Purpose: To develop an AAV dual vector gene therapy treatment for Stargardt disease that delivers elements of an ABCA4 transgene in two separate vectors. We aim to optimise recombination through the overlap region between dual vector transgenes and subsequently provide functional ABCA4 to photoreceptor outer segments in the Abca4-/-mouse model.

Methods: Overlapping ABCA4 transgenes were packaged into AAV capsids. Vector 1 contained promoter (CMV. CBA for in vitro studies and hGRK1 promoter for in vivo experiments) and the 5’ABCA4 CDS between AAV2 ITRs. Vector 2 contained the 3’ABCA4 CDS, WPRE and BGH poly A signal between AAV2 ITRs. Both vectors contained a region of ABCA4 CDS overlap and were~ 4.8 kb in size. HEK293T cells were transduced at a MOI of 10,000 per vector. Cells were harvested at various time points for analysis of ABCA4 expression. QPCRs using cDNA were normalised to actin and untransduced time point controls. Abac4-/-mice were injected subretinally with 2μl of a 1: 1 mix of AAV8 Vector 1 and Vector 2. Eyes were harvested at 2-6 weeks post-injection and the retina removed for ABCA4 expression analysis. Whole eyes were fixed and prepared for IHC assessment.

Results: Dual vector transduction of HEK293T cells lead to detection of full length ABCA4 protein by western blot analysis. QPCR analysis targeting the ABCA4 CDS revealed an increase in expression following overlap optimisation. Abca4-/-mouse retina injected with AAV8 dual vectors revealed full length ABCA4 protein in western blot analysis. IHC analysis of injected Abca4-/-mouse eyes revealed specific ABCA4 staining in the photoreceptor outer segments.

Conclusions: We show success of a dual vector strategy relying on recombination between an optimised overlap region within the ABCA4 CDS, suggesting this approach is a valid alternative to other dual vector strategies that rely on single ITR-packaging or splice donor-acceptor sites to combine two transgene fragments.