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, ultimately, 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.
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, the effect on cardiomyocytes was modest. Therefore, in this study, we aim to achieve exon skipping by developing a base editor (BE) delivered either as single AAV or as dual-AAV-system targeting the splice acceptor site (SAS) of either exon 51 or exon 53 with the benefit of avoiding double strand breaks and bypassing NHEJ repair.
Screening of various deaminase and Cas9 combinations, together with suitable sgRNAs was performed in primary kidney fibroblasts of a DMDΔ52-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 then packed as AAVs and used to transduce porcine 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 on-target editing efficacy of almost 40% and insignificant by-stander editing in 2D cell culture. In 3D engineered heart patches, where human iPSC-derived DMDΔ52 cardiomyocytes were reseeded onto a native, decellularized heart slice (extracelluar matrix), a reduction of the arrhythmic load and normalization of the effective refractory period could be observed.
This study aims to demonstrate that base editing may critically improve efficacy and safety of cardiac gene editing in DMD, a rapidly progressing disease with few effective alternate options.
