Gene therapy is a promising treatment for the muscular dystrophies. Our Center has played major roles in advancing the use of AAV for muscle disease. These include extensive characterization of dystrophin and DUX4 expression and function, the discovery that AAV can be used for systemic gene delivery to muscle, and generation of numerous small, muscle-specific expression cassettes (MSECs), micro-dystrophins (μDys) and RNAi cassettes. These advances have enabled multiple gene therapy trials for DMD, LGMDs, XL-MTM1and other muscle disorders. We have also developed and advanced many skeletal and cardiac muscle testing platforms and tools useful for muscle gene therapies, including 3D DMD human iPSC-derived engineered muscle tissues and a DMD rat model. However, current AAV- μDys therapies are not as effective as hoped, and AAV- RNAi therapies for FSHD and other dominant disorders have not advanced to the clinic. Here we propose two related aims to address limitations of AAV gene therapies: (A) Testing new AAV capsid variants for cardiac and skeletal muscles, iterative testing of novel micro-dystrophins, larger split-intein dystrophins, and MSEC designs to minimize immunogenicity and increase cardiac and skeletal muscle potency; (B) Application of enhanced cassettes and vectors for DUX4 transcript knock-down in FSHD therapy. In Aim 1, we develop vectors to improve DMD clinical interventions that have been limited by the levels and functionality of therapeutic dystrophins, and by very high vector doses that have caused serious adverse events (SAEs) in some patients due to immunological reaction to the vector and/or transgene. We will compare multiple myotropic serotypes, which show significantly increased muscle transduction to deliver novel transgene cassettes that have been designed for reduced immunogenicity and increased potency, especially in cardiac muscle. A major focus will include the testing of novel, split-intein AAV vectors to produce mini- and full-length dystrophins. We also test a dual vector strategy to recover depressed function in the heart via overexpression of an enzyme (RNR) that elevates cardiac dATP, a small molecule myosin activator. Studies of immune response to dystrophin will be augmented by screening patient blood cells for immune reactivity. In Aim 2 we combine the myotropic vectors with enhanced RNAi cassettes to advance gene therapy for FSHD. This experimental plan combines vector developments from Aim 1 with new FSHD animal models and FSHD clinical studies from Project 2. Genetic and phenotypic changes in the FLExDUX4 mouse model of FSHD will be targeted via local and systemic delivery of AAV-DUX4 RNAi, while further studies will evaluate these AAV-DUX4 RNAi vectors in the Göttingen minipig model of FSHD that is being characterized in Project 2. DUX4 gene silencing and reduction of inflammation in the minipig will be measured by MRI, muscle structural changes, and biomarkers based on clinical results from Project 2. Toge...