Full-length dystrophin restoration via targeted exon integration by AAV-CRISPR in a humanized mouse model of Duchenne muscular dystrophy.

Targeted gene-editing strategies have emerged as promising therapeutic approaches for the permanent treatment of inherited genetic diseases. However, precise gene correction and insertion approaches using homology-directed repair are still limited by low efficiencies. Consequently, many gene-editing strategies have focused on removal or disruption, rather than repair, of genomic DNA. In contrast, homology-independent targeted integration (HITI) has been reported to effectively insert DNA sequences at targeted genomic loci. This approach could be particularly useful for restoring full-length sequences of genes affected by a spectrum of mutations that are also too large to deliver by conventional adeno-associated virus (AAV) vectors. Here, we utilize an AAV-based, HITI-mediated approach for correction of full-length dystrophin expression in a humanized mouse model of Duchenne muscular dystrophy (DMD). We co-deliver CRISPR-Cas9 and a donor DNA sequence to insert the missing human exon 52 into its corresponding position within the DMD gene and achieve full-length dystrophin correction in skeletal and cardiac muscle. Additionally, as a proof-of-concept strategy to correct genetic mutations characterized by diverse patient mutations, we deliver a superexon donor encoding the last 28 exons of the DMD gene as a therapeutic strategy to restore full-length dystrophin in >20% of the DMD patient population. This work highlights the potential of HITI-mediated gene correction for diverse DMD mutations and advances genome editing toward realizing the promise of full-length gene restoration to treat genetic disease.