Restoration of vision in RPE65-deficient Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium

Abstract

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 electroretinography responses and improved psychophysical outcomes. Use of a specific RPE65 promoter and a rAAV vector that targets transgene expression specifically to the 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 À/À purebred Briard dogs and to assess the safety of gene transfer with respect to retinal morphology and function. rAAV2/4 and rAAV2/2 vectors containing similar human RPE65 promoter and cDNA cassettes were generated and administered subretinally in eight affected dogs, ages 8-30 months (n ¼ 6 with rAAV2/4, n ¼ 2 with rAAV2/2). Although fluorescein angiography and optical coherence tomography 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 postinjection, and remaining stable thereafter in all animals treated at 8-11 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, whereas 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 À/À dogs by gene replacement therapy.

Gene therapy in the second eye of RPE65-deficient dogs improves retinal function

“…Previous studies have documented the histologic appearance of lesions resulting from subretinal injections in dogs and include damage at the injection site, retinal thinning and focal defects in reattachment resulting in retinal 'ripples' . 10,11,33 The small areas of tapetal hyper-reflectivity that we detected in 3 of the 18 eyes did not preclude successful recovery of rod and cone function in these eyes. Reasons for the more diffuse tapetal hyper-reflectivity and presumed retinal thinning in the injected area of eye 2b are not clear but may reflect the potential detrimental effects of the creation of a retinal detachment.…”

“…Small foci of tapetal hyper-reflectivity were observed in 3 of 18 injected eyes, consistent with focal retinal thinning as described in a previous report. 10 The presence of the tapetum in the dog allows for detection of retinal thinning by ophthalmoscopic examination more readily than in species with no tapetum. Previous studies have documented the histologic appearance of lesions resulting from subretinal injections in dogs and include damage at the injection site, retinal thinning and focal defects in reattachment resulting in retinal 'ripples' .…”

“…In one eye (2b), a larger area of tapetal hyper-reflectivity developed, covering the majority of the treated fundus, consistent with retinal thinning as described in a previous report. 10 …”

“…6 A number of studies have shown rod and cone photoreceptor rescue using rAAV vectors to deliver a normal copy of the RPE65 gene via a subretinal injection in the RPE65 mutant Briard. [6][7][8][9][10][11][12][13] On the basis of the great success of the canine trials, phase I/II clinical trials of rAAV-RPE65 gene replacement therapy in human LCA patients have started with the first reported results showing great promise. [14][15][16] Thus far in all human patients only one eye has been treated.…”

Ocular gene therapy trials due to report this year; Keeping an eye on clinical trials in 2008

“…Several groups have demonstrated that adeno-associated virus-mediated gene replacement therapy in the Swedish Briard dog, which is homozygous for a null mutation in RPE65, can dramatically improve both retinal function and visual behaviour after a single subretinal injection of vector. [2][3][4][5] In Is there any value in running three similar trials concurrently? All the clinical trials for RPE65 will use similar adeno-associated virus vectors but the protocols have important differences in terms of vector design and inclusion criteria.…”