AAV-mediated gene therapy restores cone function in the Cnga3/Nrl double knockout mouse

Abstract

Purpose: Deletion of neural retina leucine zipper in mice (Nrl^−/−) results in the complete absence of rods and an all-cone retina. Nrl^−/− mice lack a scotopic response and have an enhanced photopic ERG amplitude. Mutations in the gene encoding the alpha-subunit of the cone cyclic nucleotide-gated (CNGA3) channel cause cone function loss in mammals and are associated with achromatopsia 2 in humans. Cnga3/Nrl double knockout (Cnga3/Nrl DKO) mice have been bred in part to mimic the cone-rich central retina in human achromatopsia 2 with CNGA3 mutations. We therefore tested whether AAV-mediated Cnga3 gene therapy targeting cones can restore cone system function/structure in this model.

Methods: At postnatal day 14, one μl of AAV5-IRBP/GNAT2-humanCnga3 vector (10¹³ particles/ml) was injected subretinally into one eye of 20 Cnga3/Nrl DKO mice. The other eye was uninjected as control. Dark- and light-adapted ERGs were recorded from 6 weeks to 6 months after injection. Six months after treatment visual acuity and contrast sensitivity were measured optokinetically. Treated and control eyes were harvested for immunohistochemical studies.

Results: In treated eyes, restored light-adapted ERG waveforms were recorded 6 weeks after injection and remained stable for at least 6 months. Cone mediated ERG amplitudes were similar as those of Nrl^−/− mice 6 months after treatment; light-adapted ERGs were unrecordable from untreated eyes. Dark-adapted ERGs were absent in both eyes. Behavioral tests showed that improved visual acuity and contrast sensitivity were found in treated eyes, compared to untreated eyes under a light environment. In untreated eyes, cone opsin staining was greatly attenuated with remaining cone opsins mis-localized to the inner retinal layers. In contrast, both M- and S-opsins were readily observed in the outer segments of treated eyes although these outer segments were much shorter than normal C57 BL/6J.

Conclusions: AAV mediated gene replacement therapy restores cone system function and halts cone degeneration in the Cnga3/Nrl DKO mouse, a cone dominant model. These results suggest that this model will be useful in testing approaches for rescuing cone function in the cone-rich macula/fovea of human achromatopsia.

Keywords: 471 color vision • 538 gene transfer/gene therapy • 585 macula/fovea