CRISPR-Cas9 mediated endogenous utrophin modulation improves Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin withno effective treatment. Upregulation of utrophin (UTRN), a paralogue of dystrophin, is a promising avenue. Here, we present a CRISPR-Cas9 strategy to raise UTRN expression in vitro and in vivo by permanently disrupting microRNA binding sites . We first screened guides RNA (gRNA) and selected the microRNA Let-7c binding site. In two and three-dimensional human DMD cultures, we observed a stable editing over-time without major off-targets significant increase of UTRN and functional improvements on calcium influx and muscle contraction. We also demonstrated that indels generated by single gRNA upregulate UTRN as efficiently as the complete deletion of the repressor domain, providing rational for the use of a single gRNA strategy. In mdx animals, microRNA Let-7c binding site disruption by systemic rAAV injection results in UTRN upregulation and histological benefits in skeletal and cardiac muscles. Our findings describe the applicability of CRISPR-Cas9 for upregulating UTRN as a therapeutic approach for DMD.