OR06

CRISPR-Cas9 mediated endogenous utrophin upregulation improves Duchenne Muscular Dystrophy

M Ralu(1) S Guiraud(1) P Galbiati(1) S Dastidar(2,3) A De Cian(4) G Ronzitti(1) F S Tedesco(2,3) M Amendola(1,5)

1:Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE; 2:University College London; 3:The Francis Crick Institute; 4:INSERM U1154, CNRS UMR7196, Museum National d'Histoire Naturelle; 5:University of Foggia

Duchenne muscular dystrophy (DMD) is an incurable progressive neuromuscular disorder due to mutations in the DMD gene, loss of dystrophin expression and muscle wasting. Dystrophin-recovery approaches only restore truncated proteins, might be immunogenic and some are mutation-dependent. Another potentially universal approach consists in upregulating utrophin (UTRN), a paralogue of dystrophin able to compensate for the deficit without inducing immunogenicity. We developed a CRISPR-Cas9 strategy to increase endogenous utrophin. We disrupted the binding site (BS) of miR-Let-7c, a known UTRN repressor, in human DMD and murine myoblasts. This induced a ~3.5-fold increase of expression. Results were confirmed in three-dimensional human DMD cultures, where editing resulted in UTRN upregulation and functional improvements. We showed the gRNA has no major off-targets. Finally, we evaluated this strategy in the mdx mouse model of Duchenne. We confirmed that miR Let-7c expression is not affected in the skeletal and cardiac muscles of this model compared to wild-type mice. We then performed intravenous injection of two recombinant adeno-associated viruses type 9 (rAAV9s) encoding for Cas9 and gRNA. DNA editing in the tibialis anterior (13%), heart (21%) and diaphragm (5%) were sufficient to upregulate UTRN expression by 1.5-2-fold. This allowed an amelioration of muscle histopathological phenotype. To increase vector delivery and editing, we are currently treating mdx mice with a more myotropic AAV. The strategy has been applied to other loci, validated in human DMD myoblasts and is currently tested in 3D organoids. These findings provide the foundations for a universal gene editing therapeutic strategy for DMD.