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Utilizing Gene Therapy Methods to Probe the Genetic Requirements to Prevent Spinal Muscular Atrophy

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Utilizing Gene Therapy Methods to Probe the Genetic Requirements to Prevent Spinal Muscular Atrophy Utilizing Gene Therapy Methods to Probe the Genetic Requirements to Prevent Spinal Muscular Atrophy _______________________________________ A Dissertation presented to the Faculty of the Graduate School at the University of Missouri _______________________________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy _____________________________________________________ by MADELINE R. MILLER Dr. Christian L. Lorson, Dissertation Supervisor JULY, 2016 The undersigned, appointed by the dean of the Graduate School, have examined the dissertation entitled Utilizing Gene Therapy Methods to Probe the Genetic Requirements to Prevent Spinal Muscular Atrophy presented by Madeline R. Miller a candidate for the degree of Doctor of Philosophy, and hereby certify that, in their opinion, it is worthy of acceptance. Professor Christian L. Lorson Professor David J. Pintel Professor Elizabeth Bryda Professor Michael Petris Professor Michael Garcia DEDICATION To my parents, Tim and Rebecca: thanks for getting me here. To my fiancé, Simon: thanks for getting me through. To many friends, family, and mentors along the way: simply, thanks. ACKNOWLEDGEMENTS I would like to thank my mentor, Dr. Christian Lorson for inviting me into a world with which I quickly fell in love. Because of you I found a study that is truly worth my passion. Because of you Mizzou truly became a home. My time in the Lorson lab was fruitful because of the comradery of its many members and unfailing patience from a few. Dr. Hans Rindt cheerfully showed me the ways of bench work and was a genuine model of the curiosity, rigor, and fun that should drive every scientist. Dr. Erik Osman did his best to prevent me from losing my pipettes, my data, and my mind. Even the most menial tasks are enjoyable with Erik in charge. I have found several unexpected role models throughout my time here as well. I immediately recognized that Dr. Dave Setzer was the kind of thorough teacher and genuine counselor that I would like to become. Dr. Elizabeth Bryda has also given me a full back-stage pass to understand the challenges and opportunities available in science and teaching. She embodies hard work while maintaining a joyful and open spirit. Finally, through my financial support I received a gracious amount of personal support and guidance from Dr. Mark Hannink and Mrs. Debbie Allen for which I am thoroughly grateful. ii TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ ii LIST OF FIGURES ......................................................................................................... viii LIST OF ABBREVIATIONS ............................................................................................. x RESEARCH ABSTRACT ............................................................................................... xiii Chapter One: Introduction .................................................................................................. 1 1: Spinal Muscular Atrophy ............................................................................................ 1 1.1: Clinical Manifestation of SMA ............................................................................ 1 Current options for SMA assistance ............................................................................ 3 Orthopedics .................................................................................................................. 3 Gastrointestinal ............................................................................................................ 4 Respiratory................................................................................................................... 4 1.2: SMA as a Genetic Disease ................................................................................... 5 Incidence of SMA ........................................................................................................ 5 Identification of the SMA locus .................................................................................. 5 Non 5q Spinal Muscular Atrophies ............................................................................. 8 Chromosome 5q ........................................................................................................... 9 1.3: SMN1 and SMN2 ............................................................................................... 10 1.4: SMN and SMA: Genotype-Phenotype Correlation ............................................ 13 1.5: SMN Molecular Function ................................................................................... 16 SMN in snRNP Biogenesis........................................................................................ 19 SMN’s role in Axons ................................................................................................. 28 2: Tools to Study Spinal Muscular Atrophy.................................................................. 32 2.1 Animal Models of SMA ...................................................................................... 32 Schizosaccharomyces pombe .................................................................................... 32 Caenorhabditis elegans .............................................................................................. 35 Drosophila melanogaster ........................................................................................... 35 Xenopus ..................................................................................................................... 38 Danio rerio ................................................................................................................. 39 Mus musculus ............................................................................................................ 42 iii Sus scroffa ................................................................................................................. 47 2.2 Other models of SMA .......................................................................................... 48 Patient fibroblast cells ............................................................................................... 48 Induced Pluripotent stem cells (iPSCs) ..................................................................... 49 2.3 Viral Gene Delivery ............................................................................................. 50 Characterization of Adeno-Associated Virus ............................................................ 50 AAV Life Cycle......................................................................................................... 53 AAV as a Vector for Gene Transfer .......................................................................... 55 2.3 Early Gene Therapy in Human Trials.................................................................. 60 2.4 SMA Gene Therapy ............................................................................................. 60 Serotype Selection ..................................................................................................... 61 Transgene Expression ................................................................................................ 62 Route of Delivery ...................................................................................................... 63 Pre-clinical Optimization of Delivery Techniques .................................................... 67 2.4 Viral Delivery to Probe SMA Biology ................................................................ 68 3. SMA Clinical Trials .................................................................................................. 70 3.1 Ionis/Biogen Nusinersen (formerly Ionis-SMNRx) ............................................ 71 3.2 AveXis-ChariSMA .............................................................................................. 72 3.3 Roche/PTC/SMAF: RG7800 ............................................................................... 73 3.4 Novartis-- LMI070 .............................................................................................. 73 3.5 Repligin/Phizer—Quinazoline............................................................................. 73 3.6 Cytokinetics/Astellas – CK-2127107 .................................................................. 74 3.7Genentech/Roche—Olesoxime (TRO19622) ....................................................... 74 Chapter Two: The Search for an Evolutionary-Guided Minimal Domain to Identify the Disease-Relevant Regions within SMN............................................................................ 78 1: Introduction ............................................................................................................... 78 SMN’s function toward motor neuron disease .......................................................... 78 1.1: Domains of SMN ................................................................................................ 80 N-Terminus/Exon 1 ................................................................................................... 83 Exon 2a ...................................................................................................................... 84 Exon 2b ...................................................................................................................... 84 iv Exon 3 .......................................................................................................................
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