730. Permanent Correction of Diverse Muscular Dystrophy Mutations in Human Cardiomyocytes by Myoediting

Abstract

Dilated cardiomyopathy (DCM) is one of the most common lethal features of Duchenne muscular dystrophy (DMD), caused by diverse mutations in the X-linked dystrophin gene (DMD). Without dystrophin, a large cytoskeletal protein, muscles degenerate, causing myopathy. Although several gene therapies have been tested, there is no curative treatment so far. In 2014, we first used CRISPR/Cas9-mediated genome editing (termed Myoediting) to correct the mutation in the germ line of mdx mice, the mouse model of DMD [Long et al. Science 345:1184-8]. Recently, we applied Myoediting to postnatal muscle tissues in mice by delivering gene editing components via a harmless adeno-associated virus. Cardiac and skeletal muscle showed progressive rescue of dystrophin protein [Long et al. Science doi: 10.1126/science.aad5725]. These studies paved the way for novel genome editing-based therapeutics in DMD. We have now advanced Myoediting to cells from human DMD patients by engineering the permanent skipping of mutant or out-of-frame exons in the genome of DMD patient-derived induced pluripotent stem cells (iPSCs). We have optimized Myoediting of DMD mutations using pools of sgRNAs to target the top 12 hot spot mutant exons. We targeted the conserved RNA splicing acceptor/donor sites of each exon. NHEJ-mediated indels efficiently abolished the splicing sites and skipped the mutated or out-of-frame exons. Based on the known DMD mutations, we established a publicly available online resource (Duchenne Skipper Database) for selecting the optimal target DMD sequences for Myoediting, which will rescue dystrophin function in 60-80% of DMD patients. We performed Myoediting on representative iPSC-derived cardiomyocytes from multiple patients with point, deletion or duplication mutations and efficiently restored dystrophin protein expression in cardiomyocytes. Rescued DMD cardiomyocyte shows enhanced function. Opportunities and obstacles in the path toward efficient and permanent elimination of the genetic cause of DMD and other DCM will be discussed.