Correction of diverse muscular dystrophy mutations in human engineered heart muscle by single-site genome editing.

Abstract

Genome editing with CRISPR/Cas9 is a promising new approach for correcting or mitigating disease-causing mutations. Duchenne muscular dystrophy (DMD) is associated with lethal degeneration of cardiac and skeletal muscle caused by more than 3000 different mutations in the X-linked dystrophin gene (<i>DMD</i>). Most of these mutations are clustered in "hotspots." There is a fortuitous correspondence between the eukaryotic splice acceptor and splice donor sequences and the protospacer adjacent motif sequences that govern prokaryotic CRISPR/Cas9 target gene recognition and cleavage. Taking advantage of this correspondence, we screened for optimal guide RNAs capable of introducing insertion/deletion (indel) mutations by nonhomologous end joining that abolish conserved RNA splice sites in 12 exons that potentially allow skipping of the most common mutant or out-of-frame <i>DMD</i> exons within or nearby mutational hotspots. We refer to the correction of DMD mutations by exon skipping as myoediting. In proof-of-concept studies, we performed myoediting in representative induced pluripotent stem cells from multiple patients with large deletions, point mutations, or duplications within the <i>DMD</i> gene and efficiently restored dystrophin protein expression in derivative cardiomyocytes. In three-dimensional engineered heart muscle (EHM), myoediting of DMD mutations restored dystrophin expression and the corresponding mechanical force of contraction. Correcting only a subset of cardiomyocytes (30 to 50%) was sufficient to rescue the mutant EHM phenotype to near-normal control levels. We conclude that abolishing conserved RNA splicing acceptor/donor sites and directing the splicing machinery to skip mutant or out-of-frame exons through myoediting allow correction of the cardiac abnormalities associated with DMD by eliminating the underlying genetic basis of the disease.