Restoration of Vision in Rpe65 –/– Briard Dogs Using a Recombinant AAV Serotype 4 Vector That Specifically Targets the Retinal Pigmented Epithelium

Abstract

Purpose: : Two studies testing gene replacement therapy using AAV–2 mediated delivery of a canine rpe65 gene have been previously reported. They both demonstrated restoration of dark– and light–adapted ERG responses and improved psychophysical outcomes in the RPE65–/– dog. As the rAAV–2 vectors used in the two reported studies produced high levels of rpe65 expression in both the RPE and photoreceptors cells, the long–term effects of which are not known, we believe that the use of a specific RPE65 promoter and rAAV vector that target transgene expression specifically to RPE might provide safer settings for the long–term therapeutic expression of RPE65. The purpose of this study was to evaluate the ability of rAAV serotype 4, that specifically targets the retinal pigmented epithelium, to restore vision in RPE65–/– dogs and to assess the safety of gene transfer with respect to retinal morphology and function.

Methods: : Recombinant rAAV2/4 vector harboring the human specific RPE65 promoter and cDNA was generated (1011 vg/ml) and administered subretinally in 6 affected dogs aged 8 to 24 months. Retinal function was testing using simultaneous bilateral full–field flash and multifocal ERGs. Vision was evaluated using behavioral testing. Fluorescein angiography and OCT imaging were performed in order to monitor retinal morphology.

Results: : Electrophysiological results demonstrated that restoration of rod and cone photoreceptor function started as soon as 15 days post–injection, was maximum at 3 months post–injection, and remained stable thereafter in all treated retinas. As assessed by the ability of the animals to avoid obstacles in dim light, vision was restored in the treated eye of all animals (untreated contralateral eye served as an internal control).

Conclusions: : For all the 6 treated RPE65–/– Briard dogs, a single dose of subretinally delivered rAAV2/4 vector carrying the human RPE65 promoter and cDNA, restored vision with no perioperative complications such as retinal detachment or uveitis.

Keywords: gene transfer/gene therapy • retina • retinal pigment epithelium