Feasibility analysis of new optogenetics tools Channelrhodopsin-XXM2.0 and Channelrhodopsin-<i>Ps</i>CatCh2.0 to restore visual function_Chinese Journal of Ocular Fundus Diseases

Authors：

Chen Fei ,  Shen Yin

Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China;

Corresponding?author：

Shen Yin, Email: yinshen@whu.edu.cn

Keywords：

Vision, ocular; Optogenetics; Channelrhodopsin-XXM2.0; Channelrhodopsin- PsCatCh2.0

DOI：

10.3760/cma.j.cn511434-20200529-00253

Video：

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Objective To explore the light sensitivity and kinetic of the new optogenetics tools Channelrhodopsin-XXM2.0 (XXM2.0) and Channelrhodopsin-PsCatCh2.0 (PsCatCh2.0), and analyze whether they could be used to restore the visual function by optogenetics.Methods Molecular biology techniques were used to link the gene fragments of XXM2.0 and PsCatCh2.0 to the vector pCIG(c)-msFoxn3 containing ampicillin resistant screening gene and reporter gene to form new plasmid pCIG(c)-msFoxn3-XXM2.0 and pCIG(c)-msFoxn3-PsCatCh2.0. The constructed plasmids were transfected into HEK 293T cells, and light responses were recorded in the whole cell mode with the HEKA patch clamp system. The photocurrent was recorded under three light intensity included 2.7×10¹⁶, 4.7×10¹⁵, and 6.4×10¹⁴ photons/(cm²·s). And then, XXM2.0 and PsCatCh2.0 were stimulated with 2.7×10¹⁶ photons/(cm²·s) and fully recovered. The opening and closing time constants were analyzed with Clampfit 10.6 software. At the same light intensity, photocurrents of XXM2.0 and PsCatCh2.0 were recorded by the light pulse stimulating of 2-32 Hz. The current attenuation was analyzed at long intervals of 4000 ms and short intervals of 200 ms after repeated stimulation. Comparisons between groups were performed by independent samples t test.Results Restriction endonuclease sites of EcoRⅠ and EcoRⅤ were successfully introduced at XXM2.0 and PsCatCh2.0 sequences. When the digestion was completed, they were ligated by T4 DNA ligase to construct new plasmids pCIG(c)-msFoxn3-XXM2.0 and pCIG (c)-msFoxn3-PsCatCh2.0, and then transfected on HEK 293T cells. The light intensity dependence was showed in XXM2.0 and PsCatCh2.0. The greater light intensity was accompanied by the greater photocurrent. Under the light intensity 6.4×10¹⁴ photons/(cm²·s) below the retinal safety threshold, large photocurrent was still generated in XXM2.0 and PsCatCh2.0 with 92.8±142.0 and 13.9±5.6 pA (t=1.24, 1.24; P=0.28, 0.29). The opening time constants of XXM2.0 and PsCatCh2.0 were 23.9±6.7 and 2.4±0.8 ms, and the closing time constants were 5803.0±568.2 and 219.9±25.6 ms. Compared with PsCatCh2.0, the opening and closing time constant of XXM2.0 were both larger than PsCatCh2.0. The differences were statistically significant (t=7.10, 31.60; P=0.00, 0.00). In terms of response frequency, XXM2.0 and PsCatCh2.0 could follow to 32 Hz high-frequency pulsed light stimulation, and all could respond to repeated light stimulation at a long (4000 ms) and a short time (200 ms) interval with the small current decay rate.Conclusion XXM2.0 and PsCatCh2.0 could be activated under light intensity with safety for the retina, and could respond to high frequency (at least 32 Hz) pulsed light stimuli with low current attenuation, which could meet the characteristics of opsins required to restore the visual function by optogenetics.

Citation： Chen Fei, Shen Yin. Feasibility analysis of new optogenetics tools Channelrhodopsin-XXM2.0 and Channelrhodopsin-PsCatCh2.0 to restore visual function. Chinese Journal of Ocular Fundus Diseases, 2020, 36(11): 838-845. doi: 10.3760/cma.j.cn511434-20200529-00253 Copy

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1. Ziccardi L, Cordeddu V, Gaddini L, et al. Gene therapy in retinal dystrophies[J]. Int J Mol Sci, 2019, 20(22): 5722. DOI: 10.3390/ijms20225722.
2. DeAngelis MM, Owen LA, Morrison MA, et al. Genetics of age-related macular degeneration (AMD)[J]. Hum Mol Genet, 2017, 26(R1): 45-50. DOI: 10.1093/hmg/ddx228.
3. Souied E, Pulido J, Staurenghi G. Autologous induced stem-cell-derived retinal cells for macular degeneration[J]. N Engl J Med, 2017, 377(8): 792. DOI: 10.1056/NEJMc1706274.
4. Garber K. RIKEN suspends first clinical trial involving induced pluripotent stem cells[J]. Nat Biotechnol, 2015, 33(9): 890-891. DOI: 10.1038/nbt0915-890.
5. Maguire AM, Simonelli F, Pierce EA, et al. Safety and efficacy of gene transfer for Leber's congenital amaurosis[J]. N Engl J Med, 2008, 358(21): 2240-2248. DOI: 10.1056/NEJMoa0802315.
6. Wood EH, Tang PH, De la Huerta I, et al. Stem cell therapies, gene-based therapies, optogenetics, and retinal prosthetics: current state and implications for the future[J]. Retina, 2019, 39(5): 820-835. DOI: 10.1097/IAE.0000000000002449.
7. Simunovic MP, Shen W, Lin JY, et al. Optogenetic approaches to vision restoration[J]. Exp Eye Res, 2019, 178: 15-26. DOI: 10.1016/j.exer.2018.09.003.
8. Khabou H, Garita-Hernandez M, Chaffiol A, et al. Noninvasive gene delivery to foveal cones for vision restoration[J/OL]. JCI Insight, 2018, 3(2): 96029[2018-01-25]. https://doi.org/10.1172/jci.insight.96029. DOI: 10.1172/jci.insight.96029.
9. Lagali PS, Balya D, Awatramani GB, et al. Light-activated channels targeted to ON bipolar cells restore visual function in retinal degeneration[J]. Nat Neurosci, 2008, 11(6): 667-675. DOI: 10.1038/nn.2117.
10. Lin B, Koizumi A, Tanaka N, et al. Restoration of visual function in retinal degeneration mice by ectopic expression of melanopsin[J]. Proc Natl Acad Sci USA, 2008, 105(41): 16009-16014. DOI: 10.1073/pnas.0806114105.
11. Garita-Hernandez M, Lampic M, Chaffiol A, et al. Restoration of visual function by transplantation of optogenetically engineered photoreceptors[J]. Nat Commun, 2019, 10(1): 4524. DOI: 10.1038/s41467-019-12330-2.
12. Soltan A, McGovern B, Drakakis E, et al. High density, high radiance $\mu$ LED matrix for optogenetic retinal prostheses and planar neural stimulation[J]. IEEE Trans Biomed Circuits Syst, 2017, 11(2): 347-359. DOI: 10.1109/TBCAS.2016.2623949.
13. Reutsky-Gefen I, Golan L, Farah N, et al. Holographic optogenetic stimulation of patterned neuronal activity for vision restoration[J]. Nat Commun, 2013, 4: 1509. DOI: 10.1038/ncomms2500.
14. 張軼, 黃熙, 張軍軍. 光遺傳學在視網膜色素變性治療中的研究進展[J]. 中華眼底病雜志, 2018, 34(6): 602-604. DOI: 10.3760/cma.j.issn.1005-1015.2018.06.018.Zhang Y, Huang X, Zhang JJ. Advances of optogenetics in the treatment of retinitis pigmentosa[J]. Chin J Ocul Fundus Dis, 2018, 34(6): 602-604. DOI: 10.3760/cma.j.issn.1005-1015.2018.06.018.
15. 沈雨濛, 邢怡橋, 沈吟. 光遺傳學技術及其在視網膜變性性疾病治療中的應用[J]. 中華眼底病雜志, 2016, 32(3): 338-342. DOI: 10.3760/cma.j.issn.1005-1015.2016.03.030.Shen YM, Xing YQ, Shen Y. The application of optogenetics in the treatment of retinal degeneration disease[J]. Chin J Ocul Fundus Dis, 2016, 32(3): 338-342. DOI: 10.3760/cma.j.issn.1005-1015.2016.03.030.
16. Sengupta A, Chaffiol A, Mace E, et al. Red-shifted channelrhodopsin stimulation restores light responses in blind mice, macaque retina, and human retina[J]. EMBO Mol Med, 2016, 8(11): 1248-1264. DOI: 10.15252/emmm.201505699.
17. Pan ZH, Ganjawala TH, Lu Q, et al. ChR2 mutants at L132 and T159 with improved operational light sensitivity for vision restoration[J/OL]. PLoS One, 2014, 9(6): 98924[2014-06-05]. http://europepmc.org/article/MED/24901492. DOI: 10.1371/journal.pone.0098924.
18. Montazeri L, El Zarif N, Trenholm S, et al. Optogenetic stimulation for restoring vision to patients suffering from retinal degenerative diseases: current strategies and future directions[J]. IEEE Trans Biomed Circuits Syst, 2019, 13(6): 1792-1807. DOI: 10.1109/tbcas.2019.2951298.
19. Cehajic-Kapetanovic J, Eleftheriou C, Allen AE, et al. Restoration of vision with ectopic expression of human rod opsin[J]. Curr Biol, 2015, 25(16): 2111-2122. DOI: 10.1016/j.cub.2015.07.029.
20. Gaub BM, Berry MH, Holt AE, et al. Optogenetic vision restoration using rhodopsin for enhanced sensitivity[J]. Mol Ther, 2015, 23(10): 1562-1571. DOI: 10.1038/mt.2015.121.
21. De Silva SR, Barnard AR, Hughes S, et al. Long-term restoration of visual function in end-stage retinal degeneration using subretinal human melanopsin gene therapy[J]. Proc Natl Acad Sci USA, 2017, 114(42): 11211-11216. DOI: 10.1073/pnas.1701589114.
22. Kleinlogel S, Feldbauer K, Dempski RE, et al. Ultra light-sensitive and fast neuronal activation with the Ca(2)+-permeable channelrhodopsin CatCh[J]. Nat Neurosci, 2011, 14(4): 513-518. DOI: 10.1038/nn.2776.
23. Ganjawala TH, Lu Q, Fenner MD, et al. Improved CoChR variants restore visual acuity and contrast sensitivity in a mouse model of blindness under ambient light conditions[J]. Mol Ther, 2019, 27(6): 1195-1205. DOI: 10.1016/j.ymthe.2019.04.002.
24. Bi A, Cui J, Ma YP, et al. Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration[J]. Neuron, 2006, 50(1): 23-33. DOI: 10.1016/j.neuron.2006.02.026.
25. Zhang Y, Ivanova E, Bi A, et al. Ectopic expression of multiple microbial rhodopsins restores ON and OFF light responses in retinas with photoreceptor degeneration[J]. J Neurosci, 2009, 29(29): 9186-9196. DOI: 10.1523/JNEUROSCI.0184-09.2009.
26. van Wyk M, Pielecka-Fortuna J, Lowel S, et al. Restoring the ON switch in blind retinas: Opto-mGluR6, a next-generation, cell-tailored optogenetic tool[J/OL]. PLoS Biol, 2015, 13(5): 1002143[2015-05-07]. https://boris.unibe.ch/74586/. DOI: 10.1371/journal.pbio.1002143.
27. Morgans CW, Zhang J, Jeffrey BG, et al. TRPM1 is required for the depolarizing light response in retinal ON-bipolar cells[J]. Proc Natl Acad Sci USA, 2009, 106(45): 19174-19178. DOI: 10.1073/pnas.0908711106.
28. Berry MH, Holt A, Salari A, et al. Restoration of high-sensitivity and adapting vision with a cone opsin[J]. Nat Commun, 2019, 10(1): 1221. DOI: 10.1038/s41467-019-09124-x.

Chinese Journal of Ocular Fundus Diseases

Feasibility analysis of new optogenetics tools Channelrhodopsin-XXM2.0 and Channelrhodopsin-PsCatCh2.0 to restore visual function

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