Compositions for use in photomodulation of intracellular g-protein signaling

Abstract

The present disclosure provides a composition used for optical control of intracellular G protein signaling, more specifically, a pharmaceutical composition, a medical material, and an experimental material, and a method for optically controlling intracellular G protein signaling.SOLUTION: To provide a pharmaceutical composition containing a nucleic acid molecule encoding either an amino acid sequence of Useda mydriiopsin or a homologous sequence thereof.SELECTED DRAWING: Figure 3

Description

[Technical Field]

[0001] The present disclosure relates to compositions for use in the optical regulation of intracellular G protein signaling, more specifically pharmaceutical compositions, medical materials, and experimental materials, as well as methods for the optical regulation of intracellular G protein signaling. [Background technology]

[0002] Retinitis pigmentosa (RIP) is a retinal disease caused by the degeneration of rod and cone photoreceptor cells due to aging or genetic factors. Patients with RIP typically experience symptoms such as night blindness, narrowed visual field, and / or decreased vision, which can eventually lead to blindness. RIP is designated as an intractable disease in Japan, affecting approximately one in 6,000 people, but no treatment has been established to date. On the other hand, in the retinas of patients with retinitis pigmentosa, it is known that even after photoreceptor cells degenerate and die, retinal cells located downstream of the photoreceptor cells, such as bipolar cells, amacrine cells, and retinal ganglion cells, remain functional. For this reason, there has been hope for the development of treatment and prevention methods for retinitis pigmentosa using optogenetic techniques that target retinal cells located downstream of the photoreceptor cells.

[0003] Patent Document 1 discloses that a therapeutic composition containing a vector containing a nucleic acid encoding human opsin is used to confer photoreceptive function to cells. However, when using human opsin, 11-cis retinal must be continuously supplied from an exogenous source to maintain the photoreceptive function of the opsin. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] International Publication No. WO2015 / 128624 Summary of the Invention [Problem to be solved by the invention]

[0005] Under these circumstances, the present inventors conducted extensive research and discovered that opsin from the coral Acropora tenuis has a photoreceptor function that can repeatedly control intracellular G protein signaling without the need for a continuous exogenous supply of 11-cis retinal. This function differs from previously reported functions of naturally occurring opsins in humans and / or vertebrates, which require an exogenous supply of 11-cis retinal, and was the first to be discovered by the present inventors. Thus, the present disclosure aims to provide a photoreaction system or photoreceptor function that utilizes Ustilago iliopsin or a nucleic acid molecule encoding said opsin, which can repeatedly control intracellular G protein signaling through photoreception (light ON / OFF) without requiring a continuous supply of 11-cis retinal from outside the opsin molecule. [Means for solving the problem]

[0006] From a first aspect, the present disclosure provides a pharmaceutical composition for treating or preventing a retinal disease, comprising: (i) Amino acid sequence of Usueda Midoriishiopsin (SEQ ID NO: 1); (ii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence (i), wherein a polypeptide consisting of said amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark; or (iii) An amino acid sequence in which one or more amino acids are substituted, added, inserted, and / or deleted in the amino acid sequence of (i) or (ii), wherein a polypeptide consisting of said amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark. The present invention provides a pharmaceutical composition comprising a nucleic acid molecule having a base sequence encoding any one of the above.

[0007] In a second aspect, the present disclosure provides use of the above-mentioned nucleic acid molecule for the manufacture of a pharmaceutical composition for treating or preventing a retinal disease.

[0008] In a third aspect, the present disclosure provides a transplant material for treating retinal diseases, comprising a culture of retinal cells containing the above-mentioned nucleic acid molecule.

[0009] In a fourth aspect, the present disclosure provides a biological membrane comprising an opsin and a G protein, wherein the opsin comprises a protein having the amino acid sequence described above.

[0010] In a fifth aspect, the present disclosure provides a method for regulating intracellular G protein signaling by light irradiation, the method comprising the step of irradiating a cell transformed with the above-mentioned nucleic acid molecule with light. [Effects of the Invention]

[0011] According to the present disclosure, it is possible to impart to a target cell a photoreceptor function that can repeatedly control intracellular G protein signaling by photoreception (ON / OFF of light). [Brief explanation of the drawings]

[0012] [Figure 1] 1 shows the absorption spectra of a pigment (opsin pigment) consisting of one of the opsins disclosed herein bound to 11-cis retinal under dark conditions and immediately after light irradiation. [Figure 2] The results of isomer analysis of retinal (oxime) before and after light irradiation are shown. "Syn" represents the syn-type isomer, "Anti" represents the anti-type isomer, "11" represents 11-cis-retinal, "AT" represents all-trans-retinal, and "9" represents 9-cis-retinal. [Figure 3] 1 shows the results of a GloSensor™ cAMP assay in cells expressing one of the opsins of the present disclosure. [Figure 4] 1 shows the results of a G protein activation assay using split luciferase in cells expressing one of the opsins of the present disclosure. DETAILED DESCRIPTION OF THE INVENTION

[0013] <Pharmaceutical Composition> In one aspect, the present disclosure provides a pharmaceutical composition for the treatment and prevention of retinal diseases, comprising: (i) the amino acid sequence of Acropora tenuis opsin (SEQ ID NO: 1); (ii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence, wherein a polypeptide consisting of the amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark; or (iii) An amino acid sequence in which one or more amino acids are substituted, added, inserted, and / or deleted in the amino acid sequence of (i) or (ii), wherein a polypeptide consisting of said amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark. The present invention provides a pharmaceutical composition (hereinafter also referred to as "the pharmaceutical composition of the present disclosure") comprising a nucleic acid molecule consisting of a base sequence encoding any one of the above.

[0014] In the present disclosure, a protein having the amino acid sequence (i), (ii), or (iii) is also referred to as the "opsin of the present disclosure."

[0015] The opsin-encoding nucleic acid molecules of the present disclosure can confer to cells a photoreceptor function that can repeatedly control intracellular G protein signaling through photoreception, and therefore the pharmaceutical compositions of the present disclosure can be effective in treating or preventing retinal diseases.

[0016] In the present disclosure, the term "retinal disease" refers to a disease that causes degeneration of retinal cells (particularly cone cells and / or rod cells). Specific examples of retinal diseases include retinitis pigmentosa, macular degeneration, diabetic retinopathy, retinal detachment, retinal dystrophy, uveitis, etc., with a retinal disease selected from the group consisting of retinitis pigmentosa, macular degeneration, diabetic retinopathy, and retinal detachment being more preferred, and retinitis pigmentosa being most preferred. The pharmaceutical composition of the present disclosure is effective in treating and / or preventing retinal diseases, more specifically, in treating and / or preventing retinal diseases selected from the group consisting of retinitis pigmentosa, macular degeneration, diabetic retinopathy, and retinal detachment, and more specifically, in treating and / or preventing age-related and / or hereditary retinitis pigmentosa.

[0017] In the present disclosure, "treatment and / or prevention of retinal disease" refers to improving one or more of the symptoms of the retinal disease or functions that have been reduced or lost due to the retinal disease, or preventing or suppressing the progression of symptoms or the reduction in functions. Here, the symptoms of the retinal disease and the functions that have been reduced or lost due to the retinal disease may be, for example, at least one selected from the group consisting of reduced contrast sensitivity, reduced visual acuity, color vision abnormalities, blurred vision, constricted visual field, photophobia, night blindness, photopsia, and floaters. In a specific embodiment, treatment and / or prevention of retinal disease refers to improving contrast sensitivity or preventing or suppressing the reduction in contrast sensitivity. In one embodiment, treatment and / or prevention of retinal disease refers to imparting to retinal cells (more specifically, retinal bipolar cells and / or retinal ganglion cells) that remain after the onset of the retinal disease the ability to repeatedly control intracellular G protein signaling through photoreception (hereinafter also referred to as "photoreceptor function"). In the present disclosure, a subject in need of "treatment and / or prevention of retinal disease" may be a vertebrate, more particularly a mammal, and more particularly a human.

[0018] In the present disclosure, the term "retinal cells" refers to any type of cell (more specifically, neuronal cells and glial cells) that constitutes the retina. In the context of treating and / or preventing retinal diseases or photoreceptor function, retinal cells refer to retinal neuronal cells, more specifically, cone cells, rod cells, bipolar cells, horizontal cells, amacrine cells (axonless cells), and retinal ganglion cells. Examples of retinal glial cells include Müller cells, astrocytes, microglia, and oligodendrocytes. Retinal neurons are preferred as cells that express the nucleic acid molecules encoding the opsins of the present disclosure contained in the pharmaceutical compositions of the present disclosure, with cone cells, rod cells, bipolar cells, horizontal cells, amacrine cells, and retinal ganglion cells being more preferred, with bipolar cells, horizontal cells, amacrine cells, and retinal ganglion cells being even more preferred, and bipolar cells and / or retinal ganglion cells being particularly preferred.

[0019] Opsins are known as photoreceptor proteins found widely in animals and microorganisms. Among them, opsins that are widely conserved in animal cells are proteins that constitute the visual pigment contained in the photoreceptor cells of the retina, and are also called visual opsins. Visual pigments are photoreceptors (also called opsin pigments) that consist of visual opsins and the chromophore 11-cis retinal or its analogs. Visual opsins are also called G protein-coupled opsins because they can be coupled to G proteins, and visual pigments are also called G protein-coupled photoreceptors. Recent advances in genome analysis technology have identified proteins with amino acid sequence similarity to visual opsins in animal genomes, some of which are called opsin-like proteins because they have opsin-like photoreceptor functions. Opsins and opsin-like proteins are known to exert photoreceptor functions in various cells and tissues, including the eye.

[0020] Opsin is a protein with seven transmembrane helices (and therefore has a seven-transmembrane structure when incorporated into a lipid bilayer), and it is known that the transmembrane structure of opsin contains a conserved lysine residue (corresponding to Lys296 in the seventh transmembrane region of bovine opsin) to which the retinal chromophore can bind. Vertebrate visual opsins bind 11-cis retinal and become active when 11-cis retinal is converted (isomerized) to all-trans retinal upon photoreception. Active visual opsins can activate G proteins and initiate intracellular G protein signaling. Because visual opsins are unstable in their active state, they eventually release all-trans retinal from the outside and are no longer able to activate G proteins. As a result, intracellular G protein signaling ceases. Subsequently, visual opsins return to their inactive, photoreceptive state by incorporating and binding extracellular 11-cis retinal. Thus, naturally occurring vertebrate visual opsins can regulate intracellular G protein signaling upon photoreception, but repeated regulation requires the replenishment of exogenous 11-cis retinal.

[0021] The opsins of the present disclosure share commonalities with naturally occurring vertebrate visual opsins in that they are incorporated into lipid bilayer membranes, are capable of binding 11-cis retinal, and convert the bound 11-cis retinal to all-trans retinal upon light reception, thereby becoming active. On the other hand, the opsin of the present disclosure differs from conventionally known naturally occurring vertebrate visual opsins in that it can maintain the binding of all-trans retinal converted from 11-cis retinal upon light reception and can convert the bound all-trans retinal back to 11-cis retinal in the dark. Specifically, the opsins of the present disclosure bind 11-cis retinal, and upon photoreception, the 11-cis retinal is converted to all-trans retinal, resulting in an active state capable of activating G proteins. The opsins of the present disclosure are stable in their active state and can continue to retain all-trans retinal. Furthermore, the opsins of the present disclosure can convert the bound all-trans retinal back to 11-cis retinal (re-isomerize) in the dark, returning to an inactive state capable of receiving light. As a result, the opsins of the present disclosure can repeatedly regulate intracellular G protein signaling through photoreception, even without the need for exogenous replenishment (i.e., continuous supply) of 11-cis retinal. In one preferred embodiment, the re-isomerization is thermal.

[0022] The opsin functions described above were first discovered in naturally occurring opsins such as Ustilago spp. The amino acid sequence of Ustilago spp. (SEQ ID NO: 1) is registered in The ReFuGe 2020 Consortium39, https: / / aten.reefgenomics.org / , under accession number aten_0.1.m1.12427.m1. The present inventors first analyzed the function of Ustilago spp. and elucidated its photoreceptor function. Thus, the opsins of the present disclosure: (i) Amino acid sequence of Usueda Midoriishiopsin (SEQ ID NO: 1); (ii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, wherein a polypeptide consisting of said amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark; or (iii) An amino acid sequence in which one or more amino acids are substituted, added, inserted, and / or deleted in the amino acid sequence of (i) or (ii), wherein a polypeptide consisting of said amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark. It is a protein having either of the following:

[0023] The sequence homology of the amino acid sequence (ii) to the amino acid sequence of SEQ ID NO: 1 can be, more specifically, 91% or more, 92% or more, 93% or more, 94% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more. In the present disclosure, "sequence homology" can be determined by analyzing two sequences using the Blast algorithm (e.g., BLAST+2.15.0; available from the National Center for Biotechnology Information (NCBI) site) with default parameters. In the present disclosure, "plurality" refers to a positive integer of 10 or less, for example, an integer of 2 to 9, 2 to 8, 2 to 7, 2 to 6, 2 to 5, or 2 to 4, in relation to amino acid substitutions, additions, insertions, and / or deletions. When amino acid substitutions are present, conservative substitutions are preferred. Conservative substitutions refer to substitutions with amino acids that have similar properties, such as acidity, basicity, polarity, or hydrophobicity / hydrophilicity, to the original amino acid. Specifically, conservative substitutions refer to substitutions between (Phe, Trp, Tyr), between (Leu, Ile, Val), between (Lys, Arg, His), between (Asp, Glu), and between (Ser, Thr).

[0024] The opsins of the present disclosure may have the same amino acid sequence as opsins of organisms other than Acropora, such as orthologues, as long as they meet the above-mentioned predetermined requirements. In addition, opsins or modified versions thereof derived from other cnidarians, specifically organisms belonging to the phylum Cnidaria and class Anthozoa, are also included in the opsins disclosed herein, as long as the polypeptides comprise an amino acid sequence that has at least 90% sequence identity with the amino acid sequence of SEQ ID NO: 1, and that can bind 11-cis retinal and convert all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark.

[0025] The details of the mechanism by which the opsins of the present disclosure convert all-trans retinal to 11-cis retinal in the dark after becoming active are unknown. However, analysis of the amino acid sequences of opsins with similar functions to the opsins of the present disclosure reveals that the amino acid residue corresponding to cysteine ​​185 in the amino acid sequence of SEQ ID NO: 1 is conserved. The inventors therefore believe that the presence of this residue may be involved in the function of the opsins of the present disclosure. Therefore, it is preferable that the amino acid residue corresponding to cysteine ​​185 in the amino acid sequence of SEQ ID NO: 1 is conserved in the opsins of the present disclosure. Furthermore, in the opsins of the present disclosure, the lysine residue to which retinal can bind is thought to be Lys294 in the amino acid sequence of SEQ ID NO: 1, which corresponds to Lys296 in bovine opsin. Therefore, it is preferable that the opsins of the present disclosure conserve Lys294 or the corresponding lysine residue.

[0026] The opsins of the present disclosure can form G protein-coupled photoreceptors with G proteins. In the present disclosure, the G protein is not particularly limited as long as it can couple with the opsin of the present disclosure and initiate intracellular signal transduction in response to activation of the opsin of the present disclosure. Whether a G protein can couple with the opsin of the present disclosure can be confirmed in advance by those skilled in the art using known methods for evaluating G protein activity. For example, if the G protein is a Gs-type or Gi-type (Gi / o-type), a method can be used to measure the intracellular cyclic AMP concentration, which changes depending on the activity of the G protein. Detailed methods are described in the Examples. The G protein may be a G protein (endogenous G protein) naturally expressed in cells expressing a nucleic acid molecule encoding the opsin of the present disclosure, or a G protein (exogenous G protein) not naturally expressed in the cells. The nucleic acid molecule encoding the exogenous G protein can be introduced into the cells before, after, or simultaneously with the introduction of the nucleic acid molecule encoding the opsin of the present disclosure. The opsins of the present disclosure preferably conserve the "NPxxY" motif (positions 300 to 304 in SEQ ID NO: 1), which may be involved in activating G proteins.

[0027] The nucleic acid molecule contained in the pharmaceutical composition of the present disclosure comprises a base sequence encoding any one of the amino acid sequences (i) to (iii) above. The nucleic acid molecule contained in the pharmaceutical composition of the present disclosure can be single-stranded or double-stranded DNA, RNA, or cDNA, but is preferably double-stranded DNA. Those skilled in the art can use available cloning techniques to prepare nucleic acid molecules suitable for transfection or transduction into cells. The base sequences of nucleic acid molecules encoding the opsins of the present disclosure are preferably codon-optimized for expression in vertebrate cells, more particularly mammalian cells, and especially human cells. The nucleic acid molecule contained in the pharmaceutical composition of the present disclosure may include a nucleic acid sequence encoding a signal peptide at the 5' end of the base sequence encoding the amino acid sequence of an opsin of the present disclosure. Specific examples of signal peptides include MDYGGALSAVGRELLFVTNPVVVNGS (SEQ ID NO: 3), MAGHSNSMALFSFSLLWLCSGVLGTEF (SEQ ID NO: 4), MGLRALMLWLLAAAGLVRESLQG (SEQ ID NO: 5), MRGTPLLLVVSLFSLLQD (SEQ ID NO: 6), KSRITSEGEYIPLDQIDINV (SEQ ID NO: 7), MTETLPPVTESAVALQAE (SEQ ID NO: 8), and MKPPLLVFIVCLLWLKDSHCA (SEQ ID NO: 9).

[0028] The nucleic acid molecule contained in the pharmaceutical composition of the present disclosure may include a promoter and various other expression control elements (e.g., terminators, enhancers) suitable for expression in a host cell at the 5' and / or 3' ends of the base sequence encoding the amino acid sequence of the opsin of the present disclosure. The promoter mediates the expression of an operably linked nucleic acid sequence. Preferably, the promoter can direct cell-specific gene expression to retinal cells (specifically, bipolar cells or retinal ganglion cells). Examples of suitable promoters can be selected from the group consisting of the L7 promoter, thy-1 promoter, recoverin promoter, calbindin promoter, human CMV promoter, GAD-67 promoter, chicken β-actin promoter, hSyn promoter, Grm6 promoter, Grm6 enhancer-SV40 promoter, 200En-mGluR500P promoter, 770En_454P (hGRM6), and 444En_454P (hGRM6) promoter. See also Japanese Patent No. 7397532 for details on bipolar cell-specific promoters / enhancers. Alternatively, the promoter may be a promoter that directs ubiquitous expression, such as a pan-neuronal promoter, an example of which is the CAG promoter.

[0029] As used herein, "operably linked" refers to the juxtaposition of genetic elements such that they are in a relationship that allows them to function in a predicted manner. For example, a promoter is operably linked to a coding region if the promoter helps initiate transcription of that coding region. Intervening residues can be present between the promoter and the coding region so long as this functional relationship is maintained.

[0030] A nucleic acid molecule comprising a base sequence encoding an opsin of the present disclosure may be contained in or incorporated into a vector. The vector backbone may be appropriately selected so that it is capable of autonomous replication in the intended host cell or capable of integration into the chromosome. The vector may also contain an appropriate selection marker (e.g., a drug resistance gene). The vector can be viral or non-viral, for example, a plasmid. Viral vectors include those derived from adenovirus, adeno-associated virus (AAV), including mutant forms, retrovirus, lentivirus, herpesvirus, vaccinia virus, MMLV, GaLV, simian immunodeficiency virus (SIV), HIV, poxvirus, and SV40. Viral vectors typically do not integrate into the genome of the retinal cells targeted for administration and can persist in an extrachromosomal state.

[0031] Viral vectors can be modified to delete any sequence. For example, in AAV, viral sequences can be modified to delete all or part of the IX gene, E1a gene, and / or E1b gene. Wild-type AAV does not require a helper virus such as adenovirus, and replication efficiency is extremely low. In recombinant adeno-associated viruses, replication and capsid genes are preferably provided in trans (in a pRep / Cap plasmid), leaving only the two ITRs of AAV to be packaged into virions, while the necessary adenoviral genes are provided by the adenovirus or another plasmid. Similar modifications can be made to lentiviral vectors.

[0032] Viral vectors are capable of entering the target host cells. However, non-viral vectors, such as plasmids, can be facilitated by the use of auxiliary agents to facilitate uptake by the target cells. Such auxiliary agents include polycationic agents such as polyethyleneimine. Alternatively, delivery systems such as liposome-based delivery systems can be used.

[0033] To introduce a nucleic acid molecule encoding an opsin of the present disclosure into a retinal cell, it is preferable to use a vector suitable as an expression vector (more specifically, an expression plasmid or an expressing viral vector). The type of expression vector and the types of regulatory elements can be appropriately selected depending on the host cell, but an AAV vector is preferred, and a self-contained adeno-associated virus (scAAV) is particularly preferred.

[0034] From another perspective, a pharmaceutical composition for treating or preventing a retinal disease may comprise a nucleic acid molecule that hybridizes under stringent conditions to a nucleic acid molecule consisting of a nucleotide sequence (e.g., SEQ ID NO: 2) encoding the amino acid sequence of any of the aforementioned (i) to (iii), wherein the polypeptide consisting of the amino acid sequence encoded by the nucleotide sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal upon photoreception back to 11-cis in the dark. Preferably, the polypeptide additionally satisfies one or more of the requirements described above for the opsin of the present disclosure. In the present disclosure, stringent conditions refer to, for example, hybridization in 5x or 6x SSC (which may contain 50% formamide) at at least 60°C (e.g., 63°C, 65°C, 68°C, or 70°C). These stringent hybridization conditions may also involve washing in 0.1 to 1x SSC at about 40 to 60°C. 1x SSC refers to a solution containing 0.15 M NaCl and 0.015 M sodium citrate. The same points as those described above regarding the nucleic acid molecule encoding the opsin of the present disclosure also apply to nucleic acid molecules that hybridize under stringent conditions with nucleic acid molecules consisting of a base sequence encoding amino acid sequences (i) to (iii). Hereinafter, nucleic acid molecules that hybridize under stringent conditions with nucleic acid molecules consisting of base sequences encoding amino acid sequences (i) to (iii) and nucleic acid molecules encoding opsin of the present disclosure are collectively referred to as "nucleic acid molecules of the present disclosure."

[0035] Pharmaceutical compositions containing the nucleic acid molecules of the present disclosure can be formulated as liquid, semi-solid, or solid forms, such as powder, gel, or paste, and are preferably in liquid form. They may further contain one or more of enzymes, excipients, stabilizers, preservatives, buffers, emulsifiers, minerals, vitamins, etc., as appropriate, as long as they do not promote or interfere with the desired therapeutic and / or prophylactic effect. A pharmaceutical composition containing a nucleic acid molecule of the present disclosure may be configured as a kit. The kit may include, in addition to the pharmaceutical composition containing a nucleic acid molecule of the present disclosure, one or more of a drug containing 11-cis retinal, a syringe or eye dropper container for administration, instructions, etc. Pharmaceutical compositions comprising the nucleic acid molecules of the present disclosure can also be administered to the eye by subretinal or intravitreal administration, in which case the pharmaceutical composition is preferably an injectable liquid, preferably provided in a syringe or the like. The effective dose of the nucleic acid molecule can be determined appropriately by those skilled in the art. In the case of subretinal administration, the effective dose is 1×10 9 ~1×10 14 or 7.5 x 10 15 , preferably 1 x 10 11 ~7.5×10 13 The number of nucleic acid molecules, vectors or viral particles may be In addition, those skilled in the art can refer to the technology described in Patent Document 1, which discloses a therapeutic composition using human-derived opsin, and the technology described in Japanese Patent No. 6757024, which discloses a composition using a chimeric rhodopsin that combines animal and microbial opsins.

[0036] The nucleic acid molecules of the present disclosure are suitable for use in the manufacture of a pharmaceutical composition for the treatment or prevention of retinal diseases. The base sequences of the nucleic acid molecules of the present disclosure are preferably codon-optimized for expression in vertebrate cells, more particularly mammalian cells, and especially human cells. The nucleic acid molecule of the present disclosure may be used in a form linked under the control of a promoter sequence operable in retinal cells (specifically, a retinal cell-specific promoter, more specifically, a bipolar cell- and / or retinal ganglion cell-specific promoter). Alternatively, the nucleic acid molecule of the present disclosure may be used by being incorporated into a vector having a promoter sequence operable in retinal cells and under the control of the promoter sequence. The nucleic acid molecules of the present disclosure, optionally together with a promoter sequence, may be maintained or have been maintained in prokaryotic (e.g., E. coli) or fungal (e.g., yeast) cells, for example, as a plasmid. Alternatively, the nucleic acid molecules of the present disclosure, optionally together with a promoter sequence, may be present in a viral vector that is maintained in a host cell. In a preferred embodiment, the nucleic acid molecule of the present disclosure is a nucleic acid molecule encoding an opsin of the present disclosure.

[0037] <Transplant material> In another aspect, the present disclosure provides a transplant material for treating retinal diseases (hereinafter also referred to as the "transplant material of the present disclosure") comprising a culture of retinal cells containing a nucleic acid molecule of the present disclosure. In one embodiment, the retinal disease is a retinal disease selected from the group consisting of retinitis pigmentosa, macular degeneration, diabetic retinopathy, and retinal detachment, hi a specific embodiment, the retinal disease is retinitis pigmentosa. The retinal cells contained in the transplant material of the present disclosure may be derived from retinal cells collected from the eye of a patient with a retinal disease, or may be pluripotent stem cells such as somatic stem cells, embryonic (ES) cells, or iPS cells, or cells obtained by differentiating progenitor cells such as retinal progenitor cells, photoreceptor progenitor cells, or bipolar cell progenitor cells into retinal cells in an appropriate differentiation-inducing medium using known methods (e.g., WO2012 / 173207). Allogeneic cells (preferably allogeneic cells) may be used, but from the viewpoint of biocompatibility, it may be preferable to use cells collected from a patient with a retinal disease and induced to differentiate as needed (autologous cells). The retinal cells contained in the transplant material of the present disclosure, which contain the nucleic acid molecule of the present disclosure and express the opsin encoded by the nucleic acid molecule, are retinal nerve cells, more specifically, one or more cells selected from the group consisting of cone cells, rod cells, bipolar cells, horizontal cells, amacrine cells (axonless cells), and retinal ganglion cells. The transplant material of the present disclosure may also contain retinal glial cells. The retinal glial cells are, for example, one or more cells selected from the group consisting of Müller cells, astrocytes, microglia, and oligodendrocytes, and preferably include Müller cells.

[0038] The implant materials of the present disclosure can be produced by transforming retinal cells with a nucleic acid molecule of the present disclosure, such that the transformed retinal cells express the opsin encoded by the nucleic acid molecule of the present disclosure. For transformation, a vector (e.g., as described above for the pharmaceutical composition) containing the nucleic acid molecule of the present disclosure and, optionally, a promoter or other expression regulatory elements can be used. Transformation may be performed on retinal cells, preferably retinal cells themselves (primary cells) collected from the eye of a patient to be transplanted, or on retinal cells that have been grown and / or passaged in culture from the primary cells. Alternatively, pluripotent stem cells or progenitor cells may be induced to differentiate into desired retinal cells, and the resulting retinal cells may be transformed. Those skilled in the art can appropriately select a transformation method from known methods depending on the cells to be transformed.

[0039] The transplant material of the present disclosure may contain a culture medium suitable for maintaining the transformed retinal cells contained in the transplant material and / or for expressing the opsin encoded by the nucleic acid molecule of the present disclosure in the transformed retinal cells. Those skilled in the art can appropriately determine and use such a culture medium and appropriate culture conditions. The transplant material of the present disclosure may also further contain extracellular matrix components secreted by the retinal cells contained in the transplant material during maintenance culture. The transplant material of the present disclosure can also be provided as a cell suspension, a cell sheet, or a retinal organoid. The implant material of the present disclosure can be implanted, for example, subretinally using a syringe needle, or can be implanted into a suitable site (eg, the site of degeneration) through an incision in the eyeball. In a preferred embodiment, the nucleic acid molecule of the present disclosure is a nucleic acid molecule encoding an opsin of the present disclosure.

[0040] <Biomembrane> In another aspect, the present disclosure provides a biological membrane comprising an opsin and a G protein of the present disclosure (hereinafter also referred to as the "biological membrane of the present disclosure"). The biological membranes of the present disclosure may be used as tools in optogenetics. The biological membrane of the present disclosure is not particularly limited as long as it is a lipid bilayer membrane (excluding the biological membrane of Acropora spp.), and may be a cell membrane or a membrane constituting a liposome. The biological membrane may be either a closed or open system. The cells are not particularly limited as long as they are excluding Acropora spp. cells, and may be, for example, prokaryotic cells, yeast cells, insect cells, or animal cells (more specifically, vertebrate cells, more specifically, mammalian cells, and more specifically, human cells). The cells may be established or immortalized, and may be, for example, HEK293 cells, COS cells, CHO cells, BHK cells, 3T3 cells, or HeLa cells. The cells may be retinal cells. In the biological membrane of the present disclosure, the opsin of the present disclosure may form a G protein-coupled photoreceptor with a G protein. When the biological membrane is a cell membrane, the G protein may be endogenous or exogenous to the cell membrane. The biological membrane of the present disclosure may contain opsins other than the opsins of the present disclosure as long as they contain the opsins of the present disclosure. When the biological membrane is a cell membrane, the opsins other than the opsins of the present disclosure may be endogenous or exogenous to the cell membrane. The biomembrane of the present disclosure may be used in an artificial biomembrane or biodevice in which membrane proteins and lipids are integrated on the surface of a solid substrate.

[0041] <Control method> In yet another aspect, the present disclosure provides a method for controlling intracellular G protein signaling by light irradiation (hereinafter also referred to as the "control method of the present disclosure"), comprising the step of irradiating a cell transformed with a nucleic acid molecule of the present disclosure with light. Because opsins encoded by the nucleic acid molecules of the present disclosure can maintain the retinal once retained within the molecule, regardless of whether it is in the 11-cis or all-trans form, the activity of the G protein coupled to the opsin can be controlled by irradiating the cell with light (by turning the light on / off and / or increasing or decreasing the amount of light), thereby controlling intracellular G protein signaling. In other words, the control method of the present disclosure allows repeated control of G protein signaling by light irradiation without requiring the opsin encoded by the nucleic acid molecule of the present disclosure to reuptake (resupply) 11-cis retinal from outside the molecule. More specifically, the control method of the present disclosure enables intracellular G protein signaling to be activated or enhanced (ON) by irradiating cells with light, and can stop or suppress (OFF) intracellular G protein signaling in the dark. The control method of the present disclosure can be used as a tool in optogenetics. Furthermore, the control method of the present disclosure can be used to elucidate and develop treatment technologies for psychiatric disorders, which have been developed in recent years, instead of methods using microbial bacteriorhodopsin.

[0042] In the control methods of the present disclosure, cells transformed with the nucleic acid molecules of the present disclosure are not limited to retinal cells, but may be any desired cells (e.g., prokaryotic cells, yeast cells, insect cells, animal cells, etc., preferably animal cells (e.g., HEK293 cells, COS cells, CHO cells, BHK cells, 3T3 cells, HeLa cells, etc.); however, Acropora cells are excluded). Transformed cells are also useful, for example, for maintaining and propagating the nucleic acid molecules of the present disclosure or for expressing and producing the opsins of the present disclosure. The nucleic acid molecule of the present disclosure used in the control method of the present disclosure may be contained in or incorporated into a vector. The vector backbone may be capable of autonomous replication in the transformed cell or may be capable of integration into the chromosome. The vector may contain an appropriate selection marker (e.g., a drug resistance gene). The vector may be a plasmid. The vector may be an expression plasmid containing a promoter suitable for expression in a host cell.

[0043] In the control method of the present disclosure, culture conditions suitable for the expression of opsin encoded by the nucleic acid molecule of the present disclosure can be appropriately determined by one skilled in the art, taking into consideration the type of host cell, vector, etc. Preferably, the culture medium contains 11-cis retinal and / or a precursor of 11-cis retinal, such as a substance that is metabolized into 11-cis retinal within cells, such as a retinoid (also called vitamin A) including retinal isomers, retinol, and retinoic acid, and / or a carotenoid (also called provitamin A) including β-carotene.

[0044] In the present disclosure, the light irradiated onto the cells includes at least a wavelength that can be received by (exogenous) opsin expressed in the cells to convert (isomerize) the bound 11-cis retinal to all-trans retinal. Other irradiation conditions, such as irradiation intensity and irradiation time, can be appropriately determined by those skilled in the art. For example, when the cells are animal cells, a culture containing the animal cells is irradiated with light containing blue light at a concentration of 50 μmol m -2 s -1By irradiating the light at the above intensity for 1 second or more, 11-cis retinal can be isomerized, and the opsin bound to retinal can become active. In a preferred embodiment, the nucleic acid molecule of the present disclosure is a nucleic acid molecule encoding an opsin of the present disclosure.

[0045] Preferably, the control method of the present disclosure further comprises the step of placing the light-irradiated cells in a dark place. In the present disclosure, "dark place" or "in the dark" does not necessarily mean a completely dark place (under complete darkness), but may mean a dark place (photon density 0) with respect to light of a wavelength to which the opsin encoded by the nucleic acid molecule of the present disclosure having at least 11-cis retinal bound thereto (particularly the opsin of the present disclosure) is sensitive / responsive, and light of a wavelength to which the opsin is not sensitive / responsive (e.g., dim red light (wavelength >690 nm)) may be irradiated. By placing the cells in the dark, the opsins expressed in the cells can be efficiently restored to an inactive state that allows them to receive light.

[0046] The disclosed biological membranes (where the biological membrane is a cell membrane) and the disclosed control methods do not include embodiments in which cells are present in vivo in humans and / or animals, unless such embodiments are not patentable under applicable law. In other words, embodiments in which cells are present in vivo in humans and / or animals may be excluded from the scope of the claims, depending on applicable law. Details of the genetic recombination / expression techniques required in connection with the present disclosure are described in standard textbooks (e.g., Ausubel, F.A. et al. (eds.) (1988), Current Protocols in Molecular Biology, John Wiley and Sons, New York, NY; Sambrook, J. et al. (1989), Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY). [Example]

[0047] The present disclosure will be explained in more detail below using examples, but the present disclosure is not limited thereto in any way.

[0048] (1) Spectroscopic analysis of retinal isomerization Materials and Methods A putative opsin gene was identified based on the genome sequence of Acropora tenuis, and a cDNA encoding the putative opsin protein was obtained by cloning (SEQ ID NO: 2). This cDNA encodes the amino acid sequence (SEQ ID NO: 1). A base sequence encoding the epitope sequence of the anti-1D4 antibody (ETSQVAPA; SEQ ID NO: 10) was added to the 3' end of this cDNA, and the resulting DNA was inserted into the multicloning site of the animal cell expression vector pUSRα (Kayada et al., Comp Biochem Physiol B Biochem Mol Biol. 110(3):599-604 1995). Cultured mammalian cells (COS-1) were transfected with the expression vector constructed above using the polyethylenimine method. The transfected cells were incubated in DMEM medium containing 10% fetal bovine serum at 37°C for 24 hours in a 5% CO2 incubator. 11-cis retinal was then added to the medium. All procedures after the addition of 11-cis retinal were performed under dim red light (>690 nm), which was previously confirmed to be insensitive to the putative opsin protein. The cells were harvested by centrifugation, and the cell membrane was solubilized using 50 mM HEPES buffer (pH 6.5) containing 0.1% n-dodecyl-β-D-maltoside (DDM) and 140 mM NaCl (Buffer A). This resulted in a crude extract containing the putative opsin pigment (putative opsin protein and retinal). The crude extract containing the putative opsin pigment was mixed with agar beads conjugated with anti-1D4 antibody, and the mixture was transferred to a spin column and washed with Buffer A. Finally, the putative opsin pigment was eluted with Buffer A containing 0.1 mg / L of 1D4 peptide. Using 300 μL of the eluate, the absorption spectrum was measured at 0 °C using a UV-Visible spectrophotometer (V-750, JASCO Corporation), and the dark spectrum of the putative opsin pigment was obtained. The same sample was then irradiated with blue light (460 nm) for 2 minutes, and the absorption spectrum was measured in the same way to obtain the post-irradiation spectrum of the putative opsin pigment. The spectral measurement results of the putative opsin pigment are shown in Figure 1.

[0049] result For the putative opsin pigment, no difference was observed between the dark spectrum and the spectrum after light exposure (Figure 1). For known visual opsins, the spectrum derived from 11-cis retinal is measured in the dark spectrum, while the spectrum derived from all-trans retinal is measured after light exposure, and differences are observed when comparing the two spectra. Due to the experimental setup, the post-illumination spectrum was measured 5 min after light irradiation, and the putative opsin pigment was left under dim red light (>690 nm) between light irradiation and measurement. Therefore, we hypothesized that re-isomerization of all-trans retinal to the original 11-cis retinal occurred while the putative opsin pigment was left under dim red light. To verify this hypothesis, we performed a more detailed analysis.

[0050] (2) HPLC analysis of retinal isomers Materials and Methods The extract containing the putative opsin pigment, prepared in the same manner as in (1), was irradiated with blue-green LED light (peak wavelength: 495 nm) on ice. Methanol and hydroxylamine were added to the pigment solution samples taken before, immediately after, and 10 minutes after light irradiation, respectively, to denature the opsin, and the retinal bound to the putative opsin pigment was oximated and separated. Furthermore, the oximated retinal was extracted from the sample by adding n-hexane and mixing. The sample taken 10 minutes after light irradiation was left under dim red light (>690 nm) for 10 minutes immediately after light irradiation.

[0051] The oxime-modified retinal extract was analyzed using HPLC (Shimadzu Corporation) under the following HPLC conditions: Detector: SPD-10A Shimadzu Measurement wavelength: 360 nm, full scale: 0.0100 AUFS Mobile phase: 15% ethyl acetate, 0.15% ethanol in n-hexane Mobile phase flow rate: 1 mL / min Column: YMC-Pack SIL column (inner diameter 6.0 mm x length 150 mm) Column temperature: 25 °C

[0052] result Figure 2 shows the results of an isomer analysis of retinal (oxime) extracted from a putative opsin pigment. The chromatograms shown in Figure 2 show, from bottom to top, the analysis results of retinal extracted from a pigment solution sample before light exposure, immediately after light exposure, and 10 minutes after light exposure. The horizontal axis of Figure 2 indicates elution time, with the peak at 7 minutes representing 11-cis retinal and the peak at 7.5 minutes representing all-trans retinal. Both peaks are syn-retinal. According to the results shown in Figure 2, before light exposure, only 11-cis retinal added to the medium was detected. Immediately after light exposure, the amount of 11-cis retinal detected decreased, while the amount of all-trans retinal detected increased. This indicates that light exposure caused retinal to be isomerized from 11-cis to all-trans within the putative opsin pigment. In the sample 10 minutes after light exposure, the amount of all-trans retinal detected decreased, and the amount of 11-cis retinal detected approached the amount detected before light exposure. These findings suggest that the putative opsin protein re-isomerizes to the 11-cis form while retaining all-trans retinal when left in the dark.

[0053] (3) Measurement of intracellular cyclic AMP concentration Materials and Methods To confirm that the putative opsin protein couples with G proteins and controls intracellular signal transduction in animal cells, we expressed the putative opsin protein in human kidney-derived cultured cells (HEK293S) that express Gi proteins, and measured the GloSensor signal. TM The time course of intracellular cyclic AMP (cAMP) concentration after light irradiation was measured using the cAMP Assay (Promega). Intracellular cAMP is a major second messenger in animal cells, and its concentration is regulated by the activity of G proteins. The DNA region (SEQ ID NO: 2) encoding the amino acid sequence of the predicted opsin protein (SEQ ID NO: 1) was inserted into the multicloning site of the animal cell expression vector pMT (manufactured by Addgene).

[0054] Human kidney-derived cultured cells (HEK293S) were seeded in a 35 mm dish or a 96-well plate and cultured overnight in DMEM / F12 medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. TM HEK293S cells were transfected with -22F plasmid (Promega) and cultured overnight, then 11-cis retinal was added and cultured overnight. All subsequent operations were performed under dim red light (>690 nm), which opsin does not respond to. The medium was supplemented with 10% FBS and 2% GloSernsor TM The medium was replaced with DMEM containing cAMP reagent (Promega) but without 11-cis retinal. The transfected cells were irradiated with blue LED light (peak wavelength 450 nm) at 25°C, and luciferase luminescence was measured using a luminometer (Promega). The amount of luciferase luminescence positively correlated with intracellular cAMP.

[0055] The light irradiation conditions are as follows: Weak light: 82.160 μmol m -2 s -1 5 seconds Strong light: 821.596 μmol m -2 s -1 1 second During each irradiation, the sample was left in the dark (under dim red light (>690 nm), to which the putative opsin protein does not react).

[0056] result Figure 3 shows the results of measuring intracellular cyclic AMP concentrations in cells transfected with a putative opsin protein. The vertical axis shows the relative luminescence intensity compared to the average luminescence intensity immediately before the first light exposure, and the horizontal axis shows time, with the first light exposure being set to 0 minutes. The downward arrowheads above the graph indicate the time of each light exposure. The seven light exposures from the first light exposure up to 40 minutes after the first light exposure represent weak light exposures, while the three light exposures from 40 minutes onwards represent strong light exposures. In HEK293S cells expressing the putative opsin protein, a decrease in the relative luminescence intensity of luciferase was observed after light irradiation. This was thought to be due to the putative opsin protein becoming activated by light irradiation, which in turn activated the coupled Gi-type G protein, which then inhibited adenylate cyclase, resulting in a decrease in intracellular cAMP levels. Therefore, the protein with the amino acid sequence of SEQ ID NO: 1 was confirmed to be an opsin from Acropora spp.

[0057] Furthermore, after light exposure, cells expressing Acropora opsin were found to rapidly increase their relative luminescence in the dark, meaning that intracellular cAMP levels rapidly recovered. This recovery of intracellular cAMP levels is thought to occur when Acropora opsin becomes inactive in the dark, inactivating Gi protein and activating adenylate cyclase. In contrast, in previous experiments using native visual opsins, rapid recovery of intracellular cAMP levels in the dark after light exposure was not observed. Furthermore, when cells expressing Usuedamyoriishiopsin were again exposed to light after intracellular cAMP levels had recovered in the dark, they again showed a decrease in relative luminescence (i.e., intracellular cAMP levels decreased). The same change was reproduced each time the cells were exposed to light and placed in the dark more than 10 times. This suggests that once Usuedamyoriishiopsin binds 11-cis retinal, it can repeatedly control intracellular G protein signaling by light exposure (in other words, photoreception) without the need for exogenous retinal replenishment.

[0058] (4) Monitoring G protein activity using split luciferase Materials and Methods To confirm that Ustilago irisiopsin can also couple with Go-type G proteins that function in retinal bipolar cells, we performed experiments using split luciferase (NanoBiT (登録商標) System (Promega). A DNA region (SEQ ID NO: 2) encoding the amino acid sequence of Ustilago iliopsin (SEQ ID NO: 1) was inserted into the multicloning site of the animal cell expression vector pMT (manufacturer: Addgene). G proteins are known to separate into two subunits upon activation. The sequences encoding the split luciferases LgBiT and SmBiT (Promega) were fused to the 3' ends of the sequences encoding the α and βγ subunits of the Go type G protein, respectively, and inserted into the multicloning site of the animal cell expression vector pcDNA3.1 (manufacturer: Invitrogen).

[0059] Human kidney-derived cultured cells (HEK293S) expressing Go protein were seeded in 35 mm dishes or 96-well plates and cultured overnight in DMEM / F12 medium containing 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator. HEK293S cells were transfected with the two expression vectors constructed above using polyethyleneimine and cultured overnight. 11-cis retinal was then added and cultured overnight. All procedures after retinal addition were performed under dim red light (>690 nm), which opsin does not react to. Transformed HEK293S cells were exposed to blue LED light (peak wavelength 450 nm) at 71.4 μmol m -2 s -1 The cells were then exposed to light for 5 seconds at 25°C and placed in the dark. Luciferase luminescence was measured using a luminometer (Promega) before and after light exposure.

[0060] result Figure 4 shows the results of monitoring G protein activity using split luciferase. The vertical axis shows the relative luminescence intensity compared to the luciferase luminescence intensity immediately before light irradiation, and the horizontal axis shows the time, with light irradiation taken as 0 minutes. As a control, the relative luminescence intensity of untransformed HEK293S cells is shown. In cells expressing Ustilago iliopsin, a decrease in luciferase luminescence was observed after light irradiation. As mentioned above, when activated, G protein dissociates into two sets of subunits (α subunit and βγ subunit), and therefore activation of G protein decreases luciferase luminescence. Based on this, it is thought that in HEK293S cells expressing Ustilago iliopsin, light irradiation activates the opsin, which then activates the Go protein. On the other hand, it can be seen that in the control cells, the relative luminescence intensity does not change significantly before and after light irradiation.

[0061] Furthermore, when HEK293S cells expressing Ustilagosinus lignin were placed in the dark after light exposure, the relative luminescence intensity rapidly recovered to the same level as before light exposure. This is thought to be due to the inactivation of Ustilagosinus ligninus in the dark, which in turn inactivates the Go protein and causes the reassembly of the two sets of Go protein subunits, resulting in an increase in luciferase luminescence. These results demonstrate that Ustilago iliopsin can couple with the Go-type G protein that functions in retinal bipolar cells, activating the Go-type G protein in response to light and inactivating it in the dark. These results demonstrate that Ustilago iliopsin and its related opsins can repeatedly control intracellular G protein signaling through light reception.

[0062] The above-described embodiments and examples are described as examples to facilitate understanding of the present invention, and the present disclosure is not limited to the specific forms and examples described in the specification or the accompanying drawings. The specific configurations, means, and methods described in the present disclosure can be replaced with many other known in the art without departing from the gist of the present disclosure.

Claims

1. A pharmaceutical composition for treating or preventing a retinal disease, comprising: (i) the amino acid sequence of SEQ ID NO: 1; (ii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence, wherein a polypeptide consisting of the amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark; or (iii) An amino acid sequence in which one or more amino acids are substituted, added, inserted, and / or deleted in the amino acid sequence of (i) or (ii), wherein a polypeptide consisting of said amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark. A pharmaceutical composition comprising a nucleic acid molecule having a base sequence encoding any one of the above.

2. The pharmaceutical composition according to claim 1, wherein the amino acid sequence (ii) or (iii) is an amino acid sequence in which the amino acid residue corresponding to cysteine ​​at position 185 in the amino acid sequence of SEQ ID NO: 1 is conserved.

3. The pharmaceutical composition of claim 1, wherein the nucleic acid molecule is contained in a viral expression vector.

4. The pharmaceutical composition according to any one of claims 1 to 3, wherein the polypeptide consisting of the amino acid sequences (i), (ii), and (iii) is capable of binding to 11-cis retinal to form a G protein-coupled photoreceptor.

5. The pharmaceutical composition according to any one of claims 1 to 3, wherein the nucleic acid molecule is linked to a promoter sequence operable in retinal cells.

6. The pharmaceutical composition according to claim 5, wherein the retinal cells are bipolar cells and / or retinal ganglion cells.

7. The pharmaceutical composition according to any one of claims 1 to 3, wherein the retinal disease is selected from the group consisting of retinitis pigmentosa, macular degeneration, diabetic retinopathy, and retinal detachment.

8. The pharmaceutical composition according to any one of claims 1 to 3, wherein the retinal disease is retinitis pigmentosa.

9. (i) the amino acid sequence of SEQ ID NO: 1; (ii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence, wherein a polypeptide consisting of the amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark; or (iii) An amino acid sequence in which one or more amino acids are substituted, added, inserted, and / or deleted in the amino acid sequence of (i) or (ii), wherein a polypeptide consisting of said amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark. Use of a nucleic acid molecule comprising a base sequence encoding any one of the above for the manufacture of a pharmaceutical composition for treating or preventing a retinal disease.

10. The use according to claim 9 , wherein the nucleic acid molecule is linked to a promoter sequence operable in retinal cells.

11. (i) the amino acid sequence of SEQ ID NO: 1; (ii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence, wherein a polypeptide consisting of the amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark; or (iii) An amino acid sequence in which one or more amino acids are substituted, added, inserted, and / or deleted in the amino acid sequence of (i) or (ii), wherein a polypeptide consisting of said amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark. A transplant material for treating retinal diseases, comprising a culture of retinal cells containing a nucleic acid molecule comprising a base sequence encoding any one of the following:

12. A biological membrane comprising an opsin and a G protein, The opsin (i) the amino acid sequence of SEQ ID NO: 1; (ii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence, wherein a polypeptide consisting of the amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark; or (iii) An amino acid sequence in which one or more amino acids are substituted, added, inserted, and / or deleted in the amino acid sequence of (i) or (ii), wherein a polypeptide consisting of said amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark. Biological membranes containing proteins (excluding the biomembrane of Acropora spp.).

13. (i) the amino acid sequence of SEQ ID NO: 1; (ii) an amino acid sequence having at least 90% sequence identity with the amino acid sequence, wherein a polypeptide consisting of the amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark; or (iii) An amino acid sequence in which one or more amino acids are substituted, added, inserted, and / or deleted in the amino acid sequence of (i) or (ii), wherein a polypeptide consisting of said amino acid sequence is capable of binding 11-cis retinal and converting all-trans retinal converted from 11-cis retinal by light reception back to 11-cis in the dark. a method for controlling intracellular G protein signaling by light irradiation, the method comprising the step of irradiating light onto a cell transformed with a nucleic acid molecule comprising a base sequence encoding any one of the above (excluding the case where the cell is an Acropora cell or the case where the cell is present in a human body).

14. The method of claim 13, wherein the cells are cultured in a nutrient medium containing 11-cis retinal and / or a precursor of 11-cis retinal.

15. The method according to claim 13 or 14, further comprising the step of placing the irradiated cells in the dark.

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