DMD is a severe genetic disorder caused by mutations in the dystrophin gene, leading to progressive muscle weakness and degeneration. One therapeutic strategy involves exon skipping, which removes specific exons during mRNA processing to restore the reading frame and enable production of a shortened but functional dystrophin protein. While adeno-associated virus (AAV)-based gene therapies have shown promise to deliver CRISPR-Cas9 into muscle tissues, their effectiveness in muscle stem cells—the stem cells responsible for muscle regeneration—has been limited. This limitation poses a major challenge to long-term therapeutic benefits, as untreated stem cells can dilute the effects of genome editing during muscle turnover.

In the study, the researchers demonstrated that lipid nanoparticle (LNP)-mediated delivery of CRISPR-Cas9 mRNA and guide RNAs, either locally or systemically, achieves efficient genome editing in muscle stem cells. Using a humanized DMD mouse model, they compared LNP-CRISPR with AAV-CRISPR following intramuscular and intravenous administration. While LNP-CRISPR induced exon skipping in Pax7-positive muscle stem cells at levels comparable to or exceeding those observed in bulk muscle tissue, AAV-CRISPR showed markedly lower activity in these regenerative cells as previously reported. Single-cell RNA sequencing revealed that Cas9 mRNA was detected in multiple cell types after LNP administration, including 38% of Pax7⁺ muscle stem cells. The uptake was partly mediated by ApoE-LDLR interactions, since pre-coating LNPs with ApoE3 enhanced editing efficiency up to 14-fold in isolated stem cells.

Importantly, the genome-editing activity produced by LNP-CRISPR sustained over time and remained resistant to muscle injury, a scenario in which AAV-CRISPR lost efficacy. When muscle damage was induced to facilitate muscle turnover, exon-skipping activity and dystrophin restoration persisted in LNP-treated mice, suggesting that edited muscle stem cells proliferate and contribute to muscle repair. Transplantation studies further confirmed that LNP-treated stem cells engraft, expand after injury, and donate dystrophin-positive nuclei to regenerating myofibers. These findings underscore the therapeutic potential of targeting muscle stem cells to achieve durable benefits in DMD patients, whose muscles undergo continuous cycles of degeneration and regeneration.

Beyond its efficacy, LNP technology offers practical advantages over viral vectors. Unlike AAV, LNPs are administrable multiple times because they do not contain protein or viral components, the root cause of susceptibility to neutralizing antibodies. However, the study also notes challenges such as transient innate immune elevation and tissue off-target delivery, which will require further investigation. Future research will focus on improving tissue specificity and exploring ways to facilitate skeletal muscle delivery systemically.

This work represents a significant step toward safe and sustainable genome-editing therapies for muscular dystrophy. By enabling efficient correction of muscle stem cells, LNP-CRISPR addresses a critical gap in current approaches and opens new avenues for treating genetic muscle diseases.

• doi: 10.1016/j.celrep.2025.116695