Transduction of photoreceptors following subretinal injections of AAV5 and helper-dependent adenovirus in human retina ex vivo and pig retina in vivo

Abstract

Purpose : Gene therapy for degenerative retinal diseases has demonstrated promise, but published studies regarding tropism and distribution, particularly in large animals and human retina, are scant for many potential therapeutic vectors. We tested the hypotheses that 1) AAV5 and helper-dependent adenoviral vectors could transduce existing photoreceptor cells and 2) distribution could be monitored by localization of GFP expression.

Methods : AAV5-GFP or hdAd5-GFP were delivered to human retinal explants ex vivo and to Yucatan miniature pigs following vitrectomy and subretinal injection in vivo. For in vivo experiments, indirect ophthalmoscopy was performed at sacrifice (e.g., 1 month post-op) to detect retinal reattachment, RPE changes, and location of GFP-positive regions. Human retina and pig eyes were fixed in 4% paraformaldehyde and sectioned for gross examination of the GFP-positive regions. Immunofluorescence staining with anti-GFP and anti-RPE65 antibody was used to detect GFP-transduced cells and RPE changes, respectively. Rhodopsin antibodies were used to visualize rods.

Results : Ex vivo transduction: at 7-days post-delivery, robust GFP expression was restricted to photoreceptor cells of the ONL in AAV5-GFP transduced human retinal explants. Although transduction of human retinal explants with hdAd5-GFP also induced GFP expression in photoreceptor cells, unlike AAV5, GFP expression was more widespread. Specifically, hdAd5 was also found to target inner retinal cells including Muller glia. In vivo transduction: All pig eyes receiving AAV5-GFP (n=6) and hdAd5-GFP (n=6) had diffuse GFP-positivity in the region of the blebs one month after treatment. Mild RPE changes were seen surrounding the blebs. Comparing H&E sections to corresponding immunofluorescence sections, there was high transduction efficiency of photoreceptors (>95%) in all animals, with GFP expression detected throughout the region of the bleb.

Conclusions : AAV5 and HD-ADV are effective viral vectors for photoreceptors following subretinal delivery to human retina ex vivo and to pig in vivo that should allow gene therapy of all inherited retinal degenerative diseases regardless of gene size.

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