ESTABLISHMENT OF RECOMBINANT ADENO-ASSOCIATED VIRUS VECTOR INTEGRATION FREQUENCY IN VITRO AND IN VIVO THESIS Presented in Partial Fulfillment of the Requirements for the Degree Master of Science in the Graduate School of The Ohio State University By Mona Odeh, BS Graduate Program in Molecular, Cellular, and Developmental Biology The Ohio State University 2012 Thesis Committee: Douglas McCarty, Ph.D., Advisor Santiago Partida-Sanchez, Ph.D. c Copyright by Mona Odeh 2012 ABSTRACT Adeno-Associated virus (AAV) is a single-stranded (ss) DNA parvovirus, that is important for the development as a gene therapy vector. Recombinant AAV (rAAV) integration of a transgene into the host cell benefits, since it remains in progeny cells. However rAAV integrates randomly into pre-existing double-stranded breaks (DSB) which poses a risk for potential mutagenesis. Earlier experiments with rAAV vectors in cell culture showed that integration was inefficient at 0.1-0.5% of infectious vector genomes, whether this happens in non-dividing cells of animal tissues (such as the liver) is unclear. Therefore to better evaluate the potential benefit or risk a clearer understanding of AAV integration frequency is needed. We wished to evaluate the frequency of rAAV vector integration in vitro as well as in vivo under the treatment of DNA damaging agents. In vitro comparison be- tween single-stranded break (SSB) and double-stranded break (DSB) agents on viral integration were made using rAAV2-GFP transduced human lung fibroblast (HFL-1) cells, additional comparison between non-dividing and dividing cells were made. We hypothesized that dividing HFL-1 cells treated with the DSB inducing agent, camp- tothecin (CPT), would have a higher integration frequency than the cells treated with a SSB inducing agent, hydrogen peroxide (H2O2), and/or were in a non-dividing state. In vivo 8 C3H/HeJ mice were transduced with rAAV8-GFP (targeting the liver) and 4 out of the 8 mice were treated with CPT. Here the hypothesis states that ii if rAAV integrates readily into pre-existing DSB, then we should see an increase of viral vector integration in CPT treated mice. Finally, we also investigated whether rAAV vector integration happens at an ear- lier or a later time point post-infection. It was previously suggested that most vector integration occurs 24-48 hrs post-infection, which is concurrent with the view that free DNA ends, i.e. from the virus, are rapidly cleared from the cell to protect the integrity of the genome. 20 C3H/HeJ mice were transduced with rAAV8-GFP and time points 2 weeks, 1 month and 6 months were investigated for rAAV integration frequency. We tested the hypothesis that viral vector integration would occur early, thus no increased vector integration frequency was expected throughout the course of the experiment. A method was developed that detects small numbers of putative integrated molecules in excess of unintegrated viral DNA in the form of monomeric or concatemerized epi- somes. The rAAV vector harbors the GFP transgene, which contains an I-Ceu1 site and a LacZ1 fragment to be used as a QPCR target. I-Ceu1 site is used to cut out only unintegrated viral vectors, since it is not found in the mouse genome. We puri- fied out the unintegrated viral DNA through a low melting point (LMP) agarose gel and detected integration with QPCR. In vitro no significant difference in virus integration was seen between cells treated with DNA damaging agents or not, nor was a difference seen between non-dividing and dividing cells. In vivo we detected as little as 1 integrated viral vector genome out of 1000 infectious viruses in the hepatocytes, resulting in an integration efficiency of 0.1%. Additionally there was no significant difference in integration efficiency e between CPT treated and untreated mice. Lastly, in the long-term experiment, we detected integration frequency ranging from 0.1-0.01%. Also no change in integra- tion frequency was seen over the time course 2 weeks to 6 months. To conclude, a iii valuable AAV integration detection method has been developed to evaluate integra- tion frequency in vitro as well as in vivo. The results suggest that DNA damaging agents do not enhance the integration efficiency of rAAV in vitro nor in vivo. Finally observing no difference in integration frequency over 6 months suggests that indeed rAAV integration occurs early post-infection. iv I dedicate this thesis to God, Lord of the Worlds, Most Magnificent, Most Merciful To my beloved husband, Dr. Shareef Dabdoub To my loving parents, Said Faleh Falah Odeh and Etidal Ahmad Dalal Odeh v ACKNOWLEDGMENTS I would like to start out by praising and thanking Almighty God, for giving me this opportunity to grow in knowledge and experience in the field of my dreams, Biology. My thanks to my beloved husband, Shareef Dabdoub, are endless. Thank you, my love, for your constant support, for your help whenever I needed it and for always encouraging me to keep going when many times I wanted to quit. Special thanks also to my mentor Dr. Douglas Mark McCarty, who with his end- less patience and guidance made me the scientist I am today. Without his financial, intellectual, and kind support I would have never made it far in the field of Biology. I would also like to thank each of my committee members; Dr. Partida-Sanchez, Dr. Peeples, Dr. Clark and last but not least Dr. Justice for their advice, continued support and believing in me when I had doubts about my abilities as a scientist. I would like to thank each member of the McCarty Lab and the Fu Lab for their invaluable input, assistance, and ideas on various research problems. Special thanks goes to Dr. Marcela Cataldi and Chelsea Bolyard who were always there to answer questions and discuss my project. Also special thanks to Kimberly Zaraspe who has helped me tremendously with tedious technical difficulties in the lab. These are the people who made the experience in the McCarty lab worthwhile. I want to thank Dave Dunaway for his help and technical support in flow cytometry. I thank Dr. David Bisaro, the Director of Molecular, Cellular and Developmental Biology vi (MCDB) graduate program, for believing in my potential as a graduate student and giving me the courage to continue on and finish with a doctorate. Lastly, a big thank you to my beautiful family. Especially to my parents, Said and Etidal Odeh for giving me a great head-start in life, stressing the importance of education and hard work, and for believing that I have the potential to make it great in this world. I thank each of my siblings; Faleh Odeh, Marwa Odeh, Marwan Odeh, Senan Odeh and Ahmad Odeh. Though they never really understood what my research was about (this thesis should give them a clue!!), they recognized the hard work that was put in, always encouraged me to keep going and cheered me up when things became difficult. I'm grateful for my family's endless love and support for me; I could not have made it without them. vii VITA 2002 - 2004 . A.S. in Biology, Cincinnati State 2004 - 2006 . B.S. in Biology, University of Cincinnati 2007 - Present . Graduate Research Associate, The Ohio State University MCDB Graduate Pro- gram April 2011 . Poster Presentation, “Effects of DNA Dou- ble Stranded Break Inducing Agent, Camp- tothecin, on rAAV Vector Integration in vivo", OSU Molecular Life Sciences Inter- disciplinary Graduate Programs Annual Symposium, Columbus, OH FIELDS OF STUDY Major Field: Molecular, Cellular and Developmental Biology Specialization: Gene Therapy and AAV Vector Biology viii TABLE OF CONTENTS Abstract . ii Dedication . iv Acknowledgments . vi Vita......................................... viii List of Figures . xi List of Tables . xii CHAPTER PAGE 1 Introduction/Background . .1 1.1 AAV Virion Genome Structure . .1 1.2 AAV Life Cycle . .5 1.3 Recombinant Adeno-Associated Virus (rAAV) . .8 1.4 rAAV and Gene Therapy . .9 1.5 rAAV Transgene Expression . 13 1.6 Self-Complementary Adeno-Associated Virus (scAAV) . 15 1.7 rAAV vector persistence in the host cell . 17 1.8 rAAV vector integration . 20 1.8.1 wt AAV . 20 1.8.2 rAAV . 23 2 Motivation and Thesis Goals . 26 3 Materials and Methods . 30 3.1 Maintenance of cells . 30 3.2 Plasmid construct . 30 3.3 Viral vector production and purification . 31 3.4 Dose response of HFL-1 cells to camptothecin (CPT) and hydrogen peroxide (H2O2)............................ 33 ix 3.5 Viral infection and drug treatment for in vitro analysis of rAAV integration . 34 3.5.1 H2O2 experiment . 34 3.5.2 CPT experiment . 35 3.5.3 Quantifying integration by flow cytometry . 36 3.6 Mice . 37 3.7 Treatment, Infection and Tissue Collection for in vivo analysis of CPT effect on viral integration . 37 3.7.1 Statistical Analysis . 37 3.8 Infection and Tissue Collection for in vivo long-term integration analysis . 38 3.9 DNA isolation and gel purification assay . 38 4 Results . 41 4.1 A novel detection assay to measure integrated rAAV viral vector . 41 4.2 The effect of DNA damaging agents on rAAV viral vector integration in vitro ................................. 44 4.2.1 Dose Response Experiment . 44 4.2.2 H2O2 treatment of transduced HFL-1 cells to determine ef- fects of rAAV integration . 50 4.2.3 CPT treatments of transduced HFL-1 cells to determine effects of rAAV integration . 53 4.3 Effects of DNA DSB inducing agent, camptothecin (CPT), on rAAV vector integration in vivo ....................... 57 4.4 Long-term in vivo study of rAAV vectors integration frequency and longevity . 62 5 Discussion . 68 6 Future Directions .