University College London The use of matrix attachment regions to enhance the in-vivo potency of rAAV vectors PhD Thesis Joanna Evette Hanley 1 DECLARATION I, Joanna Evette Hanley, 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 this thesis. Signed: Joanna Hanley Date: 06/03/2014 2 ABSTRACT Adeno-associated virus vector encoding codon optimised human factor IX (AAV-LP1-hFIXco) has demonstrated great promise for the treatment of patients with severe haemophilia B. However, in some patients treated with a high vector dose, hepatocellular toxicity was observed. To improve AAV vector potency, various scaffold/matrix attachment regions (S/MARs) were cloned at the 3’ end of a modified single-stranded (ss) AAV-LP1-hFIXco expression cassette. In a head to head comparison, a vector containing a S/MAR element from the human hypoxanthine-guanine phosphoribosyl-transferase gene in the forward orientation (ssAAV-LP1-hFIXco-HPRT-F) was found to mediate the highest levels of hFIX expression in mice. In comparison to animals transduced with a control vector containing no S/MAR, the ssAAV-LP1-hFIXco-HPRT-F transduced cohort expressed hFIX at 28-fold higher levels. This trend was reproducible in rhesus macaques where 10-fold higher FIX levels were observed following transduction with ssAAV-LP1-hFIXco-HPRT-F as compared to delivery of ssAAV-LP1-hFIXco-control vector. Through a deletion analysis, short regions from the IFNβ and HPRT S/MARs with potent enhancer activity were identified. This allowed for the in-silico elicitation of motifs with a potential role in S/MAR function and also minimised the space occupied by S/MARs within our AAV expression cassette. When cloned into a self-complementary (sc) AAV-LP1-hFIXco expression cassette, the 130bp region from the HPRT S/MAR (fragment 2b) was sufficient to enhance FIX levels in mice by 35-fold over that observed with a control self-complementary vector. Mechanistic studies showed that S/MAR elements enhanced AAV transgene expression by reducing heterochromatin marks (H3K9me2 and HP1α) in the promoter region, resulting in an increase in FIX mRNA levels by up to 20-fold. S/MARs therefore provide a novel inbuilt process for enhancing AAV mediated transgene expression by preventing epigenetic silencing of the provirus. As such, S/MARs offer the possibility to improve gene transfer to humans through using lower and potentially safer doses of AAV. 3 ACKNOWLEDGMENTS I would like to thank my supervisor, Professor Amit Nathwani, for giving me the opportunity to carry out my PhD in his research group and for his expert guidance and help towards this thesis. I have learnt so much over the past four years and this would not have been possible without the advice of such a brilliant mentor. A special thank you must also go to Cecilia Rosales and Jenny McIntosh for their constant support and contribution towards my research. I would also like to thank my colleagues and good friends Marco Della Peruta, Gordon Cheung, Maria Virgilio, Christopher Allen, Marc Davies, Pollyanna Goh, Doyoung Lee, Arnold Pizzey, Deepak Raj, Allison Dane and Gabrielle To for their help and for making my PhD studies such a pleasant experience. Thank you also to Allison Dane, Deepak Raj and Marc Davies for all the proof reading. Last but most definitely not least, I would like to thank my family for their love and support through the more stressful times. To my late father, John Eric Hanley, I would like to say thank you for encouraging me to always believe in my abilities. 4 TABLE OF CONTENTS TITLE PAGE………………………………………………………………………………………………………………………….1 DECLARATION .......................................................................................................................... 2 ABSTRACT ................................................................................................................................. 3 ACKNOWLEDGMENTS .............................................................................................................. 4 TABLE OF CONTENTS ................................................................................................................ 5 TABLE OF FIGURES ................................................................................................................... 9 LIST OF TABLES ....................................................................................................................... 11 COMMONLY USED ABBREVIATIONS ...................................................................................... 12 CHAPTER 1 INTRODUCTION ................................................................................................... 15 1.1 Haemophilia B ........................................................................................................ 15 1.2 Gene therapy using adeno-associated virus ................................................................ 16 1.3 Improving vector potency ............................................................................................ 24 1.3.1 The AAV transduction pathway ............................................................................ 24 1.3.2 Capsid modifications ............................................................................................. 28 1.3.2.1 Changing serotype/transcapsidation ............................................................. 28 1.3.2.2 Hybrid capsids ................................................................................................ 29 1.3.2.3 Epitope tagging .............................................................................................. 29 1.3.2.4 Immune escape capsids ................................................................................. 29 1.3.2.5 Empty capsid decoys ...................................................................................... 30 1.3.2.6 Capsids that bypass cellular blocks ................................................................ 31 1.3.2.7 Critique of capsid modifications .................................................................... 32 1.3.3 Cassette optimisation ........................................................................................... 32 1.3.3.1 Self complementary vectors .......................................................................... 32 1.3.3.2 Promoter selection ........................................................................................ 33 1.3.3.3 Codon-optimisation ....................................................................................... 34 1.4 Repeat induced gene silencing .................................................................................... 35 1.5 Scaffold/Matrix Attachment Regions ........................................................................... 38 1.6 Research aim ................................................................................................................ 43 1.7 Brief outline of strategy: .............................................................................................. 43 CHAPTER 2 MATERIALS AND METHODS ................................................................................ 46 2.1 Molecular biology ........................................................................................................ 46 2.1.1 Reagents ................................................................................................................ 46 5 2.1.2 Polymerase chain reaction (PCR) .......................................................................... 47 2.1.3 Restriction enzyme digests ................................................................................... 47 2.1.4 Agarose gel electrophoresis .................................................................................. 48 2.1.5 Klenow fill-in ......................................................................................................... 49 2.1.6 Alkaline phosphatase treatment ........................................................................... 49 2.1.7 Ligations ................................................................................................................ 49 2.1.8 Bacterial Transformation and expansion of Colonies ........................................... 50 2.2 Vector production ........................................................................................................ 51 2.2.1 Reagents ................................................................................................................ 51 2.2.2 Triple transfection of HEK293T cells ..................................................................... 52 2.2.3 Virus purification by affinity chromatography ...................................................... 53 2.2.4 Vector titration by Q-PCR ...................................................................................... 56 2.2.5 Vector titration by alkaline gel .............................................................................. 56 2.2.6 Coomassie Stain of Capsid Proteins ...................................................................... 58 2.3 Animal studies .............................................................................................................. 61 2.4 Assessment of human factor IX (hFIX) antigen levels .................................................. 61 2.4.1 Reagents ................................................................................................................ 61 2.4.2