Development of a near universal genome editing for Duchenne Muscular Dystrophy

Duchenne Muscular Dystrophy (DMD) is a severe, progressive, X-linked recessive disorder caused by mutations in the DMD gene. It results in either the complete absence of dystrophin or a significant reduction in dystrophin, a vital protein that stabilizes muscle cell membranes during contraction. This leads to the gradual weakening of skeletal and cardiac muscles, early loss of mobility, respiratory and cardiac failure, and premature death, usually in the third or fourth decade of life. Current standard treatments include corticosteroids to slow symptoms, but they do not address the underlying genetic cause. New therapies, such as AAV-based microdystrophin gene addition, exon-skipping oligonucleotides, and other methods to restore dystrophin, show promise but are limited by mutation specificity in many cases, the need for repeated doses in some strategies, and potential immunological risks from vectors or transgenes.

Genome editing with customizable endonucleases, especially the CRISPR/Cas9 system, where RNA guides direct Cas9 to create precise double-strand breaks, has become a promising approach for permanent genetic correction. In this study, a CRISPR-Cas9-mediated homology-independent targeted integration (HITI) strategy was developed and tested to restore functional dystrophin expression from the endogenous DMD promoter by targeting the non-coding intron 1 region. This enables insertion of a human microdystrophin transgene (hMD1) downstream of the native full-length dystrophin promoters and exon 1 elements, ensuring physiologically regulated expression and broad applicability to about 95% of DMD patients by bypassing mutations in exons 2–79.

Bioinformatics guided selection identified effective SaCas9 guide RNAs targeting human and mouse DMD intron 1, demonstrating high on-target efficiency and low predicted off-target activity. In vitro validation in HEK293T cells, Neuro2A cells, and primary DMD patient-derived myoblasts showed successful gRNA cloning, gene editing, and HITI-mediated transgene integration, as confirmed by junction PCR and Western blotting.

In vivo proof of concept was demonstrated through AAV9-mediated co-delivery into the tibialis anterior muscle of mdx mice. This led to targeted integration at the DMD intron 1 locus, microdystrophin (~137 kDa) expression driven by the endogenous promoter, sarcolemmal localization in some myofibers, and partial functional recovery. Treated muscles showed significantly greater resistance to eccentric contraction-induced force loss and decreased passive muscle length compared with controls, while peak isometric force remained largely unchanged. There was on-target editing efficiency in skeletal muscle, with no detectable off-target cleavage at the tested genomic sites.

Despite some limitations, its extensive mutational coverage, lower immunogenicity compared to repeated treatments, and compatibility with AAV delivery make it a promising candidate for future improvements through systemic administration, advanced capsid engineering, and more extensive preclinical studies. Ultimately, it paves the way for a widely applicable therapy for most individuals with DMD.