Correction of DMD in human iPSC-derived cardiomyocytes by base-editing-induced exon skipping.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene. Previously, we showed that adenine base editing (ABE) can efficiently correct a nonsense point mutation in a DMD mouse model. Here, we explored the feasibility of base-editing-mediated exon skipping as a therapeutic strategy for DMD using cardiomyocytes derived from human induced pluripotent stem cells (hiPSCs). We first generated a DMD hiPSC line with a large deletion spanning exon 48 through 54 (ΔE48-54) using CRISPR-Cas9 gene editing. Dystrophin expression was disrupted in DMD hiPSC-derived cardiomyocytes (iCMs) as examined by RT-PCR, western blot, and immunofluorescence staining. Transfection of ABE and a guide RNA (gRNA) targeting the splice acceptor led to efficient conversion of AG to GG (35.9% ± 5.7%) and enabled exon 55 skipping. Complete AG to GG conversion in a single clone restored dystrophin expression (42.5% ± 11% of wild type [WT]) in DMD iCMs. Moreover, we designed gRNAs to target the splice sites of exons 6, 7, 8, 43, 44, 46, and 53 in the mutational hotspots and demonstrated their efficiency to induce exon skipping in iCMs. These results highlight the great promise of ABE-mediated exon skipping as a promising therapeutic approach for DMD.