Optogenetic visual restoration using ChrimsonR: Photoactivation below safety radiation limit in retinal ganglion cell populations from non-human primates

Abstract

Purpose : In a parallel study (see poster Caplette et al.), we have shown that ChrimsonR (ChrR), a red shifted opsin, can induce light activation of retinal ganglion cells (RGCs) in blind rodents. In this pre-clinical primate study, we assessed whether an intravitreal injection of AAV vectors driving expression of ChrR, or ChrR fused to the fluorescent protein tdTomato (ChrR-tdT), can result in sufficient optogenetic protein expression to allow direct photoactivatation of RGCs.

Methods : Cynomolgus macaques (Macaca fascicularis) were injected intravitreally with either ChrR or ChrR-tdT cDNAs under the control of CAG promoter and vectorized by AAV2 or AAV2.7m8 (5 x 10¹¹ VG/eye). A total of 8 monkeys were injected 2 months before ex vivo recordings of RGCs electrical activity in flat-mounted retinas using a 256 multielectrode array (MEA). Natural light responses were blocked by a cocktail of synaptic blockers (L-AP4, CNQX, CPP), in order to assess photoactivity of optogenetically engineered RGCs .

Results : In vivo ophthalmic exams did not detect any anomaly at 2 months. Expression of tdT was confined to a peri-foveal ring. Only half of the macular area was used for MEA recording (the other half was used for patch clamp experiments, see poster Chaffiol et al.). A similar number of RGCs were recorded in all retinas. However, the number of cells responding to light was different according to the injected AAV vector. A greater number of light responsive cells was recorded for AAV2.7m8-ChrR-tdT, followed by AAV2-7m8-ChrR, and AAV2-ChrR-tdT. No light responsive cells were found for AAV2-ChrR. Light sensitivity was distributed in a similar fashion across the different constructs and light level requested to induce RGC firing was below radiation safety limit. Short flash durations (in a ms range) evoked instantaneous firing at high rate (>50Hz) in a majority of responsive cells.

Conclusions : We demonstrated here that the most efficient AAV vector to ontogenetically engineer RGCs in the non-human primate retina is AAV2-7m8 ChrimsonR-TdTomato. All response kinetics are compatible with an in vivo activation using an external device within the safety radiation limit. Our study indicates that this vector - named GS030 - is an excellent candidate for optogenetic therapy in patients affected by outer retinal degeneration.

This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.