Vitreal delivery of AAV vectored Cnga3 restores cone function in CNGA3-/-/Nrl-/- mice, an all-cone model of CNGA3 achromatopsia

Abstract

The CNGA3(-/-)/Nrl(-/-) mouse is a cone-dominant model with Cnga3 channel deficiency, which partially mimics the all cone foveal structure of human achromatopsia 2 with CNGA3 mutations. Although subretinal (SR) AAV vector administration can transfect retinal cells efficiently, the injection-induced retinal detachment can cause retinal damage, particularly when SR vector bleb includes the fovea. We therefore explored whether cone function-structure could be rescued in CNGA3(-/-)/Nrl(-/-) mice by intravitreal (IVit) delivery of tyrosine to phenylalanine (Y-F) capsid mutant AAV8. We find that AAV-mediated CNGA3 expression can restore cone function and rescue structure following IVit delivery of AAV8 (Y447, 733F) vector. Rescue was assessed by restoration of the cone-mediated electroretinogram (ERG), optomotor responses, and cone opsin immunohistochemistry. Demonstration of gene therapy in a cone-dominant mouse model by IVit delivery provides a potential alternative vector delivery mode for safely transducing foveal cones in achromatopsia patients and in other human retinal diseases affecting foveal function.

Figure 1.

Photopic electroretinograms (ERGs) and statistical analysis. (A) Representative photopic ERG tracings elicited at 1.4 log cd-s/m² from CNGA3^−/−/Nrl^−/− and control eyes. (B) Distribution of photopic b-wave amplitudes from each treated and untreated eyes elicited at 1.4 log cd-s/m² (mouse eyes which had one eye subretinally injected and the other eye intravitreally injected with a mixture of AAV8 (Y447, 733F)-IRBP/GNAT2-mCNGA3 and AAV2 (Y444, 500, 730F)-smCBA-GFP vectors are marked with circles). (C) Statistical analysis of photopic b-wave amplitudes elicited at 1.4 log cd-s/m²; (D) Statistical analysis of photopic b-wave implicit times at 1.4 log cd-s/m². All of photopic ERG scale bars are the same (y-axis: 50 μV/Div, x-axis: 50 ms/Div) except for NRL^−/−mice at 2 months of age (y-axis: 100 μV/Div, x-axis: 50 ms/Div). NS, no statistical difference; *P < 0.05; **P < 0.001; ***P < 0.0001. A1 = 2.5-month-old CNGA3^−/−/Nrl^−/− untreated eyes, A2 = CNGA3^−/−/Nrl^−/− eyes 2 months after AAV5 IVit injection at P14, A3 = CNGA3^−/−/Nrl^−/− eyes 2 months after AAV8 (Y447, 733F) IVit injection at P14, A4 = CNGA3^−/−/Nrl^−/− eyes 2 months after AAV8 (Y447, 733F) SR injection at P14, A5 = 2.5-month-old NRL^−/− eyes, A6 = 2.5-month-old C57 BL/6J eyes, B1–B6 groups are similar as those of A1–A6, but the time is 6/6.5 months instead of 2/2.5 months.

Figure 2.

Intravitreal delivery of AAV8 (Y447, 733)-IRBP/GNAT2-mCNGA3 restores photopic visual acuity and contrast sensitivity in CNGA3^−/−/Nrl^−/− mice. (A) Comparison of average values for photopic acuities. (B) Comparison of average values for photopic contrast sensitivities; NS, no statistical difference, *P < 0.05, **P < 0.001, ***P < 0.0001. Labels are same as those in Figure 1. A1 = 2.5-month-old CNGA3^−/−/Nrl^−/− untreated eyes, A2 = CNGA3^−/−/Nrl^−/− eyes 2 months after AAV5 IVit injection at P14, A3 = CNGA3^−/−/Nrl^−/− eyes 2 months after AAV8 (Y447, 733F) IVit injection at P14, A4 = 2.5-month-old NRL^−/− eyes, A5 = 2.5-month-old C57 BL/6J eyes; B1 = 2.5-month-old CNGA3^−/−/Nrl^−/− untreated eyes, B2 = CNGA3^−/−/Nrl^−/− eyes 2 months after AAV5 IVit injection at P14, B3 = CNGA3^−/−/Nrl^−/− eyes 2 months after AAV8 (Y447, 733F) IVit injection at P14, B4 = CNGA3^−/−/Nrl^−/− eyes 2 months after AAV8 (Y447, 733F) SR injection at P14, B5 = 2.5-month-old NRL^−/− eyes, A6 = 2.5-month-old C57 BL/6J eyes.

Figure 3.

IVit-AAV8 (Y447, 733)-mediated mCNGA3 expression leads to cone opsin preservation in treated CNGA3^−/−/Nrl^−/− eyes. (A) Six months post-IVit injection, frozen retinal sections immunostained with anti-mouse CNGA3 antibody and cone-specific PNA showing mouse CNGA3 expression in OSs of age-matched control C57 BL/6J and Nrl^−/− eyes, SR-AAV5 and IVit-AAV8 (Y447, 733) treated eyes, but not in untreated CNGA3^−/−/Nrl^−/− eye. (B) Frozen retinal sections immunostained with S- or M-opsin antibody from the same eyes as in (A) showing S- or M-opsin expression in OSs of C57 BL/6J, Nrl^−/−, SR AAV5 and IVit-AAV8 (Y447, 733) treated eyes; little S- or M-opsin staining was detected in contralateral untreated CNGA3^−/−/Nrl^−/− eye. (C) Frozen retinal section from one CNGA3^−/−/Nrl^−/− mouse immunostained with GFP antibody 2 months after treatment with a mixture of AAV8 (Y447, 733)-IRBP/GNAT2-mCNGA3 and AAV2 (Y444, 500, 730F)-smCBA-GFP (ERG responses of these two eyes marked with circles in Fig. 1B). Left panel: intravitreally injected eye, with GFP signal primarily in cells of the inner retina and photoreceptors. Right panel: subretinally injected eye with GFP expressed primarily in photoreceptors and RPE cells. RPE, retinal pigment epithelium; OS, outer segment layer; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; GCL, ganglion cell layer. Red, CNGA3, S- or M-opsin staining as indicated; green, GFP (green fluorescence protein); blue, DAPI (4′,6-diamidino-2-phenylindole dihydrochloride) stained nuclei; PNA, lectin peanut agglutinin. Scale bar = 100 μm.