Gene Therapy - A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References

GENE

THERAPY A M EDICAL D ICTIONARY , B IBLIOGRAPHY , AND A NNOTATED R ESEARCH G UIDE TO I NTERNET R EFERENCES

J AMES N. P ARKER , M.D. AND P HILIP M. P ARKER , P H .D., E DITORS

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ICON Health Publications ICON Group International, Inc. 4370 La Jolla Village Drive, 4th Floor San Diego, CA 92122 USA Copyright ©2004 by ICON Group International, Inc. Copyright ©2004 by ICON Group International, Inc. All rights reserved. This book is protected by copyright. No part of it may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without written permission from the publisher. Printed in the United States of America. Last digit indicates print number: 10 9 8 7 6 4 5 3 2 1

Publisher, Health Care: Philip Parker, Ph.D. Editor(s): James Parker, M.D., Philip Parker, Ph.D. Publisher's note: The ideas, procedures, and suggestions contained in this book are not intended for the diagnosis or treatment of a health problem. As new medical or scientific information becomes available from academic and clinical research, recommended treatments and drug therapies may undergo changes. The authors, editors, and publisher have attempted to make the information in this book up to date and accurate in accord with accepted standards at the time of publication. The authors, editors, and publisher are not responsible for errors or omissions or for consequences from application of the book, and make no warranty, expressed or implied, in regard to the contents of this book. Any practice described in this book should be applied by the reader in accordance with professional standards of care used in regard to the unique circumstances that may apply in each situation. The reader is advised to always check product information (package inserts) for changes and new information regarding dosage and contraindications before prescribing any drug or pharmacological product. Caution is especially urged when using new or infrequently ordered drugs, herbal remedies, vitamins and supplements, alternative therapies, complementary therapies and medicines, and integrative medical treatments. Cataloging-in-Publication Data Parker, James N., 1961Parker, Philip M., 1960Gene Therapy: A Medical Dictionary, Bibliography, and Annotated Research Guide to Internet References / James N. Parker and Philip M. Parker, editors p. cm. Includes bibliographical references, glossary, and index. ISBN: 0-597-83954-9 1. Gene Therapy-Popular works. I. Title.

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Disclaimer This publication is not intended to be used for the diagnosis or treatment of a health problem. It is sold with the understanding that the publisher, editors, and authors are not engaging in the rendering of medical, psychological, financial, legal, or other professional services. References to any entity, product, service, or source of information that may be contained in this publication should not be considered an endorsement, either direct or implied, by the publisher, editors, or authors. ICON Group International, Inc., the editors, and the authors are not responsible for the content of any Web pages or publications referenced in this publication.

Copyright Notice If a physician wishes to copy limited passages from this book for patient use, this right is automatically granted without written permission from ICON Group International, Inc. (ICON Group). However, all of ICON Group publications have copyrights. With exception to the above, copying our publications in whole or in part, for whatever reason, is a violation of copyright laws and can lead to penalties and fines. Should you want to copy tables, graphs, or other materials, please contact us to request permission (E-mail: [email protected]). ICON Group often grants permission for very limited reproduction of our publications for internal use, press releases, and academic research. Such reproduction requires confirmed permission from ICON Group International Inc. The disclaimer above must accompany all reproductions, in whole or in part, of this book.

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Acknowledgements The collective knowledge generated from academic and applied research summarized in various references has been critical in the creation of this book which is best viewed as a comprehensive compilation and collection of information prepared by various official agencies which produce publications on gene therapy. Books in this series draw from various agencies and institutions associated with the United States Department of Health and Human Services, and in particular, the Office of the Secretary of Health and Human Services (OS), the Administration for Children and Families (ACF), the Administration on Aging (AOA), the Agency for Healthcare Research and Quality (AHRQ), the Agency for Toxic Substances and Disease Registry (ATSDR), the Centers for Disease Control and Prevention (CDC), the Food and Drug Administration (FDA), the Healthcare Financing Administration (HCFA), the Health Resources and Services Administration (HRSA), the Indian Health Service (IHS), the institutions of the National Institutes of Health (NIH), the Program Support Center (PSC), and the Substance Abuse and Mental Health Services Administration (SAMHSA). In addition to these sources, information gathered from the National Library of Medicine, the United States Patent Office, the European Union, and their related organizations has been invaluable in the creation of this book. Some of the work represented was financially supported by the Research and Development Committee at INSEAD. This support is gratefully acknowledged. Finally, special thanks are owed to Tiffany Freeman for her excellent editorial support.

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About the Editors James N. Parker, M.D. Dr. James N. Parker received his Bachelor of Science degree in Psychobiology from the University of California, Riverside and his M.D. from the University of California, San Diego. In addition to authoring numerous research publications, he has lectured at various academic institutions. Dr. Parker is the medical editor for health books by ICON Health Publications. Philip M. Parker, Ph.D. Philip M. Parker is the Eli Lilly Chair Professor of Innovation, Business and Society at INSEAD (Fontainebleau, France and Singapore). Dr. Parker has also been Professor at the University of California, San Diego and has taught courses at Harvard University, the Hong Kong University of Science and Technology, the Massachusetts Institute of Technology, Stanford University, and UCLA. Dr. Parker is the associate editor for ICON Health Publications.

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About ICON Health Publications To discover more about ICON Health Publications, simply check with your preferred online booksellers, including Barnes & Noble.com and Amazon.com which currently carry all of our titles. Or, feel free to contact us directly for bulk purchases or institutional discounts: ICON Group International, Inc. 4370 La Jolla Village Drive, Fourth Floor San Diego, CA 92122 USA Fax: 858-546-4341 Web site: www.icongrouponline.com/health

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Table of Contents FORWARD .......................................................................................................................................... 1 CHAPTER 1. STUDIES ON GENE THERAPY......................................................................................... 3 Overview........................................................................................................................................ 3 The Combined Health Information Database................................................................................. 3 Federally Funded Research on Gene Therapy ................................................................................ 7 E-Journals: PubMed Central ....................................................................................................... 65 The National Library of Medicine: PubMed ................................................................................ 76 CHAPTER 2. NUTRITION AND GENE THERAPY ............................................................................. 121 Overview.................................................................................................................................... 121 Finding Nutrition Studies on Gene Therapy............................................................................. 121 Federal Resources on Nutrition ................................................................................................. 123 Additional Web Resources ......................................................................................................... 123 CHAPTER 3. ALTERNATIVE MEDICINE AND GENE THERAPY ...................................................... 125 Overview.................................................................................................................................... 125 National Center for Complementary and Alternative Medicine................................................ 125 Additional Web Resources ......................................................................................................... 138 General References ..................................................................................................................... 139 CHAPTER 4. DISSERTATIONS ON GENE THERAPY ........................................................................ 141 Overview.................................................................................................................................... 141 Dissertations on Gene Therapy.................................................................................................. 141 Keeping Current ........................................................................................................................ 144 CHAPTER 5. CLINICAL TRIALS AND GENE THERAPY ................................................................... 145 Overview.................................................................................................................................... 145 Recent Trials on Gene Therapy.................................................................................................. 145 Keeping Current on Clinical Trials ........................................................................................... 160 CHAPTER 6. PATENTS ON GENE THERAPY ................................................................................... 163 Overview.................................................................................................................................... 163 Patents on Gene Therapy ........................................................................................................... 163 Patent Applications on Gene Therapy ....................................................................................... 199 Keeping Current ........................................................................................................................ 232 CHAPTER 7. BOOKS ON GENE THERAPY ....................................................................................... 235 Overview.................................................................................................................................... 235 Book Summaries: Federal Agencies............................................................................................ 235 Book Summaries: Online Booksellers......................................................................................... 237 Chapters on Gene Therapy......................................................................................................... 245 Directories.................................................................................................................................. 245 CHAPTER 8. MULTIMEDIA ON GENE THERAPY ............................................................................ 247 Overview.................................................................................................................................... 247 Video Recordings ....................................................................................................................... 247 Audio Recordings....................................................................................................................... 248 Bibliography: Multimedia on Gene Therapy.............................................................................. 248 CHAPTER 9. PERIODICALS AND NEWS ON GENE THERAPY ......................................................... 251 Overview.................................................................................................................................... 251 News Services and Press Releases.............................................................................................. 251 Newsletter Articles .................................................................................................................... 256 Academic Periodicals covering Gene Therapy ........................................................................... 256 APPENDIX A. PHYSICIAN RESOURCES .......................................................................................... 259 Overview.................................................................................................................................... 259 NIH Guidelines.......................................................................................................................... 259 NIH Databases........................................................................................................................... 261 Other Commercial Databases..................................................................................................... 264

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APPENDIX B. PATIENT RESOURCES ............................................................................................... 265 Overview.................................................................................................................................... 265 Patient Guideline Sources.......................................................................................................... 265 Finding Associations.................................................................................................................. 272 APPENDIX C. FINDING MEDICAL LIBRARIES ................................................................................ 275 Overview.................................................................................................................................... 275 Preparation................................................................................................................................. 275 Finding a Local Medical Library................................................................................................ 275 Medical Libraries in the U.S. and Canada ................................................................................. 275 ONLINE GLOSSARIES................................................................................................................ 281 Online Dictionary Directories ................................................................................................... 281 GENE THERAPY DICTIONARY ............................................................................................... 283 INDEX .............................................................................................................................................. 373

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FORWARD In March 2001, the National Institutes of Health issued the following warning: "The number of Web sites offering health-related resources grows every day. Many sites provide valuable information, while others may have information that is unreliable or misleading."1 Furthermore, because of the rapid increase in Internet-based information, many hours can be wasted searching, selecting, and printing. Since only the smallest fraction of information dealing with gene therapy is indexed in search engines, such as www.google.com or others, a non-systematic approach to Internet research can be not only time consuming, but also incomplete. This book was created for medical professionals, students, and members of the general public who want to know as much as possible about gene therapy, using the most advanced research tools available and spending the least amount of time doing so. In addition to offering a structured and comprehensive bibliography, the pages that follow will tell you where and how to find reliable information covering virtually all topics related to gene therapy, from the essentials to the most advanced areas of research. Public, academic, government, and peer-reviewed research studies are emphasized. Various abstracts are reproduced to give you some of the latest official information available to date on gene therapy. Abundant guidance is given on how to obtain free-of-charge primary research results via the Internet. While this book focuses on the field of medicine, when some sources provide access to non-medical information relating to gene therapy, these are noted in the text. E-book and electronic versions of this book are fully interactive with each of the Internet sites mentioned (clicking on a hyperlink automatically opens your browser to the site indicated). If you are using the hard copy version of this book, you can access a cited Web site by typing the provided Web address directly into your Internet browser. You may find it useful to refer to synonyms or related terms when accessing these Internet databases. NOTE: At the time of publication, the Web addresses were functional. However, some links may fail due to URL address changes, which is a common occurrence on the Internet. For readers unfamiliar with the Internet, detailed instructions are offered on how to access electronic resources. For readers unfamiliar with medical terminology, a comprehensive glossary is provided. For readers without access to Internet resources, a directory of medical libraries, that have or can locate references cited here, is given. We hope these resources will prove useful to the widest possible audience seeking information on gene therapy. The Editors

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From the NIH, National Cancer Institute (NCI): http://www.cancer.gov/cancerinfo/ten-things-to-know.

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CHAPTER 1. STUDIES ON GENE THERAPY Overview In this chapter, we will show you how to locate peer-reviewed references and studies on gene therapy.

The Combined Health Information Database The Combined Health Information Database summarizes studies across numerous federal agencies. To limit your investigation to research studies and gene therapy, you will need to use the advanced search options. First, go to http://chid.nih.gov/index.html. From there, select the “Detailed Search” option (or go directly to that page with the following hyperlink: http://chid.nih.gov/detail/detail.html). The trick in extracting studies is found in the drop boxes at the bottom of the search page where “You may refine your search by.” Select the dates and language you prefer, and the format option “Journal Article.” At the top of the search form, select the number of records you would like to see (we recommend 100) and check the box to display “whole records.” We recommend that you type “gene therapy” (or synonyms) into the “For these words:” box. Consider using the option “anywhere in record” to make your search as broad as possible. If you want to limit the search to only a particular field, such as the title of the journal, then select this option in the “Search in these fields” drop box. The following is what you can expect from this type of search: •

Gene Therapy: Future Therapy for Erectile Dysfunction Source: Current Urology Reports. 2(6): 480-487. December 2001. Contact: Current Science, Inc. 400 Market Street, Suite 700, Philadelphia, PA 19106 (800) 427-1796. Fax (215) 574-2225. E-mail: [email protected]. Website: http://www.current-reports.com. Summary: Advances in molecular biological techniques, completion of the Human Genome Project, and the ensuing age of molecular medicine, in conjunction with the sum of a decades-long accumulation of knowledge of the physiology of erection and the pathophysiology of erectile dysfunction (ED, formerly called impotence) have converged to make gene therapy for ED a distinct possibility. This report highlights the goals and strategies of gene therapy for erectile dysfunction and reviews the strategies

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that initially have been employed. Both the intrinsic complexities of mechanisms responsible for ensuring normal erection and the multifactorial nature of ED ensure that there is a relatively vast number of physiologically relevant molecular targets for gene therapy. The authors note that virtually every preclinical gene therapy strategy or target examined thus far has been largely successful in easing conditions associated with compromised erectile function in vivo or in vitro. While this preclinical data is quite preliminary in many regards, the results are nonetheless quite impressive and encouraging. If similar success is obtained in clinical trials, gene therapy for ED may provide the first concrete 'proof of concept' for using gene therapy in the treatment of human smooth muscle disorders. 3 figures. 2 tables. 25 references. •

Immune Gene Therapy in Urology Source: Current Urology Reports. 3(1): 82-89. February 2002. Contact: Current Science, Inc. 400 Market Street, Suite 700, Philadelphia, PA 19106 (800) 427-1796. Fax (215) 574-2225. E-mail: [email protected]. Website: http://www.current-reports.com. Summary: Effective treatments are urgently needed for metastatic disease in bladder, prostate, and renal (kidney) cell cancer. In the past few years, several new approaches for treating these conditions have been proposed, including gene therapy. This article illustrates the recent developments in immune gene therapy that have applications in urology. A number of different strategies have been developed to accomplish urologic cancer gene therapy. Genetic immunomodulation strategies attempt to activate immune defense mechanisms against tumor cells by transfer of tumor antigens, cytokine genes, or strongly immunogenic cell surface molecules. 2 figures. 1 table. 42 references.

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Status of Gene Therapy for Erectile Dysfunction Source: Contemporary Urology. 14(10): 14, 16, 21-22, 25, 28, 30-31. October 2002. Contact: Available from Medical Economics Publishing Inc. Montvale, NJ 07645. (800) 432-4570. Summary: Erectile dysfunction (ED) is defined as the inability of a man to attain or maintain an erection long enough to complete sexual intercourse. Although medical therapy with sildenafil citrate (Viagra, Pfizer) acts by enhancing smooth muscle function, it is effective in only 50 to 65 percent of patients with ED. This article reviews the status of gene therapy for erectile dysfunction. The concept of up-regulating the function of erection-promoting enzymes is simply termed gene therapy. The authors review gene therapy for the treatment of ED, highlighting those genes that are involved in the normal process of cavernosal smooth muscle relaxation of the penis and, more specifically, those involved with the production of nitric oxide (NO), the chemical mediator of penile erections. Topics include penile structure and erectile physiology, how gene therapy works and why the penis is a likely target, current gene therapies in research or clinical application, and future directions. One sidebar offers an editorial comment on this subject. 1 figure. 1 table. 43 references.

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Impact of Gene Therapy on Dentistry: A Revisiting After Six Years Source: JADA. Journal of the American Dental Association. 133(1): 35-44. January 2002. Contact: Available from American Dental Association. ADA Publishing Co, Inc., 211 East Chicago Avenue, Chicago, IL 60611. (312) 440-2867. Website: www.ada.org.

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Summary: Gene therapy is an emerging field of biomedicine that has commanded considerable scientific and popular attention. The procedure involves the transfer of genes to patients for clinical benefit. Transferred genes can be used for either reparative or pharmacological purposes. This article summarizes the impact of gene therapy on dentistry. In 1995, the first author and a colleague described the potential impact of gene therapy on dentistry, on the basis of initial studies of gene transfer applications to salivary glands, keratinocytes and cancer cells. Their conclusion was that gene therapy would have a significant impact on the nature of dental practice within 20 years. In the past six years, remarkable progress has been made in the field of gene therapy, including seven areas relevant to dental practice: bone repair, salivary glands, autoimmune disease, pain, DNA vaccinations, keratinocytes, and cancer. While considerable problems remain, thus impeding the routine clinical use of gene transfer, gene therapy will have a pervasive and significant impact on areas of dental practice that are based in biological science. The authors conclude that by 2015, this will translate into practitioners' having a wide range of novel biological treatment options for their patients. 4 figures. 1 table. 56 references. •

Mouth is a Gateway to the Body: Gene Therapy in 21st Century Dental Practice Source: CDA Journal. California Dental Association Journal. 26(6): 455-460. June 1998. Contact: Available from California Dental Association (CDA). 1201 K Street, Sacramento, CA 95814. (916) 443-0505. Summary: This article contends that gene therapy may become an integral tool in dental practice early in the 21st century. The author discusses how gene therapy and other biological therapies are expected to be applied to oral diseases and disorders during the midpractice lifetime of today's dental students. One area of application is particularly important for oral health professions to recognize: the use of genes as pharmaceutical agents. There are certainly many oral-specific, corrective applications of gene transfer, such as repair of irradiated salivary glands or treatments of oral cancer. It is conceivable that, analogous to conventional medications taken by mouth, a number of systemic gene therapeutics may also follow the convenient route of oral delivery. The author describes the methods of gene transfer, noting use of the salivary glands; the use of gene therapeutics in the upper gastrointestinal (GI) tract; systemic gene therapy; and the role of dentists as gene therapists. 2 tables. 29 references. (AA-M).

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Inherited Liver Disease: What Role for Gene Therapy? Source: Contemporary Gastroenterology. p. 15-20, 22, 24-25. July-August 1991. Summary: This article explores the role of gene therapy in inherited liver disease. The authors note that introducing normal genes into hepatocytes may be a workable alternative to transplantation for such disorders as familial hypercholesterolemia. The genes for many of the disease-related proteins of the liver have already been cloned and efficiently expressed in cultured cells. Several promising methods have been developed to introduce these normally functioning genes into cultured hepatocytes or directly into the recipient's liver. Technical problems remaining include maintaining high levels of expression of the exogenous gene, assuring long-term survival of re-implanted hepatocytes, and minimizing potential continued harmful effects from the endogenous mutant gene. 2 figures. 2 tables. 60 references.

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Impact of Gene Therapy on Dentistry Source: JADA. Journal of the American Dental Association. 126(2): 179-189. February 1995. Summary: This article is designed to provide the dental practitioner with a general understanding of gene therapy, as well as several examples of how it is used to better manage dental and oral problems. Topics covered include a brief review of the recent advances in molecular biology; general principles of gene transfer; methods of gene transfer, including viral methods and non-viral or physical methods; the uses of gene transfer; applying gene therapy to oral cancer; gene transfer to oral mucosal keratinocytes; gene transfer to salivary glands; gene therapy; gene therapeutics; and the future of gene transfer and its impact on dentistry. The authors note that it is likely that gene transfer approaches will not be used initially for any routine care, but rather for patients whose conditions are refractory to more conventional treatment, such as people at especially high risk for caries or periodontal diseases. 6 figures. 2 tables. 33 references. (AA-M).

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Gene Therapy for Type 1 and Type 2 Diabetes Source: Diabetes Reviews. 7(2): 124-138. 1999. Contact: Available from American Diabetes Association, Inc. 1701 North Beauregard Street, Alexandria, VA 22311. (800) 232-3472. Summary: This article reviews gene therapy for type 1 and type 2 diabetes. Gene therapy can be divided into four major approaches: expansion of beta cells and beta cell precursors, gene transfer to primary beta cells, engineering of glucose responsiveness in nonbeta cells, and immunomodulation of pancreatic beta cells. The pathophysiology of type 1 and type 2 diabetes means that some gene therapy approaches can be applied to both variants of the disease, while others will be specific to one variant or the other. For example, insulin deficiency, a prominent feature of both variants, can be approached by in vivo or ex vivo insertion of genes that stimulate the growth of pancreatic beta cells or beta cell precursors. An increasing number of genes involved in the process of beta cell growth and differentiation are being discovered. Induction of differentiation in early endocrine precursors is an attractive, albeit difficult, approach for beta cell expansion. One alternative is engineering glucose responsive insulin secretion in nonbeta cells such as neuroendocrine cells and hepatocytes. Substantial progress has been made in this direction; however, in the absence of intact insulin secretory apparatus, it is difficult to achieve tight coupling between glucose stimulation and insulin secretion. In type 2 diabetes, insulin resistance increases the secretory demand on failing beta cells. Recent progress in understanding the regulation of body weight, adiposity, and insulin resistance, as well as the interaction between insulin resistance, hyperglycemia, and beta cell dysfunction, provides a new direction for gene therapy strategies to reduce insulin resistance and protect pancreatic beta cells. Finally, gene therapy may be valuable for primary prevention of autoimmune destruction of pancreatic beta cells in type 1 diabetes and for the prevention of immune rejection, recurrent autoimmunity, and apoptosis in transplanted islets. 4 figures. 1 table. 138 references. (AA-M).

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Gene Therapy and Tissue Engineering in Sports Medicine Source: Physician and Sportsmedicine. 28(2):. February 2000.

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Contact: Available from McGraw-Hill Healthcare Information. 4530 West 77th Street, Floor 3, Minneapolis, MN 55435. (800) 525-5003 or (609) 426-7070 (for subscriptions) or (952) 835-3222 (for back issues). Summary: This journal article provides health professionals with information on gene therapy and tissue engineering in sports medicine. Although treatment of sports injuries has improved during the past two decades with the use of sophisticated rehabilitation programs, novel operative approaches, and advances in biomechanical engineering, deficits in injury treatment remain because of the limited healing capacity of certain musculoskeletal system tissues. Gene therapy is a promising new option that delivers therapeutic genes into cells and tissues. The article discusses gene therapy in terms of vectors, delivery strategies, and limitations. Another promising technology is tissue engineering. This technology is based on developing biological substitutes for the repair, reconstruction, regeneration, or replacement of tissues. The article describes possible gene therapy or tissue engineering techniques for treating skeletal muscle injuries, articular cartilage damage, anterior cruciate ligament injuries, meniscal tears, and bone fractures. In addition, the article comments on future directions for gene therapy and tissue engineering techniques. 2 figures, 2 tables, and 64 references. •

Current Progress in Gene Therapy Source: American Porphyria Foundation. Winter, 1992. p. 1-3. Contact: Available from American Porphyria Foundation. P.O. Box 22712, Houston, TX 77227. (713) 266-9617. Summary: This newsletter article addresses current progress in gene therapy, with a focus on considerations for gene therapy in the treatment of porphyria. Topics include safety concerns about gene therapy, social and ethical questions, how gene therapy works, the history of gene transfer, current clinical uses of gene therapy around the world, the use of gene therapy for different types of cancer, and the long-term sideeffects of gene therapy.

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Potential of Gene Therapy for Treatment of Kidney Diseases Source: Seminars in Nephrology. 15(1): 57-69. January 1995. Contact: Available from W.B. Saunders Company. Periodicals Department, 6277 Sea Harbor Drive, Orlando, FL 32887-4800. Summary: To provide a framework for understanding the concepts and problems involved in gene therapy for the treatment of kidney diseases, the authors of this article discuss basic aspects of gene structure and regulation. Topics include gene delivery vectors, steps necessary to achieve tissue-and cell-specific expression of delivered genes, and some present and future applications of gene therapy in kidney diseases. The authors conclude that, although the potentials for gene therapy are exciting, gene therapy for human kidney diseases is a long way from being practical at this stage. 1 figure. 2 tables. 82 references. (AA-M).

Federally Funded Research on Gene Therapy The U.S. Government supports a variety of research studies relating to gene therapy. These studies are tracked by the Office of Extramural Research at the National Institutes of

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Health.2 CRISP (Computerized Retrieval of Information on Scientific Projects) is a searchable database of federally funded biomedical research projects conducted at universities, hospitals, and other institutions. Search the CRISP Web site at http://crisp.cit.nih.gov/crisp/crisp_query.generate_screen. You will have the option to perform targeted searches by various criteria, including geography, date, and topics related to gene therapy. For most of the studies, the agencies reporting into CRISP provide summaries or abstracts. As opposed to clinical trial research using patients, many federally funded studies use animals or simulated models to explore gene therapy. The following is typical of the type of information found when searching the CRISP database for gene therapy: •

Project Title: A CILIATED CELL-SPECIFIC PROMOTER FOR GENE THERAPY OF CF Principal Investigator & Institution: Ostrowski, Lawrence E.; Medicine; University of North Carolina Chapel Hill Office of Sponsored Research Chapel Hill, Nc 27599 Timing: Fiscal Year 2002; Project Start 01-APR-2002; Project End 31-MAR-2006 Summary: (provided by applicant): The long-term objective of this research is to develop a ciliated cell-specific promoter that will improve the effectiveness of gene therapy or cystic fibrosis (CF). In normal airways, the cystic fibrosis transmembrane conductance regulator (CFTR) protein is expressed primarily at the apical surface of ciliated cells and in the submucosal glands. For gene therapy of CF to be successful, the normal CFTR protein must be expressed in the proper location. However, many of the gene therapy vectors currently under investigation have no specificity for the differentiated airway epithelium. In addition, these vectors frequently use viral promoter elements or promoters of constitutively expressed genes to drive high-level expression of reporter genes. A major drawback to the use of these vectors therefore is that they may result in high levels of CFTR expression in unwanted cell types (e.g., macrophages, basal cells). These promoters may also be less efficient at providing stable, long-term expression in the non-dividing ciliated cell population. Our hypothesis is that the use of a specific promoter to direct expression of the CFTR protein to the ciliated cells located at the apical surface of the airways will correct the CF phenotype. In addition, we hypothesize that by using an endogenous promoter in an integrating vector, we will achieve stable long-term expression of the CFTR protein. The use of a ciliated cell-specific promoter will also increase the safety of gene therapy for CF by preventing potentially deleterious expression of CFTR in the wrong cell types. To test our hypothesis, we propose the following specific aims: Specific Aim 1: To identify and clone the promoter regions of ciliated cell-specific genes. Specific Aim 2: To identify the essential regulatory elements responsible for ciliated cell specific gene expression. Specific Aim 3: To demonstrate correction of the CF phenotype in both in vitro and in vivo models by targeted expression of the normal CFTR gene in ciliated cells. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

2 Healthcare projects are funded by the National Institutes of Health (NIH), Substance Abuse and Mental Health Services (SAMHSA), Health Resources and Services Administration (HRSA), Food and Drug Administration (FDA), Centers for Disease Control and Prevention (CDCP), Agency for Healthcare Research and Quality (AHRQ), and Office of Assistant Secretary of Health (OASH).

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Project Title: A HYPERTENSION

GENE-BASED

THERAPEUTIC

FOR

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PULMONARY

Principal Investigator & Institution: Brigham, Kenneth L.; Professor of Medicine; Generx+, Inc. 3200 West End Ave, Ste 500 Nashville, Tn 372011322 Timing: Fiscal Year 2001; Project Start 30-SEP-1998; Project End 31-OCT-2002 Summary: (Adapted from the Investigator's abstract) Primary pulmonary hypertension is a uniformly fatal disease of young and middle aged people; there is little understanding of the pathogenesis and no specific therapy. The principal pharmacologic therapy in current use is chronic constant intravenous infusion of the vasodilator prostanoid, prostacyclin. This therapy is effective, but requires maintenance of an intravenous catheter and continuous intravenous infusion; the only alternative is lung transplantation. The investigators showed some time ago that it is possible to transfect the lungs with the arachidonate cyclooxygenase (COX) gene and achieve selective increases in prostacyclin and PGE2 prostanoids which are potentially therapeutic for pulmonary hypertension. Studies supported by this phase I grant have documented that aerosol delivery of the COX gene as a plasmid-cationic liposome complex can decrease pulmonary vascular reactivity significantly in an animal model which is anatomically and physiologically similar to humans with no adverse effects on lung function. The investigators hypothesize that aerosol delivery of the COX gene in an expression plasmid complexed with cationic liposomes will prevent development and/or progression of the physiologic and pathologic changes of chronic pulmonary hypertension. The investigators further speculate that this therapy will prove superior to any current therapy for the treatment of patients with primary pulmonary hypertension and may provide a new therapeutic modality for treatment of a much larger group of patients with secondary pulmonary hypertension. In this Phase II application, the PI will determine: 1) effects of repeated administration of the COX gene in a plasmidcationic liposome complex by aerosol on lung function, 2) prostanoid generation and pulmonary vascular reactivity in unanesthetized sheep; 3) determine whether administration of the COX gene by aerosol in a plasmid-liposome complex will prevent development or progression of sustained pulmonary hypertension and pulmonary vascular remodeling in a well-characterized chronic air embolization model of sustained pulmonary hypertension in sheep; and 4) increase the efficiency of aerosol delivery of plasmid-cationic liposome complexes by using newer generation aerosol delivery devices and improved liposome-DNA formulations. PROPOSED COMMERCIAL APPLICATION: NOT AVAILABLE Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: AD OC TK/VAL GENE THERAPY CLINICAL CORRELATES Principal Investigator & Institution: Chung, Leland W.; Professor; Urology; Emory University 1784 North Decatur Road Atlanta, Ga 30322 Timing: Fiscal Year 2001; Project Start 11-APR-2000; Project End 31-JUL-2003 Summary: The objective of the proposed study is to obtain clinical correlates on an ongoing investigator-initiated investigational new drug (IND) at the University of Virginia. There are two major components to this proposed study. First, we request funding to test the hypothesis that an adenoviral toxic gene, Ad-OC-TK, plus an orally bioavailable Valacyclovir (Val) could induce maximal cell-kill in recurrent primary and metastatic prostate cancers. Laboratory evidence suggests that both cancer epithelium and its supporting stroma express a common protein, osteocalcin (OC). Delivering and expressing a toxic gene, herpes simplex thymidine kinase (TK), in an adenoviral

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construct could result in the most efficacious control of recurrent prostate cancer and its metastases. We propose to take advantage of the availability of cancer tissue specimens and biologic fluids such as blood and urine from patients to conduct the evaluation of the immunological status of the prostate cancer tissues obtained prior to, during, and after toxic gene therapy. Clinical correlates on apoptosis, activated partial thrombosplatin time (aPTT) and radiographic imaging in patients treated with toxic gene therapy will be obtained. Second, we request funding to expand our on-going clinical trial to treat additional prostate cancer patients with skeletal metastasis. The hypothesis to be tested is that Ad-OC-TK/Val will cause most effective tumor regression at the skeletal site. We propose to evaluate parallely the clinical correlates in biopsy tissue, blood, and urine samples obtained from patients treated with maximum doses of Ad-OC-TK. The specific aims of this proposal are: 1) to determine the level and distribution of Ad-OC-TK expression In prostate cancer cells at recurrent primary and metastatic lymph node and bone sites. A number of markers will be evaluated to assess viral distribution and delivery. Detection of other bone matrix protein expression of proteins such as OC, osteonectin (OSN), and bone sialoprotein (BSP) will be studied; 2) to assess and compare Ad-OC-TK induced anti-tumor immunity in tumor specimens obtained from recurrent primary and metastatic lymph node and bone sites. Immunohistochemistry, RT-PCR, bioassay, and enzyme linked immunoassay will be used to assess both local anti-tumor immunity and circulating anti-adenoviral antibodies; 3) to obtain clinical correlates in tumor and blood specimens obtained from patients before, during, and after Ad-OC-TK/Val therapy. Apoptotic indices, blood coagulation profiles, and radiographic imaging analyses will be evaluated; 4) to recruit at least 12 patients with minimum bone metastasis, and to treat these patients with intratumoral Ad-OC-TK/Val injection. We hope this will allow us to evaluate whether this form of gene therapy will be safe and efficacious in patients with bone metastasis. The ultimate goal of this study is to provide strong background information, which will assist us in development of future trials to incorporate "re-targeted" virus injected systemically to eradicate disseminated prostate cancer at both bone and soft tissue sites. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ADENOVIRUS LIMITATIONS AND TUMOR TARGETED GENE THERAPY Principal Investigator & Institution: O'malley, Bert W.; Professor & Chairman; Surgery; University of Maryland Balt Prof School Baltimore, Md 21201 Timing: Fiscal Year 2003; Project Start 01-JAN-2003; Project End 31-DEC-2007 Summary: (provided by applicant): Squamous cell carcinoma of the oral cavity and head and neck (HNSCC) is a devastating disease in which surgery, radiation and/or chemotherapy have not improved the 50 percent overall 5 year survival over the past 20 years. In an attempt to improve survival and reduce morbidity, gone therapy strategies are being developed for oral cancer. Despite encouraging preclinical data in many tumor types, initial clinical studies with adenovirus gene therapy have been disappointing. We posit that cellular differences exist even among head and neck cancers of the same histology that limit gone therapy responses. We further posit that variations in shared Coxsackie and adenovirus receptor (CAR) and integrin receptors play a major role in the transduction efficiency and translates to a significant variation in multi-tumor responses to adenovirus gene therapy strategies. We will test five hypotheses by addressing the following Specific Aims: 1) Determine the concentration of CAR, integrins, and FGF2 receptor on fresh human HNSCC samples and derived cell lines; 2) Establish the correlation between expression of CAR or integrin and Ad-tk anti-tumor effects and

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develop a FGF2 retargeting strategy in vitro that circumvents these limitations; 3) Quantify gene expression and therapeutic response to Ad-tk using both standard adenovirus and FGF2-R retargeted vectors in tumors established from 11NSCC lines. 4) Optimize direct linter-tumor injection therapy using circumventing treatment strategies and introduce systemic FGF2 retargeting therapy. We focus on a newly created fibroblast growth factor (FGF) conjugated adenovirus vector to develop a! Circumventing strategy that will improve gone transfer efficiency and corresponding therapeutic response. This novel FGF-2 receptor-based retargeting strategy may also allow safe and effective systemic delivery of tumor targeted adenovirus vectors. Five investigations regarding the role of adenovirus receptor and integrin expression on tumor cells will provide a platform of important gone therapy information that will lead to more effective and applicable preclinical animal studies and human clinical investigation. Adenovirus receptor or integrin testing prior to enrollment into a clinical trial may provide a means of selecting, stratifying, or assessing outcomes in head and neck cancer patients. This platform of information will also prove valuable to investigators who wish to circumvent limitations by developing and using alternative strategies such as FGF adenovirus retargeting. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ANNUAL GENE THERAPY SYMPOSIUM FOR LUNG & BLOOD DISEASES Principal Investigator & Institution: Tarantal, Alice F.; Professor; Primate Research Center; University of California Davis Sponsored Programs, 118 Everson Hall Davis, Ca 95616 Timing: Fiscal Year 2003; Project Start 01-JAN-2003; Project End 31-DEC-2007 Summary: (provided by applicant): This R13 application requests funds to partially support an annual interdisciplinary scientific symposium ("Annual Gene Therapy Symposium for Lung & Blood Diseases") on essential topics associated with gene therapy for the treatment of congenital lung and blood diseases. The goal of this twoday fall symposium will be to bring together junior and senior investigators, and students and postdoctoral fellows that study gene transfer strategies, the pathophysiology of pediatric congenital disorders, and developmental anatomy/ontogeny for the understanding and treatment of human disease. Our aim is to provide an integrated and interactive forum for the presentation of new data and novel hypotheses for gene therapy applications. The annual symposium will be organized such that investigators in gene therapy and those focusing on studies related to lung and blood diseases who do not currently have an opportunity to interact will be present to discuss and identify crucial issues for study. The first day of the symposium will focus on the most current and timely topics in gene therapy. The second day will be dedicated to the theme of the meeting, with keynote and dinner speakers selected based on their areas of expertise. The theme of the 1st Annual Symposium will be stem cells, with subsequent years focusing on the fetus and newborn, in vivo imaging, animal models, and gene expression profiling. A competitive process will be established to provide stipends to graduate students and postdoctoral fellows. Students selected will present their research findings in an informal setting of a poster session. Minorities, women, and individuals with disabilities will be sought and strongly encouraged to apply. The symposium will be held at the Primate Center at the University of California at Davis. Funding is requested for planning and organizing the meeting, and travel and housing costs for speakers and those graduate students and postdoctoral fellows selected for stipends. Matching funds will be provided by the Institution to cover other

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costs. It is anticipated that approximately 100 individuals will attend in the first year, and that this number may increase in subsequent years. The Organizing/Scientific Committee will consist of members from research disciplines that represent the integrated/educational concept of the Symposium. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CAR & ADENOVIRAL GENE THERAPY FOR DIABETIC RENAL DISEASE Principal Investigator & Institution: Bhatt, Udayan Y.; Internal Medicine; Ohio State University 1960 Kenny Road Columbus, Oh 43210 Timing: Fiscal Year 2003; Project Start 01-MAR-2003; Project End 31-JAN-2008 Summary: (provided by applicant): Replication deficient recombinant adenoviruses (rAv) are potent vectors for DNA transfer (transduction). Diabetic glomerulosclerosis is a potential target for rAv-based forms of gene therapy. The coxsackie adenovirus receptor protein (CAR) mediates rAv infection. Despite widespread use of rAv, the mechanisms of CAR-mediated viral entry into cells are not well characterized. Therefore, the scientific objective of this proposal is to define CAR receptor expression, function, and utility in controlling transduction. The project has 3 Specific Aims. Aim #1 will further characterize the expression of CAR in normal and diabetic kidney. Aim #2 will investigate the functional consequences of rAv engagement of CAR under normal and diabetic conditions. Beginning with DNA microarray analysis followed by confirmatory studies using quantitative PCR, the gene activation profile resulting from CAR engagement by rAv will be elucidated. Aim #3 will explore the clinical utility of CAR in regulating rAv mediated gene transfer. The goal of this aim is to control rAv transduction by using a doxycycline-responsive CAR transgene. Taken together, these studies will define CAR expression, function, and utility in the development of rAvbased forms of gene therapy for diabetic glomerulosclerosis. The scientific goals of this project are a natural extension of the candidate's current studies (NIH 1F32 DK1006401). The educational curriculum developed by the candidate and his sponsors will complement the scientific studies in developing a comprehensive training experience. The educational curriculum employs a multi-faceted approach consisting of didactics, seminars, and meetings. These activities provide the foundation for the applicant in the pursuit of a career as a physician scientist. The long-term career goal is to develop into a translational scientist with all of the clinical and basic investigative tools necessary to design and apply novel forms of gene therapy for kidney disease. In this regard, the candidate will continue his relationship with his current mentor, N. S. Nahman, Jr., M.D. Dr. Nahman provides an excellent role model as a clinician scientist. Chandan K. Sen, Ph.D., serves as a cosponsor on the project and brings a diverse background in the basic sciences for the candidate's training plan. Thus, the candidate's scientific plan, educational curriculum, and association with effective mentors ensure an excellent career development experience. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CARDIOVASCULAR CELL AND GENE THERAPY CONFERENCE Principal Investigator & Institution: Hajjar, Roger J.; Assistant Professor of Medicine; Massachusetts General Hospital 55 Fruit St Boston, Ma 02114 Timing: Fiscal Year 2002; Project Start 01-JUN-2002; Project End 31-MAY-2005 Summary: (provided by applicant): Cardiovascular disease is a major cause of morbidity and mortality in the United States. New treatments are being formulated based on a

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better understanding of the signaling pathways involved in the pathogenesis of cardiovascular diseases. Furthermore cell replacement therapy has recently emerged as a novel way of correcting contractile and vascular deficiencies in cardiovascular diseases. The focus of this yearly symposium will be on the use of somatic gene transfer and cell therapy in cardiovascular diseases. Targeting genes to the heart through somatic gene transfer or transplanting stem cells have the potential to alter our approach to patients with cardiovascular diseases. Gene and cell therapy allow us to test hypotheses about mechanisms of disease, and, it is hoped, tailor therapy accordingly. This symposium will bring together scientists from industry, clinicians and basic scientists. It will be a multidisciplinary meeting that should bring together people who are beginning to have regular dialogues but whose traditions have been somewhat separate Through this combination of investigators with multidisciplinary backgrounds, diverse scientific perspectives will be brought into focus on gene and cell therapy. The conference will consist of cover nine separate sessions over two and a half days. The topics of the sessions are 1) Viral vectors, 2) Delivery approaches, 3) Lessons from development, 4) Cell therapy, 5) Targeting Ischemic Heart Disease, 6) Targeting hypertrophy and growth, 7) Targeting heart failure and arrhythmias, 8) Targeting vascular disease, and 9) NIH programs and regulatory issues.The conference will be organized on a yearly basis in April. All the logistics of the first conference along with speaker commitments have been completed and the assigned date of the first conference is April 8-20, 2002. The convergence of investigators from different fields which are typically separate will hopefully foster greater collaborative efforts in gene and cell therapy and provide better understanding and treatment modalities for cardiovascular diseases. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CELL TARGETING LIGANDS AND VECTORS FOR CLL Principal Investigator & Institution: Barry, Michael A.; Associate Professor; Molecular and Human Genetics; Baylor College of Medicine 1 Baylor Plaza Houston, Tx 77030 Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2006 Summary: The inability to deliver therapeutic agents to chronic lymphocytic leukemia (CLL) cells in vitro or in vivo is a fundamental impediment to any drug or gene therapy for this disease. For CLL, gene therapy approaches to deliver immunostimulatory gene products to CLL cells hold great promise for treating this disease, however, current gene therapy vectors are not optimal for this application. In particular, most gene therapy vectors fail to effectively deliver genes into CLL cells making ex vivo approaches inefficient. By contrast, most vectors mediate robust, but non-specific gene delivery to many non-CLL cells in the body. This inability to deliver genes into CLL cells combined with a robust ability to deliver genes into the liver and immune cells makes current vectors unsafe for in vivo applications against CLL. Given that current vectors are inadequate for these CLL applications, this project proposes to develop CLL-targeting ligands and vectors to increase CLL transduction in vitro and mediate CLL-specific gene delivery in vivo. Towards this goal, the project will pursue the following Specific Aims: Specific Aim 1: To generate cell-targeting ligands against human CLL cells. Specific Aim 2. To translate CLL- targeting ligands onto adenovirus gene therapy vectors and test for improved CLL transduction in vitro. Specific Aim 3. To optimize the affinity and specificity of CLL-binding and CLL- targeting peptides. Specific Aim 4. To test the ability of CLL- targeting adenoviral vectors to mediate CLL-specific gene delivery in mouse xenografts of human CLL cells. We will use our peptide libraries to select targeting ligands against a panel of primary patient CLL cancer cells with the rationale

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that ligands generated here can be translated directly into clinical application against human tumors. As targeting ligands are identified, they will be optimized and tested for their ability to increase CLL transduction and CLL-specific transduction by adenoviral gene therapy vectors in vitro. Promising targeting vectors will then be tested in vivo in mouse xenografts for their ability to mediate human CLL-specific transduction in the context of a living organism. If successful, this work will lay the foundation for future applications of these CLL-targeting ligands and vectors for gene therapy for CLL patients through the clinical arm of the Center for Cell and Gene Therapy at Baylor College of Medicine. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: COMBINATION OF GM-CSF WITH TKR GENE THERAPY Principal Investigator & Institution: Chhikara, Madhu; Advantagene, Inc. 160 Paulson Rd Waban, Ma 02468 Timing: Fiscal Year 2003; Project Start 04-APR-2003; Project End 31-MAR-2004 Summary: (provided by applicant): Combined gene therapy (Herpes Simplex Virus Thymidine Kinase + Anti-herpetic Prodrug) and radiation therapy (TKR therapy) is a novel approach in the armamentarium against cancer. This radio-gene therapy combination creates a new spatial co-operation whereby two local treatment modalities have demonstrated enhanced local and metastatic tumor control and prolongation of survival. We have taken TKR therapy into clinical studies and currently have more than 60 patients in a Phase II trial in prostate cancer. Our corporate strategy is to add novel therapies (gene therapy) with distinct, non-additive toxicity profiles to the standard-ofcare (surgery, chemo- or radio-therapy) to enhance cancer cure or decrease treatment morbidity. TKR shows a potent systemic anti-metastatic effect, however, we believe this effect could be significantly enhanced by the addition of a cytokine that could further stimulate antigen presentation to the immune system. This Phase I application proposes to develop the reagents and single gene animal data to adequately evaluate the use of GM-CSF in combination with TKR. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: COMBINED IL-2 AND MHC CLASS II GENE THERAPY FOR CANCER Principal Investigator & Institution: Xu, Minzhen; Antigen Express, Inc. 100 Barber Ave Worcester, Ma 01606 Timing: Fiscal Year 2001; Project Start 01-AUG-2001; Project End 31-JUL-2002 Summary: Antigen Express has developed technology to force the presentation of endogenous tumor antigens in a manner that produces an effective tumor cell vaccine. The key to this strategy is the generation of tumor cells that express MHC Class II molecules in the absence of the MHC Class II associated invariant chain (Ii protein). We are now developing an in vivo gene therapy strategy for inhibition of Ii protein expression (using Ii reverse gene constructs) in tumors. As not all tumors are MHC Class II positive, we are combining Ii inhibition with IL-2 and/or MHC Class II transactivator (CIITA) gene therapy. IL-2 is advantageous in that it stimulates the expansion of antigen specific T cells and causes interferon-gamma production (which is a good inducer of MHC Class II molecules) while CIITA is a potent and direct MHC Class II inducer. We will establish the ability of IL-2 and CIITA gene therapy vectors to induce MHC Class II molecules in vivo using a murine renal cell carcinoma model and an ovarian ascites model. Selective inhibition of Ii will be accomplished using a reverse

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gene construct. Finally, we will establish the therapeutic efficacy of combined IL-2 (or CIITA) and Ii reverse construct gene therapy on established Renca tumors and MOT ascites in vivo. Success will trigger Phase II studies, wherein we will develop appropriate constructs using human gene sequences, demonstrate activity in primary human tumor samples and complete all necessary studies requisite to an IND filing. PROPOSED COMMERCIAL APPLICATION: Immunotherapy represents a novel form of cancer therapy that complements existing treatment modalities. Successful demonstration of the augmentation of tumor immunogenicity in a manner that generates a robust anti-tumor immune response will satisify a significant unmet need in modern health care. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CONFERENCE ON GENE THERAPY 2001: A GENE ODYSSEY Principal Investigator & Institution: Kay, Mark A.; Professor; Keystone Symposia Drawer 1630, 221 Summit Pl #272 Silverthorne, Co 80498 Timing: Fiscal Year 2001; Project Start 01-JAN-2001; Project End 31-DEC-2001 Summary: (taken from the application) Gene therapy approaches have made important strides in the last several years. This is in large part due to the technical advances in vector delivery approaches. A number of preclinical studies has demonstrated reduced toxicity, with prolonged and therapeutic levels of clinically relevant gene products from various tissues. This is likely to lead to early success in new clinical trials under development. However, not all problems have been solved and there are many diseases with complex pathophysiology for which gene therapy has yet to show promise. Moreover, there are new safety obstacles that have arisen that will likely preclude treating less severe medical conditions. The meeting will highlight recent successes and barriers that still challenge the gene therapy community as it relates to basic science, preclinical animal studies, and clinical trials. The meeting will be held approximately 6 months away from the American Society for Gene Therapy (ASGT) meeting and will feature important new results that have occurred during the intervals of the yearly ASGT meetings. The meeting will also offer a format for research summaries as well as new primary data for newcomers to the field. The environment will be conducive for young investigators to interact directly with more senior investigators. This interaction is likely to allow trainees to find new mentors for advanced training (e.g., senior students who wish to pursue post-doctoral training; therefore, student participation is a critical component of the meeting.The plenary speakers blend a mix of senior, middle career, and young rising stars. The senior scientists who we anticipate will have new important accomplishments in 2001 were selected to help attract trainees to the conference. Session chairs were selected from the senior leaders in the specific target areas. All of the speakers (including the younger investigators) were selected on their recent past record of superb scientific accomplishment and verbal articulation. The younger scientists were included to add more depth and present fresh new ideas. We have selected 7 more "junior" speakers who have presented outstanding data at a session during the last ASGT meeting. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: CONSUMER PERSPECTIVES ON THE PROMISE OF CF GENE THERAPY Principal Investigator & Institution: Stockdale, Alan; Education Development Center, Inc. 55 Chapel St Newton, Ma 02458

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Timing: Fiscal Year 2001; Project Start 01-JUN-2000; Project End 31-MAY-2003 Summary: (Adapted from the Investigator's Abstract): Although gene therapy has been widely publicized as an impending cure for numerous diseases, including cystic fibrosis (CF), little empirical research has been conducted to determine the impact of this publicity, and its promise of cure, on patients and their families. Such knowledge of consumer perspectives is essential for enhancing trust in the biomedical establishment, for protecting vulnerable consumer groups from potential harms, and for the design of effective consumer and clinician educational materials. The Education Development Center, Inc., in collaboration with the International Association of Cystic Fibrosis Adults (IACFA), Temple University, St. Vincent's Hospital, and Brigham and Women's Hospital propose a study to (a) assess how gene therapy research, and the publicity surrounding it, are perceived by adults with CF, parents of children with CF, and CF Center physicians; (b) determine the knowledge, beliefs and attitudes that inform consumer perceptions and felt needs; (c) discover how those needs and perceptions affect patients' and families' treatment decisions and life plans; and (d) identify the ethical and psychosocial implications of gene therapy developments on patients, families, and their physicians. The team will do a content analysis of information materials disseminated to CF patients and the public; conduct qualitative interviews with 30 adult CF patients, 30 parents of children with CF, and 30 physicians. These interviews will be used to develop a survey questionnaire to be administered to al consenting adult CF patients and parents of children with CF cared for at all 13 New England CF treatment centers (N=1595) and to IACFA members in the U.S. (N=713). Findings will be presented to an expert advisory group of CF consumers, researchers, clinicians, ethicists, and representatives of other disease perspectives, such as AIDS and cancer. This goup will assist the study team in drafting recommendations for how best to reconcile the promise with the reality of CF gene therapy and identify the possible implications for other areas of gene therapy research. Recommendations will focus on consumer and clinician education; however, they will also have relevance for a broader array of issues, such as recruitment into gene therapy trials and priorities for research and care delivery. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CORE CENTER FOR GENE THERAPY Principal Investigator & Institution: Miller, Arthur D.; Member; Pathology; University of Washington Seattle, Wa 98195 Timing: Fiscal Year 2001; Project Start 15-JAN-1994; Project End 31-DEC-2003 Summary: The Core Center for Gene Therapy at the University of Washington School of Medicine (UWSM) is under the direction of A. Dusty Miller, Ph.D. (Program Director), Affiliate Professor of Pathology and a Member of the Fred Hutchinson Cancer Research Center, and Associate Program Direction Bonnie W. Ramsey, M.D., Professor of Pediatrics. The Center brings together gene therapy research efforts at UWSM, the Fred Hutchinson Cancer Research Center, Children's Hospital and Medical Center, and the Veteran's Administration Medical Center in Seattle, and includes close ties with the Cystic Fibrosis Research Program headed by Dr. Bonnie Ramsey, and the General Clinical Research Center (GCRC) headed by John Brunzell, M.D. The Center will focus on three main areas of research: 1) development of viral vectors and procedures for the treatment of cystic fibrosis, 2) development of retroviral vectors for gene transfer and expression of therapeutic genes in hematopoietic and lymphoid cells in humans, and 3) development of retroviral vectors of gene transfer and expression of therapeutic genes in hematopoietic and lymphoid cells in humans, and 3) development of methods to

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deliver circulating proteins such as erythropoietin and clotting factors fro treatment of human disease. A total of 35 investigators who receive over $10 million in independent external support will regulatory use the core, facilities and collaborate in development of gene therapy protocols. This work will be supported by an Administrative Component and five other core facilities: Human Applications Core, Vector Development Core, Immunohistochemistry Core, Hematopoietic Cell Transduction Core, and Animal Core. In addition, the pilot and feasibility program will consist of 8 projects which are related to CF gene therapy and 2 projects which are directed towards gene therapy in other genetic disorders. The CF-related projects include the following: 1) The Biology of Adeno-Associated Virus and Vector (J. Allen and D. Miller), 2) Recruitment of the Adenoviral Pre-terminal Protein (pTP) for nuclear Import of LVDNA in Non-Dividing Cells (A. Lieber), 3) Identification of P. aeruginosa Virulence Determinants using an Invertebrate Model (C. Manoil), 4) Organ immunosuppression for donor lungs: Optimizing a safe approach using non-viral gene therapy (M. Allen), 5) Evaluation of lentiviral vectors for airway gene therapy (C. Halbert), 6) Refining nonviral gene therapy approaches for CF (M. Horowitz), 7) CFTR gene targeting by adenoassociated virus vectors (D. Russell), 8) Improving transgene expression by adenoviral vectors modified to co-express murine CD8 molecules (P. Fink). The 2 non CF-related projects include 1) Development of a coumermycin-responsive proliferation switch (C.A. Blau) and 2) A Novel approach for the modulation of host immune responses to gene modified cells (H.P. Kiem). Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: CYTOKINE REGULATION OF GENE THERAPY VECTORS Principal Investigator & Institution: Bromberg, Jonathan S.; Surgical Director; Center for Gene Therapy & Molecular Medicine; Mount Sinai School of Medicine of Nyu of New York University New York, Ny 10029 Timing: Fiscal Year 2001; Project Start 01-FEB-1998; Project End 31-JAN-2003 Summary: (Adapted from the applicant's abstract) Gene transfer offers unprecedented potential to modify physiologic phenomena. Unfortunately, insufficient and transient gene expression limit this potential. For example, the investigators have been able to use gene transfer methods to introduce immunosuppressive cytokines (IL-10, TGFb) into cardiac allografts to prolong graft survival, but have been unable to achieve permanent engraftment or tolerance. Although the use of strong viral promoters has improved efficacy somewhat, limited gene expression is still the major barrier to gene therapy. It is increasingly recognized now that specific immune responses to vector components and gene transfer products result in destruction of vectors or infected cells and are a significant impediment to stable expression. Preliminary data now demonstrate that the T lymphocyte derived cytokines IFNg and TNFa limit gene expression, without killing cells, by mechanisms involving negative regulation of transcription, especially from certain viral promoters and enhancers such as HCMVie and RSV-LTR. Since gene therapy is considered for treatment of diseases which are associated with many changes in cytokines (e.g., cancer, AIDS, transplantation, atherosclerosis, autoimmunity, ischemia-reperfusion); it is hypothesized that cytokines inhibit vector gene expression in transduced cells and that determination of the mechanisms involved in cytokineregulated gene expression will fundamentally alter the design of vectors for gene transfer and gene therapy. The objectives of the proposal are to define how cytokines regulate the expression from gene transfer vectors in order to improve strategies for gene delivery and control and thereby optimize protocols for gene therapy. The specific aims are: (1) demonstrate that TNFa and IFNg differentially regulate expression from

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selected viral and cellular promoters; (2) determine how cytokine response elements regulate transcription; (3) map the cytokine-responsive negative regulatory elements; and (4) demonstrate that manipulation of cytokine responses and response elements will improve gene transfer and gene therapy in an in vivo transplantation model. Experiments will entail the manipulation of cytokines and vector promoter-reporter constructs in vitro in primary myoblasts and the C2C12 myoblast cell line to define cytokine-initiated, promoter-dependent regulation of transcription. Promoter constructs will be transfected into murine cardiac allografts to probe cytokine initiated regulation of transferred gene expression in vivo, and to show that this regulation may be manipulated to improve the utility of gene transfer and gene therapy. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: DEVELOPMENT OF RTVP-1 GENE THERAPY FOR PROSTATE CANCER Principal Investigator & Institution: Kadmon, Dov; Professor; Baylor College of Medicine 1 Baylor Plaza Houston, Tx 77030 Timing: Fiscal Year 2002; Project Start 15-JUL-2002; Project End 31-MAY-2007 Summary: (provided by applicant): Although limitations regarding the delivery and targeted expression of potentially therapeutic genes remain, it is important to continue to work toward effective cancer gene therapy including gene therapy for prostate cancer. We have previously developed in situ adenoviral vector-mediated gene therapy for HSV-tk+GCV gene therapy. More recently, we have continued with more immunomodulatory gene therapy based approaches and a Phase I clinical trial involving adenoviral vector-delivered IL-12 gene therapy in patients with prostate cancer is pending. We recently identified a gene, RTVP-1 (related to testes-specific vespid and pathogenesis proteins), that possessed direct cytotoxic and immunomodulatory activities that could offer unique opportunities for specific gene therapy approaches. We have recently demonstrated that mouse RTVP-1 (mRTVP-1) is a direct p53 target gene and is upregulated by p53 in mouse and human prostate cancer cells. Further analysis revealed that RTVP-1 mRNA is abundant in normal mouse and human prostatic epithelial cells, but is progressively downregulated in primary tumors and has only low-level expression in metastatic tissues. In an orthotopic mouse model of metastatic prostate cancer adenoviral vector-mediated mRTVP-1 (AdmRTVP-1) expression leads to apoptosis, potent growth suppression, anti-angiogenic and antimetastatic activities, and importantly local and systemic immune response. We propose to conduct further preclinical studies to develop optimized adenoviral vector systems for RTVP-1 gene therapy. We will also evaluate specific RTVP-1 gene-modified cell-based strategies including RTVP-1 gene-modified tumor cell vaccines. We have considerable experience with alternative promoters for adenoviral vectors and will fully evaluate non-specific promoters such as CMV relative to a prostate selective promoter such as ARR2PB and a prostate cancer/endothelium targeting promoter (caveolin-1) in replication-defective and attenuated replication competent adenoviral vector systems. These preclinical studies will proceed concurrently with the initiation of a Phase I trial in which replication-defective adenoviral vectors will be used to transfer the human RTVP1 gene directly into prostate cancer in patients who will subsequently be subjected to radical prostatectomy. Comprehensive analysis of the effects of RTVP-1 locally and systemically will be completed in this clinical trial. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: DOPAMINE REGULATION IN PARKINSONIAN RAT BY GENE THERAPY Principal Investigator & Institution: Kang, Un Jung.; Associate Professor; Neurology; University of Chicago 5801 S Ellis Ave Chicago, Il 60637 Timing: Fiscal Year 2002; Project Start 01-AUG-1993; Project End 31-MAR-2007 Summary: (provided by applicant): L-3,4-dihydroxyphenylalanine (L-DOPA) is the mainstay of therapy for Parkinson's disease (PD). Chronic L-DOPA therapy is limited, however, by the development of motor response complications, such as progressively shorter duration of improvement in akinesia (wearing-off) and the appearance of LDOPA-induced abnormal involuntary movements. Innovative methods of sustained and localized central nervous system (CNS) dopamine delivery may further optimize LDOPA therapy. Such methods are being explored clinically by CNS transplantation studies with fetal dopaminergic neurons and experimentally by neuronal stem cell implants and gene therapy. Our studies during the past funding cycles have defined optimal sets of genes necessary for dopamine replacement using ex vivo gene therapy using genetically modified fibroblasts. We also developed rat behavioral models that are relevant to the akinesia of PD patients. Using akinesia behaviors, we have noted that lesion severity has a major influence on the shortening of the response duration with minor contribution by the chronic intermittent L-DOPA therapy. Therefore, studies proposed in this continuing renewal application will determine the optimal parameters of gene therapy to improve akinesia and minimize and prevent motor response complications. We will use adeno-associated virus vectors to deliver tyrosine hydroxylase and guanosine triphosphate (GTP) cyclohydrolase 1 genes. The optimal combination of anatomical targets for gene therapy to improve akinesia will be defined by examining the effects of gene therapy delivered to basal ganglia structures, such as subthalamic nucleus, substantia nigra par reticulata, that receive dopaminergic inputs, in addition to the striatum. The optimal timing to initiate dopamine replacement gene therapy to forestall development of motor response complications will also be examined. These results will have significant implications beyond dopamine replacement gene therapy proposed here and guide other therapies such as fetal dopaminergic cell transplantation, neurotrophic factor therapy, stem cell therapy, and other CNS targeted delivery systems. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: DUAL GENE THERAPY FOR HEART FAILURE Principal Investigator & Institution: Nuss, H B.; Medicine; Johns Hopkins University 3400 N Charles St Baltimore, Md 21218 Timing: Fiscal Year 2001; Project Start 30-SEP-2000; Project End 31-JAN-2002 Summary: Heart failure is a multifactorial disease, having both electrical and contractile components. Downregulation of key potassium channels and concomitant prolongation and instability of repolarization, predispose the heart to arrhythmias. Meanwhile, downregulation of the sarcoplasmic reticulurn Ca2+ ATPase and concomitant calcium handling abnormalities contribute to depressed myocardial contractility. The electrical abnormalities and the contractile abnormalities are not mutually exclusive. Alterations in the control of membrane voltage will modulate the triggered release of Ca2+ from the sarcoplasmic reticulurn and, conversely, alterations in the intracellular calcium transient will influence membrane potential. It is the interplay between the electrical and contractile abnormalities of heart failure which compounds the complexity of abnormalities and confounds the design of successful treatments. Novel antiarrhythmic

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gene therapy based upon manipulation of a select K channel gene alone to decrease susceptibility to arrhythmias may lead to depressed contractility, which is already depressed in heart failure. Conversely, genetic manipulation of a SR Ca2+ ATPase protein alone, to amplify contractility, may create a proarrhythmic substrate in a failing heart which is already predisposed to fatal arrhythmic events. Thus, monogenic strategies, based upon selective overexpression of a single gene, may not suffice to correct heart failure abnormalities because of the interplay between excitation and contraction in cardiac muscle. This proposal seeks to offset abnormalities of tachycardia, pacing- induced heart failure in rabbits using combination gene therapy: overexpression of a select K channel gene and a SR Ca2+ ATPase gene in tandem. As a prelude we will test the hypotheses that gene therapy targeted to correct the electrical abnormalities alone or the calcium handling abnormalities alone will result in adverse conditions. The proposal focuses on potassium channels and SR Ca2+ ATPase's that are highly relevant to repolarization and contractility in the human heart failure. In vivo adenoviral mediated gene transfer, cellular and cardiac electrophysiology, and quantitative modeling will be used to investigate repolarization and calcium handling with the goal of correcting the electrical and contractile abnormalities in heart failure. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: EBV BASED STRATEGIES FOR AIDS RELATED MALIGNANCIES Principal Investigator & Institution: Kenney, Shannon C.; Professor; Medicine; University of North Carolina Chapel Hill Office of Sponsored Research Chapel Hill, Nc 27599 Timing: Fiscal Year 2001; Project Start 13-FEB-1995; Project End 31-JAN-2003 Summary: AIDS patients frequently develop central nervous system (CNS) lymphomas, for which there is currently no effective treatment. These AIDS-related CNS lymphomas all carry the Epstein-Barr virus (EBV) genome and express the EBV protein, EBNA 1. The ubiquitous presence of EBV in the AIDS- related CNS lymphomas presents unique opportunities for targeting these malignant cells for destruction using gene therapy approaches. In addition, gene therapy strategies which distinguish between proliferating (tumor) versus nonproliferating (neuronal) cells, and which have already been shown to cure glioblastomas in animal models, may likewise be useful in the treatment of CNS lymphomas. In these studies, we propose to use a recently developed SCID mouse model of CNS lymphoma to develop a variety of gene therapy approaches for the potential treatment of CNS lymphomas in AIDS. In our first specific aim, we will exploit the presence of EBV in AIDS-related CNS lymphomas and insert the "suicide" gene HSV-TK (herpes simplex virus thymidine kinase), which confers ganciclovir sensitivity to cells, into a retroviral vector under the control of the EBV element, oriP. OriP, which contains both an origin of replication and a transcriptional enhancer element, requires the EBV protein EBNA 1 for function. In our second specific aim, we will insert the HSV-TK gene (under the control of the EBNA 1 dependent oriP enhancer element) into a plasmid vector and use the molecular-conjugate method to deliver DNA. The molecular conjugates will include a peptide containing the CD21 ligand (expressed on B cells) to specifically deliver the HSV-TK DNA only to tumor cells. In our third specific aim, we will attempt to induce lytic destruction of the EBV-infected lymphoma cells by using gene therapy to over-express the EBV immediate-early protein, BZLF1. Over-expression of BZLF1 is known to induce lytic EBV infection and consequent lysis of the host cell. In the final specific aim, we will examine the ability of defective HSV mutants to lyse EBV-transformed B cells in vitro, and cure EBV-induced CNS lymphomas in vivo. The studies proposed will not only be important in the

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development of an animal model system for AIDS-related CNS lymphomas, but should provide critical information regarding which therapeutic approaches are most promising for eventual human trials. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: ENHANCEMENT OF HUMAN ISLET ENGRAFTMENT BY GENE THERAPY Principal Investigator & Institution: Gaber, Osama A.; Chief; Surgery; University of Tennessee Health Sci Ctr Health Science Center Memphis, Tn 38163 Timing: Fiscal Year 2001; Project Start 15-JUN-2000; Project End 31-MAY-2004 Summary: Results of clinical islet transplantation remain disappointing despite sporadic reports of short-term insulin independence. Failure of a large proportion of islet grafts to function has been ascribed to poor islet recovery from cadaveric pancreas, lack of adequate islet vascularization following transplantation and nonspecific immune mediated islet destruction or allogenic rejection. To overcome the problem of primary nonfunction, our laboratory has developed procedures to improve viability of isolated islets including novel culture techniques. In addition, we developed a NOD-SCID mouse model that allows viability testing of islets in vivo prior to transplantation. Furthermore, we embarked on a gene immunotherapy approach to improve human islet survival in vivo. We hypothesized that transfection of human islets with TGFbeta1 will prevent structural and immune mediated destruction of islets. Reconstitution of the NOD-SCID mouse with human CD34+ cells in the presence or absence of thymic fragments, will allow us to test the impact of the genetic manipulation in a clinically relevant model. Our specific aims are to test the hypothesis that: l) transient, low level of TGFbeta1 expression by human islets transduced with Ad-RSV-TGFbeta1 vector will improve and prolong in vivo function; 2) transient TGFbeta1 expression by human islets will promote their implantation by enhancing extracellular matrix formation and neovascularization, resulting in preservation or improvement of in vivo function; 3) expression of TGFbeta1 by human islets will protect against nonspecific inflammatory destruction in vivo; and 4) expression of TGFbeta1 by human islets will protect against allogenic immune destruction. The proposed work emphasizes the utilization of human islets for genetic modification and should provide clinically relevant data regarding the structural and immunological requirement for successful engraftment. The long-term goal of this multidisciplinary research is to develop an effective gene therapy approach that could be clinically utilized to achieve successful human islet transplantation. Future experiments based on the findings of this research would allow us to examine the utility of other gene candidates or other vectors that could complement our gene therapy program in islet transplantation. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: ENZYME AND GENE THERAPY OF MPS I IN ANIMAL MODELS Principal Investigator & Institution: Neufeld, Elizabeth F.; Professor and Chair; Biological Chemistry; University of California Los Angeles 10920 Wilshire Blvd., Suite 1200 Los Angeles, Ca 90024 Timing: Fiscal Year 2001; Project Start 01-AUG-1987; Project End 31-JUL-2003 Summary: The molecular basis of Mucopolysaccharidosis I (MPS I, Hurler, Hurler/Scheie and Scheie syndromes) is mutations in the gene encoding alpha-Liduronidase, resulting in absence of enzyme activity, accumulation of undegraded glycosaminoglycans, and systemic disease. Because alpha-L-iduronidase, a lysosomal

22

Gene Therapy

enzyme, can be secreted as well as taken up by receptor-mediated endocytosis, MPS I has long been considered a prime candidate for replacement therapy. Alpha-LIduronidase provided by donor cells of hematopoietic origin (probably macrophages) is thought to be responsible for changes in disease progression that are seen after bone marrow transplantation. The course of the disease can also be altered by administration of recombinant alpha-L-iduronidase. The therapeutic effect of the enzyme previously observed in the canine MPS I model had been promising enough to generate a clinical trial in MPS I patients. But even though recombinant alpha-L-iduronidase may soon become available as a pharmaceutical, there is still a need for developing effective and long-lasting gene therapy. To have a suitable animal model, we have produced mutant mice by targeted disruption of the alpha-L-iduronidase gene. Aim 1 is to define the phenotype of the MPS I mouse model at the biochemical, pathological, behavioral and clinical levels. Aim 2 is to determine the effect of administration of human recombinant alpha-L-iduronidase on the disease phenotype, in order to provide a basis of comparison for gene-based procedures. Aim 3 is to compare transplantation of genemodified bone marrow over-expressing human alpha-L-iduronidase with transplantation of bone marrow expressing normal levels of the enzyme, for effectiveness in altering the disease phenotype. Aim 4 is to determine the effectiveness of tetracycline-inducible alpha-L-iduronidase expression in macrophages as a means of enzyme delivery to affected organs, in particular to the brain, as well as to compare it with the above procedures for ability to alter the disease phenotype. The proposed studies represent steps in our long-term program to develop treatment for patients affected with MPS I. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: EVALUATION OF CELLULAR GENE THERAPY FOR OI Principal Investigator & Institution: Niyibizi, Christopher; Associate Professor; Orthopaedic Surgery; University of Pittsburgh at Pittsburgh 350 Thackeray Hall Pittsburgh, Pa 15260 Timing: Fiscal Year 2001; Project Start 18-AUG-2000; Project End 31-JUL-2002 Summary: (Taken from the application): Ontogenesis imperfecta (0I) is a group of heritable disorders of connective tissue whose common feature is bone fragility. Most forms of OI are the result of mutations in the genes that encode proalpha1 and proalpha2 polypeptide chains of type I collagen the major protein of bone. The longterm objective of the proposal is to develop strategies using cell therapy or gene therapy for the treatment of some forms of OI and other bone related diseases. The focus of this research proposal is to utilize a mouse model of human OI (oim) that has defective synthesis of proalpha2(I) chains to evaluate the feasibility of reversing OI defects and other bone related disease by either bone marrow stromal cell transplantation or delivery of normal collagen genes to bone. The aims are: (1) to evaluate the potential of bone marrow stromal cells from normal donor mice transplanted into syngeneic OI mice to engraft, synthesize and deposit normal type I collagen in bone matrix of the recipient mice and (2), to test the feasibility of gene therapy by evaluating the potential of bone marrow stromal to be transduced with collagens and to deliver and express the genes in bone. As a prelude to this, bone marrow stromal cells will be established from the normal mice by flushing the marrow from femurs and tibias. The established bone marrow stromal cells will be transduced with a retroviral vector containing LacZ and neo~ genes (BAG-LacZ neo) prior to transplanting them to the recipient mice to aid in cell tracking. The bone marrow stromal cells established from normal mice will be injected in the femurs of the irradiated or non-irradiated OI mice and the expression of

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the proalpha2(I) chains will be determined by immunofluorescence localization using a mouse alpha2(I) antiserum and cyanogen bromide digestion of the bone collagen of the recipient mice. To test for the collagen gene expression by bone marrow stromal cells, the cells will be transduced with an adenovirus containing the mouse proalpha2(I) collagen gene and the transduced cells will be injected in the femurs of the o=s mice. The alpha2(I) collagen expression will be determined by immunofluorescence localization using the mouse proalpha2(I) and the cyanogen bromide digestion of the tissue. Future plans will involve determination of the amount of collagen made by the transplanted cells in the bones of the recipient mice and the assessment of the bone quality by radiographic, histological and biomechanical analysis of the bones of the recipient mice. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: EVALUATION OF DELIVERY SYSTEMS FOR RENAL GENE THERAPY Principal Investigator & Institution: Klotman, Mary E.; Professor and Chief; Mount Sinai School of Medicine of Cuny New York, Ny 10029 Timing: Fiscal Year 2001; Project Start 01-AUG-2000; Project End 31-JUL-2003 Summary: A number of renal diseases are either a result of a genetic defect or the result of a chronic systemic disease resulting in renal injury from both local as well as systemic production of specific mediators. Gene therapy either to replace a missing factor or to suppress the production of deleterious exogenous (virus) or endogenous mediators represents a promising new therapeutic approach for chronic renal disease. The success and safety of genetic therapy will depend on both the successful expression of the exogenous gene as well as the efficient and specific delivery of the gene to the appropriate target cell. Thus, optimum genetic constructs for expression in specific renal cell types and optimum systems for either ex vivo or in vivo delivery of an exogenous gene to the kidney need to be defined. The function of strong constitutive promoters as well as potentially more renal-specific promoters will be examined in primary cells from human and mouse kidney (including mesangial and fibroblasts as well as proximal tubule, thick ascending limb and distal tubule cells) as well as in vivo. Each of these promoters will be inserted upstream from the indicator genes, beta-gal(alone) and luciferase (with therapeutic genes), which will allow convenient tracking and quantitation of expression in vivo. Since many of the diseases resulting in renal failure are chronic in nature, a delivery system that will result in the long-term stability and expression of a therapeutic gene is desirable; best achieved through integration of the gene into the host chromosome. The defective parvovirus, adeno-associated virus (AAV), efficiently integrates into the host genome and has the added advantage of efficient transduction into non-dividing cells making it an attractive candidate for gene therapy in the kidney. Furthermore, data suggest that delivery of a therapeutic gene flanked by genetic components of AAV system delivered as DNA in a liposome might result in the efficient integration of a delivered gene in a host cell without the use of a recombinant virus. These two delivery systems will be used to define the efficiency of gene delivery in vitro to primary renal cells and in vivo (using a murine model) to renal cells. Specific issues that will be addressed will include the efficiency of delivery to specific cell types, strategies to enhance transduction particularly to non-dividing renal cells, integration characteristics of the AAV-based constructs and the potential toxicity when a gene is delivered in this vector system. A model of disease, HIV-1 associated nephropathy in the transgenic mouse will be utilized to define efficacy of gene inhibition in a pathogenic model. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Gene Therapy

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Project Title: FUSOGENIC MEMBRANE GLYCOPROTEINS FOR GENE THERAPY Principal Investigator & Institution: Vile, Richard G.; Consultant; Mayo Clinic Rochester 200 1St St Sw Rochester, Mn 55905 Timing: Fiscal Year 2001; Project Start 31-AUG-2001; Project End 31-JUL-2006 Summary: (provided by applicant): The goal of this project is to exploit the highly potent cytotoxic properties of a novel class of genes, called Fusogenic Membrane Glycoproteins (FMG), for the gene therapy of prostate cancer. Many viruses kill their target cells by causing cell fusion through binding of the viral envelope protein on an infected cell with its cellular receptor on neighboring cells. The result is the formation of large, multinucleated syncytia which eventually become non-viable and die. We have used gene transfer of the cDNAs of three different types of FMG to prostate tumor cells. The cytotoxicities of these FMG were consistently greatly superior to that of conventional suicide genes and the local bystander killing effects were at least one log greater than those of the HSVtk/Ganciclovir system. FMG tested so far kill target cells via nonapoptotic mechanisms with the concomitant induction of immune stimulatory signals such as heat shock proteins. We now hypothesize that these properties of FMGmediated tumor cell killing can be exploited, and enhanced, to generate more effective gene therapies for prostate cancer. We will characterize in detail the mechanisms by which FMG gene transfer leads to cell death to understand what regulates the efficiency of syncytial killing and how to improve it for therapeutic purposes. We will investigate how the mechanisms of syncytial killing can be enhanced in vivo to stimulate potent immune responses against tumor metastases. This will be done by constructing vectors in which additional immune stimulatory genes, such as GM-CSF, are co-expressed with FMG. In addition, we will take full advantage of collaborations within the SPORE group to investigate whether co-expression of an FMG with the sodium iodide symporter (NIS) gene can augment the cytotoxicity of FMG alone by and allowing increased tumor cell killing in combination with radioiodine treatment. We will also generate FMGinduced prostate tumor cell-dendritic cell hybrids for anti-tumor vaccination, in close collaboration with the expertise of Dr. Esteban Celis as a co-member of the SPORE group. We propose to make a series of viral vectors to transfer the cDNAs of different FMG into prostate tumor cells to identify the most effective FMG for the gene therapy of prostate cancer. Finally, we will construct retroviral and adenoviral vectors which incorporate tight transcriptional regulatory elements of the PSA promoter to allow targeting of FMG expression to prostate cells to increase the safety of these potent genes for progression to clinical trials. We propose using a GALV adenoviral vector system for a Phase I/II clinical trial in years 4 and 5 of the funding period. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: FUSOGENIC TRANSGENES

MEMBRANE

PROTEINS

AS

THERAPEUTIC

Principal Investigator & Institution: Galanis, Evanthia; Mayo Clinic Rochester 200 1St St Sw Rochester, Mn 55905 Timing: Fiscal Year 2001; Project Start 01-MAY-2000; Project End 31-MAR-2005 Summary: Measles virus exerts its cytopathic effect by cell-cell fusion which eventually leads to cell death. We have cloned the cDNA for the measles F and H fusogenic membrane glycoproteins into eukaryotic expression vectors and have shown significant cytotoxicity through induction of cell-cell fusion in different human tumor cell lines including A431 (epithelial carcinoma), C170 (colon cancer), HeLa (cervical cancer), TE671 (rhabdomyosarcoma), and the glioma cell lines U87 and U118. In addition, we

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have shown significantly higher bystander effects as compared to the herpes simplex thymidine kinase (HSV-tk) system. This proposal utilizes the fusogenic membrane proteins F and H of the measles virus to develop a clinician investigator's career in gene transfer/gene therapy. The applicant proposes to: 1) Investigate the use of fusogenic measles virus proteins F and H as novel therapeutic transgenes using the U87 and U118 glioma models. Gliomas were selected because they are highly lethal tumors despite the therapeutic use of surgery, radiation therapy, and chemotherapy. They also offer the further advantage of their limited metastatic potential that makes them appropriate targets for intratumoral gene transfer/gene therapy. We plan to a) construct retroviral vectors encoding the F and H transgenes, b) compare the developed vectors with the gold standard of cytotoxicity which is HSV-tk producing retroviral vectors in both glioma cell lines and tumor xenografts, and c) target vectors to the tumor environment by exploiting the over-expression of matrix metalloproteinases in gliomas. The longterm goal is to introduce this novel transgene system into clinical trials as a new therapeutic alternative. 2) Develop expertise to pursue an independent clinician scientist career within the area of gene transfer/gene therapy. In addition to the interaction with the mentor and co-mentors, this will be achieved through attending Mayo Graduate School courses and major scientific meetings, such as the American Association for Cancer Research and the American Society of Gene Therapy meetings. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: GAMMA HEMOGLOBINOPATHIES

GLOBIN

VECTORS

FOR

TREATMENT

OF

Principal Investigator & Institution: Persons, Derek A.; St. Jude Children's Research Hospital Memphis, Tn 381052794 Timing: Fiscal Year 2001; Project Start 01-FEB-2000; Project End 31-JAN-2004 Summary: This application is focused on the candidate's immediate career goal, which is to enhance and further his laboratory-based training to date by acquiring new skills in the development, testing and use of globin vectors designed for gene therapy approaches to the beta-chain hemoglobinopathies. With the applicant's clinical background, previous doctoral research experience and three years of post-doctoral work in the laboratory of Dr. Arthur Nienhuis at St. Jude Children's Research Hospital (SJCRH) most recently, the candidate is now entering a transitional phase in his career with the goal of becoming an independent investigator as a clinician-scientist. However, the candidate and the sponsor strongly believe that further training involving the new vectors and animal models outlined in this application will facilitate this transition and greatly enhance the potential for early success as an independent investigator. As an independent faculty member in an academic medical setting, it is the candidate's longterm career goal to continue in the area of gene therapy for hematologic disorders with specific interest in developing a research program compatible with the translation of successful preclinical gene therapy approaches to the clinic. In this application, the candidate proposes to obtain additional training and specific expertise in the development and testing of new therapeutic globin vectors with his current mentor, Dr. Arthur Nienhuis, at SJCRH. Within the Div. of Experimental Hematology in which Dr. Nienhuis is a member and Chief, there is significant expertise in retroviral and lentiviral vector development, in techniques of gene transfer into murine and human hematopoietic stem cells, in animal models of thalassemia, and in the use of the NOD/SCID murine transplant model for human stem cells. Thus, the further training the applicant requires for the execution of the proposed research is readily available. The proposed research project is based on the need for the development of improved

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Gene Therapy

globin vectors for use in a gene therapy approach to both thalassemia and sickle cell anemia. The focus of this project involves a gene addition strategy based on the hypothesis that delivery of an optimized gamma-globin gene cassette can achieve a sufficient level of expression in developing erythroid cells to reverse the thalassemic or sickle cell disease phenotype. The project contains 3 specific aims: 1) to design and test novel gamma-globin retroviral and lentiviral vectors, 2) to use a murine model of betathalassemia to model gene therapy approaches using optimized gamma-globin vectors. and 3) to characterize and use primitive hematopoietic cells from patients with betathalassemia to evaluate the therapeutic potential of optimized gamma-globin vectors. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: GENE THERAPY FOR BLOOD PROTEIN DEFICIENCIES Principal Investigator & Institution: Ponder, Katherine P.; Associate Professor of Internal Medicine; Barnes-Jewish Hospital Ms 90-94-212 St. Louis, Mo 63110 Timing: Fiscal Year 2002; Project Start 15-SEP-1996; Project End 31-DEC-2005 Summary: Hemophilia B occurs in 1:30,000 males and is associated with a life-long bleeding diathesis. Although IV injection of Factor IX can prevent or stop bleeding, this treatment is inconvenient, expensive, and can transmit infections. Hepatic gene therapy could permanently correct the clinical manifestations of hemophilia. Retroviral vectors (RV) can result in long-term and therapeutic levels of expression of coagulation factors from the liver in rodents, and are currently being used in a clinical trial for Hemophilia A in humans. However, there are two major problems that must be solved before RVmediated hepatic gene therapy will be used routinely: 1) identify ways to achieve a higher efficiency of stable gene transfer without major toxicity; and 2) identify methods for blocking an immune response to the therapeutic gene in the context of RV-mediated hepatic gene therapy. This project will address both of these issues. The first aim is to determine if delivery of an RV expressing the canine Factor IX (cFIX) cDNA into the liver can reduce the bleeding manifestations of Hemophilia B dogs obtained from a colony that rarely makes antibodies to the canine protein. This should allow us to quantify gene expression without the confounding issue of an immune response. Initial studies will use neonatal dogs, as their high baseline level of hepatocyte replication allows transduction of 9 percent of liver cells. Subsequent studies will use hepatocyte growth factor to induce replication in young adult dogs. Animals will be evaluated for cFIX levels, development of antibodies, bleeding, and for other adverse effects. The second aim will address the second major problem of RV-mediated hepatic gene therapy, that of immune responses to the therapeutic gene product. In this aim, we will try to block immune responses to the de novo expression of a transgene from an RV in mice by either performing neonatal gene transfer, or by injecting immunoinhibitory agents at the time of gene therapy in young adults. Although mice are optimal for initial studies due to cost considerations, approaches that function in inbred mice sometimes fail in outbred larger animals. We will therefore test any immunomodulatory approaches that function in mice for their efficacy in normal and Hemophilia B dogs in Aim III. Success in this project might lead to a safe, effective, and permanent therapy for Hemophilia B. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: DISORDERS

GENE

THERAPY

FOR

CHRONIC

NEURODEGENERATIVE

Principal Investigator & Institution: Castro, Maria G.; Professor; Cedars-Sinai Medical Center Box 48750, 8700 Beverly Blvd Los Angeles, Ca 90048

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Timing: Fiscal Year 2002; Project Start 01-AUG-2002; Project End 31-JUL-2007 Summary: (provided by applicant): Parkinson's disease (PD) is a chronic neurodegenerative disorder. Although we do not yet understand its cause, there is extensive degeneration of nigro-striatal DA neurons. Powerful neurotrophic factors which could be used for the treatment of PD, like GDNF, have been described recently. The ultimate goal of this proposal is to develop novel high-capacity adenoviral systems for cell-type specific, inducible, long term, stable, and non-immunogenic delivery of neuroprotective genes to the brain for both experimental transgene expression in adult animals, and for the future treatment of chronic neurodegenerative diseases such as PD and Alzheimer's disease by gene therapy. Currently the use of adenovirus vectors has been limited by the low efficiency of transcriptional promoter elements currently used, which directly leads to the need to use higher doses of vectors, and the cytotoxicity and immunogenicity of viral proteins expressed from the genornes of first generation adenoviral vectors. We now wish to develop novel cell-type specific and inducible vectors, that will allow efficient, safe, and long-term gene delivery vectors for neurological gene therapy. We will construct high-capacity helper-dependent adenoviral vectors that express no adenoviral genes. We will utilize the powerful, astrocyte specific major immediate early murine Cytomegalovirus promoter, driving novel tetracycline-dependent transcriptional activators to achieve cell-type specific and regulatable expression of GDNF. The efficacy, cell-type specificity, and inducibility of these vectors will be tested stringently to assess their capacity to deliver cell-type specific and regulatable GDNF, and also to determine any potential side effects caused either by the vectors or the long term expression of powerful neurotrophic agents. The reagents and principles established by this work will be of substantial value to those with interests in the basic and clinical neurosciences, and will lead to the development of novel, efficient, and safe approaches for the treatment of human chronic neurodegenerative diseases. This research will facilitate the development of the tools needed to achieve long-lived, safe, cell-type specific, regulatable, non-cytotoxic transgene expression, and, ultimately, for the treatment of patients suffering from chronic neurodegenerative diseases. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: GENE THERAPY FOR ERYTHROPOIETIC PROTOPORPHYRIA Principal Investigator & Institution: Mathews-Roth, Micheline M.; Associate Professor of Medicine; Brigham and Women's Hospital 75 Francis Street Boston, Ma 02115 Timing: Fiscal Year 2001; Project Start 01-AUG-1999; Project End 31-JUL-2003 Summary: Erythropoietic protoporphyria (EPP) is a genetic disease in which ferrochelatase, the enzyme that inserts iron into protoporphyrin, is defective. In EPP, protoporphyrin accumulate in erythrocytes, leaks into about 5% of patients. Since it has been demonstrated that the vast majority of the protoporphyrin found in plasma, skin and liver derives from the erythrocytes, we propose that gene therapy directed at the bone marrow could cure EPP. This would be especially beneficial for patients with severe photosensitivity or incipient liver disease resistant to pharmacological treatment. Using a mouse model of EPP (homozygous recessive mutation), we have recently demonstrated that transplantation of bone marrow from normal syngeneic Balb/C donors into irradiated EPP recipients completely cures the disease phenotype. We have also shown that retrovirus-mediated transfer of human ferrochelatase cDNA into peripheral blood BFU-E from human EPP patients corrects the specific protoporphyrinmediated fluorescence. In addition, we are developing improved gene transfer methods for hematopoietic stem cells by means of Lentiviral vectors and in vivo selection

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Gene Therapy

protocols, which will be validated and optimized in the EPP gene therapy model. Based on these preliminary results, our Specific Aims are as follows: Specific Aim 1: To demonstrate that retroviral transfer of human ferrochelatase cDNA into hematopoietic stem cells of EPP donor mice followed by their transplantation into EPP recipient mice will cure or significantly ameliorate the disease phenotype. Specific Aim 2: To compare the efficiencies of Murine Leukemia Virus-verus HIV-1 Lentivirus-based vectors to transduce murine hematopoietic stem cells and correct the EPP phenotype by bone marrow transplantation. Specific Aim 3: To evaluate the efficacy of gene therapy protocols for EPP that combine minimal myeloablation prior to bone marrow transplantation, Multi-drug Resistance (MDR) or Dihydrofolate Reductase (DHFR) retroviral vectors and corresponding in vivo selection regimens. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: GENE THERAPY FOR INHERITED JAUNDICE Principal Investigator & Institution: Roy-Chowdhury, Jayanta R.; Professor of Medicine and Molecular Gene; Medicine; Yeshiva University 500 W 185Th St New York, Ny 10033 Timing: Fiscal Year 2001; Project Start 01-AUG-1993; Project End 31-JUL-2003 Summary: Our long-term objective is to develop safe and efficient methods for liverdirected gene therapy for genetic disorders of hepatic metabolism, using inherited jaundice due to bilirubin-uridinediphospho-glucuronate glucuronosyltransferase (bilirubin-UGT) deficiency as a model target. Absence of hepatic bilirubin-UGT activity leads to accumulation of the bilirubin in plasma and consequent brain damage, resulting in the potentially lethal Crigler-Najjar syndrome type I (CN-I). Currently, liver transplantation is the only definitive therapy. Gunn rats, the animal model for CN-I, will be used for these studies. During the last four years, both viral and non-viral vehicles for delivery of normal human bilirubin-UGT genes to Gunn rat liver were devised and tested. In this continuation application, two strategies for correcting hepatic bilirubinUGT deficiency in vivo will be pursued. In the first approach, recombinant viral vectors will be used to substitute hepatic bilirubin-UGT by delivering a normal gene. Recombinant adenoviruses are very efficient in transferring genes into the liver in vivo, but the transgene expression is transient because the virus is episomal and the host immune response precludes its repeated administration. Three methods for specific tolerization of the host to adenoviral antigens, developed in our laboratory, show promise for long-term adenovirusmediated gene therapy without immunosuppression. To refine these methods for future clinical application, the mechanisms by which they induce specific tolerance will be elucidated. In addition, a much less immunogenic vector, based on the SV40 virus, will be developed and tested. The second approach will utilize RNA/DNA chimeric molecules to repair the genetic lesion in Gunn rats, using our highly efficient vectors for liver-specific nucleic acid delivery by receptor-mediated endocytosis. The gene transfer efficiency will be assessed by molecular, enzymatic and metabolic studies. Successful completion of these studies will provide a basis for gene therapies for CN-I and other inherited metabolic disorders, such as deficiency of alpha1-antitrypsin and urea cycle enzymes. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen

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Project Title: GENE THERAPY FOR LEBER CONGENITAL AMAUROSIS Principal Investigator & Institution: Hauswirth, William; Ophthalmology; University of Florida Gainesville, Fl 32611 Timing: Fiscal Year 2001; Project Start 30-SEP-2001; Project End 31-JUL-2006

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Summary: (Applicant?s Abstract) A multi-investigator, multi-center research/clinical plan is proposed to develop a viral vector-based gene therapy for RPE65 Leber congenital amaurosis (LCA), to complete preclinical safety testing for an Investigational New Drug (IND) submission to the FDA and to begin Phase I/II clinical testing. Seven coordinated modules are described, each with a distinct set of specific aims that contributes in a unique and complimentary way towards the therapeutic goal. Module 1, RPE65 Vector Production will improve AAV vector production for the LCA clinical trial and will provide research and GMP grade vectors for other modules. Module 2, RPE65 Vector improvement will enhance the in vivo efficiency and specificity of Rpe65 gene delivery/expression in RPE cells in animal models by promoter and vector modifications. Module3, RPE65 Mouse Studies will optimize the therapeutic effect of viral (AAV and Lentivirus) vector-delivered RPE65 genes and evaluate any toxic effects in the Rpe65 knock out mouse. Module 4, RPE65 Canine Studies will evaluate vector administration options on the therapeutic outcome of RPE65 gene augmentation in the RPE65 mutant dog. Module 5, RPE65 LCA Human Studies will identify RPE65 LCA patients suitable for entry into a Phase I/II gene therapy trial and develop standardized trial outcome measures. Module 6, RPE65 LCA Clinical Trial, has two aspects: 6A, Preclinical Testing and IND Development, will determine the potential for human toxicity and the range of efficacious doses of subretinal AAV-RPE65 in animal models and develop an FDA approved clinical protocol for 613; 6B, Phase IM Trial will evaluate the safety and preliminary efficacy of AAVRPE65 gene replacement therapy for RPE65 LCA-The basic science Modules 1, 2, 3, and 4. and the clinical screening Module 5 also develop information that feeds into the preclinical toxicity study, Module 6A- Data generated in the first 3 years by these modules will help guide the clinical trial design of Module 6B that is scheduled to begin in year 3/4. The University of Florida leads this collaboration with the University of Pennsylvania and Cornell University. The Universities of Iowa and Washington are subcontracting collaborators. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: GENE THERAPY FOR PROSTATE CANCER Principal Investigator & Institution: Thompson, Timothy; Associate Professor; Baylor College of Medicine 1 Baylor Plaza Houston, Tx 77030 Timing: Fiscal Year 2001; Project Start 01-JUN-2000; Project End 31-MAY-2002 Summary: It is important to develop additional therapeutic approaches for prostate cancer which can be applied separately or in conjunction with current modalities. Various strategies for gene therapy may provide therapeutic benefits for this important disease. The mouse prostate reconstitution (MPR) model system can be used as a preclinical model for gene therapy in prostate cancer. The validity of thi in vivo model for prostate cancer is well established and its unique features provide an opportunity to test important parameters of specific gene therapy protocols including; general efficacy; appropriate timing o therapy; as well s the comparative efficiency of various delivery systems. We have tested a replication- defective recombinant adenovirus carrying the Herpes Simplex Virus thymidine kinase (HSV-tk) gene followed by grancicylovir (GCV) in vivo and in vivo using cell lines derived from a ras + myc=induced mouse prostate carcinoma as well as from human prostate-cancer. Following inoculation of the mouse prostate cancer line cell into immunocompetent male hosts, we found that subcutaneous tumors in treated animals (n=5) were reduced in volume to 18% that in untreated animals (n-15). On histologic evaluation athe treated tumors demonstrated significantly higher levels of apoptosis and necrosis than control tumors. The efficacy of HSV-tk gene therapy was further demonstrated using the C57BL/6 MPR carcinogenesis model.

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Gene Therapy

Primary site lesions were injected with Ad/HSV-tk virus and the virus and the mice were treated with GCV for 6 days. In the control group (n=5), 4 of the MPRs produced poorly differentiated carcinomas (wt= 939 + 875 mg) and ! was hyperplastic (wt=77 mg). In the treated group (n-5), although malignant cells were present, extensive necrosis and growth suppression was apparent in all cases (wt+19 + 3 mg). These results demonstrate the efficacy of HSV-tk/GCV gene therapy as well as the utility of the MPR model system as a preclinical model. The metastatic MPR model using p53 knock-out mice allows extension of these studies to all aspects of clinically relevant disease. The primary site lesion, under the renal capsule, is suitable for injection of gene therapy vectors as we have done with Ad(HSV-tk and systemic factors which influence metastasis can be evaluated. The parameters we will evaluate include overall growth response of the primary tumor, number and location of metastases, apoptotic response, and development of an immune response by evaluating activation of tumor infiltrating lymphocytes as well as by evaluating the ability to reject subsequent challenge with tumor cells. We propose to use these preclinical models to test genes involved in growth suppression (e.g.,p53 and p21) a well as genes which may enhance the localized immune response (e.g., IL-2 and GM-CSF) together with in HSV-tk/GCV gene therapy protocol. The efficacy of the combination of gene therapy with anti-androgen therapy or radiothermy will also be evaluated. Phase I clinical trials will be developed for a select groups of patient based on the results of preclinical trials and after vector safety has been established. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: GENE THERAPY FOR PROSTATE CANCER Principal Investigator & Institution: Ponnazhagan, Selvarangan; Associate Professor of Molecular; Pathology; University of Alabama at Birmingham Uab Station Birmingham, Al 35294 Timing: Fiscal Year 2003; Project Start 07-FEB-2003; Project End 31-JAN-2008 Summary: (provided by applicant): The mortality associated with prostate cancer is primarily due to systemic dissemination of the disease to which conventional therapies such as surgery, androgen-depletion and chemotherapy fail to provide long-term cure. Thus, development of novel approaches is important for the treatment of both primary and metastatic prostate disease. Among the possible therapeutic targets, the tumor endothelium appears promising since endothelial cell growth during angiogenesis is crucial for tumor growth and metastasis. A majority of earlier studies using purified anti-angiogenic factors to modulate disease have been unsuccessful due to a requirement for constant administration, clinical side effects, and/or high cost. Thus, gene therapy approaches to achieve stable expression of anti-angiogenic factors in vivo have the potential to overcome many of these limitations. Recombinant adenoassociated virus (rAAV) vectors are a unique group of viruses that are less immunogenic than other viral vectors and arc integrating, hence, have greater advantage for long-term expression. We have recently shown that genetic transfer of an anti-angiogenic factor, sFlt-I abrogated the growth of human fibro sarcoma in nude mice. We also demonstrated with targeted-vectors, that high-efficiency, tumor cellspecific delivery is achievable. Further, by generating a genetically deficient transgenic adenocarcinoma mouse prostate cancer (TRAMP) model for the early growth response protein-I (Egr-l), we recently demonstrated a role for Egr-1 in delaying the progression of prostatic intra-epithelial neoplasia(PIN) to invasive carcinoma. Additional preliminary studies with rAAV encoding the anti-angiogenic factors human angiostatin, endostatin and sFlt-1 have shown protection against the growth of a human epithelial

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ovarian tumor cell line in athymic mice indicating the efficacy of AAV-mediated antiangiogenic gene therapy. Based on these results, we hypothesize that gene therapy for prostate Cancer by AAV-mediated stable expression of anti-angiogenic factors will be efficacious both as a primary therapy, and as an adjuvant. Further, development of prostate cancer-specific rAAV containing anti-angiogenic genes would not only increase targeted-transduction but also minimize the vector dose and thus any associated toxicities. The present proposal will determine the efficacy of sustained anti-angiogenic gene therapy both as a primary therapy, and as an adjuvant therapy against recurrence in the TRAMP model. A successful outcome of these studies will form the basis for the development of Phase I clinical trials. Website: http://crisp.cit.nih.gov/crisp/Crisp_Query.Generate_Screen •

Project Title: GENE THERAPY FOR THE HEMOPHILIAS Principal Investigator & Institution: Walsh, Christopher E.; Professor; Medicine; University of North Carolina Chapel Hill Office of Sponsored Research Chapel Hill, Nc 27599 Timing: Fiscal Year 2002; Project Start 01-JUL-2002; Project End 31-OCT-2002 Summary: (provided by applicant): Effective gene therapy will revolutionize the treatment of the hemophilias. Recombinant adeno-associated virus (rAAV) vectors are considered among the most promising viral vectors for hemophilia gene therapy. The non-pathogenic nature of AAV, the ability to transduce mitotic and post-mitotic cells, and the capacity for stable persistence of rAAV/transgene sequences are unique among all viral vectors. A major obstacle in the application of rAAV in gene therapy for hemophilia A (factor VIII deficiency) is the conflict of the limited packaging capacity of rAAV and the large size of the human FVIII gene. The major rate-limiting aspect of this delivery system has always been the small packaging capacity (5kb) of rAAV. Factor VIII with its large cDNA (7.0 Kb) is an excellent model to test a variety of new approaches for AAV-mediated gene transfer. Here we present compelling evidence supporting the use of AAV vectors for the expression human factor VIII gene therapy. We developed several different novel approaches for the expression of functional factor VIII. First, we developed rAAV vectors carrying a truncated version of the full-length FVIII cDNA. Removal of the B-domain sequence of factor VIII (~4.0 Kb) results in a fully functional protein (termed B-domain deleted, BDD FVIII which express therapeutic levels of functional FVIII in vivo. Despite truncation of the FVIII sequence, the use of small (