Photoresponsive modified opsins

Abstract

The present disclosure provides opsins having light cycle characteristics. More specifically, the present disclosure provides a protein comprising an amino acid sequence of opsins including a modification corresponding to the amino acid at position 188 when compared with SEQ ID NO: 1 in the amino acid sequence of opsins. The disclosure additionally provides medicaments, formulations, compositions, therapies, and the like comprising the proteins, related nucleic acid molecules, nucleic acid constructs, and / or cells.

Description

Technical Field

[0001] The present disclosure relates to a protein comprising an amino acid sequence of opsin-like, a nucleic acid molecule comprising a nucleic acid encoding the amino acid sequence of the protein, a nucleic acid construct comprising the nucleic acid molecule, a cell comprising the nucleic acid construct, and a drug comprising the cell. Background Art

[0002] Opsin is a light-receiving protein that is ubiquitous in animals and can be classified into three types according to its light response characteristics. Rhodopsin of vertebrates, one of the opsins, forms an active state by light irradiation (light stimulation), but does not return to the original dark state by light reaction or thermal reaction. On the other hand, many opsins other than animal rhodopsins such as channelrhodopsin can form an active state by light irradiation (light stimulation) and then return to the dark state by thermal reaction. Summary of the Invention

[0003] Problems to be Solved by the Invention

[0004] The inventors of the present invention found that for vertebrate rhodopsin, photocycle and photoreversibility can also be obtained by introducing one mutation at position 188. Thus, it was found that the residue at position 188 contributes to the diversification of the light response characteristics of opsin by controlling the recovery from the active state to the original dark state.

[0005] Therefore, the present disclosure provides the following solutions.

[0006] (Item X1)

[0007] A protein comprising an amino acid sequence of opsin-like, wherein the amino acid sequence of the opsin-like comprises a modification of the amino acid corresponding to position 188 when aligned with SEQ ID NO: 1.

[0008] (Item X2-1)

[0009] A protein comprising an amino acid sequence of opsin-like, wherein the amino acid sequence of the opsin-like comprises a modification of the amino acid corresponding to position 188 being G, T, S, or E when aligned with SEQ ID NO: 1.

[0010] (Item X2-2)

[0011] A protein comprising an amino acid sequence of opsin-like, wherein the amino acid sequence of the opsin-like comprises a modification of the amino acid corresponding to position 188 being G when aligned with SEQ ID NO: 1.

[0012] (Item X3)

[0013] The protein according to any one of the above items, wherein, in the above amino acid sequence, it includes the modification of the amino acid corresponding to position G188 when aligned with SEQ ID NO: 1 to cysteine.

[0014] (Item X4)

[0015] The protein according to any one of the above items, wherein the above protein does not release the photoreceptor factor and inactivate after being activated by light stimulation.

[0016] (Item X5)

[0017] The protein according to any one of the above items, wherein the above amino acid sequence includes:

[0018] 1) The amino acid sequence containing the above modified amino acid sequence in any one of the amino acid sequences shown by SEQ ID NOs: 1 to 34;

[0019] 2) The sequence other than the above modified site has at least about 80% identity with the sequence of 1), and the encoded protein has an amino acid sequence with substantially the same biological activity as the protein obtained from the sequence of 1);

[0020] 3) The sequence of 1) has one or more mutations outside the above modified site, and the encoded protein has an amino acid sequence with substantially the same biological activity as the protein obtained from the sequence of 1);

[0021] 4) The amino acid sequence containing the above modified amino acid sequence in the amino acid sequence encoded by the nucleic acid that can hybridize with the nucleic acid encoding the sequence of 1), or

[0022] 5) The amino acid sequence containing the above modified amino acid sequence in the amino acid sequence encoded by the allelic mutant of the nucleic acid encoding the sequence of 1).

[0023] (Item X6)

[0024] The protein according to any one of the above items, wherein, in the amino acid sequence of the above opsins, it further includes the modification of the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1.

[0025] (Item X7)

[0026] The protein according to any one of the above items, wherein, in the amino acid sequence of the above opsins, it further includes the modification of the amino acid E corresponding to position 122 when aligned with SEQ ID NO: 1.

[0027] (Item X8)

[0028] The protein according to any one of the above items, wherein, in the amino acid sequence of the above rhodopsin-like protein, there is further a modification of the amino acid corresponding to the 122nd position upon alignment with SEQ ID NO: 1 to glutamine.

[0029] (Item X9)

[0030] The protein according to any one of the above items, wherein the above light-receiving factor contains retinal.

[0031] (Item X9-1)

[0032] The protein according to any one of the above items, wherein, in the amino acid sequence of the above rhodopsin-like protein, there are further modifications of the amino acids corresponding to a part of the N-terminal domain (positions 1 to 11) and the extracellular third loop part (positions 279 to 285) upon alignment with SEQ ID NO: 1.

[0033] (Item X9-2)

[0034] The protein according to any one of the above items, wherein, in the amino acid sequence of the above rhodopsin-like protein, there is further a modification of the amino acids corresponding to a part of the N-terminal domain (positions 1 to 11) and the extracellular third loop part (positions 279 to 285) upon alignment with SEQ ID NO: 1 to cysteine.

[0035] (Item X9-3)

[0036] The protein according to any one of the above items, wherein, in the amino acid sequence of the above rhodopsin-like protein, there are further modifications of the amino acids corresponding to positions 2 and 282 upon alignment with SEQ ID NO: 1.

[0037] (Item X9-4)

[0038] The protein according to any one of the above items, wherein the above modifications of the amino acids corresponding to positions 2 and 282 improve the thermal stability of the above rhodopsin-like protein.

[0039] (Item X9-5)

[0040] The protein according to any one of the above items, wherein the above modifications of the amino acids corresponding to positions 2 and 282 include modifications of the amino acids corresponding to positions 2 and 282 to cysteine.

[0041] (Item X10A)

[0042] The protein according to any one of the above items, wherein the above rhodopsin-like protein is a chimeric rhodopsin.

[0043] (Project X10B)

[0044] The protein according to any one of the above items, wherein the opsin is a chimeric opsin in which the amino acid sequence that binds to a G protein in the amino acid sequence of a certain opsin is replaced with the amino acid sequence that binds to a G protein in another opsin.

[0045] (Project X10C)

[0046] The protein according to any one of the above items, wherein the opsin is a chimeric opsin in which the amino acid sequence that binds to a G protein in the amino acid sequence of a certain opsin is replaced with a functional sequence derived from another organism.

[0047] (Project X10D)

[0048] The protein according to any one of the above items, wherein the opsin is a chimeric opsin of the same or different organisms.

[0049] (Project X10E)

[0050] The protein according to any one of the above items, wherein the organism is selected from the group consisting of vertebrates, invertebrates, and microorganisms.

[0051] (Project X10F1)

[0052] The protein according to any one of the above items, wherein the opsin is a chimeric opsin of a microbial opsin and a vertebrate opsin.

[0053] (Project X10F2)

[0054] The protein according to any one of the above items, wherein the opsin is a chimeric opsin of a microbial opsin and an invertebrate opsin.

[0055] (Project X10F3)

[0056] The protein according to any one of the above items, wherein the opsin is a chimeric opsin of a vertebrate opsin and an invertebrate opsin.

[0057] (Project X10F4)

[0058] The protein according to any one of the above items, wherein the opsin is a chimeric opsin of microbial opsins.

[0059] (Project X10F5)

[0060] The protein according to any one of the above items, wherein the opsin class is a chimeric opsin of vertebrate-type opsins with each other.

[0061] (Item X10F6)

[0062] The protein according to any one of the above items, wherein the opsin class is a chimeric opsin of invertebrate-type opsins with each other.

[0063] (Item X10)

[0064] The protein according to any one of the above items, which comprises the sequence of SEQ ID NO: 1, 3 or 5.

[0065] (Item X11)

[0066] A nucleic acid molecule, which comprises a nucleic acid encoding the amino acid sequence of the protein according to any one of the above items.

[0067] (Item X12)

[0068] A nucleic acid construct, which comprises the nucleic acid molecule according to any one of the above items.

[0069] (Item X13)

[0070] A cell, which comprises the protein according to any one of the above items, the nucleic acid molecule according to any one of the above items, and / or the nucleic acid construct according to any one of the above items.

[0071] (Item X14)

[0072] A drug, which comprises the protein according to any one of the above items, the nucleic acid molecule according to any one of the above items, the nucleic acid construct according to any one of the above items, and / or the cell according to any one of the above items.

[0073] (Item X15)

[0074] The drug according to any one of the above items, which is used for visual regeneration or for preventing or treating visual impairment or diseases.

[0075] (Item 1)

[0076] A composition, which comprises an opsin class that is inactivated without releasing a light-receiving factor.

[0077] (Item 2)

[0078] The composition according to any one of the above items, wherein the opsin class causes a transient change in the concentration of cAMP due to light stimulation.

[0079] (Item 3)

[0080] The composition according to any one of the above items, wherein the transient change in cAMP concentration is a decrease in cAMP concentration.

[0081] (Item 4)

[0082] The composition according to any one of the above items, wherein the opsins include the amino acid sequence shown by any one of SEQ ID NOs: 1 to 34.

[0083] (Item 5)

[0084] The composition according to any one of the above items, wherein the opsins include G protein-coupled receptor rhodopsin.

[0085] (Item 6)

[0086] The composition according to any one of the above items, wherein the G protein-coupled receptor rhodopsin is derived from a mammal (visual rhodopsin).

[0087] (Item 7)

[0088] The composition according to any one of the above items, wherein the G protein-coupled receptor rhodopsin is a modified type.

[0089] (Item 8)

[0090] The composition according to any one of the above items, wherein the modification includes G188C when aligned with SEQ ID NO: 1.

[0091] (Item 9)

[0092] The composition according to any one of the above items, wherein the light-receiving factor includes retinal.

[0093] (Item 10)

[0094] The composition according to any one of the above items, wherein the modification further includes E122Q when aligned with SEQ ID NO: 1.

[0095] (Item 11)

[0096] The composition according to any one of the above items, wherein the modification further includes the modification of the amino acids corresponding to the 2nd and 282nd positions to cysteine when aligned with SEQ ID NO: 1.

[0097] (Item 12)

[0098] The composition according to any one of the above items, which is used for drugs.

[0099] (Item 13)

[0100] The composition according to any one of the above items, which is used for visual regeneration or for preventing or treating visual disorders or diseases.

[0101] (Item Z1)

[0102] A method for modifying or endowing visual function, which includes a step of using opsins that are inactivated without releasing a light-receiving factor.

[0103] (Item Z2)

[0104] A method for use as an optical switch, which includes a step of using opsins that are inactivated without releasing a light-receiving factor.

[0105] (Item Z3)

[0106] A method including a step of using opsins that are inactivated without releasing a light-receiving factor.

[0107] (Item A1)

[0108] A protein containing the amino acid sequence of opsins, and for the opsins, the protein has been modified to be inactivated without releasing a light-receiving factor after being activated by light stimulation, and the above opsins activate the Gs or Gq subfamily of G proteins.

[0109] (Item A1a)

[0110] The protein according to any one of the above items, wherein the above opsins activate the Gs subfamily of G proteins.

[0111] (Item A1b)

[0112] The protein according to any one of the above items, wherein the above opsins increase the cAMP concentration by activating G proteins.

[0113] (Item A1c)

[0114] The protein according to any one of the above items, wherein the above opsins are chimeric opsins formed by replacing the second and third intracellular loops of the above opsins with the second and third loops of a Gs or Gq-activated G protein-coupled receptor.

[0115] (Item A2)

[0116] The protein according to any one of the above items, wherein the modification to be inactivated without releasing a light-receiving factor after being activated by light stimulation is achieved by modifying the amino acid corresponding to position 188 when aligned with SEQ ID NO: 1.

[0117] (Project A3)

[0118] The protein according to any one of the above items, wherein the amino acid corresponding to position 188 includes G, T, S, or E.

[0119] (Project A4)

[0120] The protein according to any one of the above items, wherein the amino acid corresponding to position 188 is G.

[0121] (Project A5)

[0122] The protein according to any one of the above items, wherein it includes the modification of the amino acid corresponding to position G188 when aligned with SEQ ID NO: 1 in the above amino acid sequence to cysteine.

[0123] (Project A6)

[0124] The protein according to any one of the above items, wherein the above amino acid sequence includes:

[0125] 1) The above modified amino acid sequence is included in the amino acid sequence shown by any one of SEQ ID NOS: 1 to 34,

[0126] 2) The sequence other than the above modified site has at least about 80% identity with the sequence of 1) and the encoded protein has substantially the same biological activity as the protein obtained from the sequence of 1),

[0127] 3) One or more mutations exist outside the above modified site in the sequence of 1) and the encoded protein has substantially the same biological activity as the protein obtained from the sequence of 1),

[0128] 4) The above modified amino acid sequence is included in the amino acid sequence encoded by a nucleic acid that can hybridize with the nucleic acid encoding the sequence of 1), or

[0129] 5) The above modified amino acid sequence is included in the amino acid sequence encoded by an allelic mutant of the nucleic acid encoding the sequence of 1).

[0130] (Project A7)

[0131] The protein according to any one of the above items, wherein in the amino acid sequence of the above opsins, it further includes the modification of the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1.

[0132] (Project A8)

[0133] The protein according to any one of the above items, wherein the amino acid corresponding to the 122nd position is E.

[0134] (Item A9)

[0135] The protein according to any one of the above items, wherein in the above amino acid sequence, it includes the modification of the amino acid corresponding to the G122 position to glutamine when aligned with Sequence No. 1.

[0136] (Item A10)

[0137] The protein according to any one of the above items, wherein the above light-receiving factor contains retinal.

[0138] (Item A11-1)

[0139] The protein according to any one of the above items, wherein in the amino acid sequence of the above opsins, it further includes the modification of the amino acids corresponding to a part of the N-terminal domain (positions 1 to 11) and a part of the extracellular third loop (positions 278 to 285) when aligned with Sequence No. 1.

[0140] (Item A11-2)

[0141] The protein according to any one of the above items, wherein in the amino acid sequence of the above opsins, it further includes the modification of the amino acids corresponding to a part of the N-terminal domain (positions 1 to 11) and a part of the extracellular third loop (positions 278 to 285) to cysteine when aligned with Sequence No. 1.

[0142] (Item A11-3)

[0143] The protein according to any one of the above items, wherein in the amino acid sequence of the above opsins, it further includes the modification of the amino acids corresponding to positions 2 and 282 when aligned with Sequence No. 1.

[0144] (Item A11-4)

[0145] The protein according to any one of the above items, wherein the above modification of the amino acids corresponding to positions 2 and 282 improves the thermal stability of the above opsins.

[0146] (Item A11-5)

[0147] The protein according to any one of the above items, wherein the above modification of the amino acids corresponding to positions 2 and 282 includes the modification of the amino acids corresponding to positions 2 and 282 to cysteine.

[0148] (Item A12a)

[0149] The protein according to any one of the above items, which comprises the sequence of SEQ ID NO: 1, 3 or 5.

[0150] (Item A12)

[0151] A nucleic acid molecule, which comprises a nucleic acid encoding the amino acid sequence of the protein according to any one of the above items.

[0152] (Item A13)

[0153] A nucleic acid construct, which comprises the nucleic acid molecule according to any one of the above items.

[0154] (Item A14)

[0155] A cell, which comprises the protein according to any one of the above items, the nucleic acid molecule according to any one of the above items, and / or the nucleic acid construct according to any one of the above items.

[0156] (Item A15)

[0157] A drug, which comprises the protein according to any one of the above items, the nucleic acid molecule according to any one of the above items, and / or a cell comprising the nucleic acid construct according to any one of the above items.

[0158] (Item A16)

[0159] The drug according to any one of the above items, which is used for visual regeneration or for preventing or treating visual impairment or diseases.

[0160] In the present disclosure, it is intended to provide further combinations thereof in addition to the explicit combinations of the above 1 or more features. Regarding further embodiments and advantages of the present disclosure, those skilled in the art can realize them by reading and understanding the following detailed description as needed.

[0161] Effects of the Invention

[0162] According to the present disclosure, for vertebrate rhodopsin, it is also possible to obtain a photocycle and photoreversibility of automatically recovering dark adaptation after light irradiation, which cannot be achieved by the prior art, by introducing 1 mutation at the 188th position, and the application possibility as a optogenetic tool is confirmed. Brief Description of the Drawings

[0163] Figure 1 A diagram showing the thermal recovery of the G188C mutant of bovine rhodopsin after yellow light irradiation.

[0164] Figure 2Figure showing the light response, retinal configuration, and G protein activation of the bovine rhodopsin G188C mutant.

[0165] Figure 3 Figure showing that the recovery rate of the photocycle characteristics of the bovine rhodopsin G188C mutant is faster by introducing the E122Q mutation.

[0166] Figure 4 Figure showing that the bovine rhodopsin mutant inhibits the intracellular cAMP level induced by light.

[0167] Figure 5 Figure showing the photopigment formation of the bovine rhodopsin G188C mutant after incubation with all-trans retinal.

[0168] Figure 6 Figure showing the concentration change of cAMP measured using a chimeric opsin in which the second and third intracellular loops of the E122Q / G188C mutant of bovine rhodopsin are replaced with the second and third intracellular loops of the mouse histamine H2 receptor.

[0169] Figure 7 Figure showing that the Xenopus tropicalis Opn5m T188C mutant acquires photocycle characteristics.

[0170] Figure 8 Figure showing the concentration change of cAMP measured using the E122Q / G188C mutant of human rhodopsin.

[0171] Figure 9 Figure showing the alignment of the amino acid sequences of opsins.

[0172] Figure 9-2 : Ibid.

[0173] Figure 9-3 : Ibid.

[0174] Figure 9-4 : Ibid.

[0175] Figure 9-5 : Ibid.

[0176] Figure 9-6 : Ibid.

[0177] Figure 9-7 : Ibid.

[0178] Figure 9-8 : Ibid.

[0179] Figure 9-9 : Ibid.

[0180] Figure 10A graph showing the change in the concentration of cAMP measured using the G188C mutant of canine rhodopsin.

[0181] Figure 11 A graph showing the change in the concentration of cAMP measured using the G188C mutant of medaka rhodopsin.

[0182] Figure 12 A graph showing the change in the concentration of cAMP measured using the G6A / G188C / N2C / D282C mutant and the V337A / G188C / N2C / D282C mutant of human rhodopsin.

[0183] Figure 13 A graph showing the change in the concentration of cAMP measured using the G188C / N2C / G3C / G280C mutant, the G188C / N2C / G3C / S281C mutant, and the G188C / G3C / N282C mutant belonging to human rhodopsin.

[0184] Figure 14 A graph showing the frequency of neural activity extracted from the extracellular potential of ganglion cells (RGC) measured using a multi-electrode array (MEA). Figure 14 A shows an untreated rd1 mouse (in a blind state, so no light response can be observed). Figure 14 B shows the case where the G188C / N2C / D282C mutant of human rhodopsin was introduced using a viral vector. Figure 14 C shows the case where a substance was introduced in which the second and third intracellular loops of the G188C / N2C / D282C mutant of human rhodopsin were replaced with the second and third intracellular loops of the human histamine H2 receptor. Figure 14 D shows the case where the second and third intracellular loops of the E122Q / G188C / N2C / D282C mutant of human rhodopsin were replaced with the second and third intracellular loops of the human histamine H2 receptor.

[0185] Figure 15 A graph showing the change in the concentration of cAMP measured using the N2C / D282C mutant and the N2C / D282C / G188C mutant of bovine rhodopsin. Detailed implementation

[0186] The present disclosure will be described by showing the best mode below. Throughout this specification, unless otherwise specified, singular expressions shall be understood to also include the concepts of their plural forms. Therefore, unless otherwise specified, singular articles (e.g., "a", "an", "the", etc. in English) shall be understood to also include the concepts of their plural forms. Additionally, unless otherwise specified, the terms used in this specification shall be understood to be used in the meanings commonly used in the art. Therefore, unless otherwise defined, all technical terms and scientific and technological terms used in this specification have the same meanings as those commonly understood by those skilled in the art to which the present disclosure pertains. In case of conflict, the present specification (including the definitions) shall prevail.

[0187] (Definitions, etc.)

[0188] The definitions and / or basic technical contents of the terms specifically used in this specification will be appropriately described below.

[0189] In this specification, "opsin" or "opsins" refers to: a protein that binds to a pigment that functions as a light-receiving factor, namely retinal or its analog, and uses retinal or its analog as a chromophore.

[0190] The types of opsins vary depending on the biological species and the types of photoreceptor cells. Examples include rhodopsin and cone opsins (e.g., blue opsin, green opsin, red opsin). Additionally, in this specification, opsins include melanopsin, encephalopsin, OPN5, RGR, and peropsin, but are not limited to these. For example, the opsin of an animal is a G protein-coupled receptor (GPCR) with a seven-transmembrane structure, and forms rhodopsin that can perform a light-receiving function by binding to retinal as a pigment. Opsin is activated by light reception to activate trimeric G protein, thereby transducing the external light signal into the cell.

[0191] Opsins can be generally classified into microbial opsins (Type I opsins) and animal opsins (Type II opsins). Animal opsins can be further divided into vertebrate visual opsins, vertebrate non-visual opsins, and invertebrate opsins. In addition, vertebrate non-visual opsins and invertebrate opsins are called bistable opsins, which are opsins that include all-trans retinal as a chromophore, just like microbial opsins. Some of microbial opsins, vertebrate non-visual opsins, and invertebrate opsins are opsins that do not release retinal from the rhodopsin they form even upon light reception, and can be used as the opsins of the present disclosure. The opsins suitable for the present disclosure are opsins that do not release retinal even upon light reception, and examples include microbial opsins, vertebrate non-visual opsins, and some of invertebrate opsins, and can also include bistable opsins including insect opsins. As the opsins used in the present disclosure, even if they do not strictly belong to microbial opsins, vertebrate non-visual opsins, or invertebrate opsins, but are functional equivalents having the same functions as these suitably usable opsin types, they can also be suitably used.

[0192] The specific amino acid sequences of opsin classes are shown in SEQ ID NOs: 1 to 34, and the amino acid at position 188 thereof is as shown in Figure 9 the alignment diagram of. That is, the amino acid at position 188 refers to the amino acid corresponding to the amino acid G at position 188 of SEQ ID NO: 1 when aligned with SEQ ID NO: 1 using CLUSTRAL W (Version 2.0, released in 2007) (see Figure 9(alignment diagram), if it is other serial numbers, the specific numbers may be staggered. In serial number 2, it is G; in serial number 3, it is G; in serial number 4, it is G; in serial number 5, it is G; in serial number 6, it is G; in serial number 7, it is G; in serial number 8, it is G; in serial number 9, it is G; in serial number 10, it is G; in serial number 11, it is G; in serial number 12, it is G; in serial number 13, it is G; in serial number 14, it is G; in serial number 15, it is G; in serial number 16, it is T; in serial number 17, it is S; in serial number 18, it is T; in serial number 19, it is T; in serial number 20, it is T; in serial number 21, it is T; in serial number 22, it is S; in serial number 23, it is T; in serial number 24, it is T; in serial number 25, it is T; in serial number 26, it is E; in serial number 27, it is S; in serial number 28, it is S; in serial number 29, it is T; in serial number 30, it is S; in serial number 31, it is T; in serial number 32, it is T; in serial number 33, it is T; in serial number 34, it is T. Regarding the serial numbers not described in this specification, those skilled in the art can also understand through obtaining the alignment. Regarding the amino acid at position 122, it is the same. The amino acid at position 122 refers to: when aligned with serial number 1 using CLUSTRAL W (Version 2.0, released in 2007), the amino acid equivalent to the amino acid E at position 122 of serial number 1. Specifically, in serial number 2, it is E; in serial number 3, it is E; in serial number 4, it is E; in serial number 5, it is E; in serial number 6, it is L; in serial number 7, it is I; in serial number 8, it is I; in serial number 9, it is L; in serial number 10, it is I; in serial number 11, it is Q; in serial number 12, it is M; in serial number 13, it is L; in serial number 14, it is I; in serial number 15, it is I; in serial number 16, it is I; in serial number 17, it is I; in serial number 18, it is C; in serial number 19, it is L; in serial number 20, it is M; in serial number 21, it is F; in serial number 22, it is I; in serial number 23, it is C; in serial number 24, it is L; in serial number 25, it is M; in serial number 26, it is I; in serial number 27, it is I; in serial number 28, it is I; in serial number 29, it is I; in serial number 30, it is I; in serial number 31, it is C; in serial number 32, it is V; in serial number 33, it is M; in serial number 34, it is L. Regarding the serial numbers not described in this specification, those skilled in the art can also understand through obtaining the alignment.

[0193] In this specification, "rhodopsin" is a protein that has a pigment called retinal inside, which is activated by receiving light and visual signals are transmitted to the brain. When rhodopsin is mentioned in this specification, in addition to referring to the contained retinal, it sometimes refers to opsin (protein part). In this case, it can be interpreted interchangeably with opsin. The ion-transporting receptor rhodopsin mainly derived from microorganisms does not shed retinal even when irradiated with light, so it can be repeatedly activated by absorbing light, but it cannot activate G proteins like the G protein-coupled receptor rhodopsin mainly derived from animals.

[0194] As long as the protein of the present disclosure can achieve the purpose of the present disclosure, it can include any type of opsin, and can also include chimeric opsins. Chimeric opsins can be generated by combining a part of two or more types of opsins known in the art. Each of the combined opsin types can be derived from the same organism or from different organisms. Those skilled in the art can appropriately select suitable opsin types according to the use and / or function of the target opsin type to provide suitable chimeric opsins. At this time, the amino acid corresponding to amino acid 188 of SEQ ID NO: 1 is modified, preferably modified to cysteine, and then it can be used in the present disclosure. The scope of the present disclosure also includes those obtained by modifying, preferably modifying to cysteine, the amino acid corresponding to amino acid 188 of SEQ ID NO: 1 in any such chimeric opsin. As the opsin types that can constitute chimeric opsins, for example, rhodopsin and cone opsins (such as blue opsin, green opsin, red opsin), as well as melanopsin, encephalopsin, OPN5, RGR, and peropsin, etc. can be used. They can be microbial opsins (Type I opsins), animal opsins (Type II opsins), and can be selected from vertebrate non-visual opsins, invertebrate opsins, etc.

[0195] As an example of the chimeric opsin that can be used in the present disclosure, a chimeric opsin of a G protein-coupled receptor and an animal opsin can be mentioned. For example, a chimeric protein that includes at least a part of a mammalian opsin and at least a part of a Gs or Gq-activated G protein-coupled receptor can be cited. If the representative example is described, by fusing a part of a Gs or Gq-activated G protein-coupled receptor to a part of a mammalian opsin, Gs or Gq activity can be obtained. In one embodiment, the chimeric opsin of the present disclosure is obtained by replacing the amino acid sequence of the second intracellular loop and / or the third intracellular loop that binds to G protein in the amino acid sequence of a certain opsin type with the amino acid sequence or functional sequence of the second intracellular loop and / or the third intracellular loop of other G protein-coupled receptors. Thus, the protein of the present disclosure can activate Gi-type G protein depending on light and reduce the intracellular cAMP level, or activate Gs-type G protein depending on light to increase the intracellular cAMP level, or activate Gq-type G protein to increase the intracellular Ca 2+ level. Without wishing to be bound by theory, the chimeric protein utilized in this embodiment of the present disclosure can be expressed in mammals such as rodents and primates while maintaining sufficient activity. Therefore, it can achieve the effect of preventing and inhibiting the progression of retinal diseases, disorders or symptoms, especially the prevention or inhibition of the progression of retinitis pigmentosa, or bring the effect of improving visual cognitive behavioral functions (such as improving the light and dark discrimination function, improving the light avoidance function and / or the crisis avoidance function), or exert the effect of enhancing visual functions such as improving eyesight.

[0196] Examples of the G protein-coupled receptor rhodopsin used in the present disclosure include human rhodopsin, bovine rhodopsin, and canine rhodopsin.

[0197] In the present specification, "retinal" is a type of vitamin A1, also known as retinaldehyde or retinene, and refers to the substance that constitutes the visual substance, rhodopsin, contained in the rod cells of the retina. The retinal that binds to opsin to form rhodopsin adopts the molecular form of 11-cis-retinal, isomerizes to all-trans-retinal upon exposure to light, and dissociates from the binding with opsin. Subsequently, all-trans-retinal is restored to 11-cis-retinal in the visual cycle and binds to opsin. It has been clarified that all-trans-retinal has phototoxicity and is related to various visual-related diseases of the retina, including age-related macular degeneration, Stargardt macular dystrophy, fundus flavimaculatus, and autosomal recessive retinitis pigmentosa.

[0198] In the present specification, "analog (analogue) of retinal" refers to a compound having substantially the same properties as retinal, including naturally occurring natural-type substances or substances having modifications, additions, functional group substitutions, etc., different from the natural type. Examples of analogues of retinal include natural-type analogues such as A2-retinal (3,4-dehydroretinal), A3-retinal (3-hydroxyretinal), A4-retinal (4-hydroxyretinal), and artificial analogues such as 9-ethyl-retinal and 9-propyl-retinal. Regarding retinal analogues, reference can be made to, for example, Akimori Wada, Yakugaku Zasshi 141(4), 557-577, 2021-04-01.

[0199] In the present specification, "visual impairment" refers to any disease, disorder, or symptom related to vision, such as retinal degenerative diseases (retinitis pigmentosa, age-related macular degeneration, etc.), retinopathies (such as diabetic retinopathy, proliferative retinopathy, simple retinopathy, etc.), floaters, retinal holes, retinal detachment (such as rhegmatogenous retinal detachment, non-rhegmatogenous retinal detachment, etc.), and also includes retinitis pigmentosa, age-related macular degeneration, myopic macular degeneration, macular dystrophy, diabetic retinopathy, retinal detachment, etc. In addition, these disorders or symptoms include impairments in visual acuity, contrast sensitivity, light and dark adaptation, color vision, etc., and symptoms related thereto.

[0200] In this specification, "protein", "polypeptide", "oligopeptide" and "peptide" are used interchangeably and refer to amino acid polymers of any length. The polymer can be linear or branched, and can also be cyclic. The amino acids can be natural amino acids, unnatural amino acids, or modified amino acids. The term also includes complexes formed by the association of multiple polypeptide chains. The term also includes natural or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other operation or modification (such as conjugation with a labeling component). This definition also includes, for example, polypeptides containing analogs of one or more amino acids (such as those containing unnatural amino acids, etc.), peptidomimetic compounds (such as peptoids), and other modifications known in the art. In this specification, "amino acid" is a general term for organic compounds having an amino group and a carboxyl group. When an antibody of an embodiment of the present disclosure contains a "specific amino acid sequence", any amino acid in the amino acid sequence can be chemically modified. In addition, any amino acid in the amino acid sequence can form a salt or a solvate. In addition, any amino acid in the amino acid sequence can be of the L-form or the D-form. In such a case, the protein of an embodiment of the present disclosure can also be said to contain the above-mentioned "specific amino acid sequence". As chemical modifications received by amino acids contained in a protein in a living body, for example, N-terminal modifications (such as acetylation, myristoylation, etc.), C-terminal modifications (such as amidation, addition of glycosylphosphatidylinositol, etc.), or side-chain modifications (such as phosphorylation, addition of sugar chains, etc.) are known. They can be natural or unnatural as long as they meet the purpose of the present disclosure.

[0201] In this specification, "chimeric" (such as a protein, opsin, etc.) refers to a state in which multiple genetic information from the same or different organisms is mixed in the same entity (in this case, a protein, etc.). A chimeric protein, for example, contains gene sequences from two or more organisms. The sequence information contained in a chimeric protein can include other sequences in addition to the sequences from the organisms to be mixed.

[0202] In this specification, "polynucleotide", "oligonucleotide", "nucleic acid" and "nucleic acid molecule" are used interchangeably and refer to nucleotide polymers of any length. This term also includes "oligonucleotide derivatives" or "polynucleotide derivatives". "Oligonucleotide derivatives" or "polynucleotide derivatives" refer to oligonucleotides or polynucleotides that include derivatives of nucleotides or in which the bonds between nucleotides are unusual bonds, and can be used interchangeably. As such oligonucleotides, specifically, for example, 2'-O-methyl ribonucleotides, oligonucleotide derivatives in which the phosphodiester bond in the oligonucleotide is converted to a phosphorothioate bond, oligonucleotide derivatives in which the phosphodiester bond in the oligonucleotide is converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which the ribose and phosphodiester bond in the oligonucleotide are converted to a peptide nucleic acid bond, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced by C-5 propynyl uracil, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced by C-5 thiazole uracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced by C-5 propynyl cytosine, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced by phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA is replaced by 2'-O-propyl ribose, and oligonucleotide derivatives in which ribose in the oligonucleotide is replaced by 2'-methoxyethoxy ribose, etc. Unless otherwise stated, a specific base sequence, like the explicitly stated sequence, is also intended to include its conservatively modified variants (such as degenerate codon substitutes) and complementary sequences. It should be noted that the sequence of a nucleic acid, in addition to being called a base sequence, is also called a nucleic acid sequence, nucleotide sequence, etc., all with the same meaning. Specifically, degenerate codon substitutes can be achieved by creating a sequence in which the third position of one or more (or all) selected codons is replaced by a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). Depending on the context, "nucleic acid" in this specification can also be used interchangeably with genes, DNA such as cDNA, RNA such as mRNA, oligonucleotides, and polynucleotides. "Nucleotide" in this specification can be natural or non-natural. Nucleic acids in this specification can be DNA or RNA.

[0203] In this specification, "gene" refers to the factor that determines a genetic trait, and "gene" sometimes refers to "polynucleotide", "oligonucleotide" and "nucleic acid".

[0204] In this specification, the terms "nucleic acid construct", "construct", or "gene construct" are used interchangeably and refer to a nucleic acid molecule that contains nucleic acids isolated from genes existing in nature or nucleic acids combined and arranged in a manner not existing in nature, as well as a vector.

[0205] In this specification, the "homology" of genes refers to the degree of identity of two or more gene sequences relative to each other. Generally, having "homology" means a high degree of identity or similarity. "Identity" refers to the degree of correspondence of the sequences of the same amino acids, and "similarity" refers to the degree of correspondence of sequences including the same amino acids and amino acids with similar properties. Therefore, the higher the homology of two genes, the higher the identity or similarity of their sequences. Whether two genes have homology can be investigated by direct comparison of the sequences or, in the case of nucleic acids, by the hybridization method under stringent conditions. When directly comparing two gene sequences, the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical, and these genes have homology. Therefore, in this specification, "homolog" or "homologous gene product" refers to a protein in other species, preferably mammals, that performs the same biological function as the protein component of the complex further described in this specification. Such homologs also include "orthologous gene products" and "paralogous gene products". It should be understood that such homologs, homologous gene products, orthologous gene products, paralogous gene products, etc. can also be used as long as they meet the objectives of the present disclosure.

[0206] In this specification, an amino acid can be represented in any of its commonly known three-letter symbols or one-letter symbols recommended by the IUPAC-IUB Commission on Biochemical Nomenclature. Similarly, a nucleotide can be represented by its commonly recognized single-letter code. In this specification, the comparison of the similarity, identity, and homology of amino acid sequences and base sequences can be calculated using BLAST as a sequence analysis tool with default parameters. The search for identity can be performed, for example, using BLAST 2.2.28 (released on April 2, 2013) of NCBI (Proc. Natl. Acad. Sci. USA 90: 5873-5877, 1993). The identity value in this specification generally refers to the value obtained when aligning using the above BLAST under default conditions. Among them, in the case where a larger value is obtained by changing the parameters, the maximum value is taken as the identity value. When evaluating identity in multiple regions, the maximum value among them is taken as the identity value. Similarity is a value that takes into account similar amino acids in addition to identity. When comparing amino acid sequences using BLAST, the algorithm can use Blastp with default settings. The measurement results are quantified as similarity (Positives) or identity (Identities). The homology of amino acid sequences and base sequences can be determined by BLAST based on the algorithm of Karlin and Altschul. Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J. Mol. Biol. 215: 403-410, 1990). When analyzing base sequences by BLASTN based on BLAST, the parameters are set, for example, as score = 100 and wordlength = 12. In addition, when analyzing amino acid sequences by BLASTX based on BLAST, the parameters are set, for example, as score = 50 and wordlength = 3. When using BLAST and Gapped BLAST programs, the default parameters of each program are used. The specific methods of these analysis methods are well known (http: / / www.ncbi.nlm.nih.gov.).

[0207] The nucleic acids or proteins used in the present disclosure may include sequences obtained by substituting, deleting, and / or adding one or more amino acids or nucleotides in an amino acid or base sequence as an object. Here, "one or more" in the full-length amino acid sequence of the chimeric protein generally refers to within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids (for example, within 5 amino acids, within 3 amino acids, 1 amino acid). Additionally, in the amino acid sequence of the domain, "one or more" generally refers to within 6 amino acids, preferably within 5 amino acids, more preferably within 4 amino acids (for example, within 3 amino acids, within 2 amino acids, 1 amino acid). When maintaining the biological activity of the chimeric protein of the present disclosure, it is desirable that, among the mutated amino acid residues, they are mutated to other amino acids with conservative properties of the amino acid side chains. As examples of the properties of amino acid side chains, hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl-containing side chains (S, T, Y), amino acids with sulfur atom-containing side chains (C, M), amino acids with carboxylic acid- and amide-containing side chains (D, N, E, Q), amino acids with basic side chains (R, K, H), and amino acids with aromatic-containing side chains (H, F, Y, W) (the parentheses in each case represent the one-letter code of the amino acid) can be listed. These are also referred to as "conservative substitutions" in this specification. It should be noted that it is well-known that a protein having an amino acid sequence modified by deletion, addition, and / or substitution of one or more amino acid residues with other amino acids in a certain amino acid sequence can maintain its biological activity (Mark, D.F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M.J. & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413). Thus, in one embodiment of the present disclosure, "several" may be, for example, 10, 8, 6, 5, 4, 3, or 2, or any value less than these. The chimeric protein with deletion, etc. can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library. As an example of site-directed mutagenesis, the KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used.Antibodies with the same activity as the wild type can be selected from mutant antibodies with deletions etc. by performing various characterizations such as FACS analysis and ELISA.

[0208] In one embodiment of the present disclosure, the amino acid sequence and nucleic acid sequence of the engineered protein and / or chimeric protein of the present disclosure may have 70% or more, 80% or more, or 90% or more identity or similarity with the reference sequence. In this specification, for amino acid sequences or base sequences, "70% or more" can be, for example, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more, "80% or more" can be, for example, 80, 85, 90, 95, 96, 97, 98, 99% or more, "90% or more" can be, for example, 90, 95, 96, 97, 98, 99% or more, and can also be within the range of any two of these values. "Homology" can be calculated by methods known in the art for the proportion of homologous amino acids in two or more amino acid sequences. Before calculating the proportion, the amino acid sequences of the amino acid sequence groups to be compared are aligned, and gaps are introduced into some amino acid sequences as needed to maximize the proportion of identical amino acids. The methods for alignment, the calculation method of the proportion, the comparison method, and the computer programs related to these are known in the art (for example, BLAST, GENETYX, etc.). In the case of "identity", the proportion of identical amino acids is calculated, and in the case of "similarity", the proportion of similar amino acids is calculated. Examples of similar amino acids include amino acids that can be conservatively substituted, but are not limited to these.

[0209] As used herein, a polynucleotide that hybridizes under stringent conditions refers to conditions well known and commonly used in the art. Such a polynucleotide is obtained by using, as a probe, a polynucleotide selected from the polynucleotides of the present disclosure and performing colony hybridization, plaque hybridization, Southern blot hybridization, or the like. Specifically, it refers to a polynucleotide that can be identified by hybridizing using a filter membrane fixed with DNA derived from colonies or plaques at 65°C in the presence of 0.7 to 1.0 M NaCl and then washing the filter membrane at 65°C with a 0.1- to 2-fold concentration of SSC (saline-sodium citrate) solution (the composition of a 1-fold concentration of SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). "Stringent conditions" can be, for example, the following conditions: (1) for washing, using a low ionic strength and a high temperature (e.g., at 50°C, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate); (2) using a denaturing agent such as formamide in hybridization (e.g., at 42°C, 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5, and 750 mM sodium chloride, 75 mM sodium citrate); or (3) incubating overnight at 37°C in a solution containing 20% formamide, 5×SSC, 50 mM sodium phosphate (pH 7.6), 5×Denhardt's solution, 10% dextran sulfate, and a solution containing 20 mg / ml of denatured and sheared salmon sperm DNA, and then washing the filter membrane with 1×SSC at about 37 - 50°C. It should be noted that the formamide concentration can be 50% or higher. The washing time can be 5 minutes, 15 minutes, 30 minutes, 60 minutes, or 120 minutes, or more than these. As factors affecting the stringency of the hybridization reaction, multiple factors such as temperature and salt concentration are considered. For details, reference can be made to Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of "highly stringent conditions" are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65 - 68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide, 42°C.Hybridization can be carried out according to the methods described in experimental books such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1-38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995), etc. Here, it is preferred to exclude sequences containing only A sequences or only T sequences from the sequences hybridized under stringent conditions. Moderate stringent conditions can be easily determined by those skilled in the art based on the length of DNA, as shown in Molecular Cloning: A Laboratory Manual by Sambrook et al., 3rd Edition, Vol. 1, 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001. In addition, regarding nitrocellulose filters, it includes: a pre-washing solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridization conditions of about 50% formamide, 2×SSC - 6×SSC at about 40-50 °C (or other similar hybridization solutions such as Stark's solution in about 50% formamide at about 42 °C), and application of washing conditions of about 60 °C, 0.5×SSC, 0.1% SDS. Therefore, the polypeptides used in the present disclosure also include: polypeptides encoded by nucleic acid molecules that hybridize with nucleic acid molecules encoding the polypeptides specifically described in the present disclosure under highly or moderately stringent conditions.

[0210] In this specification, a "purified" substance or biological factor (such as a nucleic acid or a protein, etc.) refers to a substance or biological factor from which at least a part of the factors naturally associated with it have been removed. Therefore, generally, the purity of the biological factor in the purified biological factor is higher than the state in which the biological factor usually exists (i.e., concentrated). The term "purified" used in this specification means that preferably at least 75% by weight, more preferably at least 85% by weight, still more preferably at least 95% by weight, and most preferably at least 98% by weight of the same type of biological factor is present. The substances or biological factors used in this disclosure are preferably "purified" substances. The "isolated" substance or biological factor (such as a nucleic acid or a protein, etc.) used in this specification refers to a substance from which the factors naturally associated with it have been substantially removed. The term "isolated" used in this specification varies according to its purpose, and therefore it is not necessarily expressed by purity. However, if necessary, it means that preferably at least 75% by weight, more preferably at least 85% by weight, still more preferably at least 95% by weight, and furthermore most preferably at least 98% by weight of the same type of biological factor is present. The substances used in this disclosure are preferably "isolated" substances or biological factors.

[0211] In this specification, "corresponding" amino acids or nucleic acids or parts refer to amino acids or nucleotides or parts in a polypeptide molecule or a polynucleotide molecule (such as rhodopsin) that have or are predicted to have the same function as the specified amino acids or nucleotides or parts in a polypeptide or polynucleotide serving as a comparison reference. Particularly in the case of an enzyme molecule, it refers to amino acids present at the same position in the active site and making the same contribution to the catalytic activity. In the case of a complex molecule, it refers to the corresponding part (such as heparan sulfate, etc.). For example, in the case of an antisense molecule, it can be the same part in an ortholog corresponding to a specific part of the antisense molecule. Corresponding amino acids can be, for example, specific amino acids that have undergone cysteinylation, glutathioneylation, S-S bond formation, oxidation (such as oxidation of the methionine side chain), formylation, acetylation, phosphorylation, sugar chain attachment, myristoylation, etc. Alternatively, corresponding amino acids can also be amino acids responsible for dimerization. Such "corresponding" amino acids or nucleic acids can be regions or domains spanning a certain range. Therefore, such a situation is referred to as a "corresponding" region or domain in this specification. Such corresponding regions or domains are used in this disclosure for designing complex molecules.

[0212] In this specification, a "corresponding" gene (in this case, it can be a sequence or molecule of a polynucleotide encoding rhodopsin, etc.) means: a gene (in this case, it can be a sequence or molecule of a polynucleotide encoding rhodopsin, etc.) in a certain species that has or is predicted to have the same function as a specified gene in a species serving as a comparison benchmark. When there are multiple genes with such a function, they are called genes with the same evolutionary origin. Therefore, the gene corresponding to a certain gene can be an ortholog of that gene. Therefore, human rhodopsin can find corresponding rhodopsin in various other animals (especially mammals). Such corresponding genes can be identified using techniques well-known in the art. Therefore, for example, for a corresponding gene in a certain animal (such as a mouse), the gene serving as the benchmark for the corresponding gene (such as rhodopsin, etc.) can be found by searching a database containing the sequence of that animal using a specific sequence as a query sequence.

[0213] In this specification, "a part", "fragment", or "segment" means: a polypeptide or polynucleotide having a sequence length from 1 to n - 1 relative to a full-length polypeptide or polynucleotide (with a length of n). The length of the fragment can be appropriately changed according to its purpose. For example, as the lower limit of its length, in the case of a polypeptide, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, and more amino acids can be listed, and lengths represented by integers not specifically listed here (such as 11, etc.) are also suitable as the lower limit. In addition, in the case of a polynucleotide, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, and more nucleotides can be listed, and lengths represented by integers not specifically listed here (such as 11, etc.) are also suitable as the lower limit. In this specification, for example, when the full-length one functions as a marker or a target molecule, such a fragment should be understood to fall within the scope of the present disclosure as long as the fragment itself has the function of a marker or a target molecule.

[0214] According to the present disclosure, in this specification, the term "activity" refers to the function of a molecule in the broadest sense. The activity is not intended to be limiting and generally includes the biological function, biochemical function, physical function, or chemical function of a molecule. Activity includes, for example, enzyme activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, hinder, inhibit, or destabilize the function of other molecules, stability, and the ability to localize to a specific intracellular location. In applicable cases, the term also relates to the function of a protein complex in the broadest sense. In this specification, "biological activity" includes the activation of a photoreaction, etc.

[0215] In this specification, "functional equivalent" refers to any substance that has the same target function as the entity in question but has a different structure. Therefore, the functional equivalent of "rhodopsin" or its chimeras is not rhodopsin or its chimeras themselves, but mutants or variants of rhodopsin or its chimeras (such as amino acid sequence variants, etc.), which can be understood to include: those having the biological effects of rhodopsin or its chimeras, and those that can be converted into rhodopsin or its antibodies themselves or mutants or variants of the rhodopsin or its chimeras at the time of exerting their effects (such as nucleic acids encoding rhodopsin or its chimeras or mutants or variants of rhodopsin or its chimeras, and vectors, cells, etc. containing such nucleic acids). As the functional equivalent of the present disclosure, those having one or more amino acids inserted, substituted, and / or deleted in the amino acid sequence, or having one or more amino acids added to one or both ends thereof can be used. In this specification, "those having one or more amino acids inserted, substituted, and / or deleted in the amino acid sequence, or having one or more amino acids added to one or both ends thereof" means: being modified by well-known technical methods such as site-directed mutagenesis, or by natural mutations using substitutions of multiple amino acids at a natural-occurring level. The modified amino acid sequence can be, for example, an amino acid sequence formed by inserting, substituting, or deleting 1 to 30, preferably 1 to 20, more preferably 1 to 9, further preferably 1 to 5, and particularly preferably 1 to 2 amino acids, or by adding them to one or both ends. For the modified amino acid sequence, it is preferable that the amino acid sequence has one or more (preferably one or several or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of rhodopsin.

[0216] In this specification, "reagent", "agent", or "factor" (all corresponding to "agent" in English) can be used interchangeably in a broad sense, and can be any substance or other element (such as energy such as light, radiant energy, heat, electricity, etc.) as long as it can achieve the intended purpose. Examples of such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (such as DNA including cDNA, genomic DNA, etc., and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, organic low-molecules (such as hormones, ligands, signaling molecules, organic low-molecules, molecules synthesized by combinatorial chemistry, low-molecules that can be used as drugs (such as low-molecular ligands, etc.)), and their composite molecules.

[0217] When administered orally, it can be formulated into various forms such as tablets, granules, fine granules, powders, capsules, etc. and used, and can contain additives such as binders, encapsulants, excipients, lubricants, disintegrants, wetting agents, etc. commonly used in preparations. In addition to these, preparations for oral administration can be formulated into liquid preparations such as internal liquid preparations, suspensions, emulsions, syrups, etc., or can be formulated into preparations in a dry state that are redissolved at the time of use.

[0218] When administered parenterally, it can be formulated into a state contained in a unit-dose ampoule, multi-dose container or tube. In addition, it can also contain additives such as stabilizers, buffers, preservatives, isotonic agents, etc. In addition, preparations for parenteral administration can be formulated into powders that can be redissolved with an appropriate carrier (such as sterilized water) at the time of use.

[0219] Examples of parenteral administration include intravitreal administration, subretinal administration, subchoroidal administration, and intracameral administration, etc. intraocular administration; subconjunctival administration, sub-Tenon's capsule administration, and eye drop administration, etc. extraocular administration, etc. Intravitreal administration is preferred. The compositions of the present disclosure, etc. can be administered to humans by using the methods described above for treatment, prevention, progression inhibition, etc.

[0220] In this specification, "treatment" means: for a certain disease or disorder (such as vesicular transport disorder, apoptosis), when already in this state, preventing the deterioration of this disease or disorder, preferably maintaining the status quo, more preferably alleviating, and further preferably eliminating it, including having a symptom improvement effect or preventive effect on one or more symptoms of the patient's disease or concomitant diseases. Sometimes, pre-diagnosing and performing appropriate treatment is called "concomitant treatment", and the diagnostic agent used for it is called "companion diagnostic agent". The present disclosure targets genetic diseases, so genes can be detected in advance and patients can be treated.

[0221] In this specification, "therapeutic agent" broadly refers to all reagents that can treat a target condition (such as retinal degenerative diseases, etc.). In one embodiment of the present disclosure, the "therapeutic agent" can be a pharmaceutical composition containing an active ingredient and one or more pharmaceutically acceptable carriers. The pharmaceutical composition can be manufactured, for example, by mixing the active ingredient and the above carriers and using any method known in the technical field of pharmaceutics. In addition, as long as the therapeutic agent is a drug that can be used for treatment, its form of use is not limited, and it can be only the active ingredient itself, or a mixture of the active ingredient and any component. In addition, the shape of the above carrier is not particularly limited, and can be, for example, solid or liquid (such as buffer solution).

[0222] In this specification, "prevention" means: for a certain disease or disorder (e.g., retinal degeneration disease), preventing the change to this state before becoming this state. The medicaments of the present disclosure can be used for diagnosis, and if necessary, the reagents of the present disclosure such as for preventing retinal degeneration disease, etc. can be used or preventive measures can be constructed. In this specification, "preventive agent" broadly refers to all reagents that can prevent the target state (e.g., vesicle transport disorder, apoptosis, etc.).

[0223] In this specification, "kit" means: a unit that usually divides the parts to be provided (e.g., nucleic acid, nucleic acid construct, cell into which the target nucleic acid has been introduced, test drug, diagnostic drug, therapeutic drug, antibody, label, instruction manual, etc.) into two or more compartments for provision. In the case where, along with a diagnostic drug, etc., it is preferred to first administer a reagent for determining patient characteristics to determine the patient to whom administration should be made and then administer a specific drug (nucleic acid drug, etc.) only to the appropriate patient, when the diagnostic drug and the therapeutic drug are provided in combination, a kit can be prepared. Or, when the purpose is to provide a composition such as a specific unstable drug, which should not be provided in a mixed form but is preferably mixed immediately before use for reasons such as stability, the form of the kit is preferred. Advantageously, such a kit preferably has an instruction manual or a specification that describes how to use the provided parts (e.g., nucleic acid, nucleic acid construct, cell into which the target nucleic acid has been introduced, test drug, diagnostic drug, therapeutic drug) or how the reagent should be handled. When the kit is used in the form of a medicament kit in this specification, the kit usually includes an instruction manual, etc. that describes the usage methods, etc. of the test drug, diagnostic drug, therapeutic drug, antibody, etc.

[0224] "Active ingredient" in this specification means the ingredient contained in an amount required to obtain the desired therapeutic, preventive, or progression-inhibiting effect, etc. of the composition, etc. of the present disclosure, and other ingredients can be contained as long as the effect is not lower than the desired level. In addition, the drugs, compositions, etc. of the present disclosure can be formulated. In addition, the administration route of the drugs, compositions, etc. of the present disclosure can be either oral or parenteral, and can be appropriately set according to the form of the preparation, etc.

[0225] As used in this specification, the "Instructions" (including attachment materials, labels used by the US FDA, etc.) are instructions for explaining to doctors or other users how to use the methods disclosed in this application. The Instructions contain statements for guiding the detection methods, usage methods of diagnostic agents, or administration of drugs, etc. disclosed in this application. In addition, in the Instructions, as the administration site, statements for guiding oral administration or administration to the retina (e.g., by injection, etc.) may be included. The Instructions are prepared in accordance with the formats specified by the regulatory authorities of the country where this application is implemented (e.g., the Ministry of Health, Labour and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and clearly state that approval has been obtained from such regulatory authorities. The Instructions are so-called attachment materials (package insert), labels, and are usually provided in the form of paper media, but are not limited thereto. For example, they may also be provided in the form of electronic media (e.g., a homepage provided on the Internet, an e-mail).

[0226] (Preferred Embodiment)

[0227] Preferred embodiments of this application are described below. The embodiments provided below are for better understanding of this application, and it should be understood that the scope of this application is not limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate changes within the scope of this application with reference to the descriptions in this specification. In addition, it should be understood that the following embodiments of this application can be used alone or in combination with these.

[0228] In one aspect, this application provides a protein comprising the amino acid sequence of opsin, wherein the amino acid sequence of opsin includes a modification corresponding to the amino acid at position 188 when aligned with SEQ ID NO: 1.

[0229] Opsin is a photosensitive G protein-coupled receptor that is ubiquitously present in animals. All opsins have a common structural element containing seven transmembrane domains, and Lys296 of opsin (based on the bovine rhodopsin numbering system) binds retinal, a light-absorbing chromophore, via a Schiff base bond. Opsin functions in both visual and non-visual light reception and can be classified into several groups based on the amino acid sequence (Shichida and Matsuyama, 2009; Koyanagi and Terakita, 2014). Bovine rhodopsin is the most studied opsin (Yau and Hardie, 2009), which functions as a visual light-receiving protein in the retina and binds 11-cis retinal in the dark. When retinal undergoes photoisomerization to form the all-trans form, the Meta II intermediate of rhodopsin is generated and binds to the G protein. Meta II is a metastable active state that spontaneously converts to Meta III (Heck et al., 2003). In addition, when Meta II is irradiated with light, the formation of Meta III is induced instead of the original dark state (Bartl et al., 2001; Ritter et al., 2008). That is, the efficiency of restoring the active state of Meta II to the original dark state by photoreaction and thermal reaction is very low. Thus, it can be seen that vertebrate opsins are specialized for photoactivation and have the characteristics of monostable opsins. On the other hand, in mollusks and arthropods, opsin forms a stable active state of the acidic Meta state by photoisomerization of 11-cis to all-trans retinal, and the active state can be restored to the original dark state containing 11-cis retinal by photoreaction (Koyanagi and Terakita, 2014; Yau and Hardie, 2009). That is, these opsins have two stable states, a dark state and an active state, that can be interconverted by light, and are therefore called bistable opsins. In recent years, insights into the molecular properties of opsins have been accumulating, and it has been found that many members of various opsin groups are bistable opsins. It is thought that vertebrate opsins evolved from ancestral bistable opsins into monostable opsins (Shichida and Matsuyama, 2009).

[0230] Among the opsins possessed by animals, the only opsin (Opn5L1) with photocycles has a cysteine residue at position 188, and this residue is known to be the basis of the photocycles of opsins (Sato et al., 2018). Instead of binding to 11-cis retinal in the dark, Opn5L1 binds to all-trans retinal to form an active state. Upon light irradiation, retinal is photoisomerized to the 11-cis form, thereby inhibiting the G protein activation function of Opn5L1. Subsequently, a covalent bond is formed between retinal and Cys188 of the opsin, and the C11═C12 double bond in retinal becomes a single bond. Then, the C11-C12 single bond in retinal undergoes thermal rotation to dissociate the Cys188-retinal adduct, and the original dark state is regenerated. The G protein activation ability of Opn5L1 can be controlled by the combination of photoisomerization and thermal isomerization of retinal, and it can be said to be the first animal opsin whose activity is controlled by photocycles.

[0231] Comparing the amino acid sequences among opsins, the cysteine residue at position 188 is highly conserved within the Opn5L1 group and is hardly observed in other opsin groups. Thus, it can be seen that Cys188 is important for the unique photocycles of Opn5L1. On the other hand, vertebrate opsins and cone opsins, which are monostable opsins, have a glycine residue at this position ( Figure 9 ). In the present disclosure, it has been found that mutations at position 188 can confer photocycles to bovine rhodopsin. It has been clarified in the present disclosure that the G188C mutant can be converted into Meta II, which is an active state, by light irradiation and can return to the original dark state by heat. In addition, when the Meta II of the G188C mutant is irradiated with light, it can return to the original dark state. Therefore, it can be seen that the G188C mutant of bovine rhodopsin exhibits photocycles and photoreversibility, and in vertebrate opsins, the residue at position 188 controls the recovery from the active state to the original dark state.

[0232] In one embodiment of the present disclosure, as the amino acid at position 188 in the amino acid sequence of the opsin class of the present disclosure when aligned with SEQ ID NO: 1, amino acids equivalent to G, T, S, or E can be exemplified, and the modified opsin class of the present disclosure can be those obtained by modifying these amino acids.

[0233] In one embodiment of the present disclosure, it may include the modification of the amino acid equivalent to position G188 to cysteine when aligned with SEQ ID NO: 1. As the amino acid equivalent to position G188 of the opsin class used in the present disclosure, amino acids equivalent to G, T, S, or E can be exemplified as described above. By modifying any of these to cysteine, photocycles and photoreversibility can be obtained. Therefore, in one embodiment of the present disclosure, the protein of the present disclosure can be inactivated without releasing the light-receiving factor after being activated by light stimulation.

[0234] In one embodiment, as the opsin used in the present disclosure, any opsin can be used. For example, G protein-coupled receptor rhodopsin, rod opsin, cone opsin (blue opsin, green opsin, red opsin, melanopsin, neuropsin, OPN5 Panopsin, RGR, and peropsin, etc.) can be used, but are not limited to these. In one embodiment, as the opsin used in the present disclosure, those derived from vertebrates, invertebrates, or microorganisms or chimeras derived from these organisms can be cited, but are not limited to these. As vertebrates, mammals, birds, reptiles, amphibians, fish (bony fish, cartilaginous fish), or agnathans can be cited, but are not limited to these. In addition, as invertebrates, mollusks, arthropods, or cnidarians can be cited, but are not limited to these. As microorganisms, eubacteria, archaea, or fungi can be cited, but are not limited to these. In one embodiment, the opsin of the present disclosure can be derived from mammals. As G protein-coupled receptor rhodopsin, those derived from mammals can be cited, but are not limited thereto. For example, rhodopsin derived from rodents, even-toed ungulates, odd-toed ungulates, primates, carnivores, etc. can be used. For example, rhodopsin of even-toed ungulates or primates can be used, and it is advantageous that it is rhodopsin of primates. In addition, as the opsin that can be used, for example, opsin derived from cattle, humans, mice, rats, cats, dogs, pigs, sheep, horses, etc. can be cited. Among these, those derived from cattle or humans are particularly preferred.

[0235] In one embodiment, the specific amino acid sequences of the opsin of the present disclosure may include the sequences of SEQ ID NOs: 1 to 34 shown in Table 1, but are not limited to these sequences.

[0236] [Table 1-1]

[0237]

[0238] [Table 1-2]

[0239]

[0240] [Table 1-3]

[0241]

[0242] [Table 1-4]

[0243]

[0244] [Table 1-5]

[0245]

[0246] [Table 1-6]

[0247]

[0248]

Table 1-7

[0249]

[0250]

Table 1-8

[0251]

[0252] In one embodiment, the specific nucleic acid sequences of the opsin-like proteins of the present disclosure may include the sequences of SEQ ID NOs: 35 to 68 recorded in Table 2, but are not limited to these sequences.

[0253]

Table 2-1

[0254]

[0255]

Table 2-2

[0256]

[0257]

Table 2-3

[0258]

[0259]

Table 2-4

[0260]

[0261]

Table 2-5

[0262]

[0263]

Table 2-6

[0264]

[0265]

Table 2-7

[0266]

[0267]

Table 2-8

[0268]

[0269]

Table 2-9

[0270]

[0271]

Table 2-10

[0272]

[0273]

Table 2-11

[0274]

[0275]

Table 2-12

[0276]

[0277]

Table 2-13

[0278]

[0279]

Table 2-14

[0280]

[0281]

Table 2-15

[0282]

[0283]

Table 2-16

[0284]

[0285]

Table 2-17

[0286]

[0287]

Table 2-18

[0288]

[0289]

Table 2-19

[0290]

[0291]

Table 2-20

[0292]

[0293] In certain embodiments, the protein of the present disclosure is a chimeric protein that includes a portion of the G protein-coupled receptor rhodopsin and includes portions of other G protein-coupled receptors, metabotropic glutamate receptors, and adrenergic receptors, and has a seven-transmembrane structure.

[0294] In one embodiment of the present disclosure, the amino acid sequence encoding the protein of the present disclosure may include:

[0295] 1) The above-modified amino acid sequence is included in the amino acid sequence shown in any one of SEQ ID NOs: 1 to 34,

[0296] 2) Amino acid sequences in which the sequences other than the above-mentioned modified sites have at least about 80% identity with the sequence of 1) and the encoded proteins have substantially the same biological activity as the proteins obtained from the sequence of 1).

[0297] 3) Amino acid sequences in which there are one or more mutations in the sequence of 1) other than the above-mentioned modified sites and the encoded proteins have substantially the same biological activity as the proteins obtained from the sequence of 1).

[0298] 4) Amino acid sequences encoded by nucleic acids that can hybridize with the nucleic acid encoding the sequence of 1) and contain the above-mentioned modified amino acid sequences, or

[0299] 5) Amino acid sequences encoded by allelic mutants of the nucleic acid encoding the sequence of 1) and contain the above-mentioned modified amino acid sequences.

[0300] In one embodiment, the amino acid sequence encoding the protein of the present disclosure may be: an amino acid sequence in which the sequences other than the modified sites have at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or 100% identity with the sequence of 1) and the encoded proteins have substantially the same biological activity as the proteins obtained from the sequence of 1).

[0301] In one embodiment of the present disclosure, the protein of the present disclosure may contain a modification of the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1 in the amino acid sequence of opsins. As the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1, for example, an amino acid corresponding to E at position 122 can be mentioned. The modified opsins of the present disclosure can modify these amino acids.

[0302] In one embodiment of the present disclosure, it may contain a modification of the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1 to glutamine. As the amino acid corresponding to position 122 of the opsins used in the present disclosure, as described above, an amino acid corresponding to E can be mentioned. By modifying any of these to glutamine, the disintegration of Meta II can be promoted and the photocycle reaction can be accelerated.

[0303] In one embodiment of the present disclosure, the opsin-like proteins of the present disclosure may include, in the amino acid sequence, amino acid modifications corresponding to a part of the N-terminal domain (positions 1 to 11) and a part of the third extracellular loop (positions 278 to 285) when aligned with SEQ ID NO: 1. In one embodiment, the amino acids corresponding to a part of the N-terminal domain (positions 1 to 11) and a part of the third extracellular loop (positions 278 to 285) when aligned with SEQ ID NO: 1 may be modified to cysteine. For example, the amino acids corresponding to a part of the N-terminal domain (positions 1 to 11) and a part of the third extracellular loop (positions 278 to 285) when aligned with SEQ ID NO: 1 may also be the amino acids corresponding to positions 2 and 282 in the amino acid sequence of the opsin-like proteins, and preferably may include the modification of these amino acids to cysteine. By performing such modifications, the thermal stability of the opsin-like proteins of the present disclosure can be improved. In another embodiment, the amino acid sequence of the opsin-like proteins of the present disclosure may be modified as follows: modification of any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acids among the amino acids corresponding to position 1, position 2, position 3, position 4, position 5, position 6, position 7, position 8, position 9, position 10, and position 11 of the N-terminal domain when aligned with SEQ ID NO: 1; and modification of any 1, 2, 3, 4, 5, 6, 7, or 8 amino acids among the amino acids corresponding to positions 278, 279, 280, 281, 282, 283, 284, and 285 of the third extracellular loop when aligned with SEQ ID NO: 1, and preferably such 1 or more amino acids may be modified to cysteine. Additionally, in yet another embodiment, the modified amino acid sequence may preferably have an amino acid sequence with 1 or more (preferably 1 or several, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) conservative substitutions in the amino acid sequence of the opsin.

[0304] The method for obtaining nucleic acids such as DNA of the present disclosure is not particularly limited, and examples thereof include known methods such as a method of obtaining cDNA by reverse transcription from mRNA (e.g., RT-PCR method), a method of preparing from genomic DNA, a method of synthesizing by chemical synthesis, and a method of isolating from a genomic DNA library or a cDNA library (e.g., refer to Japanese Patent Laid-Open No. 11-29599).

[0305] In this specification, the chimeric protein can be prepared, for example, by using a transformant into which an expression vector containing a nucleic acid such as DNA encoding the above chimeric protein has been introduced. For example, first, the transformant is cultured under suitable conditions to synthesize the chimeric protein encoded by the DNA or the like nucleic acid. Then, by recovering the synthesized protein from the transformant or the culture solution, the chimeric protein of the present disclosure can be obtained.

[0306] More specifically, it can be prepared by inserting DNA encoding the above chimeric protein into a suitable expression vector. A "suitable vector" only needs to be able to replicate, maintain, or self-proliferate in various hosts of prokaryotes and / or eukaryotes, and can be appropriately selected according to the purpose of use. For example, in the case where a large amount of nucleic acid such as DNA is desired, a high-copy vector can be selected, and in the case where a polypeptide (chimeric protein) is desired, an expression vector can be selected. Specific examples thereof are not particularly limited, and for example, known vectors described in Japanese Patent Laid-Open No. 11-29599 can be cited.

[0307] In addition, the expression vector is not only used for synthesizing the chimeric protein, but also can be used in the compositions of the present disclosure and the like. That is, the compositions of the present disclosure and the like can contain an expression vector incorporating the nucleic acid construct of the present disclosure as an active ingredient. By directly introducing the above expression vector into the human body, it can be used for the treatment, prevention, and inhibition of progression of diseases, disorders, or symptoms of the retina. In this case, a vector that can be introduced into human cells is used. As the above vector, for example, an adeno-associated virus vector (AAV vector) or a lentiviral vector is suitable.

[0308] The method for introducing the vector can be appropriately selected according to the type of the vector, the host, and the like. Specific examples thereof are not particularly limited. For example, when bacteria are used as the host, known methods such as the protoplast method and the competent cell method can be cited (for example, refer to Japanese Patent Laid-Open No. 11-29599). In addition, when the expression vector is used as an active ingredient of the visual function restoring agent or the visual function reducing preventive agent of the present disclosure, for example, it can be introduced by injecting the above AAV vector or the like into the eye.

[0309] The host into which the expression vector is introduced only needs to be a host suitable for the expression vector and capable of transformation. Specific examples thereof are not particularly limited, and known natural cells or artificially established cells such as bacteria, yeast, animal cells, and insect cells can be cited (refer to Japanese Patent Laid-Open No. 11-29599), or animals such as humans and mice. For the culture of the transformant, in order to obtain the chimeric protein in large quantities and easily, it can be appropriately selected from known nutrient media according to the type of the transformant and the like, and the temperature, the pH of the nutrient medium, the culture time, etc. can be appropriately adjusted for culturing (for example, refer to Japanese Patent Laid-Open No. 11-29599).

[0310] In a preferred embodiment, the opsin-like proteins of the present disclosure are provided in the form of nucleic acid molecules or nucleic acid constructs containing such nucleic acid molecules, and are provided in the form of drugs for gene therapy. As the nucleic acid molecules encoding opsin-like proteins or nucleic acid constructs containing such nucleic acid molecules that can be used in this embodiment, nucleic acid molecules encoding the following proteins or nucleic acid constructs containing such nucleic acid molecules can be cited. The protein is a protein containing the amino acid sequence of an opsin-like protein, and the amino acid sequence of the opsin-like protein contains a modification corresponding to the amino acid at position 188 when aligned with SEQ ID NO: 1. As the opsin-like protein, the opsin-like proteins described in other positions of this specification can be used. In addition, in this specification, the nucleic acid molecules encoding opsins include, but are not limited to, nucleic acid molecules encoding melanopsin, neuropsin, OPN5, RGR, and peropsin. In one embodiment, the nucleic acid molecules used in the present disclosure can be nucleic acid molecules encoding microbial opsins, animal opsins, etc. More specifically, the animal opsins can further be nucleic acid molecules encoding vertebrate visual opsins, vertebrate non-visual opsins, invertebrate opsins, etc. The nucleic acid molecules that can be used in the present disclosure can be nucleic acid molecules encoding bistable opsins such as vertebrate non-visual opsins and invertebrate opsins. As the nucleic acid molecules encoding opsins used in the present disclosure, nucleic acid molecules encoding functional equivalents that strictly speaking do not belong to microbial opsins, vertebrate non-visual opsins, or invertebrate opsins but have the same function as these suitably usable opsin types can also be suitably used.

[0311] The nucleic acid molecules used in the present disclosure can be, for example, nucleic acid molecules encoding G protein-coupled receptor rhodopsin, chimeric opsins of G protein-coupled receptor rhodopsin. As the nucleic acid molecules encoding G protein-coupled receptor rhodopsin that can be used here, when derived from an animal, preferably from a mammal, the encoded protein can maintain the function of repeated activation and can obtain high activity through endogenous G proteins.

[0312] The methods for separating and purifying the chimeric proteins are not particularly limited, and known methods such as methods using solubility, methods using molecular weight differences, methods using charge, etc. can be cited (for example, refer to Japanese Patent Laid-Open No. 11-29599).

[0313] (Drug uses of opsin-like proteins)

[0314] On the other hand, the present disclosure provides a composition, a compound, a drug or a method for visual regeneration or for treating, preventing or suppressing the progression of a visual disorder or disease, which uses the following protein, a nucleic acid molecule encoding the same or a nucleic acid construct containing the nucleic acid molecule, the protein being a protein containing an amino acid sequence of opsin and containing a modification of an amino acid corresponding to the 188th position when aligned with SEQ ID NO: 1. The present disclosure can provide a method for visual regeneration of a subject or for treating, preventing or suppressing the progression of a visual disorder or disease of the subject, which includes the step of administering the protein of the present disclosure to the above-mentioned subject. In addition, the present disclosure can provide the use of the opsin of the present disclosure or a nucleic acid molecule encoding the same for manufacturing a composition or a drug for visual regeneration or for treating, preventing or suppressing the progression of a visual disorder or disease of the subject. The opsin of the present disclosure may have an activity of not releasing retinal when receiving light.

[0315] In one embodiment, examples of the disease, disorder or symptom that can be the subject of the opsin of the present disclosure, a nucleic acid molecule encoding the same or a nucleic acid construct containing the nucleic acid molecule include vesicular transport disorders, diabetes, diabetic retinopathy, myopia, macular degeneration (e.g., age-related macular degeneration), glaucoma, cataract, viral infections, corneal dystrophy, retinoblastoma, Alzheimer's disease, Parkinson's disease, lifestyle diseases, stroke, hearing loss, arrhythmia, heart failure, motor paralysis, autonomic nerve disorders, depression, anxiety disorders, urination and defecation disorders, rehabilitation, etc., but are not limited to these.

[0316] In one embodiment, the opsin of the present disclosure, a nucleic acid molecule encoding the same or a nucleic acid construct containing the nucleic acid molecule can also be formulated into a drug targeting human diseases, disorders or symptoms, or can be formulated into a drug targeting diseases, disorders or symptoms of animals other than humans.

[0317] In one embodiment, the opsin of the present disclosure, a protein containing the same, a nucleic acid molecule encoding the opsin and / or a nucleic acid construct containing the nucleic acid molecule can be provided in the form of cells containing these, and the cells can also be used as a drug.

[0318] In one embodiment, a protein of the present disclosure that includes an amino acid sequence of an opsin class and has a modification of an amino acid corresponding to position 188 when aligned with SEQ ID NO: 1 can also be used in optogenetics and can be used to control only specific neural activities in a target animal. Optogenetics refers to the following technology: by introducing a gene encoding a light-driven ion channel, pump, enzyme, etc., a light-driven protein is expressed and converted into a form capable of optically controlling target cells. Thereby, reversible, immediate, and bioorthogonal operation / control and analysis can be performed on specific cells (groups) at the millisecond time scale in a living animal. Regarding optogenetics, reference can be made to, for example, Tye, K.M.; Deisseroth, K. “Optogenetic investigation of neural circuits underlying brain disease in animal models,” Nat. Rev. Neurosci. 2012, 13, 251. doi:10.1038 / nrn3171; Deisseroth, K. “Optogenetics,” Nat. Method 2011, 8, 26. doi:10.1038 / nmeth.f.324, etc.

[0319] In another aspect of the present disclosure, the opsin class of the present disclosure or a nucleic acid molecule encoding the same can also be provided in the form of a kit combined with a companion diagnostic agent. For example, a composition containing an active ingredient of the drug of the present disclosure can be administered to the subject when a disease or disorder such as a retina-related disease occurs or is expected to occur, thereby exerting a therapeutic effect. Therefore, the composition of the present disclosure can be combined with a companion diagnostic agent for pre-diagnosing visual-related disorders, diseases, or disorders, and after diagnosing or examining the gene status of the subject or the genes carried by the subject, it can be administered only to the subject who is expected to be effective for the composition of the present disclosure.

[0320] In one aspect of the present disclosure, the composition or drug of the present disclosure can also be provided in the form of a nucleic acid drug. In one embodiment, when using the nucleic acid drug of the present disclosure for gene therapy or gene treatment, a polynucleotide can be introduced into the genome of a cell in order to restore or modify a gene and / or the expression of a gene. For example, a therapeutic method of introducing a normal gene using a vector that can be introduced into human cells through various viral vectors, etc., or other delivery systems can be used. The method of introducing the vector can be appropriately selected according to the type of the vector, host, etc. When using an expression vector as the active ingredient of the composition of the present disclosure, for example, it can be introduced by intravitreal injection of an AAV vector, etc.

[0321] For the purposes of this specification, "gene therapy" means: inserting a nucleic acid sequence (e.g., the introduced gene as defined in this specification) into the cells and / or tissues of an individual to treat a disease. The introduced gene can be a functional mutant allele that replaces or supplements a defective allele. Gene therapy also includes: inserting an introduced gene that can inhibit, reduce, or decrease the expression, activity, or function of an endogenous gene or protein such as a naturally inhibitory, i.e., unwanted or abnormal (e.g., pathogenic) gene or protein. Such an introduced gene can be exogenous. An exogenous molecule or sequence can be understood as a molecule or sequence that is not normally present in the cells, tissues, and / or individual being treated. Gene therapy is applicable to both acquired and congenital diseases.

[0322] In this specification, a "gene therapy vector" is any vector capable of delivering a polynucleotide encoding a therapeutic protein (e.g., opsins, etc.) to a host, such as a patient. In some embodiments, the gene therapy vector targets specific host cells or organs, for example, for local delivery, such as tissue-specific delivery. Typically, in local delivery, it is necessary to form a reservoir mainly in an organ, such as the liver, and / or the protein (e.g., the therapeutic protein) encoded by the mRNA translated and expressed therefrom, such as a liver reservoir for producing (and secreting) the protein. In some embodiments, the gene therapy vector is configured to deliver a polynucleotide encoding a therapeutic protein to the eye of a patient. In some embodiments, the gene therapy vector delivers a polynucleotide encoding a therapeutic protein to other tissues in a patient. In some embodiments, the gene therapy vector delivers a polynucleotide encoding a therapeutic protein to the optic nerve of a patient.

[0323] Known or future-developed gene therapy delivery vectors, whether natural or engineered, can be used in the practice of the present disclosure. In some embodiments, the gene therapy vector is a viral vector, including, for example, a virus, viral capsid, viral genome, etc. In some embodiments, the gene therapy vector is a naked polynucleotide, such as an episome. In some embodiments, the gene therapy vector comprises a polynucleotide complex. Exemplary and non-limiting polynucleotide complexes used as gene therapy vectors include lipid complexes, polymersomes, polypexes, dendrimers, inorganic nanoparticles (e.g., gold, silica, iron oxide, calcium phosphate, etc. coated with polynucleotides). In some embodiments, the gene therapy vector described in this specification comprises a combination of a viral vector, a naked polynucleotide, and a polynucleotide complex.

[0324] In one embodiment, the gene therapy vector is a viral vector. Examples of such viral vectors include retroviruses, adenoviruses, herpes simplex viruses, poxviruses, vaccinia viruses, lentiviruses, or adeno-associated viruses. In one embodiment, the gene therapy vector is an adeno-associated virus (AAV). Examples of such adeno-associated viruses include serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, or engineered or naturally selected variants thereof. In one embodiment, the polynucleotide further contains an adeno-associated virus (AAV) nucleic acid sequence. In one embodiment, the gene therapy vector is a chimeric adeno-associated virus containing gene factors from two or more serotypes. For example, an AAV vector having a rep gene derived from AAV1 and a cap gene derived from AAV2 (referred to as AAV1 / 2 or AAV RC1 / 2) can be used as a gene therapy vector for delivering the polynucleotide of the therapeutic protein of the present disclosure to cells or patient cells in need.In one embodiment, the gene therapy vector is AAV1 / 2, AAV1 / 3, AAV1 / 4, AAV1 / 5, AAV1 / 6, AAV1 / 7, AAV1 / 8, AAV1 / 9, AAV1 / 10, AAV1 / 11, AAV2 / 1, AAV2 / 3, AAV2 / 4, AAV2 / 5, AAV2 / 6, AAV2 / 7, AAV2 / 8, AAV2 / 9, AAV2 / 10, AAV2 / 11, AAV3 / 1, AAV3 / 2, AAV3 / 4, AAV3 / 5, AAV3 / 6, AAV3 / 7, AAV3 / 8, AAV3 / 9, AAV3 / 10, AAV3 / 10, AAV4 / 1, AAV4 / 2, AAV4 / 3, AAV4 / 5, AAV4 / 6, AAV4 / 7, AAV4 / 8, AAV4 / 9, AAV4 / 10, AAV4 / 11, AAV5 / 1, AAV5 / 2, AAV5 / 3, AAV5 / 4, AAV5 / 6, AAV5 / 7, AAV5 / 8, AAV5 / 9, AAV5 / 10, AAV5 / 11, AAV6 / 1, AAV6 / 2, AAV6 / 3, AAV6 / 4, AAV6 / 5, AAV6 / 7, AAV6 / 8, AAV6 / 9, AAV6 / 10, AAV6 / 10, AAV7 / 1, AAV7 / 2, AAV7 / 3, AAV7 / 4, AAV7 / 5, AAV7 / 6, AAV7 / 8, AAV7 / 9, AAV7 / 10, AAV7 / 11, AAV8 / 1, AAV8 / 2, AAV8 / 3, AAV8 / 4, AAV8 / 5, AAV8 / 6, AAV8 / 7, AAV8 / 9, AAV8 / 10, AAV8 / 11, AAV9 / 1, AAV9 / 2, AAV9 / 3, AAV9 / 4, AAV9 / 5, AAV9 / 6, AAV9 / 7, AAV9 / 8, AAV9 / 10, AAV9 / 11, AAV10 / 1, AAV10 / 2, AAV10 / 3, AAV10 / 4, AAV10 / 5, AAV10 / 6, AAV10 / 7, AAV10 / 8, AAV10 / 9, AAV10 / 11, AAV11 / 1, AAV11 / 2, AAV11 / 3, AAV11 / 4, AAV11 / 5, AAV11 / 6, AAV11 / 7, AAV11 / 8, AAV11 / 9, AAV11 / 10, their chimeric virus particles or derivatives.Gao et al., "Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy", PNAS 99(18):11854-11859, Sep. 3, 2002 is incorporated herein by reference for AAV vectors and chimeric virus particles useful as gene therapy vectors, and their construction pairs and applications.

[0325] In some embodiments, the gene therapy vector is a viral vector that has been pseudotyped (e.g., engineered) to target specific cells (e.g., retinal cells). Many of the advances in targeted gene therapy using viral vectors have been the non-recombinant (non-genetic) or recombinant (genetic) modification of viral vectors, which may result in the pseudotyping, expansion, and / or redirection of the natural tropism of the viral vector (reviewed in Nicklin and Baker (2002) Curr. Gene Ther. 2:273-93; Verheiji and Rottier (2012) Advances Virol 2012:1-15). Non-genetic approaches typically utilize an adaptor that recognizes both the wild-type (unmodified) viral surface protein and the target cell. As the viral-binding domain of the adaptor, soluble pseudoreceptors (for wild-type virus), polymers such as polyethylene glycol, and antibodies or portions thereof are used. On the other hand, as the cell-binding domain of the adaptor, natural peptides or vitamin ligands, and antibodies and portions thereof are used. For example, redirection of the viral vector to the target cell can be achieved by binding the vector:adaptor complex to a protein expressed on the surface of the target cell, such as a cell surface protein. Such approaches can be used for AAV (Bartlett et al. (1999) Nat. Biotechnol. 74:2777-2785), adenovirus (Hemminki et al. (2001) Cancer Res. 61:6377-81; van Beusechem et al. (2003) Gene Therapy 10:1982-1991; Einfeld, et al. (2001) J. Virol. 75:11284-91; Glasgow et al. (2009) PLOS One 4:e8355), herpesvirus (Nakano etal. (2005) Mol. Ther. 11:617-24), paramyxovirus (Bian et al. (2005) Cancer Gene Ther. 12:295-303; Bian et al. (2005) Int. J. Oncol. 29:1359-69), coronavirus (Haijema et al. (2003) J. Virol. 77:4528-4538; Wurdinger et al. (2005) Gene Therapy 12:1394-1404).

[0326] Conventional approaches are based on recombinant genetic alterations of viral capsid proteins and thus on recombinant genetic alterations on the surface of the viral capsid. In indirect recombinant approaches, the viral capsid is modified with a heterologous "scaffold" and then linked to an adaptor. The adaptor binds to the scaffold and the target cell. (See also Arnold et al. (2006) Mol. Ther. 5:125-132; Ponnazhagen et al. (2002) J. Virol. 76:12900-907; WO 97 / 05266) Scaffolds such as (1) Fc-binding molecules (e.g., Fc receptors, protein A, etc.) that bind to the Fc of an antibody adaptor, (2) (strept)avidin that binds to a biotinylated adaptor, (3) biotin that binds to an adaptor fused to (strept)avidin, and (4) SpyCatcher that binds to a SpyTagylated adaptor, etc., proteins that form isopeptide bonds: protein-binding pairs can be incorporated into Ad (Pereboeva et al. (2007) Gene Therapy 14:627-637; Park et al. (2008) Biochemical and Biophysical Research Communications 366:769-774; Henning et al. (2002) Human Gene Therapy 13:1427-1439; Banerjee et al. (2011) Bioorganic and Medicinal Chemistry Letters 21:4985-4988), AAV (Gigout et al. (2005) Molecular Therapy 11:856-865; Stachler et al. (2008) Molecular Therapy 16:1467-1473), and togavirus (Quetglas et al. (2010) Virus Research 153:179-196; Ohno et al. (1997) Nature Biotechnology 15:763-767; Klimstra et al. (2005) Virology 338:9-21).

[0327] In direct re-targeting regimens, the targeting ligand is altered by direct insertion or binding to the viral capsid, i.e., the protein viral capsid is modified to express a heterologous ligand. The ligand is re-directed, e.g., to bind to a receptor or marker that is preferentially or exclusively expressed on the target cell. This can be used for poxviruses (Guse et al. (2011) Expert Opinion on Biological Therapy 11:595-608; Galmiche et al. (1997) Journal of General Virology 78:3019-3027; Paul et al. (2007) Viral Immunology 20:664-671), paramyxoviruses (Nakamura and Russell (2004) Expert Opinion on Biological Therapy 4:1685-1692; Hammond et al. (2001) Journal of Virology 75:2087-2096; Galanis (2010) Clinical Pharmacology and Therapeutics 88:620-625; Blechacz and Russell (2008) Current Gene Therapy 8:162-175; Russell and Peng (2009) Current Topics in Microbiology and Immunology 330:213-241) and herpesviruses (Shah and Breakefield (2006) Current Gene Therapy 6:361-370; Campadelli-Fiume et al. (2011) Reviews in Medical Virology 21:213-226).

[0328] In some embodiments, the gene therapy vectors described in this specification comprise naked polynucleotides. For example, in some embodiments, polynucleotides encoding a therapeutic polypeptide are sometimes directly injected into an organ, intravenously, to form a depot, e.g., near the eye. Other known methods for enhancing the delivery of naked polynucleotides are not limited to the following, and include electroporation, sonoporation, gene gun using gold particles coated with polynucleotides for injection, magnetic particles, and hydrodynamic delivery.

[0329] In some embodiments, the gene therapy vectors described in this specification comprise polynucleotide complexes, e.g., including, without limitation, nanoparticles (e.g., polynucleotide self-assembled nanoparticles, polymer-based self-assembled nanoparticles, inorganic nanoparticles, lipid nanoparticles, semiconducting / metallic nanoparticles), gels and hydrogels, polynucleotide complexes containing cations and anions, microparticles, and any combination thereof, etc.

[0330] In some embodiments, the polynucleotides disclosed in this specification can be formulated into self-assembled nanoparticles. As non-limiting examples, polynucleotides can be used to prepare nanoparticles for use in polynucleotide delivery systems (e.g., see International Patent Application Publication No. 2012125987, which is incorporated herein by reference in its entirety). In some embodiments, the polynucleotide self-assembled nanoparticles can comprise the polynucleotide core and a polymer shell disclosed in this specification. The polymer shell can be any of the polymers described in this specification and is known in the art. In other embodiments, the polymer shell can be used to protect the polynucleotides within the core.

[0331] In some embodiments, these self-assembled nanoparticles are sometimes microsponges formed from long polymers, i.e., polynucleotide hairpins, which form crystalline "wrinkled" sheets and then self-assemble into microsponges. These microsponges are highly dense-packed sponge-like microparticles that act as efficient carriers capable of delivering payloads into cells. The diameter of the microsponges can range from 1 μm to 300 nm. The microsponges can be complexed with other reagents known in the art to form larger microsponges. As non-limiting examples, the microsponges are sometimes complexed with reagents for forming an outer layer and promoting cell uptake, such as polycationic polyethyleneimine (PEI), etc. The complex can form particles with a diameter of 250 nm that can remain in a stable state at high temperature (150 °C) (Grabow and Jaegar, Nature Materials 2012, 11:269-269; which is incorporated herein by reference in its entirety). Furthermore, these microsponges can sometimes provide extraordinary protection against ribonuclease degradation. In another embodiment, polymer-based self-assembled nanoparticles, such as non-limiting microsponges, etc., are sometimes fully programmed nanoparticles. The geometric shape, size, and stoichiometry of the nanoparticles can be precisely controlled to form nanoparticles most suitable for delivering payloads, such as non-limiting polynucleotides, etc.

[0332] In some embodiments, the polynucleotide can be formulated into inorganic nanoparticles (the entirety of which is incorporated herein by reference to U.S. Patent No. 8,257,745). The inorganic nanoparticles can include clay-like materials that can swell in water, but are not limited thereto. As a non-limiting example, the inorganic nanoparticles can include synthetic montmorillonite clay made of simple silicates (see, e.g., U.S. Patent Nos. 5,585,108 and 8,257,745, the entireties of which are incorporated herein by reference).

[0333] In some embodiments, the polynucleotide can be formulated with water-dispersible nanoparticles comprising a semiconducting material or a metallic material (the entirety of which is incorporated herein by reference to U.S. Patent Application Publication No. 20120228565), or can be formed with magnetic nanoparticles (the entirety of which is incorporated herein by reference to U.S. Patent Application Publication Nos. 20120265001 and 20120283503). The water-dispersible nanoparticles can be hydrophobic nanoparticles or hydrophilic nanoparticles.

[0334] In some embodiments, the polynucleotides disclosed in this specification can be encapsulated in any hydrogel known in the art that can form a gel when injected into a subject. A hydrogel is a network of hydrophilic polymer chains and sometimes exists as a colloidal gel with water as the dispersion medium. The hydrogel can include natural or synthetic polymers with high absorbency (which can contain more than 99% water). The hydrogel has a relatively high water content and thus also has a softness very similar to natural tissues. The hydrogels described in this specification can be used to encapsulate biocompatible, biodegradable, and / or porous lipid nanoparticles.

[0335] As a non-limiting example, the hydrogel can be an aptamer-functionalized hydrogel. The aptamer-functionalized hydrogel can be programmed by polynucleotide hybridization to release one or more polynucleotides. (Battig et al., J. Am. Chem. Society. 2012 134:12410-12413; the entirety of which is incorporated herein by reference). In some embodiments, the polynucleotide can be encapsulated within lipid nanoparticles, and then the lipid nanoparticles can be encapsulated within the hydrogel.

[0336] In some embodiments, the polynucleotide can be encapsulated within a fibrin gel, fibrin hydrogel, or fibrin adhesive. In another embodiment, the polynucleotide can be formulated into lipid nanoparticles or rapidly cleared lipid nanoparticles prior to encapsulation within a fibrin gel, fibrin hydrogel, or fibrin adhesive. Further, in another embodiment, the polynucleotide can be formulated into a lipid complex prior to encapsulation within a fibrin gel, hydrogel, or fibrin adhesive. Fibrin gels, hydrogels, and adhesives comprise two components, namely a fibrinogen solution and a calcium-rich thrombin solution (see, e.g., Spicer and Mikos, Journal of Controlled Release 2010.148:49-55; Kidd et al., Journal of Controlled Release 2012.157:80-85, which are hereby incorporated by reference in their entireties). The concentrations of the components of the fibrin gel, hydrogel, and / or adhesive can be varied to alter the properties of the gel, hydrogel, and / or adhesive, the mesh size of the network, and / or the degradation properties, such as, without limitation, the release properties of the fibrin gel, hydrogel, and / or adhesive (see, e.g., Spicer and Mikos, Journal of Controlled Release 2010.148:49-55; Kidd et al., Journal of Controlled Release 2012.157:80-85; Catelas et al., Tissue Engineering 2008.14:119-128, which are hereby incorporated by reference in their entireties). This feature is suitable for delivering the polynucleotides disclosed in this specification. (See, e.g., Kidd et al., Journal of Controlled Release 2012.157:80-85; Catelas et al., Tissue Engineering 2008.14:119-128, which are hereby incorporated by reference in their entireties).

[0337] In some embodiments, the polynucleotides disclosed in this specification can comprise cations or anions. In one embodiment, the formulation comprises a metal cation, such as, without limitation, Zn2+, Ca2+, Cu2+, Mg+, and combinations thereof. As a non-limiting example, the formulation can comprise a polymer and a polynucleotide complexed with a metal cation (see, e.g., U.S. Patent Nos. 6,265,389 and 6,555,525, which are hereby incorporated by reference in their entireties).

[0338] In some embodiments, the polynucleotide can be formulated into nanoparticles and / or microparticles. These nanoparticles and / or microparticles can be shaped into shapes and chemical properties of any size. As an example, the nanoparticles and / or microparticles can be fabricated using the PRINT (registered trademark) technology of LIQUIDA TECHNOLOGIES.RTM (Morrisville, N.C.) (refer to International Patent Application Publication No. 2007024323, which is incorporated herein by reference in its entirety).

[0339] In some embodiments, the polynucleotide can be formulated with nanojackets and nanoliposomes by Keystone Nano (State College, Pennsylvania). The nanojacket is made of a compound containing calcium, phosphate and a compound naturally present in the body or a compound further containing a small amount of silicate. The size of the nanojacket is sometimes from 5 to 50 nm and can be used, without limitation, to deliver hydrophilic and hydrophobic compounds such as polynucleotides, main constructs and / or polynucleotides. The nanoliposome is made of lipids, for example, without limitation, made of lipids naturally produced in the body. The size of the nanoliposome is sometimes 60-80 nm and can be used to deliver hydrophilic and hydrophobic compounds, for example, without limitation, to deliver polynucleotides, main constructs and / or polynucleotides. In one embodiment, the polynucleotide disclosed in the present specification is formulated with a nanoliposome such as a ceramide nanoliposome, but is not limited thereto.

[0340] In some embodiments, the polynucleotide, such as DNA, further contains a promoter operably linked to a nucleic acid sequence encoding a therapeutic protein. In certain embodiments, the promoter is a tissue-specific promoter that drives gene expression in a specific tissue. In one embodiment, the tissue-specific promoter is a liver-specific enhancer / promoter derived from the Serpina1 and / or TTR promoter. In another embodiment, the promoter is the CMV promoter. In another embodiment, the promoter is the ubiquitin C promoter.

[0341] In some embodiments, the polynucleotide further comprises a "nucleic acid sequence targeting a locus". The sequence targeting the locus can integrate the polynucleotide encoding the therapeutic protein into the genome of the recipient host cell. In some embodiments, the sequence targeting the locus comprises adjacent homologous arms for homologous recombination. In some embodiments, the sequence targeting the locus comprises a guide RNA sequence and a type II Cas enzyme (i.e., the CRISPR-Cas9 method) for promoting integration. In some embodiments, the sequence targeting the locus comprises a guide zinc finger nuclease (ZFN) recognition sequence for promoting integration. In some embodiments, the sequence targeting the locus comprises a transcription activator-like effector nuclease (TALEN) recognition sequence for promoting integration. Furthermore, in another embodiment, the sequence targeting the locus comprises a single residue encoding a nucleotide used by a BuD-derived nuclease for promoting integration.

[0342] In addition, in one embodiment, as a cell therapy of a cell comprising the composition of the present disclosure, a treatment method of transplanting retinal cells comprising the composition of the present disclosure can be cited. In one embodiment, the cells comprising the composition of the present disclosure can be administered together with other reagents other than cells. As such other reagents, reagents commonly used in ophthalmic treatment (e.g., steroids, antibiotics, antibacterial agents, NSAIDs) can be used. Such other reagents can be included in the cell drug of the present disclosure as a drug, or can be provided in a separately administered manner. When provided or administered separately, it is provided in the form of a kit or a combination reagent. When used in the form of a kit or a combination reagent, an instruction manual describing its usage method can be combined.

[0343] In a preferred embodiment, the present disclosure is preferably administered to a subject before or just after the onset of the above-mentioned disease, disorder or symptom, such as within 1 year, preferably within 6 months, 3 months, or 1 month after the onset (e.g., the appearance of subjective symptoms), but is not limited to these.

[0344] In one specific embodiment, the protein, nucleic acid molecule, nucleic acid construct and / or cell of the present disclosure or a drug comprising them is administered once during the treatment period. As described in the examples, it has been confirmed that the drug of the present disclosure can exert its effect by a single administration, and it is considered that the patient compliance is good.

[0345] In one specific embodiment, when the drug of the present disclosure is administered to the eye, the usage amount of the vector can be set to about 0.01×10 11 ~about 100×10 11 vg / eye dose, for example, the lower limit can be about 0.01×10 11 vg / eye, about 0.02×10 11 vg / eye, about 0.03×10 11vg / eye, approximately 0.04×10 11 vg / eye, approximately 0.05×10 11 vg / eye, approximately 0.06×10 11 vg / eye, approximately 0.07×10 11 vg / eye, approximately 0.08×10 11 vg / eye, approximately 0.09×10 11 vg / eye, approximately 0.1×10 11 vg / eye, approximately 0.2×10 11 vg / eye, approximately 0.3×10 11 vg / eye, approximately 0.4×10 11 vg / eye, approximately 0.5×10 11 vg / eye, etc., the upper limit can be approximately 2×10 11 vg / eye, approximately 3×10 11 vg / eye, approximately 4×10 11 vg / eye, approximately 5×10 11 vg / eye, approximately 6×10 11 vg / eye, approximately 7×10 11 vg / eye, approximately 8×10 11 vg / eye, approximately 9×10 11 vg / eye, approximately 10×10 11 vg / eye, approximately 15×10 11 vg / eye, approximately 20×10 11 vg / eye, approximately 30×10 11 vg / eye, approximately 40×10 11 vg / eye, approximately 50×10 11 vg / eye, approximately 100×10 11 vg / eye, etc.

[0346] In another embodiment, when the drug of the present disclosure is systemically administered (intravenously) or locally administered (intramuscularly, intracerebrally, in the inner ear, etc.), the dosage of the carrier can be set to approximately 0.1×10 11 ~approximately 1000×10 11 vg / kg, for example, the lower limit can be approximately 0.1×10 11 vg / kg, approximately 0.2×10 11 vg / kg, approximately 0.3×10 11 vg / kg, approximately 0.4×10 11 vg / kg, approximately 0.5×10 11 vg / kg, approximately 0.6×10 11 vg / kg, approximately 0.7×10 11 vg / kg, approximately 0.8×10 11vg / kg, about 0.9×10 11 vg / kg, about 1.0×10 11 vg / kg, etc., and the upper limit can be about 20×10 11 vg / kg, about 30×10 11 vg / kg, about 40×10 11 vg / kg, about 50×10 11 vg / kg, about 60×10 11 vg / kg, about 70×10 11 vg / kg, about 80×10 11 vg / kg, about 90×10 11 vg / kg, about 100×10 11 vg / kg, about 150×10 11 vg / kg, about 200×10 11 vg / kg, about 300×10 11 vg / kg, about 400×10 11 vg / kg, about 500×10 11 vg / kg, about 1000×10 11 vg / kg, etc.

[0347] (Combination)

[0348] In one aspect of the present disclosure, two or more of the opsin-like proteins, nucleic acid molecules, nucleic acid constructs, and / or cells of the present disclosure for preventing visual impairment or suppressing its progression, or drugs containing them, can be used in combination. In one embodiment, when the above-mentioned uses are combined, the same active ingredient can be used for combined uses or different active ingredients can be combined.

[0349] (General technology)

[0350] The molecular biology methods, biochemical methods, and microbiological methods used in this specification are well-known and commonly used in the art, such as those described in Current Protocols in Molecular Biology (http: / / onlinelibrary.wiley.com / book / 10.1002 / 0471142727) and Molecular Cloning: A Laboratory Manual (Fourth Edition) (http: / / www.molecularcloning.com), etc., and these are incorporated by reference into the relevant parts (which can be all) of this specification.

[0351] In this specification, "or" is used when "at least one or more" of the matters listed in the text can be adopted. The same applies to "or". When it is clearly stated in this specification that it is "within the range" of "two values", the range also includes the two values themselves.

[0352] For the references such as scientific literatures, patents, patent applications, etc. cited in this specification, in this specification, their entirety is incorporated by reference to the same extent as the specifically described content.

[0353] As described above, for ease of understanding, preferred embodiments have been shown to illustrate the present disclosure. The following describes the present disclosure based on examples. The above description and the following examples are provided for illustrative purposes only and not for the purpose of limiting the present disclosure. Therefore, the scope of the present disclosure is not limited to the specific embodiments and examples described in this specification, but is defined only by the claims.

[0354]

Examples

[0355] Examples are described below. If necessary, the handling of animals used in the following examples complies with the standards and other relevant ethical standards and guidelines stipulated by the institutions to which the applicant belongs, and is based on the Declaration of Helsinki. For reagent products, the products described in the examples are specifically used, and equivalents from other manufacturers (such as Sigma-Aldrich, Wako Pure Chemical Industries, Nacalai, R&D Systems, USCN Life Science INC, etc.) can also be used instead.

[0356] (Experimental procedures)

[0357] In the following examples, each experimental procedure is carried out as follows.

[0358] (Preparation of bovine rhodopsin mutants)

[0359] A mutant cDNA of bovine rhodopsin (accession number: AB062417) was constructed using an in - situ cloning kit (manufactured by Clontech). The wild - type (Gene ID: 509933) and mutant cDNAs of bovine rhodopsin were inserted into mammalian expression vectors pUSRα (Kayada et al., 1995) or pCAGGS (Niwa et al., 1991). HEK293T cells were identified by short tandem repeat profiling. The mycoplasma contamination of these cells was negative. The plasmid was transfected into HEK293T cells by the calcium phosphate method. After 2 days of culture, the transfected cells were collected by centrifugation and suspended in buffer A (50 mM HEPES, 140 mM NaCl, 3 mM MgCl2, pH 6.5), and 11 - cis or all - trans retinal was added to reconstitute the photopigment. These were dissolved in buffer A containing 1% dodecyl maltoside (DDM) and adsorbed onto a Rho1D4 (anti - bovine rhodopsin monoclonal antibody) affinity column to purify the pigment. After washing the column with buffer A containing 0.02% DDM, a synthetic peptide with an epitope sequence was added to elute the pigment. To purify the apoprotein of rhodopsin, the transfected cell membrane without added retinal was dissolved in buffer A containing 1% DDM and adsorbed onto a Rho1D4 affinity column.

[0360] (Spectrophotometry)

[0361] The ultraviolet - visible absorption spectra were recorded using an ultraviolet - visible spectrophotometer (UV - 2450, UV - 2400, Shimadzu Corporation). To analyze the thermal reaction of the pigment in detail, a cuvette holder with a temperature - controlled circulating water bath was used to maintain the sample at 0 °C, 20 °C, or 37 °C. For the sample, either yellow light from a 1 kW tungsten - halogen lamp (Master HILUX - HR; RIKEN) passing through a Y - 52 cut - off filter (Toshiba) or ultraviolet light passing through a UV D - 36 glass filter (AGC TECHNO GLASS) was irradiated.

[0362] To monitor the photocycle process of the G188C mutant of bovine rhodopsin, a time - resolved CCD spectrophotometer (C10000 system, Hamamatsu Photonics) (Sakai et al.) was used to obtain spectra at different times in the dark and after irradiation (170 μs, yellow light from a xenon flash lamp passing through a Y - 52 cut - off filter) from the G188C mutant sample. The temperature of the sample was maintained at 37 °C using a temperature controller (pqod, QUANTUM Northwest). The absorbance change at λmax was plotted as a function of time and fitted with a single - exponential function to obtain the time constant for recovery to the original dark state.

[0363] (Analysis of retinaldehyde isomers)

[0364] For the retinaldehyde isomers in rhodopsin samples, analysis was performed by high-performance liquid chromatography (LC-10ATvp; Shimadzu) using a silica gel column (YMC-Pack SIL, particle size 3 μm, 150 × 6.0 mm, YMC) as described above (Tsutsuie et al., 2007).

[0365] (G protein activation assay)

[0366] Activation of the Gi type of G protein was measured by GDP / GTPγS exchange of G protein using a radioactive nucleotide filtration binding assay (Yamashita et al., 2000; Yamashita et al., 2010). Giαβγ was prepared by mixing rat Giα1 expressed in Escherichia coli BL21 strain (Lee et al., 1994) and Gtβγ purified from bovine retina (Tachibanaki et al., 1997). All assay procedures were carried out at 0 °C. The assay mixture consisted of 10 nM pigment, 600 nM G protein, 50 mM HEPES (pH 7.0), 140 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.01% DDM, 1 μM [35S]GTPγS and 2 μM GDP. Bovine rhodopsin wild type and G188C mutant purified after reconstitution with 11-cis retinaldehyde were mixed with the G protein solution and stored in the dark or irradiated with yellow light (>500 nm) for 1 minute, followed by UV light irradiation for 1 minute or re-irradiation with yellow light for 1 minute. After irradiation, [35S]GTPγS solution was added to the mixture of rhodopsin and G protein to initiate the GDP / GTPγS exchange reaction. After incubation for the selected time in the dark, an aliquot (20 μl) from the sample was added to 200 μl of stop solution (20 mM Tris / Cl [pH 7.4], 100 mM NaCl, 25 mM MgCl2, 1 μM GTPγS, 2 μM GDP), immediately filtered through a nitrocellulose membrane, and [35S]GTPγS bound to the G protein was captured. The amount of bound [35S]GTPγS was quantified by measuring the membrane with a liquid scintillation counter (Tri-Carb 2910TR; PerkinElmer).

[0367] (Measurement of cAMP amount in cultured cells)

[0368] The amount of cAMP in HEK293T cells was measured using the GloSensor cAMP assay (Promega) according to the manufacturer's instructions and previous reports (Bailes and Lucas, 2013). For HEK293T cells, they were seeded in a 96-well plate at a density of 20,000 cells / well in low-serum medium (D-MEM / F12 containing 0.25% FBS). After culturing for 24 hours, the cells were transfected by the polyethyleneimine transfection method with 50 ng of rhodopsin plasmid and 50 ng of Glosensor 22F plasmid per well. After culturing overnight, the medium was replaced with an equilibration medium containing a 2% dilution of the GloSensor cAMP reagent stock solution in CO2-independent medium (Thermo Fisher Scientific), 10% FBS, and 5 μM retinal. After equilibration at room temperature for 2 hours, the luminescence from the cells was measured using a microplate reader (SpectraMax L, Molecular Devices). In the measurement of Gi activation induced by wild-type and mutant rhodopsins, the cells were first treated with 2 μM forskolin to increase the cAMP-dependent luminescence to a plateau level, and then stimulated with yellow light from a 1 kW tungsten halogen lamp passing through a Y-52 cutoff filter for 30 seconds.

[0369] (Example 1: Obtaining the photocycle characteristics of the bovine rhodopsin G188C mutant)

[0370] To analyze whether the G188C mutant of bovine rhodopsin acquired photocycle characteristics, the G188C mutant was purified after reconstitution with 11-cis retinal. Figure 1 Shows the thermal stability of wild-type bovine rhodopsin ( Figure 1 of A), G188C ( Figure 1 of B), and the G188C / N2C / D282C ( Figure 1 of C) mutants after incubation with 11-cis retinal. Absorption spectra were recorded after incubation in the dark at 37 °C for 0, 5, 10, 15, 20 minutes (curves 1-5, respectively). Figure 1 of D shows a schematic diagram of the retinal configuration change of wild-type bovine rhodopsin. The dark state, Meta II, and Meta III include 11-cis-15-trans retinal, all-trans-15-trans retinal, and all-trans-15-cis retinal, respectively. Figure 1 of E and F show the N2C / D282C ( Figure 1 of E) and G188C / N2C / D282C ( Figure 1Absorption spectra of the F) mutant. Spectra were recorded at 20 °C in the dark state (curve 1) and 0, 5, 15, 30, 60, 120 minutes after yellow light irradiation (curves 2 - 7, respectively). The inset shows the difference spectra (curves 1 - 5, respectively) obtained by subtracting the spectra measured immediately after irradiation (curves 2 of E and F) from the spectra measured after irradiation (curves 3 - 7 of E and F). Figure 1 G shows the absorption spectra of the G188C / N2C / D282C mutant measured at 37 °C. Spectra were recorded in the dark state (curve 1) and 0.1, 10, 50, 100, 1000 seconds after yellow light irradiation (curves 2 - 6, respectively). The inset shows the difference spectra (curves 1 - 4, respectively) obtained by subtracting the spectra measured immediately after irradiation (curve 2 of G) from the spectra measured after irradiation (curves 3 - 6 of G). Figure 1 H shows the isomer composition of the retinal of the G188C / N2C / D282C mutant. Chromophores were extracted from samples before light irradiation and 0, 5, 60 minutes after yellow light irradiation at 20 °C, and the retinal composition was analyzed by high performance liquid chromatography (HPLC).

[0371] It was found that the G188C mutant has a very low thermal stability compared to the wild type. That is, the G188C mutant gradually decays during incubation in the dark at 37 °C ( Figure 1 of B), whereas the wild type is extremely stable under the same conditions ( Figure 1 of A). Therefore, the thermal stability of the G188C mutant was improved and the detailed molecular properties of the mutant were analyzed. According to previous reports (Xie et al., 2003; Standfuss et al., 2007), two cysteine residues (N2C / D282C) were introduced into the mutant, and the thermal decay rate during incubation in the dark at 37 °C was measured. From the time-dependent spectral changes, it was found that the G188C / N2C / D282C mutant decays much more slowly than the G188C mutant ( Figure 1 of C). Therefore, spectral changes among the wild type, N2C / D282C, and G188C / N2C / D282C mutants were compared at 20 °C. After yellow light irradiation, the spectrum of the wild type shifted to the ultraviolet region. This indicates the formation of a Meta II intermediate containing all-trans-15 - anti-retinal ( Figure 1 of D). Subsequently, the absorbance near 470 nm increased, indicating the transition from Meta II to a Meta III intermediate containing all-trans-15 - cis-retinal ( Figure 1 of D). These spectral changes were also observed in N2C / D282C ( Figure 1E). On the other hand, the G188C / N2C / D282C mutant has an absorption maximum (λmax) at 487 nm, and its spectrum also shifts to the ultraviolet region due to yellow light irradiation, forming Meta II. Subsequently, when cultured in the dark, a decrease in absorbance in the ultraviolet region and an accompanying increase in absorbance near 485 nm were observed ( Figure 1 F). As a result of analyzing the configuration of retinal, it was found that retinal is isomerized to the all-trans form by light irradiation and then converted to the 11-cis form during incubation in the dark ( Figure 1 H). This interconversion of retinal isomers can explain the spectral changes of the G188C / N2C / D282C mutant after light irradiation. In addition, thermal recovery to the original dark state after light irradiation was also observed at 37 °C ( Figure 1 G). Furthermore, the G188C mutant showed thermal recovery of the absorption spectrum of the original dark state after light irradiation at 20 °C, confirming an increase in the amount of 11-cis retinal during the culture after light irradiation. These results indicate that the G188C mutation has acquired the ability to recover from the photoactivated state to the original dark state by heat.

[0372] Next, it was also analyzed whether other G188 mutants had acquired photocycle characteristics. It is known that the G188E and G188R mutants of human rhodopsin cannot form photopigments after reconstitution with 11-cis retinal (Sung et al., 1993). Therefore, mutant proteins were prepared by introducing 16 other mutations at position 188 of bovine rhodopsin and purifying them after reconstitution with 11-cis retinal. Photopigments were successfully detected from 8 of these mutants. Except for G188D (509 nm), the λmax of the mutants showed a blue shift relative to the wild type (500 nm) (Table 3).

[0373]

Table 3

[0374] Comparison of λ maximum in the dark state and spectral components after UV light irradiation

[0375] λmax Dark state (%) meta|| (%) meta III (%) Wild type 500 21.6 13.7 64.7 G188C 487 41.2 48.5 10.3 G188A 494 18.1 51.7 30.2 G188D 509 30.9 67.7 1.4 G188M 491 9.2 10 80.8 G188N 492 0 0 100 G188Q 493 2.4 5.5 92.1 G188S 495 14.6 37.7 47.7 G188T 488 11 2.9 86.1 G188V 486 7.3 63.4 29.3

[0376] When these mutants were irradiated with yellow light, the spectrum shifted to the ultraviolet region, forming Meta II. Subsequently, when cultured in the dark at 20 °C, each mutant showed characteristic spectral changes. However, these spectral changes were different from the large increase in absorbance near their λmax. From these results, it was found that thermal recovery to the original dark state after light irradiation was not clearly detected in these mutants. Therefore, it can be concluded that the G188C mutant specifically exhibits photocycle characteristics.

[0377] (Example 2: Acquisition of photo-reversible characteristics of bovine rhodopsin G188C mutant)

[0378] Furthermore, analyze whether the Meta II of the G188C mutant recovers to its original dark state depending on light. Figure 2 Show the light response, retinal configuration, and G protein activation of bovine rhodopsin G188C mutant. Figure 2 A and B of show the wild type of bovine rhodopsin purified after incubation with 11-cis retinal at 0 °C ( Figure 2 A of ) or G188C mutant ( Figure 2 B of ). The absorption spectra were recorded in the dark (curve 1), after irradiation with yellow light (>500 nm) (curve 2), after subsequent irradiation with ultraviolet light (360 nm) (curve 3), and after re-irradiation with yellow light (curve 4). The inset shows the spectral changes of the wild type ( Figure 2 A of ) or G188C mutant ( Figure 2 B of ) brought about by yellow light irradiation (curve 1), subsequent ultraviolet light irradiation (curve 2), and re-irradiation with yellow light (curve 3). The difference spectra were calculated from the spectra shown in Figure 2 A of and Figure 2 B of. Figure 2 C and D of show the isomer composition of retinal of the wild type ( Figure 2 C of ) and G188C mutant ( Figure 2 D of ). The chromophore was extracted from the samples before light irradiation, after yellow light irradiation, after subsequent ultraviolet light irradiation, and after re-irradiation with yellow light at 0 °C, and the retinal composition was analyzed by high performance liquid chromatography (HPLC). Figure 2 E of shows the Gi type of the G protein activation ability of the wild type. The activation ability was measured in the dark (closed circles) and after yellow light irradiation (open circles). Figure 2 F of shows the Gi type of the G protein activation ability of the G188C mutant. The activation ability was measured in the dark (closed circles), after yellow light irradiation (open circles), after subsequent ultraviolet light irradiation (open triangles), and after re-irradiation with yellow light (open diamonds). Figure 2 The data shown in E and F of were obtained at 0 °C and are shown in the form of the average ± SEM of 3 independent experiments. Figure 2 G of shows the absorption spectrum (0 °C) of the G188C / N2C / D282C mutant purified after incubation with 11-cis retinal. The spectra were recorded in the dark (curve 1), after irradiation with yellow light (>500 nm) (curve 2), after subsequent irradiation with ultraviolet light (360 nm) (curve 3), after re-irradiation with yellow light (curve 4), and after re-irradiation with ultraviolet light (curve 5). The inset shows the spectral changes caused by yellow light irradiation (curve 1), subsequent ultraviolet light irradiation (curve 2), re-irradiation with yellow light (curve 3), and re-irradiation with ultraviolet light (curve 4). The difference spectra were based on Figure 2Calculated from the spectrum shown in G.

[0379] The wild type and the G188C mutant were cooled to 0 °C to prevent the thermal reaction of Meta II, and the spectral changes induced by yellow light irradiation followed by ultraviolet light irradiation were measured. When yellow light was irradiated on the wild type, Meta II was generated, and the spectrum shifted to the visible region by subsequent ultraviolet light irradiation. The λmax (∼470 nm) was blue-shifted from the λmax of the original dark state ( Figure 2 of A). According to previous studies, this state is equivalent to Meta III ( Figure 1 of D). In addition, template absorption spectra of the dark state, Meta II, and Meta III were constructed by the previous method (Lamb, 1995; Govardovskii et al., 2000), and the difference spectrum obtained by subtracting the spectrum after yellow light irradiation from the spectrum after ultraviolet light irradiation was fitted. As a result of the fitting, it was found that Meta II generates Meta III with much higher efficiency under ultraviolet light irradiation than in the original dark state (Table 3). This spectral analysis is not inconsistent with the observation that a very limited amount of 11-cis retinal is generated from a large amount of all-trans retinal by ultraviolet light irradiation ( Figure 2 of C). From these results, it was confirmed that ultraviolet light irradiation on Meta II induced cis / trans isomerization of the C=N double bond of the Schiff base more efficiently than cis / trans isomerization of retinal.

[0380] When yellow light was irradiated on the G188C mutant, Meta II was generated, and the spectrum shifted to the visible region by subsequent ultraviolet light irradiation. The λmax was basically the same as that in the original dark state ( Figure 2 of B). By re-irradiating with yellow light, a state was formed in which the spectrum almost overlapped with the spectrum induced by the initial yellow light irradiation ( Figure 2 curve 4 in B of). The spectral changes induced by ultraviolet light irradiation and yellow light re-irradiation were mirror images of each other ( Figure 2 curves 2 and 3 in the inset of B of). Substituting the ultraviolet light-dependent spectral changes into the template spectrum indicated that ultraviolet light irradiation on Meta II formed the original dark state with much higher efficiency than Meta III (Table 3). This was also supported by the following observation: when the G188C mutant was irradiated with ultraviolet light, 11-cis retinal increased with much higher efficiency than when the wild type was irradiated with ultraviolet light. Figure 2D). These results indicate that Meta II of the G188C mutant can efficiently undergo a photoconversion to the original dark state. Moreover, bovine rhodopsin not only transduces proteins but also activates Gi / Go-type G proteins (Yamashita et al., 2000; Terakita et al., 2002), so the activation ability of the Gi-type G protein of the G188C mutant was measured. It was found through a GTPγS binding assay that the light-dependent Gi activation ability of G188C is equivalent to that of the wild type ( Figure 2 E, F). Subsequently, when the G188C mutant was irradiated with ultraviolet light, its ability was inhibited, and when irradiated with yellow light again, the ability increased ( Figure 2 F). This can be explained by the change in the absorption spectrum and the change in the retinal isomer ( Figure 2 B, D). Furthermore, it can also be shown that the G188C / N2C / D282C mutant can mutually transform between the original dark state and Meta II by irradiation with yellow light and ultraviolet light at 0 °C ( Figure 2 G). From these data, it can be seen that the G188C mutant has acquired photoreversibility between the dark state and Meta II.

[0381] In addition, the photoreactions of eight other mutants were also analyzed. It was confirmed that the spectra of these mutants all shifted to the ultraviolet region due to yellow light irradiation, and the absorbance in the visible region increased again after subsequent ultraviolet light irradiation. The difference spectra calculated before and after ultraviolet light irradiation were fitted with the spectra of the dark state, Meta II, and Meta III as templates to obtain information on the component ratios of the dark state, Meta II, and Meta III after ultraviolet light irradiation (Table 3). These results indicate that the recovery to the original dark state caused by ultraviolet light irradiation occurs most efficiently in the case of the G188C mutant.

[0382] (Example 3: Acceleration of the photocycle of the G188C mutant)

[0383] Then, it was investigated whether a change in the lifetime of Meta II would change the photocycle rate of the G188C mutant. Figure 3 It shows the acceleration of the recovery rate of the photocycle characteristics of the bovine rhodopsin G188C mutant caused by the introduction of the E122Q mutation. Figure 3 A in shows the absorption spectrum of the E122Q / G188C / N2C / D282C mutant measured at 0 °C. Spectra were recorded in the dark (curve 1), and 0, 5, and 60 minutes after irradiation with yellow light (>500 nm) (curves 2-4, respectively). The inset shows the spectra measured after irradiation ( Figure 3 curves 2-4 in A of ) minus the spectrum before irradiation ( Figure 3Differential spectra (curves 1 - 3 respectively) obtained from the curve 1 of A. Figure 3 B of shows the isomer composition of retinal in the E122Q / G188C / N2C / D282C mutant. Chromophores were extracted from samples before light irradiation, 0, 5, and 60 minutes after yellow light irradiation, and the retinal composition was analyzed by high - performance liquid chromatography (HPLC). Figure 3 C of shows the absorbance spectrum of the E122Q / G188C / N2C / D282C mutant measured at 37°C. Spectra were recorded in the dark (curve 1), 0.1, 1, 5, 10, and 50 seconds after yellow flash irradiation (curves 2 - 6 respectively). The inset shows the differential spectrum (curves 1 - 5 respectively) obtained by subtracting the spectrum before irradiation ( Figure 3 curve 1 of C) from the spectra measured after irradiation ( Figure 3 curves 2 - 6 of C). Figure 3 D of is a graph showing the comparison of the thermal recovery processes of G188C / N2C / D282C and E122Q / G188C / N2C / D282C. The differential absorbance at λmax obtained by subtracting the spectrum before irradiation from the spectra measured after irradiation shown in Figure 1 G and Figure 3 C was plotted against the elapsed time after irradiation. The time constants for the thermal recovery of the G188C / N2C / D282C and E122Q / G188C / N2C / D282C mutants to the dark state at 37°C were 57.4 seconds and 5.1 seconds respectively.

[0384] It has been reported that the E122Q mutation of vertebrate rhodopsin promotes the disintegration of Meta II and shortens the lifetime of Meta II (Imai et al., 1997; Imai et al., 2007). Therefore, the E122Q / G188C / N2C / D282C mutant was prepared, and the spectral changes after light irradiation were measured. From the results of spectral analysis and retinal isomer analysis, it was confirmed that the E122Q / G188C / N2C / D282C mutant could thermally recover to the original dark state after light irradiation at 0°C ( Figure 3 A, B). In addition, the photocycle rate of the E122Q / G188C / N2C / D282C mutant at 37°C ( Figure 3 C) was about 12 times faster than that of the G188C / N2C / D282C mutant ( Figure 3 D). Thus, by using one mutation to change the lifetime of Meta II, the high - speed photocycle reaction of the G188C mutant was successfully achieved.

[0385] (Example 4: Changes in G - protein activation ability due to photocycle characteristics)

[0386] Furthermore, it was investigated whether the photocycle characteristics obtained by the G188C mutation affect the G protein activation ability. Figure 4 It is a graph showing the inhibition of the intracellular cAMP level caused by light in the bovine rhodopsin mutant. Using the GloSensor cAMP assay, the cAMP levels of HEK293T cells transfected with N2C / D282C-( Figure 4 (A, B)), G188C / N2C / D282C-( Figure 4 (C, D)), E122Q / G188C / N2C / D282C-( Figure 4 (E, F)) and mock( Figure 4 (G)) were measured at room temperature. The cells were incubated with 5 μM 11-cis retinal for 2 hours, and then treated with 2 μM forskolin before exposure to yellow light (>500 nm). The data were normalized to the highest value before light irradiation. The detailed curves of the light-dependent cAMP level changes of N2C / D282C, G188C / N2C / D282C, and E122Q / G188C / N2C / D282C are shown in Figure 4 B, Figure 4 D, Figure 4 F, respectively.

[0387] As Figure 2 shown, at 0 °C, the light-dependent Gi activation ability of the wild type and the G188C mutant was the same, and no significant thermal recovery to the original dark state was observed in the G188C mutant. Therefore, the intracellular cAMP amount in cultured cells was measured using a cAMP biosensor (GloSensor), and a comparative study of the luminescence changes of the biosensor triggered by bovine rhodopsin was conducted. Regarding the increase in cAMP level induced by the addition of forskolin, it was found that in N2C / D282C bovine rhodopsin-transfected cells, it decreased due to yellow light irradiation( Figure 4 (A, B)), while in mock-transfected cells, it did not decrease( Figure 4 (G)), and then slowly recovered. On the other hand, it was confirmed that in cells transfected with the G188C / N2C / D282C mutant, yellow light irradiation caused the cAMP level to rapidly recover after a decrease( Figure 4 (C, D)). In addition, it was confirmed that in cells transfected with the E122Q / G188C / N2C / D282C mutant, the cAMP level induced by yellow light irradiation recovered more rapidly from the decrease( Figure 4 (E, F)). These results indicate that the photocycle characteristics obtained by the G188C mutation promote faster recovery to the original dark state and change the G protein activation profile.

[0388] (Example 5: Pigment formation upon addition of all-trans retinal caused by the G188C mutant)

[0389] It was analyzed whether the G188C mutant forms a photopigment after reconstitution with all-trans retinol. Figure 5 It is a figure showing the formation of the photopigment of the bovine rhodopsin G188C mutant after incubation with all-trans retinol. The absorption spectra of the wild type ( Figure 5 A) or the G188C mutant ( Figure 5 B) purified after adding all-trans retinol to a suspension of rhodopsin-expressing cell membranes at 0 °C are shown. The spectra were measured in the dark (curve 1), after irradiation with yellow light (>500 nm) (curve 2), after subsequent irradiation with ultraviolet light (360 nm) (curve 3), and after re-irradiation with yellow light (curve 4). The inset shows the spectral changes caused by yellow light irradiation (curve 1), subsequent ultraviolet light irradiation (curve 2), and re-irradiation with yellow light (curve 3). Figure 5 C shows the isomer composition of retinol of the G188C mutant purified after adding all-trans retinol. The chromophore was extracted from the samples after irradiation with yellow light, subsequent ultraviolet light irradiation, and re-irradiation with yellow light, and the retinol composition was analyzed by high performance liquid chromatography (HPLC). The regeneration of the photopigment when 11-cis retinol ( Figure 5 D, E) or all-trans retinol ( Figure 5 F, G) was added to the purified carrier protein of N2C / D282C is shown. Figure 5 The spectrum of D was measured before addition (curve 1) and at 0, 3, 6, 15, 30, 60, and 120 minutes after adding 1.1 μM 11-cis retinol (curves 2 - 8). Figure 5 E calculates the difference spectrum (curves 1 - 6 respectively) by subtracting the spectra measured at 3, 6, 15, 30, 60, and 120 minutes after adding 11-cis retinol ( Figure 5 curve 3 - 8 of D) from the spectrum measured just after adding 11-cis retinol ( Figure 5 curve 2 of D). Figure 5 F shows the spectra measured before adding 1.1 μM all-trans retinol (curve 1) and at 0, 0.5, 1, 2, 6, 12, and 16 hours after addition (curves 2 - 8). Regarding Figure 5 the difference spectrum of G, it is calculated by subtracting the spectrum measured just after adding all-trans retinol ( Figure 5 curve 2 of F) from the spectra measured at 0.5, 1, 2, 6, 12, and 16 hours after adding all-trans retinol ( Figure 5 curve 3 - 8 of F) (curves 1 - 6 respectively). The regeneration of the photopigment when 11-cis retinol ( Figure 5 H, I) or all-trans retinol ( Figure 5 J, K) was added to the purified carrier protein of G188C / N2C / D282C is shown. Figure 5The spectrum of H was measured before addition (curve 1) and at 0, 3, 6, 15, 30, 60, and 120 minutes after the addition of 1.1 μM 11-cis retinal (curves 2 - 8). Figure 5 I of shows the spectrum from just after the addition of 11-cis retinal ( Figure 5 curve 2 of H) minus the spectra measured at 3, 6, 15, 30, 60, and 120 minutes after the addition of 11-cis retinal ( Figure 5 curves 3 - 8 of H), which were calculated to obtain the difference spectra (curves 1 - 6, respectively). Figure 5 J of shows the spectra measured before the addition of 1.1 μM all-trans retinal (curve 1) and at 0, 0.5, 1, 2, 6, 12, and 16 hours after the addition (curves 2 - 8). Regarding Figure 5 the difference spectrum of K, it was calculated by subtracting the spectrum measured just after the addition of all-trans retinal ( Figure 5 curve 2 of J) from the spectra measured at 0.5, 1, 2, 6, 12, and 16 hours after the addition of all-trans retinal ( Figure 5 curves 3 - 8 of J) (curves 1 - 6, respectively). Figure 5 L of shows the regeneration process of the photopigments of N2C / D282C and G188C / N2C / D282C caused by the addition of all-trans retinal, which was monitored by the change in absorbance at 500 nm, as shown in Figure 5 F of and Figure 5 J of.

[0390] After adding all-trans retinal to the suspension of the rhodopsin-expressing cell membrane, the wild type and the G188C mutant were purified. In the absorption spectrum of the wild type, there were almost no peaks in the visible light region and the near-ultraviolet region ( Figure 5 A of). On the other hand, the absorption spectrum of the G188C mutant had a peak in the visible region ( Figure 5 curve 1 of B of), which did not originate from all-trans retinal but from the preferential uptake of 11-cis and 9-cis retinal ( Figure 5 C of). When the pigment was irradiated with yellow light, the retinal was converted to the all-trans form, and the spectrum shifted to the ultraviolet region. Through subsequent ultraviolet light irradiation, at 0 °C, the retinal was isomerized from the all-trans form to the 11-cis form, and the absorbance in the visible region increased again ( Figure 5 B, C of). This was very similar to the observation results of the G188C mutant purified after reconstitution with 11-cis retinal ( Figure 2 B, D of).

[0391] In addition, purified carrier proteins of N2C / D282C and G188C / N2C / D282C were prepared, and the regeneration process of the photopigment caused by the addition of 11-cis or all-trans retinal was studied. When 11-cis retinal was added to N2C / D282C and G188C / N2C / D282C, the absorbance near 505 nm ( Figure 5 D, E) and 490 nm ( Figure 5 H, I) immediately increased, indicating the formation of the 11-cis retinal-bound dark state. When all-trans retinal was added to N2C / D282C, the absorbance near 480 nm slightly increased ( Figure 5 F, G), while when all-trans retinal was added to G188C / N2C / D282C, the absorbance near 485 nm increased significantly ( Figure 5 J, K). In addition, for the addition of all-trans retinal, the regeneration ability of G188C / N2C / D282C was much higher than that of N2C / D282C ( Figure 5 L). These results indicate that the G188C mutant can form the photopigment independently not only by adding 11-cis retinal but also by adding all-trans retinal.

[0392] (Example 6: Chimeric opsin)

[0393] For bovine rhodopsin N2C / D282C, in the same manner as in the above examples, for the G188C mutant and the E122Q / G188C mutant, a chimeric opsin in which the second intracellular loop and the third intracellular loop (amino acid numbers 140-152 and 225-251 of Gene ID: 509933, respectively) were replaced with the second intracellular loop and the third intracellular loop of the human histamine H2 receptor (amino acid numbers 121-134 and 203-232 of Gene ID: 15466, respectively) was further expressed in the human cultured cell HEK293. At this time, as a probe for cAMP, a modified luciferase (Promega, GloSensor) was co-expressed so that the luminescence became higher when the cAMP concentration was high. The cultured cells were irradiated with yellow light, and the luminescence derived from the modified luciferase was compared. It was observed that: compared with bovine rhodopsin N2C / D282C in which the second intracellular loop and the third intracellular loop were replaced ( Figure 6 A), the luminescence transiently increased and then immediately decreased in those into which the G188C mutation was introduced ( Figure 6 B) and those into which the E122Q / G188C mutation was introduced ( Figure 6 C). This result indicates that by introducing the G188C mutation and the E122Q / G188C mutation, it is possible to modify the intracellular cAMP concentration to transiently increase and then recover due to light.

[0394] (Example 7: Obtaining the photocycle characteristics of the Xenopus tropicalis Opn5m T188C mutant)

[0395] Xenopus tropicalis Opn5m can bind 11-cis retinal, and its absorption spectrum shows a maximum absorption at 360 nm. When it receives ultraviolet light, the retinal isomerizes to the all-trans form, and the maximum absorption of the absorption spectrum changes to 474 nm. Furthermore, when it receives visible light, the retinal isomerizes to the 11-cis form, and the absorption spectrum that shows a maximum absorption at 360 nm is restored. That is, Opn5m is a bistable opsin (Yamashita et al., 2014). Threonine is present at position 188 of Xenopus tropicalis Opn5m, so the T188C mutant was prepared. This T188C mutant only binds the all-trans form, and its absorption spectrum shows a maximum absorption at 470 nm. When it receives visible light, whether it is at 20 degrees ( Figure 7 of a) or 37 degrees ( Figure 7 of b, Figure 7 of c), the absorption at 470 nm temporarily decreases and then recovers over time. Furthermore, during this process, the retinal changes from the all-trans form to the 11-cis form or the 13-cis form by receiving visible light, and recovers to the all-trans form over time ( Figure 7 of d). These results indicate that the Xenopus tropicalis Opn5m T188C mutant spontaneously recovers after receiving light, and it can be said that the photocycle characteristics are obtained.

[0396] (Example 8: Human rhodopsin)

[0397] For human rhodopsin N2C / N282C, in the same manner as in the example of bovine rhodopsin, the human cultured cells HEK293 were made to express the G188C mutant and the E122Q / G188C mutant. At this time, as a probe for cAMP, a modified luciferase (Promega, GloSensor) was co-expressed so that the luminescence became higher when the cAMP concentration was high. The cultured cells were irradiated with yellow light and the luminescence derived from the modified luciferase was compared. It was observed that compared with human rhodopsin N2C / N282C ( Figure 8 of A), those into which the G188C mutant was introduced ( Figure 8 of B) and those into which the E122Q / G188C mutant was introduced ( Figure 8C) Due to light irradiation, the luminescence instantaneously decreases and immediately recovers. This result indicates that by introducing the G188C mutation and the E122Q / G188C mutation, it is possible to engineer it in such a way that the intracellular cAMP concentration decreases for a short time due to light. In addition, for the human rhodopsin N2C / N282C and G188C / N2C / N282C mutants, a chimeric opsin in which the second intracellular loop and the third intracellular loop (amino acid numbers 140 to 152 and 225 to 251 of SEQ ID NO: 1, respectively) are replaced with the second intracellular loop and the third intracellular loop of the human histamine H2 receptor (amino acid numbers 121 to 134 and 204 to 233 of GeneID: 3274, respectively) is further expressed in the human cultured cell HEK293. At this time, as a probe for cAMP, a modified luciferase (Promega, GloSensor) is co-expressed so that the luminescence becomes higher when the cAMP concentration is high. The cultured cells are irradiated with yellow light, and the luminescence derived from the modified luciferase is compared. Compared with the human rhodopsin N2C / N282C Figure 8 D) into which the G188C mutation has been introduced Figure 8 E) shows that the luminescence instantaneously increases and immediately decreases due to light irradiation. This result indicates that by introducing the G188C mutation, it is possible to engineer it in such a way that the intracellular cAMP concentration increases and recovers for a short time due to light.

[0398] (Example 9: Canine rhodopsin)

[0399] For canine (Canis familiaris) rhodopsin N2C / D282C, in the same manner as in the example of bovine rhodopsin, the G188C mutant is expressed in the human cultured cell HEK293. At this time, as a probe for cAMP, a modified luciferase (Promega, GloSensor) is co-expressed so that the luminescence becomes higher when the cAMP concentration is high. The cultured cells are irradiated with yellow light, and the luminescence derived from the modified luciferase is compared. Compared with canine rhodopsin N2C / D282C Figure 10 A) into which the G188C mutation has been introduced Figure 10B) It was observed that upon light irradiation, the luminescence decreased instantaneously and then immediately recovered. This result indicates that by introducing the G188C mutation, it is possible to engineer the intracellular cAMP concentration to decrease transiently due to light. Additionally, for the canine rhodopsin N2C / D282C and G188C / N2C / D282C mutants, a chimeric opsin in which the second and third intracellular loops were replaced with those of the human histamine H2 receptor was further expressed in human cultured cells HEK293. At this time, as a probe for cAMP, a modified luciferase (Promega, GloSensor) was co-expressed such that the luminescence increased when the cAMP concentration was high. Yellow light was irradiated onto this cultured cell, and the luminescence derived from the modified luciferase was compared. Compared with the canine rhodopsin N2C / D282C ( Figure 10 C) into which the G188C mutation was introduced ( Figure 10 D) It was observed that upon light irradiation, the luminescence increased instantaneously and then immediately decreased. This result indicates that by introducing the G188C mutation, it can be engineered to increase and then recover the intracellular cAMP concentration transiently due to light.

[0400] (Example 10: Medaka rhodopsin)

[0401] For medaka (Oryzias latipes) rhodopsin N2C / E282C, in the same manner as in the example of bovine rhodopsin, the G188C mutant was expressed in human cultured cells HEK293. At this time, as a probe for cAMP, a modified luciferase (Promega, GloSensor) was co-expressed such that the luminescence increased when the cAMP concentration was high. Yellow light was irradiated onto this cultured cell, and the luminescence derived from the modified luciferase was compared. Compared with the medaka rhodopsin N2C / E282C ( Figure 11 A) into which the G188C mutation was introduced ( Figure 11 B) It was observed that upon light irradiation, the luminescence decreased instantaneously and then immediately recovered. This result indicates that by introducing the G188C mutation, it is possible to engineer the intracellular cAMP concentration to decrease transiently due to light.

[0402] (Example 11: Additional mutations other than positions 188 and 122)

[0403] At the N-terminal domain (positions 1 to 34) and C-terminal domain (starting from position 308) when aligned with SEQ ID NO: 1, conservative substitutions of amino acids were made. Specifically, the G6A mutation or V337A mutation was introduced into the human rhodopsin G188C / N2C / D282C mutant and expressed in the human-derived cultured cells HEK293. At this time, as a probe for cAMP, a modified luciferase (Promega, GloSensor) was co-expressed such that the luminescence increased when the cAMP concentration was high. The cultured cells were irradiated with yellow light, and the luminescence derived from the modified luciferase was compared. Similar to the human rhodopsin G188C / N2C / D282C mutant ( Figure 12 of A), the human rhodopsin G6A / G188C / N2C / D282C mutant ( Figure 12 of B) and the human rhodopsin V337A / G188C / N2C / D282C mutant ( Figure 12 of C) showed an instantaneous decrease and immediate recovery of luminescence due to light irradiation. This result indicates that these three mutants cause a short-term decrease in the intracellular cAMP concentration due to light. That is, even when conservative substitutions of amino acids were made in the N-terminal domain (positions 1 to 34) and C-terminal domain (starting from position 308) when aligned with SEQ ID NO: 1, the ability to cause a short-term decrease in the intracellular cAMP concentration due to light was maintained.

[0404] (Example 12: Introduction of cysteine mutations other than at positions 2 and 282)

[0405] Using combinations other than the amino acids corresponding to positions 2 and 282 when aligned with SEQ ID NO: 1, modifications to cysteine were made at the amino acids corresponding to a part of the N-terminal domain (positions 1 to 11) and a part of the extracellular third loop (positions 278 to 285). Specifically, the N2C / G3C / G280C mutation, N2C / G3C / S281C mutation or G3C / N282C mutation was introduced into the human rhodopsin G188C mutant and expressed in the human-derived cultured cells HEK293. At this time, as a probe for cAMP, a modified luciferase (Promega, GloSensor) was co-expressed such that the luminescence increased when the cAMP concentration was high. The cultured cells were irradiated with yellow light, and the luminescence derived from the modified luciferase was compared. Similar to the human rhodopsin G188C / N2C / D282C mutant ( Figure 13 of A), the human rhodopsin G188C / N2C / G3C / G280C mutant ( Figure 13 of B), the human rhodopsin G188C / N2C / G3C / S281C mutant ( Figure 13 of C) and the human rhodopsin G188C / G3C / N282C mutant (Figure 13 D) Observation revealed an instantaneous decrease and immediate recovery of luminescence due to light irradiation. This result indicates that these 4 mutants cause a short-term decrease in intracellular cAMP concentration due to light. That is, for the amino acids corresponding to a part of the N-terminal domain (positions 1 to 11) and a part of the extracellular third loop (positions 278 to 285) when aligned with SEQ ID NO: 1, even when cysteine is introduced at positions other than the combination corresponding to the amino acids at positions 2 and 282, the ability to cause a short-term decrease in intracellular cAMP concentration due to light is maintained.

[0406] (Example 13: Restoration of light-induced behavior from the retina using the G188C mutant)

[0407] A viral vector (rAAV-DJ) containing the coding sequence of the G188C mutant under the control of the CMV promoter was injected into the vitreous body of 10-week-old rd1 mice (retinitis pigmentosa blindness model mice). To introduce the gene more efficiently and widely, the AAV-DJ vector was used, and AAV-2 has been used as a benchmark for clinical applications. After 1 month, the retina was obtained. Expression of the reporter gene (EGFP) was observed in the whole retina and in both the ganglion cell layer (GCL) and the inner nuclear layer (INL). To evaluate the function of the G188C mutant ectopically induced in the mouse retina, a multi-electrode array (MEA) test capable of recording the extracellular potential of RGCs was performed. The test was carried out as Figure 14 shown. Figure 14 A of shows untreated rd1 mice. Figure 14 B of shows the case where the human rhodopsin G188C / N2C / D282C mutant was introduced using a viral vector, Figure 14 C of shows the case where the intracellular second loop and third loop of the human rhodopsin G188C / N2C / D282C mutant were replaced with the intracellular second loop and third loop of the human histamine H2 receptor, Figure 14 D of shows the case where the intracellular second loop and third loop of the human rhodopsin E122Q / G188C / N2C / D282C mutant were replaced with the intracellular second loop and third loop of the human histamine H2 receptor. These mutants are the same as those described in Example 8 and the like.

[0408] (Results)

[0409] The results are shown in Figure 14 . The untreated rd1 mice were blind, so no light response was observed ( Figure 14 A of ), and the case where the human rhodopsin G188C / N2C / D282C mutant was introduced using a viral vector ( Figure 14B) The case of introducing a mutant in which the second intracellular loop and the third intracellular loop of human rhodopsin G188C / N2C / D282C are replaced with the second intracellular loop and the third intracellular loop of human histamine H2 receptor ( Figure 14 C) The case of replacing the second intracellular loop and the third intracellular loop of human rhodopsin E122Q / G188C / N2C / D282C mutant with the second intracellular loop and the third intracellular loop of human histamine H2 receptor ( Figure 14 D) A light response was observed, and the result indicated that a visual regeneration effect was obtained by introducing the G188C mutant. (

[0410] As a result of photoreceptor degeneration, the untreated control retina did not show a response from RGCs detectable by MEA. Figure 14 A). In contrast, the treated retina reproduced a significant light-evoked response. Figure 14 B, C, D).

[0411] (Example 14: Evaluation of visual evoked potential)

[0412] To investigate whether light reception at the retina was transmitted to the visual cortex, visual evoked potential (VEP) from the visual cortex was investigated. In these experiments, rd1 mice with both eyes treated with the G188C mutant construct linked to AAV-DJ-CAGGS and a control EGFP virus (AAV-DJ-CAGGS-EGFP) were used. As a result, the control mice did not show a VEP response, while the mice given the G188C mutant construct linked to AAV-DJ-CAGGS reproduced a significant VEP response.

[0413] (Example 15: Evaluation of light-dark discrimination function)

[0414] A light-dark transition test (LDT) was performed to investigate whether the ectopic expression of the G188C mutant in the degenerated retina caused behavioral changes due to visual restoration. Rodents are nocturnal and feel uneasy in a bright environment, so they tend to stay in the dark according to their visual function, while blind animals spend half of their time in bright and dark places. Compared with untreated rd1 mutant mice, the mice treated with the G188C mutant spent significantly less time in the bright area, indicating that visual restoration was confirmed behaviorally.

[0415] (Example 16: Optogenetics <neural control>)

[0416] Intervention of cAMP is essential for inducing the branching and elongation of nerve axons. The G188C mutant was expressed in mouse hippocampal neurons, and the second intracellular loop and the third intracellular loop were replaced with the second loop and the third loop of human histamine H2 receptor as shown in Example 8, and optogenetic manipulation of the branching and elongation of nerve cell axons was attempted. As a result, compared with those not irradiated, the branching and elongation of axons were inhibited in hippocampal neurons transfected with the G188C mutant after 30 minutes of blue light irradiation. On the other hand, compared with those not irradiated, the branching and elongation of axons were promoted in hippocampal neurons transfected with the G188C mutant chimeric opsin after 30 minutes of blue light irradiation.

[0417] (Example 17: Calcium ion-variant chimeric opsin)

[0418] For bovine rhodopsin N2C / D282C, in the same manner as in Example 6, for the G188C mutant, the second intracellular loop and the third intracellular loop were further replaced with the second loop and the third loop of human α1A adrenergic receptor, and the resulting chimeric opsin was expressed in the human-derived cultured cell HEK293. At this time, as a calcium ion probe, aequorin derived from jellyfish was co-expressed so that the luminescence became higher when the calcium ion concentration was high. The cultured cells were irradiated with yellow light, and the luminescence derived from aequorin was compared. Compared with bovine rhodopsin N2C / D282C ( Figure 15 A) with the replaced second intracellular loop and third intracellular loop, those transfected with the G188C mutant ( Figure 15 B) showed an instantaneous increase and then an immediate decrease in luminescence. This result indicates that by introducing the G188C mutation, it can be modified to cause the intracellular calcium ion concentration to increase and recover only for a short time due to light.

[0419] (Note)

[0420] As described above, the present disclosure has been illustrated using preferred embodiments of the present disclosure. However, it is understood that the scope of the present disclosure is defined only by the claims. Regarding the patents, patent applications, and other documents cited in this specification, it is understood that their contents are incorporated by reference in the same manner as when the contents are specifically described in this specification. This application claims priority from Japanese Patent Application No. 2022-138707 filed with the Japan Patent Office on August 31, 2022, and the entire contents thereof are incorporated herein by reference as needed.

[0421] Industrial applicability

[0422] According to the present disclosure, by utilizing the evolutionary concept in nature that specific opsins are related to light-cycling properties, other opsins can be artificially evolved to have light-cycling properties. According to the present disclosure, there is provided a technology that can be used for the production of various molecular tools and industries (such as pharmaceuticals) based on such technologies.

[0423]

Sequence Listing Free Text

[0424] Sequence No. 1: Amino acid sequence of Homo sapiens rhodopsin NP_001372054.1

[0425] Sequence No. 2: Amino acid sequence of Mus musculus rhodopsin NP_663358.1

[0426] Sequence No. 3: Amino acid sequence of Canis lupus familiaris rhodopsin CAA50502.1

[0427] Sequence No. 4: Amino acid sequence of Gallus gallus domesticus rhodopsin NP_001384426.1

[0428] Sequence No. 5: Amino acid sequence of Oryzias latipes rhodopsin BAD99136.1

[0429] Sequence No. 6: Amino acid sequence of Homo sapiens blue cone opsin NP_001372054.1

[0430] Sequence No. 7: Amino acid sequence of Homo sapiens red cone opsin NP_064445.2

[0431] Sequence No. 8: Amino acid sequence of Homo sapiens green cone opsin NP_000504.1

[0432] Sequence No. 9: Amino acid sequence of Mus musculus UV cone opsin AAG17989.1

[0433] Sequence No. 10: Amino acid sequence of Mus musculus green cone opsin AAB64302.1

[0434] Sequence No. 11: Amino acid sequence of Gallus gallus domesticus green cone opsin AAA48786.1

[0435] Sequence No. 12: Amino acid sequence of Gallus gallus domesticus blue cone opsin AAA48633.1

[0436] Sequence No. 13: Amino acid sequence of Gallus gallus domesticus violet cone opsin AAA49141.1

[0437] Sequence No. 14: Amino acid sequence of Gallus gallus domesticus red cone opsin CAA40727.1

[0438] Sequence No. 15: Amino acid sequence of Gallus gallus domesticus non-visual opsin AAA64223.1

[0439] Sequence No. 16: Amino acid sequence of Homo sapiens Opn3 AAH36773.1

[0440] Sequence No. 17: Amino acid sequence of Homo sapiens Opn4 AAI13559.1

[0441] Sequence No. 18: Amino acid sequence of Homo sapiens Opn5 AAR21109.1

[0442] Sequence No. 19: Amino acid sequence of Homo sapiens Rgr AAA56748.1

[0443] Sequence No. 20: Amino acid sequence of Homo sapiens Rrh AAC51757.1

[0444] Sequence No. 21: Amino acid sequence of Mus musculus Opn3 AAD32670.1

[0445] Sequence No. 22: Amino acid sequence of Mus musculus Opn4 AAF24979.1

[0446] Sequence No. 23: Amino acid sequence of Mus musculus Opn5 AAR08201.1

[0447] Sequence No. 24: Amino acid sequence of Mus musculus Rgr AAC69836.1

[0448] Sequence No. 25: Amino acid sequence of Mus musculus Rrh AAC53344.1

[0449] Sequence No. 26: Amino acid sequence of Gallus gallus bankiva VAL opsin ACX32474.1

[0450] Sequence No. 27: Amino acid sequence of Gallus gallus bankiva Opn3 BAV92607.1

[0451] Sequence No. 28: Amino acid sequence of Gallus gallus bankiva TMT opsin BAV93805.1

[0452] Sequence No. 29: Amino acid sequence of Gallus gallus bankiva Opn4x ABX10830.1

[0453] Sequence No. 30: Amino acid sequence of Gallus gallus bankiva Opn4m BAL14786.1

[0454] Sequence No. 31: Amino acid sequence of Gallus gallus bankiva Opn5m BAG65738.1

[0455] Sequence No. 32: Amino acid sequence of Gallus gallus bankiva Opn5L2 BAG65739.2

[0456] Sequence No. 33: Amino acid sequence of Gallus gallus bankiva Rrh AAR02098.1

[0457] Sequence No. 34: Amino acid sequence of Gallus gallus Rgr AAR02099.1

[0458] Sequence No. 35: Nucleic acid sequence of Homo sapiens rhodopsin NM_000539.3

[0459] Sequence No. 36: Nucleic acid sequence of Mus musculus rhodopsin NM_145383.2

[0460] Sequence No. 37: Nucleic acid sequence of Canis lupus familiaris rhodopsin X71380.1

[0461] Sequence No. 38: Nucleic acid sequence of Gallus gallus rhodopsin NM_001397497.1

[0462] Sequence No. 39: Nucleic acid sequence of Oryzias latipes rhodopsin AB180742.1

[0463] Sequence No. 40: Nucleic acid sequence of Homo sapiens blue cone opsin NM_001385125.1

[0464] Sequence No. 41: Nucleic acid sequence of Homo sapiens red cone opsin NM_020061.6

[0465] Sequence No. 42: Nucleic acid sequence of Homo sapiens green cone opsin NM_000513.2

[0466] Sequence No. 43: Nucleic acid sequence of Mus musculus UV cone opsin AF190670.1

[0467] Sequence No. 44: Nucleic acid sequence of Mus musculus green cone opsin AF011389.1

[0468] Sequence No. 45: Nucleic acid sequence of Gallus gallus green cone opsin M92038.1

[0469] Sequence No. 46: Nucleic acid sequence of Gallus gallus blue cone opsin M92037.1

[0470] Sequence No. 47: Nucleic acid sequence of Gallus gallus violet cone opsin M92039.1

[0471] Sequence No. 48: Nucleic acid sequence of Gallus gallus red cone opsin X57490.1

[0472] Sequence No. 49: Nucleic acid sequence of Gallus gallus non-visual opsin U15762.1

[0473] Sequence No. 50: Nucleic acid sequence of Homo sapiens Opn3 BC036773.1

[0474] Sequence No. 51: Nucleic acid sequence of Homo sapiens Opn4 BC113558.1

[0475] Sequence No. 52: Nucleic acid sequence of Homo sapiens Opn5 AY377391.1

[0476] Sequence No. 53: Nucleic acid sequence of Homo sapiens Rgr U14910.1

[0477] Sequence No. 54: Nucleic acid sequence of Homo sapiens Rrh AF012270.1

[0478] Sequence No. 55: Nucleic acid sequence of Mus musculus Opn3 AF140241.1

[0479] Sequence No. 56: Nucleic acid sequence of Mus musculus Opn4 AF147789.1

[0480] Sequence No. 57: Nucleic acid sequence of Mus musculus Opn5 AY318865.1

[0481] Sequence No. 58: Nucleic acid sequence of Mus musculus Rgr AF076930.1

[0482] Sequence No. 59: Nucleic acid sequence of Mus musculus Rrh AF012271.1

[0483] Sequence No. 60: Nucleic acid sequence of Gallus gallus bankiva VAL opsin GQ280390.1

[0484] Sequence No. 61: Nucleic acid sequence of Gallus gallus bankiva Opn3 AB436160.1

[0485] Sequence No. 62: Nucleic acid sequence of Gallus gallus bankiva TMT opsin AB519059.1

[0486] Sequence No. 63: Nucleic acid sequence of Gallus gallus bankiva Opn4x EU124630.1

[0487] Sequence No. 64: Nucleic acid sequence of Gallus gallus bankiva Opn4m AB295599.1

[0488] Sequence No. 65: Nucleic acid sequence of Gallus gallus bankiva Opn5m AB368182.1

[0489] Sequence No. 66: Nucleic acid sequence of Gallus gallus bankiva Opn5L2 AB368183.3

[0490] Sequence No. 67: Nucleic acid sequence of Gallus gallus bankiva Rrh AY339626.1

[0491] Sequence No. 68: Nucleic acid sequence of Gallus gallus bankiva Rgr AY339627.1

Claims

1. A protein comprising an amino acid sequence of a rhodopsin-like protein, wherein the amino acid sequence of the rhodopsin-like protein comprises a modification of the amino acid corresponding to the 188th position when aligned with SEQ ID NO:

1.

2. A protein comprising an amino acid sequence of a rhodopsin-like protein, wherein the amino acid sequence of the rhodopsin-like protein comprises a modification of the amino acid corresponding to the 188th position G when aligned with SEQ ID NO:

1.

3. The protein according to claim 1 or 2, wherein In the amino acid sequence, it comprises a modification of the amino acid corresponding to the G188 position to cysteine when aligned with SEQ ID NO:

1.

4. The protein according to any one of claims 1 to 3, wherein, The protein does not release a photoreceptor factor and inactivate after being activated by light stimulation.

5. The protein according to any one of claims 1 to 4, wherein, The amino acid sequence comprises: 1) An amino acid sequence containing the modified amino acid sequence in any one of the amino acid sequences shown in SEQ ID NOs: 1 to 34, 2) A sequence other than the modified site has at least about 80% identity with the sequence of 1) and the encoded protein has substantially the same biological activity as the protein obtained from the sequence of 1), 3) An amino acid sequence having one or more mutations outside the modified site in the sequence of 1) and the encoded protein has substantially the same biological activity as the protein obtained from the sequence of 1), 4) An amino acid sequence containing the modified amino acid sequence in the amino acid sequence encoded by a nucleic acid that can hybridize with the nucleic acid encoding the sequence of 1), or 5) An amino acid sequence containing the modified amino acid sequence in the amino acid sequence encoded by an allelic mutant of the nucleic acid encoding the sequence of 1).

6. The protein according to any one of claims 1 to 5, wherein, In the amino acid sequence of the rhodopsin-like protein, it further comprises a modification of the amino acid corresponding to the 122nd position when aligned with SEQ ID NO:

1.

7. The protein according to any one of claims 1 to 6, wherein, In the amino acid sequence of the rhodopsin-like protein, it further comprises a modification of the amino acid corresponding to the 122nd position E when aligned with SEQ ID NO:

1.

8. The protein according to any one of claims 1 to 7, wherein, In the amino acid sequence of the rhodopsin-like protein, it further comprises a modification of the amino acid corresponding to the 122nd position to glutamine when aligned with SEQ ID NO:

1.

9. The protein according to claim 4, wherein The photoreceptor factor contains retinal.

10. The protein according to any one of claims 1 to 9, wherein, In the amino acid sequence of the rhodopsin-like protein, it further comprises a modification of the amino acids corresponding to a part of the N-terminal domain (positions 1 to 11) and a part of the extracellular third loop (positions 278 to 285) when aligned with SEQ ID NO:

1.

11. The protein according to any one of claims 1 to 10, wherein, In the amino acid sequence of the rhodopsin-like protein, it further comprises a modification of the amino acids corresponding to a part of the N-terminal domain (positions 1 to 11) and a part of the extracellular third loop (positions 278 to 285) to cysteine when aligned with SEQ ID NO:

1.

12. The protein according to any one of claims 1 to 11, wherein, In the amino acid sequence of the rhodopsin-like protein, it further comprises a modification of the amino acids corresponding to the 2nd and 282nd positions when aligned with SEQ ID NO:

1.

13. The protein according to claim 12, wherein, The modification of the amino acids corresponding to the 2nd and 282nd positions improves the thermal stability of the rhodopsin-like protein.

14. The protein according to claim 12 or 13, wherein, The modification of the amino acids corresponding to the 2nd and 282nd positions comprises a modification of the amino acids corresponding to the 2nd and 282nd positions to cysteine.

15. The protein according to any one of claims 1 to 14, wherein, The rhodopsin-like protein is a chimeric rhodopsin.

16. The protein according to any one of claims 1 to 14, which comprises the sequence of SEQ ID NO: 1, 3 or 5.

17. A nucleic acid molecule, which comprises a nucleic acid encoding the amino acid sequence of the protein according to any one of claims 1 to 16.

18. A nucleic acid construct, which comprises the nucleic acid molecule according to claim 17.

19. A cell, which comprises the protein according to any one of claims 1 to 16, the nucleic acid molecule according to claim 17 and / or the nucleic acid construct according to claim 18.

20. A medicament, which comprises the protein according to any one of claims 1 to 16, the nucleic acid molecule according to claim 17, the nucleic acid construct according to claim 18 and / or the cell according to claim 19.

21. The medicament according to claim 20, which is used for visual regeneration or for preventing or treating visual disorders or diseases.

22. A composition, which comprises opsins that are inactivated without releasing a light-receiving factor.

23. The composition according to claim 22, wherein The opsins cause a transient change in the concentration of cAMP due to light stimulation.

24. The composition according to claim 23, wherein, The transient change in the concentration of cAMP is a decrease in the cAMP concentration.

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