Abstract
Duchenne muscular dystrophy is an X-linked disorder typically caused by out-of-frame mutations in the DMD gene. Most of these are deletions of one or more exons, which can theoretically be corrected through CRISPR-Cas9-mediated knockin. Homology-independent targeted integration is a mechanism for achieving such a knockin without reliance on homology-directed repair pathways, which are inactive in muscle. We designed a system based on insertion into intron 19 of a DNA fragment containing a pre-spliced mega-exon encoding DMD exons 1-19, along with the MHCK7 promoter, and delivered it via a pair of AAV9 vectors in mice carrying a Dmd exon 2 duplication. Maximal efficiency was achieved using a Cas9:donor adeno-associated virus (AAV) ratio of 1:5, with Cas9 under the control of the SPc5-12 promoter. This approach achieved editing of 1.4% of genomes in the heart, leading to 30% correction at the transcript level and restoration of 11% of normal dystrophin levels. Treatment efficacy was lower in skeletal muscles. Sequencing additionally revealed integration of fragmentary and recombined AAV genomes at the target site. These data provide proof of concept for a gene editing system that could restore full-length dystrophin in individuals carrying mutations upstream of intron 19, accounting for approximately 25% of Duchenne muscular dystrophy patients.