Preventing, and suppressing progression of, retinal disease, improving visual cognitive behavioral function, and strengthening visual function

Abstract

To provide prevention and suppression of progression of retinal diseases, improvement in visual cognitive behavioral function, and strengthening of visual function.SOLUTION: A chimeric protein of two types of rhodopsins, an ion-transporting rhodopsin and a G protein-coupled receptor rhodopsin, and a nucleic acid encoding the chimeric protein are provided for the prevention and suppression of progression of retinal diseases, improvement in visual cognitive behavioral functions (e.g., improvement in light-dark determination functions, improvement in bright spot evading functions, and / or crisis avoidance functions), and enhancement of visual functions (e.g., improvement in visual acuity). The present invention also provides a method for preventing or suppressing progression of a disease, disorder or symptom of the retina, for improving a visual cognitive behavioral function (e.g., improvement in light-dark determination functions, improvement in bright spot evading functions, and / or crisis avoidance functions), or for enhancing visual functions (e.g., improvement in visual acuity), in a subject. The method comprises the step of administering an effective amount of a nucleic acid encoding a chimeric protein of an ion-transporting receptor rhodopsin and a G protein-coupled receptor rhodopsin to the subject.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to the prevention and suppression of progression of retinal diseases, the improvement of visual cognitive behavioral functions, and the enhancement of visual functions.

Background Art

[0002] Rhodopsin is a photosensitive receptor having a seven-transmembrane structure in the retinas of humans and animals, and is also applied in medicine.

Summary of the Invention

Means for Solving the Problems

[0003] The present inventors have found that a chimeric protein of two types of rhodopsin, an ion-transporting rhodopsin and a G-protein-coupled receptor rhodopsin, has effects of preventing and suppressing the progression of retinal diseases, improving visual cognitive behavioral functions, and enhancing visual functions, and have completed the present invention.

[0004] The present invention provides, for example, the following. (Item 1) A composition for preventing or suppressing the progression of a retinal disease, disorder or symptom, comprising a nucleic acid encoding a chimeric protein of an ion-transporting receptor rhodopsin and a G-protein-coupled receptor rhodopsin. (Item 2) A composition for improving visual cognitive behavioral functions (for example, improving the light-dark discrimination function, improving the light avoidance function and / or the crisis avoidance function), comprising a nucleic acid encoding a chimeric protein of an ion-transporting receptor rhodopsin and a G-protein-coupled receptor rhodopsin. (Item 3) A composition for enhancing visual functions (for example, improving visual acuity), comprising a nucleic acid encoding a chimeric protein of an ion-transporting receptor rhodopsin and a G-protein-coupled receptor rhodopsin. (Item 4) The composition according to Item 1, wherein the disease, disorder or symptom includes a retinal degenerative disease. (Item 5) The composition according to Item 1 or 4, wherein the disease, disorder or symptom is retinitis pigmentosa. (Item 6) The composition according to any one of Items 1, 4, or 5, wherein the disease, disorder, or symptom is autosomal dominant hereditary. (Item 7) The composition according to Item 1 or any one of Items 4 to 6, which is for preventing or suppressing the progression of retinitis pigmentosa. (Item 8) The composition according to Item 1 or any one of Items 4 to 7, which is characterized in that it is administered to a subject before or immediately after the onset of the disease, disorder, or symptom. (Item 9) The composition according to any one of Items 1 to 8, which is characterized in that it is administered once. (Item 10) 0.1×10 11 ~10×10 11 The composition according to any one of Items 1 to 9, which is administered at a unit dose of vg / eye. (Item 11) Among the base sequences encoding the ion transport type receptor rhodopsin, the base sequence encoding the second loop on the cytoplasmic side and / or the base sequence encoding the third loop on the cytoplasmic side are replaced with the base sequence encoding the second loop on the cytoplasmic side and / or the base sequence encoding the third loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin. The composition according to any one of Items 1 to 10. (Item 12) The composition according to any one of Items 1 to 11, wherein the ion transport type receptor rhodopsin is derived from cyanobacteria (blue bacteria). (Item 13) The composition according to any one of Items 1 to 12, wherein the G protein-coupled receptor rhodopsin is derived from a mammal. (Item 14) The composition according to any one of Items 1 to 13, wherein the chimeric protein has an amino acid sequence in which the glutamic acid corresponding to the 132nd position of the amino acid sequence of SEQ ID NO: 8 is replaced with glutamine. (Item 15) The chimeric protein is (a) The amino acid sequence described in any one of SEQ ID NOs: 1 to 4 or a fragment thereof; (b) An amino acid sequence having at least 80% identity to (a); An amino acid sequence having one or more amino acids substituted, added, and / or deleted with respect to (c), (a), or (b); having one of the following and having biological activity, or the nucleic acid encoding the chimeric protein is (A) a nucleic acid having a base sequence encoding the amino acid sequence set forth in any of SEQ ID NOs: 1 to 4 or the base sequence set forth in SEQ ID NO: 10 or a fragment thereof; (B) a nucleic acid having at least 80% identity with respect to (A); (C) a nucleic acid having one or more nucleotides substituted, added, and / or deleted with respect to (A) or (B); (D) a nucleic acid that hybridizes under stringent conditions with respect to any of (A) to (C), having one of the following and the chimeric protein having biological activity, The composition according to any one of Items 1 to 14. (Item 16) The composition according to any one of Items 1 to 15, wherein the base sequence is contained in a vector. (Item 17) A composition for preventing or suppressing the progression of a retinal disease, disorder, or symptom, comprising a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. (Item 18) A composition for improving visual recognition motor functions (e.g., improvement of light-dark discrimination function, improvement of light avoidance function, and / or crisis avoidance function), comprising a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. (Item 19) A composition for enhancing visual function (e.g., improving visual acuity), comprising a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. (Item 20) The composition according to any one of Items 17 to 19, further having the features described in any one or more of Items 4 to 16. (Item 21) A nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for preventing or suppressing the progression of a retinal disease, disorder or symptom. (Item 22) A nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for improving visual cognitive behavioral functions (e.g., improving light-dark discrimination function, improving light avoidance function and / or crisis avoidance function). (Item 23) A nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for enhancing visual function (e.g., improving visual acuity). (Item 24) A chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for preventing or suppressing the progression of a retinal disease, disorder or symptom. (Item 25) A chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for improving visual cognitive behavioral functions (e.g., improving light-dark discrimination function, improving light avoidance function and / or crisis avoidance function). (Item 26) A chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for enhancing visual function (e.g., improving visual acuity). (Item 27) A nucleic acid or protein according to any one of Items 21 to 26, further having the features described in any one or more of Items 4 to 16. (Item 28) A method for preventing or suppressing the progression of a retinal disease, disorder or symptom in a subject, the method comprising administering to the subject an effective amount of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. (Item 29) A method for improving visual cognitive behavioral functions (e.g., improving light-dark discrimination function, improving light avoidance function and / or crisis avoidance function) in a subject, the method comprising administering to the subject an effective amount of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. (Item 30) A method for enhancing visual function (for example, improving eyesight) in a subject, the method comprising administering to the subject an effective amount of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. (Item 31) A method for preventing or suppressing the progression of a retinal disease, disorder or symptom in a subject, the method comprising administering to the subject an effective amount of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. (Item 32) A method for improving visual cognitive behavioral function (for example, improving light-dark discrimination function, improving light avoidance function and / or crisis avoidance function) in a subject, the method comprising administering to the subject an effective amount of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. (Item 33) A method for enhancing visual function (for example, improving eyesight) in a subject, the method comprising administering to the subject an effective amount of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. (Item 34) The method according to any one of Items 28 to 33, further having the features described in any one or more of Items 4 to 16. (Item 35) Use of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for preventing or suppressing the progression of a retinal disease, disorder or symptom. (Item 36) Use of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for improving visual cognitive behavioral function (for example, improving light-dark discrimination function, improving light avoidance function and / or crisis avoidance function). (Item 37) Use of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for enhancing visual function (for example, improving eyesight). (Item 38) Use of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for preventing or suppressing the progression of a retinal disease, disorder or symptom. (Item 39) Use of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for improving visual recognition motor function (e.g., improvement of light-dark discrimination function, improvement of light avoidance function and / or crisis avoidance function). (Item 40) Use of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for enhancing visual function (e.g., improving visual acuity). (Item 41) Use according to any one of Items 35 to 40, further having the feature(s) described in any one or more of Items 4 to 16. (Item 42) (a) A nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 10 or a fragment thereof; (b) A nucleic acid having at least 80% identity to (a); (c) A nucleic acid having one or more nucleotides substituted, added and / or deleted with respect to (a) or (b); (d) A nucleic acid that hybridizes under stringent conditions to any one of (a) to (c), A nucleic acid having one of the above, wherein the protein encoded by the nucleic acid has biological activity.

[0005] In the present invention, it is intended that the above one or more features may be provided in further combinations in addition to the explicitly stated combinations. Further embodiments and advantages of the present invention will be recognized by those skilled in the art upon reading the following detailed description as necessary.

Effects of the Invention

[0006] The present invention provides an effect of preventing and suppressing the progression of retinal diseases, disorders or symptoms. The present invention also provides an improvement effect on visual cognitive behavioral functions (for example, improvement of light and dark discrimination function, improvement of light avoidance function and / or crisis avoidance function). The present invention further provides an enhancement effect on visual functions such as visual acuity improvement.

Brief Description of Drawings

[0007]

Figure 1

Figure 2

Figure 3

Figure 4

Modes for Carrying Out the Invention

[0008] The present invention will be described below while showing the best mode. Throughout this specification, it should be understood that the singular expressions also include the concepts of their plural forms unless otherwise specified. Therefore, singular articles (for example, in English, "a", "an", "the", etc.) should be understood to also include the concepts of their plural forms unless otherwise specified. Also, the terms used in this specification should be understood to be used in the meanings commonly used in the art unless otherwise specified. Therefore, unless otherwise defined, all technical terms and scientific and technical terms used in this specification have the same meanings as generally understood by those skilled in the art to which the present invention pertains. In case of contradiction, this specification (including the definitions) shall prevail.

[0009] (Definition) The definitions of the terms particularly used in this specification and / or the basic technical contents will be appropriately described below.

[0010] As used herein, "rhodopsin" is a protein that contains a pigment called retinal inside. When it receives light, it is activated and visual signals are transmitted to the brain. The ion transport receptor rhodopsin represented by microorganisms does not release retinal even when irradiated with light, so it can be repeatedly activated by absorbing light, but it cannot activate G proteins like the G protein-coupled receptor rhodopsin represented by animals. In contrast, the chimeric rhodopsin provided in the present disclosure, which is a chimera of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin, is considered to have enhanced functions compared to conventional rhodopsin. In particular, as the ion transport receptor rhodopsin, preferably, it can be derived from microorganisms and can be repeatedly used. Also, when using an animal-derived, preferably mammalian-derived G protein-coupled receptor rhodopsin, while retaining the function of repeated activation, high activity can be obtained through endogenous G proteins. Although not wishing to be bound by theory, the chimeric protein used in the present invention is sufficiently active and expressed in mammals such as rodents and primates as demonstrated in animal models. Therefore, it can achieve the preventive and progression-suppressing effects of retinal diseases, disorders or symptoms, particularly the prevention or progression suppression of retinitis pigmentosa, or improve visual recognition behavioral functions (e.g., improvement of light-dark discrimination function, improvement of light avoidance function and / or crisis avoidance function), or exhibit the enhancement effect of visual functions such as improvement of visual acuity. Thus, it has been actually found that a chimeric protein combining completely different types of receptors functions in these various applications. Also, in this specification, it has been confirmed that in a model system with a slow onset rate where preventive effects and progression suppression can be observed, by administering before the actual blindness state, the onset and progression of the disease can be suppressed, and the progression suppression of retinal diseases such as retinitis pigmentosa has been achieved.

[0011] As used herein, "ion transport receptor rhodopsin" refers to any rhodopsin having the function of transporting ions, and examples thereof include ion pump type receptor rhodopsin and ion channel type receptor rhodopsin.

[0012] Regarding ion-transporting receptor rhodopsin, the steric compatibility with the G protein activation loop and the membrane translocation efficiency are considered important. In particular, ion-transporting receptor rhodopsin derived from microorganisms has good steric compatibility with the G protein activation loop and membrane translocation efficiency, and among them, those belonging to the genus Gloeobacter are preferred. In particular, among the microorganisms belonging to the genus Gloeobacter, Gloeobacter violaceus is preferred. Further, rhodopsin of microorganisms belonging to the genus Gloeobacter (for example, SEQ ID NO: 8) is preferably used in combination with G protein-coupled receptor rhodopsin derived from animals, among which G protein-coupled receptor rhodopsin derived from mammals, preferably, G protein-coupled receptor rhodopsin of artiodactyls such as cows (for example, SEQ ID NO: 9), primates such as humans (for example, SEQ ID NO: 14 and SEQ ID NO: 15). Further, the genus Gloeobacter is also preferred in that it has an important property of being well expressed in both Escherichia coli, which is a prokaryote, and human cells, which are eukaryotes.

[0013] As used herein, the term "G protein-coupled receptor rhodopsin" refers to rhodopsin classified as a type of G protein-coupled receptor, which is a receptor present on the cytoplasmic membrane of eukaryotic cells or on the constituent membranes inside the cell. A G protein-coupled receptor has a seven α-helix structure that traverses the cytoplasmic membrane, with the N-terminal side outside the cell and the C-terminal side inside the cell, and is said to have three extracellular loops (Extracellular loop; ECL1 / 2 / 3) and three intracellular loops (Intracellular loop; ICL1 / 2 / 3). Rhodopsin is composed of an apoprotein and a chromophore retinal. When retinal absorbs light, it is isomerized, causing a structural change in the protein part, and driving the intracellular signal transduction system via a G protein.

[0014] As used herein, the term "retinal disease, disorder, or symptom" refers to any disease, disorder, or symptom related to the retina, including retinal degenerative diseases (such as retinitis pigmentosa, age-related macular degeneration, etc.), retinopathies (such as diabetic retinopathy, proliferative retinopathy, simple retinopathy, etc.), floaters, retinal holes, retinal detachment (such as rhegmatogenous retinal detachment, non-rhegmatogenous retinal detachment, etc.), etc. It is possible to prevent, treat, or inhibit the progression of retinitis pigmentosa, age-related macular degeneration, myopic maculopathy, macular dystrophy, diabetic retinopathy, uveitis, retinal detachment, etc. Disorders or symptoms include impairments in visual acuity, contrast sensitivity, light-dark adaptation, color vision, and related symptoms.

[0015] As used herein, the term "visual cognitive behavioral function" means that visual information recognized by a visual organ (such as an eye) functions as the behavior of the target organism. For example, it refers to the manifestation as actual behaviors such as a light-dark discrimination function, a bright-place avoidance function, and a crisis avoidance function. It is a function that can be confirmed not only by checking light sensitivity but also by actual verification using an animal model (see Example 2), etc.

[0016] As used herein, the term "light-dark discrimination function" refers to the ability or function to discriminate between light and dark. Improvement thereof means any improvement in the light-dark discrimination function. For example, in addition to the ability to perform light-dark discrimination that was previously impossible, it also includes cases where the ability to finally distinguish between light and dark is improved.

[0017] As used herein, the term "bright-place avoidance function" refers to the ability or function to move away from a light source or avoid bright light. Improvement thereof means the recovery or enhancement of the ability to avoid bright places.

[0018] As used herein, the term "crisis avoidance function" refers to the function or ability to avoid a crisis based on visual function. Improvement thereof includes not only the regeneration of the crisis avoidance ability but also an increase in the level.

[0019] As used herein, "enhancement" or "augmentation" of "visual function" refers to the improvement, enhancement or augmentation of any visual function (e.g., visual acuity, color vision, contrast sensitivity, light and dark adaptation, etc.).

[0020] As used herein, "visual acuity improvement" means that visual acuity is improved or restored. Visual acuity can be measured, for example, in humans using a visual acuity test with a Landolt ring, or with a Snellen chart or an E chart, and can be expressed as decimal visual acuity or fractional visual acuity. These can also be expressed in logMAR visual acuity. In the case of a mouse, it can be measured using a visual stimulus that manipulates the spatial frequency of a light and dark stripe pattern. Experimentally, it can also be determined by measuring visual evoked potentials.

[0021] As used herein, "retinal degenerative disease" refers to any disease caused by retinal degeneration, and examples thereof include retinitis pigmentosa, age-related macular degeneration, and the like.

[0022] As used herein, "retinitis pigmentosa" is a hereditary disease in which abnormalities are observed in the retina, and it is a disease in which photoreceptor cells and pigment epithelial cells in the retina are extensively degenerated. Three symptoms appear: night blindness (difficulty seeing in the dark), visual field constriction (narrow visual field), and decreased visual acuity. Degeneration of only rod cells among photoreceptor cells is called rod dystrophy, and degeneration of both rod cells and cone cells is called rod-cone dystrophy. Research on gene therapy, artificial retina, retinal regeneration, photoreceptor cell protection therapy, etc. is being promoted, but no treatment method has been established yet. Since it is bilateral and progressive, and many people become socially blind in their 40s at the earliest, the realization of progression suppression is of very high significance.

[0023] In this specification, "retinitis pigmentosa" includes not only autosomal recessive hereditary types but also autosomal dominant hereditary types and X-linked recessive hereditary types. The most common is the type showing autosomal recessive inheritance, which accounts for about 35% of the total. The next most common is the type showing autosomal dominant inheritance, which accounts for 10% of the total. The least common is the type showing X-linked inheritance (X-linked recessive inheritance), which accounts for about 5% of the total. In particular, the fact that the progression of autosomal dominant retinitis pigmentosa caused by rhodopsin can be suppressed should be noted as a remarkable point. In autosomal dominant retinitis pigmentosa, in addition to abnormalities in rhodopsin, peripherin (PRPH2, also known as RDS) is considered the main factor. As autosomal recessive hereditary types, genes such as EYS, rod cGMP-phosphodiesterase α and β subunits, rod cyclic nucleotide-gated cation channels, retinal guanylate cyclase, RPE65, cellular retinol-binding protein, arrestin, and usherin (USH2) are known. As X-linked retinitis pigmentosa, retinitis pigmentosa GTPase regulator (RPGR), RP2, etc. can be mentioned.

[0024] In this specification, "progression suppression" means that the progression of a certain disease (for example, retinitis pigmentosa) is suppressed. Suppression includes not only a decrease in the rate of deterioration compared to the case without treatment but also the maintenance or improvement of the disease level. If a certain disease has not occurred, it corresponds to "prevention of onset". In this specification, "onset" means the appearance of symptoms of a disease from a state where no symptoms are felt. For example, symptoms such as night blindness, visual field constriction, photophobia, decreased visual acuity, and color vision abnormalities can be mentioned as symptoms.

[0025] In this specification, "immediately after onset" refers to within a certain period from the time when the patient experiences symptoms, for example, within 1 year, within 6 months, within 3 months, etc., but is not limited to these.

[0026] As used herein, "protein", "polypeptide", "oligopeptide", and "peptide" are used interchangeably herein and refer to a polymer of amino acids of any length. This polymer may be linear, branched, or cyclic. The amino acids may be natural, non-natural, or modified amino acids. This term may also include assemblies into complexes of multiple polypeptide chains. This term also includes amino acid polymers that have been modified either naturally or artificially. 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 includes, for example, polypeptides containing one or more analogs of amino acids (e.g., including non-natural amino acids, etc.), peptide-like compounds (e.g., peptoids), and other modifications known in the art. As used herein, "amino acid" is a general term for organic compounds having an amino group and a carboxyl group. When an antibody according to an embodiment of the present invention contains a "specific amino acid sequence", any amino acid in the amino acid sequence may be chemically modified. Also, any amino acid in the amino acid sequence may form a salt or a solvate. Also, any amino acid in the amino acid sequence may be of the L-form or the D-form. Even in such cases, it can be said that the protein according to the embodiment of the present invention contains the above "specific amino acid sequence". Chemical modifications that amino acids contained in a protein undergo in vivo include, for example, N-terminal modifications (e.g., acetylation, myristoylation, etc.), C-terminal modifications (e.g., amidation, glycosylphosphatidylinositol addition, etc.), or side-chain modifications (e.g., phosphorylation, sugar chain addition, etc.). As long as the object of the present invention is satisfied, they may be natural or non-natural.

[0027] As used herein, "chimera" (protein, rhodopsin) refers to a state in which genetic information derived from different organisms is mixed in the same entity (in this case, protein, rhodopsin, 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.

[0028] As used herein, "polynucleotide", "oligonucleotide", and "nucleic acid" are used interchangeably and refer to polymers 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 bonds between nucleotides that are different from normal, and are used interchangeably. Specific examples of such oligonucleotides include, for example, 2'-O-methyl-ribonucleotides, oligonucleotide derivatives in which the phosphodiester bonds in the oligonucleotide are converted to phosphorothioate bonds, oligonucleotide derivatives in which the phosphodiester bonds in the oligonucleotide are converted to N3'-P5' phosphoramidate bonds, oligonucleotide derivatives in which the ribose and phosphodiester bonds in the oligonucleotide are converted to peptide nucleic acid bonds, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced by C-5 propynyluracil, oligonucleotide derivatives in which uracil in the oligonucleotide is replaced by C-5 thiazoleuracil, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced by C-5 propynylcytosine, oligonucleotide derivatives in which cytosine in the oligonucleotide is replaced by phenoxazine-modified cytosine, oligonucleotide derivatives in which ribose in DNA is replaced by 2'-O-propylribose, and oligonucleotide derivatives in which ribose in the oligonucleotide is replaced by 2'-methoxyethoxyribose. Unless otherwise indicated, a particular nucleotide sequence is also intended to include its conservatively modified variants (e.g., degenerate codon substitutions) and complementary sequences, as well as the explicitly shown sequences. Note that the sequence of a nucleic acid is also referred to as a base sequence, nucleic acid sequence, nucleotide sequence, etc., all of which have the same meaning.Specifically, the degenerate codon variant can be achieved by creating an array in which the third position of one or more selected (or all) codons is substituted with a mixed base and / or deoxyinosine residue (Batzer et al., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al., J. Biol. Chem. 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes 8:91-98 (1994)). As used herein, "nucleic acid" may also be used interchangeably with gene, DNA such as cDNA, RNA such as mRNA, oligonucleotide, and polynucleotide, depending on the context. As used herein, "nucleotide" may be either natural or unnatural. As used herein, the nucleic acid may be DNA or RNA.

[0029] As used herein, "gene" refers to a factor that defines a genetic trait, and "gene" may refer to "polynucleotide", "oligonucleotide", and "nucleic acid".

[0030] As used herein, the "homology" of a gene refers to the degree of identity between two or more gene sequences. Generally, having "homology" means having a high degree of identity or similarity. "Identity" refers to the corresponding degree of the same amino acid sequence, and "similarity" refers to the corresponding degree of the sequence including amino acids with similar properties in addition to the same amino acids. Therefore, the higher the homology between two genes, the higher the identity or similarity of their sequences. Whether two types of genes have homology can be examined by direct comparison of the sequences or, in the case of nucleic acids, by the hybridization method under stringent conditions. When directly comparing two gene sequences, when the DNA sequences between the gene sequences are typically at least 50% identical, preferably at least 70% identical, more preferably at least 80%, 90%, 95%, 96%, 97%, 98% or 99% identical, those genes have homology. Therefore, as used herein, "homolog" or "homologous gene product" means a protein in another species, preferably a mammal, that exhibits the same biological function as the protein component of the complex further described herein. Such homologs may also be referred to as "orthologous gene products". It is understood that such homologs, homologous gene products, orthologous gene products, etc. can also be used as long as they meet the objectives of the present invention.

[0031] Amino acids may be referred to herein by either their generally known three-letter symbols or the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides may likewise be referred to by their generally recognized one-letter codes. In this specification, comparisons of amino acid sequences and base sequence similarities, identities, and homologies are calculated using the default parameters with the BLAST sequence analysis tool. The identity search can be performed, for example, using NCBI's BLAST 2.2.28 (released on April 2, 2013) (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). The identity value in this specification usually refers to the value when aligned under the default conditions using the above BLAST. However, if a higher value is obtained by changing the parameters, the highest value shall be taken as the identity value. When identity is evaluated in multiple regions, the highest value among them shall be taken as the identity value. Similarity is a numerical value that takes into account similar amino acids in addition to identity. When comparing amino acid sequences with BLAST, the Blastp algorithm can be used with default settings. The measurement results are quantified as Positives or Identities. The homology of amino acid sequences and base sequences can be determined by 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 a base sequence by BLASTN based on BLAST, the parameters are, for example, score = 100 and wordlength = 12. When analyzing an amino acid sequence by BLASTX based on BLAST, the parameters are, for example, score = 50 and wordlength = 3. When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. The specific methods of these analysis methods are known (http: / / www.ncbi.nlm.nih.gov.).

[0032] The nucleic acids or proteins used in the present invention may include sequences in which one or more amino acids or nucleotides are substituted, deleted, and / or added in the target amino acid or base sequence. Here, in SEQ ID NOs: 1 to 4 of the full-length amino acid sequence of the chimeric protein, "one or more" usually means within 50 amino acids, preferably within 30 amino acids, more preferably within 10 amino acids (for example, within 5 amino acids, within 3 amino acids, within 1 amino acid). Also, in the amino acid sequence of the domain such as SEQ ID NOs: 5 to 7, "one or more" usually means within 6 amino acids, preferably within 5 amino acids, more preferably within 4 amino acids (for example, within 3 amino acids, within 2 amino acids, within 1 amino acid). When maintaining the biological activity of the chimeric protein of the present invention, it is desirable that the amino acid residue to be mutated is mutated to another amino acid in which the nature of the amino acid side chain is conserved. For example, as the nature of the amino acid side chain, 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 having aliphatic side chains (G, A, V, L, I, P), amino acids having hydroxyl group-containing side chains (S, T, Y), amino acids having sulfur atom-containing side chains (C, M), amino acids having carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids having base-containing side chains (R, K, H), amino acids having aromatic-containing side chains (H, F, Y, W) can be mentioned (the parentheses represent the single-letter codes of the amino acids). These are also referred to as "conservative substitutions" in this specification.It is known that a protein having an amino acid sequence modified by deletion, addition, and / or substitution of one or more amino acid residues with other amino acids for a certain amino acid sequence maintains its biological activity (Mark, D.F. et al., Proc. Natl. Acad. Sci. USA (1984) 81, 5662-5666, Zoller, M.J. & Smith, M. Nucleic Acids Research (1982) 10, 6487-6500, Wang, A. et al., Science 224, 1431-1433, Dalbadie-McFarland, G. et al., Proc. Natl. Acad. Sci. USA (1982) 79, 6409-6413). Therefore, in one embodiment of the present invention, "several" may be, for example, 10, 8, 6, 5, 4, 3, or 2, and may be less than any of those values. The chimeric protein with deletions or the like can be prepared, for example, by site-directed mutagenesis, random mutagenesis, or biopanning using an antibody phage library. As the site-directed mutagenesis method, for example, KOD-Plus-Mutagenesis Kit (TOYOBO CO., LTD.) can be used. It is possible to select an antibody having the same activity as the wild type from the mutant antibody into which deletions or the like have been introduced by performing various characterizations such as FACS analysis and ELISA.

[0033] In one embodiment of the present invention, the amino acid sequence and nucleic acid sequence of the chimeric protein of the present invention may have an identity or similarity of 70% or more, 80% or more, or 90% or more with respect to a reference sequence. In the present specification, for an amino acid sequence or a base sequence, "70% or more" may be, for example, 70, 75, 80, 85, 90, 95, 96, 97, 98, 99% or more, "80% or more" may be, for example, 80, 85, 90, 95, 96, 97, 98, 99% or more, "90% or more" may be, for example, 90, 95, 96, 97, 98, 99% or more, or may be within the range of any two of these values. "Homology" may be calculated by a method known in the art for the ratio of the number of homologous amino acids in two or more amino acid sequences. Before calculating the ratio, the amino acid sequences of the amino acid sequence groups to be compared are aligned, and gaps are introduced into a part of the amino acid sequence if necessary to maximize the ratio of identical amino acids. Methods for alignment, methods for calculating the ratio, comparison methods, and computer programs related thereto are well known in the art (for example, BLAST, GENETYX, etc.). In the case of "identity", the ratio of identical amino acids is calculated, and in the case of "similarity", the ratio of similar amino acids is calculated. Examples of similar amino acids include, but are not limited to, amino acids capable of conservative substitution.

[0034] As used herein, the term "polynucleotide that hybridizes under stringent conditions" refers to well-known conditions commonly used in the art. Such polynucleotides can be obtained by using, as a probe, a polynucleotide selected from the polynucleotides of the present invention, and employing methods such as colony hybridization, plaque hybridization, or Southern blot hybridization. Specifically, it means a polynucleotide that can be identified by performing hybridization at 65°C in the presence of 0.7 - 1.0 M NaCl using a filter immobilized with DNA derived from colonies or plaques, and then washing the filter under 65°C conditions using an SSC (saline-sodium citrate) solution at a concentration of 0.1 - 2 times (the composition of a 1× SSC solution is 150 mM sodium chloride and 15 mM sodium citrate). "Stringent conditions" can, for example, employ the following conditions: (1) using low ionic strength and high temperature for washing (e.g., at 50°C, 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate), (2) using a denaturing agent such as formamide during hybridization (e.g., at 42°C, 50% (v / v) formamide and 0.1% bovine serum albumin / 0.1% ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer at pH 6.5, and 750 mM sodium chloride, 75 mM sodium citrate), or (3) incubating overnight at 37°C in a solution containing 20% formamide, 5× SSC, 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 mg / ml of denatured and sheared salmon sperm DNA, and then washing the filter with 1× SSC at approximately 37 - 50°C. Note that the formamide concentration may be 50% or higher. The washing time may be 5, 15, 30, 60, or 120 minutes, or longer.Multiple factors, such as temperature and salt concentration, can affect the stringency of the hybridization reaction. For details, reference can be made to Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience Publishers, (1995). Examples of "highly stringent conditions" are 0.0015 M sodium chloride, 0.0015 M sodium citrate, 65 - 68 °C, or 0.015 M sodium chloride, 0.0015 M sodium citrate, and 50% formamide, 42 °C. Hybridization can be carried out according to the methods described in experimental manuals such as Molecular Cloning 2nd ed., Current Protocols in Molecular Biology, Supplement 1 - 38, DNA Cloning 1: Core Techniques, A Practical Approach, Second Edition, Oxford University Press (1995). Here, sequences that hybridize under stringent conditions preferably exclude sequences containing only the A sequence or only the T sequence. Moderately stringent conditions can be easily determined by those skilled in the art based on, for example, the length of the DNA, as shown in Sambrook et al., Molecular Cloning: A Laboratory Manual, 3rd Edition, Vol. 1, 7.42 - 7.45 Cold Spring Harbor Laboratory Press, 2001, and for nitrocellulose filters, include the use of a pre - wash solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization conditions of approximately 40 - 50 °C in approximately 50% formamide, 2×SSC - 6×SSC (or other similar hybridization solutions such as Stark's solution in approximately 50% formamide at approximately 42 °C), and washing conditions of approximately 60 °C, 0.5×SSC, 0.1% SDS.Accordingly, the polypeptides used in the present invention include polypeptides encoded by nucleic acid molecules that hybridize to nucleic acid molecules encoding the polypeptides specifically described in the present invention under highly or moderately stringent conditions.

[0035] As used herein, a "purified" substance or biological agent (e.g., a nucleic acid or protein, etc.) refers to a substance or biological agent from which at least a portion of the factors that are naturally associated with that substance or biological agent have been removed. Accordingly, generally, the purity of the biological agent in a purified biological agent is higher (i.e., concentrated) than in the state in which the biological agent normally exists. The term "purified" as used herein preferably means that 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 agent is present. The substances or biological agents used in the present invention are preferably "purified" substances. As used herein, an "isolated" substance or biological agent (e.g., a nucleic acid or protein, etc.) refers to a substance or biological agent from which the factors that are naturally associated with that substance or biological agent have been substantially removed. Since the term "isolated" as used herein varies depending on the purpose, it does not necessarily have to be expressed in terms of purity, but if necessary, preferably means that 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 agent is present. The substances used in the present invention are preferably "isolated" substances or biological agents.

[0036] As used herein, a "corresponding" amino acid, nucleic acid, or moiety refers to an amino acid, nucleotide, or moiety in a polypeptide molecule or polynucleotide molecule (e.g., rhodopsin) that has, or is predicted to have, a similar function to a given amino acid, nucleotide, or moiety in a reference polypeptide or polynucleotide. In particular, in an enzyme molecule, it refers to an amino acid that is present at a similar position in the active site and makes a similar contribution to catalytic activity. In a complex molecule, it refers to a corresponding moiety (e.g., heparan sulfate, etc.). For example, in the case of an antisense molecule, it can be a similar moiety in an ortholog corresponding to a specific moiety of the antisense molecule. A corresponding amino acid can be, for example, a specific amino acid that is subject to cysteinylation, glutathionylation, S-S bond formation, oxidation (e.g., oxidation of the methionine side chain), formylation, acetylation, phosphorylation, glycosylation, myristoylation, etc. Alternatively, a corresponding amino acid can be an amino acid responsible for dimerization. Such "corresponding" amino acids or nucleic acids can be regions or domains over a certain range. Therefore, in such cases, they are referred to herein as "corresponding" regions or domains. Such corresponding regions or domains are useful in designing complex molecules in the present invention.

[0037] As used herein, a "corresponding" gene (e.g., a polynucleotide sequence or molecule) refers to a gene (e.g., a polynucleotide sequence or molecule) that has, or is predicted to have, the same function as a given gene in a reference species in a certain species. When there are multiple genes having such a function, those having the same evolutionary origin are referred to. Thus, a gene corresponding to a certain gene may be an ortholog of that gene. Thus, human rhodopsin can find corresponding rhodopsin in other animals (especially mammals), respectively. Such corresponding genes can be identified using techniques well known in the art. Thus, for example, a corresponding gene in a certain animal (e.g., a mouse) can be found by searching a database containing the sequences of that animal using the sequences of SEQ ID NOs: 1 to 17, etc. as query sequences for the reference gene of the corresponding gene (e.g., rhodopsin, etc.).

[0038] As used herein, a "fragment" refers to a polypeptide or polynucleotide having a sequence length from 1 to n - 1 with respect to a full-length polypeptide or polynucleotide (length n). The length of the fragment can be appropriately changed according to the purpose. For example, as the lower limit of the length, in the case of a polypeptide, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50 and more amino acids can be mentioned, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be appropriate as the lower limit. Also, in the case of a polynucleotide, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50, 75, 100 and more nucleotides can be mentioned, and lengths represented by integers not specifically listed here (e.g., 11, etc.) can also be appropriate as the lower limit. It is understood that such fragments are within the scope of the present invention as long as the fragment itself also has the function as a marker or target molecule, for example, when the full-length one functions as a marker or target molecule.

[0039] In accordance with the present invention, the term "activity" refers, in the broadest sense, to the function of a molecule herein. Activity includes, but is not intended to be limiting, generally the biological, biochemical, physical, or chemical function of a molecule. Activity includes, for example, enzyme activity, the ability to interact with other molecules, and the ability to activate, promote, stabilize, inhibit, suppress, or destabilize the function of other molecules, stability, and the ability to localize to a specific intracellular location. Where applicable, the term also relates to the function of a protein complex in the broadest sense. As used herein, "biological activity" includes the activation of a photoreaction and the like.

[0040] As used herein, the term "functional equivalent" refers to anything that has the same target function but a different structure with respect to the original entity. Therefore, the functional equivalent of "rhodopsin" or its chimera is not rhodopsin or its chimera itself, but a variant or modified form of rhodopsin or its chimera (e.g., an amino acid sequence variant, etc.) that has the biological activity of rhodopsin or its chimera, and that can be changed into rhodopsin or its antibody itself or a variant or modified form of this rhodopsin or its chimera at the time of action (e.g., nucleic acids encoding rhodopsin or its chimera or a variant or modified form of rhodopsin or its chimera, and vectors, cells, etc. containing such nucleic acids). It is understood that the functional equivalents of the present invention include those in which one or more amino acids are inserted, substituted, and / or deleted in the amino acid sequence, or added to one or both ends thereof. As used herein, "insertion, substitution, and / or deletion of one or more amino acids in the amino acid sequence, or addition to one or both ends thereof" means that the modification is made by a well-known technical method such as site-directed mutagenesis or by natural mutation, or by substitution of a plurality of amino acids to the extent that can occur naturally. The modified amino acid sequence can be, for example, one in which 1 to 30, preferably 1 to 20, more preferably 1 to 9, still more preferably 1 to 5, and particularly preferably 1 to 2 amino acids are inserted, substituted, or deleted, or added to one or both ends thereof. The modified amino acid sequence preferably has an amino acid sequence in which one or more (preferably 1 or several or 1, 2, 3, or 4) conservative substitutions are made in the amino acid sequence of rhodopsin.

[0041] In this specification, "drug", "agent", or "factor" (all corresponding to "agent" in English) are used interchangeably in a broad sense and can be any substance or other element (e.g., energy such as light, radioactivity, heat, electricity, etc.) as long as the intended purpose can be achieved. Such substances include, for example, proteins, polypeptides, oligopeptides, peptides, polynucleotides, oligonucleotides, nucleotides, nucleic acids (including 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 of these, but are not limited thereto.

[0042] In the case of oral administration, it may be formulated into various forms such as tablets, granules, fine granules, powders, capsules, etc., and may contain additives such as binders, inclusion agents, excipients, lubricants, disintegrants, and wetting agents commonly used in formulations. In addition, in addition to these, the formulations for oral administration may be formulated in a liquid state such as internal aqueous solutions, suspensions, emulsions, syrups, etc., or may be formulated in a dry state that is redissolved at the time of use.

[0043] In the case of parenteral administration, it may be formulated in a state contained in a unit dose ampoule or multi-dose container or tube, and may also contain additives such as stabilizers, buffers, preservatives, isotonic agents, etc. In addition, the formulations for parenteral administration may be formulated into powders that can be redissolved with an appropriate carrier (such as sterile water) at the time of use.

[0044] Examples of parenteral administration include intravitreal administration, subconjunctival administration, intracameral administration, eye drop administration, etc., and intravitreal administration is preferred. The compositions of the present invention, etc., can be used for treatment, prevention, progression inhibition, etc. by administering to humans by the methods described above.

[0045] As used herein, "treatment" refers to, with respect to a disease or disorder (e.g., a retinal degenerative disease), preventing the progression of such a disease or disorder, preferably maintaining the current state, more preferably alleviating, and even more preferably eliminating such a disease or disorder when in such a state, and includes being able to exert a symptom-improving effect or a preventive effect on the patient's disease or one or more symptoms associated with the disease. Performing a diagnosis in advance and then administering appropriate treatment is called "companion treatment", and the diagnostic agent therefor may be called a "companion diagnostic agent". Since the present invention is directed to genetic diseases, genes may be examined in advance and patients may be treated.

[0046] As used herein, "therapeutic agent" broadly refers to any agent that can treat a target condition (e.g., a retinal degenerative disease). In one embodiment of the present invention, the "therapeutic agent" may be a pharmaceutical composition comprising an active ingredient and one or more pharmaceutically acceptable carriers. The pharmaceutical composition can be produced, for example, by mixing the active ingredient and the above carriers by any method known in the technical field of pharmaceutics. Further, the form of use of the therapeutic agent is not limited as long as it is a substance used for treatment, and it may be the active ingredient alone or a mixture of the active ingredient and any component. Also, the shape of the above carrier is not particularly limited and may be, for example, solid or liquid (e.g., a buffer solution).

[0047] As used herein, "prevention" refers to, with respect to a disease or disorder (e.g., a retinal degenerative disease), preventing the occurrence of such a state before such a state occurs. Using the agent of the present invention, a diagnosis can be made, and if necessary, the agent of the present invention can be used to prevent, for example, a retinal degenerative disease or take preventive measures. As used herein, "preventive agent" broadly refers to any agent that can prevent a target condition (e.g., a disease such as a retinal degenerative disease).

[0048] As used herein, the term "kit" refers to a unit that is usually divided into two or more compartments and in which the parts to be provided (for example, test agents, diagnostic agents, therapeutic agents, antibodies, labels, instructions, etc.) are provided. When the purpose is to provide a composition that should not be provided in a mixed state for reasons such as stability and is preferably mixed and used immediately before use, this kit form is preferred. Such a kit preferably includes instructions or a manual that describes how to use the provided parts (for example, how to use a test agent, diagnostic agent, or therapeutic agent) or how to process a reagent. When the kit is used as a reagent kit in this specification, the kit usually includes instructions that describe how to use test agents, diagnostic agents, therapeutic agents, antibodies, etc.

[0049] The "active ingredient" as used herein refers to the ingredient contained in an amount necessary to obtain effects such as treatment, prevention, or suppression of progression, which are the objectives of the composition of the present invention. As long as the effects are not impaired to less than the desired level, other ingredients may also be contained. Further, the medicament, composition, etc. of the present invention may be formulated. Also, the administration route of the medicament, composition, etc. of the present invention may be either oral or parenteral, and can be appropriately set according to the form of the preparation, etc.

[0050] As used herein, the "instructions" (including attached documents, labels used by the US FDA, etc.) describe how to use the present invention for physicians or other users. These instructions describe the detection method of the present invention, how to use the diagnostic agent, or words instructing the administration of a medicine, etc. Further, the instructions may include words instructing oral administration or administration to the retina (e.g., by injection, etc.) as the administration site. These instructions are prepared in accordance with the format specified by the regulatory agency of the country where the present invention is implemented (e.g., the Ministry of Health, Labour and Welfare in Japan, the Food and Drug Administration (FDA) in the United States, etc.), and it is specified that approval has been obtained from that regulatory agency. The instructions are so-called package inserts or labels, and are usually provided in paper form, but are not limited thereto, and can also be provided in forms such as electronic media (e.g., a homepage provided on the Internet, an e-mail).

[0051] (Preferred Embodiment) Preferred embodiments of the present invention will be described below. It is understood that the embodiments provided below are provided for better understanding of the present invention, and the scope of the present invention should not be limited to the following description. Therefore, it is obvious that those skilled in the art can make appropriate modifications within the scope of the present invention by referring to the descriptions in this specification. Further, it is understood that the following embodiments of the present invention can be used alone or in combination with each other.

[0052] (Chimeric Rhodopsin) In one aspect, the present invention provides a novel use of chimeric rhodopsin and a nucleic acid molecule. The chimeric rhodopsin used in the present invention may be any as long as the object of the present invention can be achieved. The chimeric rhodopsin used in the present invention is typically a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. To illustrate a typical example, by fusing an animal-derived G protein-coupled receptor rhodopsin to a repeatedly usable ion transport receptor rhodopsin derived from a microorganism, while retaining the repeatedly activating function of the ion transport receptor ion channel type receptor rhodopsin derived from the microorganism, a high activity via the endogenous G protein by the G protein-coupled receptor can be obtained, and excellent therapeutic, ameliorating, and preventive, progression-suppressing effects on retinal diseases, disorders, and symptoms can be achieved.

[0053] In one embodiment, as the ion transport receptor rhodopsin used in the chimeric protein of the present invention, an ion pump type receptor rhodopsin or an ion channel type receptor rhodopsin can be used. In a preferred embodiment, the ion transport receptor rhodopsin is preferably derived from a microorganism. For example, those derived from cyanobacteria (blue bacteria) are typical. For example, rhodopsins derived from microorganisms belonging to eubacteria such as the genus Gloeobacter, and eukaryotes such as the genus Volvox, the genus Chlamydomonas, and the genus Guillardia can be mentioned. Examples of the genus Gloeobacter include Gloeobacter violaceus. Examples of the genus Volvox include Volvox carteri. Examples of the genus Chlamydomonas include Chlamydomonas reinhardtii. Examples of the genus Guillardia include Guillardia theta.

[0054] In one embodiment, as the G protein-coupled receptor rhodopsin used in the chimeric protein of the present invention, those derived from animals are typical, and rhodopsin derived from rodents, artiodactyls, perissodactyls, primates, carnivores, etc. are preferred, more preferably artiodactyls or primates, and even more preferably primate rhodopsin. Further, preferred G protein-coupled receptor rhodopsins include, for example, rhodopsin derived from bovine, human, mouse, rat, cat, dog, pig, sheep, horse, etc. Among these, bovine or human-derived rhodopsin is particularly preferred.

[0055] In a specific embodiment, the chimeric protein of the present invention is a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin and has a seven-transmembrane structure. In the present invention, the chimeric protein of the ion transport receptor rhodopsin and the G protein-coupled receptor rhodopsin is preferably designed to have both the function of repeatedly activating the ion transport receptor rhodopsin and the G protein activity by the G protein-coupled receptor rhodopsin at a high level. From this viewpoint, in order to maintain the activities of both at a high level and particularly exhibit a high visual function regeneration ability, in the amino acid sequence of the ion transport receptor rhodopsin of the chimeric protein of the present invention, the amino acid sequence of the second loop on the cytoplasmic side and / or the amino acid sequence of the third loop on the cytoplasmic side is preferably replaced with the amino acid sequence of the second loop on the cytoplasmic side and / or the amino acid sequence of the third loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin. Here, the "second loop on the cytoplasmic side" and the "third loop on the cytoplasmic side" respectively refer to the second loop and the third loop located second and third from the N-terminal side among the seven loops.

[0056] In one embodiment, it is advantageous for the chimeric protein of the present invention to have an amino acid sequence in which the glutamic acid corresponding to the 132nd position of the amino acid sequence of SEQ ID NO: 8 (GR) is substituted with glutamine. Examples of the amino acid sequence with glutamine substitution include, but are not limited to, the amino acid sequences encoded by SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 10.

[0057] As a method for obtaining a nucleic acid such as DNA of the present invention, there is no particular limitation, but examples thereof include a method of obtaining cDNA by reverse transcription from mRNA (for example, RT-PCR method), a method of preparing from genomic DNA, a method of synthesizing by chemical synthesis, and known methods such as a method of isolating from a genomic DNA library or a cDNA library (for example, see Japanese Patent Application Laid-Open No. 11-29599).

[0058] In the present specification, the preparation of the chimeric protein can be carried out, for example, by using a transformant into which an expression vector containing a nucleic acid such as DNA encoding the above-described chimeric protein has been introduced. For example, first, the transformant is cultured under appropriate conditions to synthesize the chimeric protein encoded by this nucleic acid such as DNA. Then, the chimeric protein of the present invention can be obtained by recovering the synthesized protein from the transformant or the culture solution.

[0059] More specifically, it can be prepared by inserting DNA encoding the above-described chimeric protein into an appropriate expression vector. The "appropriate vector" may be any vector that can be replicated, maintained, or self-propagated in various hosts of prokaryotes and / or eukaryotes, and can be appropriately selected according to the purpose of use. For example, when it is desired to obtain a large amount of nucleic acid such as DNA, a high-copy vector can be selected, and when it is desired to obtain a polypeptide (chimeric protein), an expression vector can be selected. Specific examples thereof are not particularly limited, and for example, known vectors described in Japanese Patent Application Laid-Open No. 11-29599 can be mentioned.

[0060] In addition, the expression vector can be used not only to synthesize the chimeric protein but also in the compositions of the present invention and the like. That is, the compositions of the present invention and the like may contain, as an active ingredient, an expression vector incorporated with a nucleic acid encoding the amino acid sequence of the above-described chimeric protein. 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. As such a vector, for example, an adeno-associated virus vector (AAV vector) or a lentivirus vector is suitable.

[0061] The method for introducing the vector can be appropriately selected according to the type of the vector, the host, and the like. Specific examples thereof are not particularly limited, but for example, when bacteria are used as the host, known methods such as the protoplast method and the competent method (see, for example, JP-A-11-29599) can be mentioned. Further, when the expression vector is used as an active ingredient of the visual function regenerating agent or the visual function deterioration preventive agent of the present invention, it can be introduced, for example, by injecting the above-described AAV vector or the like into the eye.

[0062] The host into which the expression vector is introduced may be any one that is compatible with the expression vector and can be transformed. Specific examples thereof are not particularly limited, but include known natural cells or artificially established cells such as bacteria, yeast, animal cells, and insect cells (see JP-A-11-29599), or animals such as humans and mice. The culture of the transformant can be appropriately selected from known nutrient media according to the type of the transformant and the like so that the chimeric protein can be obtained in large quantities and easily, and the temperature, the pH of the nutrient medium, the culture time, and the like can be appropriately adjusted (see, for example, JP-A-11-29599).

[0063] The method for isolating and purifying the chimeric protein is not particularly limited, and examples thereof include known methods such as a method using solubility, a method using a difference in molecular weight, and a method using charge (see, for example, JP-A-11-29599).

[0064] In a specific embodiment, the chimeric protein of the present invention has any of the following amino acid sequences: (a) the amino acid sequences set forth in SEQ ID NOs: 1-4 or fragments thereof; (b) an amino acid sequence having at least 80% identity to (a); (c) an amino acid sequence having one or more amino acid substitutions, additions and / or deletions relative to (a) or (b); and has biological activity, or preferably, the chimeric protein of the present invention has an amino acid sequence encoded by a nucleic acid described in any of the following: (aa) a nucleic acid having a base sequence encoding the amino acid sequence set forth in any of SEQ ID NOs: 1-4 or the base sequence set forth in SEQ ID NO: 10 (bb) a nucleic acid having a base sequence that can hybridize under stringent conditions with a base sequence complementary to the base sequence encoding the amino acid sequence set forth in any of SEQ ID NOs: 1-4 or the base sequence set forth in SEQ ID NO: 10 (cc) a nucleic acid having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence set forth in any of SEQ ID NOs: 1-4 and having biological activity (dd) a nucleic acid consisting of a base sequence encoding an amino acid sequence having 90% or more homology with the amino acid sequence set forth in any of SEQ ID NOs: 1-4 and having biological activity; or (aaa) the base sequence set forth in SEQ ID NO: 10 or a fragment thereof; (bbb) a nucleic acid having at least 80% identity to (aaa); (ccc) a base sequence having one or more nucleotide substitutions, additions and / or deletions relative to (aaa) or (bbb); (ddd) a base sequence that hybridizes under stringent conditions to any of (aaa)-(ccc), and having a nucleic acid having biological activity of the chimeric protein.

[0065] In one embodiment, the nucleic acid encoding the chimeric protein of the present invention is preferably any of the following: (aaa) the nucleotide sequence set forth in SEQ ID NO: 10 or a fragment thereof; (bbb) a nucleic acid having at least 80% identity to (aaa); (ccc) a nucleotide sequence having one or more nucleotides substituted, added and / or deleted with respect to (aaa) or (bbb); (ddd) a nucleotide sequence that hybridizes under stringent conditions to any of (aaa) to (ccc), and the chimeric protein has biological activity.

[0066] Alternatively, as the second loop on the cytoplasmic side of the above-described G protein-coupled receptor rhodopsin, those having the amino acid sequence encoded by the nucleic acid described below are preferred.

[0067] (i) a nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 5 or 6; (ii) a nucleic acid having a nucleotide sequence that can hybridize under stringent conditions to a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 5 or 6; (iii) a nucleic acid having a nucleotide sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence set forth in SEQ ID NO: 5 or 6; (iv) a nucleic acid consisting of a nucleotide sequence encoding an amino acid sequence having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 5 or 6; Alternatively, the nucleic acid encoding the second loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin is preferably any of the following.

[0068] (i) a nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 5 or 6 (ii) A nucleic acid having a nucleotide sequence that can hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 5 or 6 (iii) A nucleic acid having a nucleotide sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence set forth in SEQ ID NO: 5 or 6 (iv) A nucleic acid consisting of a nucleotide sequence encoding an amino acid sequence having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 5 or 6 (x) A nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 11 or SEQ ID NO: 12 or a fragment thereof; (y) A nucleic acid having at least 80% identity to (x); (z) A nucleic acid having one or more nucleotides substituted, added and / or deleted with respect to (x) or (y); (w) A nucleic acid that hybridizes under stringent conditions to any of (x) to (z), and the loop has biological activity.

[0069] As the third loop on the cytoplasmic side of the above-mentioned G protein-coupled receptor rhodopsin, those having the amino acid sequence encoded by the nucleic acid described in any of the following are preferred: (l) A nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 7; (k) A nucleic acid having a nucleotide sequence that can hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 7; (m) A nucleic acid having a nucleotide sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence set forth in SEQ ID NO: 7; (n) A nucleic acid consisting of a nucleotide sequence encoding an amino acid sequence having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 7.

[0070] Alternatively, it is preferable that the nucleic acid encoding the third loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin is any of the following: (l) A nucleic acid having a nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 7; (k) A nucleic acid having a nucleotide sequence that can hybridize under stringent conditions with a nucleotide sequence complementary to the nucleotide sequence encoding the amino acid sequence set forth in SEQ ID NO: 7; (m) A nucleic acid having a nucleotide sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in the amino acid sequence set forth in SEQ ID NO: 7; (n) A nucleic acid consisting of a nucleotide sequence encoding an amino acid sequence having 90% or more homology with the amino acid sequence set forth in SEQ ID NO: 7; (xx) A nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 13 or a fragment thereof; (yy) A nucleic acid having at least 80% identity to (xx); (zz) A nucleic acid having one or more nucleotides substituted, added and / or deleted with respect to (xx) or (yy); or (ww) A nucleic acid that hybridizes under stringent conditions to any of (xx) to (zz), having the loop and the loop having biological activity.

[0071] The present invention also provides the following: (a) A nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 10 or a fragment thereof; (b) A nucleic acid having at least 80% identity to (a); (c) A nucleic acid having one or more nucleotides substituted, added and / or deleted with respect to (a) or (b); (d) A nucleic acid that hybridizes under stringent conditions to any of (a) to (c), and the protein encoded by the nucleic acid having biological activity. The present invention also provides a nucleic acid.

[0072] In this specification, representative examples of "biological activity" include the functions of G protein-coupled receptors possessed by the loop (e.g., membrane translocation efficiency). In addition, functions that can prevent and suppress the progression of retinal diseases (e.g., retinitis pigmentosa), visual cognitive behavioral functions (e.g., improvement of light / dark discrimination function, improvement of light avoidance function and / or crisis avoidance function), and functions that can exert the effect of enhancing visual acuity can be mentioned. The biological activity in the case of the loop can include, but is not limited to, functions such as three-dimensional structural compatibility and membrane translocation efficiency. Alternatively, the function of the loop may be evaluated by the function of the entire incorporated protein (here rhodopsin).

[0073] In the present invention, the chimeric protein of the present invention and the nucleic acid encoding the same have been found to be useful for preventing or suppressing the progression of retinal diseases, disorders or symptoms, improving visual cognitive behavioral functions (e.g., improvement of light / dark discrimination function, improvement of light avoidance function and / or crisis avoidance function), and enhancing visual functions such as improving visual acuity.

[0074] Among eye diseases that currently have no treatment methods, there are retinal degenerative diseases such as retinitis pigmentosa and atrophic age-related macular degeneration. There is a possibility that the fundamental treatment for these diseases will be provided by the present invention. It is said that the total number of patients worldwide exceeds 130 million. In Japan, retinitis pigmentosa ranks third among the causes of moderate blindness, and age-related macular degeneration ranks fourth. Due to the large number of patients and the severity of visual impairment, the development of treatment methods has been highly desired, but it may be solved by the present invention.

[0075] Photoreceptor cells, which are primary neurons of vision, like the central nervous system, cannot regenerate once lost. However, in retinitis pigmentosa and atrophic age-related macular degeneration, bipolar cells and retinal ganglion cells, which are secondary and tertiary neurons of vision, are preserved, which is considered to be one of the reasons for the efficacy of the present invention. The present invention is a gene transfer therapy using optogenetics that can be expected to have a safe and long-term visual regeneration effect with less invasion. Different from the conventional method of introducing a photoactivatable ion channel, by using the original more physiological light transmission pathway that utilizes the endogenous G protein signal cascade and channel, highly efficient and safe visual regeneration has become possible. The conventional method of introducing a photoactivatable ion channel was for regeneration targeting patients with already progressed retinal degeneration. However, since this method does not require the VisualCycle, a metabolic regeneration system of retinal, which is necessary for normal light transmission, an effect of suppressing the progression of retinal degeneration can also be expected. Thus, it has been demonstrated that it can be applied not only to patients with already progressed retinal degeneration but also to the prevention of progression in patients at the initial stage.

[0076] (Prevention or suppression of progression of retinal diseases, disorders or symptoms) In one aspect, the present invention provides a composition for preventing or suppressing the progression of a retinal disease, disorder or symptom, comprising a nucleic acid encoding a chimeric protein of an ion transport type receptor rhodopsin and a G protein-coupled receptor rhodopsin. The chimeric protein used in this aspect of the present invention can utilize any embodiment described in (chimeric rhodopsin). In the present invention, regarding the prevention or suppression of the progression of retinal diseases, disorders or symptoms, typified by the suppression of the progression of retinitis pigmentosa, it has been confirmed by the thinning experiment of photoreceptor cells shown in Example 1, Figures 1 and 2.

[0077] In another aspect, the present invention provides a composition for preventing or suppressing the progression of a retinal disease, disorder or condition, the composition comprising a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. The chimeric protein used in this aspect of the present invention can utilize any of the embodiments described in (chimeric rhodopsin).

[0078] In yet another aspect, there is provided a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for preventing or suppressing the progression of a retinal disease, disorder or condition, or a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for preventing or suppressing the progression of a retinal disease, disorder or condition. The chimeric protein used in this aspect of the present invention can utilize any of the embodiments described in (chimeric rhodopsin).

[0079] In yet another aspect, the present invention provides a method for preventing or suppressing the progression of a retinal disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin, or a method for preventing or suppressing the progression of a retinal disease, disorder or condition in a subject, the method comprising administering to the subject an effective amount of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. The chimeric protein used in this aspect of the present invention can utilize any of the embodiments described in (chimeric rhodopsin).

[0080] In yet another aspect, the present invention provides the use of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for preventing or suppressing the progression of a retinal disease, disorder or symptom, or the use of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for preventing or suppressing the progression of a retinal disease, disorder or symptom.

[0081] In one embodiment, the disease, disorder or symptom is a retinal degenerative disease, and among retinal degenerative diseases, for example, retinitis pigmentosa, age-related macular degeneration, etc. are preferable, and more preferably, retinitis pigmentosa is advantageous.

[0082] In a preferred embodiment, the retinitis pigmentosa targeted by the present invention is autosomal dominant, preferably RHO autosomal dominant.

[0083] In a preferred embodiment, the present invention is used for the prevention or suppression of the progression of retinitis pigmentosa.

[0084] In a preferred embodiment, the present invention is preferably administered to a subject within 1 year, preferably within 6 months, within 3 months, within 1 month after the onset of the disease, disorder or symptom, for example, after the onset (for example, when subjective symptoms appear), but is not limited thereto.

[0085] In one particular embodiment, the composition or vector of the present invention is administered once. It has been confirmed that the present invention has an effect by administering once, and it is considered that the compliance for patients is also good.

[0086] In one particular embodiment, the dosage of the vector of the present invention is 0.1×10 11 ~10×10 11 vg / eye per unit dose, for example, the lower limit is 0.01×10 11 vg / eye, 0.02×10 11 vg / eye, 0.03×1011 vg / eye, 0.04×10 11 vg / eye, 0.05×10 11 vg / eye, 0.06×10 11 vg / eye, 0.07×10 11 vg / eye, 0.08×10 11 vg / eye, 0.09×10 11 vg / eye, 0.1×10 11 vg / eye, 0.2×10 11 vg / eye, 0.3×10 11 vg / eye, 0.4×10 11 vg / eye, 0.5×10 11 It can be, for example, vg / eye, and the upper limit is 2×10 11 vg / eye, 3×10 11 vg / eye, 4×10 11 vg / eye, 5×10 11 vg / eye, 6×10 11 vg / eye, 7×10 11 vg / eye, 8×10 11 vg / eye, 9×10 11 vg / eye, 10×10 11 vg / eye, 15×10 11 vg / eye, 20×10 11 vg / eye, 30×10 11 vg / eye, 40×10 11 vg / eye, 50×10 11 It can be, for example, vg / eye, etc.

[0087] (Improvement of Visual Recognition Behavioral Function) In one aspect, the present invention provides a composition for improving visual cognitive behavioral functions (e.g., improvement of light-dark discrimination function, improvement of light avoidance function and / or crisis avoidance function) comprising a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. Any embodiment described in the section (chimeric rhodopsin) can be used for the chimeric protein used in this aspect. Also, it is understood that any embodiment described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment form used in this aspect. Functions such as improvement of visual cognitive behavioral functions (e.g., improvement of light-dark discrimination function, improvement of light avoidance function and / or crisis avoidance function) have been verified in the present invention using experimental models and can be said to have significant effects. The effects of visual cognitive behavioral functions (e.g., improvement of light-dark discrimination function, improvement of light avoidance function and / or crisis avoidance function) have been demonstrated by the results of tests using the light-dark box choice test (LDT) demonstrated in Example 2 (see Figure 3). Visual cognitive behavioral functions are functions that can be confirmed not only by confirming the light sensitivity of the visual organ but also by verifying whether they actually appear as behaviors in animal models and the like. The fact that verification was possible in experiments such as Example 2 (see Figure 3) can be said to be one of the achievements of the present invention. Improvement of visual cognitive behavioral functions includes improvement, enhancement or augmentation of visual acuity, contrast sensitivity, light-dark adaptation, color vision, etc.

[0088] In another aspect, the present invention provides a composition for improving visual cognitive behavioral functions (e.g., improvement of light-dark discrimination function, improvement of light avoidance function and / or crisis avoidance function) comprising a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. Any embodiment described in the section (chimeric rhodopsin) can be used for the chimeric protein used in this aspect. Also, it is understood that any embodiment described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment form used in this aspect.

[0089] In another aspect, the present invention provides a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for improving visual cognitive behavioral functions (for example, improvement of light and dark determination function, improvement of light avoidance function and / or crisis avoidance function). The chimeric protein used in this aspect can use any embodiment described in the section of (chimeric rhodopsin). It is understood that any embodiment described in the section of (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment modalities used in this aspect.

[0090] In yet another aspect, the present invention provides a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for improving visual cognitive behavioral functions (for example, improvement of light and dark determination function, improvement of light avoidance function and / or crisis avoidance function). The chimeric protein used in this aspect can use any embodiment described in the section of (chimeric rhodopsin). It is understood that any embodiment described in the section of (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment modalities used in this aspect.

[0091] In another aspect, the present invention provides a method for improving visual cognitive behavioral functions (for example, improvement of light and dark determination function, improvement of light avoidance function and / or crisis avoidance function) in a subject, the method comprising administering to the subject an effective amount of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. The chimeric protein used in this aspect can use any embodiment described in the section of (chimeric rhodopsin). It is understood that any embodiment described in the section of (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment modalities used in this aspect.

[0092] In yet another aspect, the present invention provides a method for improving visual cognitive behavioral functions (e.g., improving light-dark discrimination function, improving light avoidance function and / or crisis avoidance function) in a subject, the method comprising administering to the subject an effective amount of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. The chimeric protein used in this aspect can employ any of the embodiments described in the section (chimeric rhodopsin). It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment modality used in this aspect.

[0093] In yet another aspect, the present invention provides the use of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for improving visual cognitive behavioral functions (e.g., improving light-dark discrimination function, improving light avoidance function and / or crisis avoidance function). The chimeric protein used in this aspect can employ any of the embodiments described in the section (chimeric rhodopsin). It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment modality used in this aspect.

[0094] In yet another aspect, the present invention provides the use of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for improving visual cognitive behavioral functions (e.g., improving light-dark discrimination function, improving light avoidance function and / or crisis avoidance function). The chimeric protein used in this aspect can employ any of the embodiments described in the section (chimeric rhodopsin). It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment modality used in this aspect.

[0095] (Enhancement of visual function and improvement of visual acuity) In one aspect, the present invention provides a composition for improving vision, comprising a nucleic acid encoding a chimeric protein of an ion-transporting receptor rhodopsin and a G-protein coupled receptor rhodopsin. The chimeric protein used in this aspect can employ any of the embodiments described in the section (chimeric rhodopsin). It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) can be applied to the treatment modality used in this aspect. The vision-improving function has been verified in an experimental model in the present invention and can be said to have a remarkable effect. Enhancement of visual functions such as vision improvement was confirmed by the experiment of visual evoked potential VEP represented by Example 3 and FIG. 4.

[0096] In another aspect, the present invention provides a composition for enhancing visual function (for example, improving vision), comprising a chimeric protein of an ion-transporting receptor rhodopsin and a G-protein coupled receptor rhodopsin. The chimeric protein used in this aspect can employ any of the embodiments described in the section (chimeric rhodopsin). It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) can be applied to the treatment modality used in this aspect.

[0097] In yet another aspect, the present invention provides a nucleic acid encoding a chimeric protein of an ion-transporting receptor rhodopsin and a G-protein coupled receptor rhodopsin for enhancing visual function (for example, improving vision). The chimeric protein used in this aspect can employ any of the embodiments described in the section (chimeric rhodopsin). It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) can be applied to the treatment modality used in this aspect.

[0098] In yet another aspect, the present invention provides a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin for enhancing visual function (e.g., improving eyesight). Any of the embodiments described in the section (chimeric rhodopsin) can be used for the chimeric protein used in this aspect. It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment modality used in this aspect.

[0099] In yet another aspect, the present invention provides a method for enhancing visual function (e.g., improving eyesight) in a subject, the method comprising administering to the subject an effective amount of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. Any of the embodiments described in the section (chimeric rhodopsin) can be used for the chimeric protein used in this aspect. It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment modality used in this aspect.

[0100] In yet another aspect, the present invention provides a method for enhancing visual function (e.g., improving eyesight) in a subject, the method comprising administering to the subject an effective amount of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin. Any of the embodiments described in the section (chimeric rhodopsin) can be used for the chimeric protein used in this aspect. It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) is applicable to the treatment modality used in this aspect.

[0101] In yet another aspect, the present invention provides the use of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for enhancing visual function (for example, improving eyesight). The chimeric protein used in this aspect can employ any of the embodiments described in the section (chimeric rhodopsin). It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) are applicable to the treatment modalities used in this aspect.

[0102] In yet another aspect, the present invention provides the use of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for enhancing visual function (for example, improving eyesight). The chimeric protein used in this aspect can employ any of the embodiments described in the section (chimeric rhodopsin). It is understood that any of the embodiments described in the section (prevention or suppression of progression of retinal diseases, disorders or symptoms) are applicable to the treatment modalities used in this aspect.

[0103] As used herein, "or" is used when "at least one or more" of the items listed in the text can be adopted. The same applies to "or". When it is specified herein that it is "within a range of two values", the range includes the two values themselves.

[0104] References such as scientific literature, patents, patent applications, etc. cited herein are hereby incorporated by reference in their entirety to the same extent as if each were specifically set forth herein.

[0105] As described above, the present invention has been described with reference to preferred embodiments for ease of understanding. Hereinafter, the present invention will be described based on examples. However, the above description and the following examples are provided for illustrative purposes only and not for the purpose of limiting the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

Example

[0106] Examples are described below. When necessary, the handling of animals used in the following examples complied with the standards defined at Keio University and other relevant ethical standards and guidelines, and was carried out based on the Helsinki Declaration. Specifically, the reagents used were the products described in the examples, but equivalents from other manufacturers (Sigma-Aldrich, Wako Pure Chemical Industries, Ltd., Nacalai Tesque, Inc., R&D Systems, USCN Life Science INC, etc.) can also be substituted.

[0107] (Vector Preparation) DNA encoding a chimeric protein (GR / BvRh) was prepared as follows. The sequence corresponding to amino acids 137 - 145 from the N-terminus, which corresponds to the second loop on the cytoplasmic side of Gloeobacter violaceus Rhodopsin (GR) (SEQ ID NO: 8), was replaced with the sequence corresponding to amino acids 137 - 145 of bovine rhodopsin (BvRh) (SEQ ID NO: 9). Also, the sequence corresponding to amino acids 198 - 206 from the N-terminus, which corresponds to the third loop on the cytoplasmic side of GR, was replaced with the sequence corresponding to amino acids 225 - 252 of bovine rhodopsin. Furthermore, DNA encoding a chimeric protein in which the 132nd amino acid of GR, glutamic acid, was replaced with glutamine was inserted into the pCDNA3.1 vector. Alternatively, a nucleic acid having the nucleotide sequence set forth in SEQ ID NO: 10 was generated, and this was inserted into the HindIII / XbaI site of the pCDNA3.1 vector as DNA encoding a chimeric protein. The nucleotide sequence set forth in SEQ ID NO: 10 was generated as follows: The sequence corresponding to amino acids 137 - 145 from the N-terminus, which corresponds to the second loop on the cytoplasmic side of Gloeobacter violaceus Rhodopsin (GR) (SEQ ID NO: 8), was replaced with the nucleotide sequence shown in SEQ ID NO: 12, which corresponds to the second loop of bovine rhodopsin (BvRh) (encoding the amino acid sequence shown in SEQ ID NO: 6). Also, the sequence corresponding to amino acids 198 - 206 from the N-terminus, which corresponds to the third loop on the cytoplasmic side of GR, was replaced with the nucleotide sequence shown in SEQ ID NO: 13, which corresponds to the third loop of bovine rhodopsin (encoding the amino acid sequence shown in SEQ ID NO: 7). The mutants were prepared by the QuikChange method. Note that the sequence portion adopted in bovine rhodopsin exactly matches the amino acid sequence of human rhodopsin, so it can also be referred to as human rhodopsin without problem.

[0108] The EGFP or GR / BvRh gene was subcloned into the AAV2 shuttle plasmid to generate the viral expression constructs AAV2-CAGGS-EGFP-WPRE-pA (vector for EGFP expression) and AAV2-CAGGS-GR / BvRh-WPRE-pA (vector for chimeric protein expression). The viral vector packaging was performed by transfecting three types of plasmids, namely the vector plasmid, AAV vector plasmid, and adenovirus helper plasmid, into HEK293 cells. The cesium chloride method was used for the purification of the viral vector. In the vector, "ITR" is the abbreviation of "Inverted Terminal Repeat". "CAGGS" is the sequence of the CAG promoter region. "WPRE" is the abbreviation of "woodchuck hepatitis virus post-transcriptional regulatory element". "pA" means a peptide tag. "EGFP" is the abbreviation of "enhanced green fluorescent protein".

[0109] Note that all numerical values in the examples are shown as mean ± SEM.

[0110] Hereinafter, using an adeno-associated virus vector (AAV) (type 2 or DJ type) incorporating the chimeric rhodopsin gene, intravitreal injection or subretinal injection was performed on a retinitis pigmentosa model mouse (rd1 mouse or P23H mouse) under anesthesia to induce the expression of chimeric rhodopsin in retinal ganglion cells or photoreceptor cells, and the visual regeneration and preventive effects were confirmed using electroretinogram (ERG) and optical coherence tomography (OCT).

[0111] (Example 1: Experiment on thinning of photoreceptor cells) In this example, by conducting an experiment on the thinning of photoreceptor cells, it was verified whether preventive and inhibitory effects on the onset of retinal degenerative diseases such as retinitis pigmentosa could be achieved.

[0112] (Method) (Animals) The P23H mouse (Rho P23H / + ), which has been established as a retinitis pigmentosa model, was used. Specifically, Rho P23H / P23H mice purchased from Jackson Laboratory (Bar Harbor, ME, USA) were crossed with C57BL / 6J mice (purchased from CLEA Japan, Inc.). This model is suitable for observing the suppression of the progression of retinitis pigmentosa.

[0113] (Vector administration) At 0 - 3 days after birth, 0.5 μl of the AAV DJ-CAGGS-Chimeric rhodopsin (GR / BvRh)-WPRE-pA vector (the amino acid sequence of chimeric rhodopsin is SEQ ID NO: 1, and the nucleotide sequence is represented by SEQ ID NO: 10, etc.) was administered by subretinal injection at a concentration of 1.0×10 9 vg / μl (equivalent to 1.0×10 11 vg / μl in human terms). The AAV DJ-CAGGS-EGFP-WPRE-pA vector was administered in the same amount to the control group.

[0114] At 24 days old and 31 days old, retinal tomograms were taken using an SD-OCT (spectral domain-optical coherence tomography) system (Envisu R4310; Leica, Wetzlar, Germany). The mice were sedated with a mixture of three anesthetics (midazolam, medetomidine, and butorphanol tartrate were administered at 4 mg / kg, 0.75 mg / kg, and 5 mg / kg body weight, respectively). The thickness of the retinal photoreceptor layer 150 μm from the optic nerve head was measured in four directions: superior, inferior, nasal, and temporal, and compared.

[0115] Similarly, electroretinograms were measured at 30 days old and 42 days old.

[0116] After more than 8 hours of dark adaptation, the mice were sedated with a mixture of three anesthetics (midazolam, medetomidine, and butorphanol tartrate at 4 mg / kg, 0.75 mg / kg, and 5 mg / kg body weight, respectively). Stimulation was performed with a White LED for three levels of measurement: rod response (0.01 cd·s·m -2 ), mixed response (3.0 cd·s·m -2 ), and cone response (3.0 cd·s·m -2 ). (n = 6 for each). The PuREC acquisition system (Mayo, Inazawa, Japan) was used as the measuring instrument.

[0117] (Results) Results of OCT: At 24 days of age, there was no significant difference between the control group (49.6 ± 12.4 μm) and the treatment group (61.25 ± 4.44 μm), but a trend was observed (Figure 1). At 31 days of age, the photoreceptor layer was significantly better preserved in the mice transfected with the chimeric rhodopsin gene (50.7 ± 2.87 μm) compared to the control group (31.8 ± 5.15 μm) (upper panel of Figure 1).

[0118] Results of ERG: At 30 days of age, in all cases, the eyes treated with the chimeric rhodopsin gene showed a tendency for larger amplitudes (Figure 2, upper left). There were significant differences in the rod response (untreated 24.5 ± 13.2 μV, treated 124 ± 43.0 μV), mixed response (untreated 24.5 ± 13.2 μV, treated 233 ± 77.3 μV), and cone response (untreated 24.5 ± 13.2 μV, treated 176 ± 56.9 μV) in the rod response (Figure 2, lower left, upper right, lower right).

[0119] At 42 days of age, significant differences in amplitude were observed for all stimuli (Figure 2, upper left). Also, significant differences were observed in rod responses (untreated: 49.8 ± 16.6 μV, treated: 172 ± 19.6 μV), mixed responses (untreated: 118 ± 28.5 μV, treated: 295 ± 36.2 μV), and cone responses (untreated: 92.6 ± 29.2 μV, treated: 258 ± 24.1 μV) (Figure 2, lower left, upper right, lower right).

[0120] (Discussion) It is considered that the expression of the chimeric rhodopsin gene used exemplarily in the present invention produced an effect of suppressing the progression of retinal degeneration.

[0121] In the results of electroretinogram (Figure 1, upper panel), the significant difference in only rod responses from 30 days of age may reflect that retinitis pigmentosa causes damage predominantly in rods. Also, in the treated eyes, the larger amplitude at 42 days of age when degeneration should have progressed more than at 30 days of age is considered to be due to the addition of the light response by chimeric rhodopsin expression.

[0122] From the above, it was demonstrated by this example that prevention or suppression of the progression of retinal degeneration diseases such as retinitis pigmentosa (especially before onset, immediately after onset, or in the early stage of onset) is possible. Such an effect is very remarkable clinically.

[0123] (Example 2: Measurement of light and dark discrimination function) Next, the influence of the present invention on the light and dark discrimination function was measured. This is described below.

[0124] (Materials and methods) (Animals) Another retinitis pigmentosa model, rd1 mice (Pde6b rd1 / rd1 ) were used. C3H / HeJ Jcl mice having the above mutation were purchased from CLEA Japan, Inc.

[0125] (Vector administration) In blind rd1 mice after 10 weeks of age, 1 μl of the AAV DJ-CAGGS-Chimeric rhodopsin (GR / BvRh)-WPRE-pA vector prepared in the preparation example was administered by intravitreal injection at a concentration of 1.0×10 9 vg / μl (equivalent to 1.0×10 11 vg / μl in human terms). The control group was administered the same amount of the AAV DJ-CAGGS-EGFP-WPRE-pA vector (vector for EGFP expression).

[0126] (Measurement) Measurements were taken more than 4 weeks after injection when gene expression reached its peak. The mice were placed in a light-dark box (acrylic case, width: 415 mm, height: 300 mm, depth: 250 mm, divided into two parts by a partition, one part with 20 lux of light entering and the other part being a dark room connected by a 5x5 mm window). Their behavior for 10 minutes was recorded on video. The ratio of the staying time in the light area to that in the dark area was measured and compared. Normal mice avoid light and stay longer in the dark, while retinal degeneration mice have almost the same staying time in light and dark.

[0127] (Results) In healthy mice (B6), the staying time in the light area is short (0.137±0.062) because they avoid the light area. On the other hand, in blind mice (rd1), the staying time ratio is approximately 0.5 (0.48±0.052), which is about half. In contrast, in the treated mice, a significant shortening of the staying time in the light area (0.24±0.049) was observed (Figure 3).

[0128] (Discussion) The treatment in this example demonstrated the regeneration of the light-dark discrimination function and the light area avoidance function in the behavioral experiment. It was found that the ability to avoid danger was restored or conferred. From the above, this example demonstrated that the present invention can improve or restore visual cognitive functions such as the improvement of the light-dark discrimination function, the improvement of the light area avoidance function, and / or the danger avoidance function.

[0129] (Example 3: Demonstration of enhanced visual function) Next, the effect of the present invention on enhancing visual function (for example, improving eyesight) was measured by visual evoked potential (VEP). This is described below.

[0130] (Materials and Methods) (Animals) Another retinitis pigmentosa model, rd1 mice (Pde6b rd1 / rd1 ) was used. C3H / HeJ Jcl mice with the above mutation were purchased from CLEA Japan, Inc.

[0131] (Vector Administration) For rd1 mice that had become blind after 10 weeks of age, the AAV DJ-CAGGS-Chimeric rhodopsin (GR / BvRh)-WPRE-pA vector was administered at a concentration of 1.0×10 9 vg / μl (equivalent to 1.0×10 11 vg / μl in humans) at a volume of 1 μl by intravitreal injection. The same volume of the AAV DJ-CAGGS-EGFP-WPRE-pA vector was administered to the control group.

[0132] (Measurement) Visual Evoked Potential (VEP) was measured more than 4 weeks after injection when gene expression reached its peak. One week before the measurement, the mice were anesthetized with the above three types of mixed anesthesia, and a measurement electrode was implanted into the skull near the visual cortex (1.5 mm in front and 1.5 mm to the side of the lambda suture).

[0133] After the measurement was repeated under sedation with the three types of mixed anesthesia, the evoked potential in response to a flash stimulus of 0.1 cds / m 2 from a White LED placed 3 cm in front of the eyes was measured. The PuREC acquisition system (Mayo, Inazawa, Japan) was used as the measurement device.

[0134] (Results) A significant increase in amplitude was observed in the chimeric treatment mice (50.0±3.49 μV) compared to the control (35.12±3.90 μV) (Figure 4).

[0135] (Examination) A visual regeneration effect at the central level was also observed by the treatment. According to this example, it can be said that the effect of enhancing visual function (improving visual acuity) was demonstrated.

[0136] (Note) As described above, the present invention has been illustrated using preferred embodiments of the present invention, but it is understood that the scope of the present invention should be interpreted only by the claims. It is understood that the patents, patent applications, and documents cited in this specification should be incorporated by reference into this specification as if the contents themselves were specifically described in this specification.

Industrial Applicability

[0137] Medicines for preventing and suppressing the progression of retinal diseases, visual cognitive behavioral functions (for example, improving light-dark discrimination function, improving light avoidance function and / or crisis avoidance function), and enhancing visual acuity are provided. Technologies applicable in industries (such as pharmaceuticals) based on such technologies are provided.

Sequence Listing Free-Text

[0138] SEQ ID NO: 1: An example of the amino acid sequence of the chimeric rhodopsin of the present invention SEQ ID NO: 2: An example of the amino acid sequence of the chimeric rhodopsin of the present invention SEQ ID NO: 3: An example of the amino acid sequence of the chimeric rhodopsin of the present invention SEQ ID NO: 4: An example of the amino acid sequence of the chimeric rhodopsin of the present invention SEQ ID NO: 5: An example of the amino acid sequence of the second loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin of the present invention SEQ ID NO: 6: An example of the amino acid sequence of the second loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin of the present invention SEQ ID NO: 7: An example of the amino acid sequence of the third loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin of the present invention SEQ ID NO: 8: The amino acid sequence of Gloeobacter violaceus Rhodopsin (GR) Accession No. 9: Amino acid sequence of bovine rhodopsin (BvRh) Accession No. 10: An example of the nucleotide sequence of the chimeric rhodopsin of the present invention (corresponding to SEQ ID NO: 1). The start codon corresponds to nucleotides 43-45, and the stop codon corresponds to nucleotides 994-996. Accession No. 11: An example of the nucleotide sequence corresponding to the second cytoplasmic loop of the G protein-coupled receptor rhodopsin (corresponding to SEQ ID NO: 5) Accession No. 12: Another example of the nucleotide sequence corresponding to the second cytoplasmic loop of the G protein-coupled receptor rhodopsin (corresponding to SEQ ID NO: 6) Accession No. 13: An example of the nucleotide sequence corresponding to the third cytoplasmic loop of the G protein-coupled receptor rhodopsin Accession No. 14: Amino acid sequence of human rhodopsin (huRh) Accession No. 15: Nucleotide sequence of human rhodopsin (huRh) Accession No. 16: Nucleotide sequence of bovine rhodopsin (BvRh) Accession No. 17: Nucleotide sequence of Gloeobacter violaceus Rhodopsin (GR)

Claims

1. A composition for enhancing visual function, comprising a nucleic acid encoding a chimeric protein of an ion transport type receptor rhodopsin and a G protein-coupled receptor rhodopsin.

2. The composition according to claim 1, characterized in that it is administered once.

3. 0.1×10 １１ ~10×10 １１ administered at a unit dose of vg / eye, characterized in that The composition according to claim 1 or 2.

4. Among the base sequences encoding the ion transport type receptor rhodopsin, the base sequence encoding the second loop on the cytoplasmic side and / or the third loop on the cytoplasmic side is replaced with the base sequence encoding the second loop on the cytoplasmic side and / or the third loop on the cytoplasmic side of the G protein-coupled receptor rhodopsin. The composition according to any one of claims 1 to 3.

5. The composition according to any one of claims 1 to 4, wherein the ion transport type receptor rhodopsin is derived from cyanobacteria (blue bacteria).

6. The composition according to any one of claims 1 to 5, wherein the G protein-coupled receptor rhodopsin is derived from a mammal.

7. The composition according to any one of claims 1 to 6, wherein the chimeric protein has an amino acid sequence in which glutamic acid corresponding to the 132nd position of the amino acid sequence of SEQ ID NO: 8 is replaced with glutamine.

8. The chimeric protein is (a) the amino acid sequence described in any one of SEQ ID NOs: 1 to 4 or a fragment thereof; (b) an amino acid sequence having at least 80% identity to (a); (c) an amino acid sequence having one or more amino acids substituted, added and / or deleted with respect to (a) or (b); and has one of the above and has biological activity, or The nucleic acid encoding the chimeric protein is (A) a nucleic acid having a base sequence encoding the amino acid sequence described in any one of SEQ ID NOs: 1 to 4 or the base sequence described in SEQ ID NO: 10 or a fragment thereof; (B) a nucleic acid having at least 80% identity to (A); (C) a nucleic acid having one or more nucleotides substituted, added and / or deleted with respect to (A) or (B); (D) a nucleic acid that hybridizes under stringent conditions to any one of (A) to (C), and has one of the above and the chimeric protein has biological activity, The composition according to any one of claims 1 to 7.

9. The composition according to any one of claims 1 to 8, wherein the base sequence is contained in a vector.

10. The composition according to any one of claims 1 to 9, wherein the visual function enhancement includes visual acuity improvement.

11. A composition for enhancing visual function, comprising a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin.

12. The composition according to claim 11, wherein the visual function enhancement includes visual acuity improvement.

13. Use of a nucleic acid encoding a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for enhancing visual function.

14. The use according to claim 13, wherein the visual function enhancement includes visual acuity improvement.

15. Use of a chimeric protein of an ion transport receptor rhodopsin and a G protein-coupled receptor rhodopsin in the manufacture of a medicament for enhancing visual function.

16. The use according to claim 15, wherein the visual function enhancement includes visual acuity improvement.

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