Duchenne muscular dystrophy (DMD) is a severe hereditary progressive muscle-wasting disorder caused by mutations in the DMD gene that abolish dystrophin expression. Gene editing–mediated exon skipping offers durable reading frame correction at the DNA level, enabling permanent restoration of functional dystrophin protein. Nevertheless, clinical translation remains hampered by delivery challenges, particularly the restricted packaging capacity of adeno-associated virus (AAV) vectors. Many current base editors exceed the AAV size limit, necessitating dual-vector systems that markedly reduce editing efficiency and limit therapeutic applicability. Thus, the development of compact, high-performance editors that can be delivered using a single AAV vector is an urgent priority. In this study, we developed an engineered all-in-one AAV base editing therapy for DMD with high efficiency editing and potentially overcoming a critical barrier in genome editing based exon skipping therapy for DMD.
Through systemic engineering of Cas9 protein and promoters, AID cytosine base editor has been minimized to 4.7kb, which can be efficiently packaged into one AAV vector with good CMC profile. All-in-one AID CBE has been developed for DMD exon 51, exon 53, and exon 50 skipping therapy. The editing and exon skipping activity were evaluated in iPSC mytobutes and in DMD models.
In DMD iPSCs myotubes with exon 51–53 deletions, the all-in-one base editor effectively induced exon 50 skipping with editing efficiency comparable to the dual-vector system. Similarly, in humanized Dmd mouse model with exon 52 deletion, intravenous administration of all-in-one AAV9-AID CBE induced effective DNA editing (4.3%) and exon 53 skipping (13.6%) at 5E13vg/kg group. Those results suggest that all-in-one CBE system possesses its editing activity. Functional improvement was not observed in this study, likely due to low vector distribution in heart and muscles. Higher muscle specific AAV should be considered to increase the expression of CBE in muscles.
A super myotropic AAV, LD-8S05 exhibited high muscle specificity and low liver tropism, which will be applied to deliver AID CBE to treat DMD. The animal data will be expected at the upcoming conference.