Project 1: Translational and pre-clinical studies of muscular dystrophy gene therapy using AAV

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This translational research project is designed to improve current AAV vector mediated dystrophin gene therapy methods, to adapt those methods for dominant muscular dystrophies, and to enhance our clinical infrastructure to facilitate participation in future AAV gene therapy trials. Our previous efforts have involved years of collaborative studies that have significantly advanced methods and approaches for gene therapy of DMD. They have also led to approaches to begin harnessing inhibitory RNAi and gene targeting to abrogate gene expression in dominantly inherited muscular dystrophies. We have also developed approaches for systemic gene delivery using AAV vectors, and have made important advances in the study of muscle gene regulation that enable muscle-restricted gene expression and immune evasion following AAV vector delivery. The AAV/micro-dystrophin approaches are far enough along that they are entering human clinical trials to assess safety, and primarily in skeletal muscles, efficacy. The micro-clones, however, are still not fully functional and show reduced activity in cardiac muscles compared with skeletal muscles. The gene therapy approaches for FSHD have been slowed by a lack of good animal models and difficulties in adapting RNAi to AAV. Nonetheless these methods are far enough along that it is reasonable and imperative to enhance their application for optimal gene therapy of many different types of muscular dystrophy. Consequently, our specific aims focus on several of the key limitations of current approaches to gene therapy for DMD and FSHD. For DMD, we focus on enhancing approaches that will functionally target both skeletal and cardiac muscles. We will develop improved gene regulatory cassettes active in all striated muscles, as well as exclusively in skeletal or cardiac muscle. Novel mini-and micro-dystrophins with enhanced function in striated muscles will be designed and screened in multiple test systems. We will also test a promising dual vector strategy, combining dystrophin replacement (structural-based therapy) with enhanced contractile performance via increased ribonucleotide reductase (contractile augmentation therapy) to improve cardiac performance. Our second area of focus is to build upon previous AAV studies for DMD and on mouse model development for FSHD to adapt AAV methods for gene therapy of FSHD. These studies will include analysis of patterns of Dux4 expression in muscle cells. They will also focus on developing and testing AAV vector mediated tissue-specific expression of RNAi hairpins targeting Dux4 mRNA in the context of low level mRNA expression in our AAV-DUX4 mouse model. Finally, we plan to develop patient databases and natural history data focused on cardiac function with colleagues at the MDA clinics in Seattle. We will gather and organize data from ongoing cardiac imaging studies, functional readouts, natural history data and mutational spectra in the DMD patient populations served by the Seattle clinics. These studies will expand the trial readiness goals of Project 2 to include DMD patients.