Sustained long–term bilateral cone function in the degenerate retina of monocular gene therapy treated RPE65–/– dogs

Abstract

Abstract: : Purpose: Previous studies have shown that subretinal gene transfer using rAAV.RPE65 leads to significant photoreceptor functional recovery in eyes of RPE65–/– dogs. In the present study long–term clinical investigations in 3 treated affected dogs were correlated with morphologic/morphometric results. Methods: RPE65–/– dogs underwent monocular gene transfer. Funduscopy, ERG and visual behavior studies were performed regularly pre– and post–operatively. Three of the dogs were euthanized 10–18 mo following unilateral gene transfer into the vitreous (n=1) or subretinally (n=2) and eye cups fixed for EM. All clinical and laboratory procedures were performed as previously described. An untreated RPE65–/– littermate served as control. In both eyes of the 4 dogs, the cross sectional area of lipid inclusions per unit length RPE in central and peripheral fundus was measured in a minimum of 200 µm RPE length. Results: Retinal dystrophy progressed after gene transfer but visual capacity increased slightly with time. The dramatically improved scotopic ERG amplitudes initially observed in the subretinally treated eyes gradually decreased, while photopic responses slowly increased and were sustained 18 mo post–op. Photopic responses were elevated also in the fellow eye 6–15 mo post–op in all 3 treated dogs. There was almost complete elimination of lipid droplet formation in the area of subretinal vector injection. This reduction was much less when the rAAV.RPE65 was administered intravitreally. Conclusions: Progression of disease was observed funduscopically, however, functional testing showed improved vision. A long–term persistence of increased ERG photopic responses were found for both eyes following unilateral subretinal or intravitreal gene therapy. Visual improvement did not correlate well with reductions in RPE lipid droplet content. This suggests that restoration of cone function can occur with little restoration of RPE65 gene expression in the RPE.

Keywords: gene transfer/gene therapy • retinal degenerations: hereditary • microscopy: electron microscopy