OPTOGENETIC VISUAL RESTORATION USING LIGHT-SENSITIVE GQ-COUPLED NEUROPSIN (OPSIN 5)

REFERENCE TO SEQUENCE LISTING

A Sequence Listing conforming to the rules of WIPO Standard ST.26 is hereby incorporated by reference. Said Sequence Listing has been filed as an electronic document via PatentCenter encoded as XML in UTF-8 text. The electronic document, created on Apr. 11, 2024, is entitled “WO11698BSUS.xml”, and has a file size of 12,595 bytes.

INTRODUCTION

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, the GPCR undergoes a conformation change that is transmitted to heterotrimeric G proteins, which are multi-subunit complexes comprising G_α and tightly associated G_βγ subunits. The G_qproteins, a subfamily of heterotrimeric G_α subunits, couple to a class of GPCRs to mediate cellular responses to neurotransmitters, sensory stimuli, and hormones throughout the body. Their primary downstream signaling targets include phospholipase C beta (PLC-β) enzymes, which catalyze the hydrolysis of phospholipid phosphatidylinositol bisphosphate (PIP₂) into inositol trisphosphate (IP₃) and diacylglycerol (DAG). IP₃triggers the release of Ca²⁺ from intracellular stores into the cytoplasm, and Ca²⁺ together with DAG activate protein kinase C (PKC). Several tools, including chemogenetics and photoactivatable small molecules, have been developed to study the signaling mechanisms and physiological functions of G_q-coupled GPCRs and intracellular Ca2+ release.

Optogenetics uses light-responsive proteins to achieve optically-controlled perturbation of cellular activities with genetic specificity and high spatiotemporal precision. Since the early discoveries of optogenetic tools using light-sensitive ion channels and transporters, diverse technologies have been developed and now support optical interventions into intracellular second messengers, protein interactions and degradation, and gene transcription. Opto-a1AR, a creatively designed G_q-coupled rhodopsin-GPCR chimera, can induce intracellular Ca²⁺ increase in response to long-time photostimulation (60 s) (Airan et al., 2009). However, this tool has not been widely used, possibly because of its limitations associated with light sensitivity and response kinetics (Tichy et al., 2019). Most animals detect light using GPCR-based photoreceptors, which comprise both a protein moiety (opsin) and a vitamin A derivative (retinal) that functions as both a ligand and a chromophore. Several thousand opsins have been identified to date. Two recent studies, having reported G_i-based opsins from mosquito and lamprey for presynaptic terminals inhibition in neurons, elegantly demonstrated that some naturally occurring photoreceptors are suitable for use as efficient optogenetic tools. Regarding the G_qsignaling, melanopsin (Opn4) in a subset of mammalian retinal ganglion cells is a G_q-coupled opsin that mediates no-image-forming visual functions. However, HEK293 or Neuro-2a cells heterologously expressing Opn4 showed weak light responses and required additional retinal in the culture medium. Opn5 (neuropsin) and its orthologs in many vertebrates have been reported as an ultraviolet (UV)-sensitive opsin that couples to G_iproteins.

Ideal optogenetic tools are urgently needed so as to recover visual function for blind patients.

SUMMARY OF THE INVENTION

The present invention relates to an isolated light-sensitive opsin for restoring sensitivity to light of the retinal cell through activating G_qsignaling. The isolated light-sensitive opsin may be used to treat a subject suffering from damage of the external layer of the retina, photoreceptor loss or degeneration, retinal degenerative disease, loss sensitivity to light, or loss light perception, loss of vision, or blindness.

In the first place, the present invention relates to an isolated light-sensitive opsin for restoring sensitivity to light of the retinal cell through activating G_qsignaling.

In some embodiments, the light has a wavelength ranging range of 360 nm-520 nm, preferably, 450-500, more preferably, 460-480 nm, in particular, 470 nm.

In some embodiments, the isolated opsin is an isolated opsin from an organism, its homologs, its orthologs, its paralogs, fragments or variants thereof having the activity of restoring sensitivity to light of the retinal cell through activating G_qsignaling.

In some embodiments, the isolated opsin shares 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 wild type opsin in the organism, its homologs, its orthologs, its paralogs, fragments or variants thereof, and has the activity of restoring sensitivity to light of the retinal cell through activating G_qsignaling.

In some embodiments, the organism is an animal.

In some embodiments, the isolated opsin is an isolated opsin 5 (Opn5) from an animal, its homologs, its orthologs, its paralogs, fragments or variants thereof having the activity of restoring sensitivity to light of the retinal cell through activating G_qsignaling.

In some embodiments, the isolated opsin 5 (Opn5) shares 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 wild type opsin 5 (Opn5) in the animal, its homologs, its orthologs, its paralogs, fragments or variants thereof, and has the activity of restoring sensitivity to light of the retinal cell through activating G_qsignaling.

In some embodiments, the animal is a vertebrate animal.

In some embodiments, the animal is an avian, a reptile, or a fish, an amphibian, or a mammal.

In some embodiments, the animal is an avian, including but not limited to chicken, duck, goose, ostrich, emu, rhea, kiwi, cassowary, turkey, quail, chicken, falcon, eagle, hawk, pigeon, parakeet, cockatoo, macaw, parrot, perching bird (such as, song bird), jay, blackbird, finch, warbler and sparrow.

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

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

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

In some embodiments, the isolated opsin 5 (Opn5) is an isolated wild type opsin 5 (Opn5) from the chicken, or fragments or variants thereof having the activity of restoring sensitivity to light of the retinal cell through activating Gq signaling.

In some embodiments, the isolated opsin 5 (Opn5) shares 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 wild type opsin 5 (Opn5) from the chicken, and has the activity of restoring sensitivity to light of the retinal cell through activating Gq signaling.

In some embodiments, the isolated opsin 5 (Opn5) is an isolated wild type opsin 5 (Opn5) from the turtle, or fragments or variants thereof having the activity of restoring sensitivity to light of the retinal cell through activating G_qsignaling.

In some embodiments, the isolated opsin 5 (Opn5) shares 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 wild type opsin 5 (Opn5) from the turtle, and has the activity of restoring sensitivity to light of the retinal cell through activating G_qsignaling.

In some embodiments, the isolated opsin 5 (Opn5) has the amino acid sequence shown by SEQ ID NO:1 (cOpn5), or fragments or variants thereof having the activity of restoring sensitivity to light of the retinal cell through activating G_qsignaling.

In some embodiments, the isolated opsin 5 (Opn5) shares 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 by SEQ ID NO:1 (cOpn5), and has the activity of restoring sensitivity to light of the retinal cell through activating G_qsignaling.

In some embodiments, the isolated opsin 5 (Opn5) has the amino acid sequence shown by SEQ ID NO:2 (tOpn5), or fragments or variants thereof having the activity of restoring sensitivity to light of the retinal cell through activating Gq signaling.

In some embodiments, the isolated opsin 5 (Opn5) shares 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 by SEQ ID NO:2 (tOpn5), and has the activity of restoring sensitivity to light of the retinal cell through activating Gq signaling.

The isolated opsin 5 (Opn5) may be used as a convenient optogenetic tool that precisely activates intracellular G_qsignaling in a retinal cell.

The retinal cell may 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 Müller cell, an Amacrine cell, or a Methylnitrosourea.

In the second place, the present invention relates to an isolated nucleic acid encoding the isolated opsin in the first place.

In some embodiments, the isolated nucleic acid encodes the wild type opsin in the organism, its homologs, its orthologs, its paralogs, fragments or variants thereof having the activity of restoring sensitivity to light of the retinal cell through activating G_qsignaling.

In the third place, the present invention relates to a chimeric gene comprising the sequence of the isolated nucleic acid in the second place operably linked to suitable regulatory sequences.

The chimeric gene further comprises a gene encoding a marker, for example, a fluorescent protein.

In the fourth place, the present invention relates to a vector comprising the isolated nucleic acid in the second place, or the chimeric gene in the third place.

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

In some embodiments, the vector is a herpes virus simplex vector, a vaccinia virus vector, or an adenoviral vector, an adeno-associated viral vector, a retroviral vector, or an insect vector.

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

In some embodiments, the vector is an expression vector.

In some embodiments, the vector is a gene therapy vector.

In the fifth place, the present invention relates to an isolated cell or a cell culture, comprising the isolated nucleic acid in the second place, the chimeric gene in the third place, or the vector in the fourth place.

For example, expressing cOpn5 in HEK 293T cells powerfully mediates blue light-triggered, G_q-dependent Ca²⁺ increase from intracellular stores.

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

In the sixth place, the present invention relates to use of the isolated opsin in the first place, the isolated nucleic acid in the second place, the chimeric gene in the third place, the vector in the fourth place, or the isolated cell or the cell culture in the fifth place for treating or preventing a disease or a condition mediated by, or involving loss sensitivity to light of the retinal cell.

cOpn5 can be applied to retinal cells and the retinal cells may be activated by light. The light has a wavelength ranging range of 360 nm-520 nm, preferably, 450-500, more preferably, 460-480 nm, in particular, 470 nm.

For example, AAV vector expressing cOpn5-t2a-EGFP is administrated subretinal or intravitreal, and cOpn5 and EGFP are expressed in retinal ganglion cells.

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

In some embodiments, the disease or condition mediated by or involving loss sensitivity to light of the retinal cell through activating Gq signaling includes but not limited to diseases or conditions benefiting from activating retinal cells, such as 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 Müller cell, an Amacrine cell, or a Methylnitrosourea.

In some embodiments, the disease or condition includes but not limited to damage of the external layer of the retina, photoreceptor loss or degeneration, retinal degenerative disease, loss sensitivity to light, or loss light perception, loss of vision due to a deficit in light perception or sensitivity, or blindness.

In some embodiments, the Opn5 in the present invention may be used to restore sensitivity to light of the retinal cell as long as the retinal ganglion cells are not completely dead.

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

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

In some embodiments, the method further comprises applying light having a wavelength range of 360 nm-520 nm, preferably, 450-500 nm, more preferably, 460-480 nm.

In some embodiments, the method further comprises applying two-photon activation using long-wavelength (≥920 nm) light.

The isolated opsin in the present invention is sensitive to the light having a wavelength ranging 360-550 nm, preferably, 450-500, more preferably, 460-480 nm. In particular, 470 nm blue light elicits the strongest Ca²⁺ transients in cells, which means that the isolated opsin in the present invention is ultra-sensitive to the light having a wavelength of 470 nm.

The invention encompasses all combination of the particular embodiments recited herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows that cOpn5 mediates light-induced strong activation of G_qsignaling in HEK 293T cells.

FIG. 2 shows that cOpn5 couples to G_qbut not G_isignaling.

FIG. 3 shows that cOpn5 sensitively mediates optical control of G_qsignaling with high temporal and spatial resolution.

FIG. 4 shows that cOpn5 mediates more rapid and sensitive response to light than opto-a1AR, hM3Dq or opn4.

FIG. 5 shows that cOpn5 effectively mediates the activation of astrocytes.

FIG. 6 shows that health retina contains several cell layers.

FIG. 7 shows that normal mice before MNU-treated have rapid pupillary light response, and C3H/HeNCrl inbred mice do not have pupillary light response.

FIG. 8 shows EGFP in the whole retina after 4 weeks after AAV injection.

FIG. 9 shows that both MNU-treated mice and C3H/HeNCrl mice recover the pupillary light response.

FIG. 10 shows pupillary light response test.

FIG. 11 shows result of immunofluorescence.

FIG. 12 shows result of electrophysiological test.

FIG. 13 shows result of electrophysiological test.

FIG. 14 schematically shows open field avoidance test.

FIG. 15 shows the results of the open field avoidance test.

FIG. 16 shows the restoration of light sensitivity in the eye of the AAV-cOPN5 treated rd1/rd1 mice after 7 weeks (A) and 9 months (B) respectively.

DESCRIPTION OF PARTICULAR EMBODIMENTS OF THE INVENTION

In the present invention, the capacity of opsin, in particular, Opn5 orthologs from multiple species is tested and it is found that many opsins sensitively and strongly mediated light-induced activation of Gq signaling and/or activating cells. The isolated light-sensitive opsin may be used to treat a subject suffering from damage of the external layer of the retina, photoreceptor loss or degeneration, retinal degenerative disease, loss sensitivity to light, or loss light perception, loss of vision, or blindness.

Preferably, the Opn5 orthologs is chicken ortholog (cOpn5 for simplicity), or turtle ortholog (tOpn5 for simplicity).

Detailed characterizations of Opn5, in particular, cOpn5 reveal that it is super sensitivity to blue light having a wavelength of 450-500 nm, more preferably, 460-480 nm (μW/mm²-level, ˜3 orders of magnitude more sensitive than existing G_q-coupled opsin-based tools: opto-a1AR and opn4), high temporal (in response to 10 ms light pulses, ˜3 orders of magnitude more rapidly than opto-a1AR or opn4) and spatial (subcellular level) resolution, and no need of chromophore addition. In particular, endogenous retinal is sufficient and no retinal is needed to be added.

cOpn5 Mediates Optogenetic Activation of G_qSignaling and/or Activating Cells.

Specifically, in the present invention, Opn5 orthologs from chicken, turtles, humans and mice (which share 80-90% protein sequence identity from each other) are tested in order to determine whether they have the capacity to mediate blue light-induced Gq signaling activation within HEK 293T cells. Blue light for stimulation and the red intracellular calcium indicator Calbryte™ 630 AM dye are used to monitor the relative Ca²⁺ response. It is found that the Opn5 orthologs from chicken (cOpn5) and turtle (tOpn5) mediated an immediate and strong light-induced increase in Ca²⁺ signal (˜3 ΔF/F), whereas no light effect is observed from cells expressing the human or mouse Opn5 orthologs. As exemplified by the chicken ortholog, the cOpn5 co-localized with the EGFP-CAAX membrane marker, indicating that it is efficiently transported to the plasma membrane. No exogenous retinal is needed to be added to the culture media, which suggests that endogenous retinal is sufficient to render cOpn5 functional. The Ca²⁺ signals are resistant to the removal of extracellular Ca²⁺, thus indicating Ca²⁺ release from the intracellular stores. Preincubation of G_qproteins inhibitor, for example, YM-254890, a highly selective G_qproteins inhibitor, reversibly abolished the light-induced Ca²⁺ transients in both cOpn5-expressing cells. In cOpn5-, but not human OPN5-expressing cells, a light-induced increase in the level of inositol phosphate (IP1), the rapid degradation product of IP3, is detected; moreover, the extent of this increase is reduced with the treatment of YM-254890. In cOpn5-expressing cells, for example, HEK 293T cells, blue light also triggers the phosphorylation of MARCKS protein, a well-established target of PKC, in a PKC activity-dependent manner. By contrast, blue light illumination effectively reduces cAMP levels in cells expressing human and mouse Opn5 with retinal, but has no such effect in cells expressing cOpn5 without retinal. Collectively, these data support that blue light illumination enables the coupling of cOpn5 to the G_qsignaling pathway in HEK 293T cells.

cOpn5-Mediated Optogenetics is Sensitive and Precise.

Specifically, the light-activating properties of cOpn5 are characterized in the present invention. cOpn5 may be heterologously expressed in cells, for example, in HEK 293T cells. Although Opn5 is previously considered as an ultraviolet (UV)-sensitive photoreceptor, mapping with a set of wavelengths ranging 365-630 nm at a fixed light intensity of (100 μW/mm²) reveals that the 470 nm blue light elicits the strongest Ca²⁺ transients, with the UVA light (365 and 395 nm) being less effective and longer-wavelength visible light (561 nm or above) completely ineffective. The effects of different light durations on cOpn5-expressing HEK 293T cells are tested, and stimulating with brief light pulses (1, 5, 10, 20, 50 ms; 16 μW/mm2; 470 nm) shows that the Ca²⁺ response achieves the saturation mode with light duration over 10 ms. Longer light durations do not further increase the Ca²⁺ signal amplitude at this light intensity (16 μW/mm²; 470 nm). Delivering 470 nm light at different intensities shows that blue light of ˜4.8 μW/mm²and 16 μW/mm²produce about half maximum and full maximum responses, respectively. These data suggest that the light sensitivity of cOpn5 is 2-3 orders of magnitude higher than the reported values of the commonly used optogenetic tool Channelrhodopsin-2 (ChR2). Together, the results in the present invention indicate that cOpn5 could function as a single-component optogenetic tool without additional retinal, and that cOpn5 is super-sensitive to blue light for its full activation requiring low light intensity (16 μW/mm²) and short duration (10 ms).

The performance of cOpn5 to that of opto-a1AR, a chimera GPCR engineered by mixing rhodopsin with G_q-coupled adrenergic receptor is compared. Following the protocol in a previous report, it is found that very long exposure of strong illumination (60 s; 7 mW/mm²) is required to trigger a slow and small (˜0.5 ΔF/F) Ca²⁺ signal increase in opto-a1AR-expressing HEK 293T cells, and 15 s illumination is inefficient. The performance of cOpn5 to that of opn4, a natural opsin which is reported as a tool for Gq signaling activating is compared. It is found that long exposure of strong illumination (25 s; 40 mW/mm²) and additional retinal are required to trigger a slow (˜1 ΔF/F) Ca²⁺ signal increase in opn4-expressing HEK 293T cells. Therefore, compared with existing opsin-based tools (opto-a1AR and opn4), cOpn5 is much more light-sensitive (˜3 orders more sensitive), requires much shorter time exposure (10 ms vs. 60 s), and produces stronger responses.

Furthermore, the performance of cOpn5 to that of the popular G_q-coupled chemogenetic tool hM3Dq, which is activated by adding the exogenous small molecule ligand clozapine-N-oxide (CNO) is compared. Light-induced activation of cOpn5-expressing HEK 293T cells has a similar peak response amplitude of the Ca²⁺ signal as CNO-induced activation of hM3Dq-expressing HEK 293T cells. Meanwhile, cOpn5-expressing HEK 293T cells has faster and temporally more precise response, as well as more rapid recovery time than hM3Dq-expressing HEK 293T cells. These results indicate that cOpn5-mediated optogenetics are more controllable in temporal accuracy than those of hM3Dq.

cOpn5 optogenetics allows spatially precise control of cellular activity. Restricting brief light stimulation (63 ms) into a subcellular region of individual cOpn5-expressing HEK 293T cell results in the immediate activation of a single cell. Interestingly, in high cell confluence area, Ca²⁺ signals propagate to surrounding cells, thus suggesting intercellular communication among HEK 293T cells through a yet-to-identified mechanism. cOpn5 is expressed in primary astrocyte cultures prepared from the neonatal mouse brain with AAV vectors for bicistronic expression of cOpn5 and the EGFP marker protein. Using the Calbryte 630 AM dye to monitor Ca²⁺ levels, it is found that blue light illumination of cOpn5-expressing astrocytes produces strong Ca²⁺ transients (˜8 ΔF/F). When the light stimulation (63 ms) is precisely restricted to only subcellular region of an individual cOpn5-expressing astrocyte, it is observed Ca²⁺ signal propagation within the individual cell. Resembling the tests in HEK 293T cells, wave-like propagation of Ca²⁺ signals from the stimulated astrocyte that proceeded gradually to more distal, non-stimulated, astrocytes, is observed. These experiments thus demonstrate that cOpn5 optogenetics allows precise spatial control, and suggest that it may be useful to study the dynamics of astrocytic networks, which was initially discovered using neurochemical and mechanical stimulation.

Here, the present invention demonstrates the use of Opn5 of the present invention as an extremely effective optogenetic tool for restoring sensitivity to light of the retinal cell through activating Gq signaling. Previous studies have characterized mammalian Opn5 as a UV-sensitive G_i-coupled opsin; we present the surprising finding that visible blue light can induce rapid Ca²⁺ transients, IP1 accumulation, and PKC activation in Opn5-expressing, for example cOpn5-expressing or tOpn5-expressing mammalian cells.

Table 6 lists the enabling features of cOpn5 by directly comparing its response amplitudes, light sensitivity, temporal resolution, and the requirement of additional chromophores to those of other optogenetic tools. For cOpn5-expressing cells, merely 10 ms blue light pulses at the intensity of 16 μW/mm²evoke rapid increase in Ca²⁺ signals with the peak amplitudes of 3-8 ΔF/F. By contrast, prior to the present invention, it is revealed that the activation of opto-a1AR or mammalian Opn4, the two proposed optogenetic tools for Gq signaling, require ˜3-fold higher light intensity (7-40 mW/mm²) and prolonged light exposure (20-60 s) and produce only weak Ca²⁺ signals (0.25-0.5 ΔF/F). Therefore, opto-a1AR or mammalian Opn4 cannot mimic the rapid activation profiles of endogenous Gq-coupled receptors that often trigger strong Gq signaling upon subsecond application of their corresponding ligands. By contrast, recent systematic characterizations show that opto-a1AR- and Opn4-mediated optogenetic stimulations do not increase the amplitudes of Ca²⁺ signals and only mildly modulate the frequency of Ca²⁺ transients and synaptic events even after prolonged illumination (Gerasimov et al., 2021; Mederos et al., 2019).

Opn5 in the present invention, in particular, cOpn5 or tOpn5-based optogenetics also enjoys the benefit of safety and convenience. Although Opn5 from many species are reported UV-responsive (Kojima et al., 2011), cOpn5 is optimally activated by 470 nm blue light, which penetrates better than UV and avoids UV-associated cellular toxicity. Its ultra-sensitivity to light also minimizes potential heating artifact. cOpn5 or tOpn5 is strongly, and repetitively activated by light without the requirement for exogenous retinal, possibly because cOpn5 or tOpn5 is a bistable opsin that covalently binds to endogenous retinal and is thus resistant to photo bleaching (Koyanagi and Terakita, 2014; Tsukamoto and Terakita, 2010). By contrast, mammalian experiments of Opn4 requires additional retinal and have long response time and low light sensitivity. Opn5 in the present invention, in particular, cOpn5 or tOpn5 as a single-component system is particularly useful for in vivo studies as it avoids the burden of delivering a compound into the tissue during the experiment.

Opn5 optogenetics in the present invention, in particular, cOpn5 or tOpn5 optogenetics also offers some major advantages over chemogenetics and uncaging tools. It is temporally much more precise and offers single-cell or even subcellular spatial resolution. Opn5 in the present invention, in particular, cOpn5 or tOpn5 also differs from caged compound-based ‘uncaging’ tools such as caged calcium and caged IP3, since these tools require compound preloading and only partially mimic the Ca²⁺-related pathways associated with G_qsignaling and/or activating cells. There exists other ‘uncaging’ tools, such as caged glutamate and caged ATP (Ellis-Davies, 2007; Lezmy et al., 2021), that target endogenous GPCRs. However, these caged compounds require their introduction into extracellular medium or the intracellular cytoplasm, which limits their applications in behaving animals (Adams and Tsien, 1993b).

Opn5 in the present invention, in particular, cOpn5 or tOpn5, optogenetics should be particularly useful for precisely activating intracellular G_qsignaling and/or activating cells, which subsequently triggers Ca²⁺ release from intracellular stores and activates PKC. Opn5 in the present invention, in particular, cOpn5 or tOpn5, differs from current channel-based optogenetic tools, such as ChR2 or its variants, which translocate cations across the plasma membrane.

On the basis of the strong light sensitivity of the Opn5 in the present invention, the present invention further demonstrates that the Opn5 in the present invention may be used to restore sensitivity to light of the retinal cell through activating Gq signaling, and thus may be used to treat or alleviate damage of the external layer of the retina, photoreceptor loss or degeneration, retinal degenerative disease, loss sensitivity to light, or loss light perception, loss of vision due to a deficit in light perception or sensitivity, or blindness.

In some embodiments, the Opn5 in the present invention may be used to restore sensitivity to light of the retinal cell as long as the retinal ganglion cells are not completely dead.

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

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

In the present invention, opn5, OPN5, Opsin and Opn5 are used interchangeably.

The descriptions of particular embodiments and examples are provided by way of illustration and not by way of limitation. Those skilled in the art will readily recognize a variety of noncritical parameters that could be changed or modified to yield essentially similar results.

EXAMPLES
Materials and Methods

TABLE 1

Primers for cloning

V5-cOpn5 forward primer
5′-cgtgaggtaccggatcctctagaatgggcaagcccatccccaacc

ccctgctgggcctggacagcaccatgagtgggatggcatcggac-3′

(SEQ ID NO: 3)

V5-cOpn5 reverse primer
5′-tcgataagcttgatatcgaattcttagacttccagttgggttccgct-3′

(SEQ ID NO: 4)

cOpn5-T2A-eGFP for hSyn promoter
5′-tagagtcgagctcaagcttgccaccatgagtgggatggcatcggactgca-3′

forward primer
(SEQ ID NO: 5)

cOpn5-T2A-eGFP for hSyn promoter
5′-aaccgcgggccctctagagcatatgttacttgtacagctcgtccatgccg-3′

reverse primer
(SEQ ID NO: 6)

cOpn5-T2A-eGFP for GfaABCID
5′-acctccgctgctcgcggggtctagaatgagtgggatggcatcggactgca-3′

promoter forward primer
(SEQ ID NO: 7)

cOpn5-T2A-eGFP for GfaABCID
5′-tatcgataagcttgatatcgaattcttacttgtacagctcgtccatgccg-3′

promoter reverse primer
(SEQ ID NO: 8)

cOpn5-T2A-eGFP for EF1a
5′-tacattatacgaagttatggcgcgccttattacttgtacagctcgtccatg-3′

promoter forward primer
(SEQ ID NO: 9)

cOpn5-T2A-eGFP for EF1a
5′-atactttatacgaagttatgctagccaccatgagtgggatggcatcggactg-3′

promoter reverse primer
(SEQ ID NO: 10)

cOpn5-T2A-mCherry forward primer
5′-gcatcacctccgctgctcgcggggtatgagtgggatggcatcggactgca-3′

(SEQ ID NO: 11)

cOpn5-T2A-mCherry reverse primer
5′-tcaccatggtggcgaccgggggatctgggccaggattctcctcgacgtca-3′

(SEQ ID NO: 12)

TABLE 2

Recombinant DNA

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

TABLE 3

Virus Strains

Lenti-cmv-cOpn5-puro
Chinese Institute for

Brain Research, Beijing

Lenti-cmv-hOPN5-puro
Chinese Institute for

Brain Research, Beijing

Lenti-cmv-tOpn5-puro
Chinese Institute for

Brain Research, Beijing

Lenti-cmv-mOpn5-puro
Chinese Institute for

Brain Research, Beijing

Lenti-cmv- hM3Dq -puro
Chinese Institute for

Brain Research, Beijing

AAV2/9-EF1a-DIO-cOpn5-T2A-eGFP
Chinese Institute for

Brain Research, Beijing

AAV2/9-hSyn-cOpn5-T2A-eGFP
Chinese Institute for

Brain Research, Beijing

AAV2/9-Ef1a-DIO-cOpn5-T2A-eGFP
Chinese Institute for

Brain Research, Beijing

AAV2/8-GFaABC1D-cOpn5-T2A-eGFP
Chinese Institute for

Brain Research, Beijing

AAV2/8-GfaABC1D-cOpn5-T2A-mCherry
Chinese Institute for

Brain Research, Beijing

AAV2/9-EF1a-EGFP
Chinese Institute for

Brain Research, Beijing

AAV2-EF1α-DIO-GCaMP6m
Chinese Institute for

Brain Research, Beijing

AAV2/9-GfaABC1D-ATP1.0
WZ Biosciences Inc.

Cat. # YL006003-AV9

AAV9-hSyn-NES-jRGECO1a-WPRE
WZ Biosciences Inc.

Cat. # BS8-NOAAAV9

AAV2/9-mCaMKIIa-jGCaMP7b-WPRE-pA
Shanghai Taitool

Bioscience Co., Ltd

Cat. # S0712-9-H20

TABLE 4

Light excitation sources

FIGS. 1f, 1g,
470 nm
Thorlabs
M470L3

FIGS. 2c, 2d, 2e,
mounted LED

2f

FIGS. 3b, 3c;

FIGS. 4a, 4b, 4e,

4f, 4h

FIGS. 5a, 5e, 5f,

5g

FIGS. 1b, 1d, 1e;
488 nm
Nikon
A1R MP

FIGS. 2a, 2b
from microscope

FIGS. 3d, 3f, 3g;
light source

FIG. 3a
365 nm
LG3535
wavelength

mounted LED

coverage:

360-370 nm

FIG. 3a
395 nm
LG3535
wavelength

mounted LED

coverage:

390-400 nm

FIG. 3a
561 nm
Changchun New
MGL-FN-561

laser
Industries

Optoelectronics

Technology,

China

FIG. 3a
590 nm
CREE XP-E2
wavelength

mounted LED

coverage:

570-615 nm

FIG. 3a
630 nm
CREE XP-E2
wavelength

mounted LED

coverage:

615-660 nm

FIGS. 4c, 4d
515 nm
Changchun New
MGL-F-515

laser
Industries

Optoelectronics

Technology,

China

TABLE 5

Microscope equipments

FIGS. 1b, 1d, 1e;
Multiphoton
Nikon
A1R MP

FIGS. 2a, 2b
confocal

microscopes

FIGS. 3a, 3b, 3c;
Spinning Disk
Nikon
ECLIPASE Ti

FIG. 4a, 4b, 4c,

4d, 4e, 4f

FIG. 5a
Confocal laser
Zeiss
LSM 880

scanning

microscope

TABLE 6

Statistical analysis:

n per

FIG.
Conditions
group
Analysis
P value

1f
ctrl vs. light
4, 4
Tukey's multiple
P < 0.0001

comparisons test

light vs.
4, 4
Tukey's multiple
P = 0.0128

light +

comparisons test

YM-254890

1g
ctrl vs. light
4, 4
Tukey's multiple
P = 0.0096

comparisons test

light vs.
4, 4
Tukey's multiple
P = 0.0004

light +

comparisons test

staurosporine

2b
cOpn5 group:
19, 15
Tukey's multiple
P < 0.0001

light vs

comparisons test

YM-254890

cOpn5 group:
15, 11
Tukey's multiple
P < 0.0001

YM-254890 vs

comparisons test

wash

cOpn5 group:
19, 11
Tukey's multiple
P = 0.2239

light vs wash

comparisons test

tOpn5 group:
15, 17
Tukey's multiple
P < 0.0001

light vs

comparisons test

YM-254890

tOpn5 group:
17, 13
Tukey's multiple
P < 0.0001

YM-254890 vs

comparisons test

wash

tOpn5 group:
15, 13
Tukey's multiple
P = 0.9388

light vs wash

comparisons test

2d
ctrl vs. light
4, 4
Unpaired t test
P = 0.4338

2f- left
ctrl vs. light
3, 3
Tukey's multiple
P = 0.992

comparisons test

2f- Right
cOpn5 group:
4, 4
Tukey's multiple
P = 0.0223

ctrl vs. light

comparisons test

tOpn5 group:
4, 4
Tukey's multiple
P = 0.4174

ctrl vs. light

comparisons test

hOPN5 group:
4, 4
Tukey's multiple
P < 0.0001

ctrl vs. light

comparisons test

mOpn5 group:
4, 4
Tukey's multiple
P < 0.0001

ctrl vs. light

comparisons test

Example 1 cOpn5 Mediates Optogenetic Activation of G_qSignaling

Whether heterologous expression of the Opn5 orthologs from chicken, turtles, humans and mice (which share 80-90% protein sequence identity) have the capacity to mediate blue light-induced G_qsignaling activation within HEK 2931 cells is tested (FIG. 1a and table 7). Blue light for stimulation and the red intracellular calcium indicator Calbryte™ 630 AM dye are used to monitor the relative Ca²⁺ response (FIG. 1b). The Opn5 orthologs from chicken (cOpn5) and turtle (tOpn5) mediated an immediate and strong light-induced increase in Ca²⁺ signal (˜3 ΔF/F), whereas no light effect was observed from cells expressing the human or mouse Opn5 orthologs (FIG. 1d and FIG. 2a, b). As exemplified by the chicken ortholog, the cOpn5 co-localized with the EGFP-CAAX membrane marker, indicating that it was efficiently transported to the plasma membrane (FIG. 1c). No exogenous retinal is supplied to the culture media, which suggests that endogenous retinal is sufficient to render cOpn5 functional. The Ca²⁺ signals are resistant to the removal of extracellular Ca²⁺, thus indicating Ca²⁺ release from the intracellular stores (FIG. 2c). Preincubation of YM-254890, a highly selective G_qproteins inhibitor 33, reversibly abolishes the light-induced Ca²⁺ transients in both cOpn5-expressing cells (FIG. 1e). In cOpn5-, but not human OPN5-expressing cells, a light-induced increase in the level of inositol phosphate (IP1), the rapid degradation product of IP₃is detected; moreover, the extent of this increase is reduced with the treatment of YM-254890 (FIG. 1f and FIG. 2d). In cOpn5-expressing HEK 293T cells, blue light also triggers the phosphorylation of MARCKS protein, a well-established target of PKC 34, in a PKC activity-dependent manner (FIG. 1g and FIG. 2e). By contrast, blue light illumination effectively reduces cAMP levels in cells expressing human and mouse Opn5 with retinal, but has no such effect in cells expressing cOpn5 without retinal (FIG. 2f). Collectively, these data support that blue light illumination enables the coupling of cOpn5 to the G_qsignaling pathway in HEK 293T cells.

TABLE 7

Opsins and species

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

FIG. 1 Shows that cOpn5 Mediates Light-Induced Strong Activation of G_qSignaling in HEK 293T Cells.

- a, Schematic diagram of the putative 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_qprotein inhibitor.
- b, Pseudocolor images of the Ca2+ signal before and after blue light stimulation (10 s; 100 μW/mm²; 488 nm) in HEK 293T cells expressing Opn5 from three species (Gallus gallus, Homo sapiens, and Mus musculus). Scale bar, 10 μm.
- c, The Cy3-counterstained V5-cOpn5 fusion protein (red) was co-localized with the membrane-tagged EGFP-CAAX (green) in HEK 293T cells. DAPI counterstaining (blue) indicates cell nuclei. Scale bar, 10 μm.
- d, Time courses of light-evoked Ca2+ signals for cells shown in c.
- e, G_qprotein inhibitor YM-254890 (10 nM) reversibly blocked cOpn5-mediated, light-induced Ca²⁺ signals.
- f, YM suppressed the IP₁accumulation evoked by continuous light stimulation (3 min; 100 μW/mm²; 470 nm) in cOpn5-expressing HEK 293T cells (Left). ***P<0.0001, *P=0.0128; Tukey's multiple comparisons test.
- g, Phosphorylation of MARCKS in cOpn5-expressing HEK 293T cells in the control group (no light stimulation), the light stimulation group, and light+staurosporine (ST, PKC inhibitor) group. The amount of p-MARCKS in the same fraction was normalized to the amount of a-tubulin. **P=0.0096, ***P=0.0004; Tukey's multiple comparisons test.
  
  FIG. 2 Shows that cOpn5 Couples to G_qbut not G_iSignaling
- a, Pseudocolor images of the Ca2+ signal before and after blue light stimulation (10 s; 100 μW/mm2; 488 nm) in HEK 293T cells expressing Opn5 from turtle species (Chelonia mydas). Scale bar, 10 μm (left); Time courses of light-evoked Ca2+ signals for responed cells (right)
- b, Group data of the Gq protein inhibitor YM-254890 (10 nM) reversibly blocked cOpn5- and turtle Opn5-mediated, light-induced Ca2+ signals. ****P<0.0001, one way ANOVA. Error bars indicate S.E.M.
- c, Time course of Ca2+ signal with photostimulation (10 ms; 16 μW/mm2; 470 nm) without extracellular Ca2+.
- d, IP1 accumulation in human Opn5-expressing HEK 293T cells with or without light stimulation (Right). n.s., no significant difference; unpaired t test.
- e, One representative of phosphorylation of MARCKS in cOpn5-expressing HEK 293T cells in the control group (no light stimulation), the light stimulation group, and light+staurosporine group. The amount of p-MARCKS in the same fraction was normalized to the amount of a-tubulin.
- f, Light has no effect on cAMP levels (10 μM forskolin preincubation) in cOpn5-expressing HEK 293T cells without additional retinal in the medium (left panel). Right panel shows the effects of photostimulation on cAMP concentrations for HEK 293T cells expressing Opn5s from four different species following 10 μM retinal preincubation.

Error bars in d and f indicate S.E.M.

Example 2 cOpn5-Mediated Optogenetics is Sensitive and Precise

Characterizing the light-activating properties of cOpn5 heterologously expressed in HEK 293T cells is performed. Although Opn5 is previously considered as an ultraviolet (UV)-sensitive photoreceptor²⁷, mapping with a set of wavelengths ranging 365-630 nm at a fixed light intensity of (100 μW/mm²) revealed that the 470 nm blue light elicited the strongest Ca2+ transients, with the UVA light (365 and 395 nm) being less effective and longer-wavelength visible light (561 nm or above) completely ineffective (FIG. 3a). The effects of different light durations on cOpn5-expressing HEK 293T cells are tested. Stimulating with brief light pulses (1, 5, 10, 20, 50 ms; 16 μW/mm²; 470 nm) shows that the Ca2+ response achieves the saturation mode with light duration over 10 ms (FIG. 3b). Longer light durations do not further increase the Ca2+ signal amplitude at this light intensity (16 μW/mm²; 470 nm) (FIG. 4a). Delivering 470 nm light at different intensities shows that blue light of ˜4.8 μW/mm²and 16 μW/mm²produce about half maximum and full maximum responses, respectively (FIG. 3c and FIG. 4b). Therefore, the light sensitivity of cOpn5 is 3-4 orders of magnitude higher than the reported values of the light-sensitive Gq-coupled GPCRs and even 2-3 orders higher than those of the commonly used optogenetic tool Channelrhodopsin-2 (ChR2)(Lin, 2011; Zhang et al., 2006) (table 8). Together, these results indicate that cOpn5 could function as a single-component optogenetic tool without additional retinal, and that cOpn5 is super-sensitive to blue light for its full activation requiring low light intensity (16 μW/mm²) and short duration (10 ms).

TABLE 8

Comparison cOpn5 with other optogenetic tools

Need for

Wavelength

Stimulation
exogeneous
Response

λ_max(nm)
Light Sensitivity
duration
chemicals(retinal)
amplitude
model

Wild-type
470 nm
8-12
mW/mm²
2.3 ± 1.1
ms
No
steady state:
Hippocampal

ChR2 ^{1, 2}

peak current
cell culture

ratio: 0.4 ±

0.04; (731 ±

100 pA

ChR2 H134R ³
450 nm
~10 mW/mm²
0.96 ± 0.12
ms
No
4.47 nA
HEK 293T

(470 nm)

ChETA ⁴
490 nm
~10
mW/mm²
0.9 ± 0.1
ms
No
steady state:
Hippocampal

peak current
cell culture

ratio: 0.6 ±

0.04; (645 ±

47 pA

ChrimsonR ⁵
590 nm
4.6
mW/mm²
0.9 ± 0.1
ms
No
~300 pA
cultured

neurons

mouse
480 nm
1015 photons s⁻¹
>60
s
11-cis-
~0.1 (ΔF/F)
HEK293-

melanopsin

cm⁻²(500 nm)

retinaldehyde
Ca²⁺
TRPC3 cells

(Opn4) ⁶

response

ampitude

mouse
488 nm
a white
60
s
11-cis retinal
~0.25
CHO cells

melanopsin

fluorescent light

(ΔF/F) Ca²⁺

(Opn4) and

source (intensity

response

its mutants ⁷

undefined)

amplitude,

the best

mutant

Opn4^9A

hOpn4-
473 nm
7
mW/mm²
20
s
Unknown
~2 (ΔF/F)
in vivo

human

Ca²⁺ event
astrocytes

melanopsin ⁸

frequence,

but no

significant

change in

Ca²⁺

amplitude

opto-α1AR ⁹
500 nm
7
mW/mm²
60
s
No
~0.227
HEK cells

(ΔF/F) Ca²⁺

response

ampitude

opto-α1AR ¹⁰
473 nm
20 Hz, 45-ms
5
min
No
>20%
in vitro

light pulses,

increase in
astrocytes

5 mW

sIPSC

frequency

human
470 nm
40
mW/mm²
25
s
ATR
~0.646
HEK 293T

melanopsin

(ΔF/F) Ca²⁺

response

ampitude

opto-α1AR
510 nm
7
mW/mm²
60
s
No
~0.5 (ΔF/F)
HEK 293T

Ca²⁺

response

ampitude

hM3Dq

CNO
~1.6 (ΔF/F)
HEK 293T

Ca²⁺ event

frequence,

but no

significant

change in

Ca²⁺

amplitude

cOpn5
470 nm
16
μW/mm²
10
ms
No
~3.0 (ΔF/F)
HEK 293T

Ca²⁺
cells

response

amplitude

470 nm
0.026
μW/mm²
>2
s
No
~1 (ΔE/F)
HEK 293T

Ca²⁺
cells

response

amplitude

The performance of cOpn5 to that of opto-a1AR, a chimera GPCR engineered by mixing rhodopsin with G_q-coupled adrenergic receptor is compared. Following the protocol in a previous report¹⁴, it is found that very long exposure of strong illumination (60 s; 7 mW/mm²) is required to trigger a slow and small (˜0.5 ΔF/F) Ca²⁺ signal increase in opto-a1AR-expressing HEK 2931 cells, and 15 s illumination is inefficient (FIG. 4c, d). The performance of cOpn5 to that of opn4, a natural opsin which was reported as a tool for G_qsignaling activating is also compared. It is found that long exposure of strong illumination (25 s; 40 mW/mm²) and additional retinal are required to trigger a slow (˜1 ΔF/F) Ca²⁺ signal increase in opn4-expressing HEK 2931 cells (FIG. 4e, f). Therefore, compared with existing opsin-based tools (opto-a1AR and opn4), cOpn5 is much more light-sensitive (˜3 orders more sensitive), requires much shorter time exposure (10 ms vs. 60 s), and produces stronger responses.

The performance of cOpn5 to that of the popular G_q-coupled chemogenetic tool hM3Dq, which is activated by adding the exogenous small molecule ligand clozapine-N-oxide (CNO)^37-39is compared. Light-induced activation of cOpn5-expressing HEK 293T cells has a similar peak response amplitude of the Ca²⁺ signal as CNO-induced activation of hM3Dq-expressing HEK 293T cells. Meanwhile, cOpn5-expressing HEK 293T cells have faster and temporally more precise response, as well as more rapid recovery time than hM3Dq-expressing HEK 293T cells (FIG. 4g-i). These results indicate that cOpn5-mediated optogenetics are more controllable in temporal accuracy than those of hM3Dq.

cOpn5 optogenetics allows spatially precise control of cellular activity. Restricting brief light stimulation (63 ms) into a subcellular region of individual cOpn5-expressing HEK 293T cell results in the immediate activation of single cell. Interestingly, in high cell confluence area, the Ca²⁺ signals propagated to surrounding cells, thus suggesting intercellular communication among HEK 293T cells through a yet-to-identified mechanism (FIG. 3d, e). The findings are extended into primary cell cultures. cOpn5 is expressed in primary astrocyte cultures prepared from the neonatal mouse brain with AAV vectors for bicistronic expression of cOpn5 and the EGFP marker protein (FIG. 5a). Using the Calbryte 630 AM dye to monitor Ca²⁺ levels, it is found that blue light illumination of cOpn5-expressing astrocytes produces strong Ca²⁺ transients (˜8 ΔF/F) (FIG. 5b, c). If the light stimulation (63 ms) is precisely restricted to only subcellular region of an individual cOpn5-expressing astrocyte, Ca²⁺ signal propagation within the individual cell is observed (FIG. 3f). Resembling the tests in HEK 293T cells, wave-like propagation of Ca²⁺ signals from the stimulated astrocyte that proceeded gradually to more distal, non-stimulated, astrocytes is observed (FIG. 3g, h). These experiments thus demonstrate that cOpn5 optogenetics allows precise spatial control, and suggest that it may be useful to study the dynamics of astrocytic networks, which is initially discovered using neurochemical and mechanical stimulation^40,41.

FIG. 3 Shows that cOpn5 Sensitively Mediates Optical Control of G_qSignaling with High Temporal and Spatial Resolution.

- a, Schematic diagram of selected wavelengths (365, 395, 470, 515, 561, 590, and 630 nm; left panel) and the amplitudes of Ca²⁺ signal of cOpn5-expressing HEK 293T cells in response to light stimulation with different wavelengths (2 s; 100 μW/mm²; right panel). Error bars indicate S.E.M.
- b, The response magnitude under different duration of light stimulation (1, 5, 10, 20, or 50 ms; 16 μW/mm²; 470 nm). Error bars indicate S.E.M.
- c, Time course of cOpn5-mediated Ca²⁺ signals under different light intensity (0, 4.8, 8, 16, or 32 μW/mm²; 10 ms; 470 nm; for 10 ms 16 μW/mm²stimulation, 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).
- d, Images of light-induced (63 ms; 17 μW; arrow points to the stimulation region) Ca²⁺ signal propagation in cOpn5-expressing HEK 293T cells. Scale bar, 10 μm.
- e, Pseudocolor images showing the process of Ca²⁺ signal propagation across time of d (frame N/(N−1)>1). Frame interval was 500 ms and each frame is counted once.
- f, Images of light-induced Ca²⁺ signal propagation in a single cOpn5-expressing primary astrocyte stimulated in a subcellular region (stimulation size 4×4 μm²and frame interval 300 ms). Scale bar, 10 μm.
- g, Images of light-induced Ca²⁺ signal propagation in cOpn5-expressing primary astrocytes. Scale bar, 10 μm.
- h, Pseudocolor images showing process of Ca²⁺ signal propagation across time of g (frame N/(N−1)>1). Frame interval was 500 ms and each frame is counted once.
  
  FIG. 4 Shows that cOpn5 Mediates More Rapid and Sensitive Response to Light than Opto-a1AR, hM3Dq or Opn4.
- a, Time course of Ca²⁺ signal with light pulses (16 μW/mm²; 470 nm; 1, 5, 10, 20, or 50 ms).
- b, The response magnitude under different light intensities (0, 4.8, 8, 16, or 32 μW/mm²) at 10 ms, 470 nm.
- c, Pseudocolor images of the baseline and peak Ca²⁺ signals (ΔF/F0) in opto-a1AR-expressing HEK 293T cells. The medium buffer contains 10 μM all-trans-retinal. Scale bar, 30 μm.
- d, Effect of 60 s light stimulation on the Ca²⁺ in opto-a1AR-expressing HEK 293T cells (n=15 cells; upper panel) and the lack of effect by 15 s light stimulation on Ca²⁺ signals (lower panel).
- e, Pseudocolor images of the baseline and peak Ca²⁺ signals (ΔF/F0) in human OPN4-expressing HEK 293T cells. The medium buffer contains 10 μM all-trans-retinal. Scale bar, 30 μm.
- f, Effect of 25 s light stimulation on the Ca²⁺ in OPN4-expressing HEK 293T cells within 10 μM ATR (n=12 cells; red line) and the lack of effect by without ATR on Ca²⁺ signals (black panel).
- g, Effects of light stimulation on the Ca²⁺ signals in cOpn5-expressing HEK 293T cells. Upper panels show pseudocolor images of baseline and peak response. Lower panel shows the heat map of Ca²⁺ signals evoked by cOpn5-mediated optogenetic stimulation in HEK 293T cells expressing cOpn5 across 5 consecutive trials. Scale bar, 20 μm.
- h, Effect of chemogenetic stimulation on the Ca²⁺ signals in hM3Dq-expressing HEK 293T cells.
- i, Time courses of Ca²⁺ signals evoked by cOpn5-mediated optogenetic stimulation (10 s) and hM3Dq-mediated chemogenetic stimulation using CNO puff (100 nM; 10 s), respectively.
  
  FIG. 5 Shows that cOpn5 Effectively Mediates the Activation of Astrocytes.
- a, cOpn5 was expressed in cultured primary astrocytes using AAV-cOpn5-T2A-EGFP (green). Astrocyte identity was confirmed by GFAP immunostaining (red). Scale bar, 20 μm.
- b, Pseudocolor images of the baseline and peak Ca²⁺ signals following light stimulation of cOpn5-expressing astrocytes. Scale bar, 20 μm.
- c, Plot of Ca²⁺ signals and heat map representation of Ca²⁺ signals across trials (n=25 cells).

Example 3 Optogenetic Visual Restoration Using Light-Sensitive Gq-Coupled Neuropsin (Opsin 5)
Animal Model:

- 1. Health retina contains several cell layers: retinal pigment epithelium, cone photoreceptor cells, rod photoreceptor cells, horizontal cells, bipolar cells, Müller cells, Amacrine cells, Ganglion cells (FIG. 6). Methylnitrosourea (MNU) results photoreceptor (rod and cone photoreceptors) damage and then induces retinal degeneration in animals. We use MNU induce mice retinal degeneration as an animal model. Retinal degeneration induced by a single intraperitoneal injection of MNU with the dose of 60 mg/kg body weight.
- 2. C3H/HeNCrl Mice are genetic retinal degeneration models. This strain has a characteristic that homozygous for Pde6b^rd1mutation causing retinal degeneration.

We use the pupillary light response with head fixed mice to test whether the animal could sense the light, and we use AAV vectors expressing cOpn5 in mice retinal ganglion cells to rescue these two mice models. The mice recover pupillary light response demonstrates our cOpn5-mediated approach of blindness treatment.

Experiments and Results

- 1. We use camera with IR blocking to automatically acquire images of head fixed mice pupils. Adjust optical fiber to make sure the light (470 nm LED light source) shoots straight on mice pupils with the same light intensity.
- 2. Normal mice before MNU-treated have rapid pupillary light response (FIG. 7). C3H/HeNCrl inbred Mice didn't have pupillary light response (FIG. 7).
- 3. C3H/HeNCrl or MNU treated retinal degeneration mice lost functions of pupillary light response
- 4. We use AAV vector expressed cOpn5-t2a-EGFP in mice retinal ganglion cells, the image shows EGFP in the whole retina after 4 weeks after AAV injection (FIG. 8).
- 5. After cOpn5 expressed in the mice retinal ganglion cells, we do the pupillary light response test again. The MNU mice-treated recovered the pupillary light response (FIG. 9). The C3H/HeNCrl mice gain the ability of pupillary light response (FIG. 9).
- 6. FIG. 10 shows in pupillary light response test: normal mice (black solid line) pupil size rapid decrease in response to light (X-axis: time (second); Y-axis: normalized pupil size). After MNU treatment, the mice lost functions in pupillary light response test (gray solid line). When using AAV vectors expressing cOpn5 in the retinal ganglion cells (RGC) of these MNU treated mice 4 weeks later, the mice partially recovered the pupillary light response capability (middle solid line).

These results demonstrate our approach that expressing cOpn5 in animal retinal ganglion cells can recover retinal degeneration.

Example 4

Experiments description: the following table 9 is a partial list of cOpn5 orthologs from vertebrata tested in the present invention. Whole genes of all reported opsin5 orthologs from vertebrata (the vertebrates subphylum, including rotundia, cartilaginous fishes, bony fishes, Amphibia, reptila, ornitha and mammals) are synthetized, and expressed in HEK 293T cells. Calcium imaging with or without 470 nm blue light stimulation is performed to test the sensitivity of the opsin 5 orthologs in response to light. The time course of light-induced calcium signal reveal the activated degree of G_qsignaling pathway and the sensitivity of these orthologs.

TABLE 9

Entry
Entry name
Activity
Protein names
Gene names

E0R7P4
E0R7P4_XENLA

Opn5 (Opsin)
opn5.L opn5

XELAEV_18028134 mg

A0A455SGG5
A0A455SGG5_9EUPU

Opsin-5A
opn5a

A0A4Z2FX25
A0A4Z2FX25_9TELE

Opsin-5
OPN5_5 EYF80_044932

A0A4Z2FH27
A0A4Z2FH27_9TELE

Opsin-5
OPN5_4 EYF80_049299

A0A4Z2IDU8
A0A4Z2IDU8_9TELE

Opsin-5
OPN5_3 EYF80_013671

A0A4Z2H0H0
A0A4Z2H0H0_9TELE

Opsin-5
OPN5_2 EYF80_030918

A0A218USZ0
A0A218USZ0_9PASE

Opsin-5
OPN5_1 RLOC_00008660

A0A4Z2FVH4
A0A4Z2FVH4_9TELE

Opsin-5
Opn5_0 EYF80_044930

A0A4Z2HA58
A0A4Z2HA58_9TELE

Opsin-5
OPN5_0 EYF80_027087

A0A218UGP1
A0A218UGP1_9PASE

Opsin-5
OPN5_0 RLOC_00005796

G1L3V2
G1L3V2_AILME

Opsin 5
OPN5

A0A6P4X9I3
A0A6P4X9I3_PANPR

opsin-5
OPN5

A0A1S2ZDX4
A0A1S2ZDX4_ERIEU

opsin-5
OPN5

A0A2I4C032
A0A2I4C032_9TELE

opsin-5
opn5

U3JFW4
U3JFW4_FICAL

Opsin 5
OPN5

A0A2Y9NPU7
A0A2Y9NPU7_DELLE

opsin-5
OPN5

A0A1U7U6G6
A0A1U7U6G6_CARSF

opsin-5
OPN5

A0A6I9I544
A0A6I9I544_VICPA

opsin-5
OPN5

M3YLS7
M3YLS7_MUSPF

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

A0A5F9CCV1
A0A5F9CCV1_RABIT

Opsin 5
OPN5

A0A671EF51
A0A671EF51_RHIFE

Opsin 5
OPN5

A0A6P6I1D4
A0A6P6I1D4_PUMCO

opsin-5
OPN5

G3RKG7
G3RKG7_GORGO

Opsin 5
OPN5

G1NYV5
G1NYV5_MYOLU

Opsin 5
OPN5

A0A6J2L9P4
A0A6J2L9P4_9CHIR

opsin-5
OPN5

A0A2K6AXI4
A0A2K6AXI4_MACNE

Opsin 5
OPN5

A0A6J3JM90
A0A6J3JM90_SAPAP

opsin-5
OPN5

A0A452TE17
A0A452TE17_URSMA

Opsin 5
OPN5

A0A384C5D1
A0A384C5D1_URSMA

opsin-5
OPN5

A0A2K5U4B7
A0A2K5U4B7_MACFA

Opsin 5
OPN5

A0A2I3MZV4
A0A2I3MZV4_PAPAN

Opsin-5
OPN5

A0A2K5U4B3
A0A2K5U4B3_MACFA

Opsin 5
OPN5

Q6U736
OPN5_HUMAN
reviewed
Opsin-5 (G-protein coupled
OPN5 GPR136 PGR12

receptor 136) (G-protein
TMEM13

coupled receptor PGR12)

(Neuropsin) (Transmembrane

protein 13)

F6UZB2
F6UZB2_XENTR

Opsin 5
opn5

A0A4W3IAF8
A0A4W3IAF8_CALMI

G_PROTEIN_RECEP_F1_2
opn5

domain-containing protein

A0A6P7HHM6
A0A6P7HHM6_9TELE

opsin-5
opn5

H3B1A3
H3B1A3_LATCH

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

A0A4W3I3H5
A0A4W3I3H5_CALMI

G_PROTEIN_RECEP_F1_2
opn5

domain-containing protein

A0A1S3MCD5
A0A1S3MCD5_SALSA

opsin-5
opn5

A0A4W4FPG5
A0A4W4FPG5_ELEEL

G_PROTEIN_RECEP_F1_2
opn5

domain-containing protein

A0A6P7LVJ1
A0A6P7LVJ1_BETSP

opsin-5 isoform X2
opn5

A0A6P7LVE3
A0A6P7LVE3_BETSP

opsin-5 isoform X1
opn5

A0A674IKC9
A0A674IKC9_TERCA

Opsin 5
OPN5

A0A674IMF3
A0A674IMF3_TERCA

Opsin 5
OPN5

F1NEY2
F1NEY2_CHICK

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

E6P6L8
E6P6L8_DANRE

Opsin 5
opn5

A0A671TVX9
A0A671TVX9_SPAAU

Opsin 5
opn5

A0A7M4FP40
A0A7M4FP40_CROPO

Opsin 5
OPN5

A0A671TVX4
A0A671TVX4_SPAAU

Opsin 5
opn5

A0A6I9Y3G3
A0A6I9Y3G3_9SAUR

opsin-5
OPN5

G1KNV3
G1KNV3_ANOCA

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

A0A493T549
A0A493T549_ANAPP

Opsin 5
OPN5

A0A6I9HELA
A0A6I9HELA_GEOFO

opsin-5 isoform X2
OPN5

A0A218UPZ6
A0A218UPZ6_9PASE

Opsin-5
OPN5 RLOC_00008263

D8KW68
D8KW68_ZONAL

Opsin 5
OPN5

A0A663EIX5
A0A663EIX5_AQUCH

Opsin 5
OPN5

G1NNA7
G1NNA7_MELGA

Opsin 5
OPN5

A0A663EK31
A0A663EK31_AQUCH

Opsin 5
OPN5

A0A6J0Z1K0
A0A6J0Z1K0_ODOVR

opsin-5
OPN5

A0A6P3J431
A0A6P3J431_BISBI

opsin-5
OPN5

A0A2K5R3Y3
A0A2K5R3Y3_CEBIM

Opsin 5
OPN5

A0A671EF86
A0A671EF86_RHIFE

Opsin 5
OPN5 mRhiFer1_012304

A0A6I9ZSG3
A0A6I9ZSG3_ACIJB

opsin-5
OPN5

A0A2K6R8Q2
A0A2K6R8Q2_RHIRO

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

A0A4W2GZA3
A0A4W2GZA3_BOBOX

Opsin 5
OPN5

U3J4Q3
U3J4Q3_ANAPP

Opsin 5
OPN5

A0A6J2V8J5
A0A6J2V8J5_CHACN

opsin-5
opn5

A0A493T6P1
A0A493T6P1_ANAPP

Opsin 5
OPN5

A0A6J2J0L1
A0A6J2J0L1_9PASS

opsin-5
OPN5

A0A6P9CE92
A0A6P9CE92_PANGU

opsin-5
OPN5

A0A6J1V4P8
A0A6J1V4P8_9SAUR

opsin-5
OPN5

A0A288HLV3
A0A288HLV3_ANSCY

Opsin-5
OPN5

A0A151PID4
A0A151PID4_ALLMI

Opsin-5
OPN5 Y1Q_0020212

Q5RIV6
Q5RIV6_DANRE

Opsin 5 (Teleost neuropsin)
opn5

D6RDV4
D6RDV4_HUMAN

Opsin-5
OPN5

J3KPQ2
J3KPQ2_HUMAN

Opsin-5
OPN5 hCG_1642475

F6XNY7
F6XNY7_ORNAN

Opsin 5
OPN5

A0A2K6FXK2
A0A2K6FXK2_PROCO

Opsin 5
OPN5

E2RPZ0
E2RPZ0_CANLF

Opsin 5
OPN5

A0A2K6V732
A0A2K6V732_SAIBB

Opsin 5
OPN5

A0A4X2K722
A0A4X2K722_VOMUR

Opsin 5
OPN5

A0A6P5KYE6
A0A6P5KYE6_PHACI

opsin-5
OPN5

A0A2K6FXJ4
A0A2K6FXJ4_PROCO

Opsin 5
OPN5

A0A4X2JZA4
A0A4X2JZA4_VOMUR

Opsin 5
OPN5

G1SX53
G1SX53_RABIT

Opsin 5
OPN5

A0A2U3WI94
A0A2U3WI94_ODORO

opsin-5
OPN5

A0A2K6V724
A0A2K6V724_SAIBB

Opsin 5
OPN5

A0A3Q7XKC9
A0A3Q7XKC9_URSAR

opsin-5
OPN5

A0A452RBH1
A0A452RBH1_URSAM

Opsin 5
OPN5

G1QVY1
G1QVY1_NOMLE

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

G1QVX6
G1QVX6_NOMLE

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

G3SJY5
G3SJY5_GORGO

Opsin 5
OPN5

A0A7N9CSX2
A0A7N9CSX2_MACFA

Opsin 5
OPN5

A0A384B2Q9
A0A384B2Q9_BALAS

opsin-5
OPN5

A0A2K6L978
A0A2K6L978_RHIBE

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

A0A2K6AXE7
A0A2K6AXE7_MACNE

Opsin 5
OPN5

A0A2J8P0S9
A0A2J8P0S9_PANTR

Opsin 5
OPN5

W5PR22
W5PR22_SHEEP

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

F7DJ88
F7DJ88_CALJA

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

A0A2K5R3Z8
A0A2K5R3Z8_CEBIM

Opsin 5
OPN5

F6PHB6
F6PHB6_CALJA

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

M3WMC9
M3WMC9_FELCA

Opsin 5
OPN5

A0A2K5L5D5
A0A2K5L5D5_CERAT

Opsin 5
OPN5

E1BNN4
E1BNN4_BOVIN

Opsin 5
OPN5

F6RFW7
F6RFW7_MACMU

Opsin 5
OPN5

A0A2J8RKP9
A0A2J8RKP9_PONAB

Uncharacterized protein
OPN5

A0A3Q7RXX8
A0A3Q7RXX8_VULVU

opsin-5
OPN5

A0A2K5L5D9
A0A2K5L5D9_CERAT

Opsin 5
OPN5

H0WJY2
H0WJY2_OTOGA

Opsin 5
OPN5

A0A6P3ENQ6
A0A6P3ENQ6_SHEEP

opsin-5
OPN5

G3UA68
G3UA68_LOXAF

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

A0A6P5DVT1
A0A6P5DVT1_BOSIN

opsin-5
OPN5

A0A0D9RJS4
A0A0D9RJS4_CHLSB

Opsin 5
OPN5

I3LTK7
I3LTK7_PIG

Opsin 5
OPN5

A0A2K5Z564
A0A2K5Z564_MANLE

Opsin 5
OPN5

A0A5G2R7I1
A0A5G2R7I1_PIG

Opsin 5
OPN5

A0A6I9JGH7
A0A6I9JGH7_CHRAS

opsin-5
OPN5

A0A2K5Z517
A0A2K5Z517_MANLE

Opsin 5
OPN5

A0A452FM79
A0A452FM79_CAPHI

Opsin 5
OPN5

F6SJH5
F6SJH5_HORSE

Opsin 5
OPN5

A0A2R9BTW5
A0A2R9BTW5_PANPA

Opsin 5
OPN5

A0A2Y9FNI2
A0A2Y9FNI2_PHYMC

opsin-5
OPN5

A0A340WR35
A0A340WR35_LIPVE

opsin-5
OPN5

A0A6J2DJL3
A0A6J2DJL3_ZALCA

opsin-5
OPN5

A0A4X1UZM3
A0A4X1UZM3_PIG

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

A0A673TX31
A0A673TX31_SURSU

Opsin 5
OPN5

A0A341D5X7
A0A341D5X7_NEOAA

opsin-5
OPN5

A0A667FWA1
A0A667FWA1_LYNCA

Opsin 5
OPN5

A0A5B7H9S7
A0A5B7H9S7_PORTR

Opsin-5
Opn5 E2C01_063173

A0A337SC50
A0A337SC50_FELCA

Opsin 5
OPN5

H2RD19
H2RD19_PANTR

Opsin 5
OPN5

A0A2U3X849
A0A2U3X849_LEPWE

opsin-5
OPN5

G3THK6
G3THK6_LOXAF

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

A0A2U3V1E1
A0A2U3V1E1_TURTR

opsin-5
OPN5

A0A096NIY4
A0A096NIY4_PAPAN

Opsin-5
OPN5

A0A6P3PSZ2
A0A6P3PSZ2_PTEVA

opsin-5
OPN5

A0A2K5EFR2
A0A2K5EFR2_AOTNA

Opsin 5
OPN5

A0A3Q7QKC2
A0A3Q7QKC2_CALUR

opsin-5
OPN5

F7DVJ0
F7DVJ0_MONDO

Opsin 5
OPN5

A0A2K5EFU2
A0A2K5EFU2_AOTNA

Opsin 5
OPN5

A0A5F8H1F1
A0A5F8H1F1_MONDO

Opsin 5
OPN5

A0A2Y9H826
A0A2Y9H826_NEOSC

opsin-5
OPN5

G3W284
G3W284_SARHA

Opsin 5
OPN5

A0A3Q0CTY5
A0A3Q0CTY5_MESAU

opsin-5
Opn5

A0A6P5NS60
A0A6P5NS60_MUSCR

opsin-5
Opn5

H0V671
H0V671_CAVPO

Opsin 5
OPN5

I3M1B1
I3M1B1_ICTTR

Opsin 5
OPN5

Q7TQN6
Q7TQN6_RAT

G protein-coupled receptor 136
Opn5 Gpr136

(Opsin 5)

A0A287CZD4
A0A287CZD4_ICTTR

Opsin 5
OPN5

A0A1W6KZ83
A0A1W6KZ83_9RODE

Neuropsin
OPN5

A0A6I9MCW1
A0A6I9MCW1_PERMB

opsin-5
Opn5

A0A6P3EVC3
A0A6P3EVC3_OCTDE

opsin-5
Opn5

A0A1S3FD42
A0A1S3FD42_DIPOR

LOW QUALITY PROTEIN:
Opn5

opsin-5

A0A6A4VE33
A0A6A4VE33_AMPAM

Opsin-5
OPN5 FJT64_010458

A0A4P2TKU6
A0A4P2TKU6_PAROL

Neuropsin
OPN5

A0A670IDE8
A0A670IDE8_PODMU

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

A0A1U7S163
A0A1U7S163_ALLSI

opsin-5
OPN5

A0A670Y2N7
A0A670Y2N7_PSETE

Opsin 5
OPN5

K7FFW2
K7FFW2_PELSI

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

D9N3D0
D9N3D0_COTJA

Opsin 5
OPN5

Q6VZZ7
OPN5_MOUSE
reviewed
Opsin-5 (G-protein coupled
Opn5 Gpr136 Pgr12

receptor 136) (G-protein

coupled receptor PGR12)

(Neuropsin)

D8KWH6
D8KWH6_ZONAL

Opsin 5
OPN5

A0A674PPK4
A0A674PPK4_TAKRU

G_PROTEIN_RECEP_F1_2
opn5

domain-containing protein

A0A674HDZ6
A0A674HDZ6_TAEGU

G_PROTEIN_RECEP_F1_2
OPN5

domain-containing protein

H2V568
H2V568_TAKRU

G_PROTEIN_RECEP_F1_2
opn5

domain-containing protein

A0A6J0H1N3
A0A6J0H1N3_9PASS

opsin-5
OPN5

A0A672UEH1
A0A672UEH1_STRHB

Opsin 5
OPN5

A0A672UBX7
A0A672UBX7_STRHB

Opsin 5
OPN5

A0A6J0U919
A0A6J0U919_9SAUR

opsin-5
OPN5

A0A6J8E395
A0A6J8E395_MYTCO

OPN5
MCOR_46347

A0A6J7ZZ06
A0A6J7ZZ06_MYTCO

OPN5
MCOR_1439

A0A2J8RKQ7
A0A2J8RKQ7_PONAB

OPN5 isoform 1
CR201_G0050220

A0A2J8P0V4
A0A2J8P0V4_PANTR

OPN5 isoform 4
CK820_G0007353

A0A212D584
A0A212D584_CEREH

OPN5
Celaphus_00014381

Entry
Organism
Length

E0R7P4

Xenopus laevis (African clawed frog)
341

A0A455SGG5

Ambigolimax valentianus

425

A0A4Z2FX25

Liparis tanakae (Tanaka's snailfish)
178

A0A4Z2FH27

Liparis tanakae (Tanaka's snailfish)
399

A0A4Z2IDU8

Liparis tanakae (Tanaka's snailfish)
396

A0A4Z2H0H0

Liparis tanakae (Tanaka's snailfish)
153

A0A218USZ0

Lonchura striata domestica (Bengalese finch)
348

A0A4Z2FVH4

Liparis tanakae (Tanaka's snailfish)
338

A0A4Z2HA58

Liparis tanakae (Tanaka's snailfish)
311

A0A218UGP1

Lonchura striata domestica (Bengalese finch)
417

G1L3V2

Ailuropoda melanoleuca (Giant panda)
381

A0A6P4X9I3

Panthera pardus (Leopard) (Felis pardus)
353

A0A1S2ZDX4

Erinaceus europaeus (Western European hedgehog)
353

A0A2I4C032

Austrofundulus limnaeus

353

U3JFW4

Ficedula albicollis (Collared flycatcher) (Muscicapa albicollis)
357

A0A2Y9NPU7

Delphinapterus leucas (Beluga whale)
362

A0A1U7U6G6

Carlito syrichta (Philippine tarsier) (Tarsius syrichta)
354

A0A6I9I544

Vicugna pacos (Alpaca) (Lama pacos)
353

M3YLS7

Mustela putorius furo (European domestic ferret) (Mustela furo)
377

A0A5F9CCV1

Oryctolagus cuniculus (Rabbit)
366

A0A671EF51

Rhinolophus ferrumequinum (Greater horseshoe bat)
380

A0A6P6I1D4

Puma concolor (Mountain lion)
353

G3RKG7

Gorilla gorilla gorilla (Western lowland gorilla)
382

G1NYV5

Myotis lucifugus (Little brown bat)
353

A0A6J2L9P4

Phyllostomus discolor (pale spear-nosed bat)
353

A0A2K6AXI4

Macaca nemestrina (Pig-tailed macaque)
354

A0A6J3JM90

Sapajus apella (Brown-capped capuchin) (Cebus apella)
354

A0A452TE17

Ursus maritimus (Polar bear) (Thalarctos maritimus)
361

A0A384C5D1

Ursus maritimus (Polar bear) (Thalarctos maritimus)
353

A0A2K5U4B7

Macaca fascicularis (Crab-eating macaque) (Cynomolgus
354

monkey)

A0A2I3MZV4

Papio anubis (Olive baboon)
354

A0A2K5U4B3

Macaca fascicularis (Crab-eating macaque) (Cynomolgus
382

monkey)

Q6U736

Homo sapiens (Human)
354

F6UZB2

Xenopus tropicalis (Western clawed frog) (Silurana tropicalis)
345

A0A4W3IAF8

Callorhinchus milii (Ghost shark)
340

A0A6P7HHM6

Parambassis ranga (Indian glassy fish)
355

H3B1A3

Latimeria chalumnae (Coelacanth)
290

A0A4W3I3H5

Callorhinchus milii (Ghost shark)
333

A0A1S3MCD5

Salmo salar (Atlantic salmon)
328

A0A4W4FPG5

Electrophorus electricus (Electric eel) (Gymnotus electricus)
333

A0A6P7LVJ1

Betta splendens (Siamese fighting fish)
308

A0A6P7LVE3

Betta splendens (Siamese fighting fish)
365

A0A674IKC9

Terrapene carolina triunguis (Three-toed box turtle)
372

A0A674IMF3

Terrapene carolina triunguis (Three-toed box turtle)
347

F1NEY2

Gallus gallus (Chicken)
357

E6P6L8

Danio rerio (Zebrafish) (Brachydanio rerio)
352

A0A671TVX9

Sparus aurata (Gilthead sea bream)
357

A0A7M4FP40

Crocodylus porosus (Saltwater crocodile) (Estuarine crocodile)
357

A0A671TVX4

Sparus aurata (Gilthead sea bream)
353

A0A6I9Y3G3

Thamnophis sirtalis

277

G1KNV3

Anolis carolinensis (Green anole) (American chameleon)
347

A0A493T549

Anas platyrhynchos platyrhynchos (Northern mallard)
337

A0A6I9HELA

Geospiza fortis (Medium ground-finch)
354

A0A218UPZ6

Lonchura striata domestica (Bengalese finch)
304

D8KW68

Zonotrichia albicollis (White-throated sparrow)
354

A0A663EIX5

Aquila chrysaetos chrysaetos

343

G1NNA7

Meleagris gallopavo (Wild turkey)
358

A0A663EK31

Aquila chrysaetos chrysaetos

370

A0A6J0Z1K0

Odocoileus virginianus texanus

353

A0A6P3J431

Bison bison bison

353

A0A2K5R3Y3

Cebus imitator (Panamanian white-faced capuchin) (Cebus
382

capucinus imitator)

A0A671EF86

Rhinolophus ferrumequinum (Greater horseshoe bat)
354

A0A6I9ZSG3

Acinonyx jubatus (Cheetah)
353

A0A2K6R8Q2

Rhinopithecus roxellana (Golden snub-nosed monkey)
354

(Pygathrix roxellana)

A0A4W2GZA3

Bos indicus × Bos taurus (Hybrid cattle)
355

U3J4Q3

Anas platyrhynchos platyrhynchos (Northern mallard)
380

A0A6J2V8J5

Chanos chanos (Milkfish) (Mugil chanos)
355

A0A493T6P1

Anas platyrhynchos platyrhynchos (Northern mallard)
400

A0A6J2J0L1

Pipra filicauda (Wire-tailed manakin)
354

A0A6P9CE92

Pantherophis guttatus (Corn snake) (Elaphe guttata)
357

A0A6J1V4P8

Notechis scutatus (mainland tiger snake)
357

A0A288HLV3

Anser cygnoid (Swan goose)
355

A0A151PID4

Alligator mississippiensis (American alligator)
369

Q5RIV6

Danio rerio (Zebrafish) (Brachydanio rerio)
352

D6RDV4

Homo sapiens (Human)
382

J3KPQ2

Homo sapiens (Human)
353

F6XNY7

Ornithorhynchus anatinus (Duckbill platypus)
327

A0A2K6FXK2

Propithecus coquereli (Coquerel's sifaka) (Propithecus verreauxi
354

coquereli)

E2RPZ0

Canis lupus familiaris (Dog) (Canis familiaris)
380

A0A2K6V732

Saimiri boliviensis boliviensis (Bolivian squirrel monkey)
381

A0A4X2K722

Vombatus ursinus (Common wombat)
353

A0A6P5KYE6

Phascolarctos cinereus (Koala)
355

A0A2K6FXJ4

Propithecus coquereli (Coquerel's sifaka) (Propithecus verreauxi
380

coquereli)

A0A4X2JZA4

Vombatus ursinus (Common wombat)
353

G1SX53

Oryctolagus cuniculus (Rabbit)
353

A0A2U3WI94

Odobenus rosmarus divergens (Pacific walrus)
353

A0A2K6V724

Saimiri boliviensis boliviensis (Bolivian squirrel monkey)
354

A0A3Q7XKC9

Ursus arctos horribilis

353

A0A452RBH1

Ursus americanus (American black bear) (Euarctos americanus)
353

G1QVY1

Nomascus leucogenys (Northern white-cheeked gibbon)
382

(Hylobates leucogenys)

G1QVX6

Nomascus leucogenys (Northern white-cheeked gibbon)
354

(Hylobates leucogenys)

G3SJY5

Gorilla gorilla gorilla (Western lowland gorilla)
354

A0A7N9CSX2

Macaca fascicularis (Crab-eating macaque) (Cynomolgus
353

monkey)

A0A384B2Q9

Balaenoptera acutorostrata scammoni (North Pacific minke
353

whale) (Balaenoptera davidsoni)

A0A2K6L978

Rhinopithecus bieti (Black snub-nosed monkey) (Pygathrix
333

bieti)

A0A2K6AXE7

Macaca nemestrina (Pig-tailed macaque)
382

A0A2J8P0S9

Pan troglodytes (Chimpanzee)
354

W5PR22

Ovis aries (Sheep)
377

F7DJ88

Callithrix jacchus (White-tufted-ear marmoset)
382

A0A2K5R3Z8

Cebus imitator (Panamanian white-faced capuchin) (Cebus
354

capucinus imitator)

F6PHB6

Callithrix jacchus (White-tufted-ear marmoset)
354

M3WMC9

Felis catus (Cat) (Felis silvestris catus)
353

A0A2K5L5D5

Cercocebus atys (Sooty mangabey) (Cercocebus torquatus atys)
382

E1BNN4

Bos taurus (Bovine)
353

F6RFW7

Macaca mulatta (Rhesus macaque)
354

A0A2J8RKP9

Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii)
354

A0A3Q7RXX8

Vulpes vulpes (Red fox)
353

A0A2K5L5D9

Cercocebus atys (Sooty mangabey) (Cercocebus torquatus atys)
354

H0WJY2

Otolemur garnettii (Small-eared galago) (Garnett's greater
352

bushbaby)

A0A6P3ENQ6

Ovis aries (Sheep)
353

G3UA68

Loxodonta africana (African elephant)
377

A0A6P5DVT1

Bos indicus (Zebu)
360

A0A0D9RJS4

Chlorocebus sabaeus (Green monkey) (Cercopithecus sabaeus)
352

I3LTK7

Sus scrofa (Pig)
378

A0A2K5Z564

Mandrillus leucophaeus (Drill) (Papio leucophaeus)
382

A0A5G2R7I1

Sus scrofa (Pig)
353

A0A6I9JGH7

Chrysochloris asiatica (Cape golden mole)
353

A0A2K5Z517

Mandrillus leucophaeus (Drill) (Papio leucophaeus)
354

A0A452FM79

Capra hircus (Goat)
353

F6SJH5

Equus caballus (Horse)
382

A0A2R9BTW5

Pan paniscus (Pygmy chimpanzee) (Bonobo)
353

A0A2Y9FNI2

Physeter macrocephalus (Sperm whale) (Physeter catodon)
353

A0A340WR35

Lipotes vexillifer (Yangtze river dolphin)
353

A0A6J2DJL3

Zalophus californianus (California sealion)
353

A0A4X1UZM3

Sus scrofa (Pig)
378

A0A673TX31

Suricata suricatta (Meerkat)
381

A0A341D5X7

Neophocaena asiaeorientalis asiaeorientalis (Yangtze finless
353

porpoise) (Neophocaena phocaenoides subsp. asiaeorientalis)

A0A667FWA1

Lynx canadensis (Canada lynx)
376

A0A5B7H9S7

Portunus trituberculatus (Swimming crab) (Neptunus
74

trituberculatus)

A0A337SC50

Felis catus (Cat) (Felis silvestris catus)
376

H2RD19

Pan troglodytes (Chimpanzee)
382

A0A2U3X849

Leptonychotes weddellii (Weddell seal) (Otaria weddellii)
365

G3THK6

Loxodonta africana (African elephant)
360

A0A2U3V1E1

Tursiops truncatus (Atlantic bottle-nosed dolphin) (Delphinus
353

truncatus)

A0A096NIY4

Papio anubis (Olive baboon)
382

A0A6P3PSZ2

Pteropus vampyrus (Large flying fox)
353

A0A2K5EFR2

Aotus nancymaae (Ma's night monkey)
382

A0A3Q7QKC2

Callorhinus ursinus (Northern fur seal)
353

F7DVJ0

Monodelphis domestica (Gray short-tailed opossum)
346

A0A2K5EFU2

Aotus nancymaae (Ma's night monkey)
354

A0A5F8H1F1

Monodelphis domestica (Gray short-tailed opossum)
347

A0A2Y9H826

Neomonachus schauinslandi (Hawaiian monk seal) (Monachus
353

schauinslandi)

G3W284

Sarcophilus harrisii (Tasmanian devil) (Sarcophilus laniarius)
355

A0A3Q0CTY5

Mesocricetus auratus (Golden hamster)
254

A0A6P5NS60

Mus caroli (Ryukyu mouse) (Ricefield mouse)
377

H0V671

Cavia porcellus (Guinea pig)
333

I3M1B1

Ictidomys tridecemlineatus (Thirteen-lined ground squirrel)
353

(Spermophilus tridecemlineatus)

Q7TQN6

Rattus norvegicus (Rat)
534

A0A287CZD4

Ictidomys tridecemlineatus (Thirteen-lined ground squirrel)
378

(Spermophilus tridecemlineatus)

A0A1W6KZ83

Cricetulus barabensis (striped dwarf hamster)
377

A0A6I9MCW1

Peromyscus maniculatus bairdii (Prairie deer mouse)
377

A0A6P3EVC3

Octodon degus (Degu) (Sciurus degus)
353

A0A1S3FD42

Dipodomys ordii (Ord's kangaroo rat)
603

A0A6A4VE33

Amphibalanus amphitrite (Striped barnacle) (Balanus
358

amphitrite)

A0A4P2TKU6

Paralichthys olivaceus (Bastard halibut) (Hippoglossus
354

olivaceus)

A0A670IDE8

Podarcis muralis (Wall lizard) (Lacerta muralis)
358

A0A1U7S163

Alligator sinensis (Chinese alligator)
350

A0A670Y2N7

Pseudonaja textilis (Eastern brown snake)
385

K7FFW2

Pelodiscus sinensis (Chinese softshell turtle) (Trionyx sinensis)
369

D9N3D0

Coturnix japonica (Japanese quail) (Coturnix coturnix japonica)
378

Q6VZZ7

Mus musculus (Mouse)
377

D8KWH6

Zonotrichia albicollis (White-throated sparrow)
354

A0A674PPK4

Takifugu rubripes (Japanese pufferfish) (Fugu rubripes)
363

A0A674HDZ6

Taeniopygia guttata (Zebra finch) (Poephila guttata)
354

H2V568

Takifugu rubripes (Japanese pufferfish) (Fugu rubripes)
394

A0A6J0H1N3

Lepidothrix coronata (blue-crowned manakin)
351

A0A672UEH1

Strigops habroptila (Kakapo)
377

A0A672UBX7

Strigops habroptila (Kakapo)
349

A0A6J0U919

Pogona vitticeps (central bearded dragon)
348

A0A6J8E395

Mytilus coruscus (Sea mussel)
317

A0A6J7ZZ06

Mytilus coruscus (Sea mussel)
235

A0A2J8RKQ7

Pongo abelii (Sumatran orangutan) (Pongo pygmaeus abelii)
382

A0A2J8P0V4

Pan troglodytes (Chimpanzee)
353

A0A212D584

Cervus elaphus hippelaphus (European red deer)
263

Example 5
Animals:

8-16 weeks rd1/rd1 retinitis pigmentosa (RP) model mice, which were fed on a 12/12 light/dark cycle (lights off at 8 μm).

Construction of AAV Vector:

The plasmids needed to package 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):

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

Intraocular Injection of AAV into Mice:

After anesthesia, mice were injected with 1l AAV into the vitreous cavity after passing through the sclera with ultra-fine glass electrode, and the electrode was pulled out after several seconds. Follow up experiments were conducted 4 weeks after AAV injection.

Immunofluorescence:

In order to confirm whether AAV successfully infects retinal cells and compare the infection efficiency and virus specificity among various AAV subtypes, the immunofluorescence experiment is needed. After 4 weeks of AAV injection, the mouse retina was taken out and fixed in 4% paraformaldehyde for 30 minutes. The fixed and cleaned retina was embedded, and was sliced vertically with Leica cryomicrotome, with a thickness of 15 μm. The slices were washed with PBS, then sealed with 3% BSA (bovine serum albumin) at room temperature for 1 hour. Then the first anti-EGFP antibody is diluted with 3% BSA with 1:500, and incubated at 4° C. for 48 hours. After cleaning the first antibody, incubating it with the fluorescent labeled second antibody for 2 hours, pasting the stained retinal slice on the glass slide, and confocal scanning to obtain the fluorescence image after sealing. Analyzing and comparing the infection efficiency of each AAV to retinal ganglion cell (RGC), and the fluorescence intensity of EGFP, and select the AAV subtypes with high infection rate and good specificity for the next experiment.

Electrophysiological Test:

In order to further confirm whether cOPN5 maintains its physiological activity in RGC cells after successful expression of the AAV, electrophysiological experiments are needs. The AAVs having high infection rate and good specificity were injected into the eyes of rd1/rd1 (purchased from GemPharmatech Co., Ltd) mice. After 4 weeks of virus injection, the mouse retina was taken out and the retinal slice was placed in the electrophysiological recording chamber. The RGC layer of the retina was upward. In order to prevent light damage to the retina, the laser was turned off after the somatic cells expressing GFP were identified by the fluorescence microscope. The current intensity was recorded after cells were stimulated by 488 nm laser with different light intensity.

Behavior Test:

The visual receptor cells of RD1/rd1 mice have degenerated. To verify whether visual information can be transmitted to the brain through infected ganglion cells, so as to restore their lost visual function, we selected several visual function tests:

(1) Pupillary Light Reflex (PLR)

In Rd1/rd1 mice, the pupil can only respond to strong light. PLR experiment was conducted 4 weeks after injection of AAV into eyes of mice. Different intensity of light is utilized to stimulate the pupil of cOPN5 expressing mice and EGFP expressing mice to record the change degree of the pupil, and evaluate the sensitivity of mice to light through the change degree of the pupil.

(2) Open Field Avoidance Test

Normal mice will avoid open and bright spaces. This innate tendency is the basis for a simple test of their visual ability. In the experiment, the mice were placed in a lighted space, and there was also a dark shelter. The visual ability of mice was evaluated by measuring the proportion of time they spent.

Safety Test:

Long term heterologous expression of genes will have different effects on expressed tissues. Long term experiments are needed to evaluate the safety of heterologous expression, and test whether heterologous expression genes will be stably expressed in tissues for a long time. AAV was injected into the eyes for 6 months, and the above immunofluorescence, electrophysiological test and behavioral test were repeated one year later to detect the expression level of cOPN5, and whether the physiological activity changed due to long-term expression, and detect whether there is inflammatory reaction in retinal tissue.

Results:

As shown in FIG. 11, A showed expression of cOPN5 protein in retinal ganglion cells in the rd1/rd1 mouse;

- B shows microglia marker Iba1 staining of retinal slices after injection. H₂O₂-injected mice (positive control) showed strong activation of microglia. Few basal Iba1 signals were observed in the AAV-cOPN5-t2a-EGFP injected retina after 1 month injection, similar to that observed in AAV-EGFP-injected retina, AAV-cOPN5-t2a-EGFP injected retina after 10 month injection and no injection retinal. Red, Iba1; green, cOPN5 or EGFP; blue, DAPI (4′,6-diamidino-2-phenylindole) signal indicating cell nuclei. Scale bar, 50 μm;
- C shows RGC marker brn3a staining of retinal slices. Red, brn3a; green, cOPN5; blue, signal indicating cell nuclei. Scale bar, 50 μm.

D shows Fundus fluorescence imaging.

As shown in FIG. 12, A shows representative responses of RGC from C3H mice injected AAV-Copn5-t2a-EGFP during different power 488 nm laser stimulation;

- B shows representative responses of RGC from C3H mice injected AAV-Copn5-t2a-EGFP during different power 561 nm laser stimulation;
- C shows raw trace that cOpn5 mediated reliable and reproducible photoactivation of RGC;
- D and E Group data show the RGC firing rates after different power 488 nm laser stimulation, (n=6);
- F Group data show the delay time after different power 488 nm laser stimulation. (n=6)

As shown in FIG. 13, A shows representative responses of v1 neurons from C57 mice during 2s 200 lux light stimulation;

- B shows representative responses of vi neurons from C3H mice injected AAV-EGFP during 2s 200 lux light stimulation;
- C shows representative responses of vi neurons from C3H mice injected AAV-cOPN5-t2a-EGFP during 2s 200 lux light stimulation;
- D shows heat maps indicating the ROC representation of the peristimulus time histogram data from the C57 mice vi neurons that were tested 2s 200 lux light stimulation. (n=107);
- E shows heat maps indicating the ROC representation of the peristimulus time histogram data from the C3H mice injected AAV-EGFP vi neurons that were tested 2s 200 lux light stimulation. (n=133);
- F shows heat maps indicating the ROC representation of the peristimulus time histogram data from the C3H mice injected AAV-cOPN5-t2a-EGFP v1 neurons that were tested 2s 200 lux light stimulation. (n=100);
- G shows visually evoked potentials (VEPs) of C57(top), AAV-EGFP injected rd/rd mice (middle), and AAV-cOPN5-EGFP injected rd1/rd1 under 2s light illumination. (n=6).

FIG. 14 Schematically Shows Open Field Avoidance Test:

Method: The light/dark box (45×27×25 cm) was made of Plexiglas and consisted of two chambers connected by an opening (4×5 cm) located at floor level in the center of the dividing wall. The light box occupies about ⅔ of the whole light/dark box, and the dark box occupy about ⅓ of the whole light/dark box. The test field was diffusely illuminated at 200 lux. Mice were carried into the testing room in their home cage. A trial began when the mouse was placed inside the dark shelter for a 2-min habituation period, with the opening from dark to light spaces closed. The mouse was then allowed to leave the shelter and explore the illuminated field for 5 min. For each mouse, the length of time the animal spent in the light side of the box was recorded. A video camcorder located above the center of the box provided a permanent record of the behavior of the mouse. Mice were then removed from the box and returned to the home cage.

The results of the open field avoidance test were shown in FIG. 15, wherein FIG. 15A shows that after 7 weeks, the blind (rd/rd) mice spent about 80% time in the light box, and the control mice (normal mice) spent about 50% time in the light box, and the AAV-EGFP injected rd1/rd1 mice spent about 30% time in the light box; and

FIG. 15B shows that after 9 months, the blind (rd/rd) mice spent about 80% time in the light box, and the control mice (normal mice) spent about 50% time in the light box, and the AAV-EGFP injected rd1/rd1 mice spent about 20% time in the light box.

FIG. 16 shows the restoration of light sensitivity in the eye of the AAV-cOPN5 treated rd1/rd1 mice after 7 weeks (A) and 9 months (B) respectively. It found that AAV-cOPN5 treated rd1/rd1 mice (C3H_O5) have similar % pupillary constriction (area) to the normal mice (C57), and the rd1/rd1 mice (C3H_EGFP) shows almost no % pupillary constriction (area).

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OPTOGENETIC VISUAL RESTORATION USING LIGHT-SENSITIVE GQ-COUPLED NEUROPSIN (OPSIN 5)

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