Correction of a splicing defect in a mouse model of congenital muscular dystrophy type 1A using a homology-directed-repair-independent mechanism.

Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases. Prevalence is higher in neuromuscular disorders (NMDs), owing to the unusually large size and multi-exonic nature of genes encoding muscle structural proteins. Therapeutic genome editing to correct disease-causing splice-site mutations has been accomplished only through the homology-directed repair pathway, which is extremely inefficient in postmitotic tissues such as skeletal muscle. Here we describe a strategy using nonhomologous end-joining (NHEJ) to correct a pathogenic splice-site mutation. As a proof of principle, we focus on congenital muscular dystrophy type 1A (MDC1A), which is characterized by severe muscle wasting and paralysis. Specifically, we correct a splice-site mutation that causes the exclusion of exon 2 from Lama2 mRNA and the truncation of Lama2 protein in the dy2J/dy2J mouse model of MDC1A. Through systemic delivery of adeno-associated virus (AAV) carrying clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing components, we simultaneously excise an intronic region containing the mutation and create a functional donor splice site through NHEJ. This strategy leads to the inclusion of exon 2 in the Lama2 transcript and restoration of full-length Lama2 protein. Treated dy2J/dy2J mice display substantial improvement in muscle histopathology and function without signs of paralysis.