Muscle Gene Therapy

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Gene therapy offers many conceptual advantages to treat muscle diseases, especially various forms of muscular dystrophies. Many of these diseases are caused by a single gene mutation. While the traditional approaches may ameliorate some symptoms, the ultimate cure will depend on molecular correction of the genetic defect. The clinical feasibility of gene therapy has been recently demonstrated in treatment of a type of inherited blindness. By delivering a therapeutic gene to the retina, investigators were able to partially recover the vision in a disease once thought incurable. Compared to retinal gene therapy, muscle gene therapy faces a number of unique challenges. Muscle is one of the most...

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Methods Molecular Biology™ in Series Editor John M. Walker School of Life Sciences University of Hertfordshire Hatfield, Hertfordshire, AL10 9AB, UK For other titles published in this series, go to www.springer.com/series/7651
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Muscle Gene Therapy Methods and Protocols Edited by Dongsheng Duan Department of Molecular Microbiology and Immunology University of Missouri, Columbia, MO, USA
Editor Dongsheng Duan, Ph.D. Department of Molecular Microbiology and Immunology University of Missouri Columbia, MO USA duand@missouri.edu ISSN 1064-3745 e-ISSN 1940-6029 ISBN 978-1-61737-981-9 e-ISBN 978-1-61737-982-6 DOI 10.1007/978-1-61737-982-6 Springer New York Dordrecht Heidelberg London © Springer Science+Business Media, LLC 2011 All rights reserved. This work may not be translated or copied in whole or in part without the written permission of the publisher (Humana Press, c/o Springer Science+Business Media, LLC, 233 Spring Street, New York, NY 10013, USA), except for brief excerpts in connection with reviews or scholarly analysis. Use in connection with any form of information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed is forbidden. The use in this publication of trade names, trademarks, service marks, and similar terms, even if they are not identified as such, is not to be taken as an expression of opinion as to whether or not they are subject to proprietary rights. While the advice and information in this book are believed to be true and accurate at the date of going to press, neither the authors nor the editors nor the publisher can accept any legal responsibility for any errors or omissions that may be made. The publisher makes no warranty, express or implied, with respect to the material contained herein. Printed on acid-free paper Humana Press is part of Springer Science+Business Media (www.springer.com)
Preface The mechanic that would perfect his work must first sharpen his tools. Confucius (c. 551 BC–479 BC), a Chinese philosopher Give us the tools, and we will finish the job. Winston Churchill (1874–1965) Gene therapy offers many conceptual advantages to treat muscle diseases, especially vari- ous forms of muscular dystrophies. Many of these diseases are caused by a single gene mutation. While the traditional approaches may ameliorate some symptoms, the ultimate cure will depend on molecular correction of the genetic defect. The clinical feasibility of gene therapy has been recently demonstrated in treatment of a type of inherited blindness. By delivering a therapeutic gene to the retina, investigators were able to partially recover the vision in a disease once thought incurable. Compared to retinal gene therapy, muscle gene therapy faces a number of unique challenges. Muscle is one of the most abundant tissues in the body. An effective therapy will require systemic infusion and targeted muscle delivery of huge amounts of therapeutic vectors. Severe inflammation associated with muscle degeneration and necrosis may further complicate immune reactions to the viral vectors and the therapeutic gene products. Furthermore, the vast majority of our current knowledge on muscle gene therapy is obtained from rodent models. Although these proof-of-concept studies have provided the critical foundation, the results are not easily translatable to human patients. With this in mind, we compiled this collection of muscle gene therapy methods and protocols with the intention of bridging the translational gap in muscle gene therapy. The book is divided into three sections. The first section includes basic protocols for optimizing the muscle gene expression cassette and for evaluating the therapeutic out- comes. The chapters on the muscle-specific promoters and codon optimization outline strategies to generate powerful cassettes for muscle expression. Four chapters are devoted to end-point analysis. These include the use of epitope-specific antibodies, noninvasive monitoring of myofiber survival, and physiology assays of skeletal muscle and heart function. Technology breakthroughs are the driving force in muscle gene therapy. Early muscle gene transfer studies were largely performed using vectors based on retrovirus, adenovi- rus, or plasmid DNA. Inherent limitations of these vectors (such as low transduction efficiency, transient expression, and a strong immune response) suggest that they are unlikely to meet the clinical need. These traditional gene delivery vehicles have now been replaced with the robust adeno-associated viral vector (AAV), oligonucleotide-mediated exon-skipping, and novel RNA-based strategies such as microRNA and RNA interference. The second section of this book is dedicated to the new developments in muscle gene therapy technology. Two chapters describe new strategies to generate muscle-specific AAV vectors by in vivo evolution and capsid reengineering. Two chapters provide methods for optimizing exon-skipping, and three chapters detail different applications of RNA-based approaches in muscle gene therapy. v
vi Preface Considering the importance of large animal studies, it is not surprising that the bulk of the protocols are devoted to muscle gene transfer in large animals models. In the last section, ten chapters provide step-by-step guidance on muscle gene delivery in swine, ovine, canine, and nonhuman primates. Methods include local delivery, isolated limb per- fusion, myocardial gene transfer, and whole body systemic delivery. Ages range from fetal and neonatal to adult subjects. In summary, this book presents a comprehensive collection of state-of-the-art muscle gene therapy protocols from leaders in the field. I would also like to mention that this col- lection of muscle gene therapy techniques complements the recently published book enti- tled “Muscle Gene Therapy” (Duan D eds., Springer, 2010, ISBN 978-1-4419-1205-3). Together, they will serve as a valuable resources for graduate students, postdoctoral fellows, and principle investigators who are interested in muscle gene therapy. I would like to thank the contributors of each chapter for their excellent contribu- tions. There is no doubt that these hard-to-find techniques, tricks, and the hands-on experience from the leading investigators will play an important role in bench-side to bed- side translation of muscle gene therapy. I would like to thank Dr. John Walker, the series editor, for his guidance in the development of this book. I would like to thank Ms. Karen Ehlert for her administrative assistance in the final stage of preparation. I am also very grateful to the National Institutes of Health and the Muscular Dystrophy Association for the funding of muscle gene therapy studies in my laboratory. I also thank the Parent Project Muscular Dystrophy and Jesse’s Journey, The Foundation for Gene and Cell Therapy for their recent support in expanding our research in developing mus- cular dystrophy gene therapy. I am also much indebted to the patients and their families. I truly believe their dream will one day come true. Finally, I’d like to dedicate this book to boys like Mark McDonald, they are our driving force. Columbia, MO Dongsheng Duan
Contents Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . v Contributors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix Part I BasIc Methodology related to Muscle gene theraPy 1. Design and Testing of Regulatory Cassettes for Optimal Activity in Skeletal and Cardiac Muscles. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3 Charis L. Himeda, Xiaolan Chen, and Stephen D. Hauschka 2. Codon Optimization of the Microdystrophin Gene for Duchenne Muscular Dystrophy Gene Therapy. . . . . . . . . . . . . . . . . . . . . . . . . 21 Takis Athanasopoulos, Helen Foster, Keith Foster, and George Dickson 3. Monitoring Duchenne Muscular Dystrophy Gene Therapy with Epitope-Specific Monoclonal Antibodies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Glenn Morris, Nguyen thi Man, and Caroline A. Sewry 4. Methods for Noninvasive Monitoring of Muscle Fiber Survival with an AAV Vector Encoding the mSEAP Reporter Gene . . . . . . . . . . . . . . . . . . 63 Jérôme Poupiot, Jérôme Ausseil, and Isabelle Richard 5. Monitoring Murine Skeletal Muscle Function for Muscle Gene Therapy . . . . . . . . 75 Chady H. Hakim, Dejia Li, and Dongsheng Duan 6. Phenotyping Cardiac Gene Therapy in Mice . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91 Brian Bostick, Yongping Yue, and Dongsheng Duan 7. Golden Retriever Muscular Dystrophy (GRMD): Developing and Maintaining a Colony and Physiological Functional Measurements. . . . . . . . . 105 Joe N. Kornegay, Janet R. Bogan, Daniel J. Bogan, Martin K. Childers, and Robert W. Grange Part II new technology In Muscle gene theraPy 8. Directed Evolution of Adeno-Associated Virus (AAV) as Vector for Muscle Gene Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 127 Lin Yang, Juan Li, and Xiao Xiao 9. Systemic Gene Transfer to Skeletal Muscle Using Reengineered AAV Vectors . . . . 141 Jana L. Phillips, Julia Hegge, Jon A. Wolff, R. Jude Samulski, and Aravind Asokan 10. Bioinformatic and Functional Optimization of Antisense Phosphorodiamidate Morpholino Oligomers (PMOs) for Therapeutic Modulation of RNA Splicing in Muscle . . . . . . . . . . . . . . . . . . . . 153 Linda J. Popplewell, Ian R. Graham, Alberto Malerba, and George Dickson 11. Engineering Exon-Skipping Vectors Expressing U7 snRNA Constructs for Duchenne Muscular Dystrophy Gene Therapy. . . . . . . . . . . . . . . . . . . . . . . . . 179 Aurélie Goyenvalle and Kay E. Davies vii
viii Contents 12. Application of MicroRNA in Cardiac and Skeletal Muscle Disease Gene Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197 Zhan-Peng Huang, Ronald L. Neppl Jr., and Da-Zhi Wang 13. Molecular Imaging of RNA Interference Therapy Targeting PHD2 for Treatment of Myocardial Ischemia . . . . . . . . . . . . . . . . . . . . 211 Mei Huang and Joseph C. Wu 14. Lentiviral Vector Delivery of shRNA into Cultured Primary Myogenic Cells: A Tool for Therapeutic Target Validation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 223 Emmanuel Richard, Gaelle Douillard-Guilloux, and Catherine Caillaud Part III Methods for Muscle gene transfer In large anIMal Models 15. Fetal Muscle Gene Therapy/Gene Delivery in Large Animals . . . . . . . . . . . . . . . . 239 Khalil N. Abi-Nader and Anna L. David 16. Electroporation of Plasmid DNA to Swine Muscle . . . . . . . . . . . . . . . . . . . . . . . . 257 Angela M. Bodles-Brakhop, Ruxandra Draghia-Akli, Kate Broderick, and Amir S. Khan 17. Local Gene Delivery and Methods to Control Immune Responses in Muscles of Normal and Dystrophic Dogs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 265 Zejing Wang, Stephen J. Tapscott, and Rainer Storb 18. Gene Transfer to Muscle from the Isolated Regional Circulation. . . . . . . . . . . . . . 277 Mihail Petrov, Alock Malik, Andrew Mead, Charles R. Bridges, and Hansell H. Stedman 19. AAV-Mediated Gene Therapy to the Isolated Limb in Rhesus Macaques . . . . . . . . 287 Louise R. Rodino-Klapac, Chrystal L. Montgomery, Jerry R. Mendell, and Louis G. Chicoine 20. Antisense Oligo-Mediated Multiple Exon Skipping in a Dog Model of Duchenne Muscular Dystrophy . . . . . . . . . . . . . . . . . . . . . . . . 299 Toshifumi Yokota, Eric Hoffman, and Shin’ichi Takeda 21. Whole Body Skeletal Muscle Transduction in Neonatal Dogs with AAV-9 . . . . . . . 313 Yongping Yue, Jin-Hong Shin, and Dongsheng Duan 22. A Translatable, Closed Recirculation System for AAV6 Vector-Mediated Myocardial Gene Delivery in the Large Animal. . . . . . . . . . . . . . 331 JaBaris D. Swain, Michael G. Katz, Jennifer D. White, Danielle M. Thesier, Armen Henderson, Hansell H. Stedman, and Charles R. Bridges 23. Method of Gene Delivery in Large Animal Models of Cardiovascular Diseases . . . 355 Yoshiaki Kawase, Dennis Ladage, and Roger J. Hajjar 24. Percutaneous Transendocardial Delivery of Self-Complementary Adeno-Associated Virus 6 in the Canine . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 369 Lawrence T. Bish, Meg M. Sleeper, and H. Lee Sweeney Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379
Contributors KhalIl n. aBI-nader • Fetal Medicine Unit and Prenatal Cell and Gene Therapy Group, EGA Institute for Women’s Health, University College London Hospitals, London, UK Aravind Asokan • Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; Department of Genetics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA Takis Athanasopoulos • School of Biological Sciences, Royal Holloway – University of London (RHUL), Egham, Surrey, TW20 0EX, UK Jérôme Ausseil • Généthon – CNRS-UMR8587 LAMBE, 1 bis rue de l’Internationale, France lawrence t. BIsh • Department of Physiology, University of Pennsylvania School of Medicine, B400 Richards Building, 3700 Hamilton Walk, Philadelphia, USA angela M. Bodles-BraKhoP • Inovio Biomedical Corporation, 2700 Research Forest Drive, The Woodlands, TX, USA danIel J. Bogan • Department of Pathology and Laboratory Medicine and The Gene Therapy Center, School of Medicine, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA Janet r. Bogan • Department of Pathology and Laboratory Medicine and The Gene Therapy Center, School of Medicine, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA BrIan BostIcK • Department of Molecular Microbiology and Immunology, School of Medicine, The University of Missouri, One Hospital Drive, Columbia, MO, USA charles r. BrIdges • Department of Surgery, Division of Cardiovascular Surgery, University of Pennsylvania School of Medicine, BRB II/III Building, 421 Currie Boulevard, Philadelphia, PA, USA Kate BroderIcK • Inovio Biomedical Corporation, 2700 Research Forest Drive, The Woodlands, TX, USA catherIne caIllaud • Département Génétique et Développement, Institut Cochin, Université Paris Descartes, CNRS (UMR 8104), Paris, France XIaolan chen • Department of Biochemistry, University of Washington, Seattle, USA louIs g. chIcoIne • Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital and Department of Pediatrics, The Ohio State University, Columbus, OH, USA MartIn K. chIlders • Department of Neurology and Wake Forest Institute for Regenerative Medicine, School of Medicine, Wake Forest University, Winston-Salem, NC, USA ix
x Contributors anna l. davId • Fetal Medicine Unit and Prenatal Cell and Gene Therapy Group, EGA Institute for Women’s Health, University College London Hospitals, London, UK Kay e. davIes • MRC Functional Genomics Unit, Department of Physiology, Anatomy, and Genetics, University of Oxford, South Parks Road, UK george dIcKson • Institute of Biomedical and Life Sciences, South West London Academic Network, St. George’s University of London, London, UK; Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway, University of London, Egham, UK; School of Biological Sciences, Royal Holloway, University of London, Egham, UK gaelle douIllard-guIllouX • Department of Pathology, University of Pittsburgh, Pittsburgh, PA, USA ruXandra draghIa-aKlI • Inovio Biomedical Corporation, 2700 Research Forest Drive, The Woodlands, TX, USA dongsheng duan • Department of Molecular Microbiology and Immunology, School of Medicine, The University of Missouri, 1 Hospital Drive, M610, Columbia, MO, USA helen foster • Institute of Biomedical and Life Sciences, South West London Academic Network, St. George’s University of London, London, UK; Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway, University of London, Egham, UK KeIth foster • Institute of Biomedical and Life Sciences, South West London Academic Network, St. George’s University of London, London, UK; Centre for Biomedical Sciences, School of Biological Sciences, Royal Holloway, University of London, Egham, UK aurélIe goyenvalle • MRC Functional Genomics Unit, Department of Physiology, Anatomy, and Genetics, University of Oxford, South Parks Road, UK Ian r. grahaM • School of Biological Sciences, Royal Holloway, University of London, Egham, UK roBert w. grange • Department of Human Nutrition, Foods, and Exercise, College of Agriculture and Life Sciences, Virginia Tech University, Blacksburg, USA roger J. haJJar • The Cardiovascular Research Center, Mount Sinai School of Medicine, Atran Berg Laboratory Building, Floor 05, 1428 Madison Avenue New York, NY, USA chady h. haKIM • Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, 1 Hospital Drive, M610, Columbia, MO, USA stePhen d. hauschKa • Department of Biochemistry, University of Washington, Seattle, WA, USA JulIa hegge • Mirus BioCorporation, Madison WI, USA arMen henderson • Department of Surgery, Division of Cardiovascular Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA, USA charIs l. hIMeda • Department of Biochemistry, University of Washington, Seattle, WA, USA erIc hoffMan • Research Center for Genetic Medicine, Children’s National Medical Center, 111 Michigan Avenue, NW ,Washington, DC, USA
Contributors xi MeI huang • Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA; Department of Radiology, Stanford University School of Medicine, Stanford, CA, USA Zhan-Peng huang • Cardiovascular Research Division, Department of Cardiology, Children’s Hospital Boston, Harvard Medical School, 320 Longwood Avenue, Boston, MA, USA MIchael g. KatZ • Department of Surgery, Division of Cardiovascular Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA, USA yoshIaKI Kawase • The Cardiovascular Research Center, Mount Sinai School of Medicine, Atran Berg Laboratory Building, Floor 05, 1428 Madison Avenue, New York, NY, USA aMIr s. Khan • Inovio Biomedical Corporation, 2700 Research Forest Drive, The Woodlands, TX, USA Joe n. Kornegay • Department of Pathology and Laboratory Medicine and The Gene Therapy Center, School of Medicine, University of North Carolina-Chapel Hill, Chapel Hill, NC, USA dennIs ladage • The Cardiovascular Research Center, Mount Sinai School of Medicine, Atran Berg Laboratory Building, Floor 05, 1428 Madison Avenue, New York, NY, USA deJIa lI • Department of Molecular Microbiology and Immunology, School of Medicine, The University of Missouri, 1 Hospital Drive, M610, Columbia, MO, USA Juan lI • Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA alBerto MalerBa • School of Biological Sciences, Royal Holloway, University of London, Egham,UK alocK MalIK • Department of Surgery, University of Pennsylvania School of Medicine, BRB II/III Building, 421 Currie Boulevard, Philadelphia, PA, USA nguyen thI Man • Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry and Institute for Science and Technology in Medicine, Keele University, UK; Institute for Science and Technology in Medicine, Keele University, Keele, UK andrew Mead • Department of Surgery, Division of Gastrointestinal Surgery, University of Pennsylvania School of Medicine, BRB II/III Building, 421 Currie Boulevard, Philadelphia, PA, USA Jerry r. Mendell • Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital and Department of Pediatrics, The Ohio State University, Columbus, OH, USA chrystal l. MontgoMery • Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital and Department of Pediatrics, The Ohio State University, Columbus, OH, USA glenn MorrIs • Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry and Institute for Science and Technology in Medicine, Keele University, UK; Institute for Science and Technology in Medicine, Keele University, Keele, UK
xii Contributors ronald l. nePPl Jr. • Cardiovascular Research Division, Department of Cardiology, Children’s Hospital Boston, Harvard Medical School, 320 Longwood Avenue, Boston, MA, USA MIhaIl Petrov • Department of Surgery, Division of Gastrointestinal Surgery, University of Pennsylvania School of Medicine, BRB II/III Building, 421 Currie Boulevard, Philadelphia, PA, USA Jana l. PhIllIPs • Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA lInda J. PoPPlewell • School of Biological Sciences, Royal Holloway, University of London, Egham, UK JérôMe PouPIot • Généthon – CNRS-UMR8587 LAMBE, 1 bis rue de l’Internationale, France eMManuel rIchard • INSERM U876, IFR 66, Université Bordeaux 2, 146 rue Léo Saignat, Bordeaux, France IsaBelle rIchard • Généthon – CNRS-UMR8587 LAMBE, 1 bis rue de l’Internationale, France louIse r. rodIno-KlaPac • Center for Gene Therapy, The Research Institute at Nationwide Children’s Hospital and Department of Pediatrics, The Ohio State University, Columbus, OH, USA r. Jude saMulsKI • Gene Therapy Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA carolIne a. sewry • Wolfson Centre for Inherited Neuromuscular Disease, RJAH Orthopaedic Hospital, Oswestry and Institute for Science and Technology in Medicine, Keele University, UK; Institute for Science and Technology in Medicine, Keele University, Keele, UK JIn-hong shIn • Department of Molecular Microbiology and Immunology, School of Medicine, University of Missouri, Columbia, MO, USA Meg M. sleePer • Section of Cardiology, Department of Clinical Studies, Veterinary Hospital of the University of Pennsylvania, Philadelphia, PA, USA hansell h. stedMan • Department of Surgery, Division of Gastrointestinal Surgery, University of Pennsylvania School of Medicine, BRB II/III Building, 421 Currie Boulevard, Philadelphia, PA, USA raIner storB • Program in Transplantation Biology, Division of Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Medicine, University of Washington, Seattle, WA, USA JaBarIs d. swaIn • Department of Surgery, Division of Cardiovascular Surgery, University of Pennsylvania School of Medicine, Philadelphia, USA h. lee sweeney • Department of Physiology, University of Pennsylvania School of Medicine, B400 Richards Building, 3700 Hamilton Walk, Philadelphia, PA, USA shIn’IchI taKeda • Department of Molecular Therapy, National Institute of Neuroscience, National Center of Neurology and Psychiatry (NCNP), Tokyo, Japan stePhen J. taPscott • Division of Human Biology, Fred Hutchinson Cancer Research Center, Seattle, WA, USA; Department of Neurology, University of Washington, Seattle WA, USA
Contributors xiii danIelle M. thesIer • Department of Surgery, Division of Cardiovascular Surgery, University of Pennsylvania, School of Medicine, Philadelphia, PA, USA da-ZhI wang • Cardiovascular Research Division, Department of Cardiology, Children’s Hospital Boston, Harvard Medical School, 320 Longwood Avenue, Boston, MA, USA ZeJIng wang • Program in Transplantation Biology, Division of Clinical Research, Fred Hutchinson Cancer Research Center, Fairview Av N, D1-100 Seattle, WA, USA JennIfer d. whIte • Department of Surgery, Division of Cardiovascular Surgery, University of Pennsylvania School of Medicine, Philadelphia, PA, USA Jon a. wolff • Mirus BioCorporation, Madison, WI, USA JosePh c. wu • Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA; Department of Radiology, Stanford University School of Medicine, Stanford, CA, USA XIao XIao • Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA lIn yang • Division of Molecular Pharmaceutics, Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA toshIfuMI yoKota • Research Center for Genetic Medicine, Children’s National Medical Center, 111 Michigan Avenue, NW, Washington, DC, USA yongPIng yue • Department of Molecular Microbiology and Immunology, School of Medicine, The University of Missouri, One Hospital Drive, Columbia, USA
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Part I Basic Methodology Related to Muscle Gene Therapy
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Chapter 1 Design and Testing of Regulatory Cassettes for Optimal Activity in Skeletal and Cardiac Muscles Charis L. Himeda, Xiaolan Chen, and Stephen D. Hauschka Abstract Gene therapy for muscular dystrophies requires efficient gene delivery to the striated musculature and specific, high-level expression of the therapeutic gene in a physiologically diverse array of muscles. This can be achieved by the use of recombinant adeno-associated virus vectors in conjunction with muscle- specific regulatory cassettes. We have constructed several generations of regulatory cassettes based on the enhancer and promoter of the muscle creatine kinase gene, some of which include heterologous enhancers and individual elements from other muscle genes. Since the relative importance of many control elements varies among different anatomical muscles, we are aiming to tailor these cassettes for high-level expres- sion in cardiac muscle, and in fast and slow skeletal muscles. With the achievement of efficient intravas- cular gene delivery to isolated limbs, selected muscle groups, and heart in large animal models, the design of cassettes optimized for activity in different muscle types is now a practical goal. In this protocol, we outline the key steps involved in the design of regulatory cassettes for optimal activity in skeletal and cardiac muscle, and testing in mature muscle fiber cultures. The basic principles described here can also be applied to engineering tissue-specific regulatory cassettes for other cell types. Key words: Skeletal muscle, Cardiac muscle, Regulatory cassette, Muscular dystrophy, Gene therapy, Transcriptional regulation, Muscle creatine kinase 1. Introduction Duchenne muscular dystrophy (DMD) is caused by a lack of functional dystrophin, resulting in patient death due to cardiac and/or respiratory failure. Gene therapy for muscle diseases such as DMD requires efficient gene delivery to the striated muscula- ture and specific, high-level expression of the therapeutic gene in a physiologically diverse array of muscles. This can be achieved by the use of regulatory cassettes composed of enhancers and pro- moters that contain combinations of muscle-specific and ubiquitous Dongsheng Duan (ed.), Muscle Gene Therapy: Methods and Protocols, Methods in Molecular Biology, vol. 709, DOI 10.1007/978-1-61737-982-6_1, © Springer Science+Business Media, LLC 2011 3
4 Himeda, Chen, and Hauschka control elements. Recombinant adeno-associated virus (rAAV) vectors (particularly serotypes 1, 6, 7, 8, and 9, which exhibit preferential transduction of striated muscle) are well-suited for this challenge, since when combined with appropriate regulatory cassettes, they mediate high-level, long-term transgene expres- sion in striated muscle. Despite the preferential targeting of stri- ated muscle by certain AAV serotypes, the widespread dissemination of vector following systemic delivery still raises concerns about inadvertent transduction of nonmuscle tissues, which may give rise to an unwanted immune response. Thus, even though viral promoters (such as the CMV and RSV promot- ers) mediate high-level expression in skeletal and cardiac muscle (1, 2), these promoters also exhibit high activity in dendritic cells and relatively high activity in spleen and testes (1). Thus, the con- struction of high-activity, muscle-specific regulatory cassettes is a necessary goal. Since the effective genome size limit for AAV packaging is limited, miniaturization of the transcription regula- tory cassettes is another important priority for the expression of cDNAs larger than ~3.5 kb. Muscle-specific gene expression is determined by combinato- rial interactions between muscle-specific and ubiquitous trans-acting factors bound to the enhancers and promoters, and the interactions of these factors with associated protein complexes. The exact nature of these combinatorial interactions is unknown, but many of the cis elements and trans components regulating muscle-specific genes have been identified and partially characterized. The muscle creatine kinase (MCK) gene has served as a useful model of muscle-specific gene transcription since its protein prod- uct is specifically and abundantly expressed in striated muscle, and its regulatory regions have been extensively characterized. MCK is also expressed at different levels in different anatomical skeletal muscles, and in skeletal vs. cardiac muscle (3, 4). This allows for the identification of control elements and binding factors impor- tant for expression in different muscle types, as well as those spe- cific to each myogenic lineage. Importantly, the MCK enhancer and promoter synergize in all striated muscle types in vivo to give 100-fold elevated activity over that of either alone (3, 4), making the composite enhancer-promoter a useful cassette for muscle gene therapy. To date, cassettes built from regulatory regions of the MCK, skeletal a-actin, myosin heavy chain, and myosin light chain (MLC) 1/3 genes, and randomized muscle gene control elements fulfill the criteria for muscle-specific expression (1, 2, 5–9). However, these cassettes either lack high-level activity in certain muscle types, have not been studied quantitatively in different tissues, or their size exceeds the limitation for packaging into an AAV vector in conjunc- tion with the smallest microdystrophin cDNAs (~3.5 kb) that provide reasonable function in the mdx mouse model of DMD.
Design and Test Muscle Regulatory Cassettes 5 Fig. 1. MCK-based regulatory cassettes for expression in skeletal and cardiac muscle. The original cassette (WT) includes the MCK enhancer (−1256 to −1050) linked to the MCK proximal promoter (−358 to +7). The Right E-box and MEF2 site within the MCK enhancer are shown. CK7 incorporates three changes from WT: (1) deletion of 63 bp between the R E-box and MEF2 site (−1140 to −1078), (2) mutation of sequence overlapping +1 to a consensus Initiator element (Inr), and (3) insertion of 43 bp 3¢ of +7. CK8 is identical to CK7 except that it contains three copies of the modified enhancer. CK9 is identical to CK7 except that it contains three additional deletions: (D1) −1256 to −1240, (D2) −1063 to −1050, and (D3) −358 to −268. Refer to text for more details. Recently, our lab has constructed several new generations of regulatory cassettes based on the MCK enhancer and promoter, with the addition of enhancers and individual elements from other muscle genes. In designing these cassettes, we have tried to utilize existing information regarding the function of control elements and their cognate binding factors. For example, we theorized that deleting nonconserved sequences between the Right E-box and MEF2 site in the MCK enhancer (Figs. 1 and 2) might better facili- tate interactions between the myogenic regulatory factors (MyoD, Myogenin, Myf5, and MRF4) and MEF2, which are known to synergize. In addition to increasing MCK enhancer activity in skel- etal myocytes, this change also significantly reduced the size of the cassette, allowing other useful elements to be incorporated. Since the relative importance of many control elements appears to vary among different anatomical muscles, we are aim- ing to tailor regulatory cassettes for high-level expression in car- diac muscle, and in fast and slow skeletal muscle. With the achievement of efficient intravascular gene delivery to isolated limbs, selected muscle groups, and heart in a large animal model (10–12), the design of cassettes optimized for activity in different muscle types is now a practical goal. Such cassettes would be useful, not only for DMD, but for many other neuromuscular diseases as well.