Retrovirus-Mediated Gene Therapy For Farber Disease by Shobha Ramsubir A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto © Copyright by Shobha Ramsubir (2008) Retrovirus-mediated Gene Therapy for Farber Disease Doctor of Philosophy, 2008 Graduate Department of Medical Biophysics University of Toronto Shobha Ramsubir Abstract Farber disease is a rare lysosomal storage disease (LSD) caused by a deficiency of acid ceramidase (AC). Patients show a classic triad of symptoms including subcutaneous granulomas, laryngeal involvement and painful swollen joints. The most common and severe form has neurological manifestations and patients typically die by the age of two. Current treatment consists of symptomatic supportive care and allogeneic bone marrow transplantation (BMT). However, BMT has shown limited success. Gene therapy has previously been shown to be a promising treatment strategy for monogenetic diseases and has the potential to treat the underlying cause of the disease. Presented here is the first report of in vivo testing of retrovirus-mediated gene therapy strategies for the treatment of Farber disease. Retroviral vectors were engineered to overexpress AC and a cell surface marker, human CD25. Transduction with these viral vectors corrected the enzymatic defect in Farber patient cells and in vivo administration of the lentiviral vector led to long-term expression of the marking transgene as well as increased AC expression in the liver. To determine the effect of over-expression of AC, human CD34+ cells were transduced and transplanted into NOD/SCID animals. It was found that transgene-expressing cells could reconstitute the host. ii To address the neurological manifestations of Farber disease, vascular endothelial growth factor (VEGF) was investigated as an agent to transiently open the blood brain barrier for entry of lentivirus. It was found that in addition to increasing the amount of therapeutic virus in the brain, VEGF treatment also increased transduction in other organs. Further, to address the concerns of insertional mutagenesis associated with using integrating vectors, an immunotoxin-based strategy was developed as a safety system to clear transduced cells. It was found that a CD25-targeted immunotoxin could eliminate both transduced hematopoietic cells as well as tumor cells over-expressing CD25. This strategy can be employed following gene therapy should an unwanted proliferative event occur. Together, these studies represent considerable advances towards the development of a cure for Farber disease, demonstrating both therapeutic potential and also containing a built-in safety system. iii Acknowledgements This journey has been one of incredible growth for me and I have learnt so much about persistence and perseverance. I am so grateful for the friendship and support of all of my colleagues throughout the years. Washing away failed experiments with pints at the pub with all of you kept me hanging on. My heartfelt thanks go to Dr. Makoto Yoshimitsu for his mentorship, encouragement and sense of humour. I am grateful to Dr. Koji Higuchi and Dr. Takeya Sato for their continuous support and valuable advice over the years. Thanks to Renee Head for always being willing to pick up the slack, to Gillian Sleep for her help and patience in dealing with the many mice of my career and to Vanessa Rasaiah for helping with manuscripts, my thesis and things too many to mention. To the ladies that I began this journey with - Julie Symes and Miriam Mossoba - we have shared many laughs and a few tears over the years and I wish you both the best of luck in all your future endeavors. To my fellow graduate students Greg Rampersad, Sean Devine and Anton Neschadim – all the chats and laughs made coming to the lab a pleasure. You are all destined for great things. I am also grateful to the post-doctoral fellows Dr. Takahiro Nonaka, Dr. Josh Silvertown, Dr. Chris Siatskas, Dr. Nobuo Mizue, Dr. Jagdeep Singh- Walia, Dr. Severine Meyer and Dr. Chyang-Jang Lee for their helpful ideas and numerous discussions about my project and science in general. I would like to thank the individuals under whose guidance I truly grew as a scientist. Thanks to my supervisor Dr. Jeffrey Medin for the opportunity to work on this project and iv for all his help throughout the years. I am thankful to Dr. Thierry Levade for welcoming me into his lab, for teaching me and for his assistance with biochemical assays. Dr. Joe Clarke gave me the opportunity to meet a patient affected with Farber Disease and this experience truly gave me greater perspective on the importance of the research that I and other scientists do. Dr. Hans Messner always made time to review my research and progress and provided much helpful advice. I am also grateful to Dr. David Rose for his candor, mentorship and support. I am also grateful to him for his help in preparing for my defense. Finally, I would like to thank my family for their unconditional support and love. I could not have gotten here without you and I am eternally indebted. Thanks especially to my mother for always going the extra mile, to my father for his support and to my sister Lesley for always believing in me. I dedicate this thesis to you all. v Table of Contents ABSTRACT.............................................................................................................................II ACKNOWLEDGEMENTS ................................................................................................. IV LIST OF FIGURES AND TABLES................................................................................. VIII LIST OF ABBREVIATIONS .............................................................................................. IX CHAPTER 1: INTRODUCTION 1.1 LYSOSOMAL STORAGE DISEASES ...................................................................................................2 1.1.1 Overview ...........................................................................................................................................2 1.1.2 Treatment of Lysosomal Storage Diseases .......................................................................................3 1.1.3 Examples of Common Lysosomal Storage Diseases ........................................................................5 1.2 FARBER DISEASE..................................................................................................................................7 1.1.2 Disease Overview..............................................................................................................................7 1.2.2 Treatment of Farber Disease .............................................................................................................9 1.2.3 Mouse Model of Farber Disease .....................................................................................................11 1.3 ACID CERAMIDASE ............................................................................................................................12 1.3.1 Gene, Structure and Biochemistry ..................................................................................................12 1.3.2 Mutations in Farber Disease............................................................................................................13 1.3.3 Other Ceramidases ..........................................................................................................................13 1.4 CERAMIDE............................................................................................................................................14 1.4.1 Structure and Physiological Function .............................................................................................14 1.4.2 Ceramide Signaling and Apoptosis.................................................................................................16 1.5 GENE THERAPY...................................................................................................................................18 1.5.1 Methods of Gene Transfer...............................................................................................................18 1.5.2 Treatment Modalities with Viral Vectors........................................................................................21 1.5.3 Gene Therapy for Farber Disease....................................................................................................21 1.5.4 Retroviral Genotoxicity...................................................................................................................24 1.6 MARKING OF TRANSDUCED CELLS ..............................................................................................26 1.6.1 Structure and Function of CD25 .....................................................................................................26 1.6.2 Use as a Pre-selective Marker .........................................................................................................27 1.6.3 Aberrant Expression in Cancer .......................................................................................................27 1.7 CURRENT STUDY OBJECTIVES .......................................................................................................28 vi CHAPTER 2: IN VIVO DELIVERY OF HUMAN ACID CERAMIDASE VIA CORD BLOOD TRANSPLANTATION AND DIRECT INJECTION OF LENTIVIRUS AS NOVEL APPROACHES FOR THE TREATMENT