prospective, randomized clinical pilot trial: 2002 Volvo Award in clinical studies. Spine 2002;27:2662–2673. 174 Johnsson R, Stromqvist B, Aspenberg P: Randomized radiostereometric study comparing osteogenic protein-1 (BMP-7) and autograft bone in human noninstrumented posterolateral lumbar fusion: 2002 Volvo Award in clinical studies. Spine 2002;27:2654–2661. 175 Toth JM, Estes BT, Wang M, Seim HB 3rd, Scifert JL, Turner AS, Cornwall GB: Evaluation of 70/30 poly(L-lactide-co-D,L-lactide) for use as a resorbable interbody fusion cage. J Neurosurg 2002;97:423–432. 176 Kandziora F, Bail H, Schmidmaier G, Schollmeier G, Scholz M, Knispel C, Hiller T, Pflugmacher R, Mittlmeier T, Raschke M, Haas NP: Bone morphogenetic protein-2 application by a poly(D,L-lactide)-coated interbody cage: In vivo results of a new carrier for growth factors. J Neurosurg 2002;97:40–48. 177 Bobo RH, Laske DW, Akbasak A, Morrison PF, Dedrick RL, Oldfield EH: Convection-enhanced delivery of macromolecules in the brain. Proc Natl Acad Sci USA 1994;91:2076–2080. 178 Laske DW, Morrison PF, Lieberman DM, Corthesy ME, Reynolds JC, Stewart-Henney PA, Koong SS, Cummins A, Paik CH, Oldfield EH: Chronic interstitial infusion of protein to primate brain: Determination of drug distribution and clearance with single-photon emission computerized tomography imaging. J Neurosurg 1997;87:586–594. 179 Lieberman DM, Corthesy ME, Cummins A, Oldfield EH: Reversal of experimental parkinsonism by using selective chemical ablation of the medial globus pallidus. J Neurosurg 1999;90:928–934. 180 Lonser RR, Gogate N, Morrison PF, Wood JD, Oldfield EH: Direct convective delivery of macromolecules to the spinal cord. J Neurosurg 1998;89:616–622.

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181 Grossi PM, Ochiai H, Archer GE, McLendon RE, Zalutsky MR, Friedman AH, Friedman HS, Bigner DD, Sampson JH: Efficacy of intracerebral microinfusion of trastuzumab in an athymic rat model of intracerebral metastatic breast cancer. Clin Cancer Res 2003;9:5514–5520. 182 Mardor Y, Roth Y, Lidar Z, Jonas T, Pfeffer R, Maier SE, Faibel M, Nass D, Hadani M, Orenstein A, Cohen JS, Ram Z: Monitoring response to convection-enhanced taxol delivery in brain tumor patients using diffusion-weighted magnetic resonance imaging. Cancer Res 2001;61:4971–4973. 183 Santini JT Jr, Richards AC, Scheidt RA, Cima MJ, Langer RS: Microchip technology in drug delivery. Ann Med 2000;32:377–379. 184 Richards Grayson AC, Choi IS, Tyler BM, Wang PP, Brem H, Cima MJ, Langer R: Multi-pulse drug delivery from a resorbable polymeric microchip device. Nat Mater 2003;2:767–772. 185 Li Y, Shawgo RS, Tyler B, Henderson PT, Vogel JS, Rosenberg A, Storm PB, Langer R, Brem H, Cima MJ: In vivo release from a drug delivery MEMS device. J Control Release, in press.

Henry Brem, MD Departments of Neurological Surgery and Oncology The Johns Hopkins Hospital, Hunterian 817, 725 North Wolfe Street Baltimore, MD 21205 (USA) Tel. ⫹1 410 614 0477, Fax ⫹1 410 614 0478, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 499–520

Immunotherapy Strategies for Treatment of Malignant Gliomas Larry Harshynea, Phyllis Flomenbergb, David W. Andrewsa a

Department of Neurosurgery and bDepartment of Internal Medicine, Thomas Jefferson University, Philadelphia, Pa., USA

Malignant astrocytomas are primary intracranial tumors that are resistant to treatment with surgery, radiation, and chemotherapy. With rare exceptions, all of these tumors recur after initial treatment, requiring repeat surgery and adjuvant treatments. Irrespective of interventions, the median survival for WHO Grade 4 tumors (glioblastomas) remains a dismal 47 weeks [1] and despite fundamental advances in chemotherapy, little progress has been made in the treatment of malignant astrocytoma. Several factors contribute to the lack of progress in this area, including suboptimal drug delivery through the bloodbrain barrier and refractory hypoxic cell subpopulations. Drug delivery to the tumor is impeded by the blood-brain barrier, limiting the choice of CNS agents to certain lipophilic drugs. Glioma cell subpopulations are markedly heterogeneous and show phenotypic differences in their sensitivity to established drugs such as lipophilic variants of carmustine (BCNU) and cisplatin. Hypoxic glioma cell subpopulations are also refractory to therapy due to their radio- and chemo-resistant properties. Over the past decade, these issues have been addressed by intratumoral drug delivery trials (as discussed by Brem et al. in another chapter in this volume), external beam radiation, or brachytherapy, all of which have shown only marginal success. Conventional treatment failure has prompted development and utilization of novel strategies such as gene therapy, antisense therapy, and immunotherapy. In the case of immunotherapy, a number of strategies have emerged and collectively represent a promising new approach to the treatment of malignant glioma. At the same time, information about native glioma immunomodulatory capabilities may help explain why immunotherapies have failed in early human trials. This chapter will serve to review what is currently known about glioma immunobiology and review strategies currently being developed as anti-glioma

immunotherapies. We provide a review of cell-mediated immunity and current assays of immune response.

Antigen Presenting Cells and the Immune Response

The cell-mediated immune response plays a vital role in anti-tumor immunity. An effective cellular immune response requires the coordination of activated helper (CD4) and killer (CD8) T cells. T cells cannot act indiscriminately and must first be exposed to foreign antigens by antigen presenting cells (APCs). Dendritic cells (DCs) are the most potent APCs in the immune system. APCs secrete many immune activators and modifiers into the local environment, which not only augment adaptive immunity but also amplify the innate immune response; for example, activation and recruitment of other APCs, macrophages, natural killer (NK) cells, eosinophils and mast cells. Furthermore, APCs assume many different roles throughout their life and must remain extremely plastic, capable of quickly modifying their function to meet the needs of the host. Their flexible nature and central role within the immune system makes them attractive candidates as targets for therapy aimed at boosting immunity. Networks of APCs exist in nearly every organ. APCs are most prevalent in epithelial barriers that serve as the first line of defense. Increased exposure to pathogens in mucosal tissue and skin necessitate increased surveillance. While APCs were described 30 years ago in the peripheral lymphoid tissue of mice [2], APCs actually were first identified in the epidermis in 1868 by Paul Langerhans, a German anatomist, who mistook them for nerve endings. Later, they were found to be APCs and still bear his name, Langerhans cells (LC). A LC-like population of APCs also can be found in the epithelial lining of the gut [3], airways [4, 5], and reproductive tract [6, 7]. A discrete subset of interstitial APCs (iAPCs) resides in the dermis [8] and submucosa. The blood contains APCs [9–11] and APC precursors [12] which are en route to tissues. APCs exist throughout the periphery and act as sentinels for the immune system. They are extremely efficient at capturing antigens from a variety of sources including soluble proteins and peptides bound by heat shock proteins, as well as cell-associated antigens from other live or dead cells (fig. 1). Upon acquiring an antigen, APCs receive an activation signal, which occurs when they sense foreign pathogens or cells. APCs are also extremely sensitive to inflammatory cytokines. Following activation, they down-regulate the ability to acquire antigen and shift their focus towards antigen presentation and T cell stimulation. Activated APCs enter lymphatic ducts and migrate to the draining lymph nodes where they encounter large numbers of T lymphocytes. During

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Fig. 1. Immature DC efficiently capture apoptotic bodies. Equal number of DiDlabeled immature DC (red) and DiO-labeled apoptotic T cells (green) were cocultured together for 4 h and analyzed by confocal microscopy. Numerous DC-containing multiple apoptotic bodies are observed in these low and high power fields.

migration, APCs process and present captured antigens with the MHC class II complex or cross-present antigens with the MHC class I complex (fig. 2). In addition, APCs, like all nucleated cells, are able to present endogenous antigen in the context of MHC class I. CD4 T cells recognize antigens bound to MHC class II molecules, whereas CD8 T cells respond to antigens bound by MHC class I molecules. T cells recognize antigen/MHC complexes via their T cell receptors. Following stimulation through MHC/T cell receptor interactions, T cells begin to proliferate. In order to become fully functional effectors, however, T cells must receive an additional, costimulatory signal from the APC. Activated APCs express high levels of costimulatory molecules, including CD40, CD80, and CD86, all of which are capable of providing this second signal to T cells. T cells which only receive one signal, through the T cell receptor, become ‘tolerant’ and are unable to respond to antigen. The primary role of activated helper cells is to provide support to the immune system. For the most part, this helper cell support involves secretion of cytokines such as -interferon (IFN-) and interleukins (IL-2, IL-4, IL-5, IL-12), which perpetuate and direct the immune response. Helper T cells are divided into two main subsets, Th1 and Th2. IFN-, IL-2, and IL-12 are considered to be Th1 cytokines, which are proinflammatory and strengthen the

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TGF-, PGE-2 and IL-10

Tumor cell

IL-2, IL-6, IFN- TH1

TK

IL-6, IL-12

CD28

TCR

CD86

MHC I

TCR MHC II

CD40L CD40

APC

Tumor antigen

Fig. 2. Mechanism for tumor antigen loading and T cell cross-priming through antigen-presenting cells.

cell-based immune response. IL-4, IL-5, IL-6, IL-10, and TGF- belong to the Th2 family of cytokines. In contrast to Th1 cytokines, Th2 cytokines are antiinflammatory and result in the down-regulation of the cell-mediated immune response. Activated killer cells, or cytotoxic T lymphocytes (CTL) search the periphery for target cells, or cells which present the proper antigen in MHC class I molecules on their surface. Upon identifying a target cell, CTL employ one of two different mechanisms to rapidly destroy it. Target cells can be killed by the release of lytic granules containing two classes of effector proteins. One class consists of pore-forming proteins called perforins. Perforins polymerize in the target cell membrane to form tiny holes throughout the lipid bilayer. The second class of effector molecules consists of a family of serine proteases called granzymes, which set the cell death cascade in motion. While nearly all target lysis by CTL occurs via the perforin or granzyme pathway, perforin knockout mice are still able to lyse target cells. An alternative lysis pathway

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exists which involves Fas receptor/ligand interaction. Binding of Fas on the surface of target cells by CTL initiates a proteolytic cascade, which results in the apoptosis of the target cell. Several important questions remain unanswered in tumor immunology which include how more potent effector T cells could be generated, what conditions facilitate better tumor cell recognition, and what is required to achieve memory T cells that results in life-long protection against specific tumors and/or metastases.

Novel Methods for Monitoring the Immune Response in Glioma

Glioma patients may have immune dysregulation. Therefore, precise and consistent assays to assess T cell numbers and function are vital for an accurate estimation of immune competency in glioma. Quantitative and qualitative measures of immune function are therefore important criteria when designing clinical trials for glioma therapy. Antigen-specific T cells circulate through the blood and lymphatic system. In order to become activated, T cells recognize peptide bound to MHC molecules on APCs. In 1996, an innovative system for quantitating antigenspecific T cells was developed [13]. MHC class I molecules were bound to peptides and attached to a fluorescenated substrate, generating tetramers. Tetramers have proven to be a powerful tool when quantifying T cell repertoires. Tetramers permit the estimation of circulating precursor frequencies of antigen-specific T cells before therapy, and the extent of expansion following immune boosting. Tetramer assays have been broadened to include the observation of antigen-specific T cells in situ [14]. Similarly, investigators have developed CD1 tetramers to look at glycolipid reactive T cells [15] and MHC class II tetramers to monitor peptide-specific CD4 T cell populations [16]. Carboxyfluorescein diacetate succinimidyl ester is a cell-permeable dye that nonspecifically labels intracellular proteins within an intact, functioning cell [17]. As a labeled cell divides, so too does its fluorescent profile, with each daughter cell receiving approximately half of the fluorescently labeled proteins. Serial dilution of the vital dye resulting from proliferation can be tracked for up to eight to ten cycles, after which the dye becomes extinct. Carboxyfluorescein diacetate succinimidyl ester enables quantitative kinetic studies of the immune response, detailed analysis of proliferative profiles within minor subsets, and the ability to address T cell differentiation. When T cells proliferate, they also differentiate into fully armed effector cells. For CD4 helper T cells, this means production and secretion of immunomodulatory cytokines. CD8 killer T cells, on the other hand, produce cytolytic molecules. Cytokine production is most often documented utilizing

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monoclonal antibodies. Enzyme-linked immunosorbent assay (ELISA) has been the classical standard for measuring cytokine secretion [18]. An ELISA utilizes two antibodies that recognize different epitopes. A capture antibody, immobilized to one plate, binds to cytokine molecules present in a serum or supernatant sample. A second antibody is used to detect the bound cytokine in an enzyme catalyzed reaction. While ELISAs are very sensitive they are also labor intensive and only detect a single cytokine. A novel flow cytometry-based method has recently been developed by BD Biosciences (Cytokine Bead Array, CBA). The CBA achieves sensitivity comparable to an ELISA, but can detect multiple cytokines in a single sample [19]. The CBA assay is very efficient, as it generates up to seven times the amount of data in less time than a conventional ELISA with a sensitivity of CBA (⬃4 pg/ml) that rivals ELISA. The enzyme-linked immunospot assay is similar to ELISA, except that cytokines are captured onto a membrane [20] instead of a plastic plate. The ELISPOT is also a very sensitive assay (as few as 100 molecules of specific protein are detected) that capitalizes on the high concentration of cytokines in the environment immediately surrounding the producer cell. A spot forms when detector antibodies undergo a colorimetric reaction in regions of high cytokine production. The spot represents a trace of the cytokine-producing cell and vary in size depending on the amount of cytokine produced by a given cell. Flow cytometry achieves both rapid detection and semi-quantitative measurements of rare cytokine-producing cells [21, 22]. Intracellular cytokine staining with fluorescent antibodies can detect subsets of rare cells producing multiple cytokines when analyzed by flow cytometry. Stimulation cultures are inhibited by the addition of monensin or brefeldin A, which stop vesicular transport [21]. Instead of secreting cytokines, substantial quantities of cytokines accumulate within cells. Following overnight incubation, cultures are fixed, permeabilized, and probed with cytokine-specific antibodies. Subsets of cells are defined by labeling with lineage markers before fixation. A drawback is that intracellular cytokine staining kills cells and prevents one from conducting additional experiments. An alternative is cytokine capture assay, which retains many advantages of intracellular cytokine staining and also maintains cell viability. In this assay, a cytokine catch reagent is attached to the outside of the cell via CD45, which is found on all hematopoietic cells. Secreted cytokine is captured on the cell surface and then detected using a fluorescent, phycoerythrinconjugated antibody specific for cytokines. MAgnetic Cell Sorting microbead technology (Miltenyi Biotech) uses miniature magnetic beads and separation columns that are gentle to cells to obtain a highly purified population of cells in less than an hour [23]. By combining cytokine capture and magnetic selection, the authors of one study were able to purify IFN- producing, melanA-specific T cells using anti-PE microbeads [24]. This technology enables

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subsequent experiments to be performed on highly purified and well-defined populations of tumor-specific effector cells. Tetramers and enzyme-linked immunospots provide innovative ways to measure the frequency of antigen-specific CD8 T cells, but they do not assess function of these responders. While many novel methods have emerged to assess CD4 helper T cell function, antigen specific lysis remains the gold standard. Traditionally, cytolytic activity was defined by 51Chromium (51Cr) release assays [25]. Target cells were loaded with antigen, labeled with 51Cr, and cocultured with effector T cells. Destruction of target cells was indirectly measured by 51Cr release into the supernatant within 4 h of adding killer T cells. The hazards associated with radioactivity as well as high background levels due to spontaneous release of 51Cr have prompted researchers to develop novel ways to identify cell-mediated cytotoxic lysis. Several nonradioactive alternatives for antigen-specific lysis have included detecting the release of intracellular enzymes [26] or the release of fluorescenated proteins [27, 28]. Direct measures of cell death can be combined with the fluorescent protein release [29, 30] to further pinpoint the mechanism of cell death. Target cells whose membrane and intracellular protein have been labeled with fluorescent dyes (e.g., DID or PKH-27, and carboxyfluorescein diacetate succinimidyl ester, respectively) can provide a bright and narrow fluorescent profile when analyzed by flow cytometry. These dyes form stable conjugates and do not readily cross intact cell membranes, resulting in much lower background levels when compared to classical 51Cr release assays. Lysis by effector cells, however, does result in an immediate loss of fluorescence due to the extravasation of labeled proteins through the compromised membrane. Moreover, APCs that endocytose released labeled protein may interfere with the interpretation of results. To control for the latter, Sheely et al. [28] labeled the cell membrane of target cells with an additional dye to be sure that they observed a loss of fluorescence in target cells and not a gain of fluorescence in bystander APCs. Labeling of intracellular and/or membrane proteins of target cells can be combined with probes of cell death including propidium iodide [29], 7-AAD [30], or TUNEL labeling. In our hands, the fluorescent-based assays for cytolytic activity have proven to be more sensitive due to low levels of spontaneous release and have enabled direct detection of lysed target cells via high-throughput flow cytometric analysis. Two molecular immunological techniques have recently emerged to effectively monitor the immune response at the population level: gene expression arrays [31] and reverse-transcriptase-polymerase chain reaction (RT-PCR) [32–34]. With expression arrays, it is possible to look at thousands of genes and determine which are differentially expressed in tumor-specific T cells when compared to naïve T cells. Large amounts of data are generated by arrays, but

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software specifically designed to interpret and quantify an immune response is currently unavailable. Protocols are under development to evaluate global antitumor responses within individual patients. In addition to microarray profiling, RT-PCR has advanced to a level where it has become a very valuable tool with which to monitor immune responses within the circulating peripheral blood mononuclear cells (PBMC). RT-PCR provides oncologists with a molecular genetic signature of the immune response in cancer; for example, Kammula et al. [33] monitored gene expression in anti-tumor lymphocytes. Copies of CD8, IFN, GM-CSF, IL-2, and TNF- mRNA increased significantly in PBMC taken from patients who were vaccinated with tumor-derived peptides. Furthermore, copies of mRNA encoding markers of early T cell activation, including CD25 and CD69, increased shortly after immunization. There was a correlation between increasing levels of gp100 mRNA and IFN- mRNA in patients receiving the vaccine that was not observed in the control group. RT-PCR results were confirmed by tetramer analysis. This study provides a compelling evidence supporting the efficacy of RT-PCR-based monitoring of immune responses.

Immune Dysfunction in Glioma and Constraints Imposed on Proinflammatory Responses in the CNS

In the periphery, the body’s innate response to anything foreign includes a massive infiltration of inflammatory cells and proteins that aid in defense and tissue repair at the site of insult. Extensive cell death and tissue destruction often accompany an inflammatory immune response. As a host organ, the CNS poses an unique set of constraints for inflammatory responses. The brain is composed of resting phase (postmitotic) and predominantly nonregenerating cells and is enclosed within a fixed cranial vault. Recognizing the physiological relationship between volume and pressure, brain compliance cannot tolerate swelling beyond a certain point which would result from the influx of excess immune cells, nor can the host afford tissue loss, particularly in eloquent regions of the brain. An example of an autoimmune T cell-mediated destruction in the brain is acute disseminated encephalomyelitis [35]. The body has in place several physiological barriers and mechanisms to safeguard the brain from such a destructive inflammatory response. The blood-brain barrier largely restricts the trafficking of immune cells and mediators from the periphery to the brain. Furthermore, brain tissue neither contains endogenous APCs, nor does it possess conventional lymphatics to carry cells and/or antigen to local nodes. By restricting the communication between the two compartments, these measures serve to reduce the risk of a full-fledged immunological attack within the brain.

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a

b

c

d Fig. 3. a–d Photomicrographs of malignant glioma permanent sections in patients previously treated with an antisense paradigm, hematoxylin and eosin stain, 400. All four photomicrographs represent examples of acquired T cell lymphocytic infiltration after abdominal implantation of apoptotic autologous malignant glioma cells.

In the event that an activated immune cell gets past the blood-brain barrier, many cells within the brain express FasL, and Fas-bearing lymphocytes would undergo apoptosis before an immune response could be initiated. Studies have demonstrated naturally occurring cellular immune responses in glioma as well as a positive correlation between lymphocytic infiltration and median survival prognosis [36–40]. Our experience in modifying the immune response in glioma involves antisense directed against the insulin-type growth factor type one receptor (IGF-IR/AS ODN) in glioma patients [41, 42]. In this work, we measured an acquired intratumoral lymphocytic infiltration, which is six-fold higher than previously reported (see fig. 3). Using Western blot, we demonstrated IGF-I receptor down-regulation after IGF-IR/AS ODN treatment

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Table 1. Immune modulatory capabilities of gliomas Glioma modulations

Consequence

Systemic and regional depletion of Th cells Lack of MHC Class I molecules on tumor cells MHC class II expression on tumor cells TGF- production by tumor cells

Lack of antigen-specific recognition of tumor cells Loss of recognition by cytotoxic T cells

Prostaglandin E2 and IL-10 production by tumor cells Production of colony-stimulating factors Chemokine production by tumor cells Presence of IL-1 autocrine loop in tumor cells Production of IL-1RA by tumor cells

Lack of IFN- genes in tumor cells

Regional depletion of Th 1 cells (T cell anergy) Profound suppression of T-, B- and NK-cells and macrophages Suppression of Th1 and professional APC functions Activation of macrophages Chemotaxis of T cells and macrophages into tumor tissue Partial activation of T cells and macrophages Regulation of IL-1 autocrine loop; suppression of IL-1-mediated immune cascade reaction Suppression of MHC molecules and induction of cytokines

Adapted from [128].

of patient tumor cells into vitro, and tumor cell apoptosis in vivo after implantation of treated tumor cells into the host abdomen [41]. It was hypothesized that acquired lymphocytic tumor infiltration may have resulted from an immune cross-priming mechanism invoked by in vivo tumor cell apoptosis. Some studies have suggested a relationship between lymphocyte competence and clinical outcome in breast cancer [43–45]. Effects of lymphocyte competence are unknown in glioma patients, although some glioma patients are known to have impaired cell-mediated immunity (e.g., decreased cellular immunity when assessed by delayed-type cutaneous reactions) [46]. Among other possibilities, this may be due to the impairment of mitogen- and antigen-induced T cell reactivity, impaired production of IL-2, or decreased expression of the IL-2 receptor p55 chain [46]. Whether host immune deficiencies are directly related to immune modulation by gliomas remains unclear, but glioma cells are known to produce a variety of cytokines that modulate host immune reactions (table 1). Gliomas also were shown to produce FasL and confer resistance to host immunity by inducing apoptosis in Fas receptor-bearing T cells [47]. Thus,

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in addition to any immunological safeguards present in normal brain tissue, gliomas possess additional mechanisms to subvert immunological detection and attack. Their primary means of immune evasion elicits immune dysfunction. By deploying anti-inflammatory Th2 cytokines (e.g., TGF-, IL-10, PGE2) gliomas can circumvent immune responses in the brain. Although tumor-infiltrating lymphocytes (TIL) can be observed in gliomas, often there are multiple immunological defects among TIL consisting of decreased responses to mitogens and reduced killing capacity, which can be attributed to the local immunosuppressive environment. While there are no resident APCs in the brain, dendritic cells and macrophages also can be found circulating in the cerebrospinal fluid. Similarly, blood dendritic cells can extravasate from vessels within the brain, enabling transient migration through tissue. These cells are fully capable of acquiring antigen and migrating to cervical lymph nodes. The immunosuppressive environment in the brain, compounded by the presence of tumor, prevents the activation of APCs that are essential for generating effector T cells. Instead, presentation to T cells by an inactivated APC results in tolerance and anergy. By maintaining an immunosuppressive environment that affects both APC and immune effector cells, gliomas induce a response that favors immunological tolerance instead of a destructive inflammatory response. Recognizing this phenomenon, one can devise ways to overcome tumor-induced immunosuppression; when designing an anti-glioma immunotherapy, great care must be taken to generate an anti-glioma immune response, which preserves normal brain tissues in order to avoid generalized inflammation and tissue necrosis.

Current Strategies under Consideration for Immunotherapy of Gliomas

Anti-glioma immunotherapy efforts can be divided into two strategies: those that target tumors directly and those that are designed to boost the existing immune response. The first strategy uses cytokines or antibodies to neutralize molecules required for tumor survival or functioning. The second strategy may boost the immune response by enhancing NK-mediated or T cellmediated killing; for example, by vaccinating the host with tumor antigens or with autologous dendritic cells primed with tumor antigens. Cytokines are intercellular mediators, which provide an important role in coordinating and skewing the immune response [48]. Expression of many cytokines is regulated by the presence of opposing cytokines. As an example, Th1 cytokines will cause the down-regulation of Th2 cytokines. Cytokines can be administered directly inside biodegradable polymers [49], with direct infusion

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into the brain [50, 51], or with intravenously injections [52, 53]. Alternatively, cytokines can be administered with gene therapy expression vectors [54–57]. IL-2, IL-12, TNF-, and IFN- producing gene transfer vectors have exhibited limited success in treating gliomas in animal models. In a recent review of glioma clinical trials, however, cytokine administration yielded little benefit [58]. Antibodies are highly specific molecules of the immune system with long half-lives that allow them to persist within the body for extended periods of time. Tumor antigen biospecificity and stability make antibodies attractive candidates for glioma immunotherapy. Antibodies employ several modes of action to target gliomas. The structure of antibodies enable them to bind target molecules, thus preventing interaction with natural ligands such as growth factors required for glioma survival. Epidermal growth factor receptor (EGFR) and a major splice variant, EGFRvIII are often overexpressed in gliomas [59, 60] and are targets for glioma therapies [61, 62]. Vascular endothelial growth factor is another target for interference by antibody-based glioma therapy [63, 64]. In addition to blocking vital tumor growth factors, antibodies can uncloak glioma immune evasion. NK cells play an important role in innate immune detection of tumors. NK cells are capable of recognizing cells coated with antibodies and killing them, known as antibody-dependent cell-mediated cytotoxicity. Stimulating antibody-dependent cell-mediated cytotoxicity with antibodies specific for EGFR (or EGFRvIII) [65–67], vimentin [68], or ganglioside GD2 [69] has been attempted for the experimental treatment of glioma. Additional glioma targets have included CD95 (Fas) and TNFR, often referred to as ‘death receptors.’ When these receptors are bound by their respective ligands, cell death is initiated. Interestingly, death receptors play an important role in maintaining the immune-privileged status of the brain. Gliomas have been shown to down-regulate these receptors in order to gain a survival advantage; there has been some limited success in targeting this pathway in glioma therapy [70] by driving expression of Fas/TNFR [71] or transducing gliomas [72, 73]. Antibodies also can be functionalized for tumor ablation by conjugating them to toxins, drugs, or radionuclides. This kind of targeted delivery of antitumor agents provides a major advantage. Antibodies linked to ricin [74–76] or pseudomonas toxin [77, 78] have been used to treat gliomas in preclinical studies. In a similar manner, radioimmunotherapy with antibodies specific for tenascin and conjugated to 131I [79–81] and 90Y [80, 82, 83] have exhibited limited success in a clinical setting. Similar data have been reported using radioactive antibodies specific for EGFR [84–86]. The drawback is that continued exposure to irradiation, due to the persistence of antibodies in the serum, delivers a substantial dose of irradiation to unaffected organs and limits the quantity that can be safely administered.

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Fig. 4. Glioma-specific T cells respond to U118 lysate-loaded DC. PBMC were cocultured for 6 h with autologous U118 lysate-loaded or control DC (matured with IL-1, IL-6, PGE2, and TNF-). Cocultures were harvested, stained with fluorescent antibodies, and analyzed by flow cytometry. U118 lysate-loaded DC activate both CD4 and CD8 T cells. Additionally, CD8 T cells produced IFN- following antigen presentation by DC.

Strategies designed to boost the immune response include the use of tumor vaccines and/or primed APCs. The use of allogeneic cell lines for glioma vaccines has been explored in orthotopic rodent models by Ashley et al. [87]. Priming of the anti-glioma immune response using cellular vaccines is not dependent on the expression of syngeneic class I MHC molecules on the immunizing cell [88, 89]. The immune priming mechanism is optimized by generating antigens from apoptosis induced in the immunizing cells [90–92]. Our lab has explored the use of allogeneic glioma cells in cancer therapy by loading human dendritic cells with antigen and cross-priming autologous CTLs. As a preliminary finding, we detected CD8 and CD4 T cell responses to allogeneic tumor lysate-loaded dendritic cells in the peripheral blood from glioma patients (fig. 4). These T cells exhibited cytotoxicity against autologous glioma cells in vitro.

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While preliminary data suggest the value of using an APC-based immunotherapy, many basic questions remain unanswered. For example, is it best to target APCs in vivo or ex vivo? Which type of APCs generate the best anti-tumor response: immature APCs or mature APCS? If mature APCs, what are the maturation stimuli?, What is the best source of antigen to ‘feed’ APCs (apoptotic cells, tumor peptides, DNA-encoding tumor antigens)? How many APCs should be utilized for each vaccination, and how frequently should vaccinations be given? Which injection route will generate the best immunity (subcutaneous, intravenous, intramuscular, intranodal)? More clinical trials and studies comparing immunization protocols are necessary to answer these questions. It is possible to enrich mature APCs populations directly from human tissue [93, 94] or circulating PBMC [95, 96]. However, the low frequency and availability of tissue have limited these approaches. Instead, well-established methods for generating APCs in vitro have been described [97–99]. These cells resemble their in vivo counterparts, LC and iAPCs, and function similarly. Ex vivo approaches begin with the purification or enrichment of CD14 and CD34 precursors, which can differentiate into APCs in the presence of cytokines. APCs that have been loaded with antigen from various sources (e.g., glioma RNA [100], glioma peptides [101, 102], apoptotic tumor cells [103], tumor lysate [104], fusion of glioma cells and APCs [105]) all have been shown to induce anti-tumor responses. When using ex vivo APCs for therapy, one can choose the maturation stimuli given to APCs. In vitro maturation of APCs can be achieved by any number of stimuli: bacterial components (cell wall, DNA) [106], double-stranded RNA (mimicking viral infection) [107], CpG DNA [108], monocyte-conditioned medium [109], CD40L (mimicking CD4 T cell helper function) [110], and inflammatory cytokines [111, 112]. CD40L and a cytokine cocktail consisting of TNF, IL1, IL6, IFN, and PGE2 can be used to obtain clinical grade, mature APCs [112, 113]. Given the complexity of generating ex vivo GMP-grade APC vaccine, APCs also may be targeted in vivo. Some systems have utilized viral vectors to target LC in skin [114, 115]. Alternatively, APCs possess many antigen-uptake receptors, which represent feasible candidates for APC therapy. To date, most efforts have focused on one receptor in particular called Dec-205. Dec-205 is a C-type lectin (binding to oligosaccharides) and its expression is restricted solely to APCs. An antibody specific for Dec-205 has been used to successfully deliver antigen to APCs in vivo [116]. Antigen was presented in both MHC class I and class II molecules [116–118]. APCs [119–121] and tumors [122] are capable of producing exosomes, which are tiny, antigen-presenting vesicles that can induce potent anti-tumor responses [119, 122] and are under investigation in a phase I/II study involving non-small cell lung cancer patients.

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A recent study detailed an innovative, two-step approach that targeted APCs in situ. Vaccination resulted in protective, tumor-specific immunity [123]. Macrophage inflammatory protein 3, a chemotactic protein that attracts APCs, was encased in a polymer rod. Similar rods were constructed that contained tumor lysate or peptide. Macrophage inflammatory protein 3 was released in a controlled fashion and resulted in an accumulation of LC in the vicinity of the rod following subcutaneous implantation. Coimplantation of rods encasing macrophage inflammatory protein 3 and tumor lysate or peptide into mice produced potent, tumor-specific CTL activity. As a comparison, mice were vaccinated with either polymer rods or conventional ex vivo generated APCs. Similar anti-tumor responses were seen in both groups, supporting a vaccination strategy. Preclinical studies reflecting dendritic cell manipulation in glioma treatments are summarized in a review by Parney et al. [58]. In addition to glioma [102] other human trials using autologous APCs to treat tumors have been published, including lymphoma [124], melanoma [125], renal cell carcinoma [126], and prostate cancer [127]. In the case of glioma, Yu et al. [102] demonstrated that APC vaccination utilizing autologous APCs pulsed with peptides acideluted from the surface of autologous glioma cells elicited systemic cytotoxicity in 4 of 7 patients treated. This study demonstrated the feasibility, safety, and bioactivity of an autologous peptide-pulsed APC vaccine for patients with malignant gliomas. Our laboratory is currently exploring the use of a novel class of sulfones, which induce apoptosis in a variety of glioma and other cell lines, suggesting another means to prime the immune system and induce antitumor responses in glioma patients. The challenge remains not only in understanding and exploiting glioma immune modulation, but also in gaining a more thorough understanding of human immunobiology and refining efficient, quantitative means of measuring immune response.

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104 Yoshida S, Morii K, Watanabe M, Saito T, Yamamoto K, Tanaka R: The generation of anti-tumoral cells using dentritic cells from the peripheral blood of patients with malignant brain tumors. Cancer Immunol Immunother 2001;50:321–327. 105 Kikuchi T, Akasaki Y, Irie M, Homma S, Abe T, Ohno T: Results of a phase I clinical trial of vaccination of glioma patients with fusions of dendritic and glioma cells. Cancer Immunol Immunother 2001;50:337–344. 106 Kopp E, Medzhitov R: Recognition of microbial infection by Toll-like receptors. Curr Opin Immunol 2003;15:396–401. 107 Cella M, Salio M, Sakakibara Y, Langen H, Julkunen I, Lanzavecchia A: Maturation, activation, and protection of dendritic cells induced by double-stranded RNA. J Exp Med 1999;189: 821–829. 108 Hartmann G, Weiner GJ, Krieg AM: CpG DNA: A potent signal for growth, activation, and maturation of human dendritic cells. Proc Natl Acad Sci USA 1999;96:9305–9310. 109 Reddy A, Sapp M, Feldman M, Subklewe M, Bhardwaj N: A monocyte conditioned medium is more effective than defined cytokines in mediating the terminal maturation of human dendritic cells. Blood 1997;90:3640–3646. 110 Ridge JP, Di Rosa F, Matzinger P: A conditioned dendritic cell can be a temporal bridge between a CD4 T-helper and a T-killer cell. Nature 1998;393:474–478. 111 Morse MA, Zhou LJ, Tedder TF, Lyerly HK, Smith C: Generation of dendritic cells in vitro from peripheral blood mononuclear cells with granulocyte-macrophage-colony-stimulating factor, interleukin-4, and tumor necrosis factor-alpha for use in cancer immunotherapy. Ann Surg 1997;226:6–16. 112 Lee AW, Truong T, Bickham K, Fonteneau JF, Larsson M, Da Silva I, Somersan S, Thomas EK, Bhardwaj N: A clinical grade cocktail of cytokines and PGE2 results in uniform maturation of human monocyte-derived dendritic cells: Implications for immunotherapy. Vaccine 2002;20 (suppl 4):A8–A22. 113 Feuerstein B, Berger TG, Maczek C, Roder C, Schreiner D, Hirsch U, Haendle I, Leisgang W, Glaser A, Kuss O, Diepgen TL, Schuler G, Schuler-Thurner B: A method for the production of cryopreserved aliquots of antigen-preloaded, mature dendritic cells ready for clinical use. J Immunol Methods 2000;245:15–29. 114 de Gruijl TD, Luykx-de Bakker SA, Tillman BW, van den Eertwegh AJ, Buter J, Lougheed SM, van der Bij GJ, Safer AM, Haisma HJ, Curiel DT, Scheper RJ, Pinedo HM, Gerritsen WR: Prolonged maturation and enhanced transduction of dendritic cells migrated from human skin explants after in situ delivery of CD40-targeted adenoviral vectors. J Immunol 2002;169: 5322–5331. 115 Esslinger C, Chapatte L, Finke D, Miconnet I, Guillaume P, Levy F, MacDonald HR: In vivo administration of a lentiviral vaccine targets DCs and induces efficient CD8() T cell responses. J Clin Invest 2003;111:1673–1681. 116 Hawiger D, Inaba K, Dorsett Y, Guo M, Mahnke K, Rivera M, Ravetch JV, Steinman RM, Nussenzweig MC: Dendritic cells induce peripheral T cell unresponsiveness under steady state conditions in vivo. J Exp Med 2001;194:769–779. 117 Mahnke K, Guo M, Lee S, Sepulveda H, Swain SL, Nussenzweig M, Steinman RM: The dendritic cell receptor for endocytosis, DEC-205, can recycle and enhance antigen presentation via major histocompatibility complex class II-positive lysosomal compartments. J Cell Biol 2000;151: 673–684. 118 Bonifaz L, Bonnyay D, Mahnke K, Rivera M, Nussenzweig MC, Steinman RM: Efficient targeting of protein antigen to the dendritic cell receptor DEC-205 in the steady state leads to antigen presentation on major histocompatibility complex class I products and peripheral CD8 T cell tolerance. J Exp Med 2002;196:1627–1638. 119 Zitvogel L, Regnault A, Lozier A, Wolfers J, Flament C, Tenza D, Ricciardi-Castagnoli P, Raposo G, Amigorena S: Eradication of established murine tumors using a novel cell-free vaccine: Dendritic cell-derived exosomes. Nat Med 1998;4:594–600. 120 Clayton A, Court J, Navabi H, Adams M, Mason MD, Hobot JA, Newman GR, Jasani B: Analysis of antigen presenting cell derived exosomes, based on immuno-magnetic isolation and flow cytometry. J Immunol Methods 2001;247:163–174.

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David W. Andrews, MD Professor of Neurosurgery 909 Walnut Street, 2nd floor, Philadelphia, PA 19107 (USA) Tel. 1 215 503 7005, Fax 1 215 503 7007, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 521–556

Glioma-Genesis Signaling Pathways for the Development of Molecular Oncotherapy

Gurpreet S. Kapoor a, Donald M. O’Rourkea,b Departments of aNeurosurgery and bPathology and Laboratory Medicine, University of Pennsylvania School of Medicine, Philadelphia, Pa., USA

Introduction to Gliomas

Gliomas are the most common primary central nervous system (CNS) tumors that arise from astrocytes, oligodendrocytes, or their precursors. Gliomas are classified according to whether they exhibit features of astrocytic, oligodendroglial, or ependymal cells. They are graded on a scale of I, II, III, or IV according to their degree of malignancy as judged by histological features [1]. Grade IV or glioblastoma multiforme (GBM) is the most aggressive glioma [2]. GBM either arise de novo (primary) or progress to GBM from low-grade gliomas (secondary). The malignant progression of gliomas involves a stepwise accumulation of genetic alterations that generally affect either signal transduction pathways activated by receptor tyrosine kinases (RTK) such as platelet-derived growth factor/receptor (PDGF/PDGFR), fibroblast growth factor 2, insulin-like growth factor receptor, and epidermal growth factor receptor (EGF-R) [3–9], or cell cycle arrest pathways involving regulators such as CDK4, CDK6, cyclin D1, MDM2, P16INK4a, P14ARF, RB, and p53 (fig. 1) [10–17]. RTK, which play a critical role in normal cell proliferation and differentiation, have been extensively studied by various laboratories for their possible role in ‘glioma-genesis’, or the process of glial transformation. RTK constitute a family of at least twenty members containing an extracellular ligand-binding domain, a single transmembrane domain, and an intracellular cytoplasmic domain with intrinsic tyrosine kinase activity [18]. Many studies have shown aberrant signaling by RTK in a variety of cancers, including human brain tumors. Constitutive activation of RTK is one of the important features of aberrant signaling leading

Grade I and II

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Low-grade astrocytoma

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p53 mutations PDGF/R overexpression

RB mutation CDK4 amplification INK4a/ARF loss PTEN loss DMBT1/mxi loss 19q loss 11p loss

EGFR amplification EGFR mutation INK4a/ARF loss PTEN loss RB mutation

Fig. 1. Stepwise accumulation of genetic alterations in gliomagenesis. PDGF/R, platelet-derived growth factor/receptor; RB, retinoblastoma; CDK4, cyclin-dependent kinase 4; INK4a, inhibitor of cyclin-dependent kinase on chromosome 4; ARF, alternative reading frame on INK4a locus; PTEN, phosphatase/tensin homolog on chromosome 10; DMBT1, deleted in malignant brain tumors 1; EGFR, epidermal growth factor receptor.

to malignant transformation and tumor proliferation, and can occur by several mechanisms [19]. Deregulated RTK signaling can occur via gene amplification, overexpression, and activating mutations, including deletions in the extracellular domain or alterations in the RTK cytoplasmic domain. Another mechanism of aberrant RTK signaling involves the activation of autocrine growth factor and receptor loops [20]. In CNS tumors, particularly astrocytomas, the classical examples of autocrine growth factor/receptor loops involve production of PDGF, epidermal growth factor (EGF), transforming growth factor-␣ (TGF-␣), and their respective receptors [21]. Therefore, it has become imperative to focus on mitogenic and transforming signaling cascades generated by PDGF/PDGFR, EGF/EGF-R and TGF-␣/TGF␣-R autocrine loops in order to understand the molecular mechanisms underlying glioma formation, and to use these signaling modules as novel therapeutic targets.

PDGF/PDGF-R Signaling in Human and Mouse Cell Tumors

Accumulated evidence suggests that PDGF and PDGFR play a significant role in glial development and lineage commitment [22]. In cell culture, PDGF functions to block differentiation and promote proliferation of O2A glial progenitors that give rise to either oligodendrocytes or type-2 astrocytes [23, 24]. Although coexpression of PDGF and PDGFR has been shown in all brain

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tumor stages, including low-grade astrocytomas, anaplastic astrocytomas, and GBM [21, 25–27], PDGF/PDGFR overexpression has been most commonly observed in low-grade astrocytomas in association with loss-of-function of the p53 tumor suppressor [28, 29]. These observations suggest a cooperative relationship between the PDGF/PDGF-R and p53 signaling pathways. A recent study using cell culture and a transgenic mouse model showed that overexpression of PDGF in neural progenitors induced the formation of oligodendrogliomas, whereas PDGF transfer into differentiated astrocytes induced either formation of oligodendrogliomas or mixed oligoastrocytomas [30]. The observed histologies of these glial tumors were consistent with low-grade neoplasms. Collectively, these reports suggest that the PDGF mitogenic signaling loop may be an early or initiating event in driving neural precursors or differentiated glial cells to low-grade astrocytomas and/or oligodendrogliomas, prior to malignant transformation of low-grade clones. It, therefore, appears that PDGF/R alterations are most commonly observed in ‘secondary GBM’ or those malignant gliomas that arise from lower-grade tumors [26]. The PDGF family consists of four members (PDGF-A, -B, -C, and -D) which transduce signals through the PDGF-␣ and PDGF-␤ receptors. Biosynthesis and processing of PDGFs involve the formation of dimers PDGF-AA, -BB, -CC, and -DD and the heterodimer PDGF-AB [31]. Numerous studies have reported the expression of PDGF-A and -B ligands in glioblastomas, and indicate that autocrine signaling by these isoforms is required for cell survival [3, 32, 33]. Moreover, expression or alteration of PDGF-A, -B, -C and PDGFR also have been implicated in medulloblastomas and ependymomas [34, 35]. Recently it was reported that PDGF-B enhances glioma angiogenesis by stimulating vascular endothelial growth factor (VEGF) expression in tumor endothelia and promoting pericyte recruitment to neovessels [36]. A recent study in glioma cell lines and primary glioblastoma tissues using quantitative reverse transcriptasePCR also implicated PDGF-C and -D ligands in the formation of brain tumors and confirmed the existence of autocrine signaling by PDGF-A and -B in brain tumors [4]. PDGF-AA and -CC selectively bind to PDGFR␣, whereas PDGF-DD preferentially binds to PDGF␤, with PDGF-BB displaying affinity for both receptors [31, 37–39]. Binding of PDGF stabilizes PDGFR dimerization, which is followed by autophosphorylation of tyrosine residues, leading to an increased tyrosine kinase activity [40, 41] and formation of docking sites for signal relay molecules containing src homology 2 (SH2) domains. A large number of SH2 domain-containing enzymes, such as phosphatidylinositol 3-kinase, phospholipase C-␥, src tyrosine kinases, protein tyrosine phosphatase SHP-2, and GTPase activating proteins for Ras have been shown to bind to particular SH2 sites on PDGF␣- and ␤-receptors and to modulate different signaling pathways.

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PDGFR binds to other molecules such as Grb2, Grb7, Nck, and Shc, which lack enzymatic activities and have adapter functions, linking the activated receptor to downstream signaling molecules and distinct pathways. PDGFR also binds to STAT family transcription factors, which translocate to the nucleus to directly activate the transcription of genes [42].

EGF-R/ErbB Signaling Cascades in Human and Experimental Astrocytomas

EGF-R belongs to the Erb-B family of type I RTK, based on structural homology to the v-erbB oncogene carried by the avian erythroblastosis virus [43]. The family includes four members: EGF-R (also termed erbB1/HER1), neu (erbB2 or HER2), erbB3 (HER3), and erbB4 (HER4). In high-grade astrocytomas or GBM, the majority of gene amplification events involve EGF-R [8, 44, 45]. Approximately 50% of GBM, but only a small percentage of anaplastic astrocytomas, express high levels of EGF-R [8]. These observations suggest that EGF-R overexpression and/or gene alteration is a late event in glioma-genesis and is frequently observed in ‘primary’ or ‘de novo’ glioblastomas occurring in older patients [46–48]. Sequencing of the amplified EGF-R genes revealed that frequent gene rearrangements result in essentially seven classes of variant EGF-R transcripts [49]. The most common rearrangement is a genomic deletion of exons 2–7, resulting in an in-frame deletion of 801 bp of the coding sequence to generate a mutant receptor called de2–7 EGF-R, ⌬EGF-R, or EGF-RvIII which cannot bind ligand due to a truncated extracellular domain but is constitutively active [9, 49–54]. This mutant receptor also has been detected in cancers of the lung, breast, and prostate [55, 56], but not in normal tissues [54]. Amplification of EGF-R genes has been implicated in poor prognosis of patients with GBM [57], and it has been demonstrated that patients with EGFRvIII-positive GBMs have shorter life expectancies [58]. Unlike wtEGF-R, EGF-RvIII transforms NIH3T3 cells [59] and strongly enhances the tumorigenicity of human gliomas in nude mice [53, 60]. Overexpression of mutant EGF-R in astrocytes or their precursors in transgenic mice has been shown to promote the development of glioblastoma [61]. However, the novel glycine residue resulting from gene rearrangement creates a new epitope at the splice site and the tumor-specific expression of EGF-RvIII makes this mutant a potential tumor-specific target for therapy in gliomas and in other cancers [62–64]. Moreover, expression of EGF-RvIII in gliomas provides resistance to cisplatin, a commonly used chemotherapeutic agent [65], which suggests a need for EGF-RvIII-targeted inhibition in combination with chemotherapy.

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Enhanced EGF-R signaling has also been reported to cooperate with other alterations in the development of GBM. The most common alterations are those that disrupt cell cycle arrest and include the deletion of p16INK4a/p19ARF [13, 16], deletion of RB [14, 15], loss of function of p53 [14], amplification of CDK4 [17, 66], CDK6 [11], cyclin D1 [10] and MDM2 [16]. However, the associated genetic lesions that cooperate with enhanced EGF-R signaling in the development of GBM have not been completely characterized. In a recent study, it was demonstrated that expression of EGF-RvIII, but not EGF-RWT in mouse astrocytes harboring activated oncogenic Ras resulted in the formation of oligodendroglioma and mixed oligoastrocytoma tumors [67]. Many reports evaluating human tumor tissues have shown that combined loss of p16INK4a and p19ARF, but not of either p53, p16INK4a or p19ARF alone, is associated with EGF-R activation in GBM [29, 68, 69]. On the other hand, a study using a mouse model system showed that human telomerase catalytic component overexpressed in normal human astrocytes cooperates with p53/pRb inactivation and Ras pathway activation, but not PI3-kinase/Akt pathway or EGFR activation, to allow the formation of intracranial tumors strongly resembling p53/pRb pathway-deficient, telomerase-positive, Ras-activated human grade III anaplastic astrocytomas [70]. The loss of p53 in combination with an NF1 (Neurofibromatosis type I) loss leads to astrocytomas and glioblastoma formation in mice [71]. Furthermore, a study in mice showed that the combined activation of Ras and Akt in neural progenitors induces the formation of glioblastoma [72]. A more recent study showed that p16INK4a/p19ARF loss cooperates with Ras and Akt activation in astrocyte precursors and neural progenitors to generate glioblastomas of various morphologies [73], indicating that glioma genesis occurs through an intricate cooperativity between genetic alterations often involving RTK signaling pathways and cell cycle regulatory molecules. Elevated levels of activated Akt have also been associated with the loss of the tumor suppressor phosphatase/tensin (PTEN) homolog, located on chromosome 10 in many glioblastomas [74, 75, 76]. Collectively, these studies suggest that robust signaling by overexpressed EGF-R and/or loss of functional PTEN and other genes leads to the increased activation of Akt in GBM and thus increased transformation. However, it is unclear whether the specific state of glial cell differentiation plays a restrictive role in glioma progression. Recent work in mice has demonstrated that deregulation of specific genetic pathways (i.e., Ink4a/Arf inactivation and EGF-R activation) may be more important than the neural cell of origin in dictating the emergence and phenotype of malignant gliomas [77]. Other ErbB proteins have been implicated in other CNS tumors. Both ErbB2 and ErbB4 expression levels have been shown to predict the prognosis of childhood medulloblastoma and ependymoma [78, 79]. In addition to their

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pathological role in brain tumors, ErbB proteins have been implicated in many systemic human cancers such as cancer of the colon, head and neck, pancreas, lung, breast, kidney, ovary, and bladder [80]. Studies in these cancers have linked the prognosis to excessive receptor kinase activity, receptor overexpression, ligand-independent constitutive activation of receptor mutants, and/or autocrine stimulation [81–84]. Increased receptor activity leads to increased downstream signaling and enhanced cell transformation. Studies on ErbB family members have demonstrated that homodimerization and heterodimerization are the initial events in variety of cellular signals required for cell growth and differentiation of many cell types under physiological conditions [85]. Thus, upon EGF stimulation, EGF-R forms a homodimer and/or heterodimer with other family members, which leads to auto- or transphosphorylation and activation of RTK activity, recruitment of various signaling relay molecules, and initiation of variety of a intracellular signaling cascades including MAPK, PI3-kinase/Akt, PLC-␥, and STAT (signal transducers and activators of transcription). Among these, MAPK and PI3-kinase/Akt pathways have been more extensively studied in glial tumors.

TGF-␣/EGF-R Autocrine Loop in Brain Tumors

TGF-␣ is a member of the EGF family and is a potent mitogen for a number of cell types in culture. Mature TGF-␣ is a 5.5-kDa peptide [86] sharing 30% structural homology with EGF. TGF-␣ binds to EGF-R and activates RTK activity [87–89]. Binding of TGF-␣ to EGF-R initiates receptor dimerization and autophosphorylation, followed by the recruitment of src-homology 2 (SH2) domain-containing molecules, which link EGF-R to similar intracellular pathways initiated by the EGF/EGF-R loop [18, 90]. Several tumors and tumor cell lines have been shown to coexpress EGF-R and TGF-␣ [9], indicating the existence of autocrine activation loop driving tumor growth. High levels of TGF-␣ have also been reported in human glioma [68, 92, 93]. Increased TGF-␣ levels have been observed in many primary human glioblastomas and anaplastic astrocytomas [93]. The highest level of TGF-␣ expression was found in recurrent tumors, which apparently had undergone transformation from low-grade to high-grade malignant anaplastic tumors. The elevated expression of TGF-␣l has also been reported in primitive neuroectodermal brain tumors including medulloblastomas [94]. It was reported that the induction of TGF-␣ via a tetracycline inducible system (tet on/off) in the glioma cell line U1242MG resulted in an increased motility at the single cell level, suggesting that coexpression of EGF-R and TGF-␣ was capable of forming an independent autocrine locomotory loop and

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that TGF-␣ may form an important component of the glioma cell invasion machinery [95]. TGF-␣ also has been shown to decrease GFAP (marker for astrocytic differentiation) mRNA levels in U373MG glioblastoma cells. On the other hand, TGF-␣ up-regulates gene expression for nestin, a marker for undifferentiated astrocytic precursors, without affecting vimentin gene transcription. These changes in the gene expression of intermediate filament proteins were correlated with an increased motility and less stellate morphology, suggesting that TGF-␣ may be required to induce dedifferentiation in glial tumors in order to promote motility [96]. In a recent study, U1242MG cells containing a TGF-␣ inducible transgene were used to determine the effect of autocrine TGF-␣ on cell proliferation in vitro and on subcutaneous tumor growth in nude mice [97]. In this study, induction of TGF expression in the absence of tetracycline resulted in increased cell proliferation in vitro, which was inhibited by blocking EGF-R with monoclonal antibody C225 and a tyrosine kinase inhibitor (RTKI) specific to EGF-R. Similarly, U1242MG clones expressing TGF-␣ (in mice which were not fed with tetracycline) developed into tumors of varied sizes. Moreover, mice injected with the TGF-␣ expressing clones developed larger tumors than those injected with control clones. These studies clearly indicate that the TGF-␣/EGF-R autocrine loop plays an important role in glial tumor progression.

Mitogen-Activated Protein Kinase (MAPK) Signaling in Gliomas

Mitogen-activated protein kinases (MAPK) are proline-directed serinethreonine kinases, which are activated by a variety of cellular stimuli and regulate a variety of cellular processes such as proliferation, differentiation, development, and tumorigenesis. The MAPK superfamily has been clearly divided into three subgroups: extracellularly responsive kinases (p42/44MAPK or Erk1/2); c-jun N-terminal kinases (p46/54JNK) which are also known as the stress-activated protein kinases (SAPKs); and p38MAPK (also known as RK, Mxi-2, CSBP1/2, or HOG-1-related). Although the MAPK families are structurally related, they are generally activated by distinct extracellular stimuli, thus comprising a series of separate MAPK cascades. Genetic and biochemical analyses have identified the universally conserved Ras/Raf/MEK/p42/44MAPK pathway [98, 99] as one of the most ubiquitous MAPK pathways stimulated by various growth factor receptors including PDGF-R, EGF-R and other RTK. In general, receptor-ligand interaction leads to recruitment of adapter proteins such as Grb2, which brings the guanine nucleotide exchange factor Sos to the receptor to form a stable complex, which is required for the activation of monomeric membrane-bound G-protein Ras by the exchange of GDP for GTP

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Ligand Membrane

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Nucleus Elk-1, c-Myc, Ets, STAT 1/3, PPAR␥

Transcription

AFX forkhead transcription factor

Cytoskeletal organization

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Fig. 2. Receptor tyrosine kinase-induced activation of Ras/Raf/MEK/MAPK pathway. RTK, receptor tyrosine kinase; Grb2, growth factor receptor 3-binding protein; Gab1, Grb2 associated binder1; Sos, son-of-sevenless (Drosophila homolog of ras); Ras, the protein product of c-ras; Raf1, a kinase, the protein product of c-raf; MEK, MAP kinase kinase; MAPK, mitogen-activated protein ser/threo kinases; PPAR, peroxisome proliferatoractivated receptor.

(fig. 2) [100]. In addition, EGF-induced activation of Ras may be transduced via another adapter protein, Shc, which binds to activated EGF-R and becomes phosphorylated, creating an additional binding site for Grb2 [101]. Recent work indicates that the protein tyrosine phosphatase SHP-2 gets recruited to the

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EGF-R bound adapter Gab1 and facilitates Sos relocation to GTP-bound Ras [102, 103]. Studies in U87MG glioblastoma cells showed that activation of Ras/Raf/MEK/p42/44MAPK pathway by EGF-RvIII was blocked by PI3-kinase inhibitors, wortmannin and LY294002, whereas wild-type EGF-R induced Erk activation was largely unaffected, suggesting that EGF-RvIII and EGF-R wildtype preferentially use different signaling pathways to activate Ras/Raf/MEK/ p42/44MAPK pathways [104]. One of the best characterized effectors of Ras are the Raf serine/threonine kinases, which are required for the activation of the MEK/p42/44 MAPK pathway and are felt to be critical for Ras-mediated cell transformation (fig. 2) [100, 105, 106]. The other three effector pathways that have demonstrated roles in Ras transformation are those mediated by Ras activation of PI3-kinase, by guanine nucleotide exchange factors (GEFs) for the Ras-related small GTPase Ral (RalGDS), and by Rac (Tiam 1). In the classical pathway, Ras-activated Raf stimulates downstream MAPK kinase, which in turn phosphorylates MAP kinases (also named ERKs) [107–110]. MAPKs or ERKs can translocate to the nucleus to phosphorylate and activate several transcription factors for activation of growth-inducing genes (fig. 2). A recent study showed that normal human astrocytes undergo a p16INK4a-associated senescence-like growth arrest in response to sustained activation of the Ras/Raf/MEK/p42/44MAPK pathway. However, high-grade glioma cells that have dismantled p16INK4a-associated senescence-like growth arrest pathways are potentially regulated by a second p21cip1-dependent growth arrest pathway in response to sustained Ras/Raf/MEK/ p42/44MAPK pathway activation [111]. Mutated and constitutively activated forms of Ras are found in ⬃50% of all human metastatic tumors [112]. In gliomas, specific mutations affecting Ras have not been observed. However, high levels of Ras-GTP have been documented in high-grade astrocytomas [113, 114]. It was recently demonstrated that a region on chromosome 10 (10p13), which carries a Ras suppressor, was deleted in 30% of the high-grade gliomas tested and the majority of oligodendrogliomas, but not in other CNS tumors, bladder or colon tumors, or normal tissue [115]. Ectopic expression of Ras Suppressor-1 inhibited tumorigenesis of a glioblastoma cell line. Similarly, a novel Ras-related protein, Rig (i.e., Rasrelated inhibitor of cell growth) has been shown to be expressed at high levels in normal cardiac and neural tissue. However, an expression of Rig protein was frequently lost or down regulated in neural tumor-derived cell lines and in primary human astrocytomas. Moreover, ectopic Rig expression in human astrocytomas suppressed cell growth [116]. These studies suggest that high levels of Ras-GTP in high-grade astrocytomas may be due in part to a gradual accumulation of Ras suppressor protein mutations in primary glial tumors, as well as from activated RTK signaling.

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Ras transformation of mouse NIH3T3 cells results in elevated levels of cyclin D1 and accelerated G1 progression [117]. Thus, increased Ras activation leads to an increased levels of the G1-specific protein complex CDK4/cyclin D in gliomas, which drives cells into S phase and mitosis. In neurofibromatosis type I (NF1), Ras is constitutively active due to a mutation in the NF1 gene, which encodes the Ras GTPase activating protein-related protein neurofibromin required to convert active GTP-bound Ras to inactive GDP-bound Ras [118, 119]. Activation of other small G-proteins such as Rap1, which also activates the Raf1/MEK/MAPK pathway, may result in increased cell proliferation in astrocytomas [120]. Tuberin, a tuberous sclerosis complex2 gene product, regulates the activity of Rap1, via a GTPase activating protein-related domain at its C-terminus [121]. Analysis of sporadic astrocytomas and ependymomas demonstrated either increased Rap1 or reduced tuberin protein expression in 50–60% of gliomas, compared to a small percentage of schwannomas and meningiomas and none of the oligodendrogliomas studied [120], suggesting that alterations in Rap1 signaling may play an important role in the development of certain sporadic human gliomas. Ral guanine nucleotide exchange factors have been associated with Rasinduced transformation in very few cell lines [122, 123]. Their role in the development of brain tumors and other human cancers is still unclear, but may be more significant than originally thought [120]. Activated forms of Rac have been reported to induce survival in Rat1 fibroblasts and M14 melanoma cells [124, 125], but their role in glial cell survival has not been studied extensively. A recent study with primary glioma and astrocyte cell cultures showed that Rac1 regulates a major survival pathway in most glioma cells, and that suppression of Rac1 activity stimulates death in virtually all glioma cells, regardless of the mutational status [126]. However, normal astrocytes were not affected, suggesting that Rac survival pathway is specific to transformed glial cells and could be used as a potential therapeutic target for the treatment of malignant gliomas. The stress-activated protein kinases, in particular c-jun N-terminal kinases (JNKs), have been implicated in human tumorigenesis [127, 128]. It was reported that the overexpression of EGF-RvIII in mouse NIH3T3 cells leads to constitutive activation of the JNK pathway, which correlates with enhanced transformation by EGF-RvIII [129]. Moreover, a recent study showed constitutively active forms of JNK isoforms in primary glial tumors, indicating a possible association between EGF-RvIII and JNKs in the development of GBMs [130]. Previously, we showed that overexpression of transforming ErbB receptor complexes leads to constitutive activation of Erk MAPK and particularly JNK MAPKs, including JNK2, enhanced transformation, and resistance to apoptosis in primary human glioblastoma cells [131]. A single study in T98G

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glioblastoma cells showed that JNK2 is required for growth of T98G cells in non-stress conditions, and that p21cip1/waf1 may contribute to the sustained growth arrest of JNK2-depleted T98G cultures [132]. Recently, we observed that JNK pathway activation might play an important and specific role in cell migration and motility in GBMs [O’Rourke, pers. commun.]. These observations clearly indicate that JNK pathway events may play a critical role in glial tumor development and progression.

PI3-Kinase/Akt Pathway and Gliomas

The PI3-kinase/Akt pathway is one of the major survival pathways in epithelial cells. In general, activation of the PI3-kinase/Akt pathway begins with receptor activation, rapid stimulation of phosphoinositol metabolism [133, 134], and coupling of PI3-kinase to phosphorylated docking proteins such as Gab1 [135]. PI3-kinase is a phospholipid kinase composed of a regulatory subunit, p85, that contains two SH2 and one SH3 domain, and a catalytic subunit designated p110. We have recently documented that coupling of the SHP-2 protein tyrosine phosphatase to Gab1 is essential for EGF-R mediated PI3kinase activation in glioblastoma cells [136–138]. Activated PI3-kinase phosphorylates phosphatidylinositol 4,5-biphosphate (PIP2) or PtdIns(4,5)P2 via its p110 subunit to generate second messengers PtdIns(3,4)P2 (PIP2) and PtdIns(3,4,5)P3 (PIP3). PIP3 mediates membrane translocation of several signaling-proteins, such as the serine-threonine (Ser-Thr) kinases PDK1 and Akt, the docking protein Gab1, and PLC-␥ [133, 134]. PDK1 phosphorylates Akt at Thr308 [139]. It has been proposed that an unidentified protein kinase (PKC) (a hypothetical PDK2) is responsible for the phosphorylation of Akt at Ser473 leading to its complete activation. Activated Akt phosphorylates and inhibits several proapoptotic proteins such as Bad [140], and the Forkhead transcription factor [141], and GSK-3 [142] to promote cell survival. It also promotes protein synthesis by activating p70S6 kinase via mammalian target of rapamycin (mTOR) (fig. 3). Approximately 80% of all human GBM express activated Akt, which might account for increased cell survival and a worse prognosis for the patients with these tumors [76]. Overexpression of constitutively activated Akt has been shown to convert anaplastic astrocytomas to GBM in a human astrocyte glioma model [70]. A previous study demonstrated an increased association of PI3-kinase with focal adhesion kinase with sustained PI3-kinase activity, PIP3 levels, and Akt phosphorylation in glioblastoma and breast cancer cells expressing a PTEN phosphatase-inactivate mutant, suggesting a possible role for PI3-kinase in cell migration, invasion, spreading, and focal adhesions [143] (cf., ref. 144). Another study

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Fig. 3. EGFR-mediated activation and regulation of PI3-kinase/Akt pathway. PDK1/2, phosphoinositide-dependent kinase 1 and 2; GSK-3, glycogen synthase kinase-3; mTOR, mammalian target of rapamycin; FKHR, forkhead/winged helix protein; 4E-BP1, eIF4Ebinding protein 1; eIF4E, eukaryotic initiation factor 4E; p70S6K, ribosomal protein S6 kinase.

in PTEN mutant C6 glioma cells showed a positive association between increased PI3-kinase/Akt signaling and increased invasiveness and gelatinase activity, again suggesting that unchecked PI3-kinase/Akt activation in gliomas not only serves as a survival signal but might also participate in tumor motility and infiltration [145]. Previously, we reported that EGF-R transcriptionally upregulates VEGF in human glioblastoma cells via PI3-kinase-dependent pathway [146]. We

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recently observed that PTEN mutation in human glioblastoma cells cooperates with EGF-R activation to upregulate VEGF expression in a PI3-kinase/Aktdependent manner, suggesting that PI3-kinase/Akt axis constitutes an important component of the angiogenic cascade required for the development of glioblastoma multiforme [147]. Activation of PI3-kinase/Akt axis is regulated by the lipid phosphatase activity of the PTEN tumor suppressor, which blocks Akt activation by converting PIP3 to PIP2, thereby mediating cell cycle arrest and/or apoptosis [74, 148–150]. Various groups have reported that a region including the PTEN locus on the long arm of chromosome 10 is deleted in many tumors, including glioblastomas, breast, prostrate, endometrial carcinoma, and melanoma [151–153], suggesting a pathogenic role for constitutively active Akt survival pathway in these cancers. Interestingly, it has been shown that PTEN suppresses growth of U87MG glioblastoma cells by blocking cell cycle progression through G1, and this was correlated to a significant accumulation of the cell cycle kinase inhibitor p27kip1 [149], suggesting that loss of function of PTEN is one of the prerequisites for GBM development. It has also been observed that PTEN mutations are more frequent in ‘primary’ or de novo GBM, but not in secondary GBMs, which arise from lowgrade (grade II) or anaplastic astrocytomas (grade III) [154]. The observation that secondary glioblastomas contain p53 mutations as a genetic hallmark but rare PTEN mutations suggests that primary and secondary GBMs develop through distinct genetic pathways involving RTK signaling [154, 155]. Approximately 40% of GBMs are associated with deletions of the PTEN locus on chromosome 10 [156–158]. Earlier studies using mini-chromosome-transfer experiments showed that introduction of human chromosome 10q into glioblastoma cells suppressed the growth in soft agar or formation of tumors in nude mice [159]. Overexpression of PTEN in glioblastoma cells via adenovirus-mediated gene delivery generated similar results in soft agar and in nude mice [160]. Furthermore, we found that overexpression of signal regulatory protein SIRP␣1 in glioblastoma cell lines negatively regulates EGF-R mediated PI3-kinase/Akt signaling and led to reduced transformation, reduced cell migration and cell spreading, and enhanced apoptosis following DNA damage [136]. Studies are underway to establish the mechanism by which SIRP␣1 proteins mediate their effects on PI3-K/Akt activation and also to determine the factors regulating the expression of these proteins in normal astrocytes and GBMs under normal physiological conditions.

Phospholipase C-␥ Pathway and Glioma

Phospholipase C-␥ (PLC-␥) is a phosphoinositide-specific phospholipase, which plays an important role in tumor cell migration occurring via PDGF and

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Fig. 4. EGFR-induced PLC-E pathway participates in activation of different pathways. PLC-␥, phosphoinositide-specific phospholipase C-␥; DAG, diacylglycerol; IP3, inositol 1,4,5-triphosphate; CaMK, Ca2⫹/calmodulin-dependent protein kinases.

EGFR by an undefined mechanism [161]. One study showed that inhibition of PLC-␥ activation by a pharmacological inhibitor or a dominant negative PLC-␥ (PLCz) blocked glioma cell motility and invasion of fetal rat brain aggregates, suggesting PLC-␥ as a potential target for anti-invasive therapy for GBM [105]. Upon receptor activation, PLC-␥ is rapidly recruited to the receptor through the binding of its SH2 domains to pTyr sites in adapter proteins such as Gab1 [97]. Coupling to the receptor activates PLC-␥, which hydrolyzes its substrate PIP2 to generate two secondary messengers, diacylglycerol and inositol 1,4,5-triphosphate (IP3) [74]. IP3 binds to specific intracellular receptors and stimulates the release of intracellular Ca2⫹. Free calcium then binds to calmodulin, which in turn activates a family of Ca2⫹/calmodulin-dependent protein kinases (CaMK).

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Diacylglycerol and Ca2⫹ also activate members of the PKC family [3, 114]. CaMKs and PKCs in turn exert both stimulatory and inhibitory effects on downstream Ras/Raf/MEK/MAPK signaling [121] (fig. 4). Several in vitro studies have supported the involvement of PKCs in glioma cell proliferation and invasion [10, 81, 150, 162, 163]. A recent paper suggests that PKC-␣ and PKC-␧ cooperate with EGF-R in the induction of ornithine decarboxylase (ODC) to increase glioma cell proliferation [25]. Also it was shown that a scaffolding protein RACK1 mediates interaction between integrin ␤ chain and PMA-activated PKC-␧ resulting in increased focal adhesion and lamellipodia formation, indicating that PKC-␧ positively regulate integrin-dependent adhesion, spreading, and motility of human glioma cells. Another study showed that PKC-␧ differentially activated Erks at focal adhesions and was required for PMA-induced adhesion and migration of human glioma cell [164]. Use of PKC inhibitors suggest that PKCs also play fundamental role in regulating cell cycle and participating in cell survival mechanism. A recent study in human glioma cells reported that PKC-␫ and PKC-␤ II phosphoylates cyclin-dependent kinase activating kinase, suggesting their role in cell cycle regulation. Overexpression of PKC-␩ has been shown to increase proliferative capacity of glioblastoma cells and block UV- and gamma-irradiation induced apoptosis by inhibiting caspase-9 activation. These studies suggest that broad spectrum targeting of PKC isoforms may form a basis for the future glioma-therapy.

Signal Transducers and Activators of Transcription (STATs)

EGF-R activates three forms of STAT (STAT-1, -3, -5) [165], whereas PDGFR binds and activates only STAT-5 [166]. Upon receptor stimulation, STATs can be activated by phosphorylation via Janus kinase-dependent or Janus kinase-independent pathways [113, 165]. Activated STATs bind to homotypic or heterotypic STATs through their SH2 domains to form homodimers or heterodimers and translocate into the nucleus to bind to sequence-specific STATresponsive elements on DNA, and activate the transcription of specific target genes such as p21CIP1, cyclin D1, myc, Bcl-2, Bcl-xL, and caspase 1. Collectively, these target genes can regulate cell cycle progression and apoptosis (fig. 5). Of the three STATs, STAT-1 and -3 have been shown to be involved in EGFR-mediated cell cycle regulation [165]. STAT-1 has been implicated as a negative regulator of cell cycle progression and a promoter of apoptosis [37], whereas STAT-3 acts as a positive regulator of cell cycle progression with antiapoptotic activities [128, 167]. A constitutively activated STAT-3␣ has been reported in large percentage of gliomas and medulloblastomas, indicating that STAT-3 may play an important role in EGF-R mediated oncogenesis [117].

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Fig. 5. Activation of STAT pathway by EGFR or PDGFR. STAT, Signal Transducers and Activators of Transcription; Janus kinase.

Targeted Molecular Therapy for Gliomas

Increased knowledge of the structure and activating mechanisms of RTKs and distinct downstream signaling modules has substantially improved our understanding of the cellular machinery that mediates glioma-genesis and maintains the malignant phenotype of transformed glia. There has been a search for new approaches to target specific steps in the pathogenesis of high grade gliomas because treatment with conventional cytotoxic agents has shown very little progress [168]. The heterogeneous nature of malignant gliomas due to different genetic lesions makes a compelling argument for more rational targeted therapies. The molecular and pharmacotherapeutic approaches to gliomas can be broadly divided into immunotherapy (monoclonal antibodies and tumor vaccination), antisense oligonucleotides, gene therapy, and small molecules such as

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(a) Inhibiting receptor-ligand interaction

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Fig. 6. Novel strategies to inhibit aberrant RTK signaling in glial tumor cells by inhibiting receptor-ligand interactions, inhibiting the TK domain of RTK, inhibiting PI3-kinase/Akt pathway, inhibiting farnesyl transferase, inhibiting MAPK pathway, or using antisense oligonucleotides to inhibit the translation of key proteins. PDGFR, platelet-derived growth factor receptor; mRNA, messenger ribonucleic acid.

tyrosine kinase inhibitors and farnesyl transferase inhibitors. In this section, we will focus mainly on small molecule and immunotherapeutic approaches that target RTK signaling with a brief overview on anti-angiogenesis therapy (fig. 6). Immunotherapy: Monoclonal Antibodies in Glioma Therapy Because ⬃50% of GBMs coexpress high levels of EGF-R and a mutant EGF-R receptor EGF-RvIII [54, 8], generation of monoclonal antibodies (mAbs) directed against the EGF-R and/or EGF-RvIII mutant may be useful in blocking tumor progression mediated by aberrant EGF-R signaling. MAbs directed against the extracellular domain of ErbB family RTKs have proven to be an effective strategy to kill tumor cells derived from systemic epithelial cancers [169]. Well-known examples include Herceptin (Trastuzumab) against HER2 or ErbB2 and IMC-C225 (Cetuximab or Erbitux, ImClone) against ErbB1 receptor. The U.S. Food and Drug Administration (FDA) has approved Herceptin for the breast cancer treatment [170–172]. C225 is in Phase III

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clinical trials and has been shown to promote growth inhibitory effects in a variety of tumors including pancreatic, colorectal, renal and breast carcinomas [173, 174]. According to data presented at the 2003 American Association of Clinical Oncology (ASCO), 329 patients with metastatic colorectal cancer were enrolled in a randomized Phase II trial to receive either Cetuximab or a combination of cetuximab and irinotecan (CPT-11), the standard chemotherapeutic agent. The study showed that tumors shrank in 22.9% of the patients receiving two-drug regimen as compared to only 10.8% of those receiving cetuximab alone [175]. In addition, two-drug regimen patients had no signs of tumor progression for a median of 4 months, whereas patients on cetuximab alone had no tumor progression for a median of 1.5 months. These results suggest that the anti-tumor efficacy can be enhanced in most cases, when Mabs are used in combination with cytotoxic agents. A number of monoclonal antibodies have also been generated against EGFRvIII [54, 176]. The most effective MAb was the murine IgG2a Mab Y10, which recognizes both human EGF-RvIII and a murine homologue of this mutation [177]. It was also shown that incubation of EGF-RvIII-expressing cells with Y10 inhibited DNA synthesis, and cell proliferation leading to cell death. In addition, Y10 was able to mediate cell death of EGF-RvIII-positive cells in the presence of complement, as well as with both murine and human cells bearing Fc receptors [177]. Intraperitoneal injection of Y10 led to increased survival in the mice bearing subcutaneous EGF-RvIII-expressing tumors. However, it failed to increase survival in animals with intracerebral-tumors because of its inability to cross blood-brain barrier. Similarly, another Mab directed against EGF-RvIII, Mab 806, showed reduced tumor volume and increased survival of mice bearing xenografts of U87MG.EGFRvIII, LN-Z308.EGFRvIII, or A1207 EGF-RvIII gliomas, but is ineffective with mice bearing U87MG tumors, suggesting the specificity of this antibody [178, 179]. In principle, Mab have been shown to promote receptor internalization resulting in the attenuation of receptor phosphorylation and downstream signaling [51, 179]. Alternatively, monoclonal antibodies can be ‘armed’ with toxins or radionuclides [180]. An anti-EGFRvIII antibody fused to pseudomonas-exotoxin-A generates cytotoxicity in mouse fibroblasts and human-glioblastoma cells expressing EGFRvIII without affecting parental cells, suggesting that ‘armed’ monoclonal antibodies can be more specific than ‘unarmed’ or ‘naked’ antibodies [64]. Monoclonal antibodies against EGF-R and EGF-RvIII have also been used to deliver 125I to GBM cells in animal xenografts and in patients [181–184]. However, iodinated antibodies may pose a risk of killing non-neoplastic cells. A relatively new approach involves the use of immunoliposomes, in which liposomes are attached to antibody fragments, to deliver variety of cytotoxic

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agents, toxins, or even genes for therapy [180–187]. A practical limitation for clinical use of full-length MAbs in the treatment of brain tumors is the requirement for these large molecules to permeate the blood-brain barrier. This limitation is the rationale behind generation of smaller mAb fragments to treat cancers [51, 177]. Immunotherapy: Tumor Vaccination for Gliomas Recent advances in research on antigen presentation, use of specific cytokines, and T-cell-mediated cytotoxicity have given new directions for glioma immunotherapy. One of the recent approaches is the use of adoptive immunotherapy, which can broadly grouped into two categories. The first method of immunotherapy involves vaccination with dendritic cells pulsed with tumor antigens. Antigen presentation by antigen presenting cells (APCs) is a pivotal step in inducing antigen-specific immunity and is essential for tumor vaccine design. Dendritic cells have always been considered as the most potent APCs for initiating an immune response. Dendritic cells are bone-marrowderived cells, capable of presenting antigens in HLA-restricted manner. The ability to efficiently prime CD4 T-helper cells and to generate CD8 cytotoxic T-cells make dendritic cells attractive in vaccine strategies for glioma therapy. One study showed that immunizing tumor cells mixed with syngeneic spleen-derived dendritc cells significantly prolonged mean survival for rats harboring preestablished intracranial tumors [188]. Another group also reported a prolonged survival in rats harboring pre-established intracranial 9L gliomas after vaccination with bone marrow-derived dendritic cells pulsed with acid-eluted protein from 9L glioma cells [189]. Similar results were demonstrated by vaccinating mice with dendritic cells pulsed with Semliki Forest virus-mediated glioma complementary DNA [190] and dendritic cells fused to glioma cells [191]. These observations involving dendritic cell antigen presentation in animal glioma models have prompted a number of vaccine clinical trials in patients with glioma. In the first Phase I clinical trial, patients receiving an autologousperipheral blood-dendritic cells pulsed with peptides from autologous glioma cell surface showed a prolonged survival time with an increased intratumoral cytotoxicity and memory T-cell infilteration [192]. However, another Phase I clinical trial involving patients vaccinated with a novel fusion product of autologous dendritic and glioma cells did not showed statistically significant treatment-associated response to therapy [193]. However, there were no serious adverse autoimmune-responses observed in this study, indicating that dendritic cell-based immunotherapy can be used safely in humans as an adjunct to currently available glioma therapies [194]. Another immunotherapy technique uses gene-based vaccination. Genetic manipulation of tumor cells to express certain cytokines like IFN-␥, GM-CSF, or

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IL-12 has been shown to stimulate a potent immunity against tumors within the brain and provides a basis for gene-based immunotherapy [195, 196]. Vaccination of allogeneic pre-B cells expressing EGF-RvIII has been shown to produce a systemic immune response against autologous intracranial-tumor expressing the same antigen [167]. Vaccination with genetically engineered glioma cells expressing antisense molecules that block specific gene expression, such as glioma-derived immunosuppressive factors TGF-␤2 and insulin-like growth factor-1, has also been shown to suppress intracranial tumor growth [197]. Two approaches have been used to introduce cytokines intracranially with gene transfer techniques. The first approach involves direct intracranial implantation of genetically engineered tumor cells, which secrete cytokines. Cytokines such as IL-2, IL-4, GM-CSF, TNF-␣ and IFN-␥ all have been shown to demonstrate a significant survival advantage in animal models [194, 198]. The second approach involves direct cytokine gene transfer with viral vectors. Genetically engineered adenovirus and herpes simplex virus expressing cytokines have been tested and showed survival advantage, when used in experimental brain tumor models [163, 199, 200]. Despite encouraging results, there have been very few clinical trial results reported. A study involving single GBM patient showed that repeated immunization of autologous tumor cells and a genetically modified fibroblasts that secrete IL-2 promoted an antitumor response mediated in part by cytotoxic T-cells [201]. Similarly, another Phase I clinical trial involving 11 GBM patients immunized with autologous tumor cells modified with Newcastle disease virus showed noticeable peripheral immune responses, but no survival advantage over patients who received conventional combination treatment of surgery, radiotherapy, and chemotherapy [202]. Small Molecule Therapy for RTK Signaling Pathways One promising approach to inhibit aberrant RTK signaling is the generation of small-molecule drugs that selectively interfere with intrinsic tyrosine kinase activity, and thereby inhibit receptor autophosphorylation and downstream signaling cascades [203]. RTK signaling can be targeted at three main levels: the receptor itself or the PI3-kinase/Akt and/or Ras/MAPK signaling modules (fig. 4). Targeting at the Level of the Receptor: EGF-R, EGF-RvIII and PDGFR One reasonable approach to inhibit RTK signaling is to generate small molecule drugs that interfere with the function of the receptor. A number of synthetic small molecules that are tyrosine kinase inhibitors have been generated, particularly for the EGFR/ErbB family. The most advanced of these are ATP analogues of the quinazoline and pyridopyrimidine family that compete with ATP for ATP-binding sites and thus block RTK activation [204].

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Reversible inhibitors ZD-1839 (IRESSA, AstraZeneca) and OSI-774 (Tarceva, Roche/OSI) are two potent EGF-R inhibitors currently being tested in clinical trials against solid tumors [162, 205]. OSI-774 has been approved for the use in Phase III clinical trials alone or in combination with conventional chemotherapy, for non-small-cell lung and pancreatic cancer. Similarly, another reversible ErbB inhibitor, PKI166 (Novartis) and the irreversible inhibitors CI1033 (Pfizer/Warner-Lambert) and EKB-569 (Wyeth-Ayerst) also inhibit EGF-R signaling by competing with ATP for binding to the receptor [206, 207]. CI1033 has also been reported to block EGF-RvIII activation [207]. The FDA has approved STI 571 (Gleevec, Novartis), another tyrosine kinase inhibitor, which inhibits the non-RTK bcr-abl kinase, and two RTKs, PDGFR and c-Kit, for the treatment of chronic-myeloid leukemia [208]. Gleevec also inhibits the growth of GBM xenografts in vivo [209] and is currently in clinical trials for malignant gliomas. Targeting the PI3-Kinase/Akt Pathway Bi-allelic PTEN loss with consequent deregulation of the PI3-knase/Akt pathway is one of the major hallmarks of most of the GBMs [76, 158]. Since this pathway is so crucial for glioblastoma cell proliferation and survival, the PTEN/PI3-kinase/Akt pathway serves as a potential target for therapy. PI3-kinase inhibitors such as wortmannin and LY294002 are effective but also quite toxic, and it has been difficult to generate specific PI3-kinase/Akt inhibitors. An alternative approach is to inhibit FRAP/mTOR kinase, a downstream target of the PI3-kinase/Akt pathway [210], with less toxicity [211]. The immunosuppressant drug rapamycin forms a complex with the immunophilin FKB12 that binds specifically to mTOR, and inhibits its kinase activity [211, 212]. An ester analogue of Rapamycin, CCl-779 (Wyeth Ayerst) showed growth inhibition of PTEN-deficient mouse as well as human cancer cells, indicating the possibility of using mTOR inhibitors for treating PTEN-null human cancers, including glioblastomas [211]. CCl-779 is currently being evaluated in Phase I testing in human malignant gliomas. The main goal is to determine the efficacy of CCI-779 in these trials and to determine whether patient sensitivity correlates with PTEN loss and PI3-kinase/Akt activation. It is also important to determine whether EGF-R and/or EGF-RvIII expressions are associated with tumor response to therapy. Inhibition of the Ras/MAPK Pathway As discussed earlier, specific mutations affecting Ras have not been identified in human gliomas. Rather, constitutive Ras activation in GBMs mainly results from overactivation of EGF-R, EGF-RvIII, and/or PDGFR signaling. For signal-transduction, Ras must attach to the plasma membrane,

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which depends on the post-translational addition of a farnesyl group to its C-terminal end by the enzyme farnesyl transferase. Thus, farnesyl transferase inhibitors (FTIs) may be helpful in blocking Ras-mediated signaling and there has been a great deal of effort into the development of FTIs as cancer therapeutics. FTIs may also target other pathways like Rho-mediated signaling and the PI3-kinase/Akt pathway, which require farnesylation [213]. Two synthetic FTIs, SCH66336 (Schering-Plough) [214] and R115777 (Zarnestra, Janssen Research Foundation), have shown promising results in pre-clinical models, including inhibition of growth of GBM cell lines [215–217]. Moreover, preliminary findings suggest that EGFR overexpresssion in GBM cells can confer increased sensitivity to SCH66336 [216]. R115777 is in Phase I/II clinical trial evaluation involving patients with recurrent glioma [218]. We recently observed in our laboratory that overexpression of mutant EGF-RvIII in human glioblastoma cells leads to constitutive activation of the Erk MAPK kinase pathway in GBM cells, and pharmacological inhibition of this pathway in turn downregulates EGFRvIII phosphorylation and transformation [O’Rourke, manuscript submitted], indicating that the use of inhibitors of the MAPK pathway may be an alternative approach to treat EGF-RvIII-containing malignant gliomas. There are several ongoing efforts to inhibit constituents of the Ras/Raf/MEK/MAPK pathway, which may eventually achieve enhanced efficacy with reduced toxicity in the management of malignant gliomas that are often refractory to conventional therapy. Anti-Angiogenesis Therapy Malignant gliomas are among the most highly vascularized solid human tumors [161]. The invasive nature of gliomas and accompanying neovasculature results from the expression of stimulatory angiogenic factors, which induce endothelial cell proliferation and promote complex interaction between cancer cell and extracellular matrix (ECM). The most extensively studied factors include VEGFs and VEGF receptors, integrins, and metalloproteinases. In recent years, targeting these molecules to generate effective anti-angiogenic drugs has become one of the major thrusts in the field of glioma therapy. VEGF was shown to be differentially overexpressed in gliomas when compared to normal tissue [219, 220]. Furthermore, expression of VEGF has been strongly correlated to microvessel density in gliomas and meningiomas [221], indicating their potential as a target for the drug development. Antibodies against VEGF and VEGF receptors are currently being tested in Phase II/III clinical trials. It was recently reported that a recombinant humanized monoclonal antibody against VEGF, bevacizumab (AVASTIN, Genentech), given with conventional chemotherapy significantly improved survival of patients with metastatic colorectal cancers, suggesting that bevacizumab can be

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effective in treatment of VEGF-overexpressing cancers [222]. Similarly, small molecule inhibitors of VEGF-R tyrosine kinases such as SU5416, SU6668, PTK787, and ZD4190 are already under clinical investigation [223]. The second category of target molecules for angiogenesis includes integrins and metalloproteinases 2 and 9 (MMP-2 and MMP-9), which participate in ECM attachment and degradation to promote neovascularization. Integrins interact with ECM constituents through specific peptide motifs such as Arg-Gly-Asp (RGD) present on ECM proteins to facilitate attachment. Synthetic analogs of RGDs are now being considered as therapeutic tools to inhibit tumor growth and invasion. It was recently demonstrated in an in vivo mouse model and by vitro experiments that an ␣ v-integrin antogonist EMD 121974, a cyclic RGDpenta-peptide was able to induce apoptosis in ␣ v-integrin-expressing U87MG glioblastoma and DAOY medulloblastoma cell lines, suggesting a potential of using RGDs for glioma therapy [224]. Also, conditionally replicativeadenoviruses (CRAds) carrying RGD motifs are being used in pre-clinical studies to treat various cancers with antiangiogenesis approaches, including gliomas [225, 226]. Furthermore, antibodies against variety of integrins have been shown to inhibit matrigel invasion of glioma cell lines and primary cultures [227]. A recent study showed that intraperitoneal administration of IS20I, a specific inhibitor of ␣(v)␤ 3 integrin, reduces tumor growth in nude mouse bearing intracranial and subcutaneous glioma tumors [165]. Synthetic MMP inhibitors such as Marimastat, Batimastat (BB-94), AG3340, and Bay12–9566 have been used in Phase II and III clinical trials in several cancers including malignant gliomas [223]. Only recently, a large number of naturally occurring endogenous inhibitors of angiogenesis, such as platelet-derived factor 4 (PF 4), thrombospondin-1, metallospondin, tissue inhibitors of metalloproteinases (TIMPs), plasminogen activator/inhibitor, angiopoientin-2 (ANG-2), angiostatin (cleaved from plasminogen), and endostatin (cleaved from collagen XVIII) have been discovered, which in theory might be exploited in anti-angiogenesis therapy. Among them, angiostatin and endostatin are already in Phase I trials. Previous studies showed that systemic administration of angiostatin successfully treated subcutaneous and intracerebral gliomas of rat and human origin in nude mouse model [228, 229]. In addition, retroviral or adenoviral transduction of the angiostatin gene into established brain tumors in mice inhibited tumor growth effectively [166, 230, 231]. Similarly, endostatin has been successfully used systemically or by gene transfer methods to treat a variety of cancers, including fibrosarcoma, melanoma, hemangiothelioma, prostate, renal, and mammary, lung, and colon carcinomas [232]. A recent study involving immunological analysis of frozen tissues from 51 patients with astrocytic tumors (Grade 2, 3 and 4) showed an increased levels of tissue endostatin, suggesting a

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definite role of the proteins in glioma formation [233]. Overexpression of endostatin has been shown to reduce tumor growth rate by 90% in rat C6 glioma model [234]. Similarly, endostatin was shown to reduce vascularization, blood flow, and tumor growth in rat gliosarcoma model [235]. Furthermore, combined administration of endostatin and a PKC␣-inhibiting DNA enzyme improved survival rate in rats bearing intracranial malignant glioma BT(4)C, suggesting that combined treatment may represent an attractive therapeutic strategy against malignant gliomas [236]. A study in ovarian cancer cells showed that combined application of angiostatin and endostatin produced synergistic effects on tumor suppression and suggests that the molecular targets differs for the two inhibitory molecules [237]. Taken together, these studies indicate that the angiogenic machinery involves a plethora of interacting molecules, which could be exploited as potential candidates for anti-angiogenesis drug development.

Conclusions and Future Directions

Over the past decade, progress has been made in understanding the molecular pathogenesis of malignant gliomas. Empirical observations and experimental studies have led to an emerging consensus that malignant progression of gliomas occurs through either abnormal activation of signal transduction pathways down-stream of RTKs and/or disruption of cell cycle arrest pathways. It has been clearly elucidated by genetic studies and animal models that there is an intricate cooperativity between RTK signaling pathways and cell cycle regulatory molecules. Abnormal or deregulated RTK signaling can occur via gene amplification or activating mutations of EGFR, overexpression of fibroblast growth factor, FGF-R and PDGF and/or PDGFR, and overactivation of the TGF-␣/EGF-R autocrine loop. This leads to constitutive activation of downstream signaling pathways, which in part involves the PI3-kinase/Akt pathway and the Ras/Raf1/MEK/MAP kinase pathway. The elucidation of the signaling pathways activated due to different genetic alterations is now being translated into glial tumor treatment strategies. Initial efforts have focused on the use of single agents directed at specific target molecules. However, the complexity and cross talk between different signaling cascades may limit potential efficacy of targeting a single molecule. Therefore, it has become imperative to consider combinatorial treatment regimens involving either multiple inhibitors targeting different pathways or combination of these inhibitors with traditional cytotoxic drugs. Furthermore, efforts should also be made to design new strategies to determine the optimal dose and effectiveness of these agents by evaluating their molecular effects as an endpoint rather than assessing traditional maximally tolerated dose and/or overall tumor response.

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Finally, the molecular heterogeneity of GBMs posses a great challenge for any targeted molecular therapy. Therefore, it is essential to develop an expanded molecular sub-classification of GBMs. This can be achieved by using genetic approaches such as gene profiling coupled with careful biological validation and development of new biochemical and clinical markers. Future treatment strategies for malignant glial tumors will involve a fine integration between a broad-spectrum drug regimen, advances in molecular profiling of tumors and protocol design. The potential benefit of these strategies offers a hope for significant improvement in the prognosis for patients with malignant glial brain tumors.

Acknowledgments Our research is supported by grants to D.M.O. from the National Institutes of Health, the Department of Veterans Affairs (Merit Review Program), and The Brain Tumor Society.

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Donald M. O’Rourke, MD 502 Stemmler Hall, Department of Neurosurgery University of Pennsylvania School of Medicine, 36th and Hamilton Walk Philadelphia, PA 19104 (USA) Tel. ⫹1 215 898 2871, Fax ⫹1 215 898 9217, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 557–579

Oncolytic Viral Therapy for Glioma Martine L.M. Lamfersa, Therese Visteda, E. Antonio Chioccab a Molecular Neuro-Oncology Laboratories, Neurosurgery Service, Massachusetts General Hospital, Harvard Medical School, Charlestown, Mass., bDepartment of Neurological Surgery, The Ohio State University Medical Center, James Cancer Hospital and Solove Research Institute, Columbus, Ohio, USA

Introduction

Despite progress in understanding the pathogenesis and molecular characteristics of malignant gliomas, successful treatment for these tumors has not been established and survival for glioma patients has not changed in the last decades. Gene therapy has received substantial attention as a treatment option for these aggressive tumors. During the decade of the 1990s, new molecular techniques yielded gene therapy strategies for the treatment of gliomas using replication-defective viral vectors. These vectors were used as a tool to deliver a wide assortment of therapeutic genes such as apoptotic, immune stimulating, or prodrug activating genes. These developments led to the initiation of a number of clinical trials by the end of the decade. The majority of published clinical trials have been based on the thymidine kinase (TK) paradigm. Cells expressing the viral TK transgene phosphorylate the prodrug ganciclovir into a phosphorylated derivative, which inhibits the DNA replication of tumor cells. Thirteen clinical gene therapy trials for malignant glioma, involving a total of 451 patients, have been published to date [1–13]. Taken together, the results of these studies have not lived up to the expectations created by preclinical data with regard to therapeutic outcome. Reasons for this discrepancy have been examined in recent years. The inability to infect large numbers of tumor cells, the limited spread of the vector within the tumor mass, and the inefficient infection of individual glioma cells all appear to have contributed to the poor results obtained in clinical trials. Prompted by these findings and by an increasing knowledge of cancer cell genetics and tumor cell infection, a novel concept in cancer therapy was developed: viral oncolysis, or ‘virotherapy,’ using replication-competent viruses. This

approach utilizes the inherent cytopathic effects, which result from the normal life cycle of the virus to kill tumor cells. Once inside the cell, the virus expresses proteins that interfere with cellular processes to prevent apoptosis or induce cellcycle progression. Viral replication and viral protein production eventually cause lysis of the infected cells and release progeny virus that can infect neighboring cells, upon which the cycle is repeated. In this way, the virus is capable of disseminating into a solid tumor mass. The terminology ‘oncolytic virus’ refers to viruses, which preferentially replicate in tumor cells as opposed to normal cells. Some naturally attenuated oncolytic viral strains exist that appear to replicate more efficiently in cancer cells than normal tissue, whereas others acquire oncolytic qualities through genetic engineering (e.g., herpes simplex virus or HSV and adenovirus; Ad). The tumor selectivity of oncolytic viruses occurs either during infection or replication. Oncolytic viral vectors based on Ad, herpes virus, polio virus, and reovirus have been described as therapeutic agents for malignant glioma [14–18]. Current research in this field is mainly focused on improving tumor selectivity and oncolytic potency of these agents. In this chapter the latest developments and future directions in research for this type of glioma therapy are described, focusing on selectively replicating herpes- and adeno-viruses.

Herpes Virus as a Tumor-Selective Oncolytic Agent

Herpes Simplex Virus type I (HSV-1) is an enveloped, double-stranded linear DNA virus with a well-defined segmented genome (unique long and short segments) of 152 kb which encodes more than 80 genes. About half the genes are essential for viral replication while the other nonessential genes encode proteins which support the viral life cycle within the host cell. HSV-1 is a good candidate oncolytic virus for cancer therapy for the following reasons: extensive knowledge of the HSV-1 genome allows the virus to be modified to improve safety and efficacy; high-titer production is possible (typically ⬎1010 infectious particles/ml); the HSV-1 genome contains nonessential genes that can be replaced with up to 30 kb of therapeutic genes without significantly affecting virus titers or replication [19]; many nonessential genes are associated with neurovirulence; neurotropism makes HSV-1 appropriate for targeted brain tumor therapy [20]; anti-herpetic agents exist which can terminate uncontrolled viral replication [21]; and insertional mutagenesis which is associated with retroviral vectors [22] is prevented since the viral genome does not appear to integrate into the host cell’s DNA [23]. Nevertheless, the HSV-1 and oncolytic viral therapy faces some major challenges. First, genetic manipulation of the large HSV-1 genome is difficult. Second, pre-existing immunity in the population must be taken into consideration.

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HSV-1 is ubiquitous in the adult population and 50–90% of people over 30 years of age have HSV antibodies. Neurotoxicity of HSV-1 also represents a challenge since the wild-type virus is capable of propagating in neurons and glia, resulting in necrotizing and potentially fatal encephalitis. Finally, HSV-1 replicates in both dividing and nondividing cells, such as postmitotic neurons, while cancer therapy aims for selective targeting of tumor cells. In this respect, selective oncolytic viral therapy for brain tumors offers the advantage of targeting islands of replicating tumor and endothelial cells (tumor angiogenesis), amidst the nonreplicating tissue of the central nervous system. Several different strategies have been applied to generate tumor-selective HSV-1 vectors. These vectors are designed to target cells, which have altered signal-transduction pathways that promote tumorigenesis. Mutations or deletions in the viral genome are commonly introduced to eliminate the expression of specific viral proteins, which render the mutant vector dependent on the tumor cells for viral propagation. Some of these viral proteins are associated with neurotoxicity and a dual beneficial effect is obtained by mutating these genes. TK and Ribonucleotide Reductase HSV-1 Mutants Many viruses, including HSV-1, encode enzymes, which are needed for the virus to propagate in quiescent cells. Some of these proteins are similar to cellular enzymes, which are expressed in dividing and thus in cancer cells, but not in normal nondividing cells. This is the case for the enzymes TK and ribonucleotide reductase. The first attenuated HSV-1 vector designed for brain tumor therapy was targeted to the p16/Retinoblastoma (Rb) pathway by deleting the viral TK gene, making viral replication dependent on the presence of TK in tumor cells [24]. This so-called replication-conditional HSV-1 vector prolonged survival in animal brain tumor models. Neurotoxicity was retained, however, and combined with the loss of susceptibility to anti-viral therapy with acyclovir or ganciclovir, the mutants were unsuitable for clinical application. The value of attenuating HSV-1 vectors for a tumor targeting was nevertheless demonstrated. The HSV-1 equivalent to cellular RR is the ICP6 protein. HSV-1 with mutations in the ICP6 gene are targeted to tumor cells with a defect in the p16/Rb pathway due to their up-regulation of cellular RR [25]. The ICP6mutant vector has been shown to replicate with a 100-fold greater viral titer in cancer cells as compared to normal cells [26]. The vector exhibited anti-tumoral efficacy and decreased neurovirulence in animal glioma models, and is one of the two most intensively studied single mutant HSV-1 vectors. g1–34.5 Mutant HSV-1 Vectors The ␥1–34.5 gene is a major determinant of HSV-1 neurotoxicity. Further, ␥1–34.5 serves two functions which are important for viral replication. The

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protein complexes with proliferating cell nuclear antigen and thus allows for viral replication [27]. The cellular counterpart is GADD34, which inactivates proliferating cell nuclear antigen in dividing cells. The second function of ␥1–34.5 is to override host cell defence mechanisms elicited as a response to viral infection. Mutation in the ␥1–34.5 gene renders the HSV-1 vector replication conditional and at the same time neurovirulence is reduced. The ␥1–34.5 mutant is the second of the two most studied single HSV-1 mutants and shows the lowest neurovirulence of all the single mutants. A ␥1–34.5deleted mutant (designated 1716) has progressed to phase 1 clinical trial in two studies including 21 patients altogether [28, 29]. In both studies, patients with recurrent malignant glioma, refractory to conventional therapy, were treated. The HSV-1 vector was well tolerated at doses up to 105 infectious particles after stereotactic intratumoral injection, and no adverse events were reported. In the first trial, multiple injections of 1 ml total volume were performed. No viral DNA was detected by PCR in serum and buccal swab samples taken from five patients, four out of nine patients were alive at 14 months after the treatment [28]. In the second trial, patients were injected with the virus followed by tumor resection after 4–9 days, and virus replication was detected in the tumor tissue. In some patients the amount of recovered virus exceeded the input dose [29]. One thousand seven hundred and sixteen patients have also been clinically tested for advanced melanoma [30]. In 3 patients receiving multiple intranodular injections, histopathological analyses revealed tumor necrosis and HSV antigen presence in the tumor only. HSV-1 Vectors with Dual Mutations Single mutant HSV-1 vectors are potentially associated with the risk of restoring a wild-type phenotype [31]. Concern about this risk led to the design of vectors with dual mutations. The most studied combination virus is a conditionally replicating derivative of HSV-1 with both deletions of the ␥1–34.5 gene and the ICP6 gene inactivated by insertion of the ␤-galactosidase gene [32]. G207 has been tested in a phase I clinical trial in patients with recurrent glioma [33]. Twenty-one patients were included and single injections of doses up to 3 ⫻ 109 infectious particles were administered. No adverse events were observed and the virus was well tolerated to the extent that no maximum tolerated dose was achieved. Promisingly, 2 patients were still alive 4 years after the treatment. Another attenuated HSV-1 vector currently being studied intensively for cancer therapy is NV1020, which originally was developed as a herpes vaccine that never proved successful. A phase I clinical trial with NV1020 is ongoing for colorectal liver metastases, with the virus administered through the hepatic artery [34].

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Tumor-Specific Promoters for Driving HSV-1 Replication Tumor-selective replication of viral vectors can be achieved by linking viral gene transcription to promoters being expressed only in dividing or malignant cells. Myb34.5 represents a transcriptionally potentiated vector. This mutant vector places the ␥1–34.5 gene under the control of the promoter of B-Myb, a cell-cycle regulatory transcription factor which is overexpressed in cancer cells with mutations in the p16/Rb tumor suppressor pathway. Myb34.5 also carries a deletion in the ICP6 gene. Another HSV-1 mutant carries a deletion in one of the viral genes which are essential for replication, ICP4. The ICP4 gene is reintroduced into the HSV-Tk gene, which is then inactivated, and expression of ICP4 is regulated by the albumin promoter. In adults, albumin is only expressed in hepatocytes and hepatocellular carcinomas. The mutant has shown selective replication in hepatocellular carcinomas in mice, with sparing of the normal hepatocytes [35, 36]. More recently, a second ICP4-deleted vector has been generated using the calponin promoter to drive HSV-1 replication in soft tissue and bone tumors [37]. A similar transcriptionally regulated replication strategy has been described for Ad vectors (see below).

Adenovirus as Tumor-Selective Oncolytic Agent

Adenoviruses (Ad) are attractive candidates as oncolytic viruses due to their low pathogenicity and their efficiency in infecting target cells. In addition, an extensive knowledge of the Ad genome allows genetic engineering to modify wild-type viruses to improve their safety and efficacy. The Ad replicative process (fig. 1) is made up of the following steps. First, primary attachment of the Ad particle to the surface of the host cell via its fiberknob to the coxsackie and Ad receptor (CAR). The CAR-docked particles interact with ␣v integrins, which promote virus internalization by endocytosis. Inside the cell the Ad escapes from the endosome and is transported toward the cell nucleus, during which the virus particle is partially broken down. In the nucleus the viral DNA remains episomal and is transcribed. The Ad proteins encoded by the early regions of the Ad genome are expressed first, upon which the Ad genome is replicated, followed by the expression of Ad proteins encoded by the late regions. Progeny Ad particles are assembled and the induction of cell death leads to the release of Ad progeny from the cell [38–40]. For application of replication-competent Ad as oncolytic agents, efforts have been aimed at improving the safety of the virus by constructing genetically modified Ad mutants. Three approaches have been described to render Ad propagation selective for tumor cells: deletions or mutations in viral genes

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Fig. 1. Life cycle of the adenovirus.

essential for replication in nonmalignant cells; insertion of tumor-specific promoters driving viral replication (transcriptional targeting); modification of viral tropism to limit infection to tumor cells (transductional targeting). Ad E1 Gene Modifications For the first approach, it is necessary to make mutations or deletions in the E1 region of the Ad genome. This region contains two genes, E1A and E1B, which encode proteins required for a productive Ad lytic cycle [41]. E1 proteins induce cell cycle activation and inhibit apoptosis by binding regulatory proteins such as p53 and the retinoblastoma gene product (pRb) in the host cell. In theory, Ad mutants unable to inactivate p53 or pRb would propagate poorly in cells expressing these proteins, but more efficiently in tumor cells where p53 or pRb is in most cases already inactive. Using this approach, an E1B-mutated Ad, termed ONYX-015, was constructed which lacked the E1B p53-binding viral protein, intended to enable selective replication of this virus in p53-deficient cells [42]. Some controversy exists as to whether or not the deletion of E1B p53binding viral protein is the sole mechanism by which ONYX-015 has selective replication capacity in malignant cells [43, 44]. Nevertheless, this oncolytic Ad proved to be very effective in a wide range of preclinical models for various tumor types, including glioma, cervical carcinoma, laryngeal carcinoma, colorectal carcinoma, and head and neck cancer, with reports on complete regression of xenograft tumors and long-term survival [42, 45–47]. Based on these studies ONYX-015 entered phase I clinical trials and was the first Ad to reach clinical testing. Multiple trials of ONYX-015 in over 300 cancer patients, using both intratumoral and intravascular delivery techniques, have established safety and demonstrated selective activity of the virus within

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malignant tissue [48–50]. However, despite biological activity of ONYX-015, clinical benefit was not seen in most patients until viral treatment was also combined with chemotherapy in patients with head and neck cancer [51]. Currently, ONYX-015 is also under investigation in a phase I dose-escalating study in which patients with recurring malignant glioma are treated by intratumoral injections. Preliminary results indicated that there were no serious adverse events attributable to ONYX-015 administration in the brain [Chiocca et al., unpubl. observations]. On a similar theoretical basis, Ad were engineered to replicate selectively in tumor cells with lesions in the pRb pathway. This was accomplished by deleting 24 bp from the E1A gene, which abolishes the pRb-binding capacity of the E1A protein [52, 53]. This Ad mutant, known as Ad⌬24 or Addl922–947, demonstrated reduced replication in nonproliferating normal cells relative to other gene-deleted and wild type Ad. The anti-cancer efficacy of these agents was confirmed in vitro and in heterotopic xenograft animal models of human glioma and breast cancer [52, 53]. Strict tumor selectivity of E1A-mutated Ad has, however, been challenged and additional mutations may be needed to enhance pRb-selectivity [54]. Tumor-Specific Promoters Another approach to constructing tumor-selective Ad involves the substitution of a viral promoter with a promoter of a tumor-associated antigen to drive viral replication in tumor cells. The tumor-specific promoters are generally inserted into the E1A promoter region of the Ad. The rationale behind these vectors is that expression of the early viral gene E1A and therefore the whole Ad transcription program will depend on the activity of these tumor specific promoters. A number of tumor-selective oncolytic Ad have been developed utilizing this approach for a number of indications: for prostate carcinoma using the prostate-specific antigen (PSA) [55]; for breast cancer using estrogen-responsive elements [56]; and for hepatocellular carcinoma using the ␣-fetoprotein promoter [57]. Both specificity and efficacy of these vectors were demonstrated in vitro and in vivo. The PSA promoter-driven Ad, CV-706, was tested in a phase I clinical trial for prostate cancer and demonstrated clinical activity as reflected by changes in serum PSA levels [58]. Recently, Post et al. [59] described the development of a hypoxia-dependent replicative Ad using the hypoxia-inducible factor promoter to drive E1A. This virus-induced cytolysis of tumor cell lines including glioma in a hypoxia-dependent manner in cell culture. Other putative tumor-specific promoters which have been described in the context of replication-competent Ad, and which may be of interest for testing in glioma are Midkine [60], telomerase reverse transcriptase [61], and osteocalcin [62] promoters. Astrocyte-specific promoters such as glial fibrillary acidic protein or

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nestin also have been reported to drive transgenes delivered by nonreplicating Ad vectors to glioma [63, 64]. However, these approaches have thus far not been described for replication-competent Ad. Modification of Ad Tropism The third strategy to achieve tumor-targeted Ad involves the modification of Ad tropism in order to limit Ad infection to certain cell types and to improve the infection efficiency. For glioma, the first limitation for an efficient Ad infection is low levels of Ad receptor CAR expression on the tumor cell membrane [65–68]. To overcome this barrier, Ad can be retargeted towards other molecules highly expressed on the glioma cell membranes such as epidermal growth factor (EGFR) and ␣v integrins. This concept was demonstrated using bispecific antibodies directed towards EGFR on one end and the Ad fiber knob on its other end. Preincubation of the Ad vector with the bispecific antibodies resulted in CAR-independent glioma cell infection and improved the infection of primary glioma cell cultures obtained from tumor material [65, 67]. In the context of replication-competent Ad, the use of bispecific antibodies to redirect Ad cell binding will only improve the first infection round, as the progeny Ad will not have access to the bispecific antibody. This limitation may be overcome by the insertion of an expression cassette encoding the bispecific antibody into the genome of the Ad.⌬24 Ad, thereby allowing the progeny viruses to become retargeted. Using this method, the EGFR-targeted Ad.⌬24 efficiently killed primary human CAR-deficient brain tumor specimens that were refractory to the parent control virus [69]. Another approach used to target Ad to glioma cells involves the insertion of an integrin-binding peptide, arg-gly-asp into the fiber of the virus, allowing it to make its primary attachment to integrins. Using replication-deficient Ad vectors encoding luciferase, it was demonstrated that arg-gly-asp modifications drastically enhanced the infection efficiency of various tumor cells including glioma [65, 70, 71]. Arg-gly-asp-modified Ad⌬24 virus demonstrated that improved infection efficiency translates to markedly enhanced oncolysis in primary glioma cell lines and impressive anti-glioma activity in subcutaneous and intracranial glioma xenografts [66, 72]. Retargeting of oncolytic Ad to glioma cells was demonstrated by Shinoura et al. [73], where an increased cytopathic effect was found when ONYX-015 was modified with a stretch of twenty lysine residues at the C-terminus of the fiber, both on glioma cells and in subcutaneous xenografts. Alternative molecules that have proven useful for redirecting Ad attachment and entry and which may be of interest to targeting glioma are the fibroblast growth factor receptor [74], folate receptor [75], transferrin receptor [76] and vascular endothelial growth factor receptor [77].

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An alternative strategy to genetic retargeting of Ad involves the substitution of Ad fibers with those from a different Ad serotype or host range that is capable of binding to a cell surface receptor other than CAR. It has been demonstrated that such chimerics can enhance the infection of various tumor cell lines and reduce liver transduction after intravenous injections in mice [78–81]. All the above-described targeting approaches were designed to overcome low CAR expression and improve the infectability of tumor cells. However, for achieving true tumor selective infection, it is essential to eliminate binding of the virus to nontarget cells by abolishing the native tropism of Ad. Einfeld et al. [82] described the so-called doubly ablated Ad vectors which lacked both CARbinding as well as the ␣v integrin-binding capacities, and showed a dramatic reduction in the infection of healthy organ tissues. Combining this approach with tumor-specific targeting strategies is expected to greatly enhance the tumorspecificity of Ad vectors. Support for this approach comes from studies in which EGFR targeting technology was combined with doubly ablated Ad vectors. Infection of organotypic glioma spheroids and normal brain explants from the same patients with EGFR-targeted doubly ablated vectors exhibited up to 38-fold-improved tumor-to-normal brain targeting index compared to native vectors [83]. Studies in immune-competent rats with doubly ablated vectors support in vitro findings of abolished brain transduction using these vectors. However, while transduction was reduced by more than 95%, inflammation was not reduced compared to wild-type vectors, demonstrating that brain inflammation occurs independently of Ad binding and infection of cells; alternative strategies are required to circumvent this problem [84]. Replication-competent Ad with ablated CAR and integrin binding in combination with tumor-specific targeting moieties have thus far not been described; this approach would be expected to greatly improve tumor-specific infection and oncolysis of malignant tissues.

Improving Oncolytic Potency

Although preliminary data from ongoing clinical trials with oncolytic viruses provide evidence for an anti-tumor effect, it appears that often the tumor proliferation out-paces vector replication. This phenomenon can, at least in part, be explained by the fact that the viruses used have a relatively slow life cycle and are quite immunogenic. Therefore, investigators are attempting to bolster the therapeutic potential of these agents by interfering with the immune response or improving the oncolytic potency of the viruses. The latter can be achieved by combining gene transfer with other modes of anti-tumor therapy

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such as chemotherapy and radiotherapy or by insertion of other genes into the viral genomes. Evading the Anti-Viral Immune Response The immune response of the body to Ad and herpes virus follows a similar pattern, consisting of an immediate innate response and a slower adaptive response. The nonspecific early occurring immune response contributes the largest effect to the elimination of the viruses [85]. This innate response includes the activation of the complement cascade [86] and recruitment and activation of macrophages, neutrophils, and natural killer cells, which kill infected cells either directly or indirectly by secreting anti-viral cytokines and chemokines [87, 88]. The recruitment and activation of antigen-presenting cells are essential for the development of an adaptive immune response [89, 90]. The adaptive immune response is elicited when the viral antigens are presented to T-helper cells, resulting in the activation and secretion of cytokines by the T-helper cells and the maturing of B-cells into plasma cells. Plasma cells produce large amounts of high-affinity antibodies directed against the infected cells and viral proteins. Together, these responses can result in a rapid inactivation of the oncolytic viruses and impede their therapeutic efficacy. For this reason, several strategies have been developed to circumvent early inactivation of the virus by the immune system. Initially these studies were performed using replication-deficient vectors in the interest of improving and prolonging transgene expression. Partial immune ablation using cytokines or CTL4A-Ig can lead to persistent Ad gene expression in mouse lung and liver [91, 92]. The anti-cancer drugs etoposide and cyclophosphamide (CPA) also were shown to enhance intratumoral transgene expression in immunocompetent mice [93]. Production of neutralizing antibodies to Ad and cytotoxic T lymphocyte mediated lysis of virally transduced cells was significantly suppressed in these animals. In a study using herpes vectors for gene transfer to neurons in the spinal cord, the coadministration of cyclosporine A led to a more persistent transgene expression in the infected cells [94]. The inactivating role of the complement system is underscored by studies of Ikeda et al. [95] who demonstrated that rodent plasma inhibits cell transduction by Ad and herpes vectors. In vitro inactivation of complement with mild heat treatment of the serum restored transduction efficiency. In vivo, complement depletion was achieved by administering cobra venom factor prior to the intra-arterial delivery of replication-conditional (oncolytic) HSV in a rat model for multiple intracerebral tumors. Complement inactivation led to a strong increase in the initial infection efficiency of the tumors. In earlier reports, it had been demonstrated that administration of CPA enhanced the propagation of the oncolytic virus [96]. Combined treatment of oncolytic HSV with

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cobra venom factor and CPA inhibited both the innate and anti-HSV neutralizing antibody response, and their concerted action prolonged survival of rodents bearing intracerebral tumors [95]. A completely different approach with regard to the anti-viral immune response postulates that an intact immune system may actually be of benefit by eliminating virus infected tumor cells and contributing to tumor regression [97]. Various strategies to exploit this mechanism have been undertaken, including the concomitant overexpression of cytokines or growth factors (see below). Whether the immune response to the virus is helpful or harmful to the therapeutic potency of oncolytic viruses requires additional investigations into underlying mechanisms and especially into the precise time course of events. It is not inconceivable that an initial immune suppression which allows viral infection and propagation to take place, should be followed by the immune boosting to evoke an optimal vaccination response. Armed Therapeutic Viruses The next approach to improving oncolysis by replicative viruses involves the insertion of therapeutic transgenes into the viral genome. Such ‘armed’ replication-competent viruses allow the action of therapeutic proteins to be combined with anti-tumor properties of the viral infection. This approach has several possible advantages. First, due to the amplification of virus within the tumor, transgene production and spread are highly increased as compared to infection with the replication-defective vector counterparts. This concept was demonstrated for Ad using the marker gene Luciferase [98, 99] as well as for HSV using the ␤-galactosidase gene [100–102]. Marked increases in transgene expression were noted using replication-competent compared to the replication-defective vectors. In addition, transgenes that have a nonoverlapping toxicity range with the viralinduced oncolytic effects can be selected in order to maximize their therapeutic benefit. Transgenes have been inserted that encode prodrug converting enzymes, immune stimulatory molecules, and apoptosis-enhancing proteins. Several groups have constructed genetically engineered oncolytic viruses encoding a prodrug-converting enzyme. These enzymes convert nontoxic prodrugs into cytotoxic metabolites and are often soluble to allow spreading within the tumor. Using this approach, a tumor-selective herpes virus was engineered encoding the rat cytochrome P450 (CYP2B1) transgene [103]. This liver enzyme activates the prodrug CPA into an active anti-cancer and immunosuppressive metabolite [104]. Addition of CPA potentiated oncolytic effects on this HSV mutant against cultured tumor cells and subcutaneous tumor xenografts established in athymic mice [103]. The addition of Ad oncolysis to the HSV-Tk/ganciclovir (enzyme-prodrug) strategy resulted in a striking improvement in the treatment efficacy in various

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human cancer xenografts, and led to the initiation of a phase I study in patients with malignant melanoma [105–107]. In human glioma xenografts, the oncolytic effects of replicating Ad vectors and HSV-Tk/GCV also demonstrated complementary therapeutic efficacy [98]. The addition of ganciclovir to the treatment with HSV mutants, which already express HSV-Tk, enhanced the regression of syngeneic rat gliosarcomas [108]. However, contradictory evidence exists with regard to the efficacy of combined therapy with HSV-Tk, ganciclovir, and oncolytic viruses. In fact, when applied with HSV in colorectal cancer cells [109, 110] and with Ad in lung carcinoma cells [111], ganciclovir actually decreased the anti-cancer efficacy. Administration of the prodrug appears to eradicate the infected cells, thereby counteracting further virus production and viral-induced oncolysis. Therefore, this approach may require optimization of the administration schedule of prodrug to ensure that sufficient viral replication and enzyme production is maintained. In addition, the gap junctional ability of the target cells determines the contribution of the bystander effect of phosphorylated ganciclovir to the final anti-cancer efficacy [109, 110]. Multiple prodrug-activating gene therapies have been used simultaneously in combination with oncolytic Ad. A replicating Ad with double enzyme/prodrug gene therapy containing the cytosine deaminase and HSV-Tk fusion gene markedly enhanced oncolysis relative to the isolated viral effect in cancer cells [112]. The combination of HSV-Tk and CYP21 gene transfer mediated by an oncolytic HSV provided anti-tumor effects that were more significant than all other treatment combinations [113]. The second type of genes that have been inserted into the oncolytic viruses were selected to boost the immune response to the infected tumor cells by stimulating localized inflammatory and/or immune responses. In the context of Ad, this was first demonstrated using a tumor-selective virus engineered to express interferon, which strongly enhanced the anti-tumor activity compared to relevant control Ad in immune-deficient mice bearing breast carcinoma xenografts [114]. Moreover, a number of replication-competent recombinant HSVs that encode immunostimulatory molecules have been constructed. Andreansky et al. [115] demonstrated that survival of immunocompetent mice bearing intracerebral tumors could be prolonged when treated with tumor-selective HSV encoding interleukin 4 (IL-4) compared to controls and immunohistochemical analysis demonstrated the marked accumulation of inflammatory cells [115]. Similarly, oncolytic HSV mutants expressing IL-12, IL-2, or soluble B7–1 immunomodulatory molecule were found to produce survival benefit compared to control viruses in various tumor models including glioma in immunocompetent mice, by combining oncolytic and immunostimulatory effects [116–119]. Moreover, insertion of the potent immune stimulator granulocyte macrophage colony stimulating factor into an oncolytic HSV backbone improved the shrinkage or clearance of

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tumors compared to control virus. These mice were also protected against rechallenge with tumor cells [120]. These data suggest that not only can the expression of immunomodulatory molecules potentiate oncolysis, but they may also induce a level of anti-tumor immunity. Finally, insertion of transgenes may improve the oncolytic potential of the replication-competent virus itself. Opportunities for enhancing the anti-tumor potential of oncolytic viruses are at the final stage of the viral reproductive cycle that involves lysis of the host cell and release of viral progeny [121]. Oncolysis of cancer cells when compared to lysis of the natural host cells may be suboptimal due to cancer cell-specific genetic alterations. These alterations mainly affect pro- and anti-apoptotic pathways that regulate the cell cycle. Coordinated and timely overexpression of apoptotic factors concomitant with the viral replicative cycle is expected to enhance the anti-tumor potential of the oncolytic virus. This concept was demonstrated using the Ad⌬24 oncolytic Ad engineered to express p53 during late stages of viral replication and which exhibited up to more than 100-fold enhanced the oncolytic potency on human cancer cell lines of various tissue origins [122]. In another study, expression of a dominant-negative I-␬B from a selectively replicating Ad sensitized tumor cells to recombinant human tumor necrosis factor-␣ (TNF-␣)-mediated apoptosis. Using this approach it could be demonstrated that the induction of apoptosis during viral DNA replication compromised virus production, whereas apoptosis induced after virion assembly enhanced viral release from infected cells and dissemination [123]. Combination Therapy with Conventional Treatments Combination treatment with conventional therapies offers a number of advantages. First, enhanced therapeutic efficacy of dual treatments will allow the administration of lower viral doses to achieve a therapeutic effect, which is important given the fact that sufficient virus delivery to tumors remains one of the major hurdles in clinical viral (gene) therapy strategies. Furthermore, combined treatment allowing lower viral doses may also lower toxic side effects. The first studies to describe the effects of dual treatment with an oncolytic virus and conventional therapy were performed with ONYX-015 Ad. The efficacy of this agent combined with cisplatin and 5-fluorouracil was significantly greater than either agent alone in nude mouse tumor xenografts [46]. These results led to the design of a phase II trial, using these agents in patients with squamous cell carcinoma of the head and neck [51]. The results of that study mirrored the preclinical data, including the frequent occurrence of complete remissions in patients treated with combination therapy. Synergy with chemotherapeutic agents paclitaxel and docetaxel was demonstrated with the

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prostate cancer-specific oncolytic Ad, CV706, in a xenograft model of prostate cancer [124]. Synergy with chemotherapeutic agents also has been described with herpes viruses. The oncolytic effect of HSV-1716 in combination with mitomycin was synergistic in two of five non-small cell lung cancer cell lines in vitro and inhibited tumor growth more efficiently than either agent alone [125]. The combination treatment of the HSV mutant G207 and vincristine led to strongly enhanced in vitro cytotoxicity without affecting infection efficiency and replication of G207 in rhabdomyosarcoma cells. In vivo combination treatment of alveolar rhabdomyosarcoma using intravenous G207 and vincristine resulted in complete tumor regression without evidence of regrowth in 5 of 8 animals, whereas none of the animals receiving either monotherapy were cured [126]. In the context of malignant brain tumors, the combination of oncolytic viruses with radiotherapy is perhaps more relevant than with chemotherapy considering the efficacy of standard treatments. Rogulski et al. [127] have studied the anti-tumor activity of ONYX-015 in combination with radiation in colon carcinoma xenografts. ONYX-015 viral therapy combined with radiation improved tumor control beyond that of either monotherapy. Studies with PSA promoterdriven oncolytic Ad CV706 in combination with radiotherapy demonstrated an improvement in the therapeutic response in prostate cancer xenografts without increasing toxicity; a phase I study was initiated based on those data [58, 128]. In glioma, synergistic oncolytic activity of ONYX-015 with radiotherapy was demonstrated in subcutaneous xenografts [129]. Also the strong anti-tumor activity of Ad5-⌬24RGD, the integrin-targeted Ad⌬24 variant, in malignant glioma could be further enhanced with low-dose irradiation such that the same therapeutic effect was achieved with a 10-fold lower viral dose [66]. The combination therapy of oncolytic HSV and irradiation has produced varying results. Whereas a potentiating effect of irradiation on G207 viral oncolysis in cervical and colorectal cancer xenografts was found [130, 131], no enhancement of anti-tumor activity was seen when these treatment modalities were combined in subcutaneous tumor models of human and murine prostate cancer [132]. Spear et al. [133] also found complementary toxicity between irradiation and the oncolytic HSV-1 mutant for the ICP6, regardless of cell type, time, MOI, irradiation dose, or culture conditions, without any evidence of augmented apoptosis or viral replication. In human glioma xenografts, on the other hand, dual treatment with the HSV-1 ␥134.5 mutant caused a significantly greater reduction in the volume or the total regression of tumors than either irradiation or infection alone. This enhanced oncolytic effect of the combined treatment correlated with 2- to 5-fold enhanced viral replication in irradiated tumor cells compared to tumors receiving virus only [134]. These results were extended to a second study in mice bearing intracerebral tumors which received

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␥134.5-mutant virus in combination with fractionated radiotherapy. Analysis of survival data revealed that the interaction between these treatment modalities was synergistic [135]. In conclusion, results from studies of combination therapy, demonstrating an enhanced therapeutic efficacy of oncolytic viral therapy over single modality treatment, are very encouraging. Investigators are successfully combining the above described strategies with armed therapeutic viruses. A trimodal approach (lytic virus, double enzyme/prodrug gene therapy, radiation) was found to be superior to any other combination in carcinoma xenografts. Significant tumor regression and ultimately 100% tumor cure were reported [136].

Future Perspectives

Future development of oncolytic virus therapy for glioma will rely on the outcome of multiple lines of ongoing research and merging of the optimal strategies which have evolved from them. Viral engineering is leading to the development of a new generation of genetically engineered truly tumor-selective and potent oncolytic viruses. A multimodal treatment can be envisioned in which the optimal vector is combined with optimal dosing and scheduling regime of conventional therapies. Combining cytotoxic agents with differing mechanisms of action is an attractive approach to the treatment of malignant glioma. It allows independent tumor debulking by specific selectivity of each agent, resulting in broader spectrum of the oncolytic action. This broader spectrum of oncolysis is expected to be particularly more efficacious in tumors with strong heterogeneity and mutagenic potential, such as glioblastoma multiforme. The design of novel imaging techniques for oncolytic viruses which monitor the transgene expression, viral distribution, and/or replication of the virus within the tumor [137] will provide a direct insight into the in vivo biology of virus-induced tumor oncolysis and can identify potential targets for the improvement of the vectors or the route of administration. The route of administration also constitutes an important field of research. Results from clinical trials have demonstrated the limitations of stereotactic or direct intratumoral injection of vectors. Hence, investigators are studying the efficacy of alternative delivery routes to the brain such as convection-enhanced bulk-flow interstitial delivery, intrathecal and intraventricular injection, and intravascular infusion with or without the modification of the blood-tumor barrier [138]. Such new surgical approaches may improve the transduction of tumor cells, and most importantly aim to reach the infiltrated tumor cells in the surrounding normal brain.

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Ultimately, the field of oncolytic virus therapy will provide a variety of agents with anti-glioma efficacy. Similar to the conventional drug-testing programs, different oncolytic viruses may have different ranges of efficacy and toxicity. Phase I clinical trials will provide us with the answers regarding such ranges.

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109 Carroll NM, Chase M, Chiocca EA, Tanabe KK: The effect of ganciclovir on herpes simplex virus-mediated oncolysis. J Surg Res 1997;69:413–417. 110 Yoon SS, Carroll NM, Chiocca EA, Tanabe KK: Cancer gene therapy using a replicationcompetent herpes simplex virus type 1 vector. Ann Surg 1998;228:366–374. 111 Lambright ES, Amin K, Wiewrodt R, Force SD, Lanuti M, Propert KJ, Litzky L, Kaiser LR, Albelda SM: Inclusion of the herpes simplex thymidine kinase gene in a replicating adenovirus does not augment antitumor efficacy. Gene Ther 2001;8:946–953. 112 Freytag SO, Rogulski KR, Paielli DL, Gilbert JD, Kim JH: A novel three-pronged approach to kill cancer cells selectively: Concomitant viral, double suicide gene, and radiotherapy. Hum Gene Ther 1998;9:1323–1333. 113 Aghi M, Chou TC, Suling K, Breakefield XO, Chiocca EA: Multimodal cancer treatment mediated by a replicating oncolytic virus that delivers the oxazaphosphorine/rat cytochrome P450 2B1 and ganciclovir/herpes simplex virus thymidine kinase gene therapies. Cancer Res 1999;59: 3861–3865. 114 Zhang JF, Hu C, Geng Y, Selm J, Klein SB, Orazi A, Taylor MW: Treatment of a human breast cancer xenograft with an adenovirus vector containing an interferon gene results in rapid regression due to viral oncolysis and gene therapy. Proc Natl Acad Sci USA 1996;93:4513–4518. 115 Andreansky S, He B, van Cott J, McGhee J, Markert JM, Gillespie GY, Roizman B, Whitley RJ: Treatment of intracranial gliomas in immunocompetent mice using herpes simplex viruses that express murine interleukins. Gene Ther 1998;5:121–130. 116 Parker JN, Gillespie GY, Love CE, Randall S, Whitley RJ, Markert JM: Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc Natl Acad Sci USA 2000;97:2208–2213. 117 Carew JF, Kooby DA, Halterman MW, Kim SH, Federoff HJ, Fong Y: A novel approach to cancer therapy using an oncolytic herpes virus to package amplicons containing cytokine genes. Mol Ther 2001;4:250–256. 118 Wong RJ, Patel SG, Kim S, DeMatteo RP, Malhotra S, Bennett JJ, St-Louis M, Shah JP, Johnson PA, Fong Y: Cytokine gene transfer enhances herpes oncolytic therapy in murine squamous cell carcinoma. Hum Gene Ther 2001;12:253–265. 119 Todo T, Martuza RL, Dallman MJ, Rabkin SD: In situ expression of soluble B7–1 in the context of oncolytic herpes simplex virus induces potent antitumor immunity. Cancer Res 2001;61:153–161. 120 Liu BL, Robinson M, Han ZQ, Branston RH, English C, Reay P, McGrath Y, Thomas SK, Thornton M, Bullock P, Love CA, Coffin RS: ICP34.5 deleted herpes simplex virus with enhanced oncolytic, immune stimulating, and anti-tumour properties. Gene Ther 2003;10: 292–303. 121 Kruyt FA, Curiel DT: Toward a new generation of conditionally replicating adenoviruses: Pairing tumor selectivity with maximal oncolysis. Hum Gene Ther 2002;13:485–495. 122 van Beusechem VW, van den Doel PB, Grill J, Pinedo HM, Gerritsen WR: Conditionally replicative adenovirus expressing p53 exhibits enhanced oncolytic potency. Cancer Res 2002;62: 6165–6171. 123 Mi J, Li ZY, Ni S, Steinwaerder D, Lieber A: Induced apoptosis supports spread of adenovirus vectors in tumors. Hum Gene Ther 2001;12:1343–1352. 124 Yu DC, Chen Y, Dilley J, Li Y, Embry M, Zhang H, Nguyen N, Amin P, Oh J, Henderson DR: Antitumor synergy of CV787, a prostate cancer-specific adenovirus, and paclitaxel and docetaxel. Cancer Res 2001;61:517–525. 125 Toyoizumi T, Mick R, Abbas AE, Kang EH, Kaiser LR, Molnar-Kimber KL: Combined therapy with chemotherapeutic agents and herpes simplex virus type 1 ICP34.5 mutant (HSV-1716) in human non-small cell lung cancer. Hum Gene Ther 1999;10:3013–3029. 126 Cinatl J Jr, Cinatl J, Michaelis M, Kabickova H, Kotchetkov R, Vogel JU, Doerr HW, Klingebiel T, Driever PH: Potent oncolytic activity of multimutated herpes simplex virus G207 in combination with vincristine against human rhabdomyosarcoma. Cancer Res 2003;63: 1508–1514. 127 Rogulski KR, Freytag SO, Zhang K, Gilbert JD, Paielli DL, Kim JH, Heise CC, Kirn DH: In vivo antitumor activity of ONYX-015 is influenced by p53 status and is augmented by radiotherapy. Cancer Res 2000;60:1193–1196.

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128 Chen Y, DeWeese T, Dilley J, Zhang Y, Li Y, Ramesh N, Lee J, Pennathur-Das R, Radzyminski J, Wypych J, Brignetti D, Scott S, Stephens J, Karpf DB, Henderson DR, Yu DC: CV706, a prostate cancer-specific adenovirus variant, in combination with radiotherapy produces synergistic antitumor efficacy without increasing toxicity. Cancer Res 2001;61:5453–5460. 129 Geoerger B, Grill J, Opolon P, Morizet J, Aubert G, Lecluse Y, van Beusechem VW, Gerritsen VW, Kirn DH, Vassal G: Potentiation of radiation therapy by the oncolytic adenovirus dl1520 (ONYX015) in human malignant glioma xenografts. Br J Cancer, in press. 130 Blank SV, Rubin SC, Coukos G, Amin KM, Albelda SM, Molnar-Kimber KL: Replication-selective herpes simplex virus type 1 mutant therapy of cervical cancer is enhanced by low-dose radiation. Hum Gene Ther 2002;13:627–639. 131 Stanziale SF, Petrowsky H, Joe JK, Roberts GD, Zager JS, Gusani NJ, Ben-Porat L, Gonen M, Fong Y: Ionizing radiation potentiates the antitumor efficacy of oncolytic herpes simplex virus G207 by upregulating ribonucleotide reductase. Surgery 2002;132:353–359. 132 Jorgensen TJ, Katz S, Wittmack EK, Varghese S, Todo T, Rabkin SD, Martuza RL: Ionizing radiation does not alter the antitumor activity of herpes simplex virus vector G207 in subcutaneous tumor models of human and murine prostate cancer. Neoplasia 2001;3:451–456. 133 Spear MA, Sun F, Eling DJ, Gilpin E, Kipps TJ, Chiocca EA, Bouvet M: Cytotoxicity, apoptosis, and viral replication in tumor cells treated with oncolytic ribonucleotide reductase-defective herpes simplex type 1 virus (hrR3) combined with ionizing radiation. Cancer Gene Ther 2000;7: 1051–1059. 134 Advani SJ, Sibley GS, Song PY, Hallahan DE, Kataoka Y, Roizman B, Weichselbaum RR: Enhancement of replication of genetically engineered herpes simplex viruses by ionizing radiation: A new paradigm for destruction of therapeutically intractable tumors. Gene Ther 1998;5: 160–165. 135 Bradley JD, Kataoka Y, Advani S, Chung SM, Arani RB, Gillespie GY, Whitley RJ, Markert JM, Roizman B, Weichselbaum RR: Ionizing radiation improves survival in mice bearing intracranial high-grade gliomas injected with genetically modified herpes simplex virus. Clin Cancer Res 1999;5:1517–1522. 136 Rogulski KR, Wing MS, Paielli DL, Gilbert JD, Kim JH, Freytag SO: Double suicide gene therapy augments the antitumor activity of a replication-competent lytic adenovirus through enhanced cytotoxicity and radiosensitization. Hum Gene Ther 2000;11:67–76. 137 Jacobs A, Tjuvajev JG, Dubrovin M, Akhurst T, Balatoni J, Beattie B, Joshi R, Finn R, Larson SM, Herrlinger U, Pechan PA, Chiocca EA, Breakefield XO, Blasberg RG: Positron emission tomographybased imaging of transgene expression mediated by replication-conditional, oncolytic herpes simplex virus type 1 mutant vectors in vivo. Cancer Res 2001;61:2983–2995. 138 Rainov NG, Kramm CM: Vector delivery methods and targeting strategies for gene therapy of brain tumors. Curr Gene Ther 2001;1:367–383.

E. Antonio Chiocca, MD, PhD Chairman, Department of Neurological Surgery Dardinger Family Professor of Oncologic Neurosurgery Director of Neurosurgical Services The Ohio State University Medical Center James Cancer Hospital and Solove Research Institute N-1017 Doan Hall, 410 W. 10th Avenue, Columbus, OH 43210 (USA) Tel. ⫹1 614 293 9312, Fax ⫹1 614 293 4024, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 580–623

Molecular Neurosurgery in the Pituitary Gland Gene Transfer as an Adjunctive Treatment Strategy

Maria G. Castroa,b, Nelson Jovel a,b, Shyam Goverdhana a,b, Jinwei Hu a,b, John Yu c, Moneeb Ehteshamc, Xiangpeng Yuana,b, Diana Greengold a,b, Weidong Xiong a,b, Pedro R. Lowensteina,b a

Gene Therapeutics Research Institute, Cedars-Sinai Medical Center, Research Pavilion, bDepartment of Medicine, UCLA David Geffen School of Medicine, c Maxine Dunitz Neurosurgical Institute, Cedars-Sinai Medical Center, Los Angeles, Calif., USA

Introduction

Current treatments for pituitary tumors encompass medical, surgical, and radiotherapeutic methods. These therapies should not be thought to be in competition; optimum results usually are obtained when these three approaches are combined. It is important to be aware of advantages, disadvantages, and risks of each therapeutic modality. The aim of pituitary tumor treatment is the eradication of the tumor, restoration of biochemical and visual abnormalities, and preservation of pituitary function. Treatments should not pose risks, complications or the possibility of recurrence. Because no single method of treatment can attain these ideals, choice of treatments will depend on the pathology of the tumor and the availability of the appropriate skills and facilities. For example, the best surgical results occur in centers where one surgeon has particular experience with this type of surgery. In most cases, pituitary tumors are benign and respond very well to current treatment modalities, but on some occasions either the patient does not respond to the treatment, becomes intolerant to the medical therapy, or the tumor recurs or it becomes invasive. In these instances, new treatment modalities are needed. Gene therapy, the use of DNA as a drug, is an emerging modality to treat resistant

pituitary tumors, tumors that have become invasive, or tumors which recur and are difficult to treat. Expression of transgenes within the anterior pituitary (AP) gland in vitro and in vivo is possible with viral vectors. The ability to induce or repress transgene expression using small molecules such as tetracyclines with tet-responsive regulatory elements has enabled experimental paradigms to uncover the function of newly discovered genes, allow the regulation of transgenes expressed during adulthood, and specifically affect production of mRNA molecules in a cell-type specific and temporal fashion. Several molecular targets are amenable to genetic therapies to treat pituitary tumors. Once the vectors (delivery systems) are proven safe and the therapeutic targets undergo additional testing in preclinical models, it will be possible to introduce gene therapies to treat human patients harboring nonresponsive and difficult to treat pituitary tumors.

Tumors of the Pituitary Gland

The pituitary gland is responsible for regulating almost every major organ system in the body. The AP secretes hormones that control hormone production in other glands/tissues of the body such as the thyroid gland, pancreas, adrenal cortex, and gonads. Examples include thyroid-stimulating hormone which stimulates the thyroid gland; luteinizing and follicle-stimulating hormones (FSH) which stimulate the gonads and initiating sexual differentiation in the fetus and sexual development during puberty; adrenal-corticotropic hormone (ACTH) which stimulates the adrenal cortex; and prolactin (PRL) which stimulates the mammary glands and also plays a role in modulating the immune system. The posterior pituitary gland produces and secretes hormones that regulate water and salt metabolism. The posterior pituitary, an extension of the neuronal tissue of the hypothalamus, stores vasopressin and oxytocin hormones produced by the hypothalamus until they are ready to be released. Because the pituitary gland regulates such a vast array of functions within the body, diseases of the pituitary gland can have dramatic body-wide adverse effects. Tumors are one of the most common diseases affecting the AP gland. A pituitary tumor can cause damage in two ways: it may cause an endocrine malfunction or it may damage the surrounding brain tissue through mass effect. Endocrine malfunctions occur because pituitary tumors can retain the hormone production capability of their cells of origin, and tumor cells may cause a clinical syndrome by oversecretion of these hormones. They can also inhibit secretion of other pituitary hormones by compression effects. Tumors overproducing PRL, growth hormone (GH), ACTH, gonadotrophins (FSH, luteinizing hormone), or thyrotrophin often cause a typical clinical syndrome, whereas nonfunctioning tumors tend to exhibit eventual hypopituitarism, although symptoms may not be

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readily apparent. For most tumors, the degree of hypersecretion correlates with the severity of their clinical syndrome. For nonfunctioning tumors, clinical syndromes are caused by tumor mass compressing the surrounding pituitary tissue and sometimes invading brain structures. Tumors can grow large enough to exert dangerous pressure effects on surrounding tissue, causing headaches, nausea, or blindness. Functioning tumors are usually identified while in the microadenoma stage, because they present with endocrine syndromes. Nonfunctioning tumors, on the other hand, present no clinical endocrine syndromes, except for eventual hypopituitarism, and they are generally not identified until they become macroadenomas and extend outside the pituitary fossa, becoming locally invasive and causing compression and mass effects. Although endocrine syndromes often denote functioning microadenomas and pressure effects often denote nonfunctioning macroadenomas, this does not always hold true. Autopsy studies suggest that pituitary tumors affect as much as 11% of the population. While they are often described as ‘benign’ adenomas, this classification hides the extent to which these tumors can affect normal bodily function by way of inducing endocrine abnormalities and compressing surrounding pituitary and/or brain tissue. Pituitary tumors are classified by their size, with microadenomas being ⬍10 mm in diameter and macroadenomas being ⬎10 mm in diameter, by hormones they produce, and by their invasiveness into the surrounding tissue. Pituitary tumor initiation and progression are not fully understood. While brain tumor occurrence can sometimes be traced to specific genomic alterations, no such molecular abnormalities have been found to account for pituitary tumor formation. Potential causes for tumor formation include abnormalities in many types of genes, such as genes regulating growth and development, tumorsuppressor genes which inhibit cell growth and proliferation, and genes controlling programmed cell death. In addition to genomic abnormalities, tumor cell genesis may be promoted by endocrine and hypothalamic factors as well as hereditary disposition. All of these potential causes for pituitary tumor formation are being examined. Two hypotheses have been developed to address the origin of pituitary tumors. One is the hypothalamic hypothesis which proposes that pituitary adenomas are caused by abnormalities in the hypothalamus. This possibility is supported by the fact that genomic alterations seem to be nonsignificant, having only been found in a minority of cases. The other hypothesis emphasizes local factors within the pituitary, proposing that pituitary adenomas are caused by an intrinsic pituitary defect within a particular pituitary cell type, which then by clonal expansion gives rise to a pituitary adenoma/tumor. The pituitary hypothesis is the more supported of the two, with two main pieces of evidence: lack of peritumoral hyperplasia in association with pituitary tumors, and the fact that

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it is possible to effectively cure pituitary cancer by way of a complete tumor excision. Neither of these features would be expected, if hypothalamic overstimulation were the dominant tumorgenic mechanism [1]. Below we review current treatment modalities, which in the majority of cases, are very effective for treating pituitary tumors. Although most pituitary tumors are clinically well managed, there are some cases in which conventional treatments fail, and it is for these tumors that we propose the use of gene therapy as a novel approach that could be used in combination with other forms of treatment. Treatment Pituitary tumor treatment, if it is to be effective, must address both the endocrine malfunctions and oncological concerns. Both issues are adequately addressed by current treatments such as surgical therapy, pharmacological therapy, and radiation therapy. These treatments can be used alone or in conjunction with each other: surgical therapy to eliminate tumor mass, medical therapy to treat endocrine symptoms and shrink tumor mass, and radiation therapy to offset both oncological and endocrine concerns. This interdisciplinary endeavor combines aspects of each treatment into a combined therapeutic strategy. As effective as this interdisciplinary treatment strategy has been, there is room for improvement. Current strategies are limited in cases where symptoms cannot be managed. It is therefore necessary to seek out new therapies capable of managing symptoms which current treatments cannot, one that incurs fewer side effects, and which seeks to cure the disease instead of simply treating its symptoms. Surgical Management of Pituitary Adenomas Surgery is indicated in cases where there is impingement of the optic chiasma secondary to mass effect, or where medical management has failed to control the symptoms of hyperprolactinemia, hypercortisolism, or acromegaly. The choice for surgical management as well as the specific route of surgical access should be carefully evaluated in each patient. Overall, surgical therapy provides a symptomatic relief, with postoperative radiotherapy playing an important role in eliminating residual tumor and preventing recurrence. Trans-sphenoidal Approach. This is usually the preferred route of access to the sella turcica and is generally recognized as the optimal method for removing the intrasellar component of pituitary neoplasms (fig. 1). Initially described using a transnasal approach by Schloffer in 1907, this method was refined by Harvey Cushing, who utilized a sublabial incision to obtain surgical access. Subsequently, Guiot [2] and Hardy [3] introduced fluoroscopy and the operative microscope for pituitary resection, resulting in widespread popularization of this approach. More recently, the use of intraoperative navigational devices

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B Pterional (frontotemporal)

C Subfrontal

D Transethmoidal

A Subtemporal

E Transnasal trans-sphenoidal

Fig. 1. Surgical approaches to the pituitary gland. Arrows illustrate the main routes of access to the pituitary. (A) Subtemporal, (B) pterional/frontotemporal, (C) subfrontal, (D) transethmoidal, (E) transnasal/trans-sphenoidal.

and intraoperative magnetic resonance imaging have added to the efficacy of this route [4]. In addition to the surgical utility, trans-sphenoidal hypophysectomy has proven to have notable therapeutic benefits over alternate transcranial approaches, particularly with regard to improvement in tumor-related visual disturbances [5, 6] and recovery of pituitary function in cases with pre-existing hypopituitarism [7, 8]. Trans-sphenoidal surgery has proven to be a highly effective therapy, with certain reports describing greater than 90% tumor control rates in selected patients with pituitary microadenomas, although success with larger neoplasms has been more limited [9, 10]. Morbidity associated with this procedure may involve transient or permanent diabetes insipidus, CSF fistulas, CSF rhinorrhea, visual loss, stroke, and cranial nerve palsy. Potentially fatal complications can occur as a result of hypothalamic injury, sepsis secondary to CSF leak, or by intraoperative damage to carotid arteries. The occurrence of these complications is, however, very low in the hands of an experienced surgeon, with operative mortality reported at between 0.27 and 0.86%. Nonfatal minor and major complications are estimated to occur in approximately 7% of

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patients [11]. Given the relative safety of trans-sphenoidal hypophysectomy, it has become the surgical procedure of choice. However, there are certain, albeit rare, cases where the trans-sphenoidal route is contraindicated. In these scenarios, detailed below, a transcranial approach is preferred to obtain optimal tumor control and for reasons of safety. Transcranial Approach. In certain settings, the transcranial route may be preferable to the generally preferred trans-sphenoidal route (fig. 1). In particular, cases where there is extensive herniation of tumor into the middle fossa may require primary transcranial approaches to ensure adequate visualization and resection. Additionally, severely ectatic carotid arteries impinging on the midline would also preclude trans-sphenoidal access. Hence, it is very important to obtain proper MRI imaging of the major vessels in proximity to the tumor prior to deciding on a surgical approach. In some cases, the consistency of a tumor compressing the optic chiasma may not allow for adequate resection and decompression. In these cases, transcranial intervention is indicated to ensure adequate relief of pressure on the optic chiasma. One of the most common transcranial routes adopted is the pterional or frontotemporal approach. Good access is provided for the removal of laterally extending tumor, and generally the optic chiasma and carotid artery are in the line of vision. Extreme care must be taken to preserve the integrity of the carotid artery, and its ophthalmic branch which can easily be damaged during mobilization of the tumor mass. Overall, the goal with pterional access, should be adequate and judicious decompression of the optic apparatus without exposing neural or vascular structures to injury. Other transcranial routes (fig. 1) that have been utilized for access to pituitary neoplasms include the subfrontal, transethmoidal, and subtemporal approaches, which are generally less popular as they do not provide optimal access to tumor and prohibit adequate visualization of critical structures. Radiotherapy for Pituitary Adenomas It is well established that pituitary adenomas, irrespective of cellular origin, are generally sensitive to radiotherapy. Given this susceptibility, radiation therapy may be the primary treatment of choice in certain patients, especially in those cases where disease is confined to the intrasellar space with no compression of surrounding structures. In addition, radiotherapy is frequently employed postoperatively to target residual tumor and minimize tumor recurrence. The efficacy of radiotherapy in preventing tumor recurrence has been substantiated in the recent literature with a comprehensive report from Brada et al. [12] describing an 88% progression-free survival rate at 10 years following the surgery with routine use of radiotherapy in patients with nonsecretory tumors. In contrast, Turner et al. [13] reported up to 44% recurrence rates at 10 years postoperatively in a cohort of 65 patients with functionless pituitary adenomas, who did

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not receive radiotherapy. Radiation therapy has, however, not been found to be as effective in preventing the progression of functional adenomas, most likely as a result of easier detection of recurrence/progression in these cases secondary to highly sensitive measurement of a secretory marker. Conventional radiotherapy for pituitary neoplasms utilizes an external beam approach that concentrates radiation on a significant target area in the brain. Although effective at achieving tumor control, standard external beam radiotherapy has been associated with significant side effects. Up to 50% of patients develop panhypopituitarism following surgery and radiotherapy, with the rapidity of onset directly related to the radiation dose [14–16]. Additionally, the optic chiasma is sensitive to radiation, and external beam radiotherapy-induced blindness has been well documented, occurring in up to 1–2% of cases [17–20]. Given the danger of iatrogenic injury with external beam radiation, the use of precisely directed stereotactic radiosurgery has gained increasing attention [21]. This technique involves the delivery of a high dose of necrotizing radiation to a delimited target area, with a sharp fall of radiation at the target margins resulting in relatively little irradiation to surrounding structures. Current stereotactic radiosurgical approaches utilize either proton beam therapy, gamma knife surgery, or linear proton acceleration. Recent reports on the efficacy of stereotactic radiosurgery for pituitary adenomas have been mixed. Initial studies have generally focused on the use of radiosurgery on small functional adenomas [22–25]. Although significant improvements in the outcome were not documented in these studies, a recent report clearly indicates that the treatment of functional pituitary tumors with stereotactic radiosurgery results in an accelerated decline in hormone levels compared to that of conventional external beam radiotherapy [26]. The role of stereotactic radiosurgery in the management of nonfunctional adenomas is less clear, as there is currently little data describing its use in this setting. However, given the efficacy that stereotactic radiosurgery has demonstrated in the treatment of other intracranial neoplasms, and as more data related to its use in pituitary tumors becomes available, it is hoped that there will be an expanded role for this approach in the treatment of pituitary neoplasms. Medical Therapy The aim of any pituitary tumor therapy is to reduce tumor mass and normalize hormone production. Surgery, with the aim of excising tumor mass, is most often the primary treatment. After tumor mass has been reduced by surgery, medical therapy is implemented to normalize hormone production. Normalization of endocrine abnormality is important, as the oversecretion of hormones continues to effect long term health and can, in some cases, increase mortality.

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Medical therapy is tumor-specific, with certain medications being appropriate to treat a given tumor and its associated increased hormone hypersecretion. Dopamine agonists are used to treat prolactinomas (hypersecretion of PRL), and somatostasin analogues and GH antagonists are implemented to treat acromegaly (hypersecretion of GH). These tumor-specific medical therapies are based on the knowledge of releasing and inhibiting hormones and neurotransmitter pathways that regulate pituitary hormone secretion as well as the existence of particular receptors on pituitary cells that respond to such hormones and neurotransmitters. Prolactinomas are the only pituitary tumors for which medical therapy is used as a primary therapeutic modality. Dopamine agonists used to treat prolactinomas can reduce tumor mass by triggering shrinkage and reducing serum PRL levels by reducing PRL release from the pituitary. These drugs can be so effective as to be the only therapy implemented, without the need of surgery [27]. In addition to dopamine agonists, the other major class of medical agents used to treat pituitary tumors is somatostatin analogs. Somatostatin is a physiological inhibitor of GH release, making it ideal for treating patients with acromegaly. Somatostatin analogs also help to minimize tumor size, but never to a clinically useful degree. Their clinical use has been enhanced by the introduction of depot preparations of octreotide and lanreotide that can be given at 2–4 week intervals, but they must still be used long term and treatment can become expensive. Most patients have favorable responses to these drugs, but responses can vary. In treating prolactinomas, medical therapy successfully reduces tumor mass and ameliorates hormone production in about 85–90% of patients [28], but these therapies are known to elicit a high rate of side effects including, nausea and vomiting, postural hypotension and dizziness, headache, constipation, and depression. Some patients cannot tolerate these side effects, and so despite their effectiveness in treating symptoms, these drugs cannot always be used. Dopamine agonists and somatostatin analogs are not tumoricidal agents. They act by inhibiting target cells and hormone release. In doing so they can reduce, in some cases, the tumor size. Therefore, in order to be effective they must be taken indefinitely. Interruption of the dose ensures an immediate return of increased hormone levels and tumor size to pretreatment levels. These drugs may help manage symptoms by reducing tumor size and normalizing hormone production, but do not cure the disease. Gene Therapy for Pituitary Tumors There is as yet no treatment that results in a complete cure for any of the different pituitary tumor types. Current progress in endocrine assays, imaging

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techniques, trans-sphenoidal microsurgery, receptor-mediated pharmacology, and radiotherapy has led to most pituitary tumors being manageable. Nevertheless for some pituitary tumors such as large and locally invasive endocrine tumors, only partial success has been made possible. For trans-sphenoidal surgery, even if initial remission rates are positive there is a recurrence in prolactinomas, acromegaly, Cushing syndrome, and nonfunctional adenomas in 24, 8, 12 and 16% of patients respectively [29]. Furthermore, 7% of patients treated for GH-secreting tumors were left with permanent diabetes insipidus [1]. Furthermore, in terms of the correction of endocrine abnormality in functional tumors, tiny amounts of tumor residue may continue to hypersecrete hormones. Radiation therapy is the widely accepted treatment for residual tumor [30]. Although radiotherapy has been shown to reduce the risk of further tumor progression, it often damages normal pituitary tissue, frequently leading to irreversible progressive hypopituitarism [14]. Thus patients require regular screening and eventual substitution therapy with corticosteroids, GH, thyroxine, and sex steroids. The impact of stereotaxic radiosurgery remains to be determined, but it is likely that the long-term endocrine consequences will be similar. The use of dopamine agonists in reducing serum PRL levels and causing shrinkage of prolactinomas (drugs such as bromocriptine and cabergoline, or somatostasin analogs such as octreotide for the treatment of acromegaly) has a debatable success rate. Despite success in 85–90% of patients, these treatments are not curative and require lifelong administration. There is also a high rate of side effects, notably nausea and vomiting, postural hypotension and dizziness, headache and constipation [28]. Furthermore, in some cases there is resistance to dopamine agonist therapy with tumors not responding [31]. There is a niche in the treatment of pituitary tumors in which a therapy is both effective at removal of the tumor mass and corrects the endocrine dysfunction, without the need for lifelong treatment or side effects. Gene therapy strategies may provide a novel flexible way of engineering not only tumor cells to eliminate their growth [either by the delivery of ‘suicide’ genes such as HSV1-TK or by the delivery of enzymes such as tyrosine hydroxylase (TH) to agonise dopamine], but also the possibility of modifying normal surrounding cells to inhibit tumor regrowth (fig. 2). Furthermore, the development of regulatable gene therapy transcription units under the control of cell-type specific promoters may have applications in the treatment of pituitary disorders. For example, the treatment of hypopituitarism could rely upon the controlled pulsatile release of AP hormones, or regression of tumors by increasing dopamine levels using TH, where excessive expression of TH may have the same effects as conventional drug-based dopamine agonists.

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Fig. 2. Gene therapy strategy to treat pituitary tumors in humans. MRI scan shows a lobulated gonadotroph adenoma. Gene therapy could be delivered intratumorally or in the tumor bed at the time of surgical resection. symbolizes the gene therapy vectors within the tumor mass itself.

Viral Vectors for Gene Delivery

The aim of gene therapy is to introduce therapeutic genes into tissues, leading to an efficient and stable expression of the therapeutic molecules and minimizing adverse side effects. Viruses can enter cells and express genetic material in the nucleus, therefore they are generally much more efficient vectors than nonviral delivery systems. The most commonly used viral vectors for gene therapy are adenovirus (Ad), adeno-associated virus (AAV), herpes simplex virus type 1 derived vectors (HSV-1), and retrovirus/lentivirus vectors (table 1). Important parameters to be considered when comparing gene therapy vectors include: size limitations for transgene insertion, ease production of virus at high titers, transduction efficiency, ability to infect dividing and/or quiescent cells, stability of transgene expression, potential to integrate into the host chromosomes, cell-type specificity, vector-associated toxicity and immunogenicity (tables 2, 3). Ad Vectors Ads are a family of DNA virus characterized by an icosohedral, nonenveloped capsid containing a linear double-stranded genome. The human Ad,

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Table 1. Gene transfer vehicles used in gene therapy applications Castro/Jovel/Goverdhana/Hu/Yu/Ehtesham/Yuan/Greengold/Xiong/Lowenstein

Size Cloning capacity (kb) Transduction In vivo? In vitro? Long-term expression Vaccination Vector titers (pfu/ml)

Adenovirus

HD-Ad

HSV-1/r

HSV-1/a

AAV

Retrovirus

Vaccinia virus

Microinjection

Transfection

36 7.5

30–36 ⬃30

152 30

10–30 10–30

4.68 2–4.5

3.5–9.2 ⬃8

186 30

Unlimited Unlimited

Unlimited Unlimited

Yes Yes No

Yes Yes Yes

Yes Yes Yes

Yes Yes Yes

Yes Yes Yes

Yes Yes Yes

Yes Yes No

Yes Yes ?

No Yes No

Yes 1012

Yes 1011

Yes 108

Yes 108

Yes 109

Yes 107

Yes 106–108

– –

– –

590

HSV-1/r, herpes simplex type 1 recombinant vector; HSV-1/a, herpes simplex type 1 amplicon; pfu/ml, plaque-forming units per ml; HD-Ad, gutless helper-dependent adenovirus vector.

Table 2. Advantages and disadvantages of viral vectors for gene therapy Virus

Maximum capacity

Advantages

Disadvantages

Adenovirus

8 kb

Broad cell tropism, infection of dividing and nondividing cells, easy to produce at high titer

Inflammatory and immune responses, transient expression

HD-Ad

36 kb

Broad cell tropism, infection of dividing and nondividing cells, less inflammatory and cellular immune response, longer term transgene expression

Difficulty in large-scale production

AAV

5 kb, 10 kb (concatamers)

Broad cell tropism, infection of dividing and nondividing cells, integration into host genome

Difficulty in producing pure preparations at high titer, discrete immune response

HSV

30 kb

Broad cell tropism, latency in neurons, very stable

Highly toxic

Lentivirus

10 kb

Infection of dividing and nondividing cells, integration into host genome

Risk of inserting mutagensis, risk of seroconversion

serotype 2 and serotype 5, both of subclass C, are approximately 36 kb and encode genes that are classified into early (E1–E4) and late (L1–L5) viral functions, depending on whether they are expressed before or after DNA replication [33]. At one end of its genome, the nominal left end, it has an inverted terminal repeat (ITR) necessary for the initiation of viral DNA replication, and an adjacent DNA-packaging signal, while a second ITR is found at the right end. Virus infection initiates with the Ad fiber protein binding to the coxsackie virus and Ad receptor on the cell surface [33], followed by a secondary interaction between Ad penton and integrins [34]. Ad is internalized by endocytosis, triggered by the penton-integrin interaction, and escapes from the early endosome prior to the formation of lysosome. The virion translocates to the nucleus along the microtubule network, during which time there is a sequential disassembly of the Ad virion and Ad hexon remains at the nuclear membrane, while the DNA is released into the nucleus and remains as an episome [35]. The most common first-generation Ad vectors developed for the gene therapy are based on the Ad2 and Ad5 serotypes, replication made defective through deletions in the E1 region, where the transcriptional cassette can be inserted. Thus 90% of the wild-type Ad genome is retained in the vector [36]. The recombinant E1-deleted Ad vector genomes are then transfected into human 293 cells, which express the E1 proteins in trans, allowing for E1-deleted Ad

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Table 3. Possible disadvantages among gene transfer vehicles in gene therapy Vectors Adenovirus

• Host immune responses: inflammatory and cytotoxic reactions in patients and depletion of transduced cells • Host’s humoral immune responses may neutralize adenoviral vector particles during, or even before, the gene transfer processes • Not suitable for long-term expression of the transgene due to the lack of integration into host genome • Complicated vector genome

Helper-dependant adenovirus

• These vectors can only be grown in the presence of a helper virus and are not economic for production of low level contamination of helper virus • To compare with first generation adenovirus, virus titers are much lower

AAV

• • • • •

HSV-1

• Host immune responses, inflammatory cytopathogenicity and neurotoxicity reactions in patients • Complicated vector genome • Difficult to produce • HSV-1-derived vectors could potentially reactivate latent wild-type HSV-1

Retrovirus

• Random insertion of viral genome, which may possibly result in mutagenesis and activate oncogenes • Possibility of replication-competent virus formation by homologous recombination. Possible recombination with human endogenous retroviruses (HERVs) • Retroviral vector particles are rapidly degraded by the complement • Infects only dividing cells, small insert capacity (6.5 kb)

Vaccinia virus

• Widespread use of vaccinia as a live vaccine, however, depends on improving safety while achieving an even higher immune response to the recombinant protein

Microinjection

• It’s difficult to introduce DNA on a scale large enough for biochemical analysis

High titers of pure virus are difficult to obtain AAV requires a helper adeno or herpesvirus This vector system is still not well characterized Limited capacity for foreign genes (about 2–4.5 kb) Lack of specific integration for recombinant AAV vectors, which may possibly result in cell mutagenesis

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Table 3. (continued) Vectors Transfection (cationic liposomes or DNA protein complexes)

• • • • •

Targeting is not specific Low transfection efficiency Only transient expression Difficult in vivo applications Host immune responses, inflammatory reactions in patients if they express chimerical cell receptors on their surface, or in the presence of unmethylated CpG sequences of bacterial plasmid DNA

AAV: Adeno-associated virus, HSV: herpes simplex virus, HERVs: human endogenous retroviruses.

vector replication and packaging. E1-deleted Ad vectors have a number of positive characteristics, one of the most important ones is their relative ease for scale up at very high titers, above 1012 IU/ml (Infectious Units/ml). Other attractive features include the ability to infect many different cell types, both dividing and nondividing differentiated cells; having an extremely low probability of random integration into the host chromosomes [37]; and having a large cloning capacity, theoretically around 8 kb with full E1 and E3 deletions. In spite of the E1 deletion, first-generation Ad vectors have residual expression of viral genes that lead to a strong host immune response, resulting in the generation of high titer, neutralizing anti-capsid antibodies that inhibit re infection with the same serotype of Ad vector [38]. In addition, at high viral doses, residual virus gene expression leads to cellular cytotoxicity, which can result in an immune-mediated loss of the Ad vector transduced cells [39]. Injection of first-generation recombinant Ad vectors into the brain parenchyma causes acute cellular- and cytokine-mediated inflammatory responses. This does not effect transgene expression. In the presence of Ad immune responses, transgene expression for first-generation Ad is rapidly established [40, 41]. Ad induced cytotoxicity is only seen when at high vector doses of ⬎108 IU/ml are used to transduce the target tissue [40]. To overcome this limitation, a series of Ad vectors with multiple deletions have been developed. In order to propagate multiply-deleted Ad vectors, packaging cell lines must be developed that trans-complement the growth and packaging of these vectors (fig. 3). Cell lines coexpressing Ad E1 and E4 genes, the E1 and E2a (single-strand DNA-binding protein, ssDBP) genes, the EI and pre-terminal protein genes, the E1 and protease genes have been generated and used to package the E1, E4 deleted Ad vectors [42], the E1, E2a deleted Ad vectors [43], the EI, E2b-deleted Ad vectors [44], the E1 and protease-deleted

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HD-Ad vector

ITR ␺⫹

Stuffer DNA Transcription cassette

Helper virus

ITR ␺⫹

ITR

␺⫹

Stuffer DNA loxP

ITR Luc

loxP or

FRT

FRT

293 Cre or 293 FLPe cells ITR ␺⫹

ITR ␺⫺

ITR

Stuffer DNA Transcription cassette

ITR Luc

Stuffer DNA

0.01% contamination with helper virus Not packaged

Packaged

HD-Ad vector

ITR ␺⫹

HD-Ad vector

Stuffer DNA Promoter

ITR Stuffer DNA

Therapeutic poly A gene

Fig. 3. Schematic representation for the production of helper-dependent adenovirus vectors. The helper virus is an E1-deleted Ad that contains a packaging signal (␺) flanked by loxP or Frt recombination sites. The HD-Ad vector is constructed as a plasmid with transgene, stuffer DNA and the Ad cis-elements, mainly the inverted terminal repeats (ITR) and packaging signal. Upon cotransfection of the HD-Ad vector and, the helper virus into a 293-derived cell line that stably expresses the Cre and FLPe recombinase, the packaging signal of the helper virus is excised, rendering the helper virus DNA unpackagable. But the helper virus provides the Ad functions that are required for replicating the vector DNA, for producing viral structural protein, and for packaging of the vector DNA into virions. The titer of HD-Ad vectors is increased by serial passages through helper virus-infected 293-derived cell line. A purification step by CsCl centrifugation can further reduce the contamination of vector with helper virus to 0.01%.

Ad vectors [45] respectively. Deleted Ad vectors further reduced the acute toxicity and inflammatory responses and increased the transgene capacity, but had other disadvantages, such as replication accompanied by expression of Ad proteins, which reduced the length of transgene expression. New helper-dependent Ad vectors (also known as high-capacity, ‘gutless’ or ‘gutted’ vectors; HD-Ad) are devoid of all viral coding sequences [46–49]. These vectors have a minimum requirement for the extreme termini of the linear Ad

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genome, containing only those cis-acting elements for viral DNA replication and packaging, mainly the ITR sequences and packaging signal. Since these elements are contained with ⬃500 bp from the ends of the genome [50], helper-dependent vectors have the potential to range in size from a few hundred base pairs to carry up to ⬃36 kb of the foreign DNA, which is close to the size of the native Ad genome. HD-Ads are copropagated with an E1-deleted helper virus, which provides in trans all of the proteins required for the propagation of the vector. Several systems have been developed to prevent packaging of the helper viral genomes during the HD-Ad vector rescue/amplification process in order to minimize the helper virus contamination. The Cre/loxP-based system for the generation of HD-Ad involves the use of a first-generation helper virus, where the packaging signal is flanked by loxP recognition sites [51]. Infection of Creexpressing 293 cells with the helper virus results in excision of the viral packaging signal, rendering the helper virus DNA unpackagable, but still able to replicate and provide helper functions for HD-Ad vector propagation [52]. Purification by caesium chloride centrifugation is necessary to reduce the titer of the helper virus to negligible levels, typically ranging from 0.1 to 0.01% of the HD-Ad vector titer [53]. Recently, another Flp/FRT-based system has been developed. The Flp recombinase was used in place of Cre, and shown to excise the FRT-flanked packaging signal in helper virus efficiently [48, 54]. The most recent improvement to this system is the development of a new Cre-expressing cell line based on E2T, an E1 and E2a-complementary cell line. Thus an E1 and E2a double-deleted helper virus can be used with the new cell line to produce HD-Ad vector with low helper contamination, further improving the HD vector safety [55]. Compared with first-generation Ad vectors, the HD-Ad vector can efficiently transduce a wide variety of cell types from numerous species in a cell cycleindependent manner. HD-Ad vectors have the added advantage of increased cloning capacity, reduced toxicity and immune responses, and prolonged stable transgene expression in vivo [40, 56]. Limitations of HD-Ad vectors are difficulties in large-scale production and helper virus contamination. If these shortcomings can be overcome by improved viral vector production technique, the HD-Ad vector could become a key viral vector for gene therapy (table 4). Adeno-Associated Virus Vector Adeno-associated virus (AAV) is a simple, linear, single-stranded DNA parvovirus, which is nonpathogenic to humans. It is currently being developed as a gene therapy vector for the treatment of numerous diseases, such as diabetes, obesity, lactose intolerance, hemophilia B and blindness [57–59]. AAV has two genes: rep, which codes for replication and integration functions of the virus; and cap, which codes for the structural components of the virus. On either side of rep and cap are two ITRs, which define the beginning

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Table 4. How to improve existing gene therapy vectors for use in clinical trials • • • • • •

Increase efficiency of expression to allow lower doses of vector as required Prolong the duration of transgene expression Generate vectors with reproducible high titers and no contaminants Increase the area of transduction Target infection and transgene expression to allow systemic delivery Generate transcriptional switches to allow to turn therapeutic transgene expression ‘on’ or ‘off’

and the end of the virus and contain DNA sequences needed to pack the viral genome into the capsids [60]. Any sequence flanked by AAV ITR can be considered as a recombinant AAV genome for the purposes of gene therapy vector development. Wild-type AAV has the capacity to establish latency in the host cells and to integrate DNA into a 4-kb region of human chromosome 19, designated AAVS1 [61]. Recombinant AAV (rAAV) vectors achieve long-term transgene expression without major immunogenic toxic or toxicity responses, although neutralizing antibodies are generated. They can infect and integrate in a wide range of cells including dividing and resting cells. The main disadvantage of AAV is the relatively small packaging capacity, approximately 4.7 kb. Because of this size limitation, rep and cap genes were removed from first-generation AAV based vectors to make room for the therapeutic or marker genes. It was later discovered that the rep gene, or at least one of its products, the Rep68 or Rep78 protein, is required for the preferential integration of AAV [62]. Recent developments in AAV gene therapy vector construction allow the inclusion of the rep gene into an AAV vector. The packaging capacity of these vectors has been extended by harnessing the observation that AAV genomes concatemerize after transduction [63]. When two vectors, one encoding for the first half and the other encoding the second half of a protein, were transduced into cells, head-to-tail stitching of the viral genomes resulted in the reconstitution of a functional gene, effectively increasing the size of the gene that can be delivered [64]. A recombinant AAV vector carrying the angiostatin gene has been constructed and developed as an anti-angiogenesis strategy to treat malignant brain tumors in a C6 glioma rat model [65]. Intratumoral injection of a high titer AAV-angiostatin vector has yielded efficacious tumor suppression and resulted in long-term survival in 40% of the treated rats [65]. Another challenge with AAV vectors is that it has been difficult to scale up production. Herpes Simplex Virus-1 Derived Vector Herpes simplex virus-1 (HSV-1) is an enveloped double-stranded DNA virus, with a genome of 152-kb size, containing unique coding sequences

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flanked by several repeat regions. HSV-1 has been exploited for gene transfer in different in vitro and in vivo models. It has been especially useful in the CNS because of its ability to persist in a latent state in neurons. Its genome structure allows large inserts (up to 30 kb). HSV-1 vectors express transgenes stably for up to 18 months; whether similar vectors could also be used for long-term transgene expression in forebrain neurons of the striatum or neocortex remains to be determined. The major drawback, which limits their application, is vector toxicity [66]. Novel, safe and robust HSV-1 vectors called HSV amplicons, deleted of almost all genomic sequences, have been developed [67]. Retrovirus Vectors Retroviruses are enveloped single-stranded RNA viruses, which have a genome of about 7–10 kb composed of four gene regions termed gag, pol, pro, env. These gene regions encode for structural capsid protein, viral integrase and transcriptase, viral protease and envelope glycoproteins respectively. This genome also has a packaging signal and cis-acting sequences, termed long terminal repeats, which have a role in transcriptional control and integration. Current vector system based on type C retroviruses, such as the Moloney murine leukemia virus has proven to be very popular as they are relatively nonpathogenic. Upon binding to its extracellular receptor, a conformational change within its envelope glycoprotein enables the retroviral envelope to fuse with the cell membrane and allows the release of the capsid core into the cytoplasm. Once inside the cytoplasm, the single-stranded RNA genome is reverse-transcribed into a double-stranded DNA proviral genome by the viral reverse transcriptase inside the capsid. Then a preintegration complex is formed and transported into the cellular nucleus. The viral integrase can randomly integrate the proviral genome into the host chromosomal genome, where the host’s transcriptional system gives rise to the expression of viral genes [68]. Recombinant retroviral vectors are devoid of all viral genes, are replicationdefective, can carry upto 8 kb of foreign DNA, and can be engineered to have varying cell tropism. The long terminal repeats and packaging sequence are the only viral sequences that remain in the vector. To propagate recombinant retroviruses, it is necessary to provide the viral genes by packaging cell lines. With this system, it is possible to produce viral titers of 105–107 CFU/ml [69]. The primary advantage of retrovirus is their ability to stably introduce genes into cells. Genes integrated into host cell DNA, are regulated as if belonging to the cell, which enables stable transfer of the gene to subsequent daughter cells following mitosis. Levels of gene expression, however, will depend on the site of vector insertion. In a clinical application, where long-term correction of genetic defect is required, retroviral vectors offer a means to achieve this goal. The ability of retroviral vectors to easily transduce cells in vitro has led to the

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development of ex vivo therapeutic strategies, whereby retroviral vectors are used to infect peripheral blood lymphocytes or bone marrow cells. In phase-I clinical trials using such a procedure, long-term gene expression has been demonstrated [70]. The strategy of generating safe and transcriptionally regulatable vectors was the development of self-inactivating vectors, in which regulatory elements have been deleted. Upon integration, all viral promoter/enhancer activity is lost and the transcription of the transgene is under the control of a heterologous promoter [71]. The main limitation of retroviral vectors has been their inability to infect nondividing cells, meaning that tissues such as brain, eye, and pancreas are not amenable to direct in vivo gene delivery. Furthermore, on transplantation of transduced cells in the host, transcription of transgenes is often extinguished [69]. These limitations have lead researchers to find new strategies for developing vectors that can infect nondividing cells, as well as integrate into the host chromosomes. Lentivirus Vector Lentiviruses, such as human immunodeficiency virus, are part of the retrovirus family, but have acquired the capability of transducing nondividing cells, and therefore can be used to transduce cells within the nervous system [72]. The first-generation lentiviral vectors relied largely on the substitution of the viral Env protein with vesicular stomatitis virus G protein, which relieved them of their dependence on CD4, the T-cell receptor protein required for lentivirus infection. Instead, the vectors showed a wider tropism by infecting cells not known to express CD4 protein, including neurons, hepatocytes, muscle fibers, and retinal cells. Although first-generation vectors fulfilled many of the criteria of an ideal vector, they were viewed with some caution because of the possibility of recombination and generation of infectious human immunodeficiency virus. To minimize those concerns, lentiviral vectors have been made devoid of as many viral accessory genes as possible, while maintaining the key feature of infection of nondividing cells [73]. An extra feature that has improved these vectors includes the central polypurine tract, which allows internal initiation of second-strand DNA synthesis and probably aids in the transport of the preintegration complex to the nucleus [74]. Some nonhuman lentiviruses, such as simian immunodeficiency virus, feline immunodeficiency virus, and equine infectious anemia virus, have been used to generate efficient vectors capable of transducing nondividing cells. There are no clinical trials with these vectors or any other lentiviral vectors at present. Like other integrating vectors, the lentiviral vectors will have the disadvantage of nonspecific integration in the chromosomes. The duration of expression of the transgene in lentiviral vector also needs further testing [75, 76].

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Table 5. Implementation of clinical gene therapy Efficacy

Efficient delivery and expression of therapeutic transgenes which should enable revertion of the disease phenotype. Adequate persistence of transgene expression. Transgene expression should be turned ‘on’ and ‘off’ as and when it is needed. Cell-type specific expression of therapeutic genes

Safety

Elimination of adverse immune responses and other cytotoxic reactions, both local and/or systemic, within the patient. Limit spread in vivo

Clinical trials

Large scale production and quality control of vectors. Compliance with local and national gene therapy advisory committee’s regulations. The cost of the therapy versus size of patient population to be treated. Benefits to health care provision. Effects on long-term survival and overall quality of life

Among the viral vectors described, advantages and disadvantages of each vector are summarized in table 5. Several approaches have been aimed at manipulating the vectors to maximize the efficiency, safety and duration of transgene expression. With the aim of combining the most beneficial properties of several vector systems, chimeric vectors have been developed such as AAV/Ad [77] or AAV/HSV-1 [67]. Significant progress in vector development is occurring in the area of tissueor cell-specific expression. Targeting can be achieved by two strategies. The first one involves engineering viral capsids for binding specific cellular receptors that subsequently mediate viral entry. This type of engineering has been reported for Ad [78] and for AAV vectors [79]. The second strategy relies on choosing an appropriate promoter to confine the expression of the transgene to a particular cell type. Examples of this later approach include the use of the used prostatespecific antigen (PSA) or tyrosinase promoters/enhancers in HD-Ad vectors to maintain strict tissue-specific expression [80]. A dual Ad vector system, encoding for cell-type-specific and regulatable tetracycline-dependent transcription elements, have been developed. The results demonstrated that the GFAP promoter is able to restrict tetracycline-dependent transgene expression to glial cells in cell lines, primary cultures and in the CNS in vivo. The neuronal specific enolase promoter did not show neuronal restricted transgene expression in vitro, but it did so in the CNS in vivo [81]. Although tissue- or cell-specific expression will continue to command interest, we suspect that regulated expression of transgenes will become an important focus for practitioners of gene therapy. The most widely used regulation system is based on the bacterial tetracycline resistance regulation (Tet system), where transgene expression can be switched ‘on’ and ‘off’ in the absence or

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presence of tetracycline [83]. Recently, a dual Ad encoding a tetracyclineregulatable expression system was generated to control the production of TH in vivo and in an experimental therapeutic setting [83]. In the absence but not in the presence of the tetracycline analog doxycycline, TH expression was observed in AP tumor cell lines, i.e., at T20, GH3 and MMQ. In both primary AP cell cultures and the AP gland, in situ expression of TH was seen in lactotrophs, somatotrophs, corticotrophs, thyrotrophs and gonadotrophs in the absence but not in the presence of doxycycline. Furthermore, expression of TH was able to normalize the circulating PRL levels and reduce the mass of the enlarged pituitary gland [83]. These results indicated that the Tet system could be a useful tool for the regulation of the therapeutic transgene expression in vivo [81, 83].

Gene Therapy Strategies for Tumor Treatment

The majority of pituitary tumors are clinically well managed, and normally long-term survival of affected patients can be achieved. In spite of this, for some very large and locally invasive pituitary tumors, treatments are far from ideal. Gene therapy has utilized several strategies to achieve tumor cell killing. The most widely used approaches are conditional cytotoxic cell killing, inhibition of angiogenesis, expression of proapoptotic and/or tumor-suppressor genes and immune stimulation by ectopic cytokine expression and/or engineering the immune recognition of tumor cells. Conditional Cytotoxicity Approaches for Tumor Gene Therapy Conditional cytotoxicity utilizes the conversion of an inert prodrug to a pharmacologically active cytotoxic drug at the tumor site. The most commonly used approach is gene-directed enzyme prodrug therapy (GDEPT). A ‘suicide’ gene encoding the enzyme is selectively delivered to tumor cells. This enzyme must be capable of metabolizing a non or weakly toxic prodrug to an active and highly toxic drug. The toxic drug is produced either within the tumor cells or the tumor mass, allowing its local diffusion to non-tranduced tumor cells, producing a ‘bystander effect’. Thymidine kinase (TK) gene from HSV1 is the most intensely studied and used conditional cytotoxic gene [84–87]. Its substrates include the anti-herpes drug ganciclovir (GCV) which HSV1-TK monophosphorylates to an intermediate, which is subsequently phosphorylated by cellular kinases to a di- or triphosphate form that can be incorporated into DNA as nucleoside analog, and therefore cause cell death of the proliferating cells (fig. 4). Phosphorylated GCV can only kill dividing cells, as tumor cells are usually the most actively dividing cells in tissues, it can be argued that this gives the therapy a certain

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level of selectivity. HSV1-TK/GCV exhibits a strong bystander effect both in vitro and in vivo. Mouse glioma tumor model studies showed that only 10% of tumor cells needed to be transduced to achieve total tumor regression [88]. The HSV1-TK/GCV pairing has been used in human gene therapy trials depending on the basis of good preclinical data [84, 87]. In vivo gene transfer using both retroviral and Ad delivery of HSV1-TK in clinical trials have been well tolerated by the patients with recurrent brain tumors, with extension of patient survival reported [87, 89, 90]. Cytosine deaminase in combination with 5-fluorocytosine (CD/5-FC) has also been moved into human gene therapy clinical trials. Cytosine deaminase is ubiquitous within bacteria and fungi but absent from mammalian cells. This enzyme deaminates the anti-fungal drug 5-fluorocytosine into 5-fluorouracil which induces cell death by inhibiting both DNA and RNA synthesis; therefore, it only kills dividing cells. Huber et al. [91] infected a colorectal tumor cell line ex vivo, then implanted the transduced cells into mice demonstrating antitumor effects as a result of local 5-FU production. A bystander effect was also observed when mixtures of transduced and nontransduced cells were implanted. These experiments suggest that as few as 2% of the tumor cells need to be transduced to eliminate the tumor. In vivo experiments demonstrated that the combination of CD/5-FC gene therapy and radiation possessed superior anti-tumor effect in comparison to single therapy [92]. Nitroreductase is an enzyme from the E. coli strain K12 and is used in combination with the prodrug CB1954, a weak alkylating agent. Nitroreductase converts its prodrug into the 4-hydroxylamino derivative, which after acetylation via thioesters becomes a powerful alkylating agent capable of cross-linking DNA. The active drug kills both quiescent and dividing cells and shows promise for in vivo tumor cell killing [93]. Carboxypeptidase G2 is an enzyme isolated from the bacteria Pseudomonas strain RS-16, which cleaves the C-terminal glutamate moiety from benzoic acid mustard prodrugs. This cleavage, results in the release of cytotoxic nitrogen mustards, which are toxic to both quiescent and dividing cells by DNA alkylation. This enzyme was first used in antibody-directed enzyme prodrug therapies (ADEPT), targeting carboxypeptidase G2 to a colorectal tumor xenograft in athymic mice, by conjugation of the enzyme to an antibody recognizing the carcinoembryonic antigen [94], thus achieving significant antitumor activity upon the administration of prodrug. Clinical trials using this antibody targeting approach are ongoing in patients with advanced, drug resistant colorectal cancer. Recently, Ad vector-mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or glycosylphosphatidylinositol (GPI)-anchored form was developed to strengthen the efficacy of the prodrug-activating system, and more than 50%

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Hypothalamus

Pituitary stalk Promoter Posterior pituitary Cell type A tumor

Anterior pituitary

Suicide gene

Tumor cell A

Intermediate pituitary Cell type B tumor

Normal cell B

Tumor cell HSV1-TK/GCV system

E E Specific endocrine cell or tumor cell transcription genes

Prodrug activating enzyme

No prodrugactivating enzyme Cell lacking endocrine-specific transcribed or tumor-specific transcription elements

Thymidine kinase GCV

GCV-MP

GCV-TP

Triphosphorylated ganciclovir Cell toxicity

E Cell type B alive Active toxic drug

Cell death of HSV1-TK transduced and adjacent untransduced tumor cells (Bystander effect)

Nontoxic drug Reduction in tumor size

Cell type A dead

Fig. 4. Cytotoxic gene therapy approach for specific anterior pituitary adenomas. The diagram illustrates a conditional cytotoxic approach to eliminate tumor cells through delivery of suicide genes via a recombinant Ad vector. Within tumor cell A in the presence of a prodrug and specific endocrine or tumor transcription factors, transcription of the prodrug activating enzyme (E) takes place. The subsequent exposure to a nontoxic prodrug alters it to its active toxic form resulting in excessive production of toxic metabolites which ultimately leads to cell death. Within normal cell B, lacking these specific transcription factors, the expression of the conditional cytotoxic gene does not take place (i.e., HSV1-TK) and therefore the nontoxic prodrug remains innocuous to the cell. Expression of HSV1-TK will only occur within the cells that can activate transcription from the promoter, i.e., in specific

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cell kill was achievable in all of the cell lines tested following only a single exposure to the prodrug, ZD2767P [95]. Dual enzyme/prodrug combinations are currently being investigated with promising results. HSV1-TK and cytosine deaminase have been simultaneously expressed from stably transfected gliosarcoma cells implanted into nude mice and after the prodrug administration, toxicity of the double suicide system was shown to be 2–3 times higher than if the cytotoxic effects of each prodrug was purely additive [96]. These enzyme prodrug combinations are also being assessed for their effects on radiosensitization, and for their use in conjunction with radiotherapy [97]. Inhibition of Angiogenesis The inhibition of angiogenesis is thought to be a promising strategy that could lead to the development of novel anti-neoplastic therapies. The process of new blood vessel formation in growing pituitary tumors offers a potential target for gene therapy. It has been demonstrated experimentally that it is possible to use gene transfer methods in vivo to disrupt the angiogenic process in a solid tumor. The main target would be vascular endothelial growth factor (VEGF) and its receptors (VEGFR-1 or flt-1, and VEGFR-2 or flk-2), basic fibroblast growth factor and its receptor, epidermal growth factor and its receptor, and also transforming growth factor ␤ and ␣. Both antisense and dominant negative variants could be used as gene therapy approaches to target angiogenesis. Replicationdefective retroviral vectors encoding both the soluble, truncated VEGF-R2 and flk-1 have been used to study anti-angiogenesis processes. The results from that work indicate that tumors from flk-1 expressing neuroblastoma cells were less than 33% of the average volume of normal tumors after 23 days. Engineered expression of flk-1, a competitive inhibitor of VEGF, results in the production of an inhibitor of endothelial cell proliferation and migration that greatly restricts the growth of the tumor cells in vivo [98]. Selective toxicity could be achieved using this approach if the molecular targets were located preferentially within the tumor blood vessels; blocking these pathways should mainly affect proliferating cells in need of continuous blood flow for oxygen and nutrients. anterior pituitary tumor cells. In the presence of HSV1-TK, the nontoxic nucleoside analog GCV is converted to ganciclovir monophosphate (GMP), which is further phosphorylated by mammalian kinases to ganciclovir triphosphate (GTP). These phosphorylated metabolites are incorporated into replicating DNA leading to cell death of actively replicating tumor cells. This approach is further effective as adjacent nontransduced tumor cells are also destroyed through the transfer of toxic GCV metabolites. This ‘bystander’ effect produced with the HSV1-TK/GCV model has important therapeutic implications. This tumor-specific suicide gene therapy approach would be amenable to treat pituitary tumors without having a negative effect on adjacent normal anterior pituitary cells.

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The discovery of angiostatin [99] and endostatin [100] provided an antiangiogenic therapeutic strategy that not only could regress large tumors in vivo, but also maintain them quiescent at microscopic size for as long as the therapy was present. These anti-angiogenic inhibitors proved neither to be toxic, nor did they induce drug resistance. Gene therapy could provide a means for delivering endostatin and/or angiostatin locally or systematically, providing a constant supply of angiogenesis inhibitors. Ma et al. [65] have utilized a recombinant AAV vector carrying the angiostatin gene to treat the malignant brain tumor in a C6 glioma/Wistar rat model. Intratumoral injection of a high titer AAV-angiostatin vector rendered efficacious tumor suppression and resulted in long-term survival of the animals. Using this approach, only migrating vascular endothelial cells in the tumor bed should be inhibited; the remaining endothelium in the body, which is quiescent, should not be affected. This therapeutic approach will, therefore, pose a low risk to normal tissue. Proapoptotic and Tumor Suppressor Genes Other promising targets for cancer gene therapy are proapoptotic and tumor suppressor genes [101, 102]. Apoptosis is regulated by a complex cascade of proteases of the interleukin-1␤-converting enzyme family, also known as caspases. Overexpression of interleukin-1␤-converting enzyme proteases is sufficient to induce apoptosis and cell death. Abnormalities in the apoptotic cascade, such as gene deletions, mutations or aberrant gene expression, are almost always present in tumor cells, including gliomas [103]. Examples of proapoptotic-targeted therapy include the use of the potent proapoptotic molecule Fas-L(CD95L). Fas-L is a 40-kDa type II transmembrane protein, a member of the tumor necrosis factor family of cytokines. When bound to its receptor CD95 (Fas, APO-1), Fas-L, a glycosylated 45 kDa transmembrane protein that belongs to the tumor necrosis factor receptor family, it induces very quick apoptosis [105]. The mechanism by which the Fas/Fas-L system induces apoptosis has recently been elucidated and has suggested some potential therapeutic targets. Binding of Fas-L to Fas induces the formation of a death-inducing signaling complex containing the proteins FADD or RIP and RAIDD, leading to the recruitment and activation of caspase 8 or caspase 2 depending on the components of the death inducing signaling complex [105]. The activation of either caspase ultimately leads to apoptotic cell death. Upregulation of Fas/Fas-L system results in cell suicide by the cross-linking of these molecules between neighboring cells [106]. Transcriptionally targeted Ads expressing Fas-L have been recently constructed [107, 108] and used as a gene therapy approach for the treatment of intracranial glioblastoma tumor model in rats [109]. Thus, gene therapy strategies may be developed that

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directly act on the Fas/Fas-L sigal transduction pathway. Such an approach should also be developed for designing gene therapy strategies for the treatment of pituitary tumors. Mutation of the tumor suppressor gene p53 is the most common genetic alteration in cancers. Inactivation of p53 occurs early in glial tumorigenesis and mutations are commonly found in low-grade astrocytomas. Replacement of a defective p53 gene had been described for many tumor models. A recombinant Ad containing the wild-type p53 cDNA has been constructed to transfer the wild-type p53 gene into the glioblastoma cell lines expressing either a wild type or mutated p53 gene. In this experiment, the wild type p53 cells showed inhibition of proliferation and mutant p53 cells underwent apoptotic cell death [110]. In anaplastic astrocytomas, the mutations of the retinoblastoma (Rb) gene and/ or p16 gene are most common. To mimic the therapy of cancer more closely, spontaneous pituitary melanotroph tumors arising in immunocompetent Rb⫾ mice were treated with a recombinant Ad carrying RB cDNA. Intratumoral RB gene transfer decreased tumor cell proliferation, re-established innervation by growth-regulatory dopaminergic neurons, inhibited the growth of tumors, and prolonged the life span of treated animals [111]. Recently, a tumor suppressor gene, mutated in multiple advanced cancers1/phosphatase and tensin homolog (MMAC1/PTEN) was identified within chromosome 10 that is commonly mutated in human glioblastoma multiforme and several other cancer types [112]. Cheney et al. [113] have constructed a replication-defective Ad encoding MMAC1/ PTEN, and infection of MMAC1 mutated cells with this virus rendered them almost completely nontumorigenic, as compared to untreated and control cells, suggesting that in vivo gene transfer of MMAC1/PTEN could potentially be useful in cancer therapy for aggressive gliomas. A similar strategy also could be used for aggressive pituitary tumors. The pathogenesis of pituitary tumors has been extensively studied to identify proapoptotic and tumor suppressor genes, which could be used as effective transgenes for gene therapy. Mutations have been described in the G protein, Gs␣, which occurs in approximately 40% of GH secreting tumors. Rarely occurring ras mutations in invasive tumors, loss of heterozygosity on chromosome 11, and near the Rb locus on chromosome 13 have also been implicated, but not reproducibly identified. The pituitary tumor transforming gene (PTTG) was isolated from rat GH4 pituitary tumors and recently the human homolog was cloned. Human PTTG is abundantly expressed in pituitary tumors and potently transforms cells both in vitro and in vivo. Furthermore, PTTG can be used as a marker for the invasiveness of hormone secreting tumors, with more invasive tumors expressing the highest amounts of PTTG [114, 115].

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Activation of the Immune Response The role of the immune system from a therapeutic viewpoint is that once appropriately activated against tumor-specific determinants, a small activation signal can produce a long lasting body wide protection. These properties make up-regulating the immune system, an attractive target for developing gene therapy strategies for cancer. No gene delivery system or chemotherapeutic drug available has the required specificity to target all tumor cells throughout the tumor mass in cancer patients. Studies of mechanisms involved in recognition and elimination of tumor cells have shown a key role for T lymphocytes in conferring the specificity of tumor rejection. In particular CD8⫹ cytotoxic T lymphocytes were identified as an important effector cell population in the elimination of tumor cells. While responses are mainly mediated by CD8⫹ cytotoxic T lymphocytes, induction of these responses is dependent on the presence of CD4⫹ helper T cells. In addition to antigen-specific effectors, roles have also been identified for natural killer cells and nonspecific effector cells such as macrophages and eosinophils. It is now believed that both the innate and adaptive immune responses act in concert through specific signaling pathways to generate anti-tumor immune responses. The two most commonly used gene therapy approaches involve the enhancement of the host’s immune response against tumors, i.e., cytokine-based gene therapy and immune targeting of tumor cells. The underlying rationale for using cytokines in cancer patients is to increase the patient’s immune response to the autologous tumor. Cytokines may act to enhance local antigen presentation by the tumor by means of inducing expression of MHC-I antigen on the surface of tumor cells, MHC-II antigen on antigen-presenting cells (APCs) or by increasing the expression of costimulatory molecules, or the tumor antigens themselves. Local expression of cytokines also may act to enhance antigen uptake and processing by tissue APCs. These APCs subsequently migrate to secondary lymphoid tissue and induce presentation to resident T and B cells. Cytokines induce Th1 and Th2 subpopulations of helper T cells, direct activation of natural killer and cytotoxic T cells, promote differentiation of granulocyte and macrophage progenitors, and expansion of dendritic cells. The effects mediated by cytokines could result in tumor cells which are better APCs themselves, or the body’s normal APCs could become better at presenting tumor antigens and tumor cells will become targets of immune activated antitumor CTLs. The potential role of cytokines as adjuvants of the immune response has been demonstrated by various studies, which illustrate the regression of human tumors following their systemic administration. Subcutaneous implantation of genetically modified cells secreting cytokines, including IL-2, IL-4, IL-6, IL-12, INF-␣, GM-CSF and tumor necrosis factor-␣, outside of the CNS has been successful in generating cellular mediated immune response,

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while at the same time, minimizing the toxic effects associated with high dose systemic administration. To increase the immune response against cancer cells, several approaches are being developed. Tumor cells can be allogenized by the expression of a highly immunogenic molecule on the surface. Nonself MHC I antigens act as strong allo-antigens and be recognized as distinct targets by CTLs. Expression of nonself antigens on the surface of tumor cells has been achieved by coupling purified protein derivatives to the surface of cells. Another approach, recognition of pre-existing tumor antigens, can be increased by enhancing T-cell activition. The mechanisms involved in antitumor immunity are extremely complex, but it is known that in order to activate resting T cells, the antigen must be seen by the T cell in association with a MHC (class I or II). Despite the fact that many tumors express MHC molecules to present antigen, an immune response, which is capable of eliminating the tumor is rarely raised. Further interaction with a co stimulatory molecule and/or certain cytokines is still required to push the T-cell into active proliferation and differentiation. Once the T-cell is pushed from resting G0, this activated cell no longer requires costimulation to react with the target antigen. The binding of the T cells with the target cells now depends upon the upregulation of accessory binging molecules, such as CD2 and LFA-1, resulting in cytokine production. Several costimulatory molecules such as B7–1 (CD80) and B7–2 (CD-86) are involved in a pathway to both positively and negatively regulate the T cell response. Immunogene therapy utilizing the B7 gene expression is one of the most promising approaches to inhibit the tumor growth. Ando et al. [116] have investigated the difference between B7–1 and B7–2 with regard to B7 gene therapy CNS. Their findings strongly suggest that B7–1 gene therapy could effectively introduce CD (4⫹) TIL activation compared with B7–2 gene therapy. This approach could also be investigated in preclinical animal models as a preclude to its use for the treatment of aggressive pituitary tumors, which do not respond to classical treatment strategies.

Animal Models to Study Pituitary Adenomas

The development and implementation of animal models for pituitary tumors characterized over the years have proven to be an indispensable constituent in the advancement of treatment strategies and gene therapy approaches for pituitary disorders. The relevance of animal models was initially demonstrated with studies on the characterization of experimentally induced pituitary tumors in rodents [117, 118]. The establishment of a range of pituitary tumor cell lines and innovations in transgenic technology have been crucial elements

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in the development of effective animal models in understanding various types of adenomas. The most common animal models employed for in vivo experimental studies related to pituitary tumor genesis are rats and mice. However, the sheep has also proven to be a very useful in vivo model to study pituitary physiology and pathogenesis and to assess the efficiency and safety of novel exploited treatments for gene therapy. The normal sheep pituitary gland is comparable in structure and size to the human pituitary gland, thereby permitting evaluation of transgene expression upon the administration of viral mediated gene therapy. Below we discuss a number of animal models developed over the years and their prospective uses in understanding and improving treatment strategies for pituitary adenomas. Prolactinomas Existing animal models employed to study PRL-secreting adenomas include three major types: hormonally stimulated, implantation of tumor cells, and prolactinomas that are hereditary in nature. Stimulatory effects on lactotrophic proliferation within the AP gland were first demonstrated using estrogen, a powerful steroid hormone [119]. A range of estrogenic-related compounds such as subcutaneous diethylstilboestrol implants, subcutaneous estrone (E1) implants and subcutaneous implants or injections of oestradiol-17␤ were successfully shown to induce lactotroph-hyperplasia. This paradigm was established and widely utilized as an animal model to examine the pathophysiology of prolactinomas, but the drawback of this approach is that estradiol-induced hyperplasia is hormone dependent unlike neoplasms of the AP gland [120]. It is also feasible that the sustained delivery of estrogen to induce hyperplasia may abate the effectiveness of treatment. Among the conventional pituitary cell lines, the PRL/ACTH secreting 7315a cell line has led to the development of two frequently employed transplantable tumor cell lines [121]. Implantation of the MMQ cell line into adult female buffalo rats caused marked elevations in PRL levels, resulting in augmented spleen weight and noticeable white pulp hyperplasia [121]. The 235–1 cell line demonstrated specific enhanced secretion of PRL and produced tumors upon implantation into buffalo rats and nude athymic mice. The buffalo rat implanted with the PRL-only MMQ tumor cells has been demonstrated to be an effective model for the assessment of chronic hyperprolactinemia [121]. As compared to estrogen-induced ‘tumors’ and the induced transplantable tumors, spontaneous transplantable human prolactinoma of the rat has been shown to be an effective animal model that very much resembles in pathology to the human prolactinoma [122]. Transplantable tumors, SmtTW and SmtTW2 implanted by serial passages were shown to exhibit 100% transplantation success beneath the kidney capsule but only 20% subcutaneously. The perpetual

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proliferation of embedded tumors was identical to those of the primary tumors, and expected rise in PRL levels were observed while the other hormone levels remained normal. SmtTW tumors have also demonstrated their potential application in understanding adenoma pathology. Polysialylated neural cell adhesion molecule (PSA-NCAM), a vastly expressed molecule during brain and pituitary development, exhibited high expression levels in rat transplantable SmtTW pituitary tumors [124]. Peak PSA-NCAM expression was detected in SmtTW4 malignant tumors transplanted subcutaneously and below the kidney capsule that were maintained by successive tumor grafts. Expression of PSA-NCAM was not identified in low growth rate benign SmtTW2, but low expression levels were seen among high growth rate SmtTW3 transplantable tumors, suggesting that PSA-NCAM expression is an indication of the proliferation rate and malignancy of the tumor. Studies from this model raise the possibility of employing the PSA-NCAM molecule as a diagnostic marker for malignant pituitary adenomas. As this model provides features that are very similar to the human pituitary adenoma pathology, the SmtTW tumors can be of practical use in assessing the likely outcomes of novel approaches to modulate PSA-NCAM expression. Also these models could be used to test the novel gene therapy approaches. A model for prolactinomas was also developed by cross-breeding the Okamoto spontaneously hypertensive rat strain and the Koletsky rat strain [124, 125]. These strains are normally used to study hypertension and obesity. This newly developed rat strain designated as spontaneously hypertensive rat/N:Mcc-cp had consequential complications of cardiomyopathy and congestive heart failure. Despite these adverse side effects, assessment of the pituitaries revealed that 70% of rats developed prolactinomas. Aged female and male Sprague Dawley and Wistar strain rats have been shown to develop PRL-secreting pituitary tumors, with an occurrence as high as 80% in Sprague Dawley rats, identified by distinct augmented PRL levels.

GH Tumors Tumors of somatotroph cells have been developed in transgenic mice. These mice encompass the SV40 T antigen under the regulation of the bovine arginine vasopressin promoter [126]. Few cases were reported to have tumor occurrences within the intermediate lobe that were immunoreactive for ACTH and proopiomelanocortin (POMC) mRNA. Further animal models that have utilized transplantable cell lines that release GH comprise the MtT/W15 cells [122] and GH3 cells [127]. Ad expressing the HSV1-TK under the control of GH promoter was also effectively utilized in a transplantable tumor model using GH3 cells in nude mice [127]. Subcutaneous transplantation of human

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GH adenoma tissue into athymic mice was also proven to be a dependable model [128]. Cushing’s Disease Studies with transgenic murine models have demonstrated to be very useful in the generation and characterization of models of Cushing’s disease. ACTH releasing pituitary tumors developed in transgenic mice which were produced by engineering the polyoma early region promoter, coupled to a complementary DNA (cDNA) encoding polyoma large T antigen [129]. Normal morphology was recorded at 4 months of age, followed by microadenoma development at 9 months and distinct macroadenomas, up to 5 mm in dimension at 13–16 months of age. Nontransgenic immunocompetent mice with subcutaneous transplants of pituitary tumor cells produced tumors with indistinguishable morphology and ACTH immunoreactivity comparable to the parent tumor [129]. Hypercorticotropism, a hallmark of the disease, was augmented with a brief latency in mice carrying transgene pituitary transplants than in the polyoma large T antigen-1 transgenic mice alone. Considerable increases in ACTH levels were observed in transplanted mice than in clinically ill transgenic mice, indicating the effectiveness of the murine model for studying this disease. Leukemia inhibitory factor, a pleiotropic cytokine plays a key role in the regulation of the mature hypothalamic-pituitary-adrenal axis in vivo. Leukemia inhibitory factor influences corticotroph cell growth and stimulates POMC transcription in vitro. Features of Cushing’s disease were observed when transgenic mice expressing leukemia inhibitory factor under the regulation of the pituitary glycoprotein hormone ␣-subunit (␣ GSU) promoter were studied. Mice pituitary glands revealed corticotroph hyperplasia, evident somatotroph and gonadotroph hypoplasia, and numerous Rathke-like cysts edged by ciliated cells [130]. Another model that has also shown to give rise to Cushing’s disease is the overexpression of the mutated form of the neuroendocrine protein 7B2 in transgenic knockout mice [131]. Mice expressing the null mutated form of 7B2 displayed augmented levels in ACTH and corticosterone, with adrenocortical expansion and eventual acute Cushing’s syndrome. The Rb gene, a tumor suppressor gene, has been shown to causel the suppression of tumor cells reconstituted with Rb ex vivo and implanted into immunodeficient mice as well as with germline transmission of a human RB transgene into tumor-prone Rb⫾ mice. Heterozygous immunocompetent Rb⫾ mice with an intermediate lobe tumor have been used to assess spontaneously arising melanotroph tumors [111]. Tumors of this nature treated with an Ad containing the Rb cDNA suppressed tumor cell proliferation and extended the lifespan of treated animals. A feasible strategy for adenomas with nonfunctional expression of tumor suppression genes could be, to reinstate normal genes via Ad gene therapy encompassing the Rb cCDNA gene.

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Null Cell Adenoma Adenomas of this type do not synthesize AP hormones in vivo, but have shown to express gonadotrophin-related genes. Models for these adenomas were derived using transgenic mice bearing the temperature sensitive mutant of the SV40 T antigen TSA58, under the regulation of the human Follicle stimulating hormone-␤ (FSH-␤) regulatory elements [132]. Animals expressing the mutated antigen developed progressive multifocal pituitary nodules with decreasing immunoreactivity for FSH-␤ and (leutinizing hormone-␤). These animals displayed features that are comparable to those observed in human null cell adenoma, and these studies provide a good model for assessing the pathophysiology of these tumors and testing novel therapeutic modalities. Uncontrolled hormonal induction and molecular deficiencies are major factors in the consequential formation of pituitary adenomas. Animal models have provided evidence that hormonal stimulation is among the elements leading to tumor genesis. Development of cell lines have provided the backbone and still remain a crucial element in the design of effective animal models in helping us to further advance and improve our treatment strategies for adenomas. Further and better insights into pituitary adenomas are imperative, as the normal gland possess complex qualities in terms of function and structure. Some animal models may have certain drawbacks, thus additional studies on these and other tumor models would allow us to decide the optimum in vivo model in which to test novel therapeutic approaches for specific adenoma types.

The Preclinical Experience

Several methods of gene therapy have been explored for the treatment of pituitary tumors, with particular focus directed at using Ads as delivery vectors [87, 133]. Studies which aimed to use gene therapy approaches to deliver genes into AP cells include recombinant Ad vectors and HSV-1 recombinant viral vectors, which were both successfully used to deliver and target transgenes to AP cells in vitro [134, 135]. The HSV-TK gene has since been targeted at pituitary tumor cells as a conditional toxic approach to kill proliferating tumor cells in conjunction with glaciclovir (GCV). In vivo studies modeling Cushing’s disease showed that targeting TK gene expression to the ACTH-producing tumor cells yielded considerable regression of the tumors [136]. High-level expression of the TK gene was observed under control of the POMC promoter (AdPOMCGal; AdPOMCTK). This promoter also yielded good cell-type specificity, in that nonpituitary cells were unaffected by infection and expression of the therapeutic gene. In another study, Windeatt et al. [86] used recombinant Ad vectors expressing HSV-1 thymidine kinase to treat estrogen-induced lactotroph hyperplasias

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Fig. 5. Delivery of recombinant adenovirus to the anterior pituitary gland in vivo. Following anesthesia with halothane and placement of the rat in a stereotaxic frame, the skull is exposed and a hole is drilled posterior to the bregma revealing the superior sagittal sinus, and the surrounding brain. Intrapituitary injections are made using a 26-gauge-modified Hamilton syringe needle, with its tip previously ground until the opening of the needle is at the base of the tip. The needle is lowered until touching the sphenoidal bone, and making contact with the bottom of the rat equivalent of the sella turcica. This leaves the opening of the needle within the pituitary gland and adequate amounts of recombinant vector are then injected. Under these conditions of injection, the pituitary can be transduced by recombinant Ad in 100% of surgical attempts [86].

within the AP gland (fig. 5). In addition to the observation of reduced tumor size and nontoxicity of AP, a bystander effect which also destroyed untransduced tumor cells was also documented [86]. Transgene expression of HSV-1-thymidine kinase (HSV1-TK) was also restricted to lactotrophic cells using the human PRL promoter [137]. Again, the importance of cell type specificity was underscored by the resulting selective apoptosis induced in lactotrophic GH3 tumor cells. The promoters used in each of these applications have played important roles in achieving specific and efficient expression: while generally stronger promoters tend to lack cell type specificity, on the other hand, cell type-specific promoters generally achieve less efficient transgene expression [137]. Further along these lines, Lee et al. [138] used Ad vectors to target the expression of the diphtheria toxin gene to GH-producing pituitary tumor cells. Their success in this targeted suicide gene therapy approach may be useful for treating GH-secreting adenomas. In another similar approach [139], gene therapy was used to induce apoptosis of lactotropic tumor cells and suppress tumor

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formation by infecting the GH4 cells with dominant negative estrogen response mutant using an Ad vector. The regulation of gene expression and cell growth by endogenous ER was interrupted, and the cells were rendered unable to differentiate or proliferate [139]. This strategy presents another possibility for the targeted treatment of pituitary lactotroph adenomas. An alternative modality for treating PRL-secreting adenomas has also been developed. This approach harnesses the overproduction of dopamine as an inhibitor of PRL secretion and lactotroph growth, thereby reducing the growth and hypersecretion of prolactinomas. A gene therapy strategy enhanced the production of dopamine in situ by augmenting expression of tyrosine hydroxylase in the AP gland in order to speed up this rate limiting step in dopamine synthesis [83]. In addition to targeting specific cells for treatment, gene therapy techniques have developed regulatable therapeutic transgene expression (fig. 6). Smith-Arica et al. [81] devised a dual Ad system in which the expression could be regulated using an inducible tetracycline system and the transcription could also be targeted to specific brain or AP cell types. The astrocyte-specific, glial fibrillary acidic protein (GFAP) and the neuronal specific enolase promoter used were able to restrict the transgene expression to glial cells and neuronal cells in vivo, respectively. Such restriction was not so apparent for the neuronal specific enolase promoter in vitro. The most efficient transgene expression was accomplished by using an excess of the transactivator in the dual Ad system, which could be completely shut off with the administration of doxycycline (fig. 6). In 2001, Smith-Arica et al. applied this concept to lactotrophic cells in the AP gland, by reusing tetracycline-responsive transcriptional elements. Under the control of the human lactotroph-specific PRL promoter, they again achieved cell-type specificity and system inducibility using a dual Ad vector system. Besides targeting tumor cells for treatment, gene therapy has the capacity for replacing gene deficiencies. Sarac et al. [140] focused on the replacement of the chaperone protein, 7B2, the absence of which can reduce the effectiveness of the prohormone convertase PC2 and lead to effects similar to Cushing’s disease [140]. It was shown that stereotactic injection of recombinant Ad vector encoding 7B2 into a pituitary deficient in 7B2 raised 7B2 levels in the pituitary and blood, increased PC2 activity, lowered ACTH levels in the blood, and slightly increased circulating ␣ MSH levels. Additionally, blood glucose levels increased, corticosterone decreased, and survival times increased. Such results indicate that the Ad administration of 7B2 can help support 7B2 null mice and illustrate the potential for gene replacement therapy. Since Ads have been established as viable vectors for gene therapy, assessment of their safety profile is necessary. Southgate [141] studied the physiological and therapeutic implications of their use to transfer genes into the AP

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gland as well as safety issues associated with such interventions. An investigation into the duration of transgene expression, local immune responses and consequences on circulating pituitary hormone levels was conducted, as well as a review of the levels of circulating anti-Ad neutralizing antibodies and AP hormones in sera and the presence of vector genome and cellular immune infiltrates within the AP gland. Though reduced transgene expression was seen over time and virus-induced immune responses were observed, results showed no major cytotoxicity or disruption of AP hormonal functions. Additionally, endocrine function in a large animal model does not seem to be affected by stereotaxic intrapituitary delivery of such cell type-specific recombinant Ads [142]. Such findings indicate that Ad-mediated delivery to the AP gland may be a safe and effective method of treating pituitary diseases. However, in 2002, Davis [142] did report evidencing that injection of high doses of recombinant Ads into the sheep AP can lead to severe inflammation of the pituitary, and warned that histological investigations and vector dosage evaluations are important for developing safe and effective endocrine gene therapy. The development of gene therapy as a useful treatment for pituitary diseases has shown very promising results. The versatility of this technique has allowed for a wide range of applications, including tumor reduction and supplementation of deleted genes. Though further investigation of in vivo effects of gene delivery and its efficiency and safety are needed, this area of medical research offers tremendous potential for increasing new and powerful therapeutic modalities.

Conclusions

Pituitary adenomas are usually benign and are well managed with classical therapies, i.e., receptor-mediated medical therapy, surgery, and in some instances, radiotherapy. In spite of this, there are occasions where the tumors do not respond to these forms of therapy, the therapies are not well tolerated by the patients, or the tumors become invasive or recur and become very difficult to treat. This usually depends on the nature of the tumor, for example whether it is secretory or nonfunctioning, its size and also the type of hormonal cell-type giving rise to the tumor. In these instances, gene therapy can provide a novel modality to treat these tumors. and transcription does not occur. b Tet-on regulated expression. Repressor proteins bind to the TRE operator site, preventing transactivator binding and initiation of transcription. When present, Tc binds to the repressor proteins, releasing them from the operator site. rtTA transactivators are then able to bind to the open operator site and initiate transcription.

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Gene therapy could be used by itself or in combination with existing therapies, depending on the presentation of the tumor and also on the medical history of the patient. As discussed above, there are several gene therapeutic approaches, which could be very attractive for treating these tumors. New therapeutic targets together with novel, improved vector systems and genetic switches to turn therapeutic gene expression ‘on’ or ‘off’ as needed, will render these therapies safe for human trials. In spite of enormous progress in the fields of vector development, therapeutic targets and improved preclinical models, much work still needs to be done with respect to improving efficacy and safety of these gene therapies before they can be implemented to treat human patients (tables 4, 5). This field is exciting and we hope that this review will inspire scientists and clinicians to embark in the basic and translational research needed to bring these treatments to the clinic. Acknowledgements We thank the generous funding our institute receives from the Board of Governors at Cedars-Sinai Medical Center and the encouragement and support of its members. We wish to thank the unparalleled support and academic leadership of Dr. Shlomo Melmed. We are grateful to Mr. Richard Katzman for his superb administrative organizational skills and to Mr. Danny Malaniak for his encouragement, support, and commitment. Work is funded by: National Institutes of Health/National Institute of Neurological Disorders & Stroke Grants #NS42893.01, NS047298–01, U54 4NS 04–5309 to Pedro R. Lowenstein, M.D., Ph.D.; National Institutes of Health/National Institute of Neurological Disorders & Stroke Grant #NS44556.01 and TW006273–01A1 to Maria Castro, Ph.D.; the European Community Framework V; the Parkinson’s Disease Foundation; and the Linda Tallen & David Paul Kane Annual Fellowship in Cancer Gene Therapy.

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Dr. M.G. Castro Gene Therapeutics Research Institute Cedars-Sinai Medical Center, Research Pavilion Room R-5090, 8700 Beverly Boulevard, Los Angeles, CA 90048–1860 (USA) Tel. ⫹1 310 423 7302, Fax ⫹1 310 423 7308, E-Mail [email protected]

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Freese A, Simeone FA, Leone P, Janson C (eds): Principles of Molecular Neurosurgery. Prog Neurol Surg. Basel, Karger, 2005, vol 18, pp 624–644

Stem Cells for Targeting CNS Malignancy Stephen Yipa, Richard L. Sidmanb, Evan Y. Snyderb,c a

Division of Neuropathology, Department of Pathology and Laboratory Medicine, Vancouver General Hospital, University of British Columbia, Vancouver, Canada; b Department of Neurology, Harvard Medical School, Harvard Institutes of Medicine, Beth Israel-Deaconess Medical Center & Children’s Hospital, Boston, Mass., and c The Burnham Institute, Program in Developmental & Regenerative Cell Biology, La Jolla, Calif., USA

The Neural Stem Cell as a Model Somatic Stem Cell

In the early 1990s, a handful of investigators interested in fundamental neural development began to identify, within cultures obtained from the developing and mature central nervous system (CNS), cells with surprising plasticity, multipotency, and a propensity for dynamically shifting their fates [1–4]. The existence of such cells – if, indeed, they represented a population normally resident in the brain – challenged the prevailing dogma that the nervous system was rigidly and immutably constructed. Neural stem cells (NSCs), as these plastic cells came to be termed, began to garner the interest of not just the developmental community but also that of the neural repair, gene therapy, and transplant communities when it was recognized that they could be expanded in culture and reimplanted into the mammalian brain where they would reintegrate appropriately and stably express foreign genes [3]. Their abundance, multipotency, ease of manipulation and engraftability made this strategy a powerful method for CNS gene therapy and repair. In comparison to extant techniques, NSCs presented certain advantages: they were a homogeneous and relatively well-defined neural cell population that could be stored and expanded on demand, and, if necessary, genetically manipulated ex vivo to express a wide variety of foreign transgenes. These transduced genes, as well as their inherent genetic repertoire, could be imported into the CNS following transplantation almost anywhere into the developing and mature host brain. Furthermore, NSCs and their progeny possessed a capacity to

integrate not only locally at their site of implantation, and competing with and interdigitating seamlessly with endogenous cells [3, 5–8], but also more broadly [9–13]. These cells were quite migratory – particularly if implanted into germinal zones – permitting cell and gene therapy to be contemplated for disseminated, even global, CNS disease processes. In that sense, NSCs had a distinct advantage over fetal tissue and non-neural cells for cell replacement and over most viral vectors for gene delivery. Even such alternative cellular vectors as hematopoietic cells, when used for protein delivery in bone marrow transplantation paradigms [14, 15], could not efficiently circumvent the restrictions of the blood-brain barrier and integrate throughout the CNS as effectively as NSCs. A single bona fide NSC clone could take up residence in, and accommodate to, any nervous system region, permitting an economy of resources. In addition, NSCs appear to be attracted by degenerating neural tissue by local cues [7, 16, 17], replacing dead or dysfunctional cells in those regions. In pathological niches, multipotent NSCs, in response to signals still poorly understood (though probably linked to inflammatory cytokines), would shift their fate toward neural lineages most in need of repletion, even if beyond the classical developmental window for genesis of that cell type. These observations gave birth to the hypothesis that certain neurodegenerative environments recapitulate developmental cues, because NSCs responded to neurogenic cues not only during their normal embryological expression, but also when recreated by particular types of cell death. NSCs, in other words, could sense niches of neurogenesis or small areas of pathology in the brain [5, 6, 11, 16]. These observations in the CNS served as an impetus to investigators in other solid organ systems to search for ‘stem-like’ cells even within tissues generally held to be more regenerative, more forgiving, and/or more redundant than the CNS. Hence, the NSC, in effect the first solid organ stem cell isolated and exploited, served as a model for other somatic stem cells. Importantly, despite the spotlight of therapeutic promise the NSC has thrown upon itself (and other stem cells), it is critical to remember that its existence was unveiled in the course of understanding development and that, in the end, it is simply one player in a broad and exceedingly complex, interdependent, finely-tuned developmental system, one that requires fundamental developmental understanding [18, 19]. In this endeavor, the CNS continues to serve as an instructive model for the stem cell field in general [20, 21].

NSCs as a Therapeutic Tool

Knowledge of the fundamental biology of NSCs has grown significantly, which in conjunction with advances in molecular biological techniques, has

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promoted their use in the treatment of various neurological disorders [13]. NSCs can migrate over long distances to areas of injuries in the CNS followed by developmentally appropriate (temporally and regionally) differentiation [22]. Experimental evidence has demonstrated that NSCs can populate as well as repopulate developing and injured/regenerating areas of the immature and mature CNS [16, 23]. For example, transplanted NSCs can effectively integrate into abnormal brains and yield either neurons or glia (e.g., myelinating oligodendrocytes in myelin-impoverished mutants) [5, 6, 11, 24, 25]. NSCs also can serve as effective gene delivery vehicles, promoting effective changes in the phenotype of genetically based loss-of-function models of neurodegeneration such as lysosomal storage diseases [9]. Other examples abound for the multipotentiality and migratory potential of NSCs in experimental models of the diseased or traumatically injured CNS [5, 6, 24, 26]. In view of their innate ability to home in on sites of injury or dysregulated tissue, one of the newer and most promising applications is the therapeutic use of NSCs to target malignancies of the CNS [27, 28].

Sources of NSCs

NSCs may be prepared for use by a number of strategies, which have been well summarized elsewhere [29]. The most straightforward method is simply to isolate them directly from the neuroectoderm or from neuroectoderm-derived structures. Whether it is better to abstract such cells from the embryo, fetus, newborn, juvenile, or adult, remains to be determined empirically. Clearly, the younger the age of the region and the more active its neurogenic potential, the easier the process becomes. Cells isolated from the inner cell mass of the blastocyst, known as embryonic stem cells, can presumably be instructed to yield cells of neuroectodermal lineage (i.e., NSCs) in vitro when given the appropriate stimuli [30]. Several groups have argued that there exists plasticity among germ layers such that NSCs can be obtained from, for example, bone marrow mesenchyme or umbilical cord cells of mesodermal origin [31, 32]. The fundamental ability of the latter sources to yield bona fide neural elements remains quite controversial and, at best, can do it only inefficiently [29]. The goal in all stem cell-based therapy is to generate a stable, selfrenewing line of such cells that can provide an inexhaustible source of cells for experimentation. One line that has proven instructive for studying and dissecting the potential and pitfalls of somatic stem cell-based therapies in general and NSC-based therapies in particular is the murine clone C17.2. These cells, initially derived from the developing murine cerebellum and

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maintained as a single clone, have been transduced with the oncogene myc in order to insure that their stem-like state would persist in vitro. These NSCs self-renew efficiently and for prolonged passages in vitro; yet when introduced to the CNS environment, they exit the cell cycle, constitutively downregulate the ‘stem’ state-promoting gene myc (along with other genes) and differentiate into appropriate daughter lineages. The cells have been shown to be nontumorigenic even in non-immunocompetent nude mice. C17.2 cells provide an abundant and reproducible cell source for experiments that can be well controlled from passage-to-passage, condition-to-condition, and animalto-animal over prolonged periods of time [3, 17].

CNS Tumors

Gliablastoma multiforme (GBM) are highly infiltrative and invasive glial cell tumors. Median survival is one year or less for GBM and three years for anaplastic astrocytoma, an intermediate form [33]. Low-grade gliomas can transform into GBM as a result of specific genetic changes. Since Harvey Cushing’s era, surgical treatment for GBM has been largely ineffective. Radical resection, even hemispherectomy, has consistently failed due to recurrence of tumor in the contralateral, normal-looking, hemisphere [34]. Spread of tumor cells along white matter tracts and perineuronal, perivascular, or subpial spaces was initially described in the 1940s in high-grade gliomas by Scherer. These routes of dissemination are responsible for the highly infiltrative nature of glioma and also the many challenges in their surgical management [35]. In fact, Silbergeld and Chicoine [36] demonstrated the presence of neoplastic cells in histological benign brain away from the tumor mass using tissue culture technique. Taking this biological destiny to an extreme, gliomatosis cerebri is a form of glioma characterized by diffuse global infiltration and is associated with a very poor prognosis [37]. Despite advances in every facet of neurosurgical oncology, from the wide use of neuronavigational systems, advancement in real time intraoperative magnetic resonance imaging (MRI)-assisted surgery and neurofunctional imaging coupled with better intraoperative neurophysiological monitoring that have permitted almost routine use of awake craniotomy, glioma surgical outcome still remains very poor [38, 39]. In summary, the inability to effect surgical cure seems to be inherent in the underlying biology of gliomas and new approaches must be found to combat the disease in a biologically rational fashion. The potential of de-differentiated tumor cells to migrate and infiltrate surrounding normal brain structures resembles that of NSCs during normal neural development. Brain tumor cells express and secrete a variety of proteases, that

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help in breaking down the intracerebral extracellular matrix to facilitate tumor invasion and to propagate a microenvironment that is favorable for survival of the tumor [40]. Glioma cells may arise from a small existing pool of neural progenitors that have undergone neoplastic transformation. On the other hand, de-differentiation of mature glial cells could give rise to gliomas exhibiting many, but not all, of the characteristics of normal NSCs [41]. Many similarities as well as subtle differences exist between normal neurodevelopment and glioma-genesis. In fact, normal glial development and glial neoplasm formation share similar molecular genetic profiles [42]. Dai et al. [43] showed platelet-derived growth factor-induced de-differentiation of astrocytes with the resultant formation of oligodendroglioma. Singh et al. [44] found evidence of a population of CD133 positive brain tumor stem cells in primary human glioma. These cells can undergo self-renewal as well as differentiation similar to normal NSCs. Interestingly, the authors found a positive correlation between the proportion of brain tumor stem cell in tumors and their biological aggressiveness. The discovery of this population of tumor stem cells has implications in understanding the biology and clinical behavior of glioma. Glioma-genesis is associated with characteristic genetic alterations, many of which are well characterized [45–47]. Knowledge of the specifics of these alterations has permitted the re-creation of the different glioma phenotypes in rodents [48]. For example, enforced expression of the constitutively active epidermal growth factor receptor (EGF-RvIII) under the control of the S100 promoter has generated a murine model of oligodendroglioma [49]. Haploinsufficiency for either p53 or ink4/arf significantly augmented the pathogenicity and aggressiveness of the tumor, recapitulating glioma genetics in human patients. Gene mutations specific to other types of human brain tumors have also allowed for the creation of the respective animal models. Meningioma development is recapitulated in mice with nf2 gene mutation in arachnoid cells [50]. Lineage specific knockout of the nf1 gene in Schwann cells in conjunction with loss of heterozygosity of nf1 in non-neoplastic cells generated neurofibromas in mice whereas the nf1 null/inactivated p53 phenotype triggered the formation of soft tissue sarcoma resembling human neurofibromatosis 1 tumors [51, 52]. These murine brain tumor models offer a test bed for novel therapeutics as well as a system for investigating the molecular events of brain tumorigenesis [53]. Questions still remain as to whether NSC may home in on brain tumors other than gliomas. For example, chordoma is a histologically benign tumor that nevertheless carries a high degree of morbidity and mortality due to its insidious nature and the prohibitive recurrence rate despite aggressive surgical resection [54]. The question has been posed if NSCs could home in on to chordoma and play a role in its overall management, and experiments are underway to test this hypothesis. In addition, experimental evidence is still

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pending in terms of NSC migration towards intracranial metastases from extracranial tumors (e.g., lung, breast, melanoma). The therapeutic potential is vast if, indeed, NSCs exhibit tropism for these intracranial tumors of non-neural origin.

Therapeutic Uses of NSCs against Primary Brain Tumors

In 2000, Aboody et al. [17] reported that transplanted exogenous murine and human NSCs were capable of ‘homing in’ over long distances to intracerebral xenogeneic brain tumors deposited into rodent brains. The authors also demonstrated the ability of NSCs to ‘track’ tumor cells escaping from the original inoculated tumor mass and invading normal brain. Specially tagged NSCs were found adjacent to invading tumor cells that appeared to be infiltrating normal brain along white matter tracts, tumor-related endothelium, and interstitial spaces. There appeared to be a particular predilection of NSCs for tumorassociated endothelium. NSCs could be introduced either intraparenchymally or into the lateral ventricles with equal homing ability. Whether introduced into the ipsilateral or contralateral cerebral ventricles or into the ipsilateral or contralateral cerebral parenchyma, NSCs were able to migrate toward the implanted tumor and appose themselves initimately to escaping, infiltrating tumor cells even at far distances from the main tumor mass. Indeed, even NSCs injected into the tail vein demonstrated successful intracranial tumor ‘homing,’ establishing a paradigm since used by other investigators for other intracranial pathologies, e.g., models of multiple sclerosis [55]. The blood-brain barrier normally acts as an efficient barrier to the successful delivery of many therapeutic agents and dictates the limited repertoire of brain tumor chemotherapeutic agents [56, 57]. In this instance, the bloodbrain barrier did not appear to affect the migration and homing of NSCs toward intracranial pathology (as modeled by the neoplasms). This ‘glioma-tropic’ capability of NSCs bodes well for their use in clinical applications. NSCs could serve as a tumor-directed homing device expressing a variety of anti-tumor genes, including those encoding cytotoxic, anti-angiogenic, anti-mitotic, anti-migratory, immunomodulatory, pro-differentiating, and/or pro-apoptotic agents. Ultimately the goal would be for NSCs to be used to in conjunction with, and optimize, other therapeutic modalities by providing the ability to track invading cells, heretofore, the weak point of radiation, surgical, and pharmacological strategies, and complete eradication of the glioma cells [58]. Aboody et al. [17] tested this hypothesis with the use of cytosine deaminase (CD)-expressing murine NSCs. This enzyme converts the non-toxic precursor 5-fluorocytosine (5-FC) into the toxic compound 5-fluorouracil.

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Injection of 5-FC into tumor-bearing mice that had been inoculated with NSCs engineered to express CD was followed by dramatic reduction of intracranial tumor burden. An impressive bystander effect on tumor cells was noted; Barresi et al. [59] replicated this phenomenon by showing in vivo regression of implanted C6 glioma cells after co-inoculation of an immortalized neural progenitor cell line ST14A expressing CD followed by administration of 5-FC. This work gave further credence to the utility of exploiting NSCs to effect the well established but hitherto unsuccessful ‘prodrug/prodrug-converting enzyme’ strategy for destroying intracerebral malignancies. The virtue of the prodrug approach is two-fold. First, NSCs need only kill a small percentage of tumor cells to have a large impact on other tumor cells by virtue of the ‘bystander effect.’ This effect can be explained by analogy to an exploding hand-grenade around the site of dying tumor cells, which send out toxic factors that are inimical to other surrounding tumor cells. The action of CD on 5-FC creates one of the greatest bystander effects of all the pro-drug converting enzymes. Second, while no adverse effects or contribution to tumor growth from NSCs have ever been detected in these models, should such an unlikely situation arise, the CD within the NSC would cause it to self-eliminate were it to become mitotic, providing a built-in safety mechanism. As an extension of using NSCs to deliver cytolytic genes, and based on the report by Lynch et al. [60] that NSCs could be used as engraftable, mobile intracranial viral packaging lines, Herrlinger et al. [61] showed that murine NSCs, engineered to release replication-conditional herpes simplex virus thymidine kinase, were efficient in the destruction of an intracranial tumor mass as well as isolated escaping tumor microdeposits. In preliminary studies, Dr. William Weiss and colleagues at UCSF have validated the glioma-tropism of murine NSCs in a transgenic oligodendroglioma-bearing mouse model. This double transgenic mouse model is the product of mating a transgenic mouse in which a mutant EGF receptor is transcribed from the S100 promoter (S100-v-erbB) with an INK4aNull mouse, yielding progeny that almost uniformly develop tumors by 6–12 months of age [49]. These investigators showed in this model that NSCs homed in on spontaneously developing tumors. With the use of CD-expressing NSCs (clone C17.2) in pilot studies, host survival was improved. Because NSCs seemed to home in on even small ‘subclinical’ tumors whose presence was unsuspected by the investigators prior to histological examination, NSCs also may be used in tumor diagnosis if armed with tags that can be imaged in the living state. It warrants mentioning that transgenic mice that spontaneously develop brain tumors appear to provide models for studying tumor biology and therapies – including the glioma-specific migratory potential of NSCs – that far more faithfully emulate the true clinical situation in

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human and are, therefore, superior to older models that depend on the artificial implantation of glioma cell lines. Immunomodulation via the judicious use of cytokines has been shown to be extremely effective in the destruction of experimental brain tumors, either via direct cytotoxicity or the activation of immune-mediated anti-tumor effect [62–64]. Benedetti et al. [65] showed that intratumoral injection of interleukin 4 expressing murine NSCs effected radiographic tumor regression and prolongation of host survival. In addition, the authors detected the presence of NSCs several weeks post injection in the recipient animals. This evidence implied persistence of implanted NSCs and possibly persistence of anti-tumor effect in vivo. The authors also described an inherent tumor inhibitory effect exhibited by the injected stem cells. This phenomenon was first observed many years ago using murine NSC clone C17.2. Gliastatin, a membrane-associated factor isolated from these NSCs was capable of converting C6 glioma cells to phenotypically normal astrocytes simply via cell contact [66]. Exploiting the specific homing capability of NSCs to intracranial tumors, Ehtesham et al. [67] transfected murine neural progenitors ex vivo with the IL-12-gene using an adenoviral vector. Stable expression of the IL-12 protein was demonstrated in vitro and in vivo. Implantation of IL-12 expressing NSCs into tumor-bearing syngeneic mice effected tumor destruction and improved host survival. In addition, the authors showed enhanced tumor infiltration by T lymphocyte as a result of the expression of IL-12 in close proximity to the tumor mass. In a separate study, the authors transfected similarly derived murine neural progenitors with the human TRAIL gene using a replicationdeficient adenoviral vector [68]. TRAIL belongs to the tumor necrosis factor superfamily of pro-apoptotic proteins, which had previously been shown to induce apoptosis in experimental tumor models [69]. Introduction of TRAILexpressing neural stem/progenitor cells into nude mice bearing GBM xenografts was followed by eradication of tumor via induction of apoptotic cell death.

Glioma-Tropism: Molecular ‘Breadcrumbs’

The exquisite tumor-homing capability of NSCs is likely a result of multiple mechanisms. Expression of a similar repertoire of adhesion molecules by NSCs and glioma cells could effect migration to the same trophic source [70–72]. Alternatively, ‘molecular breadcrumbs’ left by the tumor cells could lead the migratory NSCs toward escaping tumor cells. Tumors could potentially secrete chemotactic factors that encourage the homing of NSCs. Brain inflammation secondary to tumor necrosis could attract NSCs directly or indirectly via

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factors secreted by microglia and leukocytes [73]. We have previously reviewed some potential tumor-homing mechanisms of NSC [28]. Chemokines are involved in the normal development of the CNS and are also implicated in various CNS pathologies [74]. The chemokine receptor CXCR4 and its ligand SDF-1 have been implicated in glioma biology [75]. Rempel et al. [76] showed that this receptor/ligand pair is over-expressed and is involved in glioma migration and neoangiogenesis. Zhou et al. [77] demonstrated expression of CXCR4 and a functional CXCR4 signaling pathway in primary glioma specimens, effecting cell survival during serum-deprivation. In addition, the authors showed CXCR4-mediated glioma chemotaxis toward a source of SDF-1. The same receptor/ligand combination has been shown to be expressed and to function in the development of early neural and glial cells [78, 79] and in migration of cells in vivo during formation of the cerebellum and cortex [80]. Recently, Rubin et al. [81] showed that a small molecule inhibitor of CXCR4 abrogates the growth of implanted glioblastoma multiforme and medulloblastoma in rodents. We have also observed, in preliminary studies, that both murine and human NSCs (the same cells that are drawn to intracranial tumors) bear CXCR4 receptors. We have found that NSCs will proliferate and migrate toward a source of SDF-1 in a Boyden chamber [E.Y. Snyder, J. Imitola, S. Kouhry, unpubl. data] and therefore we have hypothesized that one mechanism for NSC homing to intracranial gliomas is mediated by chemotaxis toward a source of SDF-. Ehtesham et al. recently showed that CXCR4 mediates glioma-topic migration of neural stem cells [124]. Malignant gliomas are among the most angiogenic of all solid tumors. Glioblastomas have elevated and deregulated expression of the highly proangiogenic cytokines vascular endothelial growth factor and basic fibroblast growth factor [82–84]. Preliminary data from J. Allport and R. Weissleder (in collaboration with us) suggest that human and murine NSCs, the same cells shown to home in to tumors, express cell surface receptors with preferential binding to tumor-derived endothelium over normal endothelium. This binding appears to be independent of CD49d binding to endothelial 4-integrin, suggesting other binding mechanisms [Allport, Weissleder, Snyder, unpubl. data]. NSCs can, therefore, migrate toward areas with active tumor-associated neoangiogenesis. Of the molecular markers selectively associated with angiogenic blood vessels in tumors, several are cell membrane-associated proteinases. With a powerful selection system in which circulating ligands that home to specific vascular beds in vivo are isolated from a phage display random peptide library, the Arap/Pasqualini team, with our participation, has identified aminopeptidase A (APA) to be upregulated and enzymatically active in new blood vessels of human tumor xenografts [126]. An APA-binding 9-amino acid peptide was identified and shown to bind specifically to APA, inhibit its enzymatic activity,

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suppress migration and proliferation of endothelial cells, inhibit in vitro and in vivo angiogenesis, home to tumor vasculature in vivo and inhibit growth of human xenografted tumors in vivo. Prolonged delivery of this and similar therapeutic peptide ligands might be achieved with NSCs. Boockvar et al. [85] observed that enhanced activation of the EGF-R, even in the absence of ligand binding, was associated with increased motility of NSCs. They showed that EGF-R activation triggered signaling events that resulted in cytoskeletal rearrangements that might explain this facilitated stem cell migration. EGF is a known mitogen in primary brain neoplasm. Over-expression of EGF and constitutive EGF-R signaling is a relatively common occurrence in high-grade glioma [86, 87]. In addition, human glioma motility and invasion is enhanced by the interaction between EGF and its receptor [88]. Relative cytokine concentration gradients in the tumor microenvironment could cause NSC migration in the direction of the EGF source i.e., location of brain tumor cells. Cellular adhesion is mediated by multiple cell surface molecules of which the cell surface glycoprotein CD44 is noteworthy because it plays an important role in normal cell migration and tumor metastasis [89]. Hyaluronic acid (HA) is a predominant extracellular component in the brain and is also a major ligand for CD44 [90]. CD44 expression is up-regulated in glioma and this has been shown to be instrumental in the migration and invasion of the neoplastic cell [91–93]. Interestingly, Delpech et al. [94] showed over-expression of HA by glioma cells, thereby creating a favorable substrate environment for glioma motility. Preliminary studies also have demonstrated the expression of CD44 in murine NSC clone C17.2 [Allport, Snyder, Weissleder, unpubl. observations]. CD44 expression appears to be regulated by the developmentally crucial cytokine leukemic inhibitory factor in human NSCs [95]. Therefore, gliomatropism exhibited by NSCs could be, in part, mediated by a CD44-HA interaction, especially in the HA-rich environment of the tumor. Recent preliminary evidence has suggested a role for NOGO-A in glioma migration [125]. NOGO-A, along with myelin-associated glycoprotein and oligodendrocyte myelin glycoprotein, binds to the common cognate receptor NOGO receptor resulting in inhibition of axonal regeneration. The inhibitory antibody IN-1 partially restores axonal regeneration in animal models [96]. Recently, a NOGO-A knockout mouse model was demonstrated to have enhanced nerve regeneration post experimental trauma [97], although results from other NOGO-A knock out mice were contradictory, suggesting possible redundancy (given the other two ligands for NOGO receptor) [98]. However, there do not appear to be deleterious effects on neural development in the NOGO-A knockout mice. It is not certain whether NOGO-A or its fellow ligands play a role in mediating glioma formation or, more provocatively, gliomatropism by NSCs.

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Smart Imaging of Intracranial Neoplasms

Instead of utilizing NSCs as carriers of therapeutic packages, Weissleder and Ntziachristos [99] have lead research in the ‘smart imaging’ of brain neoplasms using specially tagged NSCs. Tang et al. [100] demonstrated the in vivo gliomatropic migration of NSCs (clone C17.2) stably transfected with the firefly luciferase gene in a rodent model. Using bioluminescence imaging of living mice in real time, the authors showed persistent luciferase activity up to 4 weeks postinoculation of luciferase-expressing NSCs. While this expression bodes well for the persistent expression of therapeutic genes in similar situations, the technique had the further advantage of permitting the real time tracking of migratory NSCs, offering powerful insight into the in vivo behavior of NSCs in living animals. C17.2 luciferase-tagged NSCs could be visualized tracking from one side of the cerebrum to the other in order to home in and appose the implanted tumor. MRI, a commonly used imaging modality for brain tumors in humans, also can be scaled down for use in experimental animals as small as a mouse. Lewin et al. [101] labeled the same murine NSCs described above (clone C17.2) with tat peptide-derivatized magnetic nanoparticles, which renders the cells visible under MRI. Using this imaging technique the authors could track-off the movement of as few as one thousand labeled NSCs of in living rodents. One can foresee the use of MRI or bioluminescence to follow the migration of labeled therapeutic NSCs in brain tumor patients to monitor both safety and efficacy.

Future Directions and Application of NSCs in Brain Tumors

The Therapeutic Package: New Candidates NSCs offer a way to deliver a broad range of therapeutic molecules to intracranial glioma cells in a specific fashion. Immunomodulatory factors hold particular promise for use with stem cells. Ehtesham et al. [67, 68] have shown successful and efficacious delivery of IL-12 and TRAIL to implanted brain tumors. Other cytokines with demonstrated promise in the treatment of glioma could similarly be delivered by NSCs [102]. For example, IL-24 or melanoma differentiation-associated-7 induces expression of the pro-apoptotic protein BAX and suppresses the growth of glioma cells [103]. Aoki et al. [104] showed that interleukin-7 reduced the tumorigenicity of glioma cells in vivo via a T cell-mediated mechanism. Parker et al. [105] also showed immune-mediated suppression of in vivo glioma with IL-12 delivered by a herpes simplex virus vector. Utilizing NSCs as gliomatropic cytokine/drug delivery vehicles would likely significantly increase the therapeutic potential of these agents.

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NSCs can also prolong the bioavailability of a therapeutic agent in vivo. For example, the potent anti-angiogenic molecule endostatin has a relatively short half-life in vivo and intracranial bioavailability is restricted by the bloodbrain barrier. Encapsulation of cells from an embryonal kidney cell line engineered to produce endostatin encased in alginate gel beads significantly prolonged the sustained release of the protein intracranially resulting in measurable reduction in tumor-related neoangiogenesis [106]. NSCs transfected with the endostatin gene or genes for other anti-angiogenic molecules can likely serve a similar role but with the added advantage of a migratory delivery system that is specific for glioma and tumor-related endothelium, that is transcriptionally and translationally active for several weeks postintroduction, and that can track invading cells throughout the brain that might well set up new tumor foci. Herrlinger et al. [61] reported the use of NSCs in the focused delivery of engineered herpes simplex virus expressing thymidine kinase into implanted brain tumors. The authors delayed cell cycle entry and hence viral replication in the NSCs with mimosine prior to transplantation into the tumor-bearing rodent host. This, however, had no deleterious effects on the migratory potential of the NSCs. Once the cytostatic effect of mimosine had expired, viral production reinitiated in close proximity to the tumor bulk as well as escaping glioma cells with effective tumor destruction following the administration of gangciclovir to trigger the thymidine kinase-mediated cytolysis. Oncolytic viruses have recently garnered much attention in neuro-oncology, and are discussed elsewhere in this volume [107]. Multiple naturally occurring as well as engineered viruses are being considered for glioma-specific treatment [108]. Taking advantage of the prevalence of ras mutations in brain tumors, reovirus has been shown to effect selective oncolysis of glioma and medulloblastoma cells [109, 110]. Clinical trials are ongoing to assess the effectiveness of the virus in patients with GBM. Advances in the understanding of the molecular genetics of vesicular stomatitis virus and isolation of unique attenuated strains has recently propelled the field of tumor oncolytic therapy forward [111]. VSV, AV1, and AV2 effect tumor-specific lysis by taking advantage of the mutated abrogation of the interferon / signaling pathway in tumor cells. The attenuated vesicular stomatitis virus strains have favorable therapeutic indices and demonstrate the selective killing of cancer cells (including CNS tumors) from the NCI-60 tumor cell panel. Can we perhaps exploit NSCs as carriers of these lethal viruses and enhance their delivery to infiltrative glioma cells? This concept is intriguing and has yet to be attempted. Assuming NSCs have none of the genetic mutations that endow the glioma cells with growth advantage (but also susceptibility to viral-mediated oncolysis), then NSCs should be able to survive viral infection and deliver the

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lethal package to glioma cells. It is beyond the scope of this review to provide a comprehensive and up-to-date review on novel glioma therapeutics, but one can appreciate that NSCs offer a way to deliver therapeutically relevant molecules and drugs effectively to diffuse glioma in vivo. Biopolymer Scaffolds Biodegradable polymer scaffolds, composed of poly-glycolic acid or poly lactic acid or combinations of the two, offer a ‘backbone’ for the growth, differentiation, and three-dimensional structure of tissues from stem cells [112]. They also permit the stem cells and degenerating CNS environment to have prolonged contact and communication with each other. Furthermore, they may fix in space various molecules emanating both from the stem and from the tissue such that the exposure of the various cells and tissue to each of these is protracted. Teng et al. [113] used NSCs supported by a poly-glycolic acid scaffold to promote the functional recovery of severely spinal cord injured adult rats. Park et al. [25] used NSCs supported by a poly-glycolic acid scaffold to promote the growth of new, reinnervated and revascularized cerebral parenchyma within a region that otherwise would have undergone cystic cavity formation following severe hypoxic-ischemic injury and its resultant liquefactive necrosis. The scaffold, acting as a template for the implanted NSCs as well as a platform for enhancing stem cell-host interactions, significantly aided the reconstitution of the injured brain. One could imagine that NSCs engineered variously to express anti-glioma and/or regenerative cytokines could be seeded onto biosynthetic scaffolds and placed within a tumor resection cavity to enhance killing of residual tumor cells and facilitate brain repair. This is somewhat analogous to the use of carmustine-impregnated biodegradable polymer wafers or GLIADEL in brain tumor, as discussed in this volume, but is significantly more biologically dynamic and adaptive. Tagging stem cells with ‘smart imaging’ agents will allow for their monitoring in the patient post-tumor resection or biopsy. In fact, this could be used as a sensitive method to assess the amount of residual tumor and the degree of recurrence. Image Guidance and Enhanced Photodynamic Therapy Shinoda et al. [114] showed recently that vital staining of glioma with fluorescein sodium introduced intravenously during dural opening at craniotomy helped to define the stained tumor and facilitated radiographically guided total resection. Photosensitizers preferentially accumulate in neoplastic cells due to a variety of mechanisms [115]. Light at an appropriate excitation wavelength when shone on the tissue results in fluorescence within areas of photosensitizer accumulation. Photodynamic therapy (PDT) using photosensitizers already has a long history in brain tumor therapy [116, 117].

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PDT for brain tumor is still not widely used clinically for several reasons. Even though PDT offers a binary approach to relatively preferential drug uptake and photic activation, and therefore constitutes a relatively safe treatment modality, systemic side effects, such as skin photosensitivity, still persist. Yet more ominous is the problem with PDT-induced cerebral edema. These problems are exacerbated by the uptake of the photosensitizer by nonneoplastic tissues [118]. The delivery of photosensitizers could be made much more selective and efficient for the detection and destruction of glioma by exploiting the exquisite gliomatropic effect of NSCs. For example, NSCs could be preloaded with a photosensitizer or smart imaging agent (see above) prior to intracavitary implantation postresection or even intravenous injection [17]. Delivery of the photosensitizer could be made much more selective and efficient for the detection and destruction of glioma by exploiting the exquisite gliomatropic effect of NSCs. Differentiating Radiation Necrosis from Tumor Recurrence Radiation therapy of brain tumors is usually followed by the recurrence of tumor. However, tumor recurrence due to treatment failure can be confused with radiation-induced neoplasm or radiation necrosis [119]. These three entities have similar radiographical appearances and can often present a diagnostic dilemma. Rock et al. [120] recently confirmed the utility of magnetic resonance spectroscopy in differentiating pure radiation necrosis from pure tumor recurrence. The authors, however, had trouble distinguishing tissues with intermediate amounts of tumor and necrosis. Could NSCs discriminate between recurrences of primary tumor, radiation-induced neoplasm and radiation necrosis? There could be differences in the migratory pattern of NSCs to each of the above intracranial pathologies that could be made apparent with specially tagged NSCs and appropriate imaging techniques. This concept is awaiting further experimental proof. Brain Reconstruction Post-Therapy Glioma growth distorts and destroys surrounding normal neural structures. Patients who survive the initial treatment are often left with permanent neurological deficits with significant effects on their activities of daily living. This damage to adjacent structures has long-term clinical consequences especially in rare cases of curable gliomas such as pediatric pilocytic astrocytomas. NSCs might repopulate areas of injured brain, differentiate on cue in response to local environmental signals, and promote the reformation of functional interactions with the host [121]. NSCs, therefore, might have dual roles in glioma therapy: (1) delivery of anti-tumor agents to glioma cells and (2) repair of the injured brain [122]. Park et al. [25] indeed showed that injected NSCs promote and interact with endogenous repair pathways to facilitate brain repair.

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Clinical Applications

How might the biology described above someday be applied in actual clinical situations? Some scenarios are envisioned below (see also tables 1, 2.)

Gliomatosis cerebri Consider a young man with a suspected radiological diagnosis of gliomatosis cerebri who is about to undergo a biopsy and/or resection [123]. A stable, readily available line of NSCs has been transfected with genes for anti-tumor and prodifferentiation cytokines, perhaps tailored to the specific tumor in this patient. These altered NSCs are administered to the patient. The engineered cells are injected either intraventricularly or into the vicinity of the tumor following biopsy. The injected cells will then target the main lesion as well as infiltrating glioma cells followed by the delivery of the therapeutic payload. There need not be concern for immunological matching of the NSCs to the patient because immune rejection of the stem cells would probably enhance their tumor-killing ability, especially if already within the tumor mass or juxtaposed to tumor cells. The physician has the choice of transducing the cells with a therapeutic gene or tagging the cells with smartimaging agents, or both. Migration of the cells, in this way, could be followed by either bioluminescence or MRI. NSCs might also discover unsuspected small islands of tumor cells that may already have left the main tumor mass and migrated to more distant locations. The patient may undergo multiple treatments with NSCs carrying different therapeutic packages. For example, initial delivery of proapoptotic genes to effect tumor destruction can be followed by immunomodulatory genes that can elicit persistent anti-glioma immunity. The most compelling transplantation work to date has been performed with NSCs and it remains unclear whether stem cells from other organs, even if they were somehow directed to transdifferentiate into NSCs (a controversial notion), would be equally effective in tracking and destroying very infiltrative brain tumors. If they were, it is also conceivable that such stem cells could be collected from the patient (e.g., via apheresis to harvest circulating stem cells, an inefficient process, or via bone marrow aspiration to obtain hematopoietic/mesenchymal stem cells) that might then be purified and expanded ex vivo with a specially tailored cytokine cocktails and custom extracellular matrix [123]. In fact, ex vivo stimulation of the stem cells could be tailored specifically to the individual patient depending on his/her age and the robustness of the harvested stem cells. At present, only NSCs seem to be on the brink of being a promising clinical application.

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GBM Consider a woman in her late fifties who presents with a contrast-enhancing left frontal mass lesion with significant mass effect and edema. The lesion is consistent with a high-grade intra-axial tumor. NSCs undergo ex vivo expansion, therapeutic packages are loaded into the cells, and the engineered cells are seeded in a biopolymer matrix. The patient undergoes awake craniotomy for surgical debulking of the tumor with the aid of advanced neuronavigation and intraoperative neurophysiological monitoring followed by placement of the cell-laden biopolymer matrix into the resection cavity. NSCs within the matrix will seek out and destroy glioma cells; in addition, using the matrix as a physical scaffold, the NSCs will start to initiate repair pathways in conjunction with endogenous stem cells. The patient is released from the hospital 2 days after the procedure. Will We Truly See These Approaches Used in the Clinic? One has to admit that the above two scenarios seem ‘far-fetched,’ but many of the technical elements are already in place. Carefully performed preclinical and then clinical trials are essential before realization of the above pictures becomes routine practice. Initial trials should focus on confirming the in vivo gliomatropic migration of NSCs in patients with high-grade terminal brain tumors such as GBM and pediatric diffuse intrinsic glioma. Safety of NSCs should also be rigorously addressed in these trials before the introduction of therapeutic genes. Realistically, many technical hurdles have to be surpassed before clinical translation ensues. Nevertheless, NSCs offer an intriguing and promising therapeutic option. Importantly, they should be viewed not as a substitute for other approaches but as one additional weapon in a multifaceted, biologically rational arsenal for attacking brain tumors. The unique biology of the stem cell may permit them to be the ‘glue’ that holds together and helps coordinate the application of these many approaches.

References 1 2 3

4 5

Ryder EF, Snyder EY, Cepko CL: Establishment and characterization of multipotent neural cell lines using retrovirus vector-mediated oncogene transfer. J Neurobiol 1990;21:356–375. Renfranz PJ, Cunningham MG, McKay RD: Region-specific differentiation of the hippocampal stem cell line HiB5 upon implantation into the developing mammalian brain. Cell 1991;66:713–729. Snyder EY, Deitcher DL, Walsh C, Arnold-Aldea S, Hartwieg EA, Cepko CL: Multipotent neural cell lines can engraft and participate in development of mouse cerebellum. Cell 1992;68: 33–51. Reynolds BA, Weiss S: Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 1992;255:1707–1710. Snyder EY, Yoon C, Flax JD, Macklis JD: Multipotent neural precursors can differentiate toward replacement of neurons undergoing targeted apoptotic degeneration in adult mouse neocortex. Proc Natl Acad Sci USA 1997;94:11663–11668.

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Rosario CM, Yandava BD, Kosaras B, Zurakowski D, Sidman RL, Snyder EY: Differentiation of engrafted multipotent neural progenitors towards replacement of missing granule neurons in meander tail cerebellum may help determine the locus of mutant gene action. Development 1997; 124:4213–4224. Park KI, Liu S, Flax JD, Nissim S, Stieg PE, Snyder EY: Transplantation of neural progenitor and stem cells: Developmental insights may suggest new therapies for spinal cord and other CNS dysfunction. J Neurotrauma 1999;16:675–687. Zlomanczuk P, Mrugala M, de la Iglesia HO, et al: Transplanted clonal neural stem-like cells respond to remote photic stimulation following incorporation within the suprachiasmatic nucleus. Exp Neurol 2002;174:162–168. Snyder EY, Taylor RM, Wolfe JH: Neural progenitor cell engraftment corrects lysosomal storage throughout the MPS VII mouse brain. Nature 1995;374:367–370. Ourednik V, Ourednik J, Park KI, Snyder EY: Neural stem cells – A versatile tool for cell replacement and gene therapy in the central nervous system. Clin Genet 1999;56:267–278. Yandava BD, Billinghurst LL, Snyder EY: ‘Global’ cell replacement is feasible via neural stem cell transplantation: Evidence from the dysmyelinated shiverer mouse brain. Proc Natl Acad Sci USA 1999;96:7029–7034. Auerbach JM, Eiden MV, McKay RD: Transplanted CNS stem cells form functional synapses in vivo. Eur J Neurosci 2000;12:1696–1704. Park KI, Ourednik J, Ourednik V, et al: Global gene and cell replacement strategies via stem cells. Gene Ther 2002;9:613–624. Thomas ED: A history of haemopoietic cell transplantation. Br J Haematol 1999;105:330–339. Burt RK, Traynor AE, Craig R, Marmont AM: The promise of hematopoietic stem cell transplantation for autoimmune diseases. Bone Marrow Transplant 2003;31:521–524. Flax JD, Aurora S, Yang C, et al: Engraftable human neural stem cells respond to developmental cues, replace neurons, and express foreign genes. Nat Biotechnol 1998;16:1033–1039. Aboody KS, Brown A, Rainov NG, et al: Neural stem cells display extensive tropism for pathology in adult brain: Evidence from intracranial gliomas. Proc Natl Acad Sci USA 2000;97: 12846–12851. Rosenthal N: Prometheus’s vulture and the stem-cell promise. N Engl J Med 2003;349: 267–274. Hochedlinger K, Jaenisch R: Nuclear transplantation, embryonic stem cells, and the potential for cell therapy. N Engl J Med 2003;349:275–286. McKay R: Stem cells in the central nervous system. Science 1997;276:66–71. Gage FH: Mammalian neural stem cells. Science 2000;287:1433–1438. Vescovi AL, Snyder EY: Establishment and properties of neural stem cell clones: Plasticity in vitro and in vivo. Brain Pathol 1999;9:569–598. Pincus DW, Goodman RR, Fraser RA, Nedergaard M, Goldman SA: Neural stem and progenitor cells: A strategy for repair. Neurosurgery 1998;42:858–867. Ourednik J, Ourednik V, Lynch WP, Schachner M, Snyder EY: Neural stem cells display an inherent mechanism for rescuing dysfunctional neurons. Nat Biotechnol 2002;20:1103–1110. Park KI, Teng YD, Snyder EY: The injured brain interacts reciprocally with neural stem cells supported by scaffolds to reconstitute lost tissue. Nat Biotechnol 2002;20:1111–1117. Riess P, Zhang C, Saatman KE, et al: Transplanted neural stem cells survive, differentiate, and improve neurological motor function after experimental traumatic brain injury. Neurosurgery 2002;51:1043–1052; discussion 1052–1054. Burns MJ, Weiss W: Targeted therapy of brain tumors utilizing neural stem and cells. Front Biosci 2003;8:e228–e234. Yip S, Aboody KS, Burns M, et al: Neural stem cell biology may be well suited for therapies. Cancer J 2003;9:189–204. Gottlieb DI: Large-scale sources of neural stem cells. Annu Rev Neurosci 2002;25:381–407. Reubinoff BE, Itsykson P, Turetsky T, et al: Neural progenitors from human embryonic stem cells. Nat Biotechnol 2001;19:1134–1140. Kabos P, Ehtesham M, Kabosova A, Black KL, Yu JS: Generation of neural progenitor cells from whole adult bone marrow. Exp Neurol 2002;178:288–293.

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65 66

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72 73 74 75

76

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80 81 82 83

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84 Chan AS, Leung SY, Wong MP, et al: Expression of vascular endothelial growth factor and its receptors in the anaplastic progression of astrocytoma, oligodendroglioma, and ependymoma. Am J Surg Pathol 1998;22:816–826. 85 Boockvar JA, Kapitonov D, Kapoor G, et al: Constitutive EGFR signaling confers a motile phenotype to neural stem cells. Mol and Cell Neurosci 2003;in press. 86 Feldkamp MM, Lau N, Guha A: Signal transduction pathways and their relevance in human astrocytomas. J Neurooncol 1997;35:223–248. 87 Dunn IF, Heese O, Black PM: Growth factors in glioma angiogenesis: FGFs, PDGF, EGF, and TGFs. J Neurooncol 2000;50:121–137. 88 Chicoine MR, Silbergeld DL: Mitogens as motogens. J Neurooncol 1997;35:249–257. 89 Goodison S, Urquidi V, Tarin D: CD44 cell adhesion molecules. Mol Pathol 1999;52:189–196. 90 Ruoslahti E: Brain extracellular matrix. Glycobiology 1996;6:489–492. 91 Merzak A, Koocheckpour S, Pilkington GJ: CD44 mediates human glioma cell adhesion and invasion in vitro. Cancer Res 1994;54:3988–3992. 92 Akiyama Y, Jung S, Salhia B, et al: Hyaluronate receptors mediating glioma cell migration and proliferation. J Neurooncol 2001;53:115–127. 93 Bouvier-Labit C, Liprandi A, Monti G, Pellissier JF, Figarella-Branger D: CD44H is expressed by cells of the oligodendrocyte oligodendrogliomas in humans. J Neurooncol 2002;60:127–134. 94 Delpech B, Maingonnat C, Girard N, et al: Hyaluronan and hyaluronectin in the extracellular matrix of human brain tumour stroma. Eur J Cancer 1993;29A:1012–1017. 95 Wright LS, Li J, Caldwell MA, Wallace K, Johnson JA, Svendsen CN: Gene expression in human neural stem cells: Effects of leukemia inhibitory factor. J Neurochem 2003;86:179–195. 96 Filbin MT: Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS. Nat Rev Neurosci 2003;4:703–713. 97 Simonen M, Pedersen V, Weinmann O, et al: Systemic deletion of the myelin-associated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury. Neuron 2003;38:201–211. 98 Woolf CJ: No Nogo: Now where to go? Neuron 2003;38:153–156. 99 Weissleder R, Ntziachristos V: Shedding light onto live molecular targets. Nat Med 2003;9:123–128. 100 Tang Y, Shah K, Messerli SM, Snyder E, Breakefield X, Weissleder R: In vivo tracking of neural progenitor cell. Hum Gene Ther 2003;14:1247–1254. 101 Lewin M, Carlesso N, Tung CH, et al: Tat peptide-derivatized magnetic nanoparticles allow in recovery of progenitor cells. Nat Biotechnol 2000;18:410–414. 102 Marras C, Mendola C, Legnani FG, DiMeco F: Immunotherapy and biological modifiers for the treatment of malignant brain tumors. Curr Opin Oncol 2003;15:204–208. 103 Yacoub A, Mitchell C, Lister A, et al: Melanoma differentiation-associated 7 (interleukin 24) inhibits growth and enhances radiosensitivity of glioma cells in vitro and in vivo. Clin Cancer Res 2003;9:3272–3281. 104 Aoki T, Tashiro K, Miyatake S, et al: Expression of murine interleukin 7 in a murine glioma cell line results in reduced tumorigenicity in vivo. Proc Natl Acad Sci USA 1992;89:3850–3854. 105 Parker JN, Gillespie GY, Love CE, Randall S, Whitley RJ, Markert JM: Engineered herpes simplex virus expressing IL-12 in the treatment of experimental murine brain tumors. Proc Natl Acad Sci USA 2000;97:2208–2213. 106 Bjerkvig R, Read TA, Vajkoczy P, et al: Cell therapy using encapsulated cells producing endostatin. Acta Neurochir Suppl 2003;88:137–141. 107 Chiocca EA: Oncolytic viruses. Nat Rev Cancer 2002;2:938–950. 108 Rainov NG, Ren H: Oncolytic viruses for treatment of malignant brain tumours. Acta Neurochir Suppl 2003;88:113–123. 109 Wilcox ME, Yang W, Senger D, et al: Reovirus as an oncolytic agent against experimental human malignant gliomas. J Natl Cancer Inst 2001;93:903–912. 110 Yang WQ, Senger D, Muzik H, et al: Reovirus prolongs survival and reduces the frequency of leptomeningeal metastases from medulloblastoma. Cancer Res 2003;63:3162–3172. 111 Stojdl DF, Lichty BD, tenOever BR, Paterson JM, Power AT: VSV strains with defects in their ability to shutdown innate immunity are potent systemic anti-cancer agents. Cancer Cell 2003;4: 263–275.

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112 113

114 115 116 117 118 119 120

121 122 123

124

125

126

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Dr. Evan Y. Snyder, MD, PhD Department of Neurology, Harvard Medical School Harvard Institutes of Medicine, Beth Israel-Deaconess Medical Center & Children’s Hospital, Boston, MA 02115 (USA) Tel. 1 858 646 3158, Fax 1 858 713 6273, E-Mail [email protected]

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Author Index

Anderson, D.G. 5 Anderson, W.F. XI Andrews, D.W. 499 Bankiewicz, K. 213 Barone, F.C. 336 Bomback, D.A. 52 Boulis, N.M. 65 Brady, R.O. 202 Brady, R.O. Jr. 202 Brem, H. 458 Broggi, G. 270 Burchiel, K. 284 Bussone, G. 270 Castro, M.G. 580 Celix, J. 169 Chaudhary, P. 284 Chiocca, E.A. 557 Couldwell, W. 124 Daadi, M. 213 Dichter, M. 169 Ehtesham, M. 580 Fink, D. 322 Flomenberg, P. 499 Franzini, A. 270 Freese, A. 1 Gilbertson, L.G. 37 Giles, J. 154 Glorioso, J.C. 322 Gouras, G.K. 258 Goverdhana, S. 580

Grauer, J.N. 52 Greengold, D. 580 Gullans, S.R. 246 Hadaczek, P. 213 Harshyne, L. 499 Heistad, D.D. 413 House, P.A. 124 Hu, J. 580 Iwanami, A. 104 Jensen, R.V. 246 Jovel, N. 580 Janson, C. 1 Kaneko, S. 104 Kang, J.D. 37 Kapoor, G.S. 521 Kim, J. 37 Kondziolka, D. 439 Kurpad, S.N. 30 Lamfers, M.L.M. 557 Leo, B.M. 5 Leone, M. 270 Leone, P. 1 Lifshutz, J. 30 Liu, J.K. 65 Lowenstein, P.R. 580 Lunsford, L.D. IX Macdonald, R.L. 377 Mata, M. 322

Nakamura, M. 104 Niranjan, A. 439 Ogawa, Y. 104 Okano, H. 104 Okano, H.J. 104 O’Rourke, D.M. 521 Rao, G. 124 Read, S.J. 336 Samulski, R.J. 154 Sawamoto, K. 104 Scherzer, C.R. 246 Schumacher, J.M. 146 Sheehan, J. 439 Sidman, R.L. 624 Simeone, F.A. 1 Snyder, E.Y. 624 Steinmetz, M.P. 65 Telfeian, A. 169 Toyama, Y. 104 Visted, T. 557 Walker, M.H. 5 Wang, P.P. 458 Watanabe, Y. 413 Xiong, W. 580 Yip, S. 624 Yu, J. 580 Yuan, X. 580 645

Subject Index

Acid-sensing ion channels, pain reception 290, 291 Adeno-associated virus (AAV) advantages as gene therapy vector 154 brain gene transfer 127, 160–163 Canavan’s disease gene transfer 164, 165 clinical-grade vector production 155, 156 crystal structure 158–160 epilepsy gene transfer 163, 164 genome and proteins 154, 155 global gene delivery 164, 165 Huntington’s disease gene transfer 163 Parkinson’s disease gene therapy vector 164, 221–226 Parkinson’s disease gene transfer 164 parvovirus homology 159, 160 pituitary adenoma gene therapy vector 595, 596 serotypes for gene transfer 157, 158 spinal cord injury gene therapy vector 83, 88, 92 Adenovirus intervertebral disc degeneration vector 40, 41 oncolytic viral therapy for glioma advantages 561, 562 E1 gene modifications 562, 563 tropism modification 564, 565 tumor-specific promoters for driving replication 563, 564

pain gene therapy vector 326 Parkinson’s disease gene therapy vector 216, 218, 219 pituitary adenoma gene therapy vector 589, 591, 593–595, 611–613 replication 561 spinal cord injury gene therapy vector 82, 83, 85–88, 90–92 Adrenergic receptors, pain reception 290, 291 Adriamycin, polymeric drug delivery systems 479, 480 Akt glioma progression signaling 531–533 small molecule inhibitors 541 Alzheimer’s disease (AD) ␤-amyloid mutations, deposition, and therapeutic targeting 259–261, 266, 267 cerebrospinal-fluid-based therapies 258, 259, 267 economic impact 258 epidemiology 258 gene therapy prospects 265, 266 management antioxidants 265 estrogen replacement therapy studies 263, 264 excitotoxicity inhibitors 265 metal chelation therapy 264, 265 nonsteroidal anti-inflammatory drugs 264

646

protease augmentation 265 secretase inhibitors 261, 262 statins in protection 263 vaccination against ␤-amyloid 263 treatment difficulty 125 ␥-Aminobutyric acid (GABA) modulation in epilepsy treatment 171, 172 nociception 295, 296 receptor expression in epilepsy 197 Aminopeptidase A, upregulation in glioma angiogenesis 632, 633 Amyloidosis ␤-amyloid mutations, deposition, and therapeutic targeting in Alzheimer’s disease 259–261, 266, 267 intracerebral hemorrhage risks 378–380 Amyotrophic lateral sclerosis, treatment difficulty 125 Angiogenesis aminopeptidase A upregulation 632, 633 endogenous inhibitors 543 gene therapy with inhibitors 603, 604 glioma 632 inhibition therapy in glioma 542–544 inhibitor delivery using polymeric drug delivery systems 478 therapeutic angiogenesis 426–428, 450 Angiotensin-converting enzyme (ACE), gene mutations in stroke 340 Antigen-presenting cells (APCs) glioma immune response 500, 501, 509 immunotherapy manipulation 512, 513 tumor vaccines 539 Antisense gene therapy constructs 126 pain management 309–311 Apolipoprotein E, gene mutations in stroke 340, 379 Apoptosis hemorrhagic stroke 389 intervertebral disc degeneration 19, 32, 35 ischemic stroke 389, 446, 447 Arteriovenous malformation (AVM), intracerebral hemorrhage risks 380, 381 Atrial natriuretic peptide (ANP), gene mutations in stroke 341–343

Subject Index

Autoantibodies, epilepsy 177–179 Autosomal dominant polycystic kidney disease, cerebral aneurysm 383, 384 B7, gene therapy for immune response enhancement in cancer 607 Back pain economic impact 5 epidemiology 5, 30 Bcl-2 ischemic stroke gene therapy 448 spinal cord injury gene therapy 85 Bcl-XL, spinal cord injury gene therapy 85 O6-Benzylguanine, polymeric drug delivery systems 472 Bleomycin, polymeric drug delivery systems 483, 484 Blood-brain barrier (BBB) antiepileptic drug delivery 183, 184 bypassing for drug delivery 460, 461 disruption for drug delivery 459, 460 drug permeability 459, 460 Bone morphogenetic proteins (BMPs) carotid artery stenosis gene therapy with BMP-2 42 discovery 53 polymeric drug delivery systems 485, 486 receptors 54 regulation 54 spinal fusion carriers 61 clinical studies BMP-2 57–59 BMP-7 59 demineralized bone matrix 59 expression 54 gene therapy prospects 60–62 preclinical studies BMP-2 55, 56 BMP-7 56, 57 demineralized bone matrix 57 preparations for use 54, 55 Bradykinin (BK), pain mediation 300 Brain convection-enhanced delivery of drugs 127, 128 gene transfer vectors 127, 160–163

647

Brain (continued) neuroprosthetic therapies 131, 133, 134 pain gene therapy 333 stem cell therapy 129 stroke, see Hemorrhagic stroke; Ischemic stroke Brain-derived neurotrophic factor (BDNF) brain graft neuroprotection 446 spinal cord injury gene therapy 84 Brain tumors, see also specific tumors classification 458 polymeric drug delivery systems, see Polymeric drug delivery systems surgical resection 458 Buthionine sulfoximine (BSO), polymeric drug delivery systems 479 CADASIL, gene mutations and stroke 338–340 Calcitonin-gene-related peptide (CGRP) cerebral vasospasm gene therapy after hemorrhagic stroke 419 pain mediation 298 Calcium flux, smooth muscle contraction and relaxation in hemorrhagic stroke 391, 394 Camptothecin, polymeric drug delivery systems 473, 474 Canavan’s disease, adeno-associated virus for gene transfer 164, 165 Cannabinoid receptors, pain reception 289 Capsaicin receptors, pain reception 288, 289 Carbamazepine, mechanism of action 171 Carboplatin, polymeric drug delivery systems 482, 483 Carboxypeptidase G2, prodrug combination in gene therapy 601 Carmustine (BCNU), delivery using polyanhydride fibers in glioma development 464, 465 initial therapy trials 470–472 preclinical trials 465–467 recurrent glioma trials 467–469 Carotid artery stenosis, gene therapy targets 422–427 CD44, glioma expression 633 Cell transplantation therapy, see specific cells and diseases

Subject Index

Cerebrospinal fluid infusion, drug delivery 461 Cerebrovascular gene therapy, see Hemorrhagic stroke; Ischemic stroke Cholecystokinin (CCK), pain mediation 299, 300 CI1033, epidermal growth factor receptor inhibition 541 Cisplatin, polymeric drug delivery systems 483 Cluster headache (CH) diagnostic criteria 271 hypothalamic deep brain stimulation electrode implantation in first patient 274 outcomes 277–279 patient selection criteria 274–276, 279 prospects 280, 282 rationale 273 surgery 276, 277 pathophysiology 271–273, 279 pharmacotherapy 273 trigeminal nerve surgery 273 Complement, activation in hemorrhagic-stroke-induced edema 388 Computed tomography (CT), intraoperative 134 Convection-enhanced delivery (CED), drug delivery to brain 127, 128, 486, 487 Cushing’s disease, animal models 610 Cyclophosphamide, polymeric drug delivery systems 479 Cytosine deaminase, prodrug combination in gene therapy 601, 629, 630 Deep brain stimulation (DBS) cluster headache electrode implantation in first patient 274 outcomes 277–279 patient selection criteria 274–276, 279 prospects 280, 282 rationale 273 surgery 276, 277 epilepsy management 190, 191

648

historical perspective 270 indications and mechanisms of action 270, 271, 279, 280 Degenerative disc disease, see Intervertebral disc degeneration Dementia, see also Alzheimer’s disease differential diagnosis 258, 259 Dexamethasone, polymeric drug delivery systems 483 Differential display hemorrhagic stroke vasospasm differential gene expression studies 390, 391 ischemic stroke differential gene expression studies 349 Disc degeneration, see Intervertebral disc degeneration DNA microarray disease gene identification biological validation 249 error minimization 248, 249 primary screen 247 secondary screen 247, 248 shotgun microarrays 248 epilepsy gene expression and pharmacogenomics 196–199 hemorrhagic stroke vasospasm differential gene expression studies 390, 391 ischemic stroke differential gene expression studies 357, 358 neurodegeneration modifying gene analysis candidate modifier screen in animal models 252, 253 cellular profiling 250, 251 extraneuronal profiling 251, 252 linkage analysis 253, 255 neuroprotective therapy prospects 255 regional profiling 250 selective vulnerability profiles 249, 250 principles 246, 247 Ehlers-Danlos syndrome, cerebral aneurysm 383, 384 EKB-569, epidermal growth factor receptor inhibition 541 Endoglin, gene mutations in stroke 379

Subject Index

Endostatin, angiogenesis inhibition 543, 544, 604 Endothelin cerebral vasospasm antisense gene therapy after hemorrhagic stroke 420 endothelial dysfunction in hemorrhagic stroke 396, 397 Enkephalin, proenkephalin gene therapy for pain 325–333 Enzyme replacement therapy (ERT) gene therapy 209, 210 historical perspective 204–206 intracerebral injection of glucocerebrosidase 207–209 overview 202 sphingolipid storage disorders 202, 204 substrate depletion 206, 207 Epidermal growth factor (EGF), intervertebral disc degeneration treatment prospects 22, 35 Epidermal growth factor receptor (EGFR) glioma expression 633 glioma progression signaling 522–524 monoclonal antibody inhibition 538 small molecule inhibitors 540, 541 Epilepsy adeno-associated virus for gene transfer 163, 164 autoantibodies 177–179 cell transplantation therapy 191, 192 deep brain stimulation 190, 191 DNA microarray analysis of gene expression and pharmacogenomics 196–199 epidemiology 169 gamma knife radiosurgery 189 gene therapy prospects 192–195 ion channel defects and therapeutic targeting 180, 181 ketogenic diet therapy 185, 186 neuroprotection in prevention 179, 180 pharmacotherapy carbamazepine 171 drug delivery advances 181–185 drug discovery 172, 173 efficacy 169, 170 ethosuximide 172

649

Epilepsy (continued) pharmacotherapy (continued) gabapentin 172, 173 mechanisms of action 170, 171 pharmacogenetics and drug metabolism 174, 175, 196 phenobarbital 172 phenytoin 171 prospects 174 resistance mechanisms 175–177 tiagabine 173 valproic acid 171, 172 vigabatrin 173 surgical resection of lesions 186–188 vagus nerve stimulation 188 ErbB, glioma progression signaling 524–527 Ethosuximide, mechanism of action 172 Factor V, gene mutations in stroke 340 Farnesyltransferase inhibitors, Ras signaling inhibition 542 Fas ligand, gene therapy for apoptosis induction 604, 605 Fibrinogen, gene mutations in stroke 340 Fibromuscular dysplasia, cerebral aneurysm 383 5-Fluorouracil cytosine deaminase combination in gene therapy 601, 629, 630 polymeric drug delivery systems 480, 481 Gabapentin, mechanism of action 172, 173 Galanin, pain mediation 299 Gamma knife radiosurgery, epilepsy management 189 Gaucher’s disease enzyme replacement therapy 204–209 gene therapy 209, 210 Gene chip, see DNA microarray Gene therapy, see specific diseases and genes Gerstmann-Sträussler-Scheinker disease, clinical features 379, 380 Gliadel, see Carmustine Glial-derived neurotrophic factor (GDNF) brain graft neuroprotection 446 ischemic stroke gene therapy 447 nerve repair role 129, 130

Subject Index

pain mediation 302, 303 spinal cord injury gene therapy 84, 90–93 Glioblastoma multiforme (GBM) gene therapy 126 neural stem cell applications 639 progression 521 survival 499, 627 treatment difficulty 124, 125, 499, 545 Glioma, see also Glioblastoma multiforme angiogenesis 632 angiogenesis inhibition therapy 542–544 carmustine delivery using polyanhydride fibers development 464, 465 initial therapy trials 470–472 preclinical trials 465–467 recurrent glioma trials 467–469 CD44 expression 633 epidermal growth factor receptor expression 633 grading 521 immune response antigen-presenting cells 500, 501, 509 dysfunction 506–509 T cells cytolytic activity assays 504 flow cytometry of cytokine production 504, 505 gene expression analysis 505, 506 proliferation assay 503, 504 types in response 501–503 immunotherapy monoclonal antibodies 537–539 prospects 509–513 tumor vaccines 539, 540 neural stem cells cell tagging for imaging 634 glioma-genesis 628, 629 homing and therapeutic applications 629–639 prospects for therapy 634–638 radiation necrosis differentiation from tumor recurrence 637 NOGO-A expression 633, 634 oncolytic viral therapy adenovirus advantages 561, 562

650

E1 gene modifications 562, 563 tropism modification 564, 565 tumor-specific promoters for driving replication 563, 564 herpes simplex virus advantages and limitations 558, 559 dual mutation vectors 560 ␥1-34.5 mutants 559, 560 thymidine kinase and ribonucleotide reductase mutants 559 tumor-specific promoters for driving replication 561 potency improvement strategies arming with therapeutic transgenes 567–569 combination therapy 569–571 immune response evasion 566, 567 principles 557, 558 prospects 571, 572 origins 628 photodynamic therapy 637 signaling in glioma-genesis Akt 531–533 epidermal growth factor/ErbB 524–527 mitogen-activated protein kinase 527–531 phosphatidylinositol 3-kinase 531–533 phospholipase C 533–535 platelet-derived growth factor 522–524 prospects for study 544, 545 receptor tyrosine kinases 521, 522 small molecule inhibitors 540–542 STATs 535 transforming growth factor-␣ 526, 527 Heat shock protein 70 (HSP70), expression after hemorrhagic stroke 386 Heme oxygenase cerebral vasospasm gene therapy after hemorrhagic stroke 420 expression after hemorrhagic stroke 386, 387 Hemorrhagic stroke biological therapies 400, 401 etiology

Subject Index

amyloid angiopathies 378–380 arteriovenous malformation 380, 381 hypertension 377, 378 intracranial aneurysm 382–385 gene therapy approaches 413, 414 cerebral vasospasm 418–420, 422, 426, 427 injection of vectors 415–418 prospects 400 vectors 414, 415 pathophysiology apoptosis 389 cerebral vasospasm 390 direct versus indirect molecular effects 385–387 edema 387–389 endothelial dysfunction 395–397 gene expression in vasospasm 390, 391, 397, 398 inflammation 389, 390, 398, 399 ischemia 389 microvascular vasospasm 390 remodeling and fibrosis 399, 400 smooth muscle contraction 391–394 relaxation 394, 395 polymeric drug delivery systems 484, 485 Herpes simplex virus (HSV) oncolytic viral therapy for glioma advantages and limitations 558, 559 dual mutation vectors 560 ␥1-34.5 mutants 559, 560 thymidine kinase and ribonucleotide reductase mutants 559 tumor-specific promoters for driving replication 561 pain gene therapy 326–333 Parkinson’s disease gene therapy vector 215, 216 pituitary adenoma gene therapy vector 596, 597, 600–602, 611, 612 spinal cord injury gene therapy vector 81, 82 Histamine, pain mediation 302 Human immunodeficiency virus, see Lentivirus

651

Huntington’s disease (HD) adeno-associated virus for gene transfer 163 treatment difficulty 125 Hypertension genetics 378 intracerebral hemorrhage risks 377, 378 Ibuprofen, polymeric drug delivery systems 484 Immunotherapy glioma monoclonal antibodies 537–539 prospects 509–513 tumor vaccines 539, 540 polymeric drug delivery systems 475, 477, 478 Insulin-like growth factor-1 (IGF-1), intervertebral disc degeneration expression 19, 21 treatment prospects 38, 43 Intercellular adhesion molecule-1 (ICAM-1), hemorrhagic stroke gene expression 399 Interleukin-1 (IL-1) inflammatory pain 296 intervertebral disc degeneration expression 20, 33, 34 Interleukin-2 (IL-2), immunotherapy 475, 477, 540 Interleukin-8 (IL-8), pain role in intervertebral disc degeneration 18 Interleukin-12 (IL-12), immunotherapy 475, 540 Intervertebral disc degeneration biomechanics 15, 16 economic impact 5 embryology of disc development 6–9 epidemiology 5, 30 etiology 16–18 gene therapy adenoviral vectors 40, 41 combination gene therapy 45, 46 cultured cell studies 43 epidermal growth factor 22, 35 matrix metalloproteinase tissue inhibitors 43, 45, 46 overview 38–40

Subject Index

prospects 21–24, 35, 49 rabbit studies 41–43, 46, 47 transforming growth factor-␤ 22, 23, 35, 38, 42, 43, 45 immune system 40 nutrition and oxygenation 13, 15, 40 pain etiology 6, 18 pathophysiology apoptosis 19, 32, 35 collagen changes 32–34 cytokines 20, 33, 34 endplate changes 34 growth factors 21 inflammation 20, 33 matrix metalloproteinases 20, 34, 37, 38 proteoglycan changes 32, 37, 38 water loss and diffusion impairment 31, 32, 40 progression characterization 5, 6 structure and anatomy 9–13, 30, 31 treatment conventional therapy 37 prospects 21–24, 35, 49 Intracerebral hemorrhage, see Hemorrhagic stroke Intrathecal drug delivery analgesic peptides 325 epilepsy 182, 183 rationale 461 Ischemic stroke animal models clinical relevance 346–348 magnetic resonance imaging 346–348 spontaneous stroke 341–345 transgenic animals 362 brain injury gene expression neuroprotective and neurodestructive genes 362, 363 overview 345, 346 plasticity and recovery 364, 365 candidate genes 340, 341 cell transplantation therapy ex vivo gene therapy 447, 448 historical perspective 439, 440, 442, 443 immunosuppression 441, 442

652

neural stem cell transplantation 448–450 NT2 cell trials 443–445 support factors for graft survival and axonal reconnection 445–447 tissue sources and preparation for implantation 440, 441 differential gene expression studies assay variation and confidence levels 359, 360 carotid artery stenosis 422–427 confirmation 360–362 differential display 349 DNA microarray analysis 357, 358 ex vivo gene therapy 447, 448 overview 348, 349 representational differential analysis 356, 357 serial analysis of gene expression 358, 359 subtractive hybridization 356 table of genes 350–355 therapeutic angiogenesis 426–428, 450 economic impact 337 epidemiology 337, 442 gene therapy approaches 413, 414 injection of vectors 415–418 principles 365 target identification 365, 366 vectors 414, 415, 447 genetic heterogeneity 336, 340, 341 heredity studies 337, 338 preconditioning stress in brain tolerance 363, 364 prevention 337 single gene mutations CADASIL 338–340 MELAS 338–340 thrombolytic therapy 337 Kallikrein, carotid artery stenosis gene therapy 425 Ketogenic diet, epilepsy management 185, 186 KRIT1, disruption and cavernous malformation 381

Subject Index

Laser capture microdissection (LCM), DNA microarray analysis of samples 250, 251 Lentivirus Parkinson’s disease gene therapy vector 219–221 pituitary adenoma gene therapy vector 598–600 spinal cord injury gene therapy vector 80, 81, 89, 90 Leukotrienes, pain role in intervertebral disc degeneration 18 Linkage analysis, combination with DNA microarray analysis 253, 255 Liposome antiepileptic drug delivery 183, 184 DNA complexes for Parkinson’s disease gene therapy 227 immunoliposomes 538, 539 Macrophage, stimulation for spinal cord injury treatment 76, 77 Magnetic resonance imaging (MRI) intraoperative costs 135, 136 glioma surgery 627 prospects 138, 141 safety 134, 135 surgical outcome advantages 134, 135 systems 136–138 neural stem cell tagging for glioma imaging 634 stroke studies in animals 346–348 Mannitol, blood-brain barrier disruption for drug delivery 459, 460 Marfan’s syndrome, cerebral aneurysm 384 Marrow stromal cell (MSC), ischemic stroke transplantation studies 448, 449 Matrix metalloproteinases (MMPs) antisense knockdown 126 inhibition in anti-angiogenesis therapy 543 intervertebral disc degeneration expression 20, 34, 37, 38 tissue inhibitor treatment prospects 43, 45, 46 MELAS, gene mutations and stroke 338–340

653

Methotrexate, polymeric drug delivery systems 481, 482 N-methyl-D-aspartate (NMDA) nociception 294, 295 receptor role in epilepsy 173, 197, 198 Microchip, drug delivery 487 Minocycline, angiogenesis inhibition 478 Mitogen-activated protein kinase (MAPK) carotid artery stenosis antisense gene therapy 424 cerebral vasospasm antisense gene therapy after hemorrhagic stroke 420, 422 glioma progression signaling 527–531 small molecule inhibitors 541, 542 smooth muscle contraction in hemorrhagic stroke 393, 394 Mitoxantrone, polymeric drug delivery systems 474 Multidrug-resistance-associated protein (MRP), antiepileptic drug resistance role 176, 177 Multiple sclerosis, DNA microarray studies 249 Nerve growth factor (NGF) brain graft neuroprotection 446 spinal cord injury gene therapy 84, 95 Neural stem cell (NSC) adult cell localization and characterization 107, 108 chemokines in tumor homing 632 endogenous stem cells differentiation into astrocytes 105 manipulation 72, 73 remyelination studies 74 glioma cell tagging for imaging 634 glioblastoma multiforme applications 639 glioma-genesis role 628, 629 gliomatosis cerebri applications 638, 639 homing and therapeutic applications 629–634 prospects for therapy 634–639 radiation necrosis differentiation from tumor recurrence 637

Subject Index

history of study 624 integration potential 625, 626 ischemic stroke transplantation studies 448–450 isolation and culture 67, 69, 70, 105–108, 626, 627 markers 106 migration 625, 626 persistence of grafts 631 pluripotency 67, 69, 104, 626 prodrug-enzyme engineering 629–631 spinal cord injury transplantation therapy aims 110 animal studies 109–116, 118 clinical prospects 118–120 endogenous cell properties 110, 111 gene transfer 67, 68 graft survival 70 rationale 68, 69 timing 111–116, 118 tissue engineering with biopolymer scaffolds 636 Neuropeptide Y (NPY), pain mediation 299 Neurotensin (NT), pain mediation 298 Neurotrophin-3 (NT-3) brain graft neuroprotection 446 pain mediation 302 spinal cord injury gene therapy 84, 130 Neurotrophin-4/5 (NT-4/5), pain mediation 302 Nitric oxide (NO) cerebral aneurysm role 383 endothelial dysfunction in hemorrhagic stroke 395, 396 pain mediation 303 smooth muscle contraction in hemorrhagic stroke 393 Nitric oxide synthase (NOS) carotid artery stenosis gene therapy 425 cerebral vasospasm gene therapy after hemorrhagic stroke 418, 419 Nitroreductase, prodrug combination in gene therapy 601 NOGO-A, glioma expression 633, 634 Notch, gene mutations in stroke 339 NT2 cells, transplantation trials in stroke 443–445

654

Nuclear factor-␬B (NF-␬B) cerebral vasospasm antisense gene therapy after hemorrhagic stroke 420, 422 hemorrhagic stroke gene expression 399 Olfactory ensheathing cell (OEC), spinal cord injury therapy 75, 76, 96 Opioid peptides, pain mediation 297, 323, 324 OSI-774, epidermal growth factor receptor inhibition 541 Osteoconduction, definition 52 Osteogenesis, definition 53 Osteoinduction, definition 52, 53 Oxidative stress intervertebral disc degeneration role 20 neurodegenerative disease 265, 267 p53, replacement in cancer therapy 605 Paclitaxel, polymeric drug delivery systems 473 Pain analgesics 322, 323 cell transplantation therapy 324–326 definition 284, 322 gene expression regulation 285 gene therapy antisense gene therapy 309–311 brain gene transfer 333 ex vivo therapy 325 prospects 311–313 rationale and targets 304, 305 RNA interference 310 viral vectors adenovirus 326 herpes simplex virus 326–333 overview 305–308 hypothalamic deep brain stimulation for management, see Deep brain stimulation intervertebral disc degeneration 6, 18 mediators bradykinin 300 calcitonin-gene-related peptide 298 cholecystokinin 299, 300 cytokines 296 excitatory glutamate receptors 294, 295

Subject Index

galanin 299 glial-derived neurotrophic factor 302, 303 histamine 302 inhibitory GABA receptors 295, 296, 323 ion channels acid-sensing ion channels 292, 293 adenosine receptors 292 ATP-gated channels 291, 292 potassium channels 293 sodium channels 293, 294 neuropeptide Y 299 neurotensin 298 neurotrophins 302 nitric oxide 303 opioid peptides 297, 323, 324 overview 285–287 pituitary adenylyl cyclase activating peptide 303 prostaglandins 301 protein kinase C in signal transduction 304 serotonin 301, 302 somatostatin 299 substance P 297, 298 vasoactive intestinal peptide 303 neuronal receptors adrenergic receptors 290, 291 cannabinoid receptors 289 capsaicin receptors 288, 289 cholinergic receptors 291 cold receptors 289, 290 proteinase-activated receptors 290 nociceptors 284, 285, 323 Parkinson’s disease (PD) gene therapy dopamine synthesis enzymes 214 expression detection microdialysis 237 positron emission tomography 236, 237 expression regulation 228–230 ex vivo therapy 231–233 in vivo therapy 214, 215 rationale 213, 214

655

Parkinson’s disease (PD) (continued) gene therapy (continued) vectors adeno-associated virus 164, 221–226 adenovirus 216, 218, 219 comparison of viral vectors 217 herpes simplex virus 215, 216 hybrid vectors 226 lentivirus 219–221 liposome-DNA complexes 227 naked DNA 226, 227 polyplexes 228 targeting of tissues 230, 231 neural progenitor cell transplantation history 108, 109, 235, 236 stem cell therapy 129 tissue transplantation studies 108, 233–235, 439, 440 treatment difficulty 125 xeno-neurotransplantation embryonic porcine ventral mesencephalon tissue 149, 150 outcomes 150, 151 overview 146–148 patient selection 149 postoperative evaluation 150 preoperative preparation 150 surgery 150 Parvovirus adeno-associated virus homology 159, 160 crystal structure 158, 159 Peripheral nerve injury, polymeric drug delivery systems 485 P-glycoprotein, antiepileptic drug resistance role 176, 177 Phenobarbital, mechanism of action 172 Phenytoin mechanism of action 171 polymeric drug delivery systems 484 Phosphatidylinositol 3-kinase (PI3K) glioma progression signaling 531–533 small molecule inhibitors 541 Phospholipase C (PLC), glioma progression signaling 533–535

Subject Index

Photodynamic therapy (PDT), photosensitizer delivery with neural stem cells 637 Pituitary adenylyl cyclase activating peptide (PACAP), pain mediation 303 Pituitary tumors animal models Cushing’s disease 610 growth hormone tumors 609, 610 null cell adenoma 611 prolactinoma 608, 609 conventional treatment goals 583 overview 580 pharmacotherapy 586, 587 radiation therapy 585, 586, 588 surgery transcranial approach 585 transsphenoidal approach 583–585, 588 damage mechanisms 581, 582 gene mutations 605 gene therapy angiogenesis inhibition 603, 604 apoptosis induction 604, 605 conditional cytotoxicity 600, 601, 603 immune response activation 606, 607 preclinical studies 611–613, 615 prospects 615, 616 rationale 580, 581, 587, 588 tumor suppressor genes 604, 605 vectors adeno-associated virus 595, 596 adenovirus 589, 591, 593–595, 611–613 comparison of viral vectors 590–593 herpes simplex virus 596, 597, 600–602, 611, 612 lentivirus 598–600 retrovirus 597, 598 initiation 582, 583 prevalence 582 PKI166, epidermal growth factor receptor inhibition 541 Platelet-derived growth factor (PDGF) glioma progression signaling 522–524 isoforms 523

656

receptor gene therapy for carotid artery stenosis 424 small molecule inhibitors 540, 541 Polymeric drug delivery systems adriamycin delivery 479, 480 angiogenesis inhibitor delivery 478 antiepileptic drugs 184 O6-benzylguanine delivery 472 buthionine sulfoximine delivery 479 camptothecin delivery 473, 474 carboplatin delivery 482, 483 carmustine delivery using polyanhydride fibers in glioma development 464, 465 initial therapy trials 470–472 preclinical trials 465–467 recurrent glioma trials 467–469 cisplatin delivery 483 cyclophosphamide delivery 479 dexamethasone delivery 483 5-fluorouracil delivery 480, 481 hemorrhagic stroke applications 484, 485 historical perspective 461–464 immunotherapy 475, 477, 478 methotrexate delivery 481, 482 mitoxantrone delivery 474 paclitaxel delivery 473 peripheral nerve injury applications 485 phenytoin delivery 484 prospects 487–489 quinacrine delivery 474 radiosensitizer delivery 484 rationale 458, 459, 461 spinal fusion applications 485, 486 Polyplex, Parkinson’s disease gene therapy vector 228 Prostaglandins, pain mediation 301 Prosthetics, neuroprosthetic therapies 131, 133, 134 Proteinase-activated receptors (PARs), pain reception 290 Protein kinase C (PKC) pain signal transduction 304 smooth muscle contraction in hemorrhagic stroke 392, 393 Prothrombin, gene mutations in stroke 340

Subject Index

Quinacrine, polymeric drug delivery systems 474 Representational differential analysis (RDA), ischemic stroke differential gene expression studies 356, 357 Rho, smooth muscle contraction in hemorrhagic stroke 394 RMP-7, blood-brain barrier disruption for drug delivery 460 RNA interference, pain gene knockdown 310 Schwann cell, spinal cord injury therapy 76 Serial analysis of gene expression (SAGE) hemorrhagic stroke vasospasm differential gene expression studies 390, 391 ischemic stroke differential gene expression studies 358, 359 Serotonin, pain mediation 301, 302 Somatostatin, pain mediation 299 Sphingolipids enzyme replacement therapy 204–210 storage disorders 202, 204 Spinal cord injury (SCI) animal models 66, 75 axonal regeneration barriers 65, 66, 125, 126 cellular therapy endogenous stem cells differentiation into astrocytes 105 manipulation 72, 73 remyelination studies 74 fetal tissue transplantation 74, 75 functionality of grafts 71, 72 macrophage stimulation 76, 77 neural restricted precursors 70, 71 neural stem cells adult cell localization and characterization 107, 108 aims 110 animal studies 109–116, 118 clinical prospects 118–120 endogenous cell properties 100, 111 gene transfer 67, 68 graft survival 70

657

Spinal cord injury (SCI) (continued) cellular therapy (continued) neural stem cells (continued) isolation and culture 67, 69, 70, 105–108 markers 106 pluripotency 67, 69, 104 rationale 68, 69 timing 111–116, 118 olfactory ensheathing cells 75, 76, 96 overview 66 Schwann cells 76 conventional therapy 65 economic impact 65 epidemiology 65 gene therapy animal models 90, 91 Bcl-2 85 Bcl-XL 85 brain-derived neurotrophic factor 84 delivery barriers 77, 78 ex vivo gene transfer 93–96 glial-derived neurotrophic factor 84, 90–93 nerve growth factor 84, 95 neurotrophin-3 84, 130 nonviral vectors 78, 79 prospects 96 viral vectors adeno-associated virus 83, 88, 92 adenovirus 82, 83, 85–88, 90–92 herpes simplex virus 81, 82 injection 87, 88 lentivirus 80, 81, 89, 90 mortality 65 recovery assays 67 regeneration potential 104 surgical techniques 130, 131 Spinal fusion animal models 53, 54 autogenous iliac crest bone graft limitations 52 bone morphogenetic proteins carriers 61 clinical studies BMP-2 57–59 BMP-7 59

Subject Index

demineralized bone matrix 59 expression 54 gene therapy prospects 60–62 preclinical studies BMP-2 55, 56 BMP-7 56, 57 demineralized bone matrix 57 preparations for use 54, 55 graft substitutes 52 Spinal fusion, polymeric drug delivery systems 485, 486 Spine disc degeneration, see Intervertebral disc degeneration embryology of disc development 6–9 Squalamine, angiogenesis inhibition 478 Statins, Alzheimer’s disease protection 263 STATs, glioma progression signaling 535 Stroke, see Hemorrhagic stroke; Ischemic stroke Subarachnoid hemorrhage, see Hemorrhagic stroke Substance P (SP), pain mediation 297, 298 Subtractive hybridization, ischemic stroke differential gene expression studies 356 Superoxide dismutase (SOD), cerebral vasospasm gene therapy after hemorrhagic stroke 420 T cell gene therapy for immune response enhancement in cancer 606, 607 glioma response studies cytolytic activity assays 504 flow cytometry of cytokine production 504, 505 gene expression analysis 505, 506 immunotherapy prospects 509–513 proliferation assay 503, 504 types in immune response 501–503 Tiagabine, mechanism of action 173 TIE2, mutation in venous anomalies 381 Transforming growth factor-␣ (TGF-␣), glioma progression signaling 526, 527 Transforming growth factor-␤ (TGF-␤) carotid artery stenosis antisense gene therapy 424

658

intervertebral disc degeneration treatment prospects 22, 23, 35, 38, 42, 43, 45 neural stem cell expression following spinal cord injury 112 TRPM8, pain reception 289, 290 TRPV1, pain reception 288, 289 Tumor necrosis factor-␣ (TNF-␣) inflammatory pain 296 pain role in intervertebral disc degeneration 18 Vagus nerve stimulation, epilepsy management 188 Valproic acid mechanism of action 171, 172 prodrug development 182 Vascular endothelial growth factor (VEGF) carotid artery stenosis gene therapy 425, 426 inhibition in anti-angiogenesis therapy 542, 543, 603 therapeutic angiogenesis 426–428, 450

Subject Index

Vasoactive intestinal peptide (VIP), pain mediation 303 Vigabatrin, mechanism of action 173 Xeno-neurotransplantation historical perspective 147, 148 immune response and rejection 148, 149 Parkinson’s disease embryonic porcine ventral mesencephalon tissue 149, 150 outcomes 150, 151 overview 146–148 patient selection 149 postoperative evaluation 150 preoperative preparation 150 surgery 150 prospects 151, 152 safety 149 ZD-1839, epidermal growth factor receptor inhibition 541

659