Self-complementary AAV-mediated gene therapy restores cone function and prevents cone degeneration in two models of Rpe65 deficiency

Abstract

To test whether fast-acting, self-complimentary (sc), adeno-associated virus-mediated RPE65 expression prevents cone degeneration and/or restores cone function, we studied two mouse lines: the Rpe65-deficient rd12 mouse and the Rpe65-deficient, rhodopsin null ('that is, cone function-only') Rpe65(-/-)::Rho(-/-) mouse. scAAV5 expressing RPE65 was injected subretinally into one eye of rd12 and Rpe65(-/-)::Rho(-/-) mice at postnatal day 14 (P14). Contralateral rd12 eyes were injected later, at P35. Rd12 behavioral testing revealed that rod vision loss was prevented with either P14 or P35 treatment, whereas cone vision was only detected after P14 treatment. Consistent with this observation, P35 treatment only restored rod electroretinogram (ERG) signals, a result likely due to reduced cone densities at this time point. For Rpe65(-/-)::Rho(-/-) mice in which there is no confounding rod contribution to the ERG signal, cone cells and cone-mediated ERGs were also maintained with treatment at P14. This work establishes that a self-complimentary AAV5 vector can restore substantial visual function in two genetically distinct models of Rpe65 deficiency within 4 days of treatment. In addition, this therapy prevents cone degeneration but only if administered before extensive cone degeneration, thus supporting continuation of current Leber's congenital amaurosis-2 clinical trials with an added emphasis on cone subtype analysis and early intervention.

Figure 1. Temporal analysis of cone photoreceptor densities in rd12 and wild type mouse retinas

FITC-labeled Peanut Agglutinin (PNA) staining revealed that cone photoreceptors were abundant in the central and peripheral regions of the untreated, P14 rd12 mouse retina (c, d), a pattern similar to that seen in the P14, wild type mouse retina (a, b). Cones degenerate in the rd12 retina by 5 weeks of age (e), remaining only in the dorsal and temporal regions through at least 5 months (f). Arrows: fragments of RPE cells. D: dorsal; V: ventral; T: temporal; N: nasal.

Figure 2. Comparison of rod- and cone-driven functions in rd12 mice after early P14- or late P35-subretinal deliveries of a scAAV5-smCBA-hRPE65 vector

The left, middle and right columns show dark-adapted, light-adapted and 10 Hz flicker ERGs respectively. P14 treatment resulted in functional rescue to rod system as early as 4 days post injection (a). Representative dark-adapted ERGs recorded at 6 or 5 months after P14 ( left eye) and P35 (right eye) treatment showed robust restoration of the rod system functions (b and c), as indicated by significant increases of both a- and b-wave amplitudes (c) relative to untreated controls which had minimal responses. For the light-adapted ERG, P14-treatment significantly improved the b-wave amplitudes to a level about 60% of normal uninjected controls (d, e and f); whereas P35-treatment did not result in significant b-wave amplitude increase compared to untreated rd12 mice (f). Light-adapted ERG showed that both P14- and P35-treatments significantly reduced the delayed b-wave implicit time to normal levels (g). Both representative (h) and statistical (i) data from 10 Hz flicker ERGs.

Figure 3. scAAV-RPE65 treatment restores photopic vision in rd12 mice treated at P14, and scotopic vision in rd12 mice treated at P14 or P35

Under scotopic conditions, treatment with AAV-RPE65 at P14 (light grey bar, n=4 eyes, and at P35 (dark grey bar, n=3 eyes) leads to acuities (a) and contrast sensitivities (b) comparable to uninjected C57BL/6 mice (white bars, n=4 eyes). Untreated rd12 eyes (black bars, n=3) perform significantly poorer in tests of both scotopic acuity (a) and scotopic contrast sensitivity (b) than those from C57 or treated rd12 eyes. The level of vision in these untreated eyes is equivalent to no visual function. Under photopic conditions, (c) rd12 mouse eyes treated with AAV-RPE65 at P14 (light grey bar, n=4) have significantly better acuity than untreated rd12 control eyes (black bar, n=3) and maintain photopic acuity comparable to wild type C57BL/6J eyes (white bar, n=4), while eyes treated at P35 (dark grey bar, n=3 eyes) show no significant change from their untreated rd12 counterparts (black bar, n=3 eyes). (d) As with photopic contrast sensitivity, rd12 eyes treated at P14 (light grey bar, n=4 eyes), but not those treated at P35 (dark grey bar, n=3 eyes) exhibit photopic contrast sensitivity near wild type C57BL/6J levels (white bar, n=4 eyes).

Figure 4. Retinalwhole mounts of rd12 mice treated at P14 (left column) or P35 (right column) with scAAV5-hRPE65 and stained with PNA, MWL-cone opsin antibody or SWL-cone opsin antibody

PNA staining revealed that cones were abundant in the peripheral (a) and central (b) regions of the P14 treated rd12 retina. P14 treated cones were positive for both MWL cone opsin (c) and SWL cone opsin (d). Cone specific PNA staining after P35 treatment was reduced centrally (f) and was restricted to dorsal and temporal retina (e), a pattern similar to that seen in the P35 untreated, rd12 retina. P35 treated cones contained MWL cone opsin (g), but lacked detectable SWL cone opsin (h). D: dorsal; V: ventral; T: temporal; N: nasal.

Figure 5. Temporal analysis of cone photoreceptor densities in the Rpe65^−/−::Rho^−/− mouse retina

PNA staining (a, b) revealed that cone photoreceptors were abundant in the central and peripheral regions of the untreated, P14 Rpe65^−/−::Rho^−/− mouse retina, a pattern similar to that seen in the P14, wild type mouse retina (see Fig 1a, b). Cones degenerate in the Rpe65^−/−::Rho^−/−retina by 5 weeks of age (c), remaining only in the dorsal and temporal retina. D: dorsal; V: ventral; T: temporal; N: nasal. DKO: Rpe65^−/−::Rho^−/− double knockout mouse. Scale bars (a, c) = 500 μM. Scale bar (b) = 100 μM.

Figure 6. Photopic electroretinogram in Rpe65^−/−::Rho^−/−mice

Cone responses were detectable within 4 days after vector treatment (a). These responses increased in amplitude (b) and remained stable until at least P42, the latest time point tested. Treated eyes of Rpe65^−/−::Rho^−/−mice (n=5) had significantly higher photopic b-wave amplitudes than untreated eyes (c). DKO: Rpe65^−/−::Rho^−/− double knockout mouse.

Figure 7. Retinal whole mounts of Rpe65^−/−::Rho^−/− mice 4 weeks after vector treatment at P14 and stained with PNA, MWL-cone opsin antibody or SWL-cone opsin antibody

PNA staining revealed that cones were abundant in the peripheral (a) and central (b) regions of a P14 treated Rpe65^−/−::Rho^−/− retina. These cones were positive for both MWL cone opsin (c) and SWL cone opsin (d). D: dorsal; V: ventral; T: temporal; N: nasal. Scale bars (a, c, d) = 500 μM. Scale bar (b) = 100 μM.