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Tushar H. Ganjawala, Qi Lu, Mitchell D Fenner, Gary W Abrams, Zhuo-Hua Pan; An ultra light-sensitive CoChR mutant restores functional vison in a blind mouse model under ambient light conditions. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5988. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
Channelrhodopsin (ChR), such as ChR2, based optogenetics is one of the promising approaches to restore vision in photoreceptor degenerative diseases, such as retinitis pigmentosa. The development of highly light-sensitive ChRs that can restore functional vision under normal light conditions is desired. We have previously reported a strategy of improving light-sensitivity of ChR2 by optimizing its kinetics (Pan et al., 2014). Recently, a new ChR variant from Chloromonas oogama, CoChR, has been shown to exhibit larger photo-current than ChR2 (Klapoetke et al., 2014). The aim of this study is to develop more light-sensitive CoChR mutants for optogenetic vision restoration.
CoChR mutants (CoChRms) were created by site-directed mutagenesis. All CoChRs were fused with GFP and cloned into a mammalian expression vector with CMV or CAG promoter. The expression and light response properties were evaluated in HEK cells by patch-clamp recordings. ChRs in the retina was delivered by AAV2 vectors via intravitreal administration. Restoration of vision was assessed by optomotor behavior in a triple knock-out (TKO; Gnat1-/-Cnga3-/-Opn4-/-) mouse model using a LED-based homemade optomotor system or a computer-based virtual optomotor system (Prusky et al., 2004).
The photocurrent of wt-CoChR shows a peak sensitive wavelength of 480 nm and a relatively fast off-rate of 112 ± 35.6 ms. We created several more light-sensitive CoChRms by increasing the off-rate and/or improving expression. The most light-sensitive CoChR mutant, CoChR-H94E/L112C/K264T (CoChR3m), showed an off-rate of 635 ± 109 ms. Its peak photocurrent (872 ± 289 pA) evoked at a low light stimulation was over 2-fold higher than that of wt-CoChR (368 ± 140 pA). In addition, its current desensitization (12%) was markedly reduced compared to that of wt-CoChR (60%). Optomotor responses for CoChR3m-treated mice were observed at the light intensity of ~1 x 1013 photons/cm2s with a wavelength of ~470 nm. Furthermore, optomotor responses could also be elicited by using the virtual optomotor system. Visual acuity up to 0.16 cycles/degree (c/d) and contrast sensitivity up to 4 (25%) at the spatial frequency of 0.042 c/d were observed.
We developed an ultra light-sensitive CoChR mutant which enables to restore functional vision in a blind mouse model under ambient light conditions.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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