Development of an AAV-Encoded Adenine Base Editor for Duchenne Muscular Dystrophy.
Duchenne muscular dystrophy (DMD) is a devastating X-linked disorder caused by out-of-frame mutations in the DMD gene, most commonly large deletions or duplications, as well as nonsense and splice site mutations, that result in the absence of functional dystrophin protein. These mutations lead to progressive skeletal and cardiac muscle failure. In particular, exon 52 of the DMD gene represents a mutational hotspot in DMD patients. Deletion of exon 52 (DMDΔ52) disrupts the reading frame, leading to premature termination of translation. Here, we used a dual recombinant adeno-associated virus (rAAV) system employing an intein-mediated split strategy to deliver ABE8e using the PAM-less SpRY Cas9 nickase for adenine base editing of splice acceptor sites (SAS) in the dystrophin gene. Targeting the SAS of exon 51 or exon 53 in a DMDΔ52 background aims to induce exon skipping, thereby restoring the open reading frame, in effect converting the severe DMD phenotype into a milder Becker muscular dystrophy (BMD)-like phenotype. We systematically screened sgRNAs in porcine kidney fibroblasts and human embryonic kidney cells (HEK293T), identifying a guide RNA targeting the SAS of exon 53 as the most effective candidate with high on-target editing efficacies and minimal bystander editing. In human DMDΔ52 iPSC-derived cardiomyocytes cultivated as 2D monolayers, high editing efficiencies were achieved with the optimized 1:2 ratio of N-Terminus to C-Terminus. Functional assessment in 3D engineered heart patches revealed a trend toward normalization of the arrhythmic DMD phenotype with an increase in the effective refractory period (ERP) and a reduction in arrhythmic load compared with untreated DMD patches, despite lower editing efficacy in the 3D setting. These findings suggest that even modest levels of base editing-mediated exon skipping may ameliorate the DMD cardiac phenotype toward a BMD-like state, supporting the translational potential of this dual rAAV base editing approach for DMD cardiomyopathy.