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Reprogramming the Retina: Next Generation Strategies of Retinal Neuroprotection and Gene Therapy Vector Potency Assessment

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Reprogramming the Retina: Next Generation Strategies of Retinal Neuroprotection and Gene Therapy Vector Potency Assessment University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2018 Reprogramming The Retina: Next Generation Strategies Of Retinal Neuroprotection And Gene Therapy Vector Potency Assessment Devin Scott Mcdougald University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Genetics Commons, Molecular Biology Commons, and the Virology Commons Recommended Citation Mcdougald, Devin Scott, "Reprogramming The Retina: Next Generation Strategies Of Retinal Neuroprotection And Gene Therapy Vector Potency Assessment" (2018). Publicly Accessible Penn Dissertations. 3158. https://repository.upenn.edu/edissertations/3158 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3158 For more information, please contact [email protected]. Reprogramming The Retina: Next Generation Strategies Of Retinal Neuroprotection And Gene Therapy Vector Potency Assessment Abstract Mutations within over 250 known genes are associated with inherited retinal degeneration. Clinical success following gene replacement therapy for Leber’s congenital amaurosis type 2 establishes a platform for the development of downstream treatments targeting other forms of inherited and acquired ocular disease. Unfortunately, several challenges relevant to complex disease pathology and limitations of current gene transfer technologies impede the development of gene replacement for each specific form of retinal degeneration. Here we describe gene augmentation strategies mediated by recombinant AAV vectors that impede retinal degeneration in pre-clinical models of acquired and inherited vision loss. We demonstrate distinct neuroprotective effects upon retinal ganglion cell survival and function in experimental optic neuritis following AAV-mediated gene augmentation. Gene transfer of the antioxidant transcription factor, NRF2, improves RGC survival while overexpression of the pro-survival and anti- inflammatory protein, SIRT1, promotes preservation of visual function. In the context of inherited retinal disease, we show stimulation of anabolic metabolism following AKT3 gene transfer preserves photoreceptor viability and delays functional loss in a mouse model of retinitis pigmentosa. In addition to these neuroprotective strategies, we also describe an approach to improve the in vitro potency of AAV vectors that are restricted by tissue-specific regulatory elements. This strategy utilizes genome engineering based on CRISPR/Cas9 gene activation to reprogram cell lines to specifically and potently express tissue-specific promoters of interest from AAV vectors. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Cell & Molecular Biology First Advisor Jean Bennett Keywords aav, gene therapy, neuroprotection, photoreceptor, retinal degeneration, retinal ganglion cell Subject Categories Genetics | Molecular Biology | Virology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3158 REPROGRAMMING THE RETINA: NEXT GENERATION STRAEGIES OF RETINAL NEUROPROTECTION AND GENE THERAPY VECTOR POTENCY ASSESSMENT Devin S. McDougald A DISSERTATION in Cell & Molecular Biology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2018 Supervisor of Dissertation ____________________ Jean Bennett M.D., Ph.D., Professor of Ophthalmology and Cell & Developmental Biology Graduate Group Chairperson _____________________ Dan Kessler Ph.D., Chair, Cell & Molecular Biology Graduate Group Dissertation Committee Valder Arruda M.D., Ph.D. Claire H. Mitchell Associate Professor of Pediatrics Professor of Anatomy & Cell Biology Kenneth S. Shindler M.D., Ph.D. Hansell H. Stedman Associate Professor of Ophthalmology Associate Professor of Surgery i ACKNOWLEDGMENT I would have not reached this point academically, professionally, or personally without the influence of past scientific mentors. Dr. Anthony Bell Jr. at the University of Southern Mississippi presented my first opportunity to perform laboratory research. His guidance as well as patience provided an excellent foundation in molecular biology techniques, experimental design, and how to conduct oneself within a research lab. Drs. Wade Chien, Lisa Cunningham, and Thomas Friedman at the NIDCD/NIH allowed me to train in an exceptionally fast-paced and cutting edge research environment prior to entering graduate school. Observing their passion, dedication, and relentless work ethic towards improving both the fundamental understanding of human diseases and creation of novel therapeutics reinvigorated my passion for pursuit of a research-based career. I have been incredibly fortunate to carry out this work alongside a crew of exceptionally talented scientists from very diverse training backgrounds. I want to particularly acknowledge Drs. Adam Wojno, Scott Dooley, Kris Fisher, Jason Mills, and Pavitra Ramachandran for always lending their time for insightful discussions or venting sessions relevant to project design, troubleshooting, career advice, and life. Much of the work shown here could not have been performed without the technical expertise of many individuals including Zhangyong Wei, Alexandra Zezulin, Kimberly Dine, Tyler Papp, and Dr. Ken Shindler. In addition, I must thank members of the CAROT research vector core, especially Drs. Shangzhen Zhou and Jieyan Pan, for producing the AAV vectors used in these studies. I thank my thesis committee for their helpful insight and technical direction in guiding some of the studies presented in this document. The ideas and concepts behind much of this work would likely have been shot down immediately if I were under the direction of any other scientist besides Jean Bennett. I believe this is one of the many reasons Jean has sustained a highly productive scientific career: she places an incredible amount of trust in her trainees to independently develop and explore creative ideas and solutions to challenging problems in vision research. Beyond all the scientific achievements and medical milestones scattered along her career path, Jean maintains an unwavering humble, ii supportive, and congenial nature of a magnitude that I have yet to encounter elsewhere in the research community. Altogether, she is an exceptional scientist and an even better person, and I am forever grateful to her for allowing me to train and grow as a scientist in her laboratory. I must thank my family and friends for their enduring love and support throughout this very long and tiresome process. I want to specifically acknowledge Terra Kuhn, Bobby French, Steven Zhao, Stephen Bart, Chris Cali, David Walter, and Lucas Van Gorder for their friendship and overwhelming support during the best and worst of times. Finally, I must thank my parents, Glenn and Elizabeth, for their continued support through all avenues of life. iii ABSTRACT REPROGRAMMING THE RETINA: NEXT GENERATION STRATEGIES OF RETINAL NEUROPROTECTION AND GENE THERAPY VECTOR POTENCY ASSESSMENT Devin S. McDougald Jean Bennett M.D., Ph.D. Mutations within over 250 known genes are associated with inherited retinal degeneration. Clinical success following gene replacement therapy for Leber’s congenital amaurosis type 2 establishes a platform for the development of downstream treatments targeting other forms of inherited and acquired ocular disease. Unfortunately, several challenges relevant to complex disease pathology and limitations of current gene transfer technologies impede the development of gene replacement for each specific form of retinal degeneration. Here we describe gene augmentation strategies mediated by recombinant AAV vectors that impede retinal degeneration in pre-clinical models of acquired and inherited vision loss. We demonstrate distinct neuroprotective effects upon retinal ganglion cell survival and function in experimental optic neuritis following AAV-mediated gene augmentation. Gene transfer of the antioxidant transcription factor, NRF2, improves RGC survival while overexpression of the pro-survival and anti-inflammatory protein, SIRT1, promotes preservation of visual function. In the context of inherited retinal disease, we show stimulation of anabolic metabolism following AKT3 gene transfer preserves photoreceptor viability and delays functional loss in a mouse model of retinitis pigmentosa. In addition to these neuroprotective strategies, we also describe an approach to improve the in vitro potency of AAV vectors that are restricted by tissue-specific regulatory elements. This strategy utilizes genome engineering based on CRISPR/Cas9 gene activation to reprogram cell lines to specifically and potently express tissue-specific promoters of interest from AAV vectors. iv TABLE OF CONTENTS ACKNOWLEDGMENT ............................................................................................................. I ABSTRACT ............................................................................................................................... IV LIST OF FIGURES .................................................................................................................. VI CHAPTER 1 INTRODUCTION ............................................................................................. 1 CHAPTER 2 STIMULATING ANTI-INFLAMMATORY/ANTIOXIDANT PATHWAYS IN EXPERIMENTAL OPTIC NEURITIS WITH AAV-MEDIATED GENE TRANSFER ................................................................................................................
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