Duchenne Muscular Dystrophy (DMD) is the most frequent hereditary childhood myopathy leading to progressive muscle degeneration followed by respiratory and cardiac failure and, ultimately, premature death. DMD is usually caused by frameshift mutations in the Dystrophin gene which encodes a 427 kDa protein.

As previously demonstrated, 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, in this study, we aim to achieve exon skipping by developing a base editor (BE) delivered as single- or dual-AAV-system targeting the splice acceptor site (SAS) of either exon 51 or 53 with the benefit of avoiding double strand breaks.

Screening of various deaminase and Cas9 combinations, together with suitable sgRNAs was performed in primary kidney fibroblasts of a DMD pig. Finally, editing efficacies up to 17% could be observed using a dual-AAV-system containing the ABE8e-BE and spRY-Cas9 targeting the SAS of exon 53. The two most promising dual-AAV-constructs were packed as AAVs and used to transduce ex vivo heart slices, followed by transduction and editing analysis. Furthermore, AAVs containing orthologous spacers were applied to human iPSC-derived DMDΔ52 cardiomyocytes resulting in an editing efficacy of 9.2%. and an exon skip rate of 10.4%.

Finally, in vivo application of the AAVs intracoronarily to DMDΔ52 pigs is scheduled, followed by electrophysiological, molecular and histological analyses.

This study aims to demonstrate that base editing may critically improve efficacy and safety of gene editing in DMD, a rapidly progressing disease with few effective alternate options.