AAV-mediated gene therapy in the guanylate cyclase (RetGC1/RetGC2) double knockout mouse model of Leber congenital amaurosis

Abstract

Mutations in GUCY2D are associated with recessive Leber congenital amaurosis-1 (LCA1). GUCY2D encodes photoreceptor-specific, retinal guanylate cyclase-1 (RetGC1). Reports of retinal degeneration in LCA1 are conflicting; some describe no obvious degeneration and others report loss of both rods and cones. Proof of concept studies in models representing the spectrum of phenotypes is warranted. We have previously demonstrated adeno-associated virus (AAV)-mediated RetGC1 is therapeutic in GC1ko mice, a model exhibiting loss of cones only. The purpose of this study was to characterize AAV-mediated gene therapy in the RetGC1/RetGC2 double knockout (GCdko) mouse, a model lacking rod and cone function and exhibiting progressive loss of both photoreceptor subclasses. Use of this model also allowed for the evaluation of the functional efficiency of transgenic RetGC1 isozyme. Subretinal delivery of AAV8(Y733F) vector containing the human rhodopsin kinase (hGRK1) promoter driving murine Gucy2e was performed in GCdko mice at various postnatal time points. Treatment resulted in restoration of rod and cone function at all treatment ages and preservation of retinal structure in GCdko mice treated as late as 7 weeks of age. Functional gains and structural preservation were stable for at least 1 year. Treatment also conferred cortical- and subcortical-based visually-guided behavior. Functional efficiency of transgenic RetGC1 was indistinguishable from that of endogenous isozyme in congenic wild-type (WT) mice. This study clearly demonstrates AAV-mediated RetGC1 expression restores function to and preserves structure of rod and cone photoreceptors in a degenerative model of retinal guanylate cyclase deficiency, further supporting development of an AAV-based vector for treatment of LCA1.

FIG. 1.

Electroretinogram (ERG) analysis of AAV-GC1-treated GCdko mice and age-matched GCdko and WT controls. (A) Representative scotopic (top) and photopic (bottom) ERG traces from each cohort of AAV-treated GCdko mice and age matched untreated and WT controls are shown at 1 month post (left) or 1 year post (right) treatment. (B) Temporal analysis of average rod (left) and cone (right) b-wave amplitudes in 4 cohorts of AAV-treated GCdko mice relative to untreated and WT controls. GC1, guanylate cyclase-1; WT, wild-type; GCdko, retGC1/retGC2 double knockout.

FIG. 2.

OCT abnormalities in GCdko retinas and the effect of treatment. (A) Representative OCT horizontal scans ∼0.6mm to either side of the ONH to illustrate ONL thickness reduction in an untreated GCdko eye compared to an age-matched WT eye; ONL is retained in the treated eye of the same GCdko animal. ONL and RPE laminae are marked at the edge of the WT scan and ONL is highlighted on all scans (blue). (B) ONL thickness as a function of age in GCdko retinas and WT retina. Nasal-temporal OCT sections were quantified for ONL thickness in GCdko mice (black circles) and age-related WT mice (gray circles). Regression lines describe log-linear change of the parameters over time with 95% prediction intervals shown. The two data points for older WT mice in the analyses were derived from published data (Xu et al., 2009). (C) Effect of treatment delivered to one eye of GCdko mice at different ages postnatal (P, days). ONL thickness fraction change (expressed as percent of WT) in treated and untreated GCdko eyes as a function of time. Arrow and labels indicate the time of treatment; color bars, GCdko-treated eyes at various timepoints; black bars, mean (error bar, +SD) of untreated GCdko eyes at the corresponding age groups; gray dashed line at top of each graph, lower limits of WT data. (D) GCdko-treated eyes (color bars as in C) were separated into two groups (2–6 and 7–12 months post-treatment) and compared to age-related untreated eyes (black bar). Asterisk indicates significant difference between treated and untreated eyes (p<0.001). OCT, optical coherence tomography; OCT, optical coherence tomography; ONL, outer nuclear layer.

FIG. 3.

Optomotry-based analysis of subcortical function in single eye, AAV-treated GCdko mice (red) and age-matched untreated GCdko (green) and WT (black) controls. M1–M14 corresponds to the 14 mice used for testing. Right eyes, solid bars; left eyes, hatched bars. GCdko mice were treated in their right eyes only. (A) Photopic spatial frequencies and (B) contrast sensitivities of WT controls (M1–M5), AAV-treated GCdko (M6-M9), and untreated GCdko controls (M10–M14) reveal significant improvements in the cone-mediated vision of treated mice. Values within each group were averaged (C) and (D) and compared with student's t-test. P values were calculated between WT and treated GCdko mice and between treated and untreated GCdko mice (C) and (D).

FIG. 4.

Morris Water Maze-based analysis of cortical function in AAV-treated GCdko mice and age-matched untreated GCdko and WT controls. Average escape latencies of WT (black bars, n=5), untreated GCdko (red bars, n=5), and AAV-treated GCdko mice (green bars, n=5) were calculated under photopic and scotopic conditions. A final comparison was made between WT mice and AAV-treated GCdko mice that had their treated eyes sutured, followed by testing under photopic conditions (“photopic w/suture”). Error bars represent one standard deviation.

FIG. 5.

Functional efficiency of AAV-mediated RetGC1 activity in vivo. (A) RetGC activity in AAV-GC1–treated GCdko mice (closed circles), control vector AAV-GFP-treated GCdko mice (closed diamonds), age matched GCdko (open circles), and WT (open diamonds) controls at different free Ca²⁺. Values are the average of three measurements, mean±SE. In both AAV-GFP treated and untreated GCdko mice, the activity was undetectable. (B) Maximal activity of RetGC (at [Ca²⁺] free<20 nM). On the average, AAV-GC1 restored ∼63% of WT RetGC activity (0.41±0.06 SD, N=3; vs. 0.65±0.137 SD nmol cGMP/min/retina, N=8, respectively). ANOVA/Bonferroni data processing at 99% CL. (C) Ca²⁺ sensitivity of RetGC in AAV-GC1-treated GCdko mice remains the same as in WT retinas. RetGC activity was normalized by the maximal activity in each series and then averaged from all series for each genotype. The data are fitted by the Hill function, a=(a_max − a_min)/(1+([Ca]_f/Ca_1/2)^h)+a_min; the Ca_1/2=68 nM±6 SD, N=8 (wild type) and 74 nM±6 SD, N=3 (GC1-AAV), not a significant difference by t-test (p=0.18); h=1.6±0.056 (WT) and 1.52±0.053 (GC1AAV), not a significant difference by t-test (p=0.1).

FIG. 6.

Expression of RetGC1 and cone arrestin in treated GCdko and untreated contralateral controls. Retinas from a 1-year-old GCdko mouse that received AAV-GC1 treatment at P40 in one eye only were stained with antibodies raised against RetGC1 and cone arrestin. Samples were counterstained with DAPI. Untreated GCdko retina (top) lacked RetGC1 expression (A), exhibited reduced cone photoreceptor densities abnormal morphology of remaining cones (B), and outer nuclear layer (ONL) thinning (4–5 nuclei rows remaining). AAV-GC1 treated retina contained pan-retinal RetGC1 expression that was exclusive to photoreceptor outer segments (C), increased cone densities with apparently normal morphology (D), and relatively preserved ONL throughout most of the retina. Entire eyecups (top, 4×) and magnified portions of each (bottom, 40×) are shown.

FIG. 7.

Expression of GCAPs and cone opsins in AAV-treated (P40) GCdko retinas and age-matched untreated and WT controls. (A) Retinas from 17-month-old mice were immunostained for GCAP1 and GCAP2 (green), and the nuclei were counterstained with DAPI (blue); note that GCAP1 and GCAP2 are both detectable in WT or treated GCdko and undetectable in untreated GCdko. PR OS, photoreceptor outer segments, ONL, outer nuclear layer. (B) Immunoblots of 12-month-old, AAV-treated (P40) GCdko retinas and age-matched GCdko and WT controls revealed the presence of RetGC1, GCAP1, and GCAP2 in AAV-GC1-treated eyes. β-actin was used as a loading control. (C) Immunofluorescence (green) of RetGC1, S-opsin and M-opsin, and DAPI nuclear counterstain in 17-month-old mouse retinas. Note that RetGC1 and M-opsin were absent while S-opsin signal was reduced and mislocalized (white arrow) in untreated GCdko retinas. In treated retinas, GCAP1 and GCAP2 were upregulated. S-opsin was present in the inferior retina of treated GCdko mice at levels similar to WT and was found exclusively in photoreceptor outer segments. M-opsin was present at reduced levels in the superior retina of treated GCdko mice and its expression appeared restricted to sparse cone outer segments (white arrow).