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Corentin Joffrois, Gregory Gauvain, Melissa Desrosiers, Joël Chavas, José-Alain Sahel, Didier Pruneau, Deniz Dalkara, Serge A Picaud; Optogenetic visual restoration using ChrimsonR: Biophysical properties study in HEK cells using patch clamp recordings. Invest. Ophthalmol. Vis. Sci. 2016;57(12):601. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Optogenetic therapy showed great promise for vision restoration in a pathological context like retinitis pigmentosa (RP), where blindness develops due to photoreceptor degeneration. However, one of the most important caveats of such gene therapy is the spectral sensitivity (blue) of microbial opsin because very intense light are required for their activation. Here, we assessed in HEK cells biophyisical properties of ChrimsonR (ChR), a newly described red-shifted opsin developed by Ed Boyden (MIT, Cambridge, USA) and Gane Ka-Shu Wong (UA, Edmonton, Canada)
HEK293FT were chemically transfected 24h after plating and recorded via whole cell patch clamp between 24 and 72h post transfection at 36°C. In some experiments 10µM of all-trans-retinal (ATR) was supplemented to the culture medium. Two different constructs were tested: ChrimsonR-TdTomato (ChRTd) a C-ter fusion protein of ChR and TdTomato a fluorescent reporter protein, and ChR, in a bicistronic plasmid also containing GFP (ChR-cis-GFP). Photo stimulation of patch-clamped cells was conducted with a 595nm LED at varying irradiance level.
Our results confirmed ChrimsonR very fast kinetics at 36°C for activation kinetics (t10-90, average +/- sd : 5 +/- 1.5 msec and 3.75 +/- 0.72 msec, for ChRTd and ChR-cis-GFP respectively ) as well as for the deactivation kinetics (τOFF : 9.68 +/- 1.45 msec, and 8.82+/-1.19 msec, ChRTd and ChR-cis-GFP respectively ). Surprisingly, we identified an action of ATR on offset kinetics of our constructs. We measured the photocurrent amplitude and kinetics to different light intensities. Finally, we identified critical parameters of ChR photocycle by measuring photocurrent time to peak in response to very short stimulation.
This preliminary study, in a heterologous cellular model, demonstrate the interest of ChrimsonR in visual restoration and paves the way for more integrated study in rodents (see poster Caplette et al.) and in non human primates (see poster Gauvain et al. and Chaffiol et al.). It will also allow us to establish an in-silico model describing accurately ChrimsonR conductance to define stimulation protocols for transfected cells.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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