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Vesiculovirus G Protein-Based Stable Cell Lines for Continuous Lentiviral Vector Production

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Vesiculovirus G Protein-Based Stable Cell Lines for Continuous Lentiviral Vector Production Vesiculovirus G protein-based stable cell lines for continuous lentiviral vector production Maha Tijani Thesis submitted for the degree of Doctor of Philosophy Infection and Immunity University College London 2018 Declaration I, Maha Tijani, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 2 Abstract Lentiviral vectors (LV) are often pseudotyped with the envelope G protein from vesicular stomatitis virus Indiana strain (VSVind.G). However, VSVind.G based continuous LV producer cell lines have not been reported; it has been assumed that VSVind.G is fusogenic and cytotoxic. To find alternative G proteins for LV production, we investigated other vesiculovirus G proteins (VesG) from VSV New Jersey strain (VSVnj), Cocal virus (COCV), Piry virus (PIRYV), VSV Alagoas virus (VSVala), and Maraba virus (MARAV). All these VesG envelopes were used in transient transfection to produce infectious particles that were robust during concentration and freeze-thawing. We then found, surprisingly, that VSVind.G and all the other VesG proteins could be constitutively expressed in 293T cells, and showed no cytotoxicity when compared to a retroviral Env protein. These VesG expressing cells could support LV production when other components were transiently supplied. However, we showed that VesG expressing cells do not show receptor interference hence LV can superinfect their producer cells, resulting in vector genome accumulation and possible toxicity. We attempted to knock-out the low- density lipoprotein receptor (LDLR) gene on producer cells, which was reported to be the primary cell entry receptor for VSVind.G. However, only a slight reduction in LV transduction was observed in LDLR-KO cells. Hence, other methods such as using anti-retroviral drugs to block superinfection may be necessary to allow construction of stable producer cell lines. 3 Impact Statement Lentiviral vectors (LVs) are usually pseudotyped with the G protein of vesicular stomatitis virus Indiana strain (VSVind.G) due to its high physical stability. However, this G protein has been believed to be toxic when expressed constitutively in cells owing to its high fusogenicity. Therefore, well-studied alternative G proteins can provide crucial insights related to their use in clinical grade LV production. In this study, I demonstrate that other tested vesiculovirus G proteins are as stable, both physically and thermally, as the gold standard VSVind.G. Also, I show that all tested G proteins, including VSVind.G, show similar cytotoxicity to that of the negative control. Also, I demonstrate that, against the commonly accepted concept that VSVind.G cannot be expressed in cells continuously, this G protein could be expressed stably in cells for up to five months. Moreover, these stable VSVind.G expressing cells supported LV production when were supplemented with other vector components transiently in this period. Similarly, other tested G proteins could also be stably expressed in cells for at least five months and generated transient LVs during this time. These G proteins can be employed to generate stable packaging cell lines for lentiviral vector production. 4 Acknowledgements Firstly, I would like to express my sincere gratitude to my supervisors Prof Mary Collins and Dr Yasuhiro Takeuchi for the continuous support of my PhD study and their patience and motivation. I am especially grateful to Mary for her support even before I join the lab, and throughout my PhD, both academically and personally. I am also thankful to Yasu; for his friendly advice and guidance during this project. A special thanks go to Dr Giada Mattiuzzo for being always there for me when I had no one to talk to about my concerns and for her knowledge, guidance, humour, and friendship. I would like to thank NIBSC for funding this PhD, and Dr Nicola Rose for her support. My sincere thanks also go to Prof Simon Waddington and Dr Joanne Ng who provided me with an opportunity to join their team. Simon’s advice during my transfer examination helped me greatly with writing this thesis. Jo’s unconditional support, made writing this thesis to go as smoothly as possible. The enthusiasm for her research was contagious and motivational for me. I would like to thank my fellow lab mates and friends for the stimulating discussions, scientific and especially non-scientific, that brought the fun we needed in the last four years. I especially want to thank Altar Munis for his help, especially when I needed it the most. I am eternally indebted to my family for their love and encouragement throughout my life and studies. I sincerely appreciate all their hard work, love and support to provide the opportunities that enabled me to pursue my passions and goals. Last but not least, I want to thank Mehdi, who made this PhD possible, and impossible. 5 Table of contents DECLARATION .................................................................................................................................. 2 ABSTRACT ........................................................................................................................................ 3 IMPACT STATEMENT ........................................................................................................................ 4 ACKNOWLEDGEMENTS .................................................................................................................... 5 TABLE OF CONTENTS ........................................................................................................................ 6 LIST OF FIGURES ............................................................................................................................. 11 LIST OF TABLES ............................................................................................................................... 13 ABBREVIATIONS ............................................................................................................................. 14 ....................................................................................................................................................... 18 INTRODUCTION .............................................................................................................................. 18 1.1 GENE THERAPY- FROM CONCEPT TO REALITY ........................................................................................ 19 1.2 VECTORS USED FOR GENE DELIVERY .................................................................................................... 20 1.3 RETROVIRUSES ............................................................................................................................... 22 1.3.1 Gamma-retroviral vectors in clinical trials ......................................................................... 28 1.4 LENTIVIRUSES ................................................................................................................................. 31 1.4.1 HIV-1 life cycle.................................................................................................................... 31 1.4.2 Restriction factors influencing HIV-1 infection .................................................................. 42 1.4.3 Development of HIV-1 derived lentiviral vectors ............................................................... 45 1.4.4 Lentiviral vector production methods ................................................................................ 49 1.4.5 Lentiviral vectors in clinical gene therapy .......................................................................... 55 1.4.6 LV delivery for genome editing .......................................................................................... 62 1.5 RHABDOVIRUSES ............................................................................................................................ 64 1.5.1 Vesiculoviruses ................................................................................................................... 64 1.6 AIMS OF THE THESIS ........................................................................................................................ 86 6 ....................................................................................................................................................... 87 ....................................................................................................................................................... 87 MATERIALS AND METHODS ........................................................................................................... 87 2.1 CELL CULTURE ................................................................................................................................ 88 2.1.1 HEK 293T cells .................................................................................................................... 88 2.1.2 TE671 cells ......................................................................................................................... 88 2.1.3 WINPAC CELLS ............................................................................................................................ 89 2.2 GENE TRANSFER TO MAMMALIAN CELLS .............................................................................................. 90 2.2.1 Transfection of Cells for G Protein Expression ................................................................... 90 2.2.2 Transient
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