AAV-mediated Gene Therapy Restores Cone Function in the Cnga3/Nrl Double Knockout Mouse following intravitreous injection

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 enhanced photopic ERG amplitudes. Mutations in the gene encoding the alpha-subunit of the cone cyclic nucleotide-gated channel (CNGA3) cause loss of cone function in mammals and are associated with achromatopsia 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 a newly developed mutant AAV8 (Y447,733) vector can restore cone system function/structure following intravitreal (IV) injection in this model.

Methods: At postnatal day 14, one μl of AAV8 (Y447, 733)-IRBP/GNAT2-mCnga3 vector (1013 particles/ml) was injected intravitreally or subretinally (as positive control) into one eye of 10 Cnga3/Nrl DKO mice. The other eye was uninjected as control. Dark- and light-adapted ERGs were recorded at six weeks after either injection. Then treated and control eyes were harvested for immunohistochemical studies.

Results: No dark-adapted ERG waveforms were recorded in either treated or untreated Cnga3/Nrl DKO eyes. Light-adapted ERGs were also unrecordable from untreated eyes. Restored light-adapted ERG waveforms were recorded in all ten eyes at 2 months following subretinal injection. Restored light-adapted ERGs were also recorded in three of ten eyes following IV injection of the same vector; their b-wave amplitudes ranged from 50% to 100% of the average value from eyes receiving subretinal injection. Cone-opsin staining showed that both M- and S-opsin were present in eyes that showed photopic ERG restoration. Eyes with the best light-adapted ERG restoration following either subretinal or intravitreal injection showed similar cone opsin levels with both M- and S-opsins observed in the photoreceptor outer segments of treated eyes although these outer segments were shorter than those in normal C57 BL/6J mice. In untreated eyes, both M- and S-cone opsin levels were greatly attenuated or absent.

Conclusions: IV delivery of mutant AAV can restore cone function and halt cone degeneration in the Nrl/Cnga3 DKO mouse. These results suggest that this model will be useful in testing approaches for rescuing cone function in the cone-rich macula/fovea of humans with achromatopsia.

Keywords: 471 color vision • 538 gene transfer/gene therapy • 696 retinal degenerations: hereditary