https://doi.org/10.1007/s00392-025-02625-4
1Klinikum rechts der Isar der Technischen Universität München Klinik und Poliklinik für Innere Medizin I München, Deutschland; 2Technische Universität München (TUM) Kardiologie, I. Med München, Deutschland
Background: Duchenne Muscular Dystrophy (DMD) is the most frequent hereditary childhood myopathy leading to loss of ambulation in the first decade of life, followed by respiratory and cardiac failure and premature death (at a mean age around 30 years). DMD is caused by the absence of dystrophin, usually due to frameshift mutations in the dystrophin gene which encodes a 427 kDa protein.
Methods: Cas9 is a nuclease cutting precisely where guided by gRNAs, allowing for excision of e.g. an exon of the DMD gene. As previously demonstrated1, dual-AAV-delivered, split-CRISPR-Cas9-mediated excision of exon 51 can reframe the dystrophin gene in the DMDΔ52 pig model and ameliorate the muscular phenotype. However, the effect on cardiomyocytes was modest. Therefore, in this study, we aim to achieve exon skipping by 1) direct injection of AAV9-Cas9-gE51 into coronary arteries and 2) by developing a base editor (BE) targeting the splice acceptor site (SAS) of either exon 51 or exon 53.
Results: Direct injection of AAV9-Cas9-gE51 (5x1014vgs per animal) increased the level of dystrophin protein in the heart from 0% (DMD untreated) and 7% (DMD, i.v. treated = DMD iv) to 13% (DMD intracoronary application = DMD ic). Ejection fraction (50±2% in wildtype controls) improved from 33±2% (DMD untreated) and 38±2% (DMD iv) to 41±2% (DMD ic). Similarly, contraction velocity (1879±207mmHg/s, WT) was found impaired in DMD (734±145mmHg/s), but improved in DMD iv (1061±167mmHg/s), even more in DMD ic (1727±238mmHg/s, p<0.05 vs. DMD iv).
Concerning a base editing strategy for targeting the SAS of exon 51 or 53 of DMD, screening of different deaminase and Cas9 combinations, together with suitable sgRNAs was performed in primary kidney fibroblasts of a DMD pig. Finally, editing efficacies up to 17% which also resulted in a successful exon skip could be observed using a dual-AAV-system containing the ABE8e-BE and spRY-Cas9 targeting the SAS of exon 53. This combination will be applied intracoronarily to DMDΔ52 pigs.
Thus, adjustments of the application strategy (DMD ic vs. DMD iv) and choice of the editor (BE vs. nuclease) may critically improve efficacy and safety of gene editing in DMD, a rapidly progressing disease with few effective alternate options.
1https://doi.org/10.1038/s41591-019-0738-2
2https://doi.org/10.1038/s41556-022-00899-8