Splice-site defects account for about 10% of pathogenic mutations that cause Mendelian diseases1. Prevalence is higher in neuromuscular disorders (NMDs)2, 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 pathway3,4,5, which is extremely inefficient in postmitotic tissues such as skeletal muscle6. 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 paralysis7. 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 MDC1A8. 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.
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The Cohn lab members are gratefully acknowledged for their technical support and critical input in this study. We thank I. Vukobradovic and A. Fleniken (Clinical Phenotyping Core, Toronto Centre for Phenogenomics), C. Rand (Aurora Scientific), and M. Justice, J. Dowling, and J. Ruston (Genetics and Genome Biology) for their critical inputs to this study. T. Paton, S. Perreira, G. Casallo, B. Thiruvahindrapuram, W. Sung (Toronto Center for Applied Genomics), and A. Cui (Deep Genomics) are acknowledged for their support in genomic and bioinformatics analyses. This work was supported by an AFM-Telethon postdoctoral fellowship and Cure CMD (to D.U.K.); an Ermenegildo Zegna Founder's scholarship (to E.M.); a Canada Research Chair (Tier 2) in Comparative Genomics and an Early Researcher Award from the Ontario Ministry of Research, Innovation and Science (to M.D.W.); and the Canadian Institute for Health Research, Natural Sciences and Engineering Research Council of Canada, the SickKids Foundation, RS McLaughlin Foundation and Women's Auxiliary Chairs (to R.D.C.).
dy2J /dy2J mouse (7230) injected with combination of AAV9- Cas9-sgRNA1 and AAV9-Cas9-sgRNA2. The animal was injected at P2 via temporal vein and video was taken at the age of 10-weeks old. Hind limb paralysis, contracture and kyphosis are no longer observed, indicating improvement of the phenotypes after restoration of Lama2 protein.