Template-assisted sequence knockin rescues skeletal and cardiac muscle function in a deletion model of Duchenne muscular dystrophy.

Abstract

Duchenne muscular dystrophy (DMD) poses challenges in therapy design due to dystrophin's complex role in maintaining muscle function since the restoration of truncated protein products has failed to completely address the disease's pathophysiology in clinical trials. As ∼70% of patients harbor deletions, strategies enabling targeted DNA insertion to restore full-length dystrophin protein are essential. Here, we present template-assisted sequence knockin (TASK), a strategy that we employed to specifically correct the Dmd Δ52-54 mutation in a murine model. By co-delivering a repair template and the Cas9 nuclease using AAV9s, the splice-competent sequence for Dmd exons 52-54 was integrated into the residual intron 54 locus, resulting in the systemic restoration of full-length dystrophin at therapeutically relevant levels in the heart and across all skeletal muscles, leading to significant functional improvements. TASK demonstrates the highest efficiency of exogenous DNA knockin reported to date, achieving rescue of key dystrophic hallmarks in a deletion model of DMD.