483. In Vivo DMD Gene Editing in Muscles and Muscle Stem Cells of Dystrophic Mice

Duchenne muscular dystrophy (DMD) is a X-linked genetic disorder that arises from frame-disrupting mutations in the DMD gene, encoding DYSTROPHIN. Lack of DYSTROPHIN expression destabilizes muscle fiber membranes, increases susceptibility to contraction-induced injury and drives muscle degeneration. Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated but still functional protein. In this study, we develop and test a direct gene editing strategy to recover DYSTROPHIN expression in the mdx mouse model of DMD. Coupling clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases delivered via adeno-associated virus (AAV) with paired guide RNAs flanking the mutated Dmd exon 23, we demonstrate precise excision of intervening DNA and restoration of Dystrophin reading frame and protein expression in vivo in both skeletal and cardiac muscles following local or systemic delivery. DYSTROPHIN expression in AAV Dmd-CRISPR treated mdx mice was sufficient to partially recover functional deficiencies of dystrophic muscle. Finally, we demonstrate in vivo targeting of the mdx mutation in endogenous muscle stem cells, suggesting that AAV-CRISPR may provide a means to support ongoing repair of dystrophic fibers with corrected muscle precursors. This study provides proof-of-concept evidence supporting the feasibility and efficacy of in vivo genome editing to correct frame-disrupting mutations in DMD.