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bioRxiv · 10.64898/2025.12.03.692187

Tripartite AAV Systems for EYS Retinal Gene Therapy

Rhee, K.-D.·Datta, P.·Baccam, C.·Seo, S.
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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.

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Rhee, K.-D., Datta, P., Baccam, C., Seo, S.. 2025-12-05. Tripartite AAV Systems for EYS Retinal Gene Therapy. https://doi.org/10.64898/2025.12.03.692187

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