CRISPR-Cas9-mediated upregulation of utrophin ameliorates Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder caused by loss of dystrophin. Upregulating utrophin, a dystrophin paralog, is a promising gene therapy approach. Here, we present a CRISPR-Cas9-based strategy to enhance utrophin expression by disrupting repressor binding sites. Using a Cas9/guide RNA (gRNA) ribonucleoprotein complex, we disrupted several such sites in DMD myoblasts and identified microRNA Let-7c binding site as effective in relieving repression of the UTRN gene. Interestingly, Cas9-generated insertions or deletions (indels) were as effective as the complete removal of Let-7c binding site in upregulating UTRN expression, with minimal off-target effects. In a three-dimensional tissue-engineered human skeletal muscle model of DMD, this editing strategy resulted in significant utrophin upregulation and functional improvements of calcium dysregulation and muscle contraction. Finally, in mdx mice, local or systemic delivery of recombinant adeno-associated viruses encoding Cas9 and gRNA targeting the Let-7c binding site resulted in utrophin upregulation and amelioration of muscle histopathology and function. These findings provide the foundations for a mutation-independent, potentially universal gene-editing therapeutic strategy for DMD.