AAV9 Edits Muscle Stem Cells in Normal and Dystrophic Adult Mice

CRISPR editing of muscle stem cells (MuSCs) with adeno-associated virus serotype-9 (AAV9) holds promise for sustained gene repair therapy for muscular dystrophies. However, conflicting evidence exists on whether AAV9 transduces MuSCs. To rigorously address this question, we used a muscle graft model. The grafted muscle underwent complete necrosis before regenerating from its MuSCs. We injected AAV9.Cre into Ai14 mice. These mice express tdTomato upon Cre-mediated removal of a floxed stop codon. About 28%–47% and 24%–89% of Pax7⁺ MuSCs expressed tdTomato in pre-grafts and regenerated grafts (p > 0.05), respectively, suggesting AAV9 efficiently transduced MuSCs, and AAV9-edited MuSCs renewed successfully. Robust MuSC transduction was further confirmed by delivering AAV9.Cre to Pax7-ZsGreen-Ai14 mice in which Pax7⁺ MuSCs are genetically labeled by ZsGreen. Next, we co-injected AAV9.Cas9 and AAV9.gRNA to dystrophic mdx mice to repair the mutated dystrophin gene. CRISPR-treated and untreated muscles were grafted to immune-deficient, dystrophin-null NSG.mdx4cv mice. Grafts regenerated from CRISPR-treated muscle contained the edited genome and yielded 2.7-fold more dystrophin⁺ cells (p = 0.015). Importantly, increased dystrophin expression was not due to enhanced formation of revertant fibers or de novo transduction by residual CRISPR vectors in the graft. We conclude that AAV9 effectively transduces MuSCs. AAV9 CRISPR editing of MuSCs may provide enduring therapy. Satellite cells are stem cells responsible for muscle repair and maintenance. Here, Nance et al. show that adeno-associated virus serotype-9 (AAV9) efficiently transduced and edited satellite cells. Intravenous delivery of AAV9 CRISPR vectors to mdx mice, a model for Duchenne muscular dystrophy, not only corrected dystrophin gene mutation in mature muscle cells but also in satellite cells. Importantly, the muscle regenerated from the edited satellite cells contained the CRISPR-corrected dystrophin gene and expressed the edited dystrophin protein. Satellite cell editing provides a means to achieve enduring muscle gene therapy that can meet the demands of muscle injury and maintenance throughout a patient's life.