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1 Gene Therapy for Facioscapulohumeral Muscular Gene Therapy for Facioscapulohumeral Muscular Dystrophy Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Lindsay M. Wallace, B.A. Molecular, Cellular, and Developmental Biology Graduate Program The Ohio State University 2012 Dissertation Committee: Dr. Scott Q. Harper, Advisor Dr. K. Reed Clark Dr. Denis C. Guttridge Dr. Jill Rafael-Fortney 1 Copyright by Lindsay M. Wallace 2012 ABSTRACT Facioscapulohumeral muscular dystrophy is the most common inherited muscular dystrophy though no treatment exists. The lack of therapeutic development for FSHD is directly linked to insufficient understanding of how the disease is caused. The goals of the studies presented here were to gain a better understanding of the pathogenic mechanisms of FSHD and to develop targeted translational therapies to treat the disease. FSHD is associated with D4Z4 repeat contraction on human chromosome 4q35, which does not result in complete loss or mutation of any gene. Consequently, the major obstacle to discerning the underlying pathogenic mechanism is to identify the cause. Although no gene was conclusively linked to FSHD development, evidence supported a role for the D4Z4-encoded DUX4 gene. In Chapter 3, our objective was to test the in vivo myopathic potential of DUX4. We delivered DUX4 to zebrafish and mouse muscle by transposon-mediated transgenesis and adeno-associated viral vectors, respectively. We found over-expression of DUX4 caused abnormalities associated with muscular dystrophy in both animal models. This toxicity required DNA binding, since a DUX4 DNA binding domain mutant produced no abnormalities. We also found the toxic effects of DUX4 were p53-dependent. This study demonstrated the myopathic potential of DUX4 in animal muscle and provided a p53-dependent mechanism for DUX4-induced ii toxicity. Considering previous studies showed DUX4 was elevated in FSHD patient muscles, our data support the hypothesis that DUX4 over-expression contributes to FSHD development. With DUX4 as a potential target, gene silencing approaches could provide treatment for FSHD. With as many as 29 different gene mutations responsible for other dominant myopathies, gene silencing approaches could have a broad impact. Feasible mechanisms to silence dominant disease genes have lagged behind gene replacement strategies, but with the discovery of RNA interference (RNAi) and its subsequent development into a promising new gene silencing tool, the landscape has changed. In Chapter 4, our objective was to demonstrate proof-of-principle for RNAi therapy of a dominant myopathy in vivo. We tested the potential of AAV-delivered therapeutic microRNAs, targeting the human Facioscapulohumeral muscular dystrophy (FSHD) Region Gene 1 (FRG1), to correct myopathic features in mice expressing toxic levels of human FRG1 (FRG1-high mice). We found that FRG1 gene silencing improved muscle mass, strength, and histopathological abnormalities associated with muscular dystrophy in FRG1-high mice, thereby demonstrating therapeutic promise for treatment of FSHD and other dominantly inherited myopathies using RNAi. Next we applied this therapeutic strategy to FSHD by targeting DUX4. Several recent studies support an FSHD pathogenesis model involving over-expression of the myopathic DUX4 gene making it the most promising therapeutic target. In Chapter 5, we tested a pre-clinical RNAi-based DUX4 gene silencing approach as a prospective treatment for FSHD. We found that AAV vector-delivered therapeutic microRNAs corrected DUX4-associated myopathy in mouse muscle. These results provide proof-of- iii principle for RNAi therapy of FSHD through DUX4 inhibition. Together these studies have helped define the main pathogenic insult in FSHD and laid out a plausible, targeted therapy to treat the disease. iv ACKNOWLEDGMENTS First and foremost I would like to thank my mentor and advisor, Dr. Scott Harper. I do not have enough words to express all he has done for me over the years. He truly embodies both of the titles Mentor and Advisor, and I would not be where I am today if not for him. He has provided helpful advice and immense opportunities as well as unwavering confidence and support during my time with him. He encouraged me to think big and gave me all the tools to achieve my goals. He acknowledges my strengths and helps me work through my weaknesses. My professional success as a scientist is a direct reflection of his encouragement and support and for that I am truly thankful. I thank my committee members Dr. Denis Guttridge, Dr. Reed Clark, and Dr. Jill Rafael-Fortney for all of their helpful discussions in committee meetings, muscle group meetings and journal clubs. They have each provided a different perspective on my projects, the field of science in general, and how to be successful in research. Because of them I received a diverse and comprehensive education. I value what all members have taught me and thank them for pushing me to my full potential. I would also like to acknowledge those from the beginning of my scientific career. I thank Dr. Mike Robinson for taking a chance on me when I was an inexperienced undergraduate student. He introduced me to the world of research, and got me excited about science. Also thanks to Dr. Heithem El-Hodiri, who convinced me to apply to v graduate school. Without his support and encouragement I would not be in this field. I would also like to thank him for his continued support and advice during my first few years of graduate school; he has been a trusted confidant and career advisor. I would like to thank all members of the Harper Lab for helping me reach this goal. They have served as a support system both scientifically and personally, especially Sara Garwick-Coppens who has been there with me from the very beginning. I would also like to thank friends from my graduate program: Matt Murtha, Tom Bebee, Dan Deathridge, Kevin Bosse and Brian Hutzen and well as my roommates and IBGP girl friends: Amanda (Haidet) Phillips, Julie (Johnson) Moravy, Sasha Beyer, Stephanie Cush, and Amanda (Beltramini) Healan for helping me through this process and for being there during all the tough times of graduate school. Finally, I want to thank my family; (parents) Phil and Jeri Wallace, (sister) Laura (Wallace) Harrington, and (grandparents) Jim and Lois Chamberlain and Dick and Sharon Wallace for always believing in me, encouraging education and giving me everything I have needed in life to be successful. I also want to thank my fiancé, Eric Winner, for his never-ending support both personally and technically. He has literally stood by my side through late night experiments, weekend vivarium runs, last minute poster printings and every Harper Lab computer problem that has ever existed. On top of that, he has truly encouraged me to be the best person I can be, both professionally and personally, and I will be forever grateful for his role in my life. vi VITA June 2001……………Hilliard Darby High School, Hilliard, Ohio May 2005……………Miami University, Oxford, Ohio Bachelor of Arts in Microbiology Sept. 2005-present......The Ohio State University, Columbus, Ohio Doctor of Philosophy in Molecular, Cellular and Developmental Biology May 2009 ...................Oral presentation, “Developing RNAi therapy for FSHD.” American Society of Gene Therapy, 12th Annual Meeting, San Diego, CA May 2009 ...................Travel Award, American Society of Gene Therapy, 12th Annual Meeting, San Diego, CA November 2009 ..........Oral presentation, “DUX4 promotes FSHD-associated pathology in vivo.” Facioscapulohumeral Muscular Dystrophy 2009 International Research Consortium Meeting, Watertown, MA January 2010-2011 .....Nationwide Children’s Hospital Outstanding Graduate Student Award for 2010, accompanied by stipend fellowship for 2010. The Research Institute at Nationwide Children’s Hospital, Columbus, Ohio April 2010 ..................Poster presentation, “DUX4, a facioscapulohumeral muscular dystrophy (FSHD) candidate gene, causes muscle toxicity in vivo.” 9th Annual OSU Medical Center Graduate and Postgraduate Research Day, Columbus, OH. April 2010 ..................Poster presentation winner accompanied by a Travel Award, 9th Annual OSU Medical Center Graduate and Postgraduate Research Day, Columbus, OH. July 2010 ....................Oral presentation, “Developing RNAi therapy for FSHD candidate genes.” Interdisciplinary Graduate Programs in the Molecular Life Sciences Symposium, Columbus, OH vii July 2010 ....................Best parallel session talk award, Interdisciplinary Graduate Programs in the Molecular Life Sciences Symposium, Columbus, OH November 2010 ..........Travel Award, 60th Annual American Society of Human Genetics meeting, Washington, D.C. November 2010 ..........Oral presentation, “DUX4 over-expression recapitulates FSHD- associated phenotypes in vivo.” 60th Annual American Society of Human Genetics meeting, Washington, D.C. November 2010 ..........Outstanding Pre-Doctoral Basic Award, 60th Annual American Society of Human Genetics meeting, Washington, D.C. November 2010 ..........Poster presentation, “Developing RNAi therapy for FSHD candidate genes.” 60th Annual American Society of Human Genetics meeting, Washington, D.C. November 2010 ..........Oral presentation, “Targeting FRG1 as proof‐of‐principle for RNAi therapy of dominant muscular dystrophies” Facioscapulohumeral Muscular Dystrophy 2010 International Research Consortium Meeting, Watertown, MA January 2011
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