AAV5 made by rHSV complementation displays increased retinal transduction relative to AAV5 made by plasmid transfection.

Abstract

Purpose : AAV has emerged as the most promising vector for gene- based therapies to treat inherited retinal disease. To reduce immunogenicity, the ideal AAV vector prep should contain highly infectious particles and the lowest possible empty capsid load. With this in mind, different manufacturing platforms are being developed for generating large scale quantities of potent, clinical grade AAV, one of which relies on the recombinant Herpes simplex virus (rHSV) system. Notably, evidence in animal models and human clinical trials showed that AAV1 made by the rHSV system is more infectious that AAV1 made by plasmid transfection. Given that AAV5 is being developed for clinical use in the eye, we sought to determine relative levels of retinal transduction by AAV5 made by either the rHSV system or plasmid transfection.

Methods : Constructs containing the CMV promoter driving GFP and AAV2 rep-AAV5 cap were used separately to create respective rHSVs. HEK293 cells were infected with rHSVs or transfected with AAV plasmids containing the same CMV-GFP transgene, Adenoviral helper genes, AAV2 rep and AAV5 cap. Both AAV preps were purified by iodixanol density gradient and ion exchange chromatography. Vector genome titers were assessed by Q-PCR and infectivity was determined in vitro by green cell assay. Capsid were visualized by electron microscopy (EM) and Coomassie-stained SDS-PAGE. C57Bl6 mice were subretinally injected with two different doses (5x10⁸ vg and 5x10⁹ vg) of each vector. A larger cohort was then injected with 5x10⁹ vg. At 4 and 8 weeks post injection (wpi), GFP expression was assessed by fundoscopy. Mice were sacrificed at 8 wpi and retinas collected for immunoblot and qPCR of GFP protein and RNA, respectively.

Results : AAV capsids made by rHSV and transfection appeared identical under EM and had roughly the same percentage of full and empty capsids. rHSV-AAV was substantially more infectious in vitro relative to transfection-made AAV. Transduction of mouse retina was also significantly higher in rHSV-AAV treated eyes when measured in vivo and in post-mortem retina.

Conclusions : AAV5 made by the rHSV complementation system displays enhanced transduction efficiency relative to transfection-made AAV5. Future clinical development of AAV5 based retinal gene therapies may benefit from the increased potency of rHSV made AAV5.

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