Long-term effectiveness and safety of new channelrhodopsin<i> Ps</i>CatCh2.0 in the treatment of retinal degenerative diseases_Chinese Journal of Ocular Fundus Diseases

Authors：

Yu Yao ¹ , Chen Fei ¹ ,  Shen Yin ^1,2

1. Eye Center, Renmin Hospital of Wuhan University, Wuhan 430060, China;
2. Medical Research Institute, Wuhan University, Wuhan 430071, China;

Corresponding?author：

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

Keywords：

Eye diseases, hereditary; Retinal diseases; Retinitis pigmentosa; Retinal ganglion cells; PsCatCh2.0

DOI：

10.3760/cma.j.cn511434-20210607-00293

Video：

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Objective To explore the light response, retinal inflammation and apoptosis of the retinal ganglion cells (RGCs) 1 year after the new type of channelrhodopsin PsCatCh2.0 was transfected into the retina of rd1 mice. Methods Twenty-four male rd1 mice were randomly divided into rd1 experimental group and rd1 control group, 12 mice in each group. 1.5 μl of recombinant adeno-associated virus (rAAV)2/2-cytomegalovirus (CMV)-PsCatCh2.0-enhanced green fluorescent protein (EGFP) was injected into the vitreous cavity 1 mm below the corneoscleral limbus of mice in the rd1 experimental group, and the same dose of recombinant virus was injected 2 weeks later at temporal side 1 mm below the corneoscleral limbus. One year after virus injection, the light response of RGCs expressing PsCatCh2.0 was recorded by patch clamp technique; the expression of PsCatCh2.0 in the retina was evaluated by immunofluorescence staining; the transfection efficiency of recombinant virus was evaluated by the transfection efficiency of virus and the number of RGCs. Hematoxylin-eosin staining was performed to measure the inner retinal thickness. Western blotting was used to detect the protein expression of nuclear factor (NF)-κB p65 in retina; real-time quantitative polymerase chain reaction was used to detect the relative expression of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and Bax mRNA. Terminal deoxynucleotidyl transferase kit was used to observe the apoptosis of retinal cells in each group of mice. Results One year after the intravitreal injection of recombinant virus, PsCatCh2.0-expressing RGCs can still generate 30 pA photocurrent. The virus PsCatCh2.0-EGFP was mainly transfected into RGCs, and partly transfected into amacrine cells, almost no transfection was seen in bipolar and horizontal cells. There were no significant differences in the number of RGCs and thickness of the inner retina between the rd1 experimental group and the rd1 control group (F=14.35, 0.05; P＞0.05), while the rd1 experimental group NF-κB p65 protein expression, TNF-α and IL-6 mRNA quantification were significantly lower than those of rd1 control group (F=4.61, 5.91, 5.78; P＜0.05). The number of red fluorescent apoptotic cells in the retina of mice in the rd1 experimental group was less than that in the rd1 control group, and the Bax mRNA expression was lower than that in the rd1 control group, and the difference was statistically significant (F=7.52, P＜0.01). Conclusion One year after intravitreal injection of recombinant virus, the PsCatCh2.0 expressing RGCs can still generate photocurrent. Long term transfection and expression of PsCatCh2.0 has no obvious cytotoxic effect on RGCs, nor it increases the inflammatory effect of the retina of rd1 mice with retinal degeneration.

Citation： Yu Yao, Chen Fei, Shen Yin. Long-term effectiveness and safety of new channelrhodopsin PsCatCh2.0 in the treatment of retinal degenerative diseases. Chinese Journal of Ocular Fundus Diseases, 2022, 38(7): 584-592. doi: 10.3760/cma.j.cn511434-20210607-00293 Copy

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1. Francis PJ. Genetics of inherited retinal disease[J]. J R Soc Med, 2006, 99(4): 189-191. DOI: 10.1258/jrsm.99.4.189.
2. Berry MH, Holt A, Salari A, et al. Restoration of high-sensitivity and adapting vision with a cone opsin[J/OL]. Nat Commun, 2019, 10(1): 1221[2019-03-15]. https://pubmed.ncbi.nlm.nih.gov/30874546/. DOI: 10.1038/s41467-019-09124-x.
3. Simon CJ, Sahel JA, Duebel J, et al. Opsins for vision restoration[J]. Biochem Biophys Res Commun, 2020, 527(2): 325-330. DOI: 10.1016/j.bbrc.2019.12.117.
4. Wood EH, Tang PH, De La Huerta I, et al. Stem cell therapies, gene-based therapies, optogentics, and retinal prosthetics: current state and implications for the future[J]. Retina, 2019, 39(5): 820-835. DOI: 10.1097/IAE.0000000000002449.
5. 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.
6. 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.
7. 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.
8. Govorunova EG, Sineshchekov OA, Li H, et al. Characterization of a highly efficient blue-shifted channelrhodopsin from the marine alga platymonas subcordiformis[J]. J Biol Chem, 2013, 288(41): 29911-29922. DOI: 10.1074/jbc.M113.505495.
9. Dawydow A, Gueta R, Ljaschenko D, et al. Channelrhodopsin-2-XXL, a powerful optogenetic tool for low-light applications[J]. Proc Natl Acad Sci USA, 2014, 111(38): 13972-13977. DOI: 10.1073/pnas.1408269111.
10. Boyden ES, Zhang F, Bamberg E, et al. Millisecond-timescale, genetically targeted optical control of neural activity[J]. Nat Neurosci, 2005, 8(9): 1263-1268. DOI: 10.1038/nn1525.
11. Hippert C, Graca AB, Barber AC, et al. Muller glia activation in response to inherited retinal degeneration is highly varied and disease-specific[J/OL]. PLoS One, 2015, 10(3): e0120415[2015-03-20]. https://pubmed.ncbi.nlm.nih.gov/25793273/. DOI: 10.1371/journal.pone.0120415.
12. 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): e98924[2014-06-05]. https://pubmed.ncbi.nlm.nih.gov/24901492/. DOI: 10.1371/journal.pone.0098924.
13. 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.
14. 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.
15. Dowling JE. Restoring vision to the blind[J]. Science, 2020, 368(6493): 827-828. DOI: 10.1126/science.aba2623.
16. 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.
17. Ducloyer JB, Le Meur G, Cronin T, et al. Gene therapy for retinitis pigmentosa[J]. Med Sci (Paris), 2020, 36(6-7): 607-615. DOI: 10.1051/medsci/2020095.
18. Madrakhimov SB, Yang JY, Ahn DH, et al. Peripapillary intravitreal injection improves AAV-mediated retinal transduction[J]. Mol Ther Methods Clin Dev, 2020, 17: 647-656. DOI: 10.1016/j.omtm.2020.03.018.
19. Calcedo R, Morizono H, Wang L, et al. Adeno-associated virus antibody profiles in newborns, children, and adolescents[J]. Clin Vaccine Immunol, 2011, 18(9): 1586-1588. DOI: 10.1128/CVI.05107-11.
20. Naso MF, Tomkowicz B, Perry WL 3rd, et al. Adeno-associated virus (AAV) as a vector for gene therapy[J]. BioDrugs, 2017, 31(4): 317-334. DOI: 10.1007/s40259-017-0234-5.
21. Smith RH. Adeno-associated virus integration: virus versus vector[J]. Gene Ther, 2008, 15(11): 817-822. DOI: 10.1038/gt.2008.55.
22. Dudus L, Anand V, Acland GM, et al. Persistent transgene product in retina, optic nerve and brain after intraocular injection of rAAV[J]. Vision Res, 1999, 39(15): 2545-2553. DOI: 10.1016/s0042-6989(98)00308-3.
23. Kalloniatis M, Nivison-Smith L, Chua J, et al. Using the rd1 mouse to understand functional and anatomical retinal remodelling and treatment implications in retinitis pigmentosa: a review[J]. Exp Eye Res, 2016, 150: 106-121. DOI: 10.1016/j.exer.2015.10.019.
24. Zhou T, Huang Z, Sun X, et al. Microglia polarization with M1/M2 phenotype changes in rd1 mouse model of retinal degeneration[J/OL]. Front Neuroanat, 2017, 11: 77[2017-09-05]. https://pubmed.ncbi.nlm.nih.gov/28928639/. DOI: 10.3389/fnana.2017.00077.
25. Zhao L, Zabel MK, Wang X, et al. Microglial phagocytosis of living photoreceptors contributes to inherited retinal degeneration[J]. EMBO Mol Med, 2015, 7(9): 1179-1197. DOI: 10.15252/emmm.201505298.
26. Koso H, Tsuhako A, Lai CY, et al. Conditional rod photoreceptor ablation reveals Sall1 as a microglial marker and regulator of microglial morphology in the retina[J]. Glia, 2016, 64(11): 2005-2024. DOI: 10.1002/glia.23038.
27. Bringmann A, Pannicke T, Grosche J, et al. Muller cells in the healthy and diseased retina[J]. Prog Retin Eye Res, 2006, 25(4): 397-424. DOI: 10.1016/j.preteyeres.2006.05.003.
28. Zabel MK, Zhao L, Zhang Y, et al. Microglial phagocytosis and activation underlying photoreceptor degeneration is regulated by CX3CL1-CX3CR1 signaling in a mouse model of retinitis pigmentosa[J]. Glia, 2016, 64(9): 1479-1491. DOI: 10.1002/glia.23016.
29. Lawrence T. The nuclear factor NF-kappaB pathway in inflammation[J/OL]. Cold Spring Harb Perspect Biol, 2009, 1(6): a001651[2009-10-07]. https://pubmed.ncbi.nlm.nih.gov/20457564/. DOI: 10.1101/cshperspect.a001651.

Chinese Journal of Ocular Fundus Diseases

Long-term effectiveness and safety of new channelrhodopsin PsCatCh2.0 in the treatment of retinal degenerative diseases

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