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Abhishek Sengupta, Antoine Chaffiol, Emilie Macé, Romain Caplette, Maruša Lampič, Olivier Marre, Serge A Picaud, Jose Alain Sahel, Deniz Dalkara, Jens Duebel; Safe red-shifted stimulation of channelrhodopsin ‘ReaChR’ evokes visual responses in blind mice and primate retinas. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4833.
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© ARVO (1962-2015); The Authors (2016-present)
The insertion of microbial opsin into retinal neurons is a promising therapeutic option for restoring vision in retinal degeneration. Existing reports of such intervention typically involve the expression of blue light sensitive channelrhodopsin (ChR). However, it would be safer to use red-shifted wavelengths, because blue light stimulation of the retina with intensities necessary to activate ChR has a high potential of inducing photochemical damage, presenting a major bottleneck to translating optogenetic vision restoration therapy to the clinic. Here, we report the use of a red-shifted ChR (ReaChR) that we targeted to retinal ganglion cells (RGCs) in mice and ex vivo primate retinas for demonstrating red-shifted optogenetic vision restoration.
We targeted the RGCs of blind mice (rd1) with an adeno-associated virus, AAV2:hSyn-ReaChR-mCit, via the minimally invasive intravitreal injection route. ReaChR expression was monitored by in vivo fundus imaging in rd1 mice as well as confocal imaging of retinal sections. To characterize light evoked responses of ReaChR, we performed patch-clamp and population recordings (MEA) of RGCs of rd1 mice. Visually evoked cortical responses were measured, and light induced locomotory behavior of treated rd1 mice was tested in an illuminated open field. We also infected ex vivo retinas from macaque monkeys with AAV2:CAG-ReaChR-GFP, followed by anatomical and electrophysiological analysis.
Four weeks post viral injection we observed high levels of membrane-bound ReaChR expression in RGCs of rd1 mice. Stimulation with orange light at 590 nm (intensites: 1014—1017 photons cm-2 sec-1) induced robust visual responses in the retina and in the visual cortex. Furthermore, significant light induced locomotory behavior was detected in treated rd1 mice upon 590 nm illumination. Finally, by infecting ex vivo retinas of macaque monkeys (3 animals), we achieved strong ReaChR-expression in primate RGCs. Light stimulation, in the presence of L-AP4, triggered robust RGC responses matching the action spectrum of ReaChR, showing the functionality of ReaChR in primate retina.
Here, we show that AAV mediated expression of ReaChR in RGCs evoke visual responses in blind mice and primate retinas at light levels well below the safety threshold for the human eye, thus making ReaChR an excellent candidate for vision restoration therapy.
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