Gene Therapy Fully Restores Vision to the All-Cone Nrl(-/-) Gucy2e(-/-) Mouse Model of Leber Congenital Amaurosis-1

Abstract

Mutations in GUCY2D are the cause of Leber congenital amaurosis type 1 (LCA1). GUCY2D encodes retinal guanylate cyclase-1 (retGC1), a protein expressed exclusively in outer segments of photoreceptors and essential for timely recovery from photoexcitation. Recent clinical data show that, despite a high degree of visual disturbance stemming from a loss of cone function, LCA1 patients retain normal photoreceptor architecture, except for foveal cone outer segment abnormalities and, in some patients, foveal cone loss. These results point to the cone-rich central retina as a target for GUCY2D replacement. LCA1 gene replacement studies thus far have been conducted in rod-dominant models (mouse) or with vectors and organisms lacking clinical translatability. Here we investigate gene replacement in the Nrl(-/-) Gucy2e(-/-) mouse, an all-cone model deficient in retGC1. We show that AAV-retGC1 treatment fully restores cone function, cone-mediated visual behavior, and guanylate cyclase activity, and preserves cones in treated Nrl(-/-) Gucy2e(-/-) mice over the long-term. A novel finding was that retinal function could be restored to levels above that in Nrl(-/-) controls, contrasting results in other models of retGC1 deficiency. We attribute this to increased cyclase activity in treated Nrl(-/-) Gucy2e(-/-) mice relative to Nrl(-/-) controls. Thus, Nrl(-/-) Gucy2e(-/-) mice possess an expanded dynamic range in ERG response to gene replacement relative to other models. Lastly, we show that a candidate clinical vector, AAV5-GRK1-GUCY2D, when delivered to adult Nrl(-/-) Gucy2e(-/-) mice, restores retinal function that persists for at least 6 months. Our results provide strong support for clinical application of a gene therapy targeted to the cone-rich, central retina of LCA1 patients.

Figure 1.

Long-term improvements in retinal function are achieved following AAV-Gucy2e treatment. Shown are representative cone ERG traces from treated and untreated Nrl^−/−Gucy2e^−/− mice (n=16) and Nrl^−/− controls (n=8) obtained under full-spectrum, M- or S-cone-isolating conditions at 2 and 6 months of age (A), full-spectrum maximum cone b-wave amplitudes (B), and M-/S-cone-derived maximum b-wave amplitudes (C) in treated and untreated Nrl^−/−Gucy2e^−/− mice over time. Error bars in (B) and (C) represent±1 SD.

Figure 2.

Visually guided behavior testing (optokinetic reflex) reveals long-term restoration of useful vision after AAV-Gucy2e treatment. Shown are average cone-mediated spatial frequency thresholds in treated and untreated Nrl^−/−Gucy2e^−/− mice (n=16) and Nrl^−/− controls (n=8) at 3 (black bars) and 6 (gray bars) months of age. Error bars represent±1 SD.

Figure 3.

Functional efficiency of AAV-mediated retGC1 in vivo. Shown are guanylate cyclase activities (mean±SE) in AAV-Gucy2e-treated and untreated Nrl^−/−Gucy2e^−/− mice, and Nrl^−/− controls titrated with different free Ca²⁺ at 1 mM free Mg²⁺ (A) and retGC1 activities in all cohorts (mean±SE) in low (<0.01 μM) versus high (2–60 μM) Ca²⁺ at saturating Mg²⁺ (B).

Figure 4.

Cones are preserved in AAV-Gucy2e-treated Nrl^−/−Gucy2e^−/− mice over the long-term. Shown are outer nuclear layer thicknesses derived from identical retinal regions of AAV-Gucy2e-treated and untreated Nrl^−/−Gucy2e^−/− mice, and Nrl^−/− controls at 6 months of age. Values from eight points encompassing all retinal regions are plotted (A) as well as those from superior vs. inferior retina (B). Error bars represent±1 SD.

Figure 5.

Retinal function in Nrl^−/−Gucy2e^−/− mice treated at P18 with AAV-Gucy2e. Shown are average maximum cone b-wave amplitudes obtained under full-spectrum, M- or S-cone-isolating conditions in treated Nrl^−/−Gucy2e^−/− mice (n=7) (gray, striped bars) and Nrl^−/− controls (n=9) (black bars) at 2 months of age. Error bars represent±1 SD.

Figure 6.

Expression of retGC1 and other relevant phototransduction proteins in pooled retinas of treated (n=4) and untreated (n=5) Nrl^−/−Gucy2e^−/− mice and Nrl^−/− controls (n=6). Shown are semiquantitative immunoblots from whole retinal extracts probed with antibodies against retGC1, its biochemical partner GCAP1, and trafficking partner, RD3 (A) and relevant cone phototransduction proteins, M opsin, S opsin, cone transducin (CTα), and cone PDE6α′ (B). β-actin was used as loading control. Each protein was probed on three separate immunoblots and representative images are shown in (A). For densitometric analysis, expression of each protein was first normalized to β-actin on each blot. These ratios were averaged and values were then normalized to Nrl^−/− controls.

Figure 7.

Expression of retGC1 and cone opsins in retinal cross sections of AAV-Gucy2e-treated and untreated Nrl^−/−Gucy2e^−/− mice and Nrl^−/− controls. Shown are retGC1 (red) (A) M opsin (red) and S opsin (green) (B) expression in representative 40× images from all three cohorts at 6 months of age. All sections were counterstained with DAPI (blue). Scale bars=50 μm.

Figure 8.

GCAP1 expression in representative retinal cross sections of AAV-Gucy2e-treated and untreated Nrl^−/−Gucy2e^−/− mice, C57BL/6 WT, and Nrl^−/− controls. Shown in the top row are GCAP1 (green) expression in 20× images counterstained with DAPI (blue) and a secondary only control. White boxes in 20× images are magnified in the bottom row, where only GCAP1 (green) is shown. White arrows in the high-magnification image of AAV-treated Nrl^−/−Gucy2e^−/− mice denote cone outer segments with more obvious GCAP1 staining. GC, ganglion cells; INL, inner nuclear layer; IS, inner segments; no Tx, no treatment; ONL, outer nuclear layer; OS, outer segments; Tx, treatment. Scale bars=30 μm.

Figure 9.

Retinal function is restored to Nrl^−/−Gucy2e^−/− mice following treatment at P40 with AAV5-hGRK1-GUCY2D. Shown are full-spectrum maximum cone b-wave amplitudes at 2 and 6 months of age (A) and S-cone-derived maximum b-wave amplitudes at 6 months (B) in treated and untreated Nrl^−/−Gucy2e^−/− mice (n=7) and Nrl^−/− controls (n=8). Error bars represent±1 SD.