Optogenetic restoration of vision by light-sensitive GQ-coupled neuroopsin (opsin 5)

Abstract

This disclosure relates to q An isolated light-sensitive opsin is provided that rapidly, reversibly and precisely restores the sensitivity of retinal cells to light by activating signal transduction.

Description

[Background technology]

[0001] G protein-coupled receptors (GPCRs) regulate many intracellular signaling pathways and represent some of the most intensively studied drug target points (Hauser et al., 2017). After ligand binding, GPCRs undergo a conformational change and transmit it to heterotrimeric G proteins, which then convert the G α G tightly bound to the subunit βγ It is a multisubunit complex containing the heterotrimeric G α The G subfamily of subunits q The 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 points include the phospholipase Cβ (PLC-β) enzyme, which catalyzes the hydrolysis of the phospholipid phosphatidylinositol diphosphate (PIP2) to inositol triphosphate (IP3) and diacylglycerol (DAG). IP3 is released from intracellular stored Ca 2+ into the cytoplasm, causing Ca 2+ G activates protein kinase C (PKC) together with DAG. q Coupled GPCRs and intracellular Ca 2+ To study the signaling mechanisms and physiological functions of release, we developed several tools, including chemical genetics and photoactivatable small molecules.

[0002] Optogenetics utilizes light-responsive proteins to achieve perturbations to optical control of cellular activity with genetic specificity and high spatiotemporal precision. Since the early discovery of optogenetic tools using light-sensitive ion channels and transporters, a variety of techniques have been developed that now support optical intervention in second messengers, protein interactions and degradation, as well as gene transcription within cells. Creatively designed G q The coupled rhodopsin-GPCR chimera Opt-a1AR increased intracellular Ca in response to prolonged light stimulation (60 s). 2+It can induce an increase in the amount of opsin produced by the retinal nucleus (Airan et al., 2009). However, such tools have not been widely used, probably due to limitations related to light sensitivity and kinetics (Tichy et al., 2019). Most animals detect light using GPCR-based photoreceptors, where the photoreceptor contains a protein moiety (opsin) and a vitamin A derivative (retinal) that functions as both ligand and chromophore. Thousands of opsins have been identified so far. Two recent studies have demonstrated the use of GPCRs from mosquitoes and lampreys in the inhibition of presynaptic terminals of neurons. i The use of G-based opsins has been reported and it is readily demonstrated that certain naturally occurring photoreceptors are suitable for use as efficient optogenetic tools. q Regarding signal transduction, melanin (Opn4) in a subpopulation of mammalian retinal ganglion cells is a G q Opn4 is a conjugated opsin. It mediates visual functions that do not form images. However, HEK293 or Neuro-2a cells heterologously expressing Opn4 show weak light responses and require additional retinal in the medium. Opn5 (glialin) and its orthologues in many vertebrates have been reported to be ultraviolet (UV)-sensitive opsins coupled to Gi proteins.

[0003] Ideal optogenetic tools are urgently needed to restore visual function in blind patients. Summary of the Invention [Problem to be solved by the invention]

[0004] The present invention is q The present invention relates to an isolated light-sensitive opsin that is used to restore sensitivity of retinal cells to light by activating signal transduction, which can be used to treat subjects suffering from damage to the outer retina, loss or degeneration of photoreceptors, retinal degenerative disease, loss of sensitivity or loss of light perception, vision loss or blindness.

[0005] In a first aspect, the present invention provides a method for the preparation of a medicament for the treatment of a cancer, comprising: q The present invention relates to an isolated light-sensitive opsin that is used to restore the sensitivity of retinal cells to light by activating signal transduction.

[0006] In some 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, and especially a wavelength of 470 nm.

[0007] In some embodiments, the isolated opsin is an isolated opsin, a homolog thereof, an ortholog thereof, a paralog thereof, a fragment or a variant thereof, derived from an organism, q It has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

[0008] In some 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 or a variant thereof in the organism, and is G q It has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

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

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

[0011] In some 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 a wild-type opsin 5 (Opn5), a homolog thereof, an ortholog thereof, a paralog thereof, a fragment or a variant thereof in the animal, and q It has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

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

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

[0014] In some embodiments, the animal is a bird, including, but not limited to, chicken, duck, goose, ostrich, emu, rhea, kiwi, cassowary, turkey, quail, chicken, falcon, hawk, peregrine falcon, pigeon, parakeet, crowned cockatoo, macaw, parrot, passerine (e.g., songbird), jay, blackbird, finch, warbler, and sparrow.

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

[0016] In some embodiments, the animal is a fish, including but not limited to catfish, eels, sharks, and swordfish.

[0017] In some embodiments, the animal is an amphibian, including but not limited to toads, frogs, newts, and salamanders.

[0018] In some embodiments, the isolated Opsin 5 (Opn5) is an isolated wild-type Opsin 5 (Opn5) from a chicken or a fragment or variant thereof, q It has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

[0019] In some 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 wild-type opsin 5 (Opn5) from the chicken, and q It has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

[0020] In some embodiments, the isolated Opsin 5 (Opn5) is an isolated wild-type Opsin 5 (Opn5) from a sea turtle or a fragment or variant thereof, q It has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

[0021] In some 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 wild-type opsin 5 (Opn5) from the sea turtle, and q It has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

[0022] In some embodiments, the isolated opsin 5 (Opn5) has the amino acid sequence set forth in SEQ ID NO:1 (cOpn5) or a fragment or variant thereof, q It has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

[0023] In some 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 is q It has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

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

[0025] In some 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 is q It has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

[0026] The isolated opsin 5 (Opn5) is a transcription factor that regulates intracellular G q It can be used as a convenient optogenetic tool to precisely activate signal transduction.

[0027] The retinal cell can be a photoreceptor cell, a retinal rod cell, a retinal cone cell, a retinal ganglion cell, a bipolar cell, a ganglion cell, a horizontal cell, a multipolar neuron, a Muller cell, an amacrine cell, or a methylnitrosourea cell.

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

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

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

[0031] The chimeric gene further comprises a gene encoding a marker (eg, a fluorescent protein).

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

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

[0034] In some 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 recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVS, AAVO or AAV10.

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

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

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

[0039] For example, expression of cOpn5 in HEK293T cells prevents the release of Ca from intracellular accumulations triggered by blue light. 2+ G q It potently mediates increased dependence.

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

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

[0042] cOpn5 can be administered to retinal cells and the retinal cells can be activated by light, the light having 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, a cOpn5-t2a-EGFP-expressing AAV vector is administered subretinally or intravitreally to express cOpn5 and EGFP in retinal ganglion cells.

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

[0045] In some embodiments, the disease or condition mediated by or related to loss of sensitivity of retinal cells to light is G q These include, but are not limited to, diseases or conditions that would benefit from restoring retinal cell sensitivity to light by activating signal transduction.

[0046] In some embodiments, the disease or condition mediated by or related to loss of sensitivity of retinal cells to light is G q These include, but are not limited to, diseases or conditions that would benefit from activation of retinal cells, such as photoreceptor cells, retinal rod cells, retinal cone cells, retinal ganglion cells, bipolar cells, ganglion cells, horizontal cells, multipolar neurons, Muller cells, amacrine cells, or methylnitrosourea, by activating signal transduction.

[0047] In some embodiments, the disease or condition includes, but is not limited to, damage to the outer retina, loss or degeneration of photoreceptors, retinal degenerative diseases, loss of sensitivity to light or loss of light perception, loss of vision due to insufficient light perception or sensitivity, or blindness.

[0048] In some embodiments, Opn5 in the present invention can be used to restore retinal cell sensitivity to light, so long as the retinal ganglion cells have not completely died.

[0049] In some embodiments, Opn5 in the present invention can be used to treat or prevent diseases associated with the degeneration and / or death of retinal ganglion cells (RGCs).

[0050] In some 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.

[0051] In some embodiments, the method further comprises applying light having a wavelength in the range of 360 nm to 550 nm, preferably 450 to 500 nm, and more preferably 460 to 480 nm.

[0052] In some embodiments, the method further comprises performing two-photon activation using long wavelength (≧920 nm) light.

[0053] The isolated opsins described in the present invention are photosensitive to wavelengths in the range of 360-550 nm, preferably 450-500 nm, and more preferably 460-480 nm. Specifically, blue light at 470 nm is the strongest Ca receptor in cells. 2+ A transient was evoked, which means that the isolated opsin described in this invention is hypersensitive to light of 470 nm wavelength.

[0054] The present invention includes all combinations of specific embodiments described herein. [Brief description of the drawings]

[0055] [Figure 1] We show that cOpn5 mediates robust activation of light-induced Gq signaling in HEK293T cells. [Diagram 2] We show that cOpn5 couples to Gq but not Gi signaling. [Diagram 3]We show that cOpn5 mediates sensitive optical control of Gq signaling with high spatiotemporal resolution. [Figure 4] 1 shows that cOpn5 mediates a more rapid and sensitive response to light than opto-a1AR, hM3Dq or opn4. [Diagram 5] We show that cOpn5 efficiently mediates astrocyte activation. [Figure 6] A healthy retina contains several cell layers. [Figure 7] It is shown that normal mice prior to MNU treatment have a rapid pupillary light response, whereas C3H / HeNCrl mice are born without a pupillary light response. [Figure 8] Shown is EGFP in the whole retina 4 weeks after AAV injection. [Figure 9] 1 shows that both MNU-treated and C3H / HeNCrl mice had restored pupillary light responses. [Figure 10] 1 shows a pupillary light response test. [Figure 11] Immunofluorescence results are shown. [Figure 12] The results of electrophysiological studies are shown. [Figure 13] The results of electrophysiological studies are shown. [Figure 14] 1 shows a schematic of the open field avoidance test. [Figure 15] The results of the open field avoidance test are shown. [Figure 16] 1 shows the restoration of light sensitivity in the eyes of AAV-cOPN5-treated rd1 / rd1 mice after 7 weeks (A) and 9 months (B), respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0056] In this study, we tested the ability of opsins from multiple species, particularly Opn5 orthologues, to mediate light-induced G qThe isolated light-sensitive opsins are found to sensitively and potently mediate signaling activation and / or cellular activation. The isolated light-sensitive opsins can be used to treat subjects suffering from outer retinal damage, photoreceptor loss or degeneration, retinal degenerative disease, loss of light sensitivity or loss of light perception, vision loss or blindness.

[0057] Preferably, said Opn5 orthologue is the chicken orthologue (abbreviated as cOpn5) or the sea turtle orthologue (abbreviated as tOpn5).

[0058] Detailed characterization of Opn5, especially cOpn5, has revealed that it has an ultra-high sensitivity (μW / mm 2 Level, existing G q Conjugated opsin-based tools are approximately three orders of magnitude more sensitive than opto-a1AR and opn4), have high temporal (response to a 10 ms light pulse, approximately three orders of magnitude faster than opto-a1AR or opn4) and spatial (subcell level) resolution, and do not require the addition of chromophores. Specifically, endogenous retinal is sufficient and no added retinal is required.

[0059] cOpn5 is G q Mediating optogenetic activation of signal transduction and / or activation of cells Specifically, we tested Opn5 orthologs from chicken, sea turtle, human and mouse (which share 80-90% protein sequence identity with each other) and found that they suppress blue light-induced G signaling in HEK293T cells. q The cells are tested to determine whether they have the ability to mediate signal transduction activation. Blue light is used as the stimulus, and the red intracellular calcium indicator Calbryte TM Monitoring relative Ca using 630AM dye 2+ The Opn5 orthologues from chicken (cOpn5) and sea turtle (tOpn5) respond to Ca 2+cOpn5 mediates an immediate and strong light-induced increase in signal transduction (approximately 3ΔF / F) whereas no light effect is observed in cells expressing human or mouse Opn5 orthologues. As exemplified with the chicken orthologue, cOpn5 colocalizes with the EGFP-CAAX membrane marker, indicating that it is efficiently transported to the plasma membrane. There is no need to add exogenous retinal to the medium, suggesting that endogenous retinal is sufficient to enable cOpn5 to function. Ca 2+ The signal is extracellular Ca 2+ This is because the Ca2+ receptor is resistant to removal from intracellular stores. 2+ The release of G q Protein inhibitors (e.g., YM-254890, highly selective G q Preincubation of the α-Ca protein inhibitor inhibits light-induced Ca 2+ In cells expressing cOpn5 but not human OPN5, a light-induced increase in the levels of inositol phosphate (IP1), a rapid degradation product of IP3, was detected, and furthermore, treatment with YM-254890 reduced the magnitude of such an increase. In cOpn5-expressing cells (e.g., HEK293T cells), blue light also triggers phosphorylation of MARCKS protein (a putative PKC target point) in a PKC activity-dependent manner. In contrast, in the presence of retinal, blue light irradiation efficiently reduces cAMP levels in cells expressing human and mouse Opn5, but in the absence of added retinal, blue light irradiation has no such effect in cells expressing cOpn5. Taken together, these data suggest that blue light irradiation reduces the levels of cOpn5 and G in HEK293T cells. q This supports the idea that it enables coupling with signal transduction pathways.

[0060] cOpn5-mediated optogenetics is sensitive and precise Specifically, the present invention characterizes the light-activation properties of cOpn5. cOpn5 can be heterologously expressed in cells (e.g., HEK293T cells). Opn5 has previously been thought to be an ultraviolet (UV)-sensitive photoreceptor, but it is not yet fully understood to be a photoreceptor that is sensitive to light at a fixed light intensity (100 μW / mm 2 ) plotting a series of wavelengths in the range of 365-630 nm, blue light at 470 nm is the most intense Ca 2+ We show that cOpn5 induces a transient, whereas UVA light (365 and 395 nm) is less efficient and longer wavelength visible light (561 nm or longer) has no effect at all. We tested the effect of various light durations on cOpn5-expressing HEK293T cells, using short light pulses (1, 5, 10, 20, 50 ms, 16 μW / mm 2 , 470 nm) stimulated Ca when the light duration exceeded 10 ms. 2+ This shows that the response reaches the saturated mode. 2 , 470 nm), even with prolonged light duration, 2+ The signal amplitude was increased by 4.8 μW / mm2 at different intensities. 2 and 16 μW / mm 2 It can be seen that 100 μW / mm blue light produces approximately half-maximal and maximal responses, respectively. These data indicate that the light sensitivity of cOpn5 is 2-3 orders of magnitude higher than reported for the common optogenetic tool, channelrhodopsin-2 (ChR2). Taken together, our results demonstrate that cOpn5 can function as a single-component optogenetic tool without the addition of retinal, that cOpn5 is ultrasensitive to blue light, and that low light intensities (16 μW / mm) are required for its full activation. 2 ) and short duration (10 ms) are required.

[0061] The performance of cOpn5 was compared with that of opto-a1AR, which binds rhodopsin to G q A chimeric GPCR engineered by mixing with an opto-α1AR-coupled adrenergic receptor. We followed previously reported protocols to measure slow and small (~0.5ΔF / F) Ca in opto-α1AR-expressing HEK293T cells.2+ To induce a signal increase, very long and intense irradiation (60 s, 7 mW / mm 2 ) is required, and 15 seconds of exposure is ineffective. The performance of cOpn5 is compared with that of opn4, and opn4 is q A natural opsin that has been reported to be a tool for signal transduction activation. Slow (~1ΔF / F) Ca in opn4-expressing HEK293T cells 2+ To induce a signal increase, strong irradiation (25 s, 40 mW / mm 2 ) and additional retinal are required. Thus, compared to existing opsin-based tools (opto-a1AR and opn4), cOpn5 is much more light sensitive (about three orders of magnitude more sensitive) and requires much shorter exposure times (10 ms versus 60 s) to generate a stronger response.

[0062] Furthermore, we compared the performance of cOpn5 with that of a general G q We compare the performance of the coupled chemical genetics tool hM3Dq, where hM3Dq is activated by the addition of the exogenous small molecule ligand clozapine-N-oxide (CNO). Light-induced activation of cOpn5-expressing HEK293T cells and CNO-induced activation of hM3Dq-expressing HEK293T cells show similar Ca 2+ The peak response amplitude of the signal. At the same time, compared with hM3Dq-expressing HEK293T cells, cOpn5-expressing HEK293T cells show faster and more temporally precise responses and faster recovery times. These results indicate that cOpn5-mediated optogenetics is more controllable in terms of temporal precision than hM3Dq.

[0063] cOpn5 optogenetics allows precise spatial control of cellular activity. Brief light stimulation (63 ms) restricted to subcellular regions of individual cOpn5-expressing HEK293T cells results in immediate activation of individual cells. Interestingly, in highly confluent regions, Ca 2+The signal propagates to surrounding cells, thereby suggesting intercellular communication between HEK293T cells by a yet unidentified mechanism. cOpn5 is expressed using an AAV vector for bicistronic expression of cOpn5 and EGFP marker proteins in primary astrocyte cultures prepared from neonatal mouse brains. Ca was detected using Calbryte 630 AM dye. 2+ We monitored the Ca levels and found that illumination of cOpn5-expressing astrocytes with blue light induced a strong Ca 2+ When light stimulation (63 ms) was precisely restricted to the subcellular region of an individual cOpn5-expressing astrocyte, a Ca transient (approximately 8ΔF / F) was observed. 2+ We observe that the signal is propagated within individual cells. Similar to the study in HEK293T cells, Ca signals are transmitted from stimulated astrocytes to more distal unstimulated astrocytes. 2+ A gentle wave-like propagation of the signal is observed. Thus, these experiments confirm that precise spatial control is possible with cOpn5 optogenetics and suggest that it may be useful to study the dynamics of the astrocytic networks we first discovered using neurochemical and mechanical stimulation.

[0064] Here, the present invention relates to q Here we demonstrate the use of Opn5 as a highly effective optogenetic tool to restore the sensitivity of retinal cells to light by activating signaling. Previous studies have shown that mammalian Opn5 mediates the UV-sensitive G i In mammalian cells that express Opn5 (e.g., expressing cOpn5 or expressing tOpn5), which has been characterized as a conjugated opsin, visible blue light induces rapid Ca 2+ We present the surprising finding that IL-1 can induce transients, IP1 accumulation and PKC activation.

[0065] Table 6 shows the enabling properties of cOpn5 by directly comparing response amplitude, light sensitivity, temporal resolution and the need for additional chromophores between cOpn5 and other optogenetic tools. In cOpn5-expressing cells, an intensity of 16 μW / mm 2 Only a 10 ms blue light pulse induced Ca 2+ In contrast, prior to the present invention, opto-a1AR or mammalian Opn4(G q The activation of the two proposed optogenetic tools for signal transduction required approximately three times the light intensity (7–40 mW / mm 2 ) and requires long exposure times (20–60 s), and is sensitive to weak Ca 2+ It is clear that opto-a1AR or mammalian Opn4 only produces a signal (0.25-0.5ΔF / F). Therefore, opto-a1AR or mammalian Opn4 does not produce endogenous G q It is not possible to mimic the rapid activation profile of endogenous G-coupled receptors, where q Coupled receptors typically exhibit strong G responses within subseconds of application of the corresponding ligand. q In contrast, recent systematic characterization has demonstrated that opto-a1AR and Opn4-mediated optogenetic stimulation induces Ca 2+ The amplitude of the signal did not increase, even after prolonged irradiation. 2+ showing that it only slightly modulates the frequency of transient and synaptic events ( Gerasimov et al., 2021 ; Mederos et al., 2019 ).

[0066] Opn5, especially cOpn5 or tOpn5-based optogenetics of the present invention also has the advantages of safety and convenience. Although Opn5 of many species has been reported to be UV-responsive (Kojima et al., 2011), cOpn5 is optimally activated by blue light at 470 nm, and the penetration of blue light is superior to that of UV, avoiding UV-related cytotoxicity. The super-sensitivity to light also minimizes potential heating artifacts. cOpn5 or tOpn5 is potently and reproducibly activated by light without the need for exogenous retinal, which is likely because cOpn5 or tOpn5 is a bistable opsin that is covalently bound to endogenous retinal, and therefore is resistant to photobleaching (Koyanagi and Terakita, 2014; Tsukamoto and Terakita, 2010). In contrast, mammalian experiments with Opn4 require additional retinal, have a long response time, and are less photosensitive. The Opn5 of the present invention, particularly cOpn5 or tOpn5, is particularly useful for in vivo studies as a single component system, since it avoids the burden of delivering compounds to tissues during experiments.

[0067] Compared to chemical genetics and uncaging tools, the Opn5 optogenetics of the present invention, particularly cOpn5 or tOpn5 optogenetics, further offers several important advantages: it is much more precise in time and provides single-cell or sub-cellular spatial resolution. The Opn5 of the present invention, particularly cOpn5 or tOpn5, is also different from caged compound-based "uncaging" tools, such as caged calcium and caged IP3, which require preloading of compounds and do not require G q Ca associated with signal transduction and / or cell activation 2+This is because they only partially mimic the relevant pathways. Other "uncaging" tools exist, such as caged glutamate and caged ATP (Ellis-Davies, 2007; Lezmy et al., 2021), which target endogenous GPCRs. However, these caged compounds require introduction into the extracellular medium or into the cytoplasm within cells, limiting their application in behaving animals (Adams and Tsien, 1993b).

[0068] The optogenetics of Opn5, particularly cOps5 or tOpn5, of the present invention can be used to detect intracellular G q It is particularly suitable for signal transduction and / or precise activation of cells, followed by the release of accumulated intracellular Ca 2+ Opn5 of the present invention, particularly cOpn5 or tOpn5, is distinct from current channel-based optogenetic tools, such as ChR2 or variants thereof, which transport cations across the cell membrane.

[0069] Based on the strong photosensitivity of the Opn5 of the present invention, the present invention further relates to the Opn5 of the present invention. q It is demonstrated that by activating signal transduction, it can be used to restore the sensitivity of retinal cells to light and thus can be used to treat or alleviate damage to the outer retina, loss or degeneration of photoreceptors, retinal degenerative disease, loss of sensitivity to light or loss of light perception, loss of vision due to insufficient light perception or sensitivity, or blindness.

[0070] In some embodiments, Opn5 of the present invention can be used to restore the sensitivity of retinal cells to light, so long as the retinal ganglion cells have not completely died.

[0071] In some 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).

[0072] In some embodiments, Opn5 of 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.

[0073] In the present invention, cOpn5, cOPN5, O5 and chicken opn5m are used interchangeably.

[0074] In the present invention, opn5, OPN5, opsin and Opn5 are used interchangeably.

[0075] The descriptions of specific embodiments and examples are provided by way of illustration and not limitation, as those skilled in the art will readily recognize that a variety of non-critical parameters can be changed or modified to produce substantially similar results.

[0076] Working Example material and method: [Table 1]

[0077] [Table 2]

[0078] [Table 3]

[0079] [Table 4]

[0080] [Table 5]

[0081] [Table 6]

[0082] Example 1.G q cOpn5 mediates optogenetic activation of signaling Heterologous expression of Opn5 orthologs from chicken, sea turtle, human and mouse (which share 80–90% protein sequence identity with each other) suppressed blue light-induced G signaling in HEK293T cells. q The ability of the cells to mediate signaling activation was tested using blue light stimulation and red intracellular calcium indicator Calbryte (Figure 1a and Table 7). TM Relative Ca using 630AM dye 2+ The Opn5 orthologues from chicken (cOpn5) and sea turtle (tOpn5) were used to monitor the Ca response (Figure 1b). 2+ cOpn5 mediates an immediate and strong light-induced increase in signal (approximately 3ΔF / F), whereas no light effect is observed in cells expressing human or mouse Opn5 orthologues (Fig. 1d and Fig. 2a,b). As exemplified by the chicken orthologue, cOpn5 colocalizes with the EGFP-CAAX membrane marker, indicating that it is efficiently transported to the plasma membrane (Fig. 1c). No exogenous retinal was added to the medium, indicating that endogenous retinal is sufficient to drive cOpn5. Ca 2+ The signal is extracellular Ca 2+ Ca from intracellular stores is resistant to removal of 2+ The release of YM-254890 (highly selective G q Protein Inhibitors 33 Preincubation of cOpn5 with 100 μg / mL of ... 2+The light-induced increase in the levels of inositol phosphate (IP1), a rapid degradation product of IP3, was detected in cOpn5 (but not human OPN5)-expressing cells, and the magnitude of the increase was reduced by treatment with YM-254890 (Fig. 1f and Fig. 2d). In cOpn5-expressing HEK293T cells, blue light also inhibited the activation of MARCKS protein (a known target point of PKC) in a PKC activity-dependent manner. 34 ) phosphorylation (Fig. 1g and Fig. 2e). In contrast, blue light irradiation efficiently reduces cAMP levels in human and mouse Opn5-expressing cells in the presence of retinal, but has no such effect in cOpn5-expressing cells without retinal (Fig. 2f). Taken together, these data suggest that blue light irradiation reduces G q Supporting the coupling of cOpn5 to signal transduction pathways.

[0083] [Table 7]

[0084] Figure 1. cOpn5 inhibits light-induced G signaling in HEK293T cells. q We show that it mediates potent activation of signal transduction. a. Schematic of putative intracellular signaling in response to light-induced cOpn5 activation. PLC: phospholipase C; PIP2: phosphatidylinositol-4,5-bisphosphate; IP3: inositol-1,4,5-triphosphate; IP1: inositol monophosphate; DAG: diacylglycerol; PKC: protein kinase C; YM-254890: selective G q Protein inhibitors.

[0085] b. Three species of Opn5-expressing HEK293T cells (Gallus gallus, Homo sapiens, and Mus musculus) were stimulated with blue light (10 s, 100 μW / mm 2 , 488 nm) before and after Ca 2+Pseudocolor image of the signal. Scale bar, 10 μm.

[0086] c. Cy3 counterstained V5-cOpn5 fusion protein (red) colocalizes with membrane tagged EGFP-CAAX (green) in HEK293T cells. DAPI counterstain (blue) supports the cell nuclei. Scale bar, 10 μm.

[0087] dc shows light-induced Ca 2+ The signal change over time is shown.

[0088] eG q The protein inhibitor YM-254890 (10 nM) inhibited cOpn5-mediated light-induced Ca 2+ Reversibly blocks the signal.

[0089] f. In cOpn5-expressing HEK293T cells, YM was stimulated with continuous light (100 μW / mm for 3 min). 2 , 470 nm) (left). ***P<0.0001, *P=0.0128, Tukey's multiple comparison test.

[0090] g. Phosphorylation of MARCKS in cOpn5-expressing HEK293T cells in the control group (no light stimulation), light stimulation group, and light + staurosporine (ST, PKC inhibitor) group. The amount of p-MARCKS in the same fraction is normalized to the amount of α-tubulin. **P=0.0096, ***P=0.0004, Tukey's multiple comparison test.

[0091] Figure 2 shows that cOpn5 is G q Signal transduction is coupled to G i This indicates that it is not coupled to signal transduction. a. Opn5-expressing HEK293T cells from a marine turtle species (Chelonia mydas) were stimulated with blue light (10 s, 100 μW / mm 2 , 488nm) before and after Ca 2+ Pseudocolor images of the signal. Scale bar, 10 μm (left); evoked Ca in responding cells.2+ Signal change over time (right).

[0092] bG q The protein inhibitor YM-254890 (10 nM) inhibited cOpn5 and sea turtle Opn5-mediated light-induced Ca 2+ Group data for reversibly blocked signal. ****P<0.0001, one-way ANOVA. Error bars indicate SEM.

[0093] c. Extracellular Ca 2+ In the absence of 2 , 470 nm) 2+ Signal change over time.

[0094] d. IP1 accumulation in human Opn5-expressing HEK293T cells with or without light stimulation with oil (right). ns, not significant difference, unpaired t test.

[0095] e. Representative results of phosphorylation of MARCKS in cOpn5-expressing HEK293T cells in the control group (no light stimulation), the light stimulation group, and the light + staurosporine group. The amount of p-MARCKS in the same fraction is normalized to the amount of α-tubulin.

[0096] f. Light has no effect on cAMP levels in cOpn5-expressing HEK293T cells without the addition of retinal in the medium (10 μM forskolin preincubation) (left). Right panel shows the effect of light stimulation on cAMP concentrations in four different species of Opn5-expressing HEK293T cells after 10 μM retinal preincubation.

[0097] Error bars in d and f indicate SEM.

[0098] Example 2. Sensitive and accurate cOpn5-mediated optogenetics To characterize the light-activation properties of cOpn5 heterologously expressed in HEK293T cells. Opn5 is a previously identified ultraviolet (UV)-sensitive photoreceptor. 27 However, at a fixed light intensity (100 μW / mm 2 ) plotting a series of wavelengths in the range 365-630 nm, blue light at 470 nm is the strongest Ca 2+ Whereas UVA light (365 and 395 nm) was less efficient and longer wavelength visible light (561 nm or longer) had no effect at all (Figure 3a). We tested the effect of different light durations on cOpn5-expressing HEK293T cells. Short light pulses (1, 5, 10, 20, 50 ms, 16 μW / mm 2 , 470 nm) stimulates Ca when the light duration exceeds 10 ms. 2+ The response reaches saturation mode (Figure 3b). 2 , 470 nm), even with prolonged light duration, 2+ The signal amplitude is not further increased (Figure 4a). Irradiation with 470 nm light at different intensities results in a signal amplitude of approximately 4.8 μW / mm 2 and 16 μW / mm 2 Blue light at 1000 nm produces approximately half and maximal responses, respectively (Figure 3c and Figure 4b). Thus, the light sensitivity of cOpn5 is consistent with that of the light-sensitive G q These values ​​are 3-4 orders of magnitude higher than reported values ​​for coupled GPCRs and even 2-3 orders of magnitude higher than those for channelrhodopsin-2 (ChR2), a commonly used optogenetic tool (Lin, 2011; Zhang et al., 2006) (Table 8). Taken together, these results demonstrate that cOpn5 can function as a single-component optogenetic tool without the addition of retinal and that low light intensities (16 μW / mm) are required for full activity. 2 ) and short duration (10 ms), indicating that cOpn5 is hypersensitive to blue light.

[0099] [Table 8-1] [Table 8-2]

[0100] The performance of cOpn5 was compared with that of opto-a1AR, where opto-a1AR is a rhodopsin-G q This is a chimeric GPCR engineered by mixing a GPCR with a GPCR-coupled adrenergic receptor. 14 We performed Ca ion channel analysis in opto-a1AR-expressing HEK293T cells according to the protocol described in the literature. 2+ A very long and intense exposure (60 s, 7 mW / mm) was required to induce a slow and small (~0.5 ΔF / F) increase in signal. 2 ) was required for the cells to be exposed to light, whereas 15 seconds of illumination was found to be ineffective (Fig. 4c, d). The performance of cOpn5 was compared to that of opn4, where opn4 is the native opsin and G q It has been reported to be a tool used to activate signal transduction. Prolonged exposure to intense radiation (25 s, 40 mW / mm 2 ) and additional retinal, Ca in opn4-expressing HEK293T cells 2+ It is found to induce a slow (~1ΔF / F) increase in signal (Figure 4e,f). Thus, compared to existing opsin-based tools (opto-a1AR and opn4), cOpn5 is much more light sensitive (about three orders of magnitude more sensitive), requires much shorter exposure times (10 ms versus 60 s), and generates a stronger response.

[0101] Furthermore, we compared the performance of cOpn5 with that of a general G q Compare the performance of the conjugated chemical genetics tool hM3Dq, the latter of which is activated by the addition of the exogenous small molecule ligand clozapine-N-oxide (CNO) 37~39 Light-induced activation of cOpn5-expressing HEK293T cells and CNO-induced activation of hM3Dq-expressing HEK293T cells were observed in similar Ca 2+with the peak value response amplitude of the signal. At the same time, compared with hM3Dq-expressing HEK293T cells, cOpn5-expressing HEK293T cells exhibit faster and more temporally precise responses and faster recovery times (Figure 4g-Figure 4i). These results indicate that cOpn5-mediated optogenetics is more controllable than hM3Dq in terms of temporal precision.

[0102] cOpn5 optogenetics allows precise spatial control of cellular activity. Restricting brief light stimulation (63 ms) to subcellular regions of individual cOpn5-expressing HEK293T cells results in immediate activation of individual cells. Interestingly, in highly confluent regions, Ca 2+ The signal propagates to surrounding cells, suggesting the presence of intercellular communication between HEK293T cells by a yet unidentified mechanism (Figure 3d, e). The findings are also extended to primary cell cultures. In primary astrocyte cultures prepared from neonatal mouse brains, cOpn5 is expressed using an AAV vector for bicistronic expression of cOpn5 and EGFP marker proteins (Figure 5a). Calbryte 630AM dye was used to measure Ca 2+ When blue light was irradiated onto cOpn5-expressing astrocytes, a strong Ca 2+ We found that the Ca transients were positive (~8ΔF / F) (Figure 5b, c). When light stimulation (63 ms) was precisely restricted to the subcellular regions of individual cOpn5-expressing astrocytes, the Ca transients were positive (~8ΔF / F) (Figure 5b, c). 2+ We observed that the signal was propagated within individual cells (Fig. 3f). Similar to the study in HEK293T cells, Ca signals were transmitted from stimulated astrocytes to more distal unstimulated astrocytes. 2+ A gentle wave-like propagation of the signal is observed (Fig. 3g, h). Thus, these experiments confirm that precise spatial control is possible with cOpn5 optogenetics and suggest that it may be useful to study the dynamics of the astrocytic networks originally discovered using neurochemical and mechanical stimulation. 40、41 .

[0103] Figure 3 shows that cOpn5 mediates G q We demonstrate that it mediates highly sensitive optical control of signal transduction. a. Ca in HEK293T cells expressing selected wavelengths (365, 395, 470, 515, 561, 590 and 630 nm, left panel) and cOpn5 in response to light stimulation at different wavelengths. 2+ Signal amplitude (2s, 100μW / mm 2 Schematic diagram of the 3D-cell hybridization reaction (right panel). Error bars indicate SEM.

[0104] b. Different light stimulation durations (1, 5, 10, 20 or 50 ms, 16 μW / mm 2 , 470 nm). Error bars indicate SEM.

[0105] c. cOpn5-mediated Ca2+ expression under different light intensities 2+ Signal change over time (0, 4.8, 8, 16 or 32 μW / mm 2 , 10ms, 470nm, 10ms 16μW / mm 2 For stimulation, 10% peak value activity = 1.36 ± 0.55 s, 90% peak value activity = 2.37 ± 0.87 s, decay time τ = 18.66 ± 4.98 s, mean ± SEM, n = 10 cells).

[0106] d. Light-induced (63 ms, 17 μW, arrows indicate the stimulation area) Ca in cOpn5-expressing HEK293T cells. 2+ Image of signal propagation. Scale bar, 10 μm.

[0107] Time course of Ca 2+ Pseudocolor image showing signal propagation (frame N / (N-1)>1). The frame interval is 500 ms and each frame is counted once.

[0108] f. Light-induced Ca in individual cOpn5-expressing primary astrocytes stimulated in the subcellular region 2+ Image of signal propagation (stimulus size is 4 × 4 μm 2(The frame interval was 300 ms.) Scale bar, 10 μm.

[0109] g. Light-induced Ca in cOpn5-expressing primary astrocytes 2+ Image of signal propagation. Scale bar, 10 μm.

[0110] Ca concentration in hg 2+ Pseudocolor image showing signal propagation (frame N / (N-1)>1). The frame interval is 500 ms and each frame is counted once.

[0111] FIG. 4 shows that cOpn5 mediates a more rapid and sensitive response to light than opto-a1AR, hM3Dq or opn4. a. Light pulse (16 μW / mm 2 , 470 nm, 1, 5, 10, 20 or 50 ms) 2+ Signal change over time.

[0112] b. Different light intensities (0, 4.8, 8, 16 or 32 μW / mm) at 470 nm for 10 ms. 2 ) response amplitude under

[0113] c. Baseline and peak Ca values ​​in opto-a1AR-expressing HEK293T cells 2+ Pseudocolor image of signal (ΔF / F0). Culture buffer contains 10 μM all-trans retinal. Scale bar, 30 μm.

[0114] d. Ca in opto-a1AR-expressing HEK293T cells 2+ Effects of 60 s of light stimulation on Ca (n=15 cells, upper panel). 15 s of light stimulation increased Ca 2+ It does not affect the signal (see below).

[0115] e. Baseline and peak Ca values ​​in human OPN4-expressing HEK293T cells 2+Pseudocolor image of signal (ΔF / F0). Culture buffer contains 10 μM all-trans retinal. Scale bar, 30 μm.

[0116] f. In opn4-expressing HEK293T cells, Ca 2+ Effect of 25 s of light stimulation under 10 uM ATR on Ca in the absence of ATR (n=12 cells, red line). 2+ No effect on the signal (black).

[0117] g. Ca in cOpn5-expressing HEK293T cells 2+ Effects of light stimulation on the signal. Top panel shows pseudocolor images of baseline and peak value responses. Bottom panel shows Ca responses induced by cOpn5-mediated optogenetic stimulation in cOpn5-expressing HEK293T cells over five consecutive trials. 2+ Signal heatmaps are shown. Scale bar, 20 μm.

[0118] h. Ca in hM3Dq-expressing HEK293T cells 2+ Effects of chemogenetic stimulation on signaling.

[0119] i. Ca induced by cOpn5-mediated optogenetic stimulation (10 s) and hM3Dq-mediated chemogenetic stimulation using CNO puffs (100 nM, 10 s), respectively. 2+ Signal change over time.

[0120] FIG. 5 shows that cOpn5 efficiently mediates astrocyte activation. AAV-cOpn5-T2A-EGFP (green) is used to express cOpn5 in cultured primary astrocytes. Astrocyte identity is confirmed by GFAP immunostaining (red). Scale bar, 20 μm.

[0121] b. Baseline and peak Ca values ​​in cOpn5-expressing astrocytes after light stimulation 2+ Pseudocolor image of the signal. Scale bar, 20 μm.

[0122] c.Ca 2+ Diagram of the signal and Ca across trials 2+ Heatmap of signals (n=25 cells).

[0123] Example 3. Photosensitive G q Optogenetic restoration of vision using conjugated neuroopsin (opsin 5) Animal models: 1. A healthy retina contains several cell layers, including retinal pigment epithelium, cone photoreceptors, rod photoreceptors, horizontal cells, bipolar cells, Muller cells, amacrine cells, and ganglion cells (Figure 6). Methylnitrosourea (MNU) also causes damage to photoreceptors (rod and cone photoreceptors) in animals, and subsequently induces retinal degeneration. We use MNU-induced 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.

[0124] 2. C3H / HeNCrl mice are a genetic model of retinal degeneration. The strain expresses Pde6b, which causes retinal degeneration. rd1 The mutation is characterized as being homozygous.

[0125] We use the pupillary light response of head-fixed mice to test whether the animals can perceive light, and we rescue these two mouse models using an AAV vector to express cOpn5 in mouse retinal ganglion cells. Restoration of the pupillary light response in mice validates our method for treating cOpn5-mediated blindness.

[0126] Experiments and Results: 1. We use a camera with IR blocking capability to automatically capture images of the pupils of head-fixed mice. Adjust the optical fiber to ensure that the light (470 nm LED light source) hits the mouse pupils directly with the same light intensity.

[0127] 2. Normal mice before MNU treatment had a rapid pupillary light response (Figure 7). C3H / HeNCrl mice were born without pupillary light response (Figure 7).

[0128] 3. Retinal degeneration mice treated with C3H / HeNCrl or MNU lose pupillary light response function.

[0129] 4. We used an AAV vector expressing cOpn5-t2a-EGFP in mouse retinal ganglion cells, and images show EGFP in the entire retina 4 weeks after AAV injection (Figure 8).

[0130] 5. After expressing cOpn5 in mouse retinal ganglion cells, we performed the pupil light response test again. Treated MNU mice regained pupil light response (Figure 9). C3H / HeNCrl mice acquired the ability to respond to pupil light (Figure 9).

[0131] 6. Figure 10 shows that in the pupil light response test, the pupil size of normal mice (solid black line) rapidly decreases in response to light (X-axis: time (sec), Y-axis: normalized pupil size). After MNU treatment, the mice lose function in the pupil light response test (solid grey line). After 4 weeks of transfection with an AAV vector expressing cOpn5 in the retinal ganglion cells (RGCs) of these MNU-treated mice, the mice partially regain their pupil light response ability (solid middle line).

[0132] These results demonstrate that our method of expressing cOpn5 in retinal ganglion cells in animals can reverse retinal degeneration.

[0133] Example 4 Experimental Description: Table 9 below is a partial list of cOpn5 orthologs from vertebrata that were tested in the present invention. The complete genes of all reported opsin 5 orthologs from vertebrata (including rotundia, cartilaginous fish, bony fish, amphibians, reptiles, birds and mammals) are synthesized and expressed in HEK293T cells. To test the sensitivity of opsin 5 orthologs to light, calcium imaging is performed with or without 470 nm blue light stimulation. The time course of light-induced calcium signal is monitored by G q We will clarify the degree of differential activation of signaling pathways and the sensitivity of these orthologs.

[0134] [Table 9-1] [Table 9-2] [Table 9-3] [Table 9-4] [Table 9-5] [Table 9-6] [Table 9-7] [Table 9-8] [Table 9-9] [Table 9-10] [Table 9-11] [Table 9-12] [Table 9-13]

[0135] Example 5 animal: House 8-16 week-old rd1 / rd1 retinitis pigmentosa (RP) model mice under a 12 / 12 light / dark cycle (lights off at 8 pm).

[0136] AAV vector construction: Plasmids required for packaging of the AAV virus include pAAV-mSNCG-chicken opn5m-t2a-EGFP, pAAV-mSNCG-chicken opn5m-t2a-mcherry, pAAV-mSNCG-chicken opn5m and pAAV-mSNCG-EGFP.

[0137] Adeno-associated virus (AAV) packaging and production: Recombinant AAVs are prepared by co-transfection of plasmids. AAV2.7M8 and AAV2 / 8 subtypes are packaged separately. Both of them include mSNCG-chicken opn5m-t2a-EGFP, mSNCG-chicken opn5m-t2a-mcherry, mSNCG-chicken opn5m and mSNCG-EGFP.

[0138] AAV injected into the mouse eye: After anesthesia, 1 μl of AAV is injected into the vitreous cavity of the mouse after passing the sclera with a fine glass electrode, and the electrode is removed after a few seconds. Follow-up experiments are performed 4 weeks after AAV injection.

[0139] Immunofluorescence: Immunofluorescence experiments need to be performed to confirm whether AAV can successfully infect retinal cells and compare the infection efficiency and virus specificity between various subtypes. Four weeks after AAV injection, the mouse retina is removed and fixed in 4% paraformaldehyde for 30 minutes. The fixed and washed retina is embedded and cut vertically to a thickness of 15 μm using a Leica glass knife maker. The sections are washed with PBS and then sealed with 3% BSA (bovine serum albumin) at room temperature for 1 hour. Then the anti-EGFP primary antibody is diluted 1:500 in 3% BSA and incubated at 4 °C for 48 hours. After washing the primary antibody, it is incubated with a fluorescently labeled secondary antibody for 2 hours, and the stained retinal section is transferred to a slide glass, and after sealing, confocal scanning is performed to obtain a fluorescent image. The infection efficiency of each AAV on retinal ganglion cells (RGC) and the fluorescence intensity of EGFP are analyzed and compared, and the AAV subtype with high infection rate and good specificity is selected for the next stage of experiments.

[0140] Electrophysiological testing: After successful expression of AAV, electrophysiological experiments need to be performed to further confirm whether cOPN5 maintains its physiological activity in RGC cells. AAV with high infection rate and good specificity is injected intraocularly into rd1 / rd1 (purchased from GemPharmatech Co., Ltd) mice. Four weeks after virus injection, the mouse retina is removed and the retinal slice is placed in the electrophysiological recording chamber. The RGC layer of the retina faces upward. To prevent photodamage to the retina, the laser is turned off after identifying GFP-expressing somatic cells with a fluorescent microscope. The current intensity is recorded after stimulating the cells with a 488 nm laser of different light intensities.

[0141] Behavioral testing: Visual receptor cells in Rd1 / rd1 mice have already degenerated, and several visual function tests are selected to verify whether visual information can be transmitted to the brain via infected ganglion cells to restore lost visual function.

[0142] (1) Pupillary light reflex (PLR) In Rd1 / rd1 mice, the pupils only react to strong light. Four weeks after AAV is injected into the mouse eye, PLR experiments are performed. Different intensities of light are used to stimulate the pupils of cOPN5-expressing mice and EGFP-expressing mice, and the degree of pupil change is recorded to evaluate the sensitivity of the mice to light through the degree of pupil change.

[0143] (2) Open field avoidance test Normal mice avoid open, lighted spaces. This natural tendency forms the basis of a simple test for their visual ability. In an experiment, mice are placed in a lighted space, where a dark shelter is also present. Their visual ability is assessed by measuring the percentage of time the mice spend there.

[0144] Safety Testing: Long-term heterologous expression of genes has various effects on expressed tissue.Long-term experiments are required to evaluate the safety of heterologous expression and test whether heterologous expression genes are stably expressed in tissue for a long time.AAV is injected into the eye for 6 months, and the above immunofluorescence, electrophysiological test and behavioral test are repeated after 1 year to detect whether the expression level of cOPN5 and the physiological activity change due to long-term expression, and detect the presence of inflammatory reaction in retinal tissue.

[0145] result: As shown in FIG. 11, A shows the expression of cOPN5 protein in retinal ganglion cells of rd1 / rd1 mice; B shows staining of the microglial marker Iba1 in retinal sections after injection. Mice injected with H2O2 (positive control) show strong microglial activity. In AAV-cOPN5-t2a-EGFP-injected retinas, basal Iba1 signal was barely observed 1 month after injection, which is similar to that observed in AAV-EGFP-injected retinas, AAV-cOPN5-t2a-EGFP-injected retinas for 10 months, and uninjected retinas. Red indicates Iba1, green indicates cOPN5 or EGFP, and blue indicates DAPI (4',6-diimidyl-2-phenylindole) signal of cell nuclei. Scale bar, 50 μm. C shows staining of retinal sections for the RGC marker brn3a. Red indicates brn3a, green indicates cOPN5, and blue indicates nuclear signal. Scale bar, 50 μm. D shows fundus fluorescence imaging.

[0146] As shown in FIG. 12, A shows the representative responses of RGCs from C3H mice injected with AAV-Copn5-t2a-EGFP during 488 nm laser stimulation at different powers; B shows representative responses of RGCs from C3H mice injected with AAV-Copn5-t2a-EGFP during 561 nm laser stimulation at different powers; C shows raw tracing of cOpn5 mediating reliable and reproducible photoactivation of RGCs. Group data in D and E show the excitation rate of RGCs after 488 nm laser stimulation at different powers (n = 6). Group data in F show the delay time following 488 nm laser stimulation at different powers (n=6).

[0147] As shown in FIG. 13, A shows a representative response of a v1 neuron from a C57 mouse during 2 seconds of 200 lux light stimulation; B shows a representative response of a v1 neuron from a C3H mouse injected with AAV-EGFP during 2 s of 200 lux light stimulation. C shows a representative response of a v1 neuron from a C3H mouse injected with AAV-cOPN5-t2a-EGFP during 2 s of 200 lux light stimulation. The heatmap shown in D shows the ROC plot of peristimulus time histogram data from v1 neurons of C57 mice, which were tested using a 2 second 200 lux light stimulus (n=107). The heatmap shown in E shows the ROC plot of peri-stimulus time histogram data from v1 neurons of AAV-EGFP-injected C3H mice, which were tested using a 2-second 200 lux light stimulus (n=133). The heatmap shown in F shows the ROC plot of peri-stimulus time histogram data from v1 neurons of C3H mice injected with AAV-cOPN5-t2a-EGFP, which were tested using a 2 second 200 lux light stimulus (n=100); G shows visual evoked potentials (VEPs) from C57 (top), rd / rd mice injected with AAV-EGFP (middle), and rd1 / rd1 mice injected with AAV-cOPN5-EGFP under 2 seconds of light illumination.

[0148] FIG. 14 shows a schematic of the open field avoidance test. Methods: The light-dark box (45 x 27 x 25 cm) was made of organic glass and consisted of two chambers connected by a ground-level opening (4 x 5 cm) in the center of the partition. The light box occupied 2 / 3 of the total light-dark box, and the dark box occupied 1 / 3 of the total light-dark box. The test arena was diffusely illuminated at 200 lux. Mice were brought into the test room in their home cages. After a 2-minute acclimatization period for the mice to the dark shelter (in this case, the opening from the dark space to the light space was closed), the test began. The mice were then removed from the shelter and allowed to explore the illuminated area for 5 minutes. For each mouse, the length of time the animal spent on the illuminated side of the box was recorded. A video camera placed above the center of the box permanently recorded the mouse's behavior. The mice were then removed from the box and returned to their home cages.

[0149] The results of the open field avoidance test are shown in FIG. 15, where FIG. 15A shows that after 7 weeks, blind (rd / rd) mice spent about 80% of their time in the light box, control mice (normal mice) spent about 50% of their time in the light box, and rd1 / rd1 mice injected with AAV-EGFP spent about 30% of their time in the light box. Figure 15B shows that after 9 months, blind (rd / rd) mice spent approximately 80% of their time in the light box, control mice (normal mice) spent approximately 50% of their time in the light box, and rd1 / rd1 mice injected with AAV-EGFP spent approximately 20% of their time in the light box.

[0150] Figure 16 shows the recovery of light sensitivity in the eyes of AAV-cOPN5-treated rd1 / rd1 mice after 7 weeks (A) and 9 months (B), respectively. AAV-cOPN5-treated rd1 / rd1 mice (C3H_O5) are found to have a % pupil constriction (area) similar to that of normal mice (C57), whereas rd1 / rdl mice (C3H_EGFP) show little % pupil constriction (area).

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Claims

1. 1. An isolated light-sensitive opsin, comprising: G ｑ An isolated opsin that restores the sensitivity of retinal cells to light by activating signal transduction.

2. The isolated opsin is an isolated opsin, a homolog thereof, an ortholog thereof, a paralog thereof, a fragment thereof, or a variant thereof, derived from an organism, ｑ 2. The isolated opsin described in claim 1, characterized in that it has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

3. 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 said organism, ｑ 2. The isolated opsin described in claim 1, characterized in that it has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

4. The isolated opsin is an isolated opsin 5 (Opn5) from an animal, a homolog thereof, an ortholog thereof, a paralog thereof, a fragment thereof, or a mutant thereof, ｑ 2. The isolated opsin described in claim 1, characterized in that it has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

5. 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 5 (Opn5), a homolog thereof, an ortholog thereof, a paralog thereof, a fragment thereof, or a variant thereof in the animal, ｑThe isolated opsin described in claim 4, characterized in that it has an activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

6. The isolated opsin of claim 2 , wherein the organism is a vertebrate.

7. The vertebrate is a bird, a reptile, a fish, an amphibian or a mammal, Preferably, the animal is a bird, including, but not limited to, chicken, duck, goose, ostrich, emu, rhea, snipe, cassowary, turkey, quail, chicken, falcon, hawk, peregrine falcon, pigeon, parakeet, crowned cockatoo, macaw, parrot, passerine (e.g., songbird), jay, blackbird, finch, warbler, and sparrow; or Preferably, the animal is a reptile, including, but not limited to, lizards, snakes, alligators, sea turtles, crocodiles and tortoises; or Preferably, the animal is a fish, including, but not limited to, catfish, eels, sharks and swordfish; or 7. The isolated opsin of claim 6, wherein the animal is preferably an amphibian, including, but not limited to, toads, frogs, newts and salamanders.

8. The isolated Opsin 5 (Opn5) is an isolated wild-type Opsin 5 (Opn5) derived from chicken or a fragment or mutant thereof, ｑ has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction, or 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 wild-type opsin 5 (Opn5) derived from chicken, and ｑThe isolated opsin described in claim 4, characterized in that it has an activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

9. The isolated Opsin 5 (Opn5) is an isolated wild-type Opsin 5 (Opn5) from a sea turtle or a fragment or mutant thereof, ｑ has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction, or 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 wild-type opsin 5 (Opn5) from a sea turtle, ｑ The isolated opsin described in claim 4, characterized in that it has an activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

10. The isolated opsin 5 (Opn5) has the amino acid sequence shown in SEQ ID NO: 1 (cOpn5) or a fragment or variant thereof, ｑ has the activity of restoring the sensitivity of retinal cells to light by activating signal transduction, or 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 shown in SEQ ID NO:1 (cOpn5), and ｑ The isolated opsin described in claim 4, characterized in that it has an activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

11. The isolated opsin 5 (Opn5) has the amino acid sequence shown in SEQ ID NO:2 (tOpn5) or a fragment or variant thereof, ｑhas the activity of restoring the sensitivity of retinal cells to light by activating signal transduction, or 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 shown in SEQ ID NO:2 (tOpn5), and ｑ The isolated opsin described in claim 4, characterized in that it has an activity of restoring the sensitivity of retinal cells to light by activating signal transduction.

12. 2. The isolated opsin of claim 1, wherein the light has a wavelength of 360 nm to 520 nm, preferably 450 to 500 nm, more preferably 460 to 480 nm, in particular 470 nm.

13. The isolated opsin of claim 1, wherein the retinal cell is a photoreceptor cell, a retinal rod cell, a retinal cone cell, a retinal ganglion cell, a bipolar cell, a ganglion cell, a horizontal cell, a multipolar neuron, a Muller cell, an amacrine cell, or a methylnitrosourea cell.

14. 1. An isolated nucleic acid comprising: An isolated nucleic acid, characterized in that it encodes an isolated opsin according to any one of claims 1 to 13.

15. A chimeric gene, The isolated nucleic acid sequence according to claim 14, wherein the nucleic acid sequence is operably linked to a suitable regulatory sequence, Preferably, the chimeric gene further comprises a gene encoding a marker, for example a fluorescent protein.

16. A vector comprising: A vector, characterized in that it comprises the isolated nucleic acid according to claim 14 or the chimeric gene according to claim 15.

17. The vector according to claim 16, characterized in that the vector is a eukaryotic vector, a prokaryotic expression vector, a viral vector or a yeast vector.

18. 18. The vector according to claim 17, characterized in that 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, preferably the vector is a recombinant AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVS, AAVO or AAV10.

19. The vector according to claim 16, characterized in that the vector is an expression vector or a gene therapy vector.

20. 1. An isolated cell or cell culture comprising: An isolated cell or cell culture, characterized in that it comprises an isolated nucleic acid according to claim 14, a chimeric gene according to claim 15 or a vector according to any one of claims 16 to 19.

21. G ｑ Use of the isolated opsin of any one of claims 1 to 13, the isolated nucleic acid of claim 14, the chimeric gene of claim 15, the vector of any one of claims 16 to 19 or the isolated cell or cell culture of claim 20 in the treatment or prevention of a disease or condition mediated by or related to loss of retinal cell sensitivity to light, by activating signal transduction.

22. G in subjects ｑ A method for treating or preventing a disease or condition mediated by or related to loss of retinal cell sensitivity to light by activating signal transduction, comprising: A method comprising administering an isolated opsin according to any one of claims 1 to 13, an isolated nucleic acid according to claim 14, a chimeric gene according to claim 15, a vector according to any one of claims 16 to 19, or an isolated cell or cell culture according to claim 20.

23. The disease or condition mediated by or related to the loss of sensitivity of retinal cells to light is G ｑ The use of claim 21 or the method of claim 22, including, but not limited to, diseases or conditions that would benefit from restoring the sensitivity of retinal cells to light by activating signal transduction.

24. The use of claim 21 or the method of claim 22, wherein the disease or condition mediated by or related to loss of sensitivity of retinal cells to light includes a disease or condition that benefits from activation of retinal cells, wherein the retinal cells are, for example, photoreceptor cells, retinal rod cells, retinal cone cells, retinal ganglion cells, bipolar cells, ganglion cells, horizontal cells, multipolar neurons, Muller cells, amacrine cells or methylnitrosourea.

25. 23. The use of claim 21 or the method of claim 22, wherein the disease or condition comprises damage to the outer retina, loss or degeneration of photoreceptors, retinal degenerative disease, loss of sensitivity to light or loss of light perception, loss of vision due to insufficient light perception or sensitivity, and / or blindness.

26. The disease or condition includes, but is not limited to, diseases associated with degeneration and / or death of retinal ganglion cells (RGCs); Preferably, the disease or condition comprises retinitis pigmentosa (RP), macular degeneration, age-related macular degeneration (AMD), autosomal dominant optic atrophy (ADOA) and / or glaucoma.

27. 23. The method of claim 22, wherein the method comprises subretinal or intravitreal administration of a cOpn5-expressing AAV vector, preferably wherein the AAV vector further expresses a fluorescent protein.

28. 23. The method according to claim 22, characterized in that the method further comprises applying blue light in the wavelength range of 360 nm to 550 nm, preferably 450 to 500 nm, more preferably 460 to 480 nm, especially 470 nm.

29. 23. The method of claim 22, further comprising performing two-photon activation using light having a wavelength of > 920 nm.

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