Gene therapeutic prospects in early onset of severe retinal dystrophy: restoration of vision in RPE65 Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.

Previous studies have tested gene replacement therapy in RPE65 deficient dogs using recombinant adeno-associated virus 2/2 (rAAV2/2), -2/1 or -2/5 mediated delivery of the RPE65 gene. They all documented restoration of dark- and light-adapted ERG responses and improved psychophysical outcomes. Use of a specific RPE65 promoter and a rAAV vector that targets transgene expression specifically to the retinal pigmented epithelium (RPE) may, however, provide a safer setting for the long-term therapeutic expression of RPE65. Subretinal injection of rAAV2 pseudotyped with serotype 4 (rAAV2/4) specifically targets the RPE. The purpose of our study was to evaluate a rAAV2/4 vector carrying a human RPE65cDNA driven by a human RPE65 promoter, for the ability to restore vision in RPE65-/- purebred Briard dogs. Recombinant rAAV2/4 and rAAV2/2 vectors containing similar human RPE65 promoter and cDNA cassettes were generated and administered subretinally in 8 affected dogs, ages 8 to 30 months (n = 6 with rAAV2/4, n = 2 with rAAV2/2). Although fluorescein angiography and OCT examinations displayed retinal abnormalities in treated retinas, electrophysiological analysis demonstrated that restoration of rod and cone photoreceptor function started as soon as 15 days post-injection, reaching maximal function at 3 months post-injection, and remaining stable thereafter in all animals treated at 8 to 11 months of age. As assessed by the ability of these animals to avoid obstacles in both dim and normal light, functional vision was restored in the treated eye, while the untreated contralateral eye served as an internal control. The dog treated at a later age (30 months) did not recover retinal function or vision, suggesting that there might be a therapeutic window for the successful treatment of RPE65 -/- dogs by gene replacement therapy.