Abstract
Purpose:
Because the majority of inherited retinal diseases are caused by mutations in photoreceptor (PR)-specific genes, there is great need to develop PR-targeted gene therapies. Development of a less invasive surgical procedure is also warranted, particularly when an underlying genetic defect results in fragile retina prone to further damage upon surgically induced retinal detachment. Retinal degeneration patients would benefit from vectors that possess an enhanced ability to transduce photoreceptors following intravitreal delivery. The purpose of our study was to develop an in vivo assay to quantify the relative abilities of novel AAV vectors to transduce photoreceptors following intravitreal delivery to mouse.
Methods:
One month old heterozygote Rho-GFP mice received trans-scleral, intravitreal injections (1.5 ul, 1E12vg/ml) of AAV2, AAV5 or AAV8-based vectors (with or without tyrosine/threonine capsid mutations). 4 weeks post injection, mCherry expression was visualized by fundoscopy, retinas were dissociated and FACS analysis was used to quantify the percentage of cells that were GFP positive (rods), mCherry positive (any retinal cells transduced with AAV) and both GFP and mCherry positive (rods transduced by AAV). Percentage of mCherry positive photoreceptors= (% of cells both GFP and mCherry positive cells/% of total photoreceptors).
Results:
Fundoscopy and FACS showed that mCherry expression varied with the serotype injected. Photoreceptor transduction was enhanced following injection with tyrosine/threonine capsid mutants. Retinas injected with AAV2 had 1.7% mCherry positive PRs, retinas injected with AAV2 Y-F QM and AAV2 Y-F QM + T-V had 6.1% and 21.8% mCherry positive PRs, respectively.
Conclusions:
We have established a reliable in vivo assay for scoring the relative photoreceptor transduction efficiencies of novel AAV vectors following delivery to the vitreous. This assay will be used to determine the optimal serotype(s) with which to treat models of inherited retinal disease. Furthermore, it will support development of AAV-based clinical vectors for the treatment of various forms of photoreceptor-mediated inherited retinal disease following a surgically less invasive intravitreal injection.
Keywords: 538 gene transfer/gene therapy •
648 photoreceptors •
763 vitreous