AAV-mediated delivery of ABCA4 to the photoreceptors of Abca4^-/- mice using an overlapping dual vector strategy

Abstract

Purpose : Gene therapy development for disorders caused by mutations in large genes is an advancing field. A dual vector approach for delivering the large gene split in two parts and carried in two separate adeno-associated viral (AAV) vectors is feasible, but given the likely reduction in transduction efficiency associated with this strategy, achieving therapeutic levels of protein in target cells has to date remained challenging. Here we reveal that a customized overlapping dual vector strategy can successfully deliver robust levels of ABCA4 protein to the photoreceptor outer segments of the Abca4^-/- mouse as assessed by immunohistochemistry.

Methods : Overlapping transgenes were packaged in AAV8 Y733F capsids. The upstream transgene contained the human rhodopsin kinase (GRK1) promoter and an upstream portion of the ABCA4 CDS. The downstream transgene contained a downstream portion of the ABCA4 CDS, Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) and polyA signal (pA). Both the upstream and downstream transgenes carried a region of ABCA4 CDS overlap. Abca4^-/- mice received a 2 µl sub retinal injection at 4-5 weeks of age containing a 1:1 mix of upstream and downstream vectors (1 x 10e13 gc/ml). Eyes were harvested 6 weeks post-injection for immunohistochemical (IHC) assessments. Eyes were fixed, frozen and sectioned prior to IHC staining. Staining for Hcn1 was used as a photoreceptor inner segment marker.

Results : Uninjected Abca4^-/- and injected controls did not stain for Abca4/ABCA4 in the photoreceptor outer segments. However, dual vector treated eyes did show robust ABCA4 staining in the photoreceptor outer segments. No off-target ABCA4 expression was observed in other cell types.

Conclusions : An overlapping dual vector approach using the AAV8 Y733F serotype generates readily detectable levels of therapeutic protein in the photoreceptor outer segment structures.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.