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 Cas9/gRNA we disrupted several miR BS in DMD myoblasts and selected the Let-7c BS has crucial for UTRN repression. Interestingly, Cas9/gRNA generated indels were as efficient as complete removal of Let-7c BS in upregulating UTRN expression, without major off-targets. In 3D human DMD myoblasts, Cas9/gRNA 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 based therapeutic strategy for DMD. ### Competing Interest Statement S.G and M.A are inventors on a patent application related to this work filed by the Genethon (PCT/EP2021/076882). FST has received speaker and consultancy honoraria from Takeda, Sanofi Genzyme and Aleph Farms (via UCL Consultants). All other authors declare that they have no competing interests.