Adeno-associated virus (AAV)-based gene therapy has been shown to be safe and effective in numerous animal models and clinical trials for various ophthalmic diseases. Stargardt disease (STGD1; MIM #248200) is the most common autosomal recessive macular dystrophy disease, and the most common form is caused by mutations in the ABCA4 gene, a gene with 6.8 kb coding sequence. Split intein approaches increase the capacity of dual AAV gene therapy, but at the cost of reduced protein expression, which may be insufficient to achieve a therapeutic effect. In this study, we designed various dual split intein ABCA4 vectors and showed that the efficiency of expression of full-length ABCA4 protein is dependent on combinations of types and split sites of the intein system. The most efficient vectors were identified through in vitro screening, and a novel dual AAV8-ABCA4 vector was constructed and subsequently proven to express full-length ABCA4 protein at a high level, reducing bisretinoid formation and correcting the visual function of ABCA4-knockout mice. Furthermore, we evaluated therapeutic effects of different dosages by subretinal injection in mice model. Both therapeutic effects and safety were guaranteed under the treatment of 1.00 × 109 GC/eye. These results support the optimized dual AAV8-ABCA4 approach in future clinical translation for treatment of Stargardt disease.
Keywords: AAV; Stargardt disease (STGD1); gene therapy; split inteins.