Optogenetic visual restoration with photosensitive gq-coupled opsin (opsin 5)

Abstract

The present disclosure provides an isolated photosensitive opsin that rapidly, reversibly, and precisely restores light sensitivity to retinal cells by activating Gq signaling.

Description

BACKGROUND

[0001] G protein-coupled receptors (GPCRs) modulate many intracellular signaling pathways and represent some of the most intensively studied drug targets (Hauser et al., 2017). Upon ligand binding, GPCRs undergo a conformational change and transmit it to a heterotrimeric G protein, a multi-subunit complex comprising a G α protein tightly bound to a G βγ protein subunit. G q proteins, a subfamily of heterotrimeric G α proteins, couple to a class of GPCRs and mediate cellular responses to neurotransmitters, sensory stimuli, and hormones throughout the body. Their main downstream signaling target includes the phospholipase C beta (PLC-b) enzyme, which catalyzes the hydrolysis of the phospholipid phosphatidylinositol bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 triggers the release of intracellular stored Ca 2+ into the cytoplasm, and Ca 2+ along with DAG activates protein kinase C (PKC). Several tools including chemical genetics and optically activatable small molecules have been developed to study the signaling mechanisms and physiological functions of G q coupled GPCRs and intracellular Ca 2+ release.

[0002] Optogenetics utilizes light-responsive proteins to achieve genetically specific and high spatiotemporal precision for the optical control of cellular activities. Since the early discovery of optogenetic tools using light-sensitive ion channels and transporters, a wide variety of techniques have been developed that now support optical intervention of intracellular second messengers, protein interactions and degradation, and gene transcription. Opt-a1AR, a creatively designed G q coupled rhodopsin-GPCR chimera, can induce intracellular Ca 2+ release in response to long light stimuli (60 s) (Airan et al., 2009). However, this tool has not been widely used, possibly due to its limitations related to light sensitivity and response kinetics (Tichy et al., 2019). Most animals use GPCR-based photoreceptors for light detection, where the photoreceptor contains a protein fraction (opsin) and a vitamin A derivative (retinal) that acts both as a ligand and as a chromophore. Several thousand opsins have been identified so far. Two recent studies reported the use of G i opsins from mosquitoes and lampreys for presynaptic terminal inhibition in neurons, briefly demonstrating that certain naturally occurring photoreceptors are suitable for use as efficient optogenetic tools. Regarding G qSignaling, melanopsin (Opn4) in a subset of mammalian retinal ganglion cells is a G q protein-coupled opsin that mediates non-image-forming visual functions. However, HEK293 or Neuro-2a cells heterologously expressing Opn4 show weak light responses and require additional retinal in the culture medium. Opn5 (neurotrypsin) and its orthologs in many vertebrates have been reported to be G i protein-coupled ultraviolet (UV)-sensitive opsins.

[0003] There is an urgent need for an ideal optogenetic tool to restore visual function in blind patients. SUMMARY

[0004] The present invention relates to an isolated light-sensitive opsin for restoring the sensitivity of retinal cells to light by activating G q signaling. The isolated light-sensitive opsin can be used to treat a subject having a damage to the outer layer of the retina, loss or degeneration of photoreceptors, retinal degenerative disease, loss of sensitivity to light or loss of light perception, visual loss or blindness.

[0005] In a first aspect, the present invention relates to an isolated light-sensitive opsin for restoring the sensitivity of retinal cells to light by activating G q signaling.

[0006] In certain embodiments, the light has a wavelength in the range of 360 nm to 520 nm, preferably 450 to 500 nm, more preferably 460 to 480 nm, in particular 470 nm.

[0007] In certain embodiments, the isolated opsin is an isolated opsin from an organism, a homolog thereof, an ortholog thereof, a paralog thereof, a fragment thereof, or a variant thereof, and has an activity of restoring the sensitivity of retinal cells to light by activating Gq signaling.

[0008] In certain embodiments, the isolated opsin has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a wild-type opsin, a homolog thereof, an ortholog thereof, a paralog thereof, a fragment thereof, or a variant thereof in the organism, and has an activity of restoring the sensitivity of retinal cells to light by activating Gq signaling.

[0009] In certain embodiments, the organism is an animal.

[0010] In certain embodiments, the isolated opsin is an isolated opsin 5 (Opn5), a homolog thereof, an ortholog thereof, a paralog thereof, a fragment or variant thereof from an animal and has an activity of restoring light sensitivity to retinal cells by activating Gq signaling.

[0011] In certain embodiments, the isolated opsin 5 (Opn5) is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the wild-type opsin 5 (Opn5), a homolog thereof, an ortholog thereof, a paralog thereof, a fragment or variant thereof in the animal and has an activity of restoring light sensitivity to retinal cells by activating Gq signaling.

[0012] In certain embodiments, the animal is a vertebrate.

[0013] In certain embodiments, the animal is a bird, a reptile, or a fish, an amphibian, or a mammal.

[0014] In certain embodiments, the animal is a bird, including but not limited to a chicken, a duck, a goose, an ostrich, an emu, a rhea, a crane, a cassowary, a turkey, a quail, a chicken, a falcon, an eagle, a hawk, a pigeon, a budgerigar, a cockatoo, a macaw, a parrot, a passerine (e.g., a songbird), a jay, a thrush, a sparrow, a bunting, and a sparrow.

[0015] In certain embodiments, the animal is a reptile, including but not limited to a lizard, a snake, an alligator, a turtle, a crocodile, and a terrapin.

[0016] In certain embodiments, the animal is a fish, including but not limited to a catfish, an eel, a shark, and a swordfish.

[0017] In certain embodiments, the animal is an amphibian, including but not limited to a toad, a frog, a newt, and a salamander.

[0018] In certain embodiments, the isolated opsin 5 (Opn5) is an isolated wild-type opsin 5 (Opn5) or a fragment or variant thereof from a chicken and has an activity of restoring light sensitivity to retinal cells by activating Gq signaling.

[0019] In certain embodiments, the isolated opsin 5 (Opn5) is at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the wild-type opsin 5 (Opn5) from a chicken and has an activity of restoring light sensitivity to retinal cells by activating Gq signaling.

[0020] In certain embodiments, the isolated opsin 5 (Opn5) is an isolated wild-type opsin 5 (Opn5) from a turtle or a fragment or variant thereof, and has an activity of restoring light sensitivity of retinal cells by activating Gq signaling.

[0021] In certain embodiments, the isolated opsin 5 (Opn5) has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 1 (cOpn5), and has an activity of restoring light sensitivity of retinal cells by activating Gq signaling.

[0022] In certain embodiments, the isolated opsin 5 (Opn5) has an amino acid sequence set forth in SEQ ID NO: 2 (tOpn5) or a fragment or variant thereof, and has an activity of restoring light sensitivity of retinal cells by activating Gq signaling.

[0023] In certain embodiments, the isolated opsin 5 (Opn5) has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 2 (tOpn5), and has an activity of restoring light sensitivity of retinal cells by activating Gq signaling.

[0024] In certain embodiments, the isolated opsin 5 (Opn5) has an amino acid sequence set forth in SEQ ID NO: 2 (tOpn5) or a fragment or variant thereof, and has an activity of restoring light sensitivity of retinal cells by activating Gq signaling.

[0025] In certain embodiments, the isolated opsin 5 (Opn5) has at least 70%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the amino acid sequence set forth in SEQ ID NO: 2 (tOpn5), and has an activity of restoring light sensitivity of retinal cells by activating Gq signaling.

[0026] The isolated opsin 5 (Opn5) can be used as a convenient optogenetic tool to precisely activate intracellular Gq signaling in retinal cells. q

[0027] The retinal cells can be photoreceptor cells, rod cells, cone cells, retinal ganglion cells, bipolar cells, ganglion cells, horizontal cells, multipolar neurons, Muller cells, amacrine cells, or methyl-nitrosourea.​

[0028] In a second aspect, the present application relates to an isolated nucleic acid encoding the isolated opsin of the first aspect.

[0029] In certain embodiments, the isolated nucleic acid encodes a wild-type opsin, a homolog thereof, an ortholog thereof, a paralog thereof, a fragment thereof, or a variant thereof in an organism, which has an activity of restoring the sensitivity of the retinal cells to light by activating Gq signaling.

[0030] In a third aspect, the present application relates to a chimeric gene comprising a sequence of the isolated nucleic acid of the second aspect operably linked to a suitable regulatory sequence.

[0031] The chimeric gene further comprises a gene encoding a marker, such as a fluorescent protein.

[0032] In a fourth aspect, the present application relates to a vector comprising the isolated nucleic acid of the second aspect or the chimeric gene of the third aspect.

[0033] The vector is a eukaryotic vector, a prokaryotic expression vector, a viral vector, or a yeast vector.

[0034] In certain embodiments, the vector is a herpes simplex virus vector, a vaccinia virus vector, or an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or an insect vector.

[0035] Preferably, the vector is a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVS, AAV0, or AAV10.

[0036] In certain embodiments, the vector is an expression vector.

[0037] In certain embodiments, the vector is a gene therapy vector.

[0038] In a fifth aspect, the present application relates to an isolated cell or cell culture comprising the isolated nucleic acid of the second aspect, the chimeric gene of the third aspect, or the vector of the fourth aspect.

[0039] For example, expression of cOpn5 in HEK 293T cells potently mediates blue light-triggered release of G q Ca 2+ dependent increase.

[0040] For example, optogenetic activation of cOpn5-expressing astrocytes induces massive ATP release in the mouse brain.

[0041] In a sixth aspect, the present application relates to the use of the isolated opsin of the first aspect, the isolated nucleic acid of the second aspect, the chimeric gene of the third aspect, the vector of the fourth aspect, or the isolated cell or cell culture of the fifth aspect, for the treatment or prevention of a disease or condition mediated by or involving a loss of sensitivity to light by retinal cells.

[0042] The cOpn5 can be administered to retinal cells, and the retinal cells can be activated by light. The light has a wavelength in the range of 360 nm to 520 nm, preferably 450 to 500 nm, more preferably 460 to 480 nm, in particular 470 nm.

[0043] For example, AAV vectors expressing cOpn5-t2a-EGFP are administered subretinally or intravitreally, expressing cOpn5 and EGFP in retinal ganglion cells.

[0044] In a seventh aspect, the present application relates to a method of treating or preventing a disease or condition mediated by or involving a loss of sensitivity to light by retinal cells in a subject, the method comprising administering the isolated opsin of the first aspect, the isolated nucleic acid of the second aspect, the chimeric gene of the third aspect, the vector of the fourth aspect, or the isolated cell or cell culture of the fifth aspect.

[0045] In certain embodiments, the disease or condition mediated by a loss of sensitivity to light by retinal cells includes, but is not limited to, a disease or condition that benefits from restoring the sensitivity to light by retinal cells through activation of Gq signaling.

[0046] In certain embodiments, the disease or condition mediated by a loss of sensitivity to light by retinal cells includes a disease or condition that benefits from activating retinal cells, such as photoreceptor cells, rod cells, cone cells, retinal ganglion cells, bipolar cells, ganglion cells, horizontal cells, multipolar neurons, Muller cells, amacrine cells, or methyl methylnitrosourea.

[0047] In certain embodiments, the disease or condition includes damage to the outer layers of the retina, loss or degeneration of photoreceptors, retinal degenerative diseases, loss of sensitivity to light or loss of light perception, vision loss caused by light perception or insufficient sensitivity, or blindness.

[0048] In certain embodiments, the Opn5 in the present application can be used to restore the sensitivity to light by retinal cells, as long as the retinal ganglion cells are not completely dead.

[0049] In certain embodiments, the Opn5 in the present application can be used for treating or preventing a disease associated with degeneration and / or death of retinal ganglion cells (RGCs).

[0050] In certain embodiments, the Opn5 in the present application can be used for treating or preventing retinitis pigmentosa (RP), macular degeneration, age-related macular degeneration (AMD), autosomal dominant optic atrophy (ADOA), and / or glaucoma.

[0051] In certain embodiments, the method further comprises applying light with a wavelength range of 360-550 nm, preferably 450-500 nm, more preferably 460-480 nm.

[0052] In certain embodiments, the method further comprises using long wavelength (>920 nm) light for two-photon activation.

[0053] The isolated opsin described in the present application is sensitive to light with a wavelength in the range of 360-550 nm, preferably 450-500 nm, more preferably 460-480 nm. Specifically, blue light at 470 nm elicits the strongest Ca 2+ transients in the isolated opsin described in the present application, meaning that the isolated opsin described in the present application is hypersensitive to light with a wavelength of 470 nm.

[0054] The present application encompasses all combinations of the specific embodiments described herein. BRIEF DESCRIPTION OF DRAWINGS

[0055] Figure 1 It is shown that cOpn5 mediates light-induced G q signaling activation in HEK 293T cells.

[0056] Figure 2 It is shown that cOpn5 couples to G q signaling but not to G i signaling.

[0057] Figure 3 It is shown that cOpn5 sensitively mediates optical control of G q signaling with high spatiotemporal resolution.

[0058] Figure 4 It is shown that cOpn5 mediates a faster and more sensitive response to light than opto-a1AR, hM3Dq or opn4.

[0059] Figure 5 It is shown that cOpn5 effectively mediates activation of astrocytes.

[0060] Figure 6The diagram shows that a healthy retina contains several cell layers.

[0061] Figure 7 The results showed that normal mice before MNU treatment had a rapid pupillary light response, while C3H / HeNCrl inbred mice did not have a pupillary light response.

[0062] Figure 8 EGFP in the entire retina is shown 4 weeks after AAV injection.

[0063] Figure 9 The results showed that both MNU-treated mice and C3H / HeNCrl mice recovered their pupillary light response.

[0064] Figure 10 The pupil light response test is shown.

[0065] Figure 11 The results of immunofluorescence are shown.

[0066] Figure 12 The results of the electrophysiological tests are shown.

[0067] Figure 13 The results of the electrophysiological tests are shown.

[0068] Figure 14 The diagram illustrates an open field avoidance test.

[0069] Figure 15 The results of the open field avoidance test are shown.

[0070] Figure 16 The recovery of light sensitivity in the eyes of AAV-cOPN5-treated rd1 / rd1 mice was shown at 7 weeks (A) and 9 months (B). Detailed Implementation

[0071] In this invention, the capabilities of opsins from multiple species, particularly Opn5 orthologs, were tested, and it was found that many opsins sensitively and strongly mediate the activation and / or activation of cells by light-induced Gq signaling. The isolated photosensitive opsins can be used to treat subjects suffering from damage to the outer retina, loss or degeneration of photoreceptors, retinal degenerative diseases, loss of light sensitivity or light perception, visual loss, or blindness.

[0072] Preferably, the Opn5 ortholog is a chicken ortholog (abbreviated as cOpn5) or a sea turtle ortholog (abbreviated as tOpn5).

[0073] Detailed characterization of Opn5, especially cOpn5, reveals its ultrasensitivity (μW / mm²) to blue light with wavelengths of 450–500 nm, more preferably 460–480 nm.2 horizontal, compared to existing G q Coupled to the visual protein, opto-a1AR and opn4 are highly sensitive ~3 orders of magnitude), have high temporal (respond to 10 ms light pulses ~3 orders of magnitude faster than opto-a1AR or opn4) and spatial (subcellular level) resolution, and do not require the addition of a chromophore. Specifically, endogenous retinal is sufficient, no addition of retinal is required. cOpn5 mediates G q photogenetic activation of signaling and / or activation of cells

[0074] Specifically, in the present invention, Opn5 orthologs from chicken, turtle, human and mouse (which have 80-90% protein sequence identity to each other) were tested to determine if they have the ability to mediate blue light-induced Gq signaling activation in HEK 293T cells. Blue light and red intracellular calcium indicator Calbryte TM 630 AM dye was used to monitor relative Ca 2+ response. It was found that Opn5 orthologs from chicken (cOpn5) and turtle (tOpn5) mediated immediate and strong light-induced increases in Ca 2+ signals (~3 AF / F), whereas no light effect was observed in cells expressing human or mouse Opn5 orthologs. As exemplified by the chicken ortholog, cOpn5 co-localized with EGFP-CAAX membrane marker, indicating that it was efficiently transported to the plasma membrane. No exogenous retinoids needed to be added to the culture medium, indicating that endogenous retinoids were sufficient for cOpn5 to function. Ca 2+ signals were resistant to removal of extracellular Ca 2+ therefore indicating that Ca 2+ was released from intracellular stores. In both cOpn5-expressing cells, G q protein inhibitors (e.g. YM-254890, a highly selective G q protein inhibitor) pre-incubation reversibly abolished light-induced Ca 2+Transient. A light-induced increase in the level of inositol phosphate (IP1), a rapid degradation product of IP3, was detected in cells expressing cOpn5 but not human OPN5; furthermore, treatment with YM-254890 reduced the extent of this increase. In cOpn5-expressing cells (e.g., HEK 293T cells), blue light also triggered phosphorylation of the MARCKS protein (a recognized PKC target) in a PKC activity-dependent manner. In contrast, in the presence of retinaldehyde, blue light irradiation effectively reduced cAMP levels in cells expressing human and mouse Opn5, but this effect was absent in cOpn5-expressing cells without exogenous retinaldehyde. In conclusion, these data support that blue light irradiation can induce a photosensitive reaction between cOpn5 and GAMP in HEK 293T cells. q Signal conduction path coupling.

[0075] cOpn5-mediated optogenetics is sensitive and precise.

[0076] Specifically, the photoactivation properties of cOpn5 were characterized in this invention. cOpn5 can be heterologously expressed in cells (e.g., HEK 293T cells). Although Opn5 was previously considered a UV-sensitive photoreceptor, at a fixed light intensity (100 μW / mm²), its photoactivation properties were... 2 The following plot, using a set of wavelengths in the 365–630 nm range, reveals that blue light at 470 nm induces the strongest Ca2+. 2+ The light intensity was transient, while UVA light (365 and 395 nm) was less efficient, and longer wavelengths of visible light (561 nm or above) were completely ineffective. The effects of different light durations on HEK 293T cells expressing cOpn5 were tested, with short light pulses (1, 5, 10, 20, 50 ms; 16 μW / mm²) applied. 2 Stimulation at 470nm showed that Ca2+ was effective when the illumination duration exceeded 10ms. 2+ The response reached saturation mode. At this illumination intensity (16 μW / mm²), 2 At 470nm, longer illumination durations do not further increase Ca 2+ Signal amplitude. Displayed by projecting 470nm light at different intensities, ~4.8μW / mm. 2 and 16μW / mm 2 Blue light produced approximately half-maximal and maximum responses, respectively. These data indicate that the photosensitivity of cOpn5 is 2–3 orders of magnitude higher than that of rhodopsin-2 (ChR2), a commonly reported optogenetic tool. In summary, the results of this invention demonstrate that cOpn5 can function as a single-component optogenetic tool without additional retinal, and that cOpn5 is hypersensitive to blue light, requiring low light intensity (16 μW / mm²) for complete activation.2 ) and short duration (10 ms).

[0077] The performance of cOpn5 was compared to that of opto-a1AR, a chimeric GPCR engineered by mixing a rhodopsin with a q adrenergic receptor coupled to G 2 . Following a previously reported protocol, it was found that very long exposure to strong illumination (60 s; 7 mW / mm 2+ ) was required to trigger a slow and small (~0.5 AF / F) increase in Ca 2 signals in HEK 293T cells expressing opto-a1AR. Long exposure to strong illumination (25 s; 40 mW / mm 2+ ) and additional retinal were required to trigger a slow (~1 AF / F) increase in Ca q signals in HEK 293T cells expressing opn4. Thus, cOpn5 is much more photosensitive (3 orders of magnitude higher in sensitivity) than existing opsin-based tools (opto-a1AR and opn4), requires much shorter exposure times (10 ms vs. 60 s), and produces a stronger response.

[0078] Furthermore, the performance of cOpn5 was compared to that of the popular G 2+ coupled chemical-genetic tool hM3Dq, which is activated by the addition of the exogenous small molecule ligand clozapine-N-oxide (CNO). The light-induced activation of HEK 293T cells expressing cOpn5 had a similar peak response amplitude of Ca 2+ signals compared to the CNO-induced activation of HEK 293T cells expressing hM3Dq. At the same time, HEK 293T cells expressing cOpn5 had a faster and more temporally precise response, as well as a faster recovery time, compared to HEK 293T cells expressing hM3Dq. These results indicate that cOpn5-mediated optogenetics is more controllable in terms of temporal accuracy than hM3Dq.

[0079] cOpn5 optogenetics allows for spatially precise control over cellular activity. Restricting a brief light stimulus (63 ms) to a subcellular region of a single HEK 293T cell expressing cOpn5 resulted in the immediate activation of the single cell. Interestingly, in a region where cells were highly packed, Ca 2+The signal propagates to surrounding cells, indicating that there is intercellular communication between HEK 293T cells through a yet unidentified mechanism. In primary astrocyte cultures prepared from neonatal mouse brains, cOpn5 was expressed using an AAV vector for bicistronic expression of the cOpn5 and EGFP marker proteins. Ca 2+ transients (~8 AF / F) were observed. When the light stimulus (63 ms) was precisely confined to a subcellular region of a single cOpn5-expressing astrocyte, Ca 2+ transients (~8 AF / F) were observed. When the light stimulus (63 ms) was precisely confined to a subcellular region of a single cOpn5-expressing astrocyte, Ca 2+ signals propagate within a single cell. Similar to the tests in HEK 293T cells, Ca 2+ signals propagate within a single cell. Similar to the tests in HEK 293T cells, Ca

[0080] Here, the present invention demonstrates the use of Opn5 of the present invention as an extremely effective optogenetic tool for restoring the sensitivity of retinal cells to light by activating Gq signaling. Previous studies have characterized mammalian Opn5 as a UV-sensitive G i coupled opsin; we present a surprising finding that in mammalian cells expressing Opn5 (e.g., expressing cOpn5 or expressing tOpn5), blue light can induce rapid Ca 2+ transients, IP1 accumulation, and PKC activation.

[0081] Table 6 lists the empowering features of cOpn5 by directly comparing the response amplitude, light sensitivity, temporal resolution, and requirement for additional chromophores of cOpn5 with other optogenetic tools. For cells expressing cOpn5, only a 10 ms blue light pulse of 16 μW / mm 2 intensity elicits rapid increases in Ca 2+ signals with peak amplitudes of 3-8 AF / F. In contrast, prior to the present invention, activation of opto-a1AR or mammalian Opn4, two proposed optogenetic tools for Gq signaling, required ~3-fold higher light intensities (7-40 mW / mm 2 ) and prolonged light exposure (20-60 s) and only produced weak Ca 2+signals (0.25-0.5 AF / F). Thus, opto-a1AR or mammalian Opn4 cannot mimic the fast activation profile of endogenous Gq-coupled receptors, which typically trigger strong Gq signaling upon subsecond application of their respective ligands. In contrast, recent systematic characterizations show that opto-a1A and Opn4-mediated optogenetic stimulation does not increase the amplitude of the signal and only modestly modulates the frequency of Ca 2+ signals, and only modestly modulates the frequency of Ca 2+ transients and synaptic events (Gerasimov et al., 2021; Mederos et al., 2019).

[0082] The opn5, in particular cOpn5 or tOpn5, based optogenetics of the present application also has the advantages of safety and convenience. Although Opn5 from many species is reported to be UV responsive (Kojima et al., 2011), cOpn5 is optimally activated by 470 nm blue light, which has better penetration than UV and avoids UV-related cytotoxicity. Its hypersensitivity to light also minimizes potential heating artifacts. cOpn5 or tOpn5 is strongly and reproducibly activated by light, and does not require exogenous retinal, which is likely because cOpn5 or tOpn5 is a bistable opsin that covalently binds to endogenous retinal, and thus is resistant to photobleaching (Koyanagi and Terakita, 2014; Tsukamoto and Terakita, 2010). In contrast, mammalian experiments with Opn4 require additional retinal, and have long response times and low light sensitivity. The opn5, in particular cOpn5 or tOpn5, of the present application is particularly useful for in vivo studies as a single-component system, as it avoids the burden of delivering a compound to a tissue during the experiment.

[0083] The opn5, in particular cOpn5 or tOpn5, optogenetics of the present application also offers some important advantages over chemical genetics and uncaging tools. It is much more precise in time, and provides single-cell or even subcellular spatial resolution. The opn5, in particular cOpn5 or tOpn5, of the present application is also different from “uncaging” tools based on caged compounds, such as caged calcium and caged IP3, because these tools require preloading of the compound, and only partially mimic the G q signaling and / or activate cell-associated Ca 2+Related pathways. Other “uncaging” tools exist, such as caged glutamate and caged ATP targeting endogenous GPCRs (Ellis-Davies, 2007; Lezmy et al., 2021). However, these caged compounds need to be introduced into the extracellular medium or cytoplasm within the cell, which limits their application in behaving animals (Adams and Tsien, 1993b).

[0084] The optronic of Opn5, particularly cOps5 or tOpn5 in the present invention is particularly suitable for precisely activating intracellular G q signaling and / or activating cells, which subsequently trigger the release of Ca 2+ and activation of PKC. The Opn5, particularly cOpn5 or tOpn5 in the present invention is different from current channel-based optogenetic tools, such as ChR2 or its variants that transfer cations across the plasma membrane.

[0085] On the basis of the strong light sensitivity of Opn5 in the present invention, it is further demonstrated that Opn5 in the present invention can be used to restore the sensitivity of retinal cells to light by activating Gq signaling, and thus can be used to treat or alleviate retinal outer layer damage, loss of photoreceptors or degeneration, retinal degenerative diseases, loss of sensitivity to light or loss of light perception, vision loss caused by light perception or insufficient sensitivity, or blindness.

[0086] In certain embodiments, Opn5 in the present invention can be used to restore the sensitivity of retinal cells to light as long as the retinal ganglion cells are not completely dead.

[0087] In certain embodiments, Opn5 in the present invention can be used to treat or prevent diseases associated with degeneration and / or death of retinal ganglion cells (RGCs).

[0088] In certain embodiments, Opn5 in the present invention can be used to treat or prevent retinitis pigmentosa (RP), macular degeneration, age-related macular degeneration (AMD), autosomal dominant optic atrophy (ADOA), and / or glaucoma.

[0089] In the present invention, cOpn5, cOPN5, O5 and chicken opn5m can be used interchangeably.

[0090] In the present invention, opn5, OPN5, opsin and Opn5 can be used interchangeably.

[0091] By way of illustration and not limitation, descriptions of specific embodiments and examples are provided. Those skilled in the art will readily recognize that various noncritical parameters can be changed or modified to yield essentially similar results.

[0092] Example

[0093] Materials and methods:

[0094] Table 1: Primers used for cloning

[0095]

[0096] Table 2: Recombinant DNA

[0097] pcDNA3.1-opto-a1AR-EYFP Addgene plasmid #20947 EGFP-CAAX Gift from Yulong Li pLJM1-EGFP Addgene plasmid #19319 pAAV-GfaABC1D-hM3D(Gq)-mCherry Addgene plasmid #50478 pAAV-EF1a-DIO-eGFP-WPRE-pA N / A pAAV-hSyn-GOI N / A pLJM1-cmv-cOpn5 N / A pLJM1-cmv-tOpn5 N / A pLJM1-cmv-hOPN5 N / A pLJM1-cmv-mOpn5 N / A pLJM1-cmv-V5-Opn5 N / A pLJM1-cmv-cOpn5-T2A-eGFP N / A PAAV-hSyn-cOpn5-T2A-eGFP-WPR-pA N / A PAAV-GfaABC1D-cOpn5-T2A-eGFP-WPR-pA N / A pAAV-EF1a-DIO-cOpn5-T2A-eGFP-WPRE-pA N / A PAAV-GfaABC1D-cOpn5-T2A-mCherry-WPR-pA N / A

[0098] Table 3: Virus strains

[0099]

[0100] Table 4: Photoexcitation Sources

[0101]

[0102]

[0103] Table 5: Microscope Equipment

[0104]

[0105] Table 6: Statistical Analysis

[0106]

[0107]

[0108] Example 1. cOpn5-mediated G q Optogenetic activation of signal transduction

[0109] The study tested whether heterologous expression of Opn5 orthologs from chickens, turtles, humans, and mice (which share 80-90% protein sequence identity) could mediate blue light-induced Gamma globulin production in HEK 293T cells. q The ability to activate signal transduction ( Figure 1 (a and Table 7). Stimulation was performed using blue light, with the red intracellular calcium indicator Calbryte used. TM 630AM dye to monitor relative Ca 2+ response( Figure 1 b). Opn5 orthologs from chickens (cOpn5) and turtles (tOpn5) mediate Ca2+. 2+ The signal showed an immediate and strong light-induced increase (~3ΔF / F), while no light effect was observed in cells expressing human or mouse Opn5 orthologs. Figure 1 d and Figure 2 a, Figure 2b). cOpn5 co-localizes with the EGFP-CAAX membrane marker, as exemplified by the chicken ortholog, indicating that it is efficiently transported to the plasma membrane Figure 1 c). No exogenous retinoids were added to the culture medium, indicating that endogenous retinoids are sufficient for cOpn5 functionality. Ca 2+ signals are tolerated by the cells, thus indicating that Ca 2+ is released from intracellular stores 2+ ( Figure 2 c). Pre-incubation with YM-254890, a highly selective G q protein inhibitor 33 , reversibly abolishes light-induced Ca 2+ transients in both cells expressing cOpn5 Figure 1 e). In cells expressing cOpn5 (but not human OPN5), a light-induced increase in the levels of IP3 rapid-degradation product, inositol phosphate (IP1), is detected; moreover, treatment with YM-254890 reduces the extent of this increase Figure 1 f and Figure 2 d). In HEK 293T cells expressing cOpn5, blue light also triggers phosphorylation of the MARCKS protein (a recognized target of PKC 34 ) in a PKC activity-dependent manner Figure 1 g and Figure 2 e). In contrast, in cells expressing human and mouse Opn5 in the presence of retinal, blue light irradiation effectively reduces cAMP levels, but this effect is not observed in cells expressing cOpn5 in the absence of retinal Figure 2 f). Altogether, these data support the ability of blue light irradiation to couple cOpn5 to G q signaling pathways in HEK 293T cells.

[0110] Table 7: Opsins and species

[0111] Alias Species Chicken Opn5 cOpn5 Gallus gallus GenBank NM_001130743.1 Turtle Opn5 tOpn5 Chelonia mydas GenBank XM_007068312.4 Human Opn5 hOPN5 Homo sapiens GenBank AY377391.1 Mouse Opn5 mOpn5 Mus musculus GenBank NM_181753.4

[0112] Figure 1 showing that cOpn5 mediates strong activation of light-induced G q signaling in HEK 293T cells.

[0113] a, schematic representation of the intracellular signaling in response to light-induced cOpn5 activation. PLC: phospholipase C; PIP2: phosphatidylinositol-4,5-bisphosphate; IP3: inositol-1,4,5-trisphosphate; IP1: inositol monophosphate; DAG: diacylglycerol; PKC: protein kinase C; YM-254890: a selective G q protein inhibitor. ​

[0114] b. In HEK 293T cells expressing Opn5 from three species (Gallus gallus, Homo sapiens, and Musmusculus), under blue light stimulation (10 s; 100 μW / mm²), 2 Before and after Ca (488nm) 2+ A pseudo-color image of the signal. Scale bar, 10 μm.

[0115] c. In HEK 293T cells, Cy3-counted V5-cOpn5 fusion protein (red) co-localizes with the membrane tag EGFP-CAAX (green). DAPI counterstaining (blue) indicates the cell nucleus. Scale bar, 10 μm.

[0116] Figures d and c show the light-evoked Ca2+ in cells. 2+ The time-course variation of the signal.

[0117] e, G q The protein inhibitor YM-254890 (10 nM) reversibly blocks cOpn5-mediated light-induced Ca2+. 2+ Signal.

[0118] f, In HEK 293T cells expressing cOpn5, YM inhibition was achieved by continuous light stimulation (3 min; 100 μW / mm). 2 IP1 accumulation evoked at 470nm (left). ***P<0.0001, *P=0.0128; Tukey's multiple comparison test.

[0119] g. Phosphorylation of MARCKS in cOpn5-expressing HEK 293T cells in the control group (no light stimulation), the light-stimulated group, and the light + staphylococcalin (ST, a PKC inhibitor) group. The amount of p-MARCKS in each fraction was normalized relative to the amount of α-tubulin. **P = 0.0096, ***P = 0.0004; Tukey's multiple comparison test.

[0120] Figure 2 This shows cOpn5 coupled to G q Signal conduction, but not coupled to G i Signal Transduction

[0121] a, In HEK 293T cells expressing Opn5 from a sea turtle species (Chelonia mydas), under blue light stimulation (10 s; 100 μW / mm²), 2 Before and after Ca (488nm) 2+ A pseudo-color image of the signal. Scale bar, 10 μm (left); Ca2+ in response to light-evoked signals in cells.2+ Temporal changes in the signal (right).

[0122] b, The Gq protein inhibitor YM-254890 (10 nM) reversibly blocked cOpn5- and turtle Opn5-mediated light-induced Ca 2+ Group data for the signal. ****P < 0.0001, one-way ANOVA. Error bars indicate S.E.M.

[0123] c, In the absence of extracellular Ca 2+ , light stimulation (10 ms; 16 μW / mm2; 470 nm) produced Ca 2+ signals. Temporal changes in the signal.

[0124] d, IP1 accumulation in HEK 293T cells expressing human Opn5 with or without light stimulation (right). n.s., not significant; unpaired t-test.

[0125] e, One representative result for MARCKS phosphorylation in HEK 293T cells expressing cOpn5 in control (no light stimulation), light stimulation, and light + staurosporine groups. The amount of p-MARCKS was normalized to the amount of a-tubulin in the same lane.

[0126] f, Light had no effect on cAMP levels in HEK 293T cells expressing cOpn5 in the absence of added retinal in the culture medium (10 μM forskolin pre-incubation) (left panel). The right panel shows the effect of light stimulation on cAMP concentration in HEK 293T cells expressing Opn5 from four different species after 10 μM retinal pre-incubation.

[0127] Error bars in d and f indicate S.E.M.

[0128] Example 2. cOpn5-mediated optogenetics is sensitive and precise

[0129] The light-activated properties of cOpn5 heterologously expressed in HEK 293T cells were characterized. Although Opn5 was previously thought to be a ultraviolet (UV)-sensitive photoreceptor 27 , plotting a range of wavelengths from 365 to 630 nm at a fixed light intensity (100 μW / mm 2 ) revealed that blue light at 470 nm elicited the strongest Ca 2+ transients, with UVA light (365 and 395 nm) being less efficient, and longer wavelength visible light (561 nm or above) being completely ineffective Figure 3a). The effect of different light duration on cOpn5 expressing HEK 293T cells was tested. Stimulation with short light pulses (1, 5, 10, 20, 50 ms; 16 μW / mm 2 ; 470 nm) showed that the Ca 2+ response reached a saturation pattern Figure 3 b). At this light intensity (16 μW / mm 2 ; 470 nm) longer light duration did not further increase the Ca 2+ signal amplitude Figure 4 a). Pulsing 470 nm light at different intensities showed that ~4.8 μW / mm 2 and 16 μW / mm 2 blue light produced about half and the maximal response, respectively Figure 3 c and Figure 4 b). Thus, the photosensitivity of cOpn5 is 3-4 orders of magnitude higher than the values reported for light-sensitive Gq-coupled GPCRs and even 2-3 orders of magnitude higher than the value for the commonly used optogenetic tool channelrhodopsin-2 (ChR2) (Lin, 2011; Zhang et al., 2006) (Table 8). In summary, these results show that cOpn5 can function as a single-component optogenetic tool without additional retinal and that cOpn5 is hypersensitive to blue light as it requires low light intensities (16 μW / mm 2 ) and short durations (10 ms) for full activation.

[0130] Table 8: Comparison of cOpn5 with other optogenetic tools

[0131]

[0132]

[0133] The performance of cOpn5 was compared to that of opto-a1AR, a chimeric GPCR engineered by mixing aopsin with a G q protein-coupled adrenergic receptor. Following a previously reported protocol 14 , it was found that very long exposure to strong illumination (60 s; 7 mW / mm 2 ) was required to trigger a slow and small (~0.5 ΔF / F) increase in Ca 2+ signals in HEK 293T cells expressing opto-a1AR, with 15 s of illumination being ineffective Figure 4 c, Figure 4d). Comparing the performance of cOpn5 with opn4, a natural opsin reported to be a tool for Gq signaling activation. It was found that long exposure to strong illumination (25 s; 40 mW / mm 2 ) and additional retinal were required to trigger a slow (~1 AF / F) increase in Ca 2+ signals in HEK 293T cells expressing opn4. Figure 4 e, Figure 4 f). Thus, cOpn5 is much more photosensitive (3 orders of magnitude higher in sensitivity) than existing opsin-based tools (opto-a1AR and opn4), requires much shorter exposure times (10 ms vs. 60 s), and produces stronger responses.

[0134] Furthermore, the performance of cOpn5 was compared with that of the popular G q coupled chemo-genetic tool hM3Dq, which activates 37-39 Ca 2+ signals in HEK 293T cells expressing cOpn5 were similar to CNO-induced activation of HEK 293T cells expressing hM3Dq. At the same time, HEK 293T cells expressing cOpn5 had faster and temporally more precise responses, and faster recovery times Figure 4 g Figure 4 i) than HEK 293T cells expressing hM3Dq. These results indicate that cOpn5-mediated optogenetics is more controllable in terms of temporal accuracy than hM3Dq.

[0135] cOpn5 optogenetics allows precise spatial control of cellular activity. Restricting a brief light stimulus (63 ms) to a subcellular region of a single HEK 293T cell expressing cOpn5 resulted in immediate activation of the single cell. Interestingly, the Ca 2+ signal propagated to surrounding cells, indicating that intercellular communication exists between HEK 293T cells through a yet unidentified mechanism Figure 3 d, Figure 3 e). The finding was extended to primary cell cultures. In primary astrocyte cultures prepared from the brains of neonatal mice, cOpn5 was expressed using an AAV vector for bicistronic expression of cOpn5 and the EGFP marker protein Figure 5 a). Ca 2+horizontal, that illumination with blue light to cOpn5 expressing astrocytes generates strong Ca 2+ transients (~8 ΔF / F) Figure 5 b, Figure 5 c) When the light stimulus (63 ms) is precisely confined to a subcellular region of a single cOpn5 expressing astrocyte, Ca 2+ signals are observed to propagate within a single cell Figure 3 f) Similar to the tests in HEK 293T cells, Ca 2+ signals are observed to propagate in a wave-like fashion from the stimulated astrocyte to more distal, non-stimulated astrocytes Figure 3 g, Figure 3 h) Thus, these experiments demonstrate that cOpn5 optogenetics allows precise spatial control and suggest that the dynamics of astrocytic networks initially discovered using neurochemical and mechanical stimuli 40,41 may be useful.

[0136] Figure 3 show that cOpn5 sensitively mediates G q optical control of G-protein signaling with high temporal and spatial resolution.

[0137] a, Schematic of the amplitude of the Ca 2+ signals (2 s; 100 μW / mm 2 ; right panel) of cOpn5 expressing HEK 293T cells in response to light stimuli of different wavelengths. Error bars indicate S.E.M.

[0138] b, Amplitude of the response at different light stimulus durations (1, 5, 10, 20 or 50 ms; 16 μW / mm 2 ; 470 nm). Error bars indicate S.E.M.

[0139] c, Time course of the cOpn5 mediated Ca 2+ signals at different light intensities (0, 4.8, 8, 16 or 32 μW / mm 2 ; 10 ms; 470 nm; for 10 ms 16 μW / mm 2 stimulus 10% peak activation = 1.36 ± 0.55 s; 90% peak activation = 2.37 ± 0.87 s; decay time τ = 18.66 ± 4.98 s, mean ± S.E.M.; n = 10 cells).

[0140] d, Light-induced (63 ms; 17 μW; arrow points to the stimulated region) Ca2+ Image of signal propagation. Scale bar, 10 pm.

[0141] e, Ca 2+ Pseudocolor image of signal propagation across time (frames N / (N-1) > 1). Frame interval, 500 ms, counted once per frame.

[0142] f, Light-induced Ca 2+ propagation in individual primary astrocytes expressing cOpn5, stimulated in subcellular regions. Scale bar, 10 pm. 2 Frame interval, 300 ms). Scale bar, 10 pm.

[0143] g, Light-induced Ca 2+ propagation in primary astrocytes expressing cOpn5. Scale bar, 10 pm.

[0144] h, Ca 2+ Pseudocolor image of signal propagation across time (frames N / (N-1) > 1). Frame interval, 500 ms, counted once per frame.

[0145] Figure 4 cOpn5 mediates faster and more sensitive responses to light than opto-a1AR, hM3Dq or opn4.

[0146] a, Time course of Ca 2 signal using light pulses (16 pW / mm 2+ ; 470 nm; 1, 5, 10, 20 or 50 ms).

[0147] b, Amplitude of response at different light intensities (0, 4.8, 8, 16 or 32 pW / mm 2 ) at 10 ms, 470 nm.

[0148] c, Pseudocolor image of baseline and peak Ca 2+ signals (AF / F0) in HEK 293T cells expressing opto-a1AR. Culture medium buffer contained 10 pM all-trans retinal. Scale bar, 30 pm.

[0149] d, Effect of 60 s light stimulation on Ca 2+ (n = 15 cells; top panel) and 15 s light stimulation had no effect on Ca 2+ signals (bottom panel) in HEK 293T cells expressing opto-a1AR.

[0150] e, Baseline and peak Ca 2+Pseudo-color image of the signal (ΔF / F0). The culture medium buffer contained 10 μΜ all-trans retinal. Scale bar, 30 μιη.

[0151] f, Effect of 25 s light stimulation on Ca 2+ signals in HEK 293T cells expressing OPN4 at 10 uM ATR (n = 12 cells; red line), without ATR (black plot) had no effect on Ca 2+ signals.

[0152] g, Effect of light stimulation on Ca 2+ signals in HEK 293T cells expressing cOpn5. Top panel shows pseudo-color images of baseline and peak responses. Bottom panel shows heat map of Ca 2+ signals evoked by optogenetic stimulation mediated by cOpn5 across 5 consecutive trials in HEK 293T cells expressing cOpn5. Scale bar, 20 μιη.

[0153] h, Effect of chemo genetic stimulation on Ca 2+ signals in HEK 293T cells expressing hM3Dq.

[0154] i, Time course of Ca 2+ signals evoked by optogenetic stimulation (10 s) and chemo genetic stimulation (100 nM; 10 s) mediated by hM3Dq, respectively.

[0155] Figure 5 shows that cOpn5 effectively mediates activation of astrocytes.

[0156] a, Expression of cOpn5 in cultured primary astrocytes using AAV-cOpn5-T2A-EGFP (green). Identity of astrocytes was confirmed by GFAP immunostaining (red). Scale bar, 20 μιη.

[0157] b, Pseudo-color images of baseline and peak Ca 2+ signals in astrocytes expressing cOpn5 following light stimulation. Scale bar, 20 μιη.

[0158] c, Plot of Ca 2+ signals and heat map of Ca 2+ signals across trials (n = 25 cells).

[0159] Example 3. Animal model of optogenetic visual restoration using a light-sensitive Gq-coupled neuro photopsin (Opn5):

[0160] 1. Healthy retina includes several cell layers: retinal pigment epithelium, cone photoreceptors, rod photoreceptors, horizontal cells, bipolar cells, Muller cells, amacrine cells, ganglion cells Figure 6 ) Methyl-nitrosourea (MNU) causes photoreceptor (rod and cone photoreceptors) damage in animals, then induces retinal degeneration. We use MNU to induce retinal degeneration in mice as an animal model. Retinal degeneration is induced by a single intraperitoneal injection of MNU at a dose of 60 mg / kg body weight.

[0161] 2. C3H / HeNCrl mice are a genetic model of retinal degeneration. This strain is characterized by a Pde6b rd1 mutation that is homozygous for retinal degeneration.

[0162] We use the pupillary light response of head-fixed mice to test whether animals can perceive light, we use AAV vectors to express cOpn5 in mouse retinal ganglion cells to rescue both mouse models. The restoration of pupillary light response in mice confirms our cOpn5-mediated treatment for blindness.

[0163] Experiments and results:

[0164] 1. We use a camera with IR blocking function to automatically acquire images of the pupils of head-fixed mice. Adjust the optical fiber to ensure that the light (470 nm LED light source) is directly irradiated on the pupils of mice with the same light intensity.

[0165] 2. Normal mice before MNU treatment have fast pupillary light responses Figure 7 ). C3H / HeNCrl mice are born without pupillary light responses Figure 7 ).

[0166] 3. C3H / HeNCrl or MNU-treated retinal degenerative mice lose pupillary light response function.

[0167] 4. We use AAV vectors to express cOpn5-t2a-EGFP in mouse retinal ganglion cells, images show EGFP in the entire retina 4 weeks after AAV injection Figure 8 ).

[0168] 5. After expressing cOpn5 in mouse retinal ganglion cells, we perform the pupillary light response test again. Treated MNU mice restore pupillary light responses Figure 9 ). C3H / HeNCrl mice gain the ability to pupillary light responses Figure 9 ).

[0169] 6. Figure 10The pupil light response test is shown: the pupil size of normal mice (black solid line) rapidly decreases in response to light (X-axis: time (sec); Y-axis: normalized pupil size). After MNU treatment, mice lose function in the pupil light response test (gray solid line). When using AAV vectors to express cOpn5 in the retinal ganglion cells (RGCs) of these MNU-treated mice, the mice partially recover the ability of pupil light response (middle solid line) after 8 weeks.

[0170] These results demonstrate that our method of expressing cOpn5 in animal retinal ganglion cells can restore retinal degeneration.

[0171] Example 4

[0172] Experimental description: Table 9 below is a partial list of cOpn5 orthologs from the subphylum Vertebrata tested in the present invention. The full genes of all reported opsin 5 orthologs from the subphylum Vertebrata (vertebrates including rotundia, cartilaginous fishes, bony fishes, amphibians, reptiles, birds, and mammals) were synthesized and expressed in HEK 293T cells. Calcium imaging was performed with or without 470 nm blue light stimulation to test the sensitivity of opsin 5 orthologs to light. The light-induced calcium signal time course reveals the degree of activation of the Gq signaling pathway and the sensitivity of these orthologs.

[0173] Table 9:

[0174]

[0175]

[0176]

[0177]

[0178]

[0179]

[0180]

[0181]

[0182]

[0183] Example 5

[0184] Animals:

[0185] 8-16 week rd1 / rd1 retinitis pigmentosa (RP) model mice were raised under 12 / 12 light / dark cycle (lights off at 8 pm).

[0186] Construction of AAV vectors:

[0187] Plasmids required for packaging AAV virus include pAAV-mSNCG-chicken opn5m-t2a-EGFP, pAAV-mSNCG-chicken opn5m-t2a-mcherry, pAAV-mSNCG-chicken opn5m, and pAAV-mSNCG-EGFP. Packaging and production of adeno-associated virus (AAV):

[0188] Recombinant AAVs were prepared by co-transfection of plasmids. AAV2.7M8 and AAV2 / 8 subtypes were packaged, respectively. Both of them include mSNCG-chicken opn5m-t2a-EGFP, mSNCG-chicken opn5m-t2a-mcherry, mSNCG-chicken opn5m, and mSNCG-EGFP.

[0189] Intraocular injection of AAV into mice

[0190] After anesthesia, 1 μΐ of AAV was injected into the vitreous cavity of the mouse with a superfine glass electrode through the sclera, and the electrode was pulled out a few seconds later. The follow-up experiment was performed 4 weeks after AAV injection.

[0191] Immunofluorescence:

[0192] To confirm whether AAV successfully infected retinal cells and compare the infection efficiency and virus specificity between various subtypes, immunofluorescence experiments were required. After 4 weeks of AAV injection, the mouse retina was removed and fixed in 4% paraformaldehyde for 30 minutes. The fixed and cleaned retina was embedded and vertically sectioned with a Leica cryostat at a thickness of 15 μιη. The sections were washed with PBS and then sealed with 3% BSA (bovine serum albumin) at room temperature for 1 hour. Then the anti-EGFP primary antibody was diluted with 3% BSA at 1:500 and incubated at 4°C for 48 hours. After washing the primary antibody, it was incubated with a fluorescently labeled secondary antibody for 2 hours, and the stained retinal sections were transferred to a glass slide, sealed after sealing, and scanned by confocal to obtain fluorescence images. The infection efficiency of each AAV on retinal ganglion cells (RGCs) and the fluorescence intensity of EGFP were analyzed and compared, and the AAV subtype with high infection rate and good specificity was selected for the next step experiment.

[0193] Electrophysiological testing:

[0194] ​To further confirm whether cOPN5 maintains its physiological activity in RGC cells after successful AAV expression, electrophysiological experiments were performed. AAV, with high infectivity and good specificity, was injected into the eyes of rd1 / rd1 mice (purchased from GemPharmatech Co., Ltd.). Four weeks after viral injection, the mouse retinas were removed, and retinal slices were placed in the electrophysiological recording chamber. The RGC layer of the retina was facing upwards. To prevent light damage to the retina, the laser was turned off after identifying GFP-expressing somatic cells using fluorescence microscopy. Current intensities were recorded after stimulating the cells with 488 nm laser light of varying intensities.

[0195] Behavioral testing:

[0196] The visual receptor cells in Rd1 / rd1 mice have degenerated. To verify whether visual information can be transmitted to the brain through infected ganglion cells to restore their lost visual function, we selected several visual function tests:

[0197] (1) Pupillary light reflex (PLR)

[0198] In Rd1 / rd1 mice, the pupils only respond to strong light. PLR experiments were performed 4 weeks after AAV injection into the mouse eyes. The pupils of mice expressing cOPN5 and EGFP were stimulated with light of different intensities to record the degree of pupillary changes, and these changes were used to assess the mice's light sensitivity.

[0199] (2) Open field avoidance test

[0200] Normal mice avoid open, bright spaces. This innate tendency forms the basis for a simple test of their visual abilities. In this experiment, mice were placed in a brightly lit space, with a dark sanctuary also provided. Their visual abilities were assessed by measuring the proportion of time the mice spent in this dark space.

[0201] Safety testing:

[0202] Long-term heterologous expression of genes has different effects on the expressing tissues. Long-term experiments are needed to assess the safety of heterologous expression and test whether the heterologously expressed gene is stably expressed in tissues over a long period. AAV was injected into the eye for 6 months, and the above immunofluorescence, electrophysiological and behavioral tests were repeated one year later to detect whether the expression level and physiological activity of cOPN5 changed due to long-term expression, and to detect whether there was an inflammatory response in the retinal tissue.

[0203] Results:

[0204] like Figure 11 As shown, Figure 11A shows expression of cOPN5 protein in retinal ganglion cells of rd1 / rd1 mice;

[0205] Figure 11 B shows microglia marker Ibal staining of retinal sections post-injection. Mice injected with H202 (positive control) show strong activation of microglia. In retinas injected with AAV-cOPN5-t2a-EGFP, little basal Ibal signal was observed 1 month post-injection, similar to that observed in retinas injected with AAV-EGFP, retinas injected with AAV-cOPN5-t2a-EGFP 10 months post-injection, and in uninjected retinas. Red, Ibal; green, cOPN5 or EGFP; blue, DAPI (4',6-diamidino-2-phenylindole) signal indicating nuclei. Scale bar, 50 pm;

[0206] Figure 11 C shows RGC marker brn3a staining of retinal sections. Red, brn3a; green, cOPN5; blue, signal indicating nuclei. Scale bar, 50 pm;

[0207] Figure 11 D shows fundus fluorescence imaging.

[0208] As Figure 12 shown in Figure 12 A shows representative responses of RGCs from C3H mice injected with AAV-Copn5-t2a-EGFP during 488 nm laser stimulation at different powers;

[0209] Figure 12 B shows representative responses of RGCs from C3H mice injected with AAV-Copn5-t2a-EGFP during 561 nm laser stimulation at different powers;

[0210] Figure 12 C shows raw traces demonstrating reliable and repeatable light activation of RGCs mediated by cOpn5;

[0211] Figure 12 D and Figure 12 E show group data of firing rates of RGCs after 488 nm laser stimulation at different powers (n = 6);

[0212] Figure 12 F shows group data of latency times of RGCs after 488 nm laser stimulation at different powers (n = 6).

[0213] As Figure 13 shown in Figure 13A shows representative responses of v1 neurons from C57 mice during 2s 200lux light stimulation;

[0214] Figure 13 B shows representative responses of v1 neurons from C3H mice injected with AAV-EGFP during 2s 200lux light stimulation;

[0215] Figure 13 C shows representative responses of v1 neurons from C3H mice injected with AAV-cOPN5-t2a-EGFP during 2s 200lux light stimulation;

[0216] Figure 13 D shows a heatmap indicating ROC plots of peristimulus time histogram data from v1 neurons from C57 mice tested using 2s 200lux light stimulation (n=107);

[0217] Figure 13 E shows a heatmap indicating ROC plots of peristimulus time histogram data from v1 neurons from C3H mice injected with AAV-EGFP tested using 2s 200lux light stimulation (n=133);

[0218] Figure 13 F shows a heatmap indicating ROC plots of peristimulus time histogram data from v1 neurons from C3H mice injected with AAV-cOPN5-t2a-EGFP tested using 2s 200lux light stimulation (n=100);

[0219] Figure 13 G shows visual evoked potentials (VEPs) in C57 (top), rd / rd mice injected with AAV-EGFP (middle) and rd1 / rd1 injected with AAV-cOPN5-EGFP (bottom) in response to 2s light.

[0220] Figure 14 The open field avoidance test is schematically shown:

[0221] Methods: The light / dark box (45 x 27 x 25 cm) was made of organic glass and consisted of two compartments connected by an opening (4 x 5 cm) at floor level in the center of the dividing wall. The light compartment occupied approximately 2 / 3 of the entire light / dark box and the dark compartment approximately 1 / 3 of the entire light / dark box. The test arena was diffusely illuminated at 200 lux. Mice were brought into the test room in their home cage. The test was started after the mice were placed in the dark shelter for an adaptation period of 2 minutes (at which time the opening from the dark to the light compartment was closed). Thereafter the mice were allowed to leave the shelter and explore the illuminated area for 5 min. For each mouse the length of time the animal spent in the lighted side of the box was recorded. A video camera located above the center of the box provided permanent recording of the mouse behavior. The mice were then removed from the box and returned to their home cage.

[0222] The results of the open field avoidance test are shown in Figure 15 wherein Figure 15 A shows that after 7 weeks, blind (rd / rd) mice spent about 80% of the time in the light compartment, control mice (normal mice) spent about 50% of the time in the light compartment, and rd1 / rd1 mice injected with AAV-EGFP spent about 30% of the time in the light compartment; and

[0223] Figure 15 B shows that after 9 months, blind (rd / rd) mice spent about 80% of the time in the light compartment, control mice (normal mice) spent about 50% of the time in the light compartment, and rd1 / rd1 mice injected with AAV-EGFP spent about 20% of the time in the light compartment.

[0224] Figure 16 The recovery of light sensitivity of the eye of rd1 / rd1 mice treated with AAV-cOPN5 is shown after 7 weeks (A) and 9 months (B), respectively. It was found that AAV-cOPN5 treated rd1 / rd1 mice (C3H_O5) had a similar % pupil constriction (area) as normal mice (C57), while rd1 / rd1 mice (C3H_EGFP) showed almost no % pupil constriction (area).

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Claims

1. Use of an isolated photoreceptive opsin for the manufacture of a medicament, said photoreceptive opsin restoring the sensitivity of retinal cells to light by activating Gq signaling, wherein said medicament is for treating a disease or condition mediated by, or involving, a loss of sensitivity of retinal cells to light, by activating Gq signaling, wherein said photoreceptive opsin is an isolated opsin 5 from chicken, cOpn5, or from turtle, tOpn5, wherein cOpn5 has GenBank Accession Number NM_001130743.1 and tOpn5 has GenBank Accession Number XM_007068312.4, wherein the retinal ganglion cells involved in said disease or condition are not completely dead, wherein said disease or condition is an outer retinal damage, a loss or degeneration of photoreceptors, a retinal degenerative disease.

2. Use of an isolated nucleic acid encoding the isolated opsin of claim 1 for the manufacture of a medicament, wherein said medicament is for treating a disease or condition mediated by, or involving, a loss of sensitivity of retinal cells to light, by activating Gq signaling, wherein the retinal ganglion cells involved in said disease or condition are not completely dead, said disease or condition is an outer retinal damage, a loss or degeneration of photoreceptors, a retinal degenerative disease.

3. Use of a chimeric gene consisting of the isolated nucleic acid sequence of claim 2 operably linked to suitable regulatory sequences, or of the chimeric gene consisting of the isolated nucleic acid sequence of claim 2 and a gene encoding a marker operably linked to suitable regulatory sequences, for the manufacture of a medicament, wherein said medicament is for treating a disease or condition mediated by, or involving, a loss of sensitivity of retinal cells to light, by activating Gq signaling, wherein the retinal ganglion cells involved in said disease or condition are not completely dead, said disease or condition is an outer retinal damage, a loss or degeneration of photoreceptors, a retinal degenerative disease.

4. The use according to claim 3, wherein said marker is a fluorescent protein.

5. Use of a vector comprising the isolated nucleic acid according to claim 2 or the chimeric gene according to any one of claims 3 to 4, for the manufacture of a medicament, wherein said medicament is for treating a disease or condition mediated by, or involving, a loss of sensitivity of retinal cells to light, by activating Gq signaling, wherein the retinal ganglion cells involved in said disease or condition are not completely dead, said disease or condition is an outer retinal damage, a loss or degeneration of photoreceptors, a retinal degenerative disease.

6. The use according to claim 5, wherein said vector is a eukaryotic vector, a prokaryotic expression vector or a viral vector.

7. The use according to claim 5, wherein said vector is a yeast vector.

8. The use of claim 6, wherein the vector is a herpes simplex virus vector, a vaccinia virus vector, an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, or an insect vector.

9. The use of claim 6, wherein the vector is a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVS, AAV0, or AAV10.

10. The use of claim 5, wherein the vector is an expression vector or a gene therapy vector.

11. Use of an isolated cell or cell culture comprising the isolated nucleic acid of claim 2, the chimeric gene of any one of claims 3-4, or the vector of any one of claims 5-10, in the manufacture of a medicament for treating a disease or condition mediated by, or involving, a loss of sensitivity to light by retinal cells, wherein the retinal ganglion cells involved in the disease or condition are not completely dead, the disease or condition is an outer layer of the retina damage, photoreceptor loss or degeneration, retinal degenerative disease, by activating Gq signaling.

12. The use of claim 1, 2, 3, 5, or 11, wherein the disease or condition is retinitis pigmentosa, macular degeneration.

13. The use of claim 1, 2, 3, 5, or 11, wherein the disease or condition is age-related macular degeneration.

14. The use of claim 1, 2, 3, 5, or 11, wherein the treatment comprises subretinal or intravitreal administration of an AAV vector expressing cOpn5.

15. The use of claim 14, wherein the AAV vector further expresses a fluorescent protein.

Images