Light-responsive modified opsin

Abstract

The present disclosure provides an opsin that has a light cycle characteristic. Specifically, the present disclosure provides a protein including an amino acid sequence of an opsin, wherein the amino acid sequence of the opsin includes a modification of the amino acid corresponding to position 188 when aligned with SEQ ID NO:1. The present disclosure also provides a medicine, preparation, composition, treatment method, and the like that include said protein and / or a nucleic acid molecule, a nucleic acid construct, and / or cells related to said protein.

Description

Photoresponsive modified opsins

[0001] The present disclosure relates to proteins comprising the amino acid sequence of opsins, nucleic acid molecules comprising nucleic acids encoding the amino acid sequence of the proteins, nucleic acid constructs comprising the nucleic acid molecules, cells comprising the nucleic acid constructs, and pharmaceuticals comprising the cells.

[0002] Opsins are photoreceptor proteins found universally in animals and are classified into three types based on their light response characteristics. Vertebrate rhodopsin, a type of opsin, becomes active when exposed to light (light stimulus) but does not return to its original dark state by either light or heat. On the other hand, many opsins other than animal rhodopsins, such as channelrhodopsins, become active when exposed to light (light stimulus) and can then return to their dark state by heat.

[0003] The inventors have found that introducing a single mutation at position 188 into vertebrate rhodopsins also results in the acquisition of photocycle and photoreversibility, and have therefore found that the residue at position 188 contributes to the diversification of the light response properties of opsins by controlling the recovery from the active state to the original dark state.

[0004] Accordingly, the present disclosure provides the following: (Item X1) A protein comprising the amino acid sequence of an opsin, which includes a modification of the amino acid corresponding to position 188 in the opsin amino acid sequence when aligned with SEQ ID NO: 1. (Item X2-1) A protein comprising the amino acid sequence of an opsin, which includes a modification of the amino acid corresponding to G, T, S, or E at position 188 in the opsin amino acid sequence when aligned with SEQ ID NO: 1. (Item X2-2) A protein comprising the amino acid sequence of an opsin, which includes a modification of the amino acid corresponding to G at position 188 in the opsin amino acid sequence when aligned with SEQ ID NO: 1. (Item X3) The protein according to any one of the above items, which includes a modification of the amino acid corresponding to G at position 188 in the amino acid sequence when aligned with SEQ ID NO: 1 to cysteine ​​in the amino acid sequence. (Item X4) The protein according to any one of the above items, which is activated by a light stimulus and then inactivated without releasing a photoreceptor. (Item X5) The protein according to any one of the above items, wherein the amino acid sequence comprises: 1) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 34, the amino acid sequence comprising the modification; 2) an amino acid sequence in which the sequence other than the modification 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 in which the sequence of 1) has one or more mutations other than the modification site, and the encoded protein has substantially the same biological activity as the protein obtained from the sequence of 1); 4) an amino acid sequence encoded by a nucleic acid to which a nucleic acid encoding the sequence of 1) hybridizes, the amino acid sequence comprising the modification; or 5) an amino acid sequence encoded by an allelic variant of the nucleic acid encoding the sequence of 1), the amino acid sequence comprising the modification. (Item X6) The protein according to any one of the above items, further comprising a modification of the amino acid at position 122 in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1.(Item X7) The protein according to any one of the above items, further comprising a modification of an amino acid corresponding to E at position 122 in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1. (Item X8) The protein according to any one of the above items, further comprising a modification of an amino acid corresponding to 122 in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1 to glutamine. (Item X9) The protein according to any one of the above items, wherein the photoreceptor factor comprises retinal. (Item X9-1) The protein according to any one of the above items, further comprising a modification of amino acids corresponding to amino acids belonging to a part of the N-terminal domain (positions 1 to 11) and a part of the third extracellular loop (positions 279 to 285) in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1. (Item X9-2) The protein according to any one of the above items, further comprising a modification to cysteine ​​of amino acids corresponding to amino acids belonging to a part of the N-terminal domain (positions 1 to 11) and a part of the third extracellular loop (positions 279 to 285) in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1. (Item X9-3) The protein according to any one of the above items, further comprising a modification to amino acids corresponding to positions 2 and 282 in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1. (Item X9-4) The protein according to any one of the above items, wherein the modification of amino acids corresponding to positions 2 and 282 improves the thermal stability of the opsin. (Item X9-5) The protein according to any one of the above items, wherein the modification of amino acids corresponding to positions 2 and 282 includes a modification to cysteine ​​of amino acids corresponding to positions 2 and 282. (Item X10A) The protein according to any one of the above items, wherein the opsin is a chimeric opsin. (Item X10B) The protein according to any one of the preceding items, wherein the opsins are chimeric opsins in which an amino acid sequence of a certain opsin that binds to a G protein is replaced with an amino acid sequence of another opsin that binds to a G protein.(Item X10C) The protein according to any one of the above items, wherein the opsins are chimeric opsins in which the amino acid sequence of a certain opsin that binds to a G protein has been replaced with a functional sequence derived from another organism. (Item X10D) The protein according to any one of the above items, wherein the opsins are chimeric opsins from the same organism or a different organism. (Item X10E) The protein according to any one of the above items, wherein the organism is selected from the group consisting of vertebrates, invertebrates, and microorganisms. (Item X10F1) The protein according to any one of the above items, wherein the opsins are chimeric opsins between a microbial opsin and a vertebrate opsin. (Item X10F2) The protein according to any one of the above items, wherein the opsins are chimeric opsins between a microbial opsin and an invertebrate opsin. (Item X10F3) The protein according to any one of the above items, wherein the opsins are chimeric opsins between a vertebrate opsin and an invertebrate opsin. (Item X10F4) The protein according to any one of the above items, wherein the opsins are chimeric opsins between microbial opsins. (Item X10F5) The protein according to any one of the above items, wherein the opsins are chimeric opsins between vertebrate opsins. (Item X10F6) The protein according to any one of the above items, wherein the opsins are chimeric opsins between invertebrate opsins. (Item X10) The protein according to any one of the above items, comprising the sequence of SEQ ID NO: 1, 3, or 5. (Item X11) A nucleic acid molecule comprising a nucleic acid encoding the amino acid sequence of the protein according to any one of the above items. (Item X12) A nucleic acid construct comprising the nucleic acid molecule according to any one of the above items. (Item X13) A cell comprising 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. (Item X14) A pharmaceutical comprising 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.(Item X15) The pharmaceutical according to any one of the above items, for restoring vision or preventing or treating visual disorders or diseases. (Item 1) A composition comprising opsins that inactivate without releasing photoreceptor factors. (Item 2) The composition according to any one of the above items, wherein the opsins cause a transient change in cAMP concentration in response to light stimulation. (Item 3) The composition according to any one of the above items, wherein the transient change in cAMP concentration is a decrease in cAMP concentration. (Item 4) The composition according to any one of the above items, wherein the opsins comprise an amino acid sequence represented by any one of SEQ ID NOs: 1 to 34. (Item 5) The composition according to any one of the above items, wherein the opsins comprise a G protein-coupled receptor rhodopsin. (Item 6) The composition according to any one of the above items, wherein the G protein-coupled receptor rhodopsin is derived from a mammal (visual rhodopsin). (Item 7) The composition according to any one of the above items, wherein the G protein-coupled receptor rhodopsin is a modified type. (Item 8) The composition according to any one of the above items, wherein the modification comprises G188C when aligned with SEQ ID NO: 1. (Item 9) The composition according to any one of the above items, wherein the photoreceptor comprises retinal. (Item 10) The composition according to any one of the above items, wherein the modification further comprises E122Q when aligned with SEQ ID NO: 1. (Item 11) The composition according to any one of the above items, wherein the modification further comprises modifying the amino acids corresponding to positions 2 and 282 when aligned with SEQ ID NO: 1 to cysteine. (Item 12) The composition according to any one of the above items for use in medicine. (Item 13) The composition according to any one of the above items for restoring vision or preventing or treating visual disorders or diseases. (Item Z1) A method for modifying or imparting visual function, comprising using opsins that inactivate the photoreceptor without releasing it. (Item Z2) A method of using opsins as an optical switch, which includes a step of using opsins that inactivate a photoreceptor without releasing it. (Item Z3) A method of using opsins that inactivate a photoreceptor without releasing it.(Item A1) A protein comprising the amino acid sequence of an opsin, wherein the opsin has been modified so that it is inactivated without releasing a photoreceptor after activation by a light stimulus, and the opsin activates the Gs or Gq subfamily of G proteins. (Item A1a) The protein according to any one of the above items, wherein the opsin activates the Gs subfamily of G proteins. (Item A1b) The protein according to any one of the above items, wherein the opsin activates a G protein, thereby increasing cAMP levels. (Item A1c) The protein according to any one of the above items, wherein the opsin is a chimeric opsin in which the intracellular second and third loops of the opsin are replaced with those of a Gs- or Gq-activated G protein-coupled receptor. (Item A2) The protein according to any one of the above items, wherein the modification that inactivates the photoreceptor without releasing it after activation by light stimulation is achieved by modifying the amino acid corresponding to position 188 in the amino acid sequence when aligned with SEQ ID NO: 1. (Item A3) The protein according to any one of the above items, wherein the amino acid corresponding to position 188 comprises G, T, S, or E. (Item A4) The protein according to any one of the above items, wherein the amino acid corresponding to position 188 is G. (Item A5) The protein according to any one of the above items, wherein the amino acid corresponding to position G188 in the amino acid sequence when aligned with SEQ ID NO: 1 is modified to cysteine.(Item A6) The protein according to any one of the above items, wherein the amino acid sequence comprises: 1) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 34, which contains the modification; 2) an amino acid sequence other than the modification site that has at least about 80% identity with the sequence of 1), and wherein 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 other than the modification site in the sequence of 1), and wherein the encoded protein has substantially the same biological activity as the protein obtained from the sequence of 1); 4) an amino acid sequence encoded by a nucleic acid to which a nucleic acid encoding the sequence of 1) hybridizes, which contains the modification; or 5) an amino acid sequence encoded by an allelic variant of the nucleic acid encoding the sequence of 1), which contains the modification. (Item A7) The protein according to any one of the above items, further comprising a modification of the amino acid at position 122 in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1. (Item A8) The protein according to any one of the above items, wherein the amino acid corresponding to position 122 is E. (Item A9) The protein according to any one of the above items, wherein the amino acid corresponding to position G122 in the amino acid sequence when aligned with SEQ ID NO: 1 is modified to glutamine. (Item A10) The protein according to any one of the above items, wherein the photoreceptor factor comprises retinal. (Item A11-1) The protein according to any one of the above items, further comprising modifications of amino acids corresponding to amino acids belonging to a part of the N-terminal domain (positions 1 to 11) and a part of the extracellular third loop (positions 278 to 285) in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1. (Item A11-2) The protein according to any one of the preceding items, further comprising, in the amino acid sequence of the opsin, amino acids corresponding to amino acids belonging to a part of the N-terminal domain (positions 1 to 11) and amino acids belonging to a part of the extracellular third loop (positions 278 to 285) when aligned with SEQ ID NO: 1, being modified to cysteine.(Item A11-3) The protein according to any one of the above items, further comprising modifications of amino acids corresponding to positions 2 and 282 in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1. (Item A11-4) The protein according to any one of the above items, wherein the modifications of the amino acids corresponding to positions 2 and 282 improve the thermal stability of the opsin. (Item A11-5) The protein according to any one of the above items, wherein the modifications of the amino acids corresponding to positions 2 and 282 include modifying the amino acids corresponding to positions 2 and 282 to cysteine. (Item A12a) The protein according to any one of the above items, comprising the sequence of SEQ ID NO: 1, 3, or 5. (Item A12) A nucleic acid molecule comprising a nucleic acid encoding the amino acid sequence of the protein according to any one of the above items. (Item A13) A nucleic acid construct comprising the nucleic acid molecule of any one of the above items. (Item A14) A cell comprising the protein described in any one of the above items, the nucleic acid molecule described in any one of the above items, and / or the nucleic acid construct described in any one of the above items. (Item A15) A pharmaceutical comprising a cell comprising the protein described in any one of the above items, the nucleic acid molecule described in any one of the above items, and / or the nucleic acid construct described in any one of the above items. (Item A16) The pharmaceutical described in any one of the above items for restoring vision or for preventing or treating visual disorders or diseases.

[0005] It is contemplated that the present disclosure may provide one or more of the above-described features in combinations other than those explicitly stated. Still further embodiments and advantages of the present disclosure will be recognized by those skilled in the art upon reading and understanding the following detailed description, if necessary.

[0006] According to the present disclosure, by introducing a single mutation at position 188 into vertebrate rhodopsin, it is possible to acquire a photocycle and photoreversibility in which the rhodopsin automatically returns to dark adaptation after light irradiation, something that was not possible with conventional technology, thereby confirming its applicability as an optogenetics tool.

[0007] Figure 1 shows the thermal recovery of the G188C mutant of bovine rhodopsin after irradiation with yellow light. Figure 2 shows the light response, retinal localization, and G protein activation of the G188C mutant of bovine rhodopsin. Figure 3 shows the increased recovery rate of the photocycle characteristics of the G188C mutant of bovine rhodopsin due to the introduction of the E122Q mutation. Figure 4 shows the suppression of intracellular cAMP levels by light using the G188C mutant of bovine rhodopsin. Figure 5 shows the formation of photopigments in the G188C mutant of bovine rhodopsin after incubation with all-trans retinal. Figure 6 is a graph showing the change in cAMP concentration measured using a chimeric opsin in which the intracellular second and third loops of the E122Q / G188C mutant of bovine rhodopsin were replaced with those of the mouse histamine H2 receptor. Figure 7 shows the acquisition of photocycle properties in the Xenopus tropicalis Opn5m T188C mutant. Figure 8 is a graph showing changes in cAMP concentration measured using the E122Q / G188C mutant of human rhodopsin. Figure 9 is an alignment of the amino acid sequences of opsins. Ibid. Ibid. Ibid. Ibid. Ibid. Ibid. Ibid. Ibid. Ibid. Figure 10 is a graph showing changes in cAMP concentration measured using the G188C mutant of canine rhodopsin. Figure 11 is a graph showing changes in cAMP concentration measured using the G188C mutant of medaka rhodopsin. Figure 12 is a graph showing changes in cAMP concentration measured using the G6A / G188C / N2C / D282C mutant and the V337A / G188C / N2C / D282C mutant of human rhodopsin. Figure 13 is a graph showing changes in cAMP concentration measured using the G188C / N2C / G3C / G280C mutant, G188C / N2C / G3C / S281C mutant, and G188C / G3C / N282C mutant of human rhodopsin. Figure 14 is a graph showing the frequency of neural activity extracted from the extracellular potential of ganglion cells (RGCs) measured using a multielectrode array (MEA). Figure 14A shows an untreated rd1 mouse (no light response is observed due to blindness).Figure 14B shows the results when the human rhodopsin G188C / N2C / D282C mutant was introduced using a viral vector. Figure 14C shows the results when the human rhodopsin G188C / N2C / D282C mutant, in which the intracellular loops 2 and 3 were replaced with those of the human histamine H2 receptor, was introduced. Figure 14D shows the results when the human rhodopsin E122Q / G188C / N2C / D282C mutant, in which the intracellular loops 2 and 3 were replaced with those of the human histamine H2 receptor. Figure 15 is a graph showing the changes in cAMP concentration measured using the N2C / D282C and N2C / D282C / G188C mutants of bovine rhodopsin.

[0008] The present disclosure will now be described with reference to the best mode. Throughout this specification, singular expressions should be understood to include the plural concept unless otherwise specified. Therefore, singular articles (e.g., "a," "an," "the," etc. in English) should be understood to include the plural concept unless otherwise specified. Furthermore, it should be understood that terms used in this specification are used in the sense commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. In the event of conflict, the present specification (including definitions) will prevail.

[0009] (Definitions, etc.) The definitions of terms particularly used in this specification and / or basic technical content will be explained as appropriate below.

[0010] As used herein, "opsin" or "opsins" refers to a protein that binds to retinal or an analog thereof, a pigment that functions as a photoreceptor, and uses retinal or an analog thereof as a chromophore.

[0011] The type of opsin varies depending on the species and the type of photoreceptor cell, but examples include rod opsins and cone opsins (e.g., blue opsin, green opsin, red opsin). Furthermore, as used herein, opsins include, but are not limited to, melanopsin, encephalopsin, OPN5, RGR, and peropsin. For example, animal opsins are G protein-coupled receptors (GPCRs) with seven transmembrane domains, and constitute rhodopsin, which exhibits photoreceptive ability upon binding to the pigment retinal. Opsins activate trimeric G proteins upon photoreception, thereby transmitting external light signals into cells.

[0012] Opsins can be broadly classified into microbial opsins (Type I opsins) and animal opsins (Type II opsins), and animal opsins can be further classified into vertebrate visual opsins, vertebrate non-visual opsins, and invertebrate opsins. Furthermore, vertebrate non-visual opsins and invertebrate opsins are called bistable opsins, and include opsins that, like microbial opsins, have all-trans retinal as a chromophore. Microbial opsins, vertebrate non-visual opsins, and some invertebrate opsins are opsins that do not release retinal from the rhodopsin that constitutes them upon receiving light, and can be used as opsins in the present disclosure. Opsins that can be advantageously used in the present disclosure are opsins that do not release retinal upon receiving light, and examples thereof include microbial opsins, some vertebrate non-visual opsins, and invertebrate opsins, as well as bistable opsins, including insect opsins. The opsins used in the present disclosure may be advantageously used even if they do not strictly fall under the category of microbial opsins, vertebrate non-visual opsins, or invertebrate opsins, as long as they are functional equivalents that have the same functions as these advantageously used types of opsins.

[0013] Specific amino acid sequences of opsins are as shown in SEQ ID NOS: 1 to 34, and the 188th amino acid is as shown in the alignment diagram in Figure 9. That is, the 188th amino acid corresponds to G, the 188th amino acid of SEQ ID NOS: 1, when aligned with SEQ ID NOS: 1 using CLUSTRAL W (Version 2.0, released in 2007) (see the alignment diagram in Figure 9). For other SEQ ID NOS, the specific number may differ, and may be G in SEQ ID NOS: 2, G in SEQ ID NOS: 3, G in SEQ ID NOS: 4, G in SEQ ID NOS: 5, G in SEQ ID NOS: 6, G in SEQ ID NOS: 7, G in SEQ ID NOS: 8, G in SEQ ID NOS: 9, G in SEQ ID NOS: 10, G in SEQ ID NOS: 11, G in SEQ ID NOS: 12, G in SEQ ID NOS: 13, G in SEQ ID NOS: 14, G in SEQ ID NOS: 15, G in SEQ ID NOS: 16, G in SEQ ID NOS: 17, G in SEQ ID NOS: 18, G in SEQ ID NOS: 19, G in SEQ ID NOS: 20, G in SEQ ID NOS: 21, G in SEQ ID NOS: 22, G in SEQ ID NOS: 23, G in SEQ ID NOS: 24, G in SEQ ID NOS: 25, G in SEQ ID NOS: 26, G in SEQ ID NOS: 27, G in SEQ ID NOS: 28, G in SEQ ID NOS: 29, G in SEQ ID NOS: 30, G in SEQ ID NOS: 31, G in SEQ ID NOS: 32, G in SEQ ID NOS in SEQ ID NO: 17, S in SEQ ID NO: 18, T in SEQ ID NO: 19, T in SEQ ID NO: 20, T in SEQ ID NO: 21, S in SEQ ID NO: 22, T in SEQ ID NO: 23, T in SEQ ID NO: 24, T in SEQ ID NO: 25, E in SEQ ID NO: 26, S in SEQ ID NO: 27, S in SEQ ID NO: 28, T in SEQ ID NO: 29, S in SEQ ID NO: 30, T in SEQ ID NO: 31, T in SEQ ID NO: 32, T in SEQ ID NO: 33, T in SEQ ID NO: 34, and those skilled in the art can also understand sequence numbers not described herein by aligning the sequence numbers.The same applies to the 122nd amino acid, which is CLUSTERAL with SEQ ID NO: 1. 1. The term "E" refers to the amino acid corresponding to the 122nd amino acid in SEQ ID NO: 1 when aligned using RIBA.W (Version 2.0, released in 2007), specifically, E in SEQ ID NO: 2, E in SEQ ID NO: 3, E in SEQ ID NO: 4, E in SEQ ID NO: 5, L in SEQ ID NO: 6, I in SEQ ID NO: 8, I in SEQ ID NO: 9, I in SEQ ID NO: 10, Q in SEQ ID NO: 11, M in SEQ ID NO: 12, L in SEQ ID NO: 13, I in SEQ ID NO: 14, I in SEQ ID NO: 15, I in SEQ ID NO: 16, I in SEQ ID NO: 17, C in SEQ ID NO: 18, L in SEQ ID NO: 19, M in SEQ ID NO: 20, F in SEQ ID NO: 21, I in SEQ ID NO: 22, C in SEQ ID NO: 23, L in SEQ ID NO: 24, M in SEQ ID NO: 25, I in SEQ ID NO: 26, I in SEQ ID NO: 27, I in SEQ ID NO: 28, I in SEQ ID NO: 29, I in SEQ ID NO: 30, I in SEQ ID NO: 31, C in SEQ ID NO: 32, V in SEQ ID NO: 33, M in SEQ ID NO: 34. SEQ ID NOs not described herein can also be understood by those skilled in the art by aligning the amino acids.

[0014] As used herein, "rhodopsin" refers to a protein containing a pigment called retinal, which is activated by exposure to light and transmits a visual signal to the brain. In this specification, the term "rhodopsin" not only refers to the inclusion of retinal, but also refers to opsin (the protein portion), in which case it is interpreted interchangeably with opsin. Ion transport receptor rhodopsins, typically derived from microorganisms, do not release retinal even when irradiated with light, so they can be repeatedly activated by absorbing light. However, unlike G protein-coupled receptor rhodopsins, typically derived from animals, they cannot activate G proteins.

[0015] The proteins of the present disclosure may include any opsins, including chimeric opsins, as long as they achieve the objectives of the present disclosure. Chimeric opsins can be generated by combining portions of two or more opsins known in the art. The combined opsins may be derived from the same organism or different organisms. Those skilled in the art can select appropriate opsins depending on the intended use and / or function of the opsins to provide appropriate chimeric opsins. In this case, the amino acid corresponding to amino acid 188 of SEQ ID NO: 1 may be modified, preferably to cysteine, for use in the present disclosure. The present disclosure also encompasses any such chimeric opsins in which the amino acid corresponding to amino acid 188 of SEQ ID NO: 1 has been modified, preferably to cysteine. Opsins that can constitute a chimeric opsin may be, for example, rod opsins and cone opsins (e.g., blue opsin, green opsin, red opsin), melanopsin, encephalopsin, OPN5, RGR, and peropsin, or may be microbial opsins (Type I opsins) or animal opsins (Type II opsins), and may be selected from vertebrate non-visual opsins and invertebrate opsins.

[0016] Examples of chimeric opsins that can be used in the present disclosure include chimeric opsins of a G protein-coupled receptor and an animal-type opsin, such as chimeric proteins containing at least a portion of a mammalian opsin and at least a portion of a Gs- or Gq-activated G protein-coupled receptor. A typical example is fusing a portion of a Gs- or Gq-activated G protein-coupled receptor to a portion of a mammalian opsin, thereby achieving Gs or Gq activity. In one embodiment, the chimeric opsin of the present disclosure has the amino acid sequence of the intracellular loop 2 and / or intracellular loop 3 that binds to a G protein, among the amino acid sequences of a certain opsin, replaced with the amino acid sequence of the intracellular loop 2 and / or intracellular loop 3 of another G protein-coupled receptor, or a functional sequence. As a result, the protein of the present disclosure can light-dependently activate a Gi-type G protein to lower intracellular cAMP levels, light-dependently activate a Gs-type G protein to increase intracellular cAMP levels, or light-dependently activate a Gq-type G protein to increase intracellular Ca. ２＋ 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 retaining sufficient activity, thereby achieving the effects of preventing and inhibiting the progression of retinal diseases, disorders, or symptoms, particularly preventing or inhibiting the progression of retinitis pigmentosa, or bringing about improvements in visual cognitive behavioral function (e.g., improvement in light / dark judgment function, improvement in photoaversion function, and / or danger avoidance function), or exerting the effect of enhancing visual function, such as improving visual acuity.

[0017] G protein-coupled receptor rhodopsins used in the present disclosure include human rhodopsin, bovine rhodopsin, and canine rhodopsin.

[0018] As used herein, "retinal" refers to a type of vitamin A1, also known as retinaldehyde or retinene, which is a component of rhodopsin, a visual pigment contained in the rod photoreceptors of the retina. The retinal that binds to opsin to form rhodopsin has the molecular form of 11-cis-retinal, which is isomerized to all-trans-retinal upon exposure to light and releases its bond with opsin. All-trans-retinal then returns to 11-cis-retinal in the visual cycle and binds to opsin. All-trans-retinal is phototoxic and has been suggested to be involved in various vision-related diseases, including age-related macular degeneration, Stargardt's disease, fundus flava, and recessive retinitis pigmentosa.

[0019] As used herein, the term "retinal analog" refers to a compound having substantially the same properties as retinal, including both naturally occurring forms and those that have modifications, additions, functional group substitutions, or other alterations that differ from the natural form. Examples of retinal analogs include natural analogs such as A2-retinal (3,4-dehydroretinal), A3-retinal (3-hydroxyretinal), and A4-retinal (4-hydroxyretinal), as well as artificial analogs such as 9-ethyl-retinal and 9-propyl-retinal. For information on retinal analogs, see, for example, Akimori Wada, Journal of Pharmaceutical Sciences 141 (4), 557-577, April 1, 2021.

[0020] As used herein, the term "visual disorder" refers to any disease, disorder, or symptom related to vision, such as the retina, and includes retinal degenerative diseases (retinitis pigmentosa, age-related macular degeneration, etc.), retinopathies (e.g., diabetic retinopathy, proliferative retinopathy, simple retinopathy, etc.), floaters, retinal breaks, retinal detachment (e.g., rhegmatogenous retinal detachment, non-rhegmatogenous retinal detachment, etc.), and also includes retinitis pigmentosa, age-related macular degeneration, myopic maculopathy, macular dystrophy, diabetic retinopathy, retinal detachment, etc. Furthermore, these disorders or symptoms include disorders of visual acuity, contrast sensitivity, light-dark adaptation, color vision, etc., and symptoms related thereto.

[0021] As used herein, the terms "protein," "polypeptide," "oligopeptide," and "peptide" are used interchangeably to refer to polymers of amino acids of any length. The polymers may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified. The term also encompasses those assembled into complex polypeptide chains. The term also encompasses naturally occurring or artificially modified amino acid polymers. Such modifications include, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (e.g., conjugation with a labeling component). This definition also encompasses, for example, polypeptides containing one or more analogs of an amino acid (e.g., including non-natural amino acids), peptide-like compounds (e.g., peptoids), and other modifications known in the art. As used herein, "amino acid" refers collectively to organic compounds containing an amino group and a carboxyl group. When an antibody according to an embodiment of the present disclosure comprises a "specific amino acid sequence," any amino acid in the amino acid sequence may be chemically modified. Furthermore, any amino acid in the amino acid sequence may form a salt or solvate. Furthermore, any amino acid in the amino acid sequence may be L- or D-form. Even in such cases, the protein according to the embodiment of the present disclosure can be said to contain the above-mentioned "specific amino acid sequence." Known chemical modifications that amino acids contained in proteins undergo in vivo include, for example, N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), and side chain modifications (e.g., phosphorylation, glycosylation, etc.). Natural or non-natural amino acids may be used as long as they satisfy the objectives of the present disclosure.

[0022] As used herein, the term "chimera" (e.g., a protein, opsin, etc.) refers to a mixture of multiple genetic sequences derived from the same or different organisms in the same entity (in this case, a protein, etc.). A chimeric protein, for example, contains a mixture of gene sequences derived from two or more organisms. The sequence information contained in a chimeric protein may include sequences other than those derived from the organisms being mixed.

[0023] As used herein, the terms "polynucleotide," "oligonucleotide," "nucleic acid," and "nucleic acid molecule" are used interchangeably to refer to a polymer of nucleotides of any length. This term also includes "oligonucleotide derivatives" or "polynucleotide derivatives." "Oligonucleotide derivatives" or "polynucleotide derivatives" refer to oligonucleotides or polynucleotides that contain derivatives of nucleotides or have unusual internucleotide bonds, and are used interchangeably. Specific examples of such oligonucleotides include 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which a phosphodiester bond in an oligonucleotide has been converted to a phosphorothioate bond, oligonucleotide derivatives in which a phosphodiester bond in an oligonucleotide has been converted to an N3'-P5' phosphoramidate bond, oligonucleotide derivatives in which a ribose and a phosphodiester bond in an oligonucleotide have been converted to a peptide nucleic acid bond, oligonucleotide derivatives in which uracil in an oligonucleotide has been substituted with C-5 propynyl uracil, oligonucleotide derivatives in which uracil in an oligonucleotide has been substituted with C-5 thiazole uracil, oligonucleotide derivatives in which cytosine in an oligonucleotide has been substituted with C-5 propynyl cytosine, oligonucleotide derivatives in which cytosine in an oligonucleotide has been substituted with phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA has been substituted with 2'-O-propyl ribose, and oligonucleotide derivatives in which ribose in an oligonucleotide has been substituted with 2'-methoxyethoxy ribose. Unless otherwise specified, a particular base sequence is also intended to encompass its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly indicated sequence. Note that a nucleic acid sequence may be referred to as a base sequence, a nucleic acid sequence, a nucleotide sequence, or the like, all of which have the same meaning.Specifically, degenerate codon substitutions can be achieved by creating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and / or deoxyinosine residues (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, the term "nucleic acid" used herein may be interchangeable with DNA such as gene or cDNA, RNA such as mRNA, oligonucleotide, and polynucleotide. As used herein, a "nucleotide" may be either natural or non-natural. As used herein, a nucleic acid may be DNA or RNA.

[0024] As used herein, the term "gene" refers to a factor that determines a genetic trait, and "gene" may refer to a "polynucleotide," an "oligonucleotide," and a "nucleic acid."

[0025] As used herein, "nucleic acid construct," "construct," or "gene construct" are used interchangeably and are nucleic acid molecules that comprise nucleic acids that are isolated from naturally occurring genes or that are combined and juxtaposed in a manner that does not occur in nature, and a vector.

[0026] As used herein, "homology" of genes refers to the degree of identity between two or more gene sequences. Generally, "homology" refers to a high degree of identity or similarity. "Identity" refers to the degree of correspondence between identical amino acid sequences, and "similarity" refers to the degree of correspondence between sequences, including identical amino acids as well as amino acids with similar properties. Therefore, the higher the homology between two genes, the higher the identity or similarity between their sequences. Whether two genes are homologous can be determined by direct sequence comparison or, in the case of nucleic acids, by hybridization under stringent conditions. When directly comparing two gene sequences, the genes are homologous if the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, and more preferably at least 80%, 90%, 95%, 96%, 97%, 98%, or 99% identical. Thus, as used herein, a "homologue" or "homologous gene product" refers to a protein in another species, preferably a mammal, that performs the same biological function as a protein component of a complex as further described herein. Such homologues also encompass "orthologous gene products" and "paralogous gene products," and it is understood that such homologues, homologous gene products, orthologous gene products, paralogous gene products, etc., can also be used so long as they are consistent with the purposes of the present disclosure.

[0027] Amino acids may be referred to herein by either their commonly known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may also be referred to by their commonly accepted one-letter codes. Herein, comparisons of similarity, identity, and homology between amino acid sequences and base sequences are calculated using the sequence analysis tool BLAST with default parameters. Identity searches can be performed, for example, using NCBI's BLAST 2.2.28 (published April 2, 2013) (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Identity values ​​herein generally refer to values ​​obtained when aligned using the above-mentioned BLAST under default conditions. However, if a higher value is obtained by changing the parameters, the highest value is used as the identity value. When identity is evaluated in multiple regions, the highest value among them is used as the identity value. Similarity is a numerical value that takes into account not only identity but also similar amino acids. When comparing amino acid sequences with BLAST, the algorithm Blastp can be used with default settings. Measurement results are quantified as Positives or Identities. The homology of amino acid sequences or nucleotide sequences can be determined using the BLAST algorithm by 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 nucleotide sequences using BLASTN based on BLAST, parameters are set, for example, as score = 100 and wordlength = 12. When analyzing amino acid sequences using BLASTX based on BLAST, 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. Specific techniques for these analysis methods are known (http: / / www.ncbi.nlm.nih.gov.).

[0028] The nucleic acids or proteins used in the present disclosure may include sequences in which one or more amino acids or nucleotides have been substituted, deleted, and / or added to the target amino acid or base sequence. Here, "one or more" in the full-length amino acid sequence of the chimeric protein typically refers to 50 amino acids or less, preferably 30 amino acids or less, and more preferably 10 amino acids or less (e.g., 5 amino acids or less, 3 amino acids or less, or 1 amino acid or less). Furthermore, in the amino acid sequence of a domain, "one or more" typically refers to 6 amino acids or less, preferably 5 amino acids or less, and more preferably 4 amino acids or less (e.g., 3 amino acids or less, 2 amino acids or less, or 1 amino acid or less). To maintain the biological activity of the chimeric protein of the present disclosure, it is desirable that the mutated amino acid residue be mutated to another amino acid whose amino acid side chain properties are conserved. For example, the properties of the amino acid side chains include 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-containing side chains (C, M), amino acids with carboxylic acid- and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic-containing side chains (H, F, Y, W) (the characters in parentheses represent the single-letter symbols of the amino acids). These are also referred to herein as "conservative substitutions." It is known that proteins having an amino acid sequence modified by deletion, addition, and / or substitution of one or more amino acid residues with other amino acids maintain their 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).Therefore, in one embodiment of the present disclosure, "several" may be, for example, 10, 8, 6, 5, 4, 3, or 2, or any of these values ​​or less. Chimeric proteins with deletions or other modifications can be produced, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library. Site-directed mutagenesis can be performed using, for example, the KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.). Antibodies with activity similar to that of the wild-type antibody can be selected from mutant antibodies with deletions or other modifications by performing various characterization methods such as FACS analysis and ELISA.

[0029] In one embodiment of the present disclosure, the amino acid sequence and nucleic acid sequence of the modified protein and / or chimeric protein of the present disclosure may have 70% or more, 80% or more, or 90% or more identity or similarity to a reference sequence. As used herein, with respect to an amino acid sequence or nucleotide sequence, "70% or more" may mean, for example, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more, "80% or more" may mean, for example, 80, 85, 90, 95, 96, 97, 98, 99% or more, and "90% or more" may mean, for example, 90, 95, 96, 97, 98, 99% or more, or may be within a range of any two of these values. "Homology" may be calculated by the percentage of homologous amino acids in two or more amino acid sequences according to methods known in the art. Before calculating the percentage, the amino acid sequences of the amino acid sequences to be compared are aligned, and gaps are introduced into some of the amino acid sequences if necessary to maximize the percentage of identical amino acids. Methods for alignment, percentage calculation, comparison, and related computer programs are well known in the art (e.g., BLAST, GENETYX, etc.). In the case of "identity," the percentage of identical amino acids is calculated, and in the case of "similarity," the percentage of similar amino acids is calculated. Similar amino acids include, but are not limited to, amino acids that can be conservatively substituted.

[0030] As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to conditions commonly used in the art. Such polynucleotides can be obtained by colony hybridization, plaque hybridization, Southern blot hybridization, or the like, using a polynucleotide selected from the polynucleotides disclosed herein as a probe. Specifically, the term refers to a polynucleotide that can be identified by hybridizing a filter onto which colony- or plaque-derived DNA has been immobilized in the presence of 0.7 to 1.0 M NaCl at 65°C, followed by washing the filter at 65°C using a 0.1 to 2x SSC (saline-sodium citrate) solution (a 1x SSC solution has a composition of 150 mM sodium chloride and 15 mM sodium citrate). For example, the following conditions can be used as "stringent conditions." (1) using low ionic strength and high temperature for washing (e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C); (2) using a denaturing agent such as formamide during hybridization (e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5, and 750 mM sodium chloride, 75 mM sodium citrate at 42°C); or (3) overnight incubation at 37°C in a solution containing 20% ​​formamide, 5x SSC, 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 mg / ml denatured sheared salmon sperm DNA, followed by washing the filter with 1x SSC at about 37-50°C. The formamide concentration may be 50% or more, and the washing time may be 5, 15, 30, 60, or 120 minutes or more.Several factors, such as temperature and salt concentration, are thought to affect the stringency of a hybridization reaction, and for details, see 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, and 65-68°C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide at 42°C. See "Highly stringent conditions" in Molecular Cloning, 2nd ed., for more information on hybridization. This can be performed in accordance with the methods described in experimental manuals such as "Current Protocols in Molecular Biology, Supplement 1-38," and "DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995)." Here, sequences that hybridize under stringent conditions preferably exclude sequences containing only A sequences or only T sequences. Moderately stringent conditions can be easily determined by those skilled in the art, for example, based on the length of the DNA, and are described in Sambrook et al., "Molecular Cloning: A Laboratory Manual," Vol. 3, Vol. 1, 7.42-7.45 Cold Spring Harbor Laboratory Press, 2001, and includes the use of, for nitrocellulose filters, a pre-wash solution of 5xSSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of about 50% formamide, 2xSSC-6xSSC (or other similar hybridization solutions such as Stark's solution in about 50% formamide at about 42°C) at about 40-50°C, and wash conditions of 0.5xSSC, 0.1% SDS at about 60°C.Thus, polypeptides as used in this disclosure also include polypeptides encoded by nucleic acid molecules that hybridize under high or moderate stringency conditions to nucleic acid molecules encoding the polypeptides specifically described in this disclosure.

[0031] As used herein, a "purified" substance or biological factor (e.g., nucleic acid, protein, etc.) refers to a substance or biological factor from which at least a portion of naturally associated factors has been removed. Therefore, typically, the purity of the biological factor in a purified biological factor is higher (i.e., concentrated) than in the state in which the biological factor normally exists. As used herein, the term "purified" means that preferably at least 75% by weight, more preferably at least 85% by weight, even 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 substance or biological factor used in the present disclosure is preferably a "purified" substance. As used herein, an "isolated" substance or biological factor (e.g., nucleic acid, protein, etc.) refers to a substance or biological factor from which naturally associated factors have been substantially removed. The term "isolated" as used herein does not necessarily refer to purity, as this may vary depending on the purpose, but where necessary, preferably means that at least 75%, more preferably at least 85%, even more preferably at least 95%, and 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.

[0032] As used herein, a "corresponding" amino acid, nucleic acid, or portion refers to an amino acid or nucleotide in a polypeptide or polynucleotide molecule (e.g., rhodopsin) that has or is predicted to have the same function as a given amino acid, nucleotide, or portion in a polypeptide or polynucleotide that is used as a reference for comparison. In particular, in the case of an enzyme molecule, this refers to an amino acid that is located in a similar position in the active site and contributes similarly to catalytic activity, and in the case of a composite molecule, it refers to a corresponding portion (e.g., heparan sulfate, etc.). For example, in the case of an antisense molecule, this may be a similar portion in an ortholog that corresponds to a specific portion of the antisense molecule. A corresponding amino acid may be, for example, a specific amino acid that is cysteinylated, glutathionylated, forms an S-S bond, oxidized (e.g., oxidation of the methionine side chain), formylated, acetylated, phosphorylated, glycosylated, myristylated, or the like. Alternatively, a corresponding amino acid may be an amino acid responsible for dimerization. Such a "corresponding" amino acid or nucleic acid may be a region or domain spanning a certain range. Therefore, in such cases, it is referred to herein as a "corresponding" region or domain. Such corresponding regions or domains are useful in the present disclosure when designing composite molecules.

[0033] As used herein, a "corresponding" gene (which in this case may be a polynucleotide sequence or molecule encoding an opsin or the like) refers to a gene (which in this case may be a polynucleotide sequence or molecule encoding an opsin or the like) that has or is predicted to have the same function in a given species as a given gene in a species used as a reference for comparison. When multiple genes with such a function exist, the term refers to genes that share the same evolutionary origin. Thus, a gene corresponding to a given gene may be its ortholog. Therefore, for each human opsin, a corresponding opsin can be found in other animals (particularly mammals). Such corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in a given animal (e.g., a mouse), or a reference gene (e.g., an opsin) for the corresponding gene, can be found by using a specific sequence as a query sequence to search a database containing sequences from that animal.

[0034] As used herein, the terms "portion," "fragment," or "fragment" refer to a polypeptide or polynucleotide having a sequence length of 1 to n-1 relative to the full-length polypeptide or polynucleotide (length n). The length of the fragment can be varied appropriately depending on the purpose. For example, the lower limit of the length for a polypeptide can be 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, or more amino acids, and lengths represented by integers not specifically recited herein (e.g., 11) may also be suitable as lower limits. Furthermore, for polynucleotides, the lower limit can be 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100, or more nucleotides, and lengths represented by integers not specifically recited herein (e.g., 11) may also be suitable as lower limits. As used herein, such fragments are understood to fall within the scope of the present disclosure, for example, if the full-length fragment functions as a marker or target molecule, as long as the fragment itself also functions as a marker or target molecule.

[0035] In accordance with the present disclosure, the term "activity" as used herein refers to the function of a molecule in the broadest sense. Activity generally includes, but is not limited to, the biological, biochemical, physical, or chemical function of a molecule. Activity includes, for example, enzymatic activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the function of other molecules, stability, and the ability to localize to a specific subcellular location. Where applicable, the term also relates to the function of a protein complex in the broadest sense. As used herein, "biological activity" includes, for example, activation of a light reaction.

[0036] As used herein, the term "functional equivalent" refers to any entity that has the same intended function as the original entity but a different structure. Therefore, functional equivalents of "opsin" or its chimera are understood to include not only opsin or its chimera itself, but also mutants or variants of opsin or its chimera (e.g., amino acid sequence variants, etc.) that have the biological action of opsin or its chimera, as well as opsin or its antibody itself, or a mutant or variant of this opsin or its chimera, when acting (e.g., nucleic acids encoding opsin or its chimera, or opsin or its chimeric mutant or variant, and vectors, cells, etc. containing such nucleic acids). Functional equivalents of the present disclosure can include insertions, substitutions, and / or deletions of one or more amino acids in the amino acid sequence, or additions to one or both termini. As used herein, the phrase "insertion, substitution, and / or deletion of one or more amino acids in an amino acid sequence, or addition to one or both termini thereof" refers to a modification that involves substitution of a number of amino acids, such as those that would occur naturally, by well-known techniques such as site-directed mutagenesis, or by natural mutation. The modified amino acid sequence may be one in which, for example, 1 to 30, preferably 1 to 20, more preferably 1 to 9, even more preferably 1 to 5, and particularly preferably 1 to 2 amino acids have been inserted, substituted, or deleted, or added to one or both termini thereof. The modified amino acid sequence may preferably have one or more (preferably one or several, or 1, 2, 3, or 4) conservative substitutions in the amino acid sequence of opsin.

[0037] As used herein, the terms "drug," "agent," or "factor" (all of which correspond to the English term "agent") are used interchangeably in a broad sense and may refer to any substance or other element (e.g., energy such as light, radioactivity, heat, or electricity) that can achieve the intended purpose. Examples of such substances include, but are not limited to, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (e.g., DNA such as cDNA and genomic DNA, and RNA such as mRNA), polysaccharides, oligosaccharides, lipids, small organic molecules (e.g., hormones, ligands, signaling substances, small organic molecules, molecules synthesized by combinatorial chemistry, small molecules that can be used as pharmaceuticals (e.g., small molecule ligands), etc.), and composite molecules thereof.

[0038] For oral administration, the formulation may be formulated into various forms such as tablets, granules, fine granules, powders, capsules, etc., and may contain additives commonly used in formulations such as binders, encapsulating agents, excipients, lubricants, disintegrants, wetting agents, etc. In addition to these, formulations for oral administration may be formulated in liquid form such as oral solutions, suspensions, emulsions, syrups, etc., or may be formulated in dry form to be redissolved when used.

[0039] For parenteral administration, the formulation may be contained in a unit-dose ampule, a multi-dose container, or a tube, and may also contain additives such as stabilizers, buffers, preservatives, isotonicity agents, etc. For parenteral administration, the formulation may be formulated into a powder that can be redissolved in an appropriate carrier (sterilized water, etc.) at the time of use.

[0040] Examples of parenteral administration include intraocular administration such as intravitreal administration, subretinal administration, subchoroidal administration, and intracameral administration, and extraocular administration such as subconjunctival administration, sub-Tenon administration, and eye drop administration, with intravitreal administration being preferred. The composition of the present disclosure can be administered to humans by the methods described above and used for treatment, prevention, progression suppression, and the like.

[0041] As used herein, "treatment" refers to preventing, preferably maintaining, more preferably alleviating, and even more preferably eliminating, a disease or disorder (e.g., vesicle transport disorder or apoptosis) from worsening when that condition occurs, and includes the potential for symptom improvement or prevention of a patient's disease or one or more symptoms associated with the disease. Preliminary diagnosis followed by appropriate treatment is referred to as "companion treatment," and diagnostic agents used for this purpose are sometimes referred to as "companion diagnostic agents." Since the present disclosure targets genetic diseases, patients may be treated after genetic testing in advance.

[0042] As used herein, the term "therapeutic agent" broadly refers to any drug capable of treating a target condition (e.g., retinal degenerative disease, etc.). In one embodiment of the present disclosure, the "therapeutic agent" may be a pharmaceutical composition containing an active ingredient and one or more pharmacologically acceptable carriers. The pharmaceutical composition can be prepared, for example, by mixing the active ingredient with the carrier using any method known in the technical field of pharmaceuticals. Furthermore, the therapeutic agent may be in any form used for treatment, and may be the active ingredient alone or a mixture of the active ingredient with any other ingredient. Furthermore, the shape of the carrier is not particularly limited, and may be, for example, a solid or liquid (e.g., a buffer solution).

[0043] As used herein, "prevention" refers to preventing a certain disease or disorder (e.g., retinal degenerative disease) from occurring before that state is reached. Diagnosis can be performed using the agent of the present disclosure, and, if necessary, the agent of the present disclosure can be used to prevent, for example, retinal degenerative disease, or other preventative measures can be taken. As used herein, the term "prophylactic drug (agent)" broadly refers to any drug that can prevent a target condition (e.g., vesicle transport disorder, apoptosis, etc.).

[0044] As used herein, the term "kit" refers to a unit containing the components to be provided (e.g., nucleic acids, nucleic acid constructs, cells transfected with a nucleic acid of interest, test agents, diagnostic agents, therapeutic agents, antibodies, labels, instructions, etc.), typically separated into two or more compartments. A kit may be used to provide a combination of a diagnostic agent and a therapeutic agent, such as a companion diagnostic agent, in which it is preferable to first identify the patient to whom the drug should be administered using a reagent to determine the patient's characteristics and then administer a specific drug (e.g., a nucleic acid drug) only to the appropriate patient. Alternatively, this kit format is preferred when providing a composition that, for stability reasons, should not be provided as a mixture but is preferably mixed immediately before use, such as a particular unstable drug. Such a kit preferably includes instructions or instructions describing how to use the components to be provided (e.g., nucleic acids, nucleic acid constructs, cells transfected with a nucleic acid of interest, test agents, diagnostic agents, therapeutic agents), or how to handle the reagents. When the "kit" is used herein as a reagent kit, the kit typically includes instructions describing how to use the test agent, diagnostic agent, therapeutic agent, antibody, etc.

[0045] As used herein, the term "active ingredient" refers to an ingredient contained in a composition, etc. of the present disclosure in an amount necessary to achieve the intended effect of treatment, prevention, or progression inhibition, and other ingredients may also be contained as long as the effect is not impaired to below the desired level. Furthermore, the medicament, composition, etc. of the present disclosure may be formulated. Furthermore, the route of administration of the medicament, composition, etc. of the present disclosure may be either oral or parenteral, and can be appropriately determined depending on the form of the formulation, etc.

[0046] As used herein, "instructions" (including package inserts and labels used by the U.S. FDA) are written instructions to a physician or other user on how to use the present disclosure. The instructions include instructions on how to use the detection method, diagnostic reagent, or administer a medicine, etc., of the present disclosure. The instructions may also include instructions for oral or intraretinal administration (e.g., by injection) as the administration site. The instructions are prepared in accordance with a format specified by the regulatory agency of the country in which the present disclosure is implemented (e.g., the Ministry of Health, Labor and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and clearly state that they have been approved by the regulatory agency. The instructions are so-called package inserts or labels, and are typically provided in paper form, but are not limited thereto and may also be provided in the form of, for example, electronic media (e.g., a homepage provided on the Internet, email, etc.).

[0047] (Preferred Embodiments) Preferred embodiments of the present disclosure will be described below. The embodiments provided below are provided for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure in light of the description herein. It is also understood that the following embodiments of the present disclosure can be used alone or in combination.

[0048] In one aspect, the present disclosure provides a protein comprising the amino acid sequence of an opsin, the protein comprising a modification of the amino acid corresponding to position 188 in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1.

[0049] Opsins are light-sensitive G protein-coupled receptors ubiquitously present in animals. All opsins share common structural elements, including seven transmembrane domains, and bind the light-absorbing chromophore retinal to Lys296 (based on the bovine rhodopsin numbering system) via a Schiff base bond. Opsins function in both visual and non-visual photoreception and are classified into several groups based on their amino acid sequence (Shichida and Matsuyama, 2009; Koyanagi and Terakita, 2014). Bovine rhodopsin is the most studied opsin (Yau and Hardie, 2009), functioning as a visual photoreceptor protein in the retina and binding 11-cis retinal in the dark. Photoisomerization of retinal to the all-trans form generates a meta II intermediate of rhodopsin, which binds to the G protein. Meta II is a metastable, active state that spontaneously converts to meta III (Heck et al., 2003). Furthermore, light irradiation of meta II induces the formation of meta III rather than the original dark state (Bartl et al., 2001; Ritter et al., 2008). Thus, the active meta II is highly inefficient in returning to its original dark state via photoreaction or heat. These findings suggest that vertebrate opsins are specialized for light activation and are characterized as monostable opsins. On the other hand, opsins from mollusks and arthropods form a stable active acid meta state by photoisomerization from 11-cis to all-trans retinal, and the active state can be converted back to the original dark state containing 11-cis retinal by light (Koyanagi and Terakita, 2014; Yau and Hardie, 2009). Thus, these opsins are known as bistable opsins because they have two stable states, the dark state and the active state, which can be interconverted by light.Recent advances in knowledge about the molecular properties of opsins have revealed that many members of various opsin groups are bi-stable opsins, suggesting that vertebrate opsins evolved from ancestral bi-stable opsins to mono-stable opsins (Shichida and Matsuyama, 2009).

[0050] Opn5L1, the only animal opsin with photocycling properties, contains a cysteine ​​residue at position 188, which is known to underlie the photocycling reaction of opsins (Sato et al., 2018). In the dark, Opn5L1 binds to all-trans retinal rather than 11-cis retinal, forming an active state. Upon light exposure, retinal is photoisomerized to the 11-cis form, suppressing Opn5L1's ability to activate G proteins. Subsequently, a covalent bond is formed between retinal and Cys188 of opsin, converting the C11=C12 double bond of retinal to a single bond. Thermal rotation of the C11-C12 single bond in retinal then dissociates the Cys188-retinal adduct, restoring the original dark state. The ability of Opn5L1 to activate G proteins is controlled by a combination of photoisomerization and thermal isomerization of retinal, making it the first animal opsin whose activity is controlled by a photocycle reaction.

[0051] Comparing the amino acid sequences of opsins reveals that the cysteine ​​residue at position 188 is highly conserved in the Opn5L1 family but is rarely found in other opsin families, indicating that Cys188 is important for the unique photocycling reaction of Opn5L1. On the other hand, monostable opsins such as vertebrate opsins and cone opsins have a glycine residue at this position (Figure 9). In this disclosure, we have found that mutations at position 188 can confer photocycling properties to bovine rhodopsin. We have demonstrated that the G188C mutant converts to the active meta II state upon light irradiation and can be thermally restored to its original dark state. Furthermore, light irradiation of the meta II of the G188C mutant can restore it to its original dark state. Therefore, the G188C mutant of bovine rhodopsin exhibits photocycling properties and photoreversibility, indicating that in vertebrate opsins, the residue at position 188 controls the restoration of the active state to its original dark state.

[0052] In one embodiment of the present disclosure, the amino acid at position 188 in the amino acid sequence of the opsins of the present disclosure, when aligned with SEQ ID NO: 1, can include an amino acid corresponding to G, T, S, or E, and the modified opsins of the present disclosure can have these amino acids modified.

[0053] In one embodiment of the present disclosure, the amino acid corresponding to position G188 when aligned with SEQ ID NO: 1 can be modified to cysteine. The amino acid corresponding to position G188 of the opsins used in the present disclosure can be, as described above, an amino acid corresponding to G, T, S, or E. Modification of any of these to cysteine ​​can confer photocycling properties and photoreversibility. Thus, in one embodiment of the present disclosure, the protein of the present disclosure can be inactivated after activation by light stimulation without releasing the photoreceptor.

[0054] In one embodiment, the opsins used in the present disclosure may be any opsins, including, but not limited to, G protein-coupled receptor rhodopsin, rod opsin, cone opsin (blue opsin, green opsin, red opsin, melanopsin, encephalopsin, OPN5 panopsin, RGR, and peropsin). In one embodiment, the opsins used in the present disclosure may be derived from, but are not limited to, vertebrates, invertebrates, or microorganisms, or chimeras derived from these organisms. Examples of vertebrates include, but are not limited to, mammals, birds, reptiles, amphibians, fish (bony fish, cartilaginous fish), or jawless fish. Examples of invertebrates include, but are not limited to, mollusks, Examples of opsins include, but are not limited to, eubacteria, archaea, and fungi. In one embodiment, the opsins of the present disclosure can be derived from mammals. Examples of G protein-coupled receptor rhodopsins include, but are not limited to, those derived from mammals, such as rodents, artiodactyls, perissodactyls, primates, and carnivorans. For example, rhodopsins from artiodactyls or primates may be advantageous, with primate rhodopsins being preferred. Examples of opsins that can be used include those derived from cattle, humans, mice, rats, cats, dogs, pigs, sheep, horses, and the like. Of these, those derived from cattle or humans are particularly preferred.

[0055] In one embodiment, specific amino acid sequences of the opsins of the present disclosure include, but are not limited to, the sequences of SEQ ID NOs: 1 to 34 listed in Table 1.

[0056] In one embodiment, specific nucleic acid sequences of the opsins of the present disclosure include, but are not limited to, the sequences of SEQ ID NOs: 35 to 68 listed in Table 2.

[0057] In certain embodiments, the proteins of the present disclosure are chimeric proteins that include a portion of the G protein-coupled receptor rhodopsin and a portion of another G protein-coupled receptor, a metabotropic glutamate receptor, or an adrenergic receptor, and have a seven-transmembrane structure.

[0058] In one embodiment of the present disclosure, the amino acid sequence encoding the protein of the present disclosure can include: 1) an amino acid sequence represented by any one of SEQ ID NOs: 1 to 34, which contains the modification; 2) an amino acid sequence in which the sequence other than the modification site has at least about 80% identity to 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 in which the sequence of 1) has one or more mutations other than the modification site, and the encoded protein has substantially the same biological activity as the protein obtained from the sequence of 1); 4) an amino acid sequence encoded by a nucleic acid to which a nucleic acid encoding the sequence of 1) hybridizes, which contains the modification; or 5) an amino acid sequence encoded by an allelic variant of a nucleic acid encoding the sequence of 1). In one embodiment, the amino acid sequence encoding the protein of the present disclosure can be an amino acid sequence in which the sequence excluding the modification site has 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 to the sequence of 1), and the encoded protein has substantially the same biological activity as the protein obtained from the sequence of 1).

[0059] In one embodiment of the present disclosure, a protein of the present disclosure can include a modification of the amino acid corresponding to position 122 in the amino acid sequence of an opsin when aligned with SEQ ID NO: 1. An example of the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1 is the amino acid corresponding to E at position 122. Modified opsins of the present disclosure can have these amino acids modified.

[0060] In one embodiment of the present disclosure, the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1 can be modified to glutamine. As described above, the amino acid corresponding to position 122 of the opsins used in the present disclosure can include the amino acid corresponding to E. Modifying any of these amino acids to glutamine can promote the decay of meta II and speed up the photocycle reaction.

[0061] In one embodiment of the present disclosure, the opsins of the present disclosure can include modifications of amino acids corresponding to the amino acids belonging to a portion of the N-terminal domain (positions 1 to 11) and the amino acids belonging to a portion of the extracellular loop 3 (positions 278 to 285) when aligned with SEQ ID NO: 1. In one embodiment, the amino acids corresponding to the amino acids belonging to a portion of the N-terminal domain (positions 1 to 11) and the amino acids belonging to a portion of the extracellular loop 3 (positions 278 to 285) when aligned with SEQ ID NO: 1 can be modified to cysteine. For example, the amino acids corresponding to the amino acids belonging to a portion of the N-terminal domain (positions 1 to 11) and the amino acids belonging to a portion of the extracellular loop 3 (positions 278 to 285) when aligned with SEQ ID NO: 1 can be the amino acids corresponding to positions 2 and 282 in the opsin amino acid sequence when aligned with SEQ ID NO: 1, and preferably these amino acids can be modified to cysteine. Such modifications can enhance the thermal stability of the opsins of the present disclosure. In other embodiments, the opsins of the present disclosure can have modifications in their amino acid sequence to any one, two, three, four, five, six, seven, eight, nine, ten, or eleven of the amino acids at positions 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the N-terminal domain when aligned with SEQ ID NO: 1, and any one, two, three, four, five, six, seven, or eight of the amino acids at positions 278, 279, 280, 281, 282, 283, 284, and 285 of the extracellular loop 3 when aligned with SEQ ID NO: 1, and preferably, such one or more amino acids can be modified to cysteine.In still yet other embodiments, the modified amino acid sequence may preferably be an amino acid sequence that has one or more (preferably one or several, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11) conservative substitutions in the amino acid sequence of opsin.

[0062] Methods for obtaining nucleic acids such as DNA of the present disclosure are not particularly limited, and include known methods such as a method of obtaining cDNA by reverse transcription from mRNA (e.g., RT-PCR), 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 (see, for example, Japanese Patent Application Laid-Open No. 11-29599).

[0063] In the present specification, chimeric proteins can be prepared, for example, by using a transformant into which an expression vector containing a nucleic acid such as DNA encoding the chimeric protein has been introduced. For example, the transformant is first cultured under appropriate conditions to synthesize the chimeric protein encoded by the nucleic acid such as DNA. The synthesized protein can then be recovered from the transformant or the culture medium to obtain the chimeric protein of the present disclosure.

[0064] More specifically, the chimeric protein can be prepared by inserting DNA encoding the chimeric protein into an appropriate expression vector. A "suitable vector" is any vector that can maintain replication or self-replicate in various prokaryotic and / or eukaryotic hosts, and can be selected appropriately depending on the intended use. For example, a high-copy vector can be selected when a large amount of nucleic acid such as DNA is to be obtained, and an expression vector can be selected when a polypeptide (chimeric protein) is to be obtained. Specific examples include, but are not limited to, known vectors such as those described in Japanese Patent Application Laid-Open No. 11-29599.

[0065] Furthermore, expression vectors can be used not only to synthesize chimeric proteins but also in compositions of the present disclosure. That is, compositions of the present disclosure may contain, as an active ingredient, an expression vector incorporating the nucleic acid construct of the present disclosure. By directly introducing such an expression vector into a human, it can be used for the treatment, prevention, and suppression of progression of retinal diseases, disorders, or symptoms. In this case, a vector that can be introduced into human cells is used. Suitable examples of such vectors include adeno-associated virus vectors (AAV vectors) and lentivirus vectors.

[0066] The method for introducing the vector can be appropriately selected depending on the type of vector and host, etc. Specific examples thereof include, but are not limited to, known methods such as the protoplast method and the competent method when bacteria are used as hosts (see, for example, JP-A-11-29599). Furthermore, when an expression vector is used as an active ingredient of the agent for regenerating visual function or the agent for preventing visual function decline of the present disclosure, the vector can be introduced, for example, by intraocular injection of the above-mentioned AAV vector, etc.

[0067] The host into which the expression vector is introduced may be any host that is compatible with the expression vector and can be transformed, and specific examples thereof include, but are not limited to, known natural cells or artificially established cells such as bacteria, yeast, animal cells, and insect cells (see JP 11-29599 A), or animals such as humans and mice. The transformant can be cultured in an appropriate nutrient medium selected from known nutrient media depending on the type of transformant, and the temperature, pH of the nutrient medium, culture time, etc. can be adjusted appropriately so that the chimeric protein can be obtained easily in large quantities (see, for example, JP 11-29599 A).

[0068] In one preferred embodiment, the opsins of the present disclosure are provided as nucleic acid molecules or nucleic acid constructs containing the nucleic acid molecules, and are used as pharmaceuticals for gene therapy. Examples of nucleic acid molecules encoding opsins or nucleic acid constructs containing the nucleic acid molecules that can be used in this embodiment include nucleic acid molecules encoding proteins containing the amino acid sequence of opsins, which contain a modification of the amino acid corresponding to position 188 in the amino acid sequence of the opsins when aligned with SEQ ID NO: 1, or nucleic acid constructs containing the nucleic acid molecules. Opsins described elsewhere herein can be used. Furthermore, opsin-encoding nucleic acid molecules herein include, but are not limited to, nucleic acid molecules encoding melanopsin, encephalopsin, OPN5, RGR, and peropsin. In one embodiment, the nucleic acid molecule used in the present disclosure may be a nucleic acid molecule encoding a microbial opsin, an animal opsin, or the like. More specifically, the animal opsin may further be a nucleic acid molecule encoding a vertebrate visual opsin, a vertebrate non-visual opsin, an invertebrate opsin, or the like. Nucleic acid molecules that can be used in the present disclosure may be nucleic acid molecules that encode bistable opsins, such as vertebrate non-visual opsins and invertebrate opsins. Even if the opsin-encoding nucleic acid molecules used in the present disclosure do not strictly fall under the category of microbial opsins, vertebrate non-visual opsins, or invertebrate opsins, they can still be used advantageously as long as they encode functional equivalents that have the same function as these advantageously used types of opsins.

[0069] The nucleic acid molecule used in the present disclosure may be, for example, a nucleic acid molecule encoding a G protein-coupled receptor rhodopsin or a chimeric opsin of a G protein-coupled receptor rhodopsin. When an animal-derived, preferably mammal-derived, nucleic acid molecule encoding a G protein-coupled receptor rhodopsin is used here, the encoded protein can maintain its function of repeated activation while achieving high activity mediated by an endogenous G protein.

[0070] Methods for isolating and purifying chimeric proteins are not particularly limited, and include known methods such as methods utilizing solubility, methods utilizing differences in molecular weight, and methods utilizing charge (see, for example, Japanese Patent Application Laid-Open No. 11-29599).

[0071] (Medicinal Uses of Opsins) In another aspect, the present disclosure provides compositions, compounds, medicaments, or methods for restoring vision or treating, preventing, or inhibiting the progression of a visual disorder or disease, using a protein comprising the amino acid sequence of an opsin, the protein comprising a modification of the amino acid corresponding to position 188 in the amino acid sequence of the opsin when aligned with SEQ ID NO: 1, a nucleic acid molecule encoding the protein, or a nucleic acid construct comprising the nucleic acid molecule. The present disclosure may also provide a method for restoring vision in a subject or treating, preventing, or inhibiting the progression of a visual disorder or disease in a subject, the method comprising administering a protein of the present disclosure to the subject. The present disclosure may also provide use of the opsins of the present disclosure, or the nucleic acid molecule encoding them, for the manufacture of a composition or medicament for restoring vision or treating, preventing, or inhibiting the progression of a visual disorder or disease in a subject. The opsins of the present disclosure may have the activity of not releasing retinal upon light reception.

[0072] In one embodiment, diseases, disorders, or symptoms that can be targeted by the opsins of the present disclosure, or nucleic acid molecules encoding them, or nucleic acid constructs containing the nucleic acid molecules, include, but are not limited to, vesicle transport disorders, diabetes, diabetic retinopathy, myopia, macular degeneration (e.g., age-related macular degeneration), glaucoma, cataracts, viral infections, corneal dystrophy, retinoblastoma, Alzheimer's disease, Parkinson's disease, lifestyle-related diseases, stroke, hearing loss, arrhythmia, heart failure, motor paralysis, autonomic nervous system disorders, depression, anxiety neurosis, urinary and defecation disorders, and rehabilitation.

[0073] In one embodiment, the opsins of the present disclosure, or the nucleic acid molecules encoding them, or the nucleic acid constructs containing the nucleic acid molecules, can be used as medicines for human diseases, disorders, or conditions, or for non-human animal diseases, disorders, or conditions.

[0074] In one embodiment, the opsins of the present disclosure, proteins containing them, or nucleic acid molecules encoding the opsins, and / or nucleic acid constructs containing the nucleic acid molecules can be provided as cells containing them, and these cells can be used as pharmaceuticals.

[0075] In one embodiment, a protein comprising the amino acid sequence of an opsin disclosed herein, which includes a modification of the amino acid corresponding to position 188 in the opsin amino acid sequence when aligned with SEQ ID NO: 1, can be used in optogenetics to control specific neural activity in a target animal. Optogenetics is a technology that involves introducing genes encoding light-activated ion channels, pumps, enzymes, etc. to express light-activated proteins, thereby converting target cells into a form that can be light-controlled. This enables reversible, instantaneous, and bioorthogonal manipulation / control and analysis of specific cell(s) on a millisecond timescale in a living animal. For more information on optogenetics, see, 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.

[0076] In another aspect of the present disclosure, the opsins of the present disclosure or the nucleic acid molecules encoding them can be provided as a kit in combination with a companion diagnostic. For example, a composition containing the active ingredient of the pharmaceutical of the present disclosure can be administered to a subject suffering from a disease or disorder such as a retinal-related disease, or when such a disease or disorder is expected to occur, to exert a therapeutic effect. Therefore, the composition of the present disclosure can be combined with a companion diagnostic for pre-diagnosing a vision-related disorder, disease, or disorder, and the subject's genetic status or genes can be diagnosed or tested, and then administered only to subjects for whom the composition of the present disclosure is expected to be effective.

[0077] In one aspect of the present disclosure, the composition or medicament of the present disclosure can also be provided as a nucleic acid drug. In one embodiment, when gene therapy or gene therapy is performed using the nucleic acid drug of the present disclosure, a polynucleotide can be introduced into the genome of a cell to restore or modify a gene and / or gene expression. For example, a therapy can be used in which a normal gene is introduced using a vector that can be introduced into human cells, such as various viral vectors, or other delivery systems. The method of vector introduction can be appropriately selected depending on the type of vector and host, and when an expression vector is used as the active ingredient of the composition of the present disclosure, it can be introduced, for example, by intraocular injection of an AAV vector.

[0078] As used herein, "gene therapy" is the insertion of a nucleic acid sequence (e.g., a transgene, as defined herein) into an individual's cells and / or tissues to treat a disease. A transgene can be a functional mutant allele that replaces or supplements a defective allele. Gene therapy also includes the insertion of a transgene that inhibits, reduces, or decreases the expression, activity, or function of an endogenous gene or protein, such as a naturally inhibitory, i.e., undesirable or abnormal (e.g., pathogenic), gene or protein. Such a transgene may be exogenous. An exogenous molecule or sequence is understood to be a molecule or sequence that is not normally present in the cells, tissues, and / or individual being treated. Both acquired and congenital diseases are amenable to gene therapy.

[0079] As used herein, a "gene therapy vector" refers to any vector capable of delivering a polynucleotide encoding a therapeutic protein (e.g., opsins, etc.) to a host, e.g., a patient. In some embodiments, the gene therapy vector targets a specific host cell or organ, e.g., for localized delivery, e.g., tissue-specific delivery. Typically, localized delivery requires the protein (e.g., a therapeutic protein) encoded by the mRNA to be translated and expressed primarily in and / or by an organ, e.g., the liver, thereby forming a depot, e.g., a liver depot, for protein production (and secretion). In some embodiments, the gene therapy vector is configured to deliver a therapeutic protein polynucleotide to the patient's eye. In some embodiments, the gene therapy vector delivers a therapeutic protein-encoding polynucleotide to other tissues in the patient. In some embodiments, the gene therapy vector delivers a therapeutic protein-encoding polynucleotide to the patient's optic nerve.

[0080] Any known or future-developed gene therapy delivery vector, whether naturally occurring or engineered, can be used in the practice of the present disclosure. In some embodiments, the gene therapy vector is a viral vector, e.g., comprising a virus, a viral capsid, a viral genome, etc. In some embodiments, the gene therapy vector is a naked polynucleotide, e.g., an episome. In some embodiments, the gene therapy vector comprises a polynucleotide complex. Exemplary, non-limiting polynucleotide complexes for use as gene therapy vectors include lipoplexes, polymersomes, polypex, dendrimers, inorganic nanoparticles (e.g., polynucleotide-coated gold, silica, iron oxide, calcium phosphate, etc.). In some embodiments, the gene therapy vectors described herein comprise a combination of a viral vector, a naked polynucleotide, and a polynucleotide complex.

[0081] In one embodiment, gene therapy vector is a viral vector, including retrovirus, adenovirus, herpes simplex virus, poxvirus, vaccinia virus, lentivirus, or adeno-associated virus.In one embodiment, gene therapy vector is an adeno-associated virus (AAV), including serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, and AAV11, or their engineered or naturally selected variants.In one embodiment, polynucleotide also contains adeno-associated virus (AAV) nucleic acid sequence.In one embodiment, gene therapy vector is a chimeric adeno-associated virus that contains genetic elements from two or more serotypes. For example, an AAV vector having the rep gene from AAV1 and the cap gene from AAV2 (referred to as AAV1 / 2 or AAV RC1 / 2) may be used as a gene therapy vector to deliver a therapeutic protein polynucleotide of the present disclosure to a cell or cells of a patient in need thereof.In one embodiment, the gene therapy vector is selected from the group consisting of 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, A AV8 / 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, chimeric virions or derivatives thereof.Gao et al., "Novel adeno-associated viruses from rhesus monkeys as vectors for human gene therapy," PNAS 99(18): 11854-11859, September 3, 2002, is incorporated herein by reference for its description of AAV vectors and chimeric virions useful as gene therapy vectors, and their construction and use.

[0082] 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 advances in targeted gene therapy using viral vectors can be summarized as non-recombinant (non-genetic) or recombinant (genetic) modifications of viral vectors, resulting in pseudotyping, expansion, and / or retargeting of the viral vector's natural tropism. (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 adapters that recognize both wild-type (unmodified) viral surface proteins and target cells. Soluble pseudoreceptors (for wild-type viruses), polymers such as polyethylene glycol, and antibodies or portions thereof have been used as the virus-binding domain of adapters, while natural peptide or vitamin ligands, and antibodies or portions thereof have been used as the cell-binding domain of the adapters. For example, retargeting of a viral vector to a target cell can be achieved by binding the vector:adapter complex to a protein expressed on the surface of the target cell, such as a cell surface protein.Such approaches have included the use of 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 et al. (2005) Mol. Ther. 11:617-24), and paramyxovirus (Bian et al. (2005) Cancer Gene Ther. 12:295-303; Bian et al. (2005) Int. J. Oncol. 29:1359-69), and coronaviruses (Haijema et al. (2003) J. Virol. 77:4528-4538; Wurdinger et al. (2005) Gene Therapy 12:1394-1404).

[0083] A common approach is to genetically modify the viral capsid protein by recombination, thus modifying the surface of the viral capsid. In the indirect recombinant approach, the viral capsid is modified with a heterologous "scaffold" and then linked to an adapter. The adapter binds to the scaffold and to the target cell.(Arnold et al. (2006) Mol. Ther. 5:125-132; Ponnazhagen et al. (2002) J. Virol. 76:12900-907; see also WO 97 / 05266.) Scaffolds, such as (1) Fc-binding molecules (e.g., Fc receptors, protein A, etc.) that bind to the Fc of antibody adapters, (2) (strept)avidin that binds to biotinylated adapters, (3) biotin that binds to adapters fused to (strept)avidin, and (4) protein:protein binding pairs that form isometric peptide bonds, such as SpyCatcher that binds to SpyTagged adapters, have been used to construct 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. (2008) Biochemical and Biophysical Research Communications 366: 769-774). 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).

[0084] In direct recombinant targeting approaches, a targeting ligand is directly inserted into or attached to the viral capsid, i.e., the protein viral capsid is modified to express a heterologous ligand that redirects, e.g., binds, to a receptor or marker that is preferentially or exclusively expressed on the target cell. 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).

[0085] In some embodiments, the gene therapy vectors described herein comprise naked polynucleotides. For example, in some embodiments, a polynucleotide encoding a therapeutic polypeptide may be injected intravenously, for example, near the eye, directly into an organ to form a depot. Additional well-known methods for enhancing the delivery of naked polynucleotides include, but are not limited to, electroporation, sonoporation, the use of a gene gun to eject polynucleotide-coated gold particles, magnetic particles, and hydrodynamic delivery.

[0086] In some embodiments, the gene therapy vectors described herein comprise polynucleotide complexes, including, but not limited to, nanoparticles (e.g., polynucleotide self-assembled nanoparticles, polymer-based self-assembled nanoparticles, inorganic nanoparticles, lipid nanoparticles, semiconducting / metallic nanoparticles), gels and hydrogels, polynucleotide complexes with cations and anions, microparticles, and any combination thereof.

[0087] In some embodiments, the polynucleotides disclosed herein can be formulated as self-assembled nanoparticles. As a non-limiting example, the polynucleotides can be used to create nanoparticles that can be used in delivery systems for polynucleotides (see, for example, International Patent Application Publication No. 2012125987, incorporated herein by reference in its entirety). In some embodiments, the polynucleotide self-assembled nanoparticles can include a polynucleotide core disclosed herein and a polymer shell. The polymer shell can be any of the polymers described herein and known in the art. In additional embodiments, the polymer shell can be used to protect the polynucleotide within the core.

[0088] In some embodiments, these self-assembled nanoparticles can be microsponges formed from long polymeric polynucleotide hairpins that are formed into crystalline "pleated" sheets and then self-assembled into microsponges. These microsponges are densely packed, sponge-like microparticles that can function as efficient carriers and deliver cargo to cells. Microsponges can be 1 μm to 300 nm in diameter. Microsponges can be complexed with other agents known in the art to form larger microsponges. As a non-limiting example, microsponges can be complexed with agents such as polycationic polyethyleneimine (PEI) to form an outer layer and promote cellular uptake. This complex can form 250 nm diameter particles that can remain stable at high temperatures (150°C) (Grabow and Jaegar, Nature Materials 2012, 11:269-269; incorporated herein by reference in its entirety). Furthermore, these microsponges may provide an extraordinary degree of protection from degradation by ribonucleases. In another embodiment, polymer-based self-assembled nanoparticles, such as, but not limited to, microsponges, may be fully programmable nanoparticles. The geometry, size, and stoichiometry of the nanoparticles can be precisely controlled to create optimal nanoparticles for delivering cargo, such as, but not limited to, polynucleotides.

[0089] In some embodiments, polynucleotides can be formulated into inorganic nanoparticles (see U.S. Pat. No. 8,257,745, which is incorporated herein by reference in its entirety). Inorganic nanoparticles can include, but are not limited to, water-swellable clay-like materials. As a non-limiting example, inorganic nanoparticles can include synthetic smectite clays made from simple silicates (see, e.g., U.S. Pat. Nos. 5,585,108 and 8,257,745, each of which is incorporated herein by reference in its entirety).

[0090] In some embodiments, polynucleotides may be formulated in water-dispersible nanoparticles comprising semiconductive or metallic materials (U.S. Patent Application Publication No. 20120228565, which is incorporated herein by reference in its entirety), or formed in magnetic nanoparticles (U.S. Patent Application Publication Nos. 20120265001 and 20120283503, which are incorporated herein by reference in their entirety). The water-dispersible nanoparticles may be hydrophobic or hydrophilic.

[0091] In some embodiments, the polynucleotides disclosed herein can be encapsulated in any hydrogel known in the art that can form a gel when injected into a subject. Hydrogels are networks of hydrophilic polymer chains and can be found as colloidal gels in which water is the dispersion medium. Hydrogels can include highly absorbent (capable of containing more than 99% water) natural or synthetic polymers. Due to their significant water content, hydrogels also possess flexibility very similar to that of natural tissue. The hydrogels described herein can be used to encapsulate biocompatible, biodegradable, and / or porous lipid nanoparticles.

[0092] As a non-limiting example, the hydrogel may be an aptamer-functionalized hydrogel. The aptamer-functionalized hydrogel may be programmed to release one or more polynucleotides using polynucleotide hybridization (Battig et al., J. Am. Chem. Society. 2012 134:12410-12413; incorporated herein by reference in its entirety). In some embodiments, the polynucleotides may be encapsulated within lipid nanoparticles, which may then be encapsulated within the hydrogel.

[0093] In some embodiments, polynucleotides may be encapsulated in fibrin gels, fibrin hydrogels, or fibrin glue. In other embodiments, polynucleotides may be formulated into lipid nanoparticles or rapid-clearing lipid nanoparticles before encapsulation in fibrin gels, fibrin hydrogels, or fibrin glue. In yet other embodiments, polynucleotides may be formulated as lipoplexes before encapsulation in fibrin gels, hydrogels, or fibrin glue. Fibrin gels, hydrogels, and glues include two components: a fibrinogen solution and a calcium-enriched 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, each of which is incorporated by reference in its entirety). The concentrations of the components of the fibrin gel, hydrogel, and / or adhesive can be varied to change the properties of the gel, hydrogel, and / or adhesive, the mesh size of the network, and / or the degradation characteristics, including, but not limited to, changing the release characteristics 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, each of which is incorporated by reference in its entirety). This characteristic can be advantageous when used to deliver the polynucleotides disclosed herein.(See, e.g., Kidd et al. Journal of Controlled Release 2012. 157:80-85; Catelas et al. Tissue Engineering 2008. 14:119-128, each of which is incorporated by reference in its entirety.)

[0094] In some embodiments, the polynucleotides disclosed herein may contain cations or anions. In one embodiment, the formulation includes a metal cation, such as, but not limited to, Zn2+, Ca2+, Cu2+, Mg+, and combinations thereof. As a non-limiting example, the formulation may include a polymer and a polynucleotide complexed with a metal cation (see, e.g., U.S. Patent Nos. 6,265,389 and 6,555,525, each of which is incorporated herein by reference in its entirety).

[0095] In some embodiments, polynucleotides may be formulated into nanoparticles and / or microparticles. These nanoparticles and / or microparticles may be formed into any size, shape, and chemistry. As an example, nanoparticles and / or microparticles may be made using PRINT® technology by LIQUIDA TECHNOLOGIES.RTM. (Morrisville, NC) (see International Patent Application Publication No. 2007024323, incorporated herein by reference in its entirety).

[0096] In some embodiments, polynucleotides may be formulated in nanojackets and nanoliposomes by Keystone Nano (State College, PA). Nanojackets are made from compounds naturally found in the body, including calcium, phosphate, or compounds that also contain small amounts of silicate. Nanojackets can range in size from 5 to 50 nm and can be used to deliver hydrophilic and hydrophobic compounds, such as, but not limited to, polynucleotides, primary constructs, and / or polynucleotides. Nanoliposomes are made from lipids, including, but not limited to, lipids that occur naturally in the body. Nanoliposomes can range in size from 60 to 80 nm and can be used to deliver hydrophilic and hydrophobic compounds, including, but not limited to, polynucleotides, primary constructs, and / or polynucleotides. In one aspect, the polynucleotides disclosed herein are formulated in nanoliposomes, such as, but not limited to, ceramide nanoliposomes.

[0097] In some embodiments, the polynucleotide, e.g., DNA, also contains a promoter operably linked to the nucleic acid sequence encoding the 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 serpinal and / or TTR promoter. In other embodiments, the promoter is a CMV promoter. In other embodiments, the promoter is a ubiquitin C promoter.

[0098] In some embodiments, the polynucleotide also comprises a "locus targeting nucleic acid sequence." The locus targeting sequence allows for integration of the polynucleotide encoding the therapeutic protein into the genome of the recipient host cell. In some embodiments, the locus targeting sequence comprises flanking homology arms to enable homologous recombination. In some embodiments, the locus targeting sequence comprises a guide RNA sequence and a type II Cas enzyme (i.e., CRISPR-Cas9 method) to drive integration. In some embodiments, the locus targeting sequence comprises a guide zinc finger nuclease (ZFN) recognition sequence to drive integration. In some embodiments, the locus targeting sequence comprises a transcription activator-like effector nuclease (TALEN) recognition sequence to drive integration. In yet other embodiments, the locus targeting sequence comprises a single residue pair nucleotide code used by a BuD-derived nuclease to drive integration.

[0099] In one embodiment, the cell therapy using cells comprising the composition of the present disclosure includes a therapy involving transplanting retinal cells comprising the composition of the present disclosure. In one embodiment, the cells comprising the composition of the present disclosure may be administered together with an additional drug in addition to the cells. Such additional drugs may include drugs commonly used in ophthalmic treatments (e.g., steroids, antibiotics, antibacterial substances, and NSAIDs). Such additional drugs may be included in the cell medicine of the present disclosure as a pharmaceutical, or may be provided in a form to be administered separately. When provided or administered separately, such drugs may be provided as a kit or combination drug. When used as a kit or combination drug, such drugs may be combined with an accompanying document or the like that describes how to use the drugs.

[0100] In a preferred embodiment, the present disclosure is administered to a subject before or immediately after the onset of the above-mentioned disease, disorder, or condition, for example, within one year, preferably within six months, three months, or one month after the onset (e.g., the appearance of subjective symptoms), but is not limited thereto.

[0101] In one specific embodiment, the protein, nucleic acid molecule, nucleic acid construct, and / or cell of the present disclosure, or a medicament containing them, is administered once during a treatment period. As described in the Examples, it has been confirmed that the medicament of the present disclosure exerts its effects with a single administration, and patient compliance is thought to be good.

[0102] In one specific embodiment, when the medicament etc. of the present disclosure is administered to the eye, the amount of the vector used is about 0.01 × 10 １１ ~Approx. 100×10 １１ The unit dose can be, for example, about 0.01 x 10 vg / eye. １１ vg / eye, approximately 0.02×10 １１ vg / eye, approximately 0.03×10 １１ vg / 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 x 10 １１ vg / eye, approximately 0.4×10 １１ vg / eye, approximately 0.5×10 １１ vg / eye, with an upper limit of about 2×10 １１ vg / eye, approx. 3 x 10 １１ vg / eye, approx. 4 x 10 １１ vg / eye, approx. 5 x 10 １１ vg / eye, approx. 6 x 10 １１ vg / eye, approximately 7x10 １１ vg / eye, approx. 8 x 10 １１ vg / eye, approx. 9 x 10 １１ vg / eye, approximately 10 x 10 １１ vg / eye, approx. 15 x 10 １１ vg / eye, approx. 20 x 10 １１ vg / eye, approx. 30 x 10 １１ vg / eye, approx. 40 x 10 １１ vg / eye, approx. 50 x 10 １１ vg / eye, approximately 100 x 10１１ vg / eye, etc.

[0103] In another embodiment, when the medicament etc. of the present disclosure is administered systemically (intravenously) or locally (intramuscularly, intracerebrally, inner ear, etc.), the amount of vector used is about 0.1 × 10 １１ ~Approx. 1000×10 １１ The unit dose can be in the range of vg / kg, for example, the lower limit is about 0.1 x 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 １１ vg / kg, approximately 0.9×10 １１ vg / kg, approximately 1.0×10 １１ vg / kg, with an upper limit of about 20×10 １１ vg / kg, approximately 30×10 １１ vg / kg, approximately 40×10 １１ vg / kg, approximately 50×10 １１ vg / kg, approximately 60×10 １１ vg / kg, approximately 70×10 １１ vg / kg, approximately 80×10 １１ vg / kg, approximately 90×10 １１ vg / kg, approximately 100×10 １１ vg / kg, approximately 150×10 １１ vg / kg, approximately 200×10 １１ vg / kg, approximately 300×10 １１ vg / kg, approximately 400×10 １１ vg / kg, approximately 500×10 １１ vg / kg, approximately 1000×10 １１ vg / kg, etc.

[0104] In one aspect of the present disclosure, two or more of the opsins disclosed herein, or proteins, nucleic acid molecules, nucleic acid constructs, and / or cells comprising the same, or pharmaceuticals comprising them, can be used in combination for the prevention or slowing of progression of visual impairment. In one embodiment, when the above-mentioned uses are used in combination, the same active ingredient can be used for the combined use, or different active ingredients can be used in combination.

[0105] (General Techniques) The molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described, for example, 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), the relevant portions of which (possibly in their entirety) are incorporated herein by reference.

[0106] In this specification, "or" is used when "at least one or more" of the items listed in the sentence can be employed. The same applies to "alternative." In this specification, when it is specified that "within a range" of "two values," the range also includes the two values ​​themselves.

[0107] All references cited herein, including scientific literature, patents, patent applications, and the like, are incorporated by reference in their entirety to the same extent as if each were specifically set forth.

[0108] The present disclosure has been described above by showing preferred embodiments for ease of understanding. The present disclosure will be described below based on examples. However, the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present disclosure. Therefore, the scope of the present disclosure is not limited to the embodiments or examples specifically described herein, but is limited only by the scope of the claims.

[0109] Examples are described below. Where necessary, the handling of animals used in the following examples complied with the standards established by the applicant's institution and other relevant ethical standards and guidelines, and was carried out in accordance with the Declaration of Helsinki. While the reagents used were specifically those listed in the examples, equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Nakarai, R&D Systems, USCN Life Science INC, etc.) can also be used.

[0110] (Experimental Procedures) In the following examples, each experimental procedure was carried out as follows.

[0111] (Construction of bovine rhodopsin mutants) Mutant bovine rhodopsin cDNA (accession number: AB062417) was constructed using the In-Fusion cloning kit (Clontech). Wild-type (Gene ID: 509933) and mutant bovine rhodopsin cDNAs were inserted into the mammalian expression vector pUSRα (Kayada et al., 1995) or pCAGGS (Niwa et al., 1991). HEK293T cells were authenticated by short tandem repeat profiling. The cells were negative for mycoplasma contamination. The plasmid was transfected into HEK293T cells using 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). 11-cis or all-trans retinal was added to reconstitute the photopigment. The cells were solubilized in Buffer A containing 1% dodecyl maltoside (DDM) and purified by adsorption onto a Rho1D4 (anti-bovine rhodopsin monoclonal antibody) affinity column. After washing the column with Buffer A containing 0.02% DDM, the dye was eluted by adding a synthetic peptide containing the epitope sequence. To purify rhodopsin apoprotein, transfected cell membranes without added retinal were solubilized in Buffer A containing 1% DDM and adsorbed onto a Rho1D4 affinity column.

[0112] (Spectroscopic Measurements) UV-visible absorption spectra were recorded using a UV-visible spectrophotometer (UV-2450, UV-2400, Shimadzu Corporation). To analyze the thermal response of the pigments in detail, samples were maintained at 0°C, 20°C, or 37°C using a cell holder equipped with a temperature-controlled circulating water bath. The samples were irradiated with either yellow light from a 1kW tungsten halogen lamp (Master HILUX-HR; RIKEN) filtered through a Y-52 cutoff filter (Toshiba) or ultraviolet light filtered through a UV D-36 glass filter (AGC Technoglass).

[0113] To monitor the photocycle of the G188C mutant of bovine rhodopsin, a time-resolved CCD spectrophotometer (C10000 system, Hamamatsu Photonics) was used (Sakai et al.). Spectra were acquired from the G188C mutant sample in the dark and at different time points after irradiation (170 μs, yellow light from a xenon flash lamp passed through a Y-52 cutoff filter). The temperature of the sample was maintained at 37°C using a temperature controller (pqod, QUANTUM Northwest). The change in absorbance at λmax was plotted as a function of time and fitted with a monoexponential function to determine the time constant for recovery to the original dark state.

[0114] (Retinal isomer analysis) Retinal isomers in the rhodopsin samples were analyzed by high-performance liquid chromatography (LC-10ATvp; Shimadzu) using a silica column (YMC-Pack SIL, particle size 3 μm, 150 × 6.0 mm, YMC) as previously described (Tsutsui et al., 2007).

[0115] (G Protein Activation Assay) Activation of Gi-type G proteins was measured by GDP / GTPγS exchange of G proteins using a radionucleotide filter 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) with Gtβγ purified from bovine retina (Tachibanaki et al., 1997). All assay procedures were performed at 0°C. The assay mixture consisted of 10 nM dye, 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. Purified bovine rhodopsin wild-type and G188C mutants reconstituted with 11-cis retinal were mixed with the G protein solution and either stored in the dark or irradiated with yellow light (>500 nm) for 1 minute, followed by UV light for 1 minute, or re-irradiated with yellow light for 1 minute. After irradiation, the [35S]GTPγS solution was added to the rhodopsin and G protein mixture to initiate the GDP / GTPγS exchange reaction. After incubation in the dark for selected times, aliquots (20 μl) were removed from the samples and added to 200 μl of stop solution (20 mM Tris / Cl [pH 7.4], 100 mM NaCl, 25 mM MgCl, 1 μM GTPγS, 2 μM GDP) and immediately filtered through a nitrocellulose membrane to trap [35S]GTPγS bound to the G protein. The amount of bound [35S]GTPγS was quantified by measuring the membranes in a liquid scintillation counter (Tri-Carb 2910 TR; PerkinElmer).

[0116] cAMP levels in cultured cells were measured using the GloSensor cAMP assay (Promega) according to the manufacturer's instructions and a previous report (Bailes and Lucas, 2013). HEK293T cells 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 24 hours of culture, the cells were transfected with 50 ng of rhodopsin plasmid and 50 ng of Glosensor 22F plasmid per well using the polyethylenimine transfection method. After overnight incubation, the cells were replaced with equilibration medium containing a 2% dilution of GloSensor cAMP reagent stock solution, 10% FBS, and 5 μM retinal in CO2-independent medium (Thermo Fisher Scientific). After equilibration at room temperature for 2 hours, luminescence from the cells was measured using a microplate reader (SpectraMax L, Molecular Devices). For measurements of Gi activation by wild-type and mutant rhodopsins, cells were first treated with 2 μM forskolin to increase cAMP-dependent luminescence to a plateau level, followed by a 30-second stimulation with yellow light from a 1 kW tungsten halogen lamp through a Y-52 cutoff filter.

[0117] Example 1: Acquisition of photocycling properties in the G188C mutant of bovine rhodopsin To analyze whether the G188C mutant of bovine rhodopsin has acquired photocycling properties, the G188C mutant was purified after reconstitution with 11-cis retinal. Figure 1 shows the thermal stability of wild-type (Figure 1A), G188C (Figure 1B), and G188C / N2C / D282C (Figure 1C) mutants of bovine rhodopsin purified after incubation with 11-cis retinal. Absorption spectra were recorded after incubation in the dark at 37°C for 0, 5, 10, 15, and 20 minutes (curves 1 to 5, respectively). Figure 1D shows a schematic diagram of the retinal conformational change in wild-type bovine rhodopsin. The dark state, meta II, and meta III contain 11-cis-15-antiretinal, all-trans-15-antiretinal, and all-trans-15-synretinal, respectively. Figures 1E and 1F show the absorption spectra of purified bovine rhodopsin N2C / D282C (Fig. 1E) and G188C / N2C / D282C (Fig. 1F) mutants after incubation with 11-cis retinal. Spectra were recorded at 20°C in the dark (curve 1) and at 0, 5, 15, 30, 60, and 120 minutes after yellow light irradiation (curves 2–7, respectively). Insets show difference spectra (curves 1–5, respectively), obtained by subtracting the spectrum immediately after irradiation (curve 2 in E and F) from the spectrum measured after irradiation (curves 3–7 in E and F). Figure 1G shows the absorption spectrum of the G188C / N2C / D282C mutant measured at 37°C. Spectra were recorded in the dark (curve 1) and at 0.1, 10, 50, 100, and 1000 seconds after yellow light irradiation (curves 2–6, respectively). The insets show difference spectra (curves 1–4, respectively) obtained by subtracting the spectrum immediately after irradiation (curve 2 in G) from the spectrum measured after irradiation (curves 3–6 in G). Figure 1H shows the isomeric composition of retinal in the G188C / N2C / D282C mutant. Chromophore was extracted from samples before light irradiation and at 0, 5, and 60 minutes after yellow light irradiation at 20°C, and the retinal composition was analyzed by high-performance liquid chromatography (HPLC).

[0118] The G188C mutant was found to have significantly lower thermal stability than the wild-type. Specifically, the G188C mutant gradually decayed during incubation in the dark at 37°C (Fig. 1B), whereas the wild-type was extremely stable under the same conditions (Fig. 1A). Therefore, we improved the thermal stability of the G188C mutant and analyzed its detailed molecular characteristics. Following previous reports (Xie et al., 2003; Standfuss et al., 2007), we introduced two cysteine ​​residues (N2C / D282C) into the mutant and measured the thermal decay rate upon incubation at 37°C in the dark. The time-dependent spectral changes indicated that the G188C / N2C / D282C mutant decayed much more slowly than the G188C mutant (Fig. 1C). Therefore, we compared the spectral changes at 20°C between the wild-type, N2C / D282C, and G188C / N2C / D282C mutants. After irradiation with yellow light, the spectrum of the wild-type mutant shifted toward the ultraviolet region, suggesting the formation of a meta II intermediate containing all-trans-15-anti retinal (Fig. 1D). Subsequently, the absorbance around 470 nm increased, indicating a transition from meta II to a meta III intermediate containing all-trans-15-syn retinal (Fig. 1D). These spectral changes were also observed in the N2C / D282C mutant (Fig. 1E). On the other hand, the G188C / N2C / D282C mutant had an absorption maximum (λmax) at 487 nm, and its spectrum also shifted toward the ultraviolet region upon irradiation with yellow light, indicating the formation of meta II. After incubation in the dark, a decrease in absorbance in the ultraviolet region and a corresponding increase in absorbance around 485 nm were observed (Fig. 1F). Analysis of retinal configuration revealed that retinal isomerized to the all-trans form upon light exposure, which subsequently converted to the 11-cis form upon incubation in the dark (Figure 1H). This interconversion of retinal isomers could explain the spectral changes after light exposure in the G188C / N2C / D282C mutant. Thermal recovery of the G188C mutant to its original dark state after light exposure was also observed at 37°C (Figure 1G). Furthermore, the G188C mutant showed thermal recovery of its absorption spectrum to its original dark state after light exposure at 20°C, confirming that the amount of 11-cis retinal increased during incubation after light exposure.These results indicated that the G188C mutation led to the acquisition of the ability to thermally restore the original dark state from the light-activated state.

[0119] We then analyzed whether other G188 mutants had acquired photocycle properties. The G188E and G188R mutants of human rhodopsin are known to be unable to form photopigments after reconstitution with 11-cis retinal (Sung et al., 1993). Therefore, we introduced 16 other mutations at position 188 of bovine rhodopsin and prepared purified mutant proteins after reconstitution with 11-cis retinal. We successfully detected photopigments in eight of these mutants. The λmax of the mutants was blue-shifted from the wild-type (500 nm), except for G188D (509 nm) (Table 3).

[0120]

[0121] Irradiation of these mutants with yellow light shifted their spectra to the ultraviolet region, resulting in the formation of Meta II. When subsequently cultured in the dark at 20°C, each mutant exhibited characteristic spectral changes. However, these spectral changes were distinct from the significant increase in absorbance around their λmax. These results indicated that thermal recovery to the original dark state after light irradiation was not clearly detected in these mutants. Therefore, we concluded that the photocycle characteristics were specifically observed in the G188C mutant.

[0122] (Example 2: Acquisition of Photoreversible Properties of Bovine Rhodopsin G188C Mutant) We further analyzed whether the meta II of the G188C mutant reverts to its original dark state in a light-dependent manner. Figure 2 shows the light response, retinal positioning, and G protein activation of the bovine rhodopsin G188C mutant. Figures 2A and 2B show the absorption spectra of purified wild-type (Fig. 2A) or G188C mutant (Fig. 2B) bovine rhodopsin after incubation with 11-cis retinal at 0°C. 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), and after re-irradiation with yellow light (curve 4). The insets show the spectral changes of the wild-type (Fig. 2A) or G188C mutant (Fig. 2B) upon irradiation with yellow light (curve 1), subsequent irradiation with ultraviolet light (curve 2), and subsequent re-irradiation with yellow light (curve 3). Difference spectra were calculated from the spectra shown in Figures 2A and 2B. Figures 2C and 2D show the isomeric composition of retinal in wild-type (Figure 2C) and G188C mutant (Figure 2D). Chromophores were extracted from samples before light exposure, after yellow light exposure, after subsequent UV exposure, and after re-exposure to yellow light at 0°C, and the retinal composition was analyzed by high-performance liquid chromatography (HPLC). Figure 2E shows the Gi form of G protein activation activity in the wild-type. Activation activity was measured in the dark (closed circles) and after exposure to yellow light (open circles). Figure 2F shows the Gi form of G protein activation activity in the G188C mutant. Activation activity was measured in the dark (closed circles), after exposure to yellow light (open circles), after subsequent UV exposure (open triangles), and after re-exposure to yellow light (open diamonds). Data shown in Figures 2E and 2F were obtained at 0°C and are presented as the mean ± SEM of three independent experiments. Figure 2G shows the absorption spectrum (0°C) of the purified G188C / N2C / D282C mutant 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 UV irradiation (360 nm) (curve 3), after re-irradiation with yellow light (curve 4), and after re-irradiation with UV (curve 5). The insets show the spectral changes resulting from irradiation with yellow light (curve 1), subsequent UV irradiation (curve 2), subsequent UV irradiation (curve 3), and subsequent UV irradiation (curve 4). Difference spectra were calculated based on the spectra shown in Figure 2G.

[0123] The wild-type and G188C mutant were cooled to 0°C to prevent the thermal reaction of meta II, and the spectral changes induced by yellow light and subsequent UV irradiation were measured. Irradiation of the wild-type with yellow light produced meta II, and subsequent UV irradiation shifted the spectrum into the visible region, with a blue shift in λmax (∼470 nm) from that of the original dark state (Fig. 2A). Previous studies have shown that this state is equivalent to meta III (Fig. 1D). We also constructed template absorption spectra for the dark state, meta II, and meta III, modeled using conventional methods (Lamb, 1995; Govardovskii et al., 2000), and fitted the difference spectrum by subtracting the spectrum after UV irradiation from that after yellow light irradiation. The fitting results indicated that UV irradiation of meta II produced meta III much more efficiently than the original dark state (Table 3). This spectral analysis is consistent with the observation that UV irradiation produces a limited amount of 11-cis retinal from the abundant all-trans retinal (Fig. 2C). These results confirm that UV irradiation of Meta II induces syn / anti isomerization of the C=N double bond of the Schiff base more efficiently than cis / trans isomerization of retinal.

[0124] Irradiation of the G188C mutant with yellow light produced meta II, whose spectrum shifted to the visible region upon subsequent UV irradiation, with a λmax nearly identical to that of the original dark state (Fig. 2B). Re-irradiation with yellow light produced a state whose spectrum nearly overlapped with that induced by the initial yellow light irradiation (curve 4 in Fig. 2B). The spectral changes induced by UV irradiation and re-irradiation with yellow light were mirror images of each other (curves 2 and 3 in the inset of Fig. 2B). Fitting of the UV-dependent spectral changes to the template spectrum demonstrated that UV irradiation of meta II produced the original dark state much more efficiently than meta III (Table 3). This was supported by the observation that UV irradiation of the G188C mutant increased 11-cis retinal more efficiently than UV irradiation of the wild-type (Fig. 2D). These results suggest that meta II in the G188C mutant can be efficiently photoconverted back to the original dark state. Next, because bovine rhodopsin can activate not only transducin but also Gi / Go-type G proteins (Yamashita et al., 2000; Terakita et al., 2002), we measured the ability of the G188C mutant to activate Gi-type G proteins. GTPγS binding assays revealed that the light-dependent Gi activation ability of G188C was comparable to that of the wild-type (Fig. 2E,F). Subsequently, UV irradiation of the G188C mutant suppressed the activity, whereas re-irradiation with yellow light increased the activity (Fig. 2F). This can be explained by changes in the absorption spectrum and retinal isomers (Fig. 2B,D). Furthermore, the G188C / N2C / D282C mutant was also able to interconvert between the original dark state and meta II upon exposure to yellow and UV light at 0°C (Fig. 2G). These data demonstrate that the G188C mutant has acquired photoreversibility between the dark state and meta II.

[0125] We also analyzed the photoresponse of eight other mutants. In all of these mutants, the spectrum shifted to the ultraviolet region upon exposure to yellow light, and subsequent exposure to UV light increased the absorbance in the visible region again. By fitting the difference spectra calculated before and after UV exposure to the dark state, meta II, and meta III spectra as templates, we obtained information on the ratios of the dark state, meta II, and meta III components after UV exposure (Table 3). These results indicate that UV-induced recovery to the original dark state occurs most efficiently in the G188C mutant.

[0126] (Example 3: Acceleration of Photocycling in the G188C Mutant) Next, we investigated whether altering the lifetime of meta II alters the photocycling rate of the G188C mutant. Figure 3 shows the acceleration of the recovery rate of the photocycling properties of the G188C mutant of bovine rhodopsin by the introduction of the E122Q mutation. Figure 3A shows the absorption spectrum of the E122Q / G188C / N2C / D282C mutant measured at 0°C. The 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 insets show the difference spectra (curves 1-3, respectively) obtained by subtracting the spectrum before irradiation (curve 1 in Figure 3A) from the spectrum measured after irradiation (curves 2-4 in Figure 3A). Figure 3B shows the isomeric composition of retinal in the E122Q / G188C / N2C / D282C mutant. Chromophore extraction was performed on samples taken before light exposure and at 0, 5, and 60 minutes after yellow light exposure, and the retinal composition was analyzed by high-performance liquid chromatography (HPLC). Figure 3C shows the absorbance spectra of the E122Q / G188C / N2C / D282C mutant measured at 37°C. Spectra were recorded in the dark (curve 1) and at 0.1, 1, 5, 10, and 50 seconds after yellow flash exposure (curves 2–6, respectively). The insets show the difference spectra (curves 1–5, respectively) obtained by subtracting the pre-exposure spectrum (curve 1 in Figure 3C) from the post-exposure spectrum (curves 2–6 in Figure 3C). Figure 3D is a graph comparing the thermal recovery processes of G188C / N2C / D282C and E122Q / G188C / N2C / D282C. The differential absorbance at λmax, obtained by subtracting the pre-irradiation spectrum from the post-irradiation spectrum (Fig. 1G and Fig. 3C), was plotted against the time elapsed after irradiation. The time constants for thermal recovery to the dark state at 37°C for the G188C / N2C / D282C and E122Q / G188C / N2C / D282C mutants were 57.4 and 5.1 seconds, respectively.

[0127] The E122Q mutation in vertebrate rhodopsin has been reported to accelerate meta II decay and shorten its lifetime (Imai et al., 1997; Imai et al., 2007). Therefore, we constructed an E122Q / G188C / N2C / D282C mutant and measured its spectral changes after light exposure. Spectral and retinal isomer analyses confirmed that the E122Q / G188C / N2C / D282C mutant thermally recovered to its original dark state after light exposure at 0°C (Fig. 3A, B). Furthermore, the photocycling rate of the E122Q / G188C / N2C / D282C mutant at 37°C (Fig. 3C) was approximately 12-fold faster than that of the G188C / N2C / D282C mutant (Fig. 3D). Thus, by changing the lifetime of meta II with a single mutation, we succeeded in speeding up the photocycling reaction of the G188C mutant.

[0128] (Example 4: Changes in G Protein Activation Ability Due to Photocycling Properties) We further investigated whether the acquisition of photocycling properties by the G188C mutation affects G protein activation ability. Figure 4 shows the light-induced suppression of intracellular cAMP levels by bovine rhodopsin mutants. cAMP levels in HEK293T cells transfected with N2C / D282C- (Figures 4A and 4B), G188C / N2C / D282C- (Figures 4C and 4D), E122Q / G188C / N2C / D282C- (Figures 4E and 4F), and mock (Figure 4G) were measured at room temperature using the GloSensor cAMP assay. 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). Data were normalized to the maximum point before light irradiation. Detailed profiles of light-dependent changes in cAMP levels in N2C / D282C, G188C / N2C / D282C, and E122Q / G188C / N2C / D282C are shown in Figures 4B, 4D, and 4F, respectively.

[0129] As shown in Figure 2, light-dependent Gi activation was comparable between the wild-type and G188C mutant at 0°C, and significant thermal recovery to the original dark state was not observed in the G188C mutant. Therefore, we measured intracellular cAMP levels in cultured cells using a cAMP biosensor (GloSensor) and compared the changes in biosensor luminescence triggered by bovine rhodopsin. The increase in cAMP levels induced by the addition of forskolin was attenuated by yellow light exposure in N2C / D282C bovine rhodopsin-transfected cells (Figure 4A, B), but not in mock-transfected cells (Figure 4G), showing a gradual recovery thereafter. In contrast, the G188C / N2C / D282C mutant-transfected cells rapidly recovered after the decrease in cAMP levels induced by yellow light exposure (Figure 4C, D). Furthermore, we confirmed that E122Q / G188C / N2C / D282C mutant-transfected cells recovered more rapidly from the decrease in cAMP levels induced by yellow light irradiation (Fig. 4E, F). These results suggest that the acquisition of photocyclic properties by the G188C mutation promotes a rapid recovery to the original dark state and alters the G protein activation profile.

[0130] (Example 5: Pigment formation by the G188C mutant upon addition of all-trans retinal) We analyzed whether the G188C mutant forms a photopigment after reconstitution with all-trans retinal. Figure 5 shows the photopigment formation of the G188C mutant of bovine rhodopsin after incubation with all-trans retinal. Absorption spectra of purified wild-type (Figure 5A) or G188C mutant (Figure 5B) were shown after addition of all-trans retinal to a suspension of rhodopsin-expressing cell membranes at 0°C. 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 insets show the spectral changes upon irradiation with yellow light (curve 1), subsequent irradiation with ultraviolet light (curve 2), and subsequent re-irradiation with yellow light (curve 3). Figure 5C shows the isomeric composition of retinal in purified G188C mutants after the addition of all-trans retinal. The chromophore was extracted from samples after yellow light irradiation, subsequent UV irradiation, and re-irradiation with yellow light, and the retinal composition was analyzed by high-performance liquid chromatography (HPLC). The regeneration of photopigment upon addition of 11-cis retinal (Figures 5D and E) or all-trans retinal (Figures 5F and G) to purified N2C / D282C apoprotein is shown. The spectra in Figure 5D were measured before addition (curve 1) and at 0, 3, 6, 15, 30, 60, and 120 min (curves 2–8) after the addition of 1.1 μM 11-cis retinal. Figure 5E shows the difference spectra (curves 1–6, respectively) calculated by subtracting the spectra measured 3, 6, 15, 30, 60, and 120 minutes after the addition of 11-cis retinal (curves 3–8 in Figure 5D) from the spectrum measured immediately after the addition of 11-cis retinal (curve 2 in Figure 5D). Figure 5F shows the spectra measured before the addition of 1.1 μM all-trans retinal (curve 1) and 0, 0.5, 1, 2, 6, 12, and 16 hours after the addition (curves 2–8). Figure 5G shows the difference spectra (curves 1–6, respectively) calculated by subtracting the spectrum measured immediately after the addition of all-trans retinal (curve 2 in Figure 5F) from the spectra measured 0.5, 1, 2, 6, 12, and 16 hours after the addition of all-trans retinal (curves 3–8 in Figure 5F).The regeneration of photopigment after addition of 11-cis retinal (Figures 5H and 5I) or all-trans retinal (Figures 5J and 5K) to purified G188C / N2C / D282C apoprotein is shown. The spectra in Figure 5H were measured before addition (curve 1) and at 0, 3, 6, 15, 30, 60, and 120 min (curves 2–8) after addition of 1.1 μM 11-cis retinal. Figure 5I shows the difference spectra (curves 1–6, respectively) calculated by subtracting the spectra measured 3, 6, 15, 30, 60, and 120 min after addition of 11-cis retinal (curves 3–8 in Figure 5H) from the spectrum measured immediately after addition of 11-cis retinal (curve 2 in Figure 5H). Figure 5J shows the spectra measured before (curve 1) and at 0, 0.5, 1, 2, 6, 12, and 16 h after the addition of 1.1 μM all-trans retinal (curves 2–8). The difference spectra in Figure 5K were calculated by subtracting the spectrum measured immediately after the addition of all-trans retinal (curve 2 in Figure 5J) from the spectra measured 0.5, 1, 2, 6, 12, and 16 h after the addition of all-trans retinal (curves 3–8 in Figure 5J) (curves 1–6, respectively). Figure 5L shows the regeneration of the N2C / D282C and G188C / N2C / D282C photopigments by the addition of all-trans retinal, as shown in Figures 5F and 5J, as monitored by the change in absorbance at 500 nm.

[0131] After adding all-trans retinal to a suspension of rhodopsin-expressing cell membranes, wild-type and G188C mutant rhodopsin were purified. The absorption spectrum of the wild-type rhodopsin showed almost no peaks in the visible and near-UV regions (Fig. 5A). In contrast, the absorption spectrum of the G188C mutant showed a peak in the visible region (Fig. 5B, curve 1), which was attributed to the predominant incorporation of 11-cis and 9-cis retinal rather than all-trans retinal (Fig. 5C). Irradiation of this pigment with yellow light converted retinal to the all-trans form, shifting the spectrum to the UV region. Subsequent UV light irradiation at 0°C caused retinal isomerization from the all-trans form to the 11-cis form, again increasing the absorbance in the visible region (Fig. 5B, C). This was very similar to the findings of the G188C mutant purified after reconstitution with 11-cis retinal (Fig. 2B, D).

[0132] We also prepared purified apoproteins of N2C / D282C and G188C / N2C / D282C and investigated the regeneration of photopigments by the addition of 11-cis or all-trans retinal. Addition of 11-cis retinal to N2C / D282C and G188C / N2C / D282C immediately increased the absorbance at around 505 nm (Fig. 5D, E) and 490 nm (Fig. 5H, I), respectively, indicating the formation of an 11-cis retinal-bound dark state. Addition of all-trans retinal to N2C / D282C slightly increased the absorbance at around 480 nm (Fig. 5F, G), whereas addition of all-trans retinal to G188C / N2C / D282C significantly increased the absorbance at around 485 nm (Fig. 5J, K). Furthermore, the regeneration ability of G188C / N2C / D282C upon addition of all-trans retinal was much higher than that of N2C / D282C (Fig. 5L). These results indicate that the G188C mutant can independently form photopigments upon addition of not only 11-cis retinal but also all-trans retinal.

[0133] (Example 6: Chimeric Opsins) As in the above Examples, the G188C and E122Q / G188C mutants of bovine rhodopsin N2C / D282C were further modified by substituting the intracellular second and third loops (amino acids 140-152 and 225-251, respectively, of Gene ID: 509933) with those of the mouse histamine H2 receptor (amino acids 121-134 and 203-232, respectively, of Gene ID: 15466). These chimeric opsins were expressed in human-derived cultured HEK293 cells. Modified luciferase (Promega, GloSensor) was co-expressed as a cAMP probe, so that luminescence increased with increasing cAMP concentrations. The cultured cells were irradiated with yellow light, and the luminescence derived from the modified luciferase was compared. Compared with bovine rhodopsin N2C / D282C (Fig. 6A) in which the intracellular loops 2 and 3 were substituted, the G188C mutation (Fig. 6B) and the E122Q / G188C mutation (Fig. 6C) showed a transient increase in luminescence followed by a rapid decrease in luminescence. These results indicate that the introduction of the G188C and E122Q / G188C mutations allows the intracellular cAMP concentration to be altered so that it can be transiently increased by light and then restored to its original state.

[0134] (Example 7: Acquisition of photocycle properties in Xenopus tropicalis Opn5m T188C mutant) Xenopus tropicalis Opn5m binds 11-cis retinal, and its absorption spectrum showed an absorption maximum at 360 nm. When it received ultraviolet light, retinal isomerized to the all-trans form, and the absorption maximum of the absorption spectrum shifted to 474 nm. Furthermore, when it received visible light, retinal isomerized to the 11-cis form, returning the absorption spectrum to the absorption maximum at 360 nm. In other words, Opn5m was a bistable opsin (Yamashita et al., 2014). Because Xenopus tropicalis Opn5m has threonine at position 188, a T188C mutant was generated. This T188C mutant bound only all-trans retinal, and its absorption spectrum showed an absorption maximum at 470 nm. Upon exposure to visible light, the absorption at 470 nm initially decreased at both 20°C (Fig. 7a) and 37°C (Fig. 7b, 7c), then recovered over time. Furthermore, during this process, retinal converted from the all-trans form to the 11-cis or 13-cis form upon exposure to visible light, then returned to the all-trans form over time (Fig. 7d). These results indicate that the Xenopus tropicalis Opn5mT188C mutant spontaneously reverts to its original form after photoreception, suggesting that it has acquired photocycling properties.

[0135] Example 8: Human Rhodopsin. Similar to the example using bovine rhodopsin, the G188C and E122Q / G188C mutants of human rhodopsin N2C / N282C were expressed in human-derived cultured HEK293 cells. Modified luciferase (Promega, GloSensor) was co-expressed as a cAMP probe, so that luminescence increased with increasing cAMP concentrations. The cultured cells were irradiated with yellow light, and the luminescence derived from the modified luciferase was compared. Compared to human rhodopsin N2C / N282C (Figure 8A), the G188C mutant (Figure 8B) and the E122Q / G188C mutant (Figure 8C) showed a transient decrease in luminescence upon light irradiation, followed by a rapid recovery. These results demonstrate that the introduction of the G188C and E122Q / G188C mutations can modify intracellular cAMP concentrations to decrease in a short time when exposed to light. Furthermore, for the N2C / N282C and G188C / N2C / N282C human rhodopsin mutants, the intracellular second and third loops (amino acids 140-152 and 225-251, respectively, of SEQ ID NO: 1) were further substituted with those of the human histamine H2 receptor (amino acids 121-134 and 204-233, respectively, of Gene ID: 3274). These chimeric opsins were expressed in human-derived cultured HEK293 cells. A modified luciferase (Promega, GloSensor) was co-expressed as a cAMP probe, allowing for increased luminescence at higher cAMP concentrations. The cultured cells were irradiated with yellow light, and the luminescence derived from the modified luciferase was compared. Compared with human rhodopsin N2C / N282C (Fig. 8D) in which the intracellular loops 2 and 3 were substituted, the one with the G188C mutation (Fig. 8E) showed a transient increase in luminescence upon light irradiation, followed by a rapid decrease. This result indicates that the introduction of the G188C mutation can modify the intracellular cAMP concentration so that it can be increased for a short time by light and then restored to its original state.

[0136] Example 9: Canine Rhodopsin. Similar to the example using bovine rhodopsin, the G188C mutant of canine (Canis familiaris) rhodopsin N2C / D282C was expressed in human-derived cultured cells HEK293. Modified luciferase (Promega, GloSensor) was co-expressed as a cAMP probe, so that luminescence increased with increasing cAMP concentrations. The cultured cells were irradiated with yellow light, and the luminescence derived from the modified luciferase was compared. Compared to canine rhodopsin N2C / D282C (FIG. 10A), the G188C mutation-introduced cells (FIG. 10B) exhibited a transient decrease in luminescence upon light irradiation, which then quickly recovered. These results demonstrate that the introduction of the G188C mutation can modify the intracellular cAMP concentration to be reduced by light for a short period of time. Furthermore, for the canorhodopsin N2C / D282C and G188C / N2C / D282C mutants, the intracellular second and third loops were further substituted with those of the human histamine H2 receptor, resulting in chimeric opsins expressed in human-derived cultured cells HEK293. Modified luciferase (Promega, GloSensor) was co-expressed as a cAMP probe, resulting in increased luminescence at high cAMP concentrations. These cultured cells were irradiated with yellow light, and the luminescence derived from the modified luciferase was compared. Compared to canorhodopsin N2C / D282C (Figure 10C) with the substituted intracellular second and third loops, the G188C mutation (Figure 10D) transiently increased luminescence upon light irradiation, followed by a rapid decrease. These results demonstrate that the introduction of the G188C mutation can modify intracellular cAMP concentrations so that they can be briefly increased by light and then restored to their original state.

[0137] Example 10: Medaka rhodopsin. Medaka (Oryzias latipes) rhodopsin N2C / E282C was expressed in human-derived cultured cells HEK293, similar to the example using bovine rhodopsin. Modified luciferase (Promega, GloSensor) was co-expressed as a cAMP probe, so that luminescence increased with increasing cAMP concentrations. These cultured cells were irradiated with yellow light, and the luminescence derived from the modified luciferase was compared. Compared to medaka rhodopsin N2C / E282C (FIG. 11A), the G188C mutation-introduced rhodopsin (FIG. 11B) exhibited a transient decrease in luminescence upon light irradiation, which then quickly recovered. These results demonstrate that the introduction of the G188C mutation can modify the intracellular cAMP concentration to be reduced by light for a short period of time.

[0138] (Example 11: Additional Mutations Other Than Positions 188 and 122) When aligned with SEQ ID NO: 1, conservative amino acid substitutions were made in the N-terminal domain (positions 1 to 34) and the C-terminal domain (positions 308 and beyond). Specifically, a G6A mutation or a V337A mutation was introduced into human rhodopsin G188C / N2C / D282C mutant, and the mutant was expressed in human-derived cultured cells HEK293. In this case, modified luciferase (Promega, GloSensor) was co-expressed as a cAMP probe, so that luminescence increased with increasing cAMP concentrations. 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 12A), the human rhodopsin G6A / G188C / N2C / D282C mutant (Figure 12B) and the human rhodopsin V337A / G188C / N2C / D282C mutant (Figure 12C) showed a transient decrease in luminescence upon light irradiation, followed by a rapid recovery. These results indicate that these three mutants reduce intracellular cAMP concentrations in a short period of time by light. This indicates that, when aligned with SEQ ID NO: 1, conservative amino acid substitutions in the N-terminal domain (positions 1 to 34) and the C-terminal domain (positions 308 and beyond) retain the ability to reduce intracellular cAMP concentrations in a short period of time by light.

[0139] Example 12: Introduction of cysteine ​​mutations other than those at positions 2 and 282. When aligned with SEQ ID NO: 1, amino acids belonging to part of the N-terminal domain (positions 1 to 11) and part of the extracellular loop 3 (positions 278 to 285) were mutated to cysteine ​​in combinations other than those corresponding to positions 2 and 282. Specifically, N2C / G3C / G280C mutations, N2C / G3C / S281C mutations, or G3C / N282C mutations were introduced into human rhodopsin G188C mutant, and the mutant was expressed in human-derived cultured cells HEK293. Modified luciferase (Promega, GloSensor) was co-expressed as a cAMP probe, so that luminescence increased with increasing cAMP concentrations. 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 13A), the human rhodopsin G188C / N2C / G3C / G280C mutant (Figure 13B), the human rhodopsin G188C / N2C / G3C / S281C mutant (Figure 13C), and the human rhodopsin G188C / G3C / N282C mutant (Figure 13D) showed a transient decrease in luminescence upon light irradiation, followed by a rapid recovery. These results indicate that these four mutants reduce the intracellular cAMP concentration in a short period of time by light. In other words, the introduction of cysteines other than those at positions 2 and 282 into the amino acids corresponding to part of the N-terminal domain (positions 1 to 11) and part of the extracellular loop 3 (positions 278 to 285) when aligned with SEQ ID NO: 1 still retains the ability to reduce the intracellular cAMP concentration in a short period of time by light.

[0140] Example 13: Restoration of light-evoked activity from the retina by the G188C mutant. A viral vector (rAAV-DJ) containing the G188C mutant coding sequence under the control of a CMV promoter was injected into the vitreous of 10-week-old rd1 mice (a mouse model of retinitis pigmentosa blindness). The AAV-DJ vector was adopted for more efficient and widespread gene transfer, with AAV-2 used as a benchmark, as it has already been clinically applied. Retinas were harvested one month later. Expression of the reporter gene (EGFP) was observed throughout the retina and in both the ganglion cell layer (GCL) and inner nuclear layer (INL). To evaluate the function of the ectopically induced G188C mutant in the mouse retina, a multielectrode array (MEA) experiment, which can record extracellular potentials from RGCs, was performed. The experiment was performed as shown in Figure 14. Figure 14A shows an untreated rd1 mouse. Figure 14B shows the results when the human rhodopsin G188C / N2C / D282C mutant was introduced using a viral vector, Figure 14C shows the results when the human rhodopsin G188C / N2C / D282C mutant in which the intracellular loops 2 and 3 were substituted with those of the human histamine H2 receptor was introduced, and Figure 14D shows the results when the human rhodopsin E122Q / G188C / N2C / D282C mutant in which the intracellular loops 2 and 3 were substituted with those of the human histamine H2 receptor. These mutants are the same as those described in Example 8, etc.

[0141] (Results) The results are shown in Figure 14. In untreated rd1 mice, no light response was observed due to blindness (Figure 14A). However, when the human rhodopsin G188C / N2C / D282C mutant was introduced using a viral vector (Figure 14B), when a human rhodopsin G188C / N2C / D282C mutant in which the second and third intracellular loops were replaced with those of the human histamine H2 receptor was introduced (Figure 14C), or when the human rhodopsin E122Q / G188C / N2C / D282C mutant in which the second and third intracellular loops were replaced with those of the human histamine H2 receptor (Figure 14D), a light response was observed. These results indicate that the introduction of the G188C mutant can achieve visual regeneration. As a result of photoreceptor degeneration, control retinas without treatment showed no response from RGCs detected by MEA (Fig. 14A). In contrast, treated retinas repeatedly showed clear light-evoked responses (Fig. 14B, C, D).

[0142] Example 14: Evaluation of visual evoked potentials To investigate whether light reception in the retina is transmitted to the visual cortex, visual evoked potentials (VEPs) from the visual cortex are examined. In these experiments, rd1 mice treated in both eyes with the G188C mutant construct linked to AAV-DJ-CAGGS and a control EGFP virus (AAV-DJ-CAGGS-EGFP) are used. As a result, no VEP response is obtained in the control mice, but a significant VEP response is reproduced in mice administered the G188C mutant construct linked to AAV-DJ-CAGGS.

[0143] Example 15: Evaluation of light-dark recognition function A light-dark box locomotion test (LDT) was performed to investigate whether ectopic expression of the G188C mutant in the degenerated retina leads to behavioral changes consistent with visual recovery. Rodents are nocturnal and feel anxious in bright environments, so they tend to stay in dark places consistent with visual function, whereas blind animals spend approximately half their time in light and half in dark places. Mice treated with the G188C mutant significantly reduced the time spent in the light place compared to untreated rd1 mutant mice, indicating visual recovery in behavior.

[0144] (Example 16: Optogenetics <Neural Control>) The mediation of cAMP is essential for the induction of axon branching and elongation. The G188C mutant and a chimeric opsin in which the intracellular second and third loops are replaced with those of the human histamine H2 receptor as in Example 8 are expressed in mouse hippocampal neurons, and optical manipulation of axon branching and elongation of neurons is attempted. As a result, axon branching and elongation are suppressed in hippocampal neurons introduced with the G188C mutant and irradiated with blue light for 30 minutes compared to those not irradiated. On the other hand, axon branching and elongation are promoted in hippocampal neurons introduced with the G188C mutant chimeric opsin and irradiated with blue light for 30 minutes compared to those not irradiated.

[0145] (Example 17: Calcium Ion-Modified Chimeric Opsin) As in Example 6, a chimeric opsin was expressed in human-derived cultured cells HEK293, in which the G188C mutant bovine rhodopsin N2C / D282C was further modified by substituting the intracellular second and third loops with those of the human α1A-adrenergic receptor. In this case, aequorin derived from Aequorea victoria was co-expressed as a calcium ion probe, so that luminescence increased at high calcium ion concentrations. The cultured cells were irradiated with yellow light, and the luminescence derived from aequorin was compared. Compared to bovine rhodopsin N2C / D282C with substitutions in the intracellular second and third loops ( FIG. 15A ), the G188C mutation ( FIG. 15B ) transiently increased luminescence, which then immediately decreased. This result demonstrates that the introduction of the G188C mutation can modify the intracellular calcium ion concentration so that it can be increased only briefly by light and then restored to its original state.

[0146] (Note) As described above, the present disclosure has been illustrated using preferred embodiments thereof, but it is understood that the scope of the present disclosure should be construed solely by the scope of the claims. It is understood that the patents, patent applications, and other documents cited in this specification are incorporated by reference into this specification in their entirety as if the contents themselves were specifically set forth herein. This application claims priority to Japanese Patent Application No. 2022-138707, filed with the Japan Patent Office on August 31, 2022, the entire contents of which are incorporated herein by reference as necessary.

[0147] According to the present disclosure, the mechanisms of natural evolution that led to the development of photocycling properties in certain opsins can be utilized to artificially evolve other opsins to have photocycling properties, providing techniques that can be used to create various molecular tools and in industries (e.g., pharmaceuticals) that rely on such technologies.

[0148] SEQ ID NO: 1: Amino acid sequence of Homo sapiens rhodopsin NP_001372054.1 SEQ ID NO: 2: Amino acid sequence of Mus musculus rhodopsin NP_663358.1 SEQ ID NO: 3: Amino acid sequence of Canis familiaris rhodopsin CAA50502.1 SEQ ID NO: 4: Amino acid sequence of Gallus gallus rhodopsin NP_001384426.1 SEQ ID NO: 5: Amino acid sequence of Oryzias latipes rhodopsin BAD99136.1 SEQ ID NO: 6: Amino acid sequence of Homo sapiens blue cone opsin NP_001372054.1 SEQ ID NO: 7: Amino acid sequence of Homo sapiens red cone opsin NP_064445.2 SEQ ID NO: 8: Amino acid sequence of Homo sapiens green cone opsin NP_000504.1 SEQ ID NO: 9: Mus SEQ ID NO: 10: Amino acid sequence of Mus musculus UV cone opsin AAG17989.1 SEQ ID NO: 10: Amino acid sequence of Mus musculus green cone opsin AAB64302.1 SEQ ID NO: 11: Amino acid sequence of Gallus gallus green cone opsin AAA48786.1 SEQ ID NO: 12: Amino acid sequence of Gallus gallus blue cone opsin AAA48633.1 SEQ ID NO: 13: Amino acid sequence of Gallus gallus violet cone opsin AAA49141.1 SEQ ID NO: 14: Amino acid sequence of Gallus gallus red cone opsin CAA40727.1 SEQ ID NO: 15: Amino acid sequence of Gallus gallus pinopsin AAA64223.1 SEQ ID NO: 16: Amino acid sequence of Homo sapiens Opn3 AAH36773.1 SEQ ID NO: 17: Homo sapiens Opn4 SEQ ID NO: 18: Amino acid sequence of AAI13559.1 Homo sapiens Opn5 SEQ ID NO: 19: Amino acid sequence of Homo sapiens Rgr AAA56748.1 SEQ ID NO: 20: Amino acid sequence of Homo sapiens Rrh AAC51757.SEQ ID NO: 21: Amino acid sequence of Mus musculus Opn3 AAD32670.1 SEQ ID NO: 22: Amino acid sequence of Mus musculus Opn4 AAF24979.1 SEQ ID NO: 23: Amino acid sequence of Mus musculus Opn5 AAR08201.1 SEQ ID NO: 24: Amino acid sequence of Mus musculus Rgr AAC69836.1 SEQ ID NO: 25: Amino acid sequence of Mus musculus Rrh AAC53344.1 SEQ ID NO: 26: Amino acid sequence of Gallus gallus VAL opsin ACX32474.1 SEQ ID NO: 27: Amino acid sequence of Gallus gallus Opn3 BAV92607.1 SEQ ID NO: 28: Amino acid sequence of Gallus gallus TMT opsin BAV93805.1 SEQ ID NO: 29: Amino acid sequence of Gallus gallus Opn4x ABX10830.1 SEQ ID NO: 30: Amino acid sequence of Gallus gallus Opn4m BAL14786.1 SEQ ID NO: 31: Amino acid sequence of Gallus gallus Opn5m BAG65738.1 SEQ ID NO: 32: Amino acid sequence of Gallus gallus Opn5L2 BAG65739.2 SEQ ID NO: 33: Amino acid sequence of Gallus gallus Rrh AAR02098.1 SEQ ID NO: 34: Amino acid sequence of Gallus gallus Rgr AAR02099.1 SEQ ID NO: 35: Nucleic acid sequence of Homo sapiens rhodopsin NM_000539.3 SEQ ID NO: 36: Nucleic acid sequence of Mus musculus rhodopsin NM_145383.2 SEQ ID NO: 37: Nucleic acid sequence of Canis familiaris rhodopsin X71380.1 SEQ ID NO: 38: Gallus gallus rhodopsin SEQ ID NO: 39: Nucleic acid sequence of Oryzias latipes rhodopsin AB180742.1 SEQ ID NO: 40: Nucleic acid sequence of Homo sapiens blue cone opsin NM_001385125.1 SEQ ID NO: 41: Homo sapiens red cone opsin NM_020061.6 nucleic acid sequence SEQ ID NO: 42: Nucleic acid sequence of Homo sapiens green cone opsin NM_000513.2 SEQ ID NO: 43: Nucleic acid sequence of Mus musculus UV cone opsin AF190670.1 SEQ ID NO: 44: Nucleic acid sequence of Mus musculus green cone opsin AF011389.1 SEQ ID NO: 45: Nucleic acid sequence of Gallus gallus green cone opsin M92038.1 SEQ ID NO: 46: Nucleic acid sequence of Gallus gallus blue cone opsin M92037.1 SEQ ID NO: 47: Nucleic acid sequence of Gallus gallus violet cone opsin M92039.1 SEQ ID NO: 48: Nucleic acid sequence of Gallus gallus red cone opsin X57490.1 SEQ ID NO: 49: Nucleic acid sequence of Gallus gallus pinopsin U15762.1 SEQ ID NO: 50: Homo SEQ ID NO: 51: Nucleic acid sequence of Homo sapiens Opn4 BC113558.1 SEQ ID NO: 52: Nucleic acid sequence of Homo sapiens Opn5 AY377391.1 SEQ ID NO: 53: Nucleic acid sequence of Homo sapiens Rgr U14910.1 SEQ ID NO: 54: Nucleic acid sequence of Homo sapiens Rrh AF012270.1 SEQ ID NO: 55: Nucleic acid sequence of Mus musculus Opn3 AF140241.1 SEQ ID NO: 56: Nucleic acid sequence of Mus musculus Opn4 AF147789.1 SEQ ID NO: 57: Nucleic acid sequence of Mus musculus Opn5 AY318865.1 SEQ ID NO: 58: Nucleic acid sequence of Mus musculus Rgr AF076930.1 SEQ ID NO: 59: Mus musculus Rrh AF012271.1 nucleic acid sequence SEQ ID NO: 60: Gallus gallus VAL opsin GQ280390.1 nucleic acid sequence SEQ ID NO: 61: Gallus gallus Opn3 AB436160.1 nucleic acid sequence SEQ ID NO: 62: Gallus gallus TMT opsin AB519059.1 nucleic acid sequence SEQ ID NO: 63: Gallus gallus Opn4x EU124630.SEQ ID NO: 64: Nucleic acid sequence of Gallus gallus Opn4m AB295599.1 SEQ ID NO: 65: Nucleic acid sequence of Gallus gallus Opn5m AB368182.1 SEQ ID NO: 66: Nucleic acid sequence of Gallus gallus Opn5L2 AB368183.3 SEQ ID NO: 67: Nucleic acid sequence of Gallus gallus Rrh AY339626.1 SEQ ID NO: 68: Nucleic acid sequence of Gallus gallus Rgr AY339627.1

Claims

1. A protein comprising an amino acid sequence of opsins, wherein the amino acid sequence of said opsins includes a modification of the amino acid corresponding to position 188 when aligned with SEQ ID NO:

1.

2. The protein according to claim 1, wherein the amino acid sequence of the opsins includes a modification of the amino acid corresponding to G at position 188 when aligned with SEQ ID NO:

1.

3. The protein according to claim 1, comprising the modification of the amino acid corresponding to position G188 in the aforementioned amino acid sequence to cysteine ​​when aligned with SEQ ID NO:

1.

4. The protein according to claim 1, wherein the protein is activated by light stimulation and then inactivated without releasing a photoreceptor factor.

5. The aforementioned amino acid sequence is, 1) An amino acid sequence in which the above modification is included in the amino acid sequence represented by any one of the sequence numbers 1 to 34, 2) The amino acid sequence other than the modified site has at least about 80% identity with the sequence in 1), and the encoded protein has substantially the same biological activity as the protein obtained from the sequence in 1), 3) An amino acid sequence having one or more mutations in addition to the modification site in the sequence of 1), and the encoded protein having substantially the same biological activity as the protein obtained from the sequence of 1), 4) In an amino acid sequence encoded by a nucleic acid that hybridizes with the nucleic acid encoding the sequence of 1), the amino acid sequence includes the modification, or 5) Amino acid sequences encoded by allele variants of nucleic acids encoding the sequence of 1), including the above modification, The protein according to claim 1, comprising:

6. Furthermore, the protein according to claim 1, comprising a modification of the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1 in the amino acid sequence of the opsins.

7. Furthermore, the protein according to claim 1, wherein the amino acid sequence of the opsins includes a modification of the amino acid corresponding to E at position 122 when aligned with SEQ ID NO:

1.

8. Furthermore, the protein according to claim 1, wherein the amino acid sequence of the opsins includes modification of the amino acid corresponding to position 122 when aligned with SEQ ID NO: 1 to glutamine.

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

10. Furthermore, the protein according to claim 1, wherein the amino acid sequence of the opsins includes modifications to amino acids that, when aligned with SEQ ID NO: 1, correspond to a portion of the N-terminal domain (positions 1 to 11) and a portion of the extracellular third loop (positions 278 to 285).

11. Furthermore, the protein according to claim 1, wherein the amino acid sequence of the opsins includes modification of amino acids to cysteine, which correspond to a portion of the N-terminal domain (positions 1 to 11) and a portion of the extracellular third loop (positions 278 to 285) when aligned with SEQ ID NO:

1.

12. Furthermore, the protein according to claim 1, wherein the amino acid sequence of the opsins includes modifications to the amino acids corresponding to positions 2 and 282 when aligned with SEQ ID NO:

1.

13. The protein according to claim 12, wherein the modifications to the amino acids corresponding to the 2nd and 282nd positions improve the thermal stability of the opsins.

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

15. The protein according to claim 1, wherein the opsins are chimeric opsins.

16. The protein according to claim 1, comprising the sequence of sequence number 1, 3, or 5.

17. A nucleic acid molecule comprising a nucleic acid encoding the amino acid sequence of the protein described in claim 1.

18. A nucleic acid construct comprising the nucleic acid molecule described in claim 17.

19. A cell comprising 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 pharmaceutical product comprising a protein according to any one of claims 1 to 16, a nucleic acid molecule according to claim 17, and / or a nucleic acid construct according to claim 18.

21. A pharmaceutical product comprising the cells described in Claim 19.

22. The pharmaceutical product according to claim 20, for the purpose of restoring vision, or for the prevention or treatment of visual impairment or disease.

23. A composition comprising opsins that inactivate photoreceptor factors without releasing them.

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

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