Tripartite AAV Systems for EYS Retinal Gene Therapy

ABSTRACT Mutations in the Eyes Shut Homolog (EYS) gene are a leading cause of autosomal recessive retinitis pigmentosa, a progressive retinal degenerative disease for which no effective treatment currently exists. However, the large size of the EYS coding sequence (∼9.4 kb) exceeds the packaging limit of adeno-associated virus (AAV) vectors, posing a major barrier to gene replacement therapy. To address this challenge, we developed a tripartite AAV vector system that enables delivery and reconstitution of the full-length EYS gene using a Cre-lox-based unidirectional DNA recombination strategy, Uni-STAR (Uni-directional and Site-specific Transgene Assembly by Recombination). The system consists of three AAV constructs carrying discrete EYS segments flanked by engineered, non-compatible lox sites that drive ordered and unidirectional recombination in target cells. We validated this system in vitro by demonstrating successful reconstitution and expression of full-length EYS protein in HEK293T cells. In vivo, subretinal co-injection of the three AAV vectors into mouse eyes led to precise reconstitution and expression of full-length EYS protein in the retina. These findings establish the feasibility of using a tripartite AAV system to deliver the complete EYS gene and provide a foundation for future therapeutic development targeting EYS-associated retinal degenerations. Competing Interest Statement S.S. and P.D. are co-inventors on a patent application (19/124,578, filed by the University of Iowa) titled Method to deliver large genes using virus and a DNA recombination system. The remaining authors declare no competing interests.