AAV-mediated Long Term Rescue of Cone Function in the CNGA3^-/-Nrl^-/- Mice Following Intravitreal 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^-/- mice have been bred in part to mimic the cone-rich central retina in human achromatopsia 2 with CNGA3 mutations. Here we tested whether mutant AAV8 (Y447, 733F) vector can persistently restore cone system function/structure following intravitreal 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 into one eye of each CNGA3^-/-Nrl^-/- mouse. The other eye was uninjected to serve as a control. Dark- and light-adapted ERGs were recorded at six months after either injection. Then treated and control eyes were harvested for immunohistochemical studies.

Results: At six months following intravitreal injection, rod-related scotopic ERG waveforms were recorded in either treated or untreated Cnga3/Nrl DKO eyes. At a flash intensity of 1.4 log cd^.s/m², the average b-wave amplitudes of cone-related photopic ERGs were 63.76±24.05 μV in treated CNGA3^-/-Nrl^-/- eyes, 12.35±5.733 μV in untreated CNGA3^-/-Nrl^-/- eyes, and 137.2 ± 25.94 μV in age-matched Nrl^-/- control eyes. There were significant differences between them. Eyes with the best light-adapted ERG restoration showed similar cone opsin levels as Nrl^-/- control eyes with abundant M- and S-opsins observed in the cone outer segments. In untreated eyes, both M- and S-cone opsins were absent. Optomotor testing showed improved cone-driven visual acuity and contrast sensitivity in the treated CNGA3^-/-Nrl^-/- eyes, compared with those in the untreated eyes.

Conclusions: Intravitreal delivery of mutant AAV can restore cone function and halt cone degeneration for at least six months in the CNGA3^-/-Nrl^-/- mice. These results serve as a baseline for studying the intravitreal injection-mediated gene replacement therapy for human achromatopsia.