Long term visual restoration using optogenetic engineering of retinal ganglion cells with AAV2.7m8- ChrimsonR-tdTomato

Abstract

Purpose : Retinitis Pigmentosa (RP) is a collection of inherited dystrophies with similar clinical features resulting in the progressive loss of photoreceptors leading to blindness. Although gene therapy is currently in clinical trial for hereditary dystrophies, the considerable genetic heterogeneity in RP preclude the rapid development of treatments for patients. In this pre-clinical study we are considering an alternative to classic gene therapy and propose to use viral vector to deliver a microbial opsin, ChrimsonR, to optically control surviving retinal ganglion cells. We previously selected AAV2.7m8-ChrimsonR-tdTomato as the most potent of our viral constructs. We investigated here in non-human primates the treatment efficacy after long term (6 month) expression of our transgene.

Methods : Non-human primates (cynomolgus macaques) were injected intra vitrealy with AAV2.7m8-ChrimsonR-tdTomato under the CAG promoter. A total of 8 monkeys were injected, with three viral doses at least 6 months before ex vivo RGC recordings on 256 electrode multielectrode arrays (MEA). Natural light responses were blocked by a cocktail of synaptic blockers (L-AP4, CNQX, CPP). Immune response following virus injection was evaluated with in-vivo imaging (Optical Coherence Tomography and fundus), and transfection efficiency was estimated on fixed tissue by confocal microscopy.

Results : When examining the non-human primates by slit lamp and SLO-OCT, none of the injected primates developed significant immune response or sign of morphological alteration based on the fundus images. Our result indicates that the highest viral dose was optimal for best efficacy in term of light sensitivity, for full field or discreet spot stimuli, and number of infected cells. However, the medium viral dose was sufficient to produce a significant activity in the infected retinas, whereas the lowest dose did not produce cell activity even if some RGCs displayed td-Tomato fluorescence.

Conclusions : While our results demonstrate stable expression of ChrimsonR-td-Tomato in non human primates on a 6 month period post-infection, they also provide evidence for functional efficacy at doses, which could represent the therapeutic dose range for the future clinical trial.

This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.