OR04

CRISPR activation of utrophin as a mutation-independent approach for Duchenne Muscular Dystrophy therapy

P Galbiati(1,2) M Ralu(1,2) M Amendola(1,2,3)

1:Genethon, UMR_S951, Inserm, Univ Evry, Université Paris Saclay, EPHE; 2:Universite´ Paris-Saclay, Université d'Évry; 3:University of Foggia

Duchenne muscular dystrophy (DMD) is a severe genetic disorder affecting 1 in 5.000 newborn males. It is caused by mutations in the DMD gene, leading to dystrophin deficiency and progressive muscle degeneration. Current dystrophin-based therapies, such as exon-skipping, AAV-delivered mini/micro-dystrophin, and CRISPR editing, aim to restore truncated dystrophin forms. However, these approaches can elicit immune responses and some are mutation-specific, limiting their therapeutic scope. An alternative approach, potentially applicable to all DMD patients regardless of their genetic mutation, involves upregulating utrophin (UTRN), a structural and functional paralogue of dystrophin. UTRN reactivation has shown therapeutic potential in preclinical models, improving the dystrophic phenotype without triggering immune responses. Here, we employed CRISPR activation (CRISPRa) systems with catalytically inactive Cas9 (dCas9) fused to transcriptional activators or epigenetic modulators, enabling targeted UTRN activation without DNA cleavage. Using in silico chromatin analysis of ENCODE muscle tissue data, we identified 12 candidate sgRNAs targeting the UTRN-A promoter and distal enhancer (DUE). Testing dCas9-activators with each single sgRNAs in HEK cells, alongside a positive control sgRNA for β-globin, we achieved up to a 5-fold increase in UTRN mRNA. Combinations of sgRNAs and editors are currently being evaluated in immortalized myoblasts from DMD patients. Given the inefficiency of plasmid transfection in these cells, we are comparing the delivery of activators via FACS sorting of plasmid-transfected myoblasts and nucleofection of in vitro-transcribed mRNA to enhance delivery efficiency. Our findings highlight UTRN-targeted CRISPRa as a promising mutation-independent therapy for DMD, offering broader applicability and reduced immunogenicity compared to dystrophin-based approaches.