Restoration of Cone Function in a Mouse Model of Achromatopsia by rAAV Mediated Promoter Targeted Expression of Cone Specific –Transducin

Abstract

Abstract: : Purpose: Complete achromatopsia is a genetic defect in cone function resulting in central vision loss affecting 1 in 33,000 Americans, for which there is no corrective therapy. Here we describe rAAV gene therapy in the cone photoreceptor function loss 3 (Gnat2^cpfl3) mouse, a model of complete achromatopsia type 4. The Gnat2^cpfl3 mouse has a missense mutation in the GNAT2 gene, which encodes cone specific α–Transducin, producing an inactive G–protein and hence a nonfunctional cone phototransduction cascade. Methods: A normal mouse GNAT2 cDNA was cloned into an AAV vector under control of the human red cone opsin promoter (pTR PR 2.1–GNAT2) and packaged into serotype 5 capsids. Two litters of 10 and 11 Gnat2^cpfl3 mice respectively, were injected subretinally with 0.5 µl of GNAT2 virus (8x10¹³ particles/ml) in their right eye on postnatal day 23 (P23) or P29. The second litter of 11 mice also received 0.5 µl of pTR PR 2.1–GFP control virus in their left eyes. Both eyes of all 21 experimental cpfl3 mice, along with controls eyes, were analyzed electroretinographically at 1 and 7 months post injection. Eight months post injection some retinas were then analyzed by electron microscopy and immunohistochemistry. Results: The Gnat2^cpfl3 mouse has no recordable photopic (cone mediated) ERG and a normal scotopic (rod) ERG for at least 8 months. After 1 month, 19 out of 21 treated eyes responded to the therapy, with 17 of the 19 responders corrected to normal or above normal photopic ERG amplitudes. Seven months post–therapy, 18 of 20 treated eyes had recordable photopic ERG signals, with 15 of the 18 responders remaining within the normal range. Staining for cone transducin 8 months post injection revealed treated Gnat2^cpfl3 and wildtype retinas have similar staining patterns, whereas untreated Gnat2^cpfl3 retinas are distinctly different. Electron microscopy reveals no frank differences in morphology between untreated, treated, and wildtype mice, consistent with the non–progressive nature of the human form of the disease. Conclusions: Our data demonstrate restoration of cone ERG and α–Transducin staining distribution after a single subretinal injection of an rAAV vector carrying the GNAT2 cDNA under the control of a cone specific promoter. To our knowledge this study is the first to show cone targeted rescue of a retinal disease by gene therapy.

Keywords: gene transfer/gene therapy • photoreceptors • color vision