Optogenetic visual restoration using ChrimsonR: Photocurrent analysis in primate retinal ganglion cells by using two-photon guided patch-clamp recording

摘要

Purpose : There is still no therapy for blind patients affected by outer retinal degeneration, such as retinitis pigmentosa (RP). Our strategy is to express optogenetic light sensors in retinal ganglion cells (RGCs) and assess feasibility of this approach in non human primates (NHP). An AAV vector was used to deliver the light-sensitive microbial opsin ChrimsonR, in order to transform light insensitive RGCs into photoactivable cells. Different vectors and parameters were tested in this pre-clinical study. Methods : Cynomolgus macaques (Macaca fascicularis) were injected intravitreally with AAV2-7m8-ChrimsonR-tdTomato under CAG promoter (GS030) and other constructs, at 5x1011 vg/eye. Two months after injection, retinal light responses from fluorescent RGCs were evaluated by using 2-photon guided patch-clamp recording. Therefore, retinas were dissected and flat-mounted in a recording chamber superfused with oxygenated Amesa?? medium. Intrinsic light responses from photoreceptors were blocked pharmacologically with L-AP4. We analyzed RGCs photocurrents in response to different stimulus light-intensity, wavelength, duration and frequency. Results : Two-photon imaging showed that the expression in the macaque retinas was essentially restricted to the perifoveal area. After selecting vector constructs with the highest expression level, patch-clamp recordings were performed from Chrimson-tdTomato-expressing RGCs from four animals. We observed robust photocurrents with fast kinetics at light-intensity below the safety radiation limit. Stimulation at different wavelengths showed that the action spectrum peaked at 575 nm. Finally, using flicker stimulation at increasing frequencies, we show that photocurrents were modulated at high temporal resolution (50Hz). Conclusions : Here, we demonstrate that Chrimson-tdTomato, vectorized by AAV2.7m8 (GS030) is an efficient optogenetic tool in the NHP retina. Our results show that it can be activated with red-shifted wavelengths at intensities below radiation safety limit. Moreover, we demonstrate that light responses can be triggered at a temporal resolution that is sufficient for vision restoration. Thus, GS030 is developed in combination with a photo-stimulation device, as an optogenetic therapy of blindness. This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.