Therapeutic suppression of mutant SOD1 by AAV9-mediated gene therapy approach in Amyotrophic Lateral Sclerosis Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Shibi B Likhite, M.S. Graduate Program in Molecular, Cellular and Developmental Biology The Ohio State University 2014 Dissertation Committee: Dr. Brian K Kaspar, Advisor Dr. Arthur Burghes Dr. Stephen Kolb Dr. Christine Beattie Copyright by Shibi B Likhite 2014 Abstract Amyotrophic Lateral Sclerosis is one of the most common, adult-onset neurodegenerative disorder, characterized by progressive and fatal loss of motor neurons in spinal cord, motor cortex and brainstem which results into muscular paralysis and ultimate respiratory failure leading to death. Dominant mutations in Superoxide Dismutase 1 (SOD1) gene are one of the frequent causes of familial ALS. Mutant SOD1 confers cell autonomous as well as non cell autonomous toxicity towards motor neurons in ALS. Therefore, along with motor neurons, surrounding non-neuronal cells like astrocytes, microglia and oligodendrocytes play important role in the pathogenesis of familial ALS. Non cell autonomous toxicity towards the motor neurons is also evident in sporadic ALS cases. Thus, suggesting that reduction of motor neuron toxicity from multiple cell types within the CNS is required to achieve the maximum therapeutic benefits in ALS. Transgenic removal of mutant SOD1 from motor neurons and astrocytes significantly delays the disease onset and progression of ALS mice with significant extension in survival. Here, we determined the feasibility and efficacy of post-natal downregulation of mutant SOD1 via AAV9-mediated shRNA delivery in two distinct mouse models of ALS. Our results show that AAV9-mediated delivery of SOD1 shRNA before or even after the disease onset results in significant extension in the lifespan of ii SOD1G93A and SOD1G37R mice. We also show that AAV9 SOD1 shRNA administration is safe and well-tolerated in wild-type mice. Moreover, widespread spinal cord transduction and efficient SOD1 reduction in non-human primates provide strong basis for the translation of AAV9 mediated SOD1 shRNA delivery approach to the human settings. Furthermore, in accordance with the guidelines established by the Food & Drug Administration (FDA), USA, we show successful construction of clinical AAV SOD1 shRNA plasmid with non-coding stuffer sequence, replacing the GFP expression cassette. We show that in vitro administration of clinical AAV SOD1 shRNA vector into human cells results into efficient downregulation of SOD1 protein levels with lack of GFP expression as well as any unnatural transcript production from the stuffer. Here, we show successful generation of clinical AAV SOD1 shRNA vector that can be utilized directly in human clinical trials for ALS. Microglia are one of the key players involved in non-cell autonomous toxicity in ALS pathogenesis. Transgenic removal of mutant SOD1 from microglia has confirmed their role in the disease progression. Transgenic inhibition of NF-κB pathway in ALS microglia has also confirmed their role in neuroinflammation induced motor neuron death in ALS. However, none of the currently available approaches are able to target the microglia post-nataly. In Chapter 4, we show that conditioning of endstage microglia from SOD1G93A mice with exogenous Galectin 1, a lectin binding carbohydrate, results in reduction of their neurotoxic potential towards the motor neurons. We also show that Galectin 1 treatment of ALS microglia reduces the secretion of proinflammatory cytokines iii like TNF-α. Finally, our results show that Galectin 1 exerts its immunomodulatory effects on ALS microglia via inhibition of NF-κB pathway. As astrocytes act as a source of endogenous Galectin 1 and remain to be one of the cell types readily targeted by AAV9, our results suggest a plausible approach for post-natal immunomodulation of ALS microglia by AAV9-mediated overexpression of exogenous Galectin 1. iv Dedication This document is dedicated to my parents. v Acknowledgments I am incredibly thankful to my mentor, Dr. Brian Kaspar for his constant support, guidance and confidence in me. His enthusiasm and passion for research and his untethered dedication and commitment to science have alwaysinspired me to be persistant, perseverant and do better and meaningful science. I cannot thank him enough for spending his time and energy in training me to be an independent researcher. With the intense training under his supervision, I feel ready for any future challenges. I would like to thank my committee members, Dr. Arthur Burghes, Dr. Stephen Kolb and Dr. Christine Beattie. Their invaluable critics and meticulous suggestions have certainly helped shape my scientific career. The strong basic and translational research background of Dr. Burghes and Dr. Beattie blended with Dr. Kolb’s clinical expertise helped me comprehend the big picture of science. I feel honored and extremely obliged to have these elite scientists looking over my shoulders. Being an international student, I feel extremely lucky to be one of “the Kaspars”! It was never just a research lab for me but was my family outside India! Every member of Kaspar lab, past or current, holds my deepest respect and gratification for making it the best lab I could have ever asked for. I would like to thank Dr. Kevin Foust for his constant advice and feedback throughout my graduate training. Being easily approachable and patient enough, he really helped me build confidence in myself and my work. I would also like to thank our senior lab members Dr. Kathrin Meyer, Dr. Laura Ferraiuolo and Dr.Carlos Miranda. I certainly owe a big part of my successful graduate career to Kathrin and Laura for their constant support and encouragement. With their vast knowledge of literature, skeptical thinking and relentless work ethics, both Kathrin and Laura are and will always be a role model for me. More importantly, for me, they not only acted as two scientific pillars in the lab but also as social and emotional strongholds. I really hope that I could be as humble and approachable to my colleagues as both of them were for me. I will always be in their debt for all the love and support. With them around, I certainly did not feel homesick that often! vi I want to thank our technicians Lyndsey Braun, Leah Schmelzer and Che Best a million times. Without their help and support, it was impossible to complete my graduate work. I would especially like to thank Leah Schmelzer for helping me out with the tremendous amount of mouse work. She not only helped me in scientific endeavors but also lend me an ear when I needed. With her objective and practical thinking, she did act as my elder sister. I cannot imagine my graduate work here in the Kaspar laboratory without my colleagues Sungwon Song and Ashley Frakes. I would like to thank them for their constant feedback and constructive criticism which kept the work environment always innovative and thought-provoking. Having such friends definitely made all the stress and hardships of graduate life bearable. I also want to thank our new members, Jillian Liu and Anton Blatnik for constant discussions and debates. Trying to answer the continuous string of questions coming from them has certainly helped me to be prompt, precise and up to date with the literature. Also, their never-ending interest in research made the lab environment innovative and exciting. I would like to thank all the visiting scholars, Olivia Michels, David Baker and Dr.Alessandra Govoni. It was a great experience to learn and exchange both scientific and non-scientific knowledge with them. I am also thankful to have Dr. Nicolas Wein as a colleague and a friend whom I could turn to if there was any problem whatsoever. I would also like to thank my friends outside the lab who have played a very important part in keeping me socially sane. I would like to extend special thanks to my friend Wenyan Jiang for always being there to support me in tough times. I would also like to thank my friends Balasubramani Hariharan and Krishna Patel for their support and patience with me. Finally, I would like to thank my parents, Balkrishna Likhite and Surekha Likhite. I would not have been here if it was not for their constant support, encouragement and confidence in me. I feel extremely obliged with all the energy, efforts and time they’ve spent on me. Their lessons of hard work, perseverance and humbleness have helped me complete this intense task of graduate study. I hope I could keep them happy and make them proud with my future endeavors. vii Vita 1995-2001 ...................... Secondary School Certificate, N.M.V High School, Pune, Maharashtra, India 2001-2003 ...................... Higher Secondary Certificate, N.M.V. Jr. College, Pune, Maharashtra, India 2003-2006 ...................... Bachelor of Science in Microbiology, M.E.S. Abasaheb Garware College of Science, Art & Commerce, Pune, Maharashtra, India 2006-2008 ..................... Master of Science in Biochemistry, The M.S. University of Baroda, Vadodara, Gujarat, India 2009-Present ................. Doctor of Philosophy in Molecular, Cellular & Developmental Biology, The Ohio State University, Columbus, Ohio, USA 2008-2009 ..................... Junior Research Fellow, Advanced Centre for Treatment, Research and Education in Cancer (ACTREC), Navi Mumbai, Maharashtra, India. 2007................................ Lectureship-NET award,