Optically-triggered oxidative stress in mouse RPE as a model of geographic atrophy in AMD

Abstract

Purpose : Efforts to develop novel therapies for geographic atrophy (GA) in age-related macular degeneration (AMD) are limited by the paucity of preclinical animal models. Here, we developed a model of GA using viral transduction of retinal pigment epithelium (RPE) with fluorescent reactive oxygen species-generating proteins (RGPs) in mouse eyes to enable focal, titratable acceleration of oxidative stress in RPE mitochondria.

Methods : We performed subretinal injections of an AAV8 vector expressing KillerRed, mCherry, or control GFP under an RPE-specific Bestrophin (VMD2) promoter and a mitochondrial targeting sequence into the eyes of wild-type C57BL/6J mice. At 3 weeks after viral transduction, we focally exposed a 500x500μm² square region of the retina to 300-700μW of 561nm light over 20 minutes using a custom scanning-laser ophthalmoscopy (SLO) system to active the fluorescent RGPs and induce RPE oxidative stress. Structural changes in the retina over 4 weeks were measured in vivo using optical coherence tomography (OCT) and ex vivo by immunostaining with antibodies to rhodopsin to assess rod outer-segments and cone arrestin to assess cone density on histological sections, and ZO-1 to assess the RPE mosaic on RPE flatmounts.

Results : AAV8-mediated expression of KillerRed in RPE resulted in global outer retinal and RPE disruption prior to light exposure, while expression of mCherry and GFP showed intact retinal architecture at baseline. Optical activation of AAV8-mCherry triggered a focal area of outer retinal and RPE atrophy resembling GA, with 12 ± 4% loss in outer nuclear layer (ONL) thickness, 32 ± 9% loss in photoreceptor outer segment (OS) length, and 15 ± 7% loss in RPE thickness, while focal activation of AAV8-GFP did not produce any detectable retinal damage. Immunohistochemistry revealed focal atrophy, with 29 ± 4% reduction in rod outer segments and 17 ± 6% reduction in cone density, and RPE disruption and loss within the area of light exposure.

Conclusions : Optogenetic activation of AAV8-mediated mCherry expression in RPE using SLO generates focal outer retinal and RPE atrophy in mouse eyes, and may serve as an inducible model of oxidative stress and GA. This model could be adapted to nonhuman primates for preclinical testing of novel therapies for atrophic AMD.

This is a 2021 ARVO Annual Meeting abstract.