CRISPR-Cas9 mediated endogenous utrophin upregulation improves Duchenne Muscular Dystrophy

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin. Upregulation of utrophin (UTRN), a dystrophin paralogue, is a promising therapeutic avenue. Here, we present a CRISPR-Cas9-mediated strategy to increase utrophin expression by disrupting microRNA (miR) binding sites (BS). Using a Cas9/gRNA ribonucleoprotein (RNP) complex we disrupted several miR BS in DMD myoblasts and selected the Let-7c BS has crucial for UTRN repression. Interestingly, Cas9/gRNA indels were as efficient as the complete removal of Let-7c BS in upregulating UTRN expression, without any major off-targets. In three-dimensional human DMD cultures, Cas9/gRNA-mediated editing resulted in significant utrophin upregulation and functional improvements of calcium dysregulation and muscle contraction. Finally, Let-7c BS disruption in mdx animals by systemic rAAVs mediated delivery of Cas9 and gRNA resulted in utrophin upregulation and amelioration of the muscle histopathological phenotype. These findings provide the foundations for a universal (mutation-independent) gene editing therapeutic strategy for DMD.