28. Mouse and Pig Photoreceptor Transduction Mediated by Triple AAV Vectors

Abstract

Purpose: The majority of inherited blinding diseases are caused by mutations in genes expressed specifically in photoreceptors (PRs). However the size of several of these genes exceeds the DNA cargo capacity of AAV vectors, to date the safest and most effective gene therapy vectors in the retina. We have previously demonstrated that dual AAV vectors expand AAV PR transfer capacity to about 9 kb. Our aim is to further expand this to around 13.5 kb using a triple AAV vector system. Methods: To assess the percentage of PR transduction mediated by three independent vectors, we initially injected subretinally in 4 week-old C57/BL6 mice single AAV8 vectors encoding either EGFP, dsRed or beta-gal reporter proteins. Then, in order to evaluate the PR transduction efficiency of triple AAV vectors we generated a reporter EGFP-dsRed fusion protein under the control of either the ubiquitous CMV or the PR-specific IRBP(interphotoreceptor retinoid-binding protein) promoters. The corresponding EGFP-dsRed expression cassettes were either included in single or triple AAV8 vectors (Fig1Fig1) which were administered by subretinal injection to C57/BL6 mice or large white pigs. Direct fluorescence, Western blot, and ELISA analysis were used to evaluate transgene expression. Results: In mice injected with three independent AAV8 vectors encoding for the different EGFP, dsRed and beta-gal reporter proteins, the percentage of PR co-transduced was 12%. In mouse and pig eyes injected with triple and single AAV8 vectors encoding EGFP-dsRed, full length protein expression was confirmed by Western Blot analysis independently of the CMV or IRBP promoters used; importantly, 75% of mouse and 65% of pig eyes injected with triple IRBP vectors showed bands of the expected size. Quantification in mice of EGFP-dsRed protein expression from triple AAV8 vectors mediated by either the CMV or the IRBP promoter shows 30% and 1%, respectively, of the levels observed with a single AAV8. Interestingly, in pigs, the levels of EGFP-dsRed protein expression from triple AAV8-IRBP vectors were 40% ratio of those observed with a single AAV8. Conclusions: Our results show that triple AAV vectors, which increase AAV transfer capacity up to 13.5 kb, transduce mouse and pig PR at levels that are 1% and 40% respectively of those obtained with single AAV vectors. This bode well for further testing this platform in animal models of inherited retinal degenerations.View Large Image | Download PowerPoint Slide