Retinoic acid related orphan receptor α is a genetic modifier that rescues retinal degeneration in a mouse model of Stargardt disease and Dry AMD

Abstract

Degeneration of the macula is associated with several overlapping diseases including age-related macular degeneration (AMD) and Stargardt Disease (STGD). Mutations in ATP Binding Cassette Subfamily A Member 4 (ABCA4) are associated with late-onset dry AMD and early-onset STGD. Additionally, both forms of macular degeneration exhibit deposition of subretinal material and photoreceptor degeneration. Retinoic acid related orphan receptor α (RORA) regulates the AMD inflammation pathway that includes ABCA4, CD59, C3 and C5. In this translational study, we examined the efficacy of RORA at attenuating retinal degeneration and improving the inflammatory response in Abca4 knockout (Abca4^-/-) mice. AAV5-hRORA-treated mice showed reduced deposits, restored CD59 expression and attenuated amyloid precursor protein (APP) expression compared with untreated eyes. This molecular rescue correlated with statistically significant improvement in photoreceptor function. This is the first study evaluating the impact of RORA modifier gene therapy on rescuing retinal degeneration. Our studies demonstrate efficacy of RORA in improving STGD and dry AMD-like disease.

Fig. 1. RORA-regulated gene pathways.

String pathway analysis demonstrates the relationship between ABCA4, RORA and CD59 in the inflammatory response and complement gene pathways regulated by RORA. Each node depicts a protein encoded by a gene, and each line represents an association between proteins. Node colors represent specific RORA-regulated pathways: angiogenesis is depicted in red, neuroprotection using indigo, the inflammatory response in light green, the complement cascade in dark green, lipofuscin formation in brown, oxidative stress in orange, lipid metabolism in yellow, phototransduction in pink and neurogenesis in mauve. Known interactions are shown using blue (curated databases), bright green (proteins mentioned in publications) or pink lines (experimentally determined proteins), while predicted interactions are shown in green (proteins in close proximity) or dark blue lines (co-occurring proteins).

Fig. 2. Reduced blue autofluorescence (BAF) (488 nm) in the fundus of AAV5-hRORA treated Abca4^−/− mice.

A Fundus BAF images show increased AF levels in untreated Abca4^−/− mice compared with 129S1 (control) retinas, and a reduction in BAF in Abca4^−/− eyes treated with AAV5-hRORA at all three doses. B Quantification of autofluorescence as measured by mean gray levels shows a reduction in the BAF levels of Abca4^−/− eyes treated with either a low, mid or high dose of AAV5-hRORA at 1 month (1M), 3M and 6M post-treatment compared with untreated eyes at all ages studied. Mean ± SEM, n ≥ 5 biological replicates.

Fig. 3. Increased scotopic b-wave amplitudes in AAV5-hRORA treated Abca4^−/− mice.

A Untreated 1M and 2M Abca4^−/− mice display a similar peak scotopic b-wave amplitude as control 129S1 mice. At 2M, eyes treated with AAV5-hRORA show a slight increase in the peak scotopic b-wave amplitude. There is a similar increase in peak scotopic b-wave amplitude at 4M and 7M in eyes treated with AAV5-hRORA compared with untreated eyes. B A statistically significant increase in scotopic b-wave amplitude was observed in AAV5-hRORA high dose-treated Abca4^−/− retinas at the highest intensity steps compared with untreated eyes at all ages tested (p < 0.05 to 0.0001). C The peak percent recovery of the baseline a-wave amplitude after photobleaching for 5 min at 400 lux exhibits a statistically significant increase in mid and high dose treated eyes compared with untreated eyes. Mean ± SEM, n ≥ 5 biological replicates.

Fig. 4. No adverse effects in Abca4^−/− retinas treated with AAV5-hRORA.

A Histological analysis did not show a difference in the thickness of the retinal layers in the treated retinas compared with untreated eyes and the control background strain. B Quantification of the ONL layer number between untreated and AAV5-hRORA-treated Abca4^−/− mice showed no statistically significant change. GCL ganglion cell layer, INL inner nuclear layer, ONL outer nuclear layer; Mean ± SEM, n ≥ 5 biological replicates. Scale bars 50 µm.

Fig. 5. Restored expression of CD59 in Abca4^−/− mice treated with the low, mid and high doses of AAV5-hRORA.

A CD59 expression is observed in the inner/outer segments (IS, OS) (white arrows) of the photoreceptors and the retinal pigment epithelium (RPE) (insets, closed white arrowheads) in 4M and 7M 129S1 control retinas that is lacking in the untreated Abca4^−/− IS/OS region and RPE. CD59 expression is restored in AAV5-hRORA treated Abca4^−/− eyes in the IS/OS region (white arrows) and the RPE (insets, closed white arrowheads) at all three doses. Open arrowheads indicate autofluorescence in blood vessels. B There is a statistically significant increase in CD59 mean fluorescence intensity in both the photoreceptor IS/OS region (p < 0.0001) and the RPE (p < 0.01 to 0.0001) in Abca4^−/− eyes treated with all doses of AAV5-hRORA compared with untreated Abca4^−/− eyes. Mean ± SEM; WT, n ≥ 3 biological replicates; Abca4^−/−, n ≥ 5 biological replicates. Scale bars 100 µm.

Fig. 6. Reduced expression of amyloid precursor protein (APP) in AAV5-hRORA-treated Abca4^−/− mice.

APP expression (green) was observed in the sub-RPE region of untreated Abca4^−/− eyes at 7M (2nd panel, outlined by dashed lines, arrows), while expression was attenuated in Abca4^−/− retinas treated with the mid dose of AAV5-hRORA. n = 5 biological replicates. Scale bars 100 µm.