Somatic gene editing ameliorates skeletal and cardiac muscle failure in pig and human models of Duchenne muscular dystrophy

Frameshift mutations in the DMD gene, encoding dystrophin, cause Duchenne muscular dystrophy (DMD), leading to terminal muscle and heart failure in patients. Somatic gene editing by sequence-specific nucleases offers new options for restoring the DMD reading frame, resulting in expression of a shortened but largely functional dystrophin protein. Here, we validated this approach in a pig model of DMD lacking exon 52 of DMD (DMD Delta 52), as well as in a corresponding patient-derived induced pluripotent stem cell model. In DMD Delta 52 pigs(1), intramuscular injection of adeno-associated viral vectors of serotype 9 carrying an intein-split Cas9 (ref. (2)) and a pair of guide RNAs targeting sequences flanking exon 51 (AAV9-Cas9-gE51) induced expression of a shortened dystrophin (DMD Delta 51-52) and improved skeletal muscle function. Moreover, systemic application of AAV9-Cas9-gE51 led to widespread dystrophin expression in muscle, including diaphragm and heart, prolonging survival and reducing arrhythmogenic vulnerability. Similarly, in induced pluripotent stem cell-derived myoblasts and cardiomyocytes of a patient lacking DMD Delta 52, AAV6-Cas9-g51-mediated excision of exon 51 restored dystrophin expression and amelioreate skeletal myotube formation as well as abnormal cardiomyocyte Ca2+ handling and arrhythmogenic susceptibility. The ability of Cas9-mediated exon excision to improve DMD pathology in these translational models paves the way for new treatment approaches in patients with this devastating disease. CRISPR-Cas9-mediated gene editing restores dystrophin expression in both pig and human induced pluripotent stem cell models of Duchenne muscular dystrophy, with beneficial effects on skeletal muscle and cardiac function.