Modified channelrhodopsin

Abstract

The present invention addresses the problem of providing a modified channelrhodopsin having high ion permeability (photoreactivity). The problem is solved by substituting, of the three extracellular domains of a channelrhodopsin derived from volvox, the third extracellular domain from the N-terminal side, with a corresponding extracellular domain of channelrhodopsin-2 derived from Chlamydomonas reinhardtii.

Description

[Technical Field]

[0001] The present invention relates to modified channelrhodopsins, and more particularly to modified channelrhodopsins with high ion permeability (light reactivity). [Background technology]

[0002] It is well known that research is being conducted worldwide to restore visual function using optogenetics, a technique in which light is applied to neurons that have been transfected with genes to express a light-responsive protein (channelrhodopsin), thereby controlling the cellular response. The present inventors have also reported in Patent Document 1 a modified channelrhodopsin in which the N-terminal region of Volvox carteri-derived channelrhodopsin was replaced with the N-terminal region of Chlamydomonas reinhardtii-derived channelrhodopsin-1, thereby improving the expression efficiency on the cell membrane. This modified channelrhodopsin is capable of opening ion channels and inducing excitation when exposed to light, and the present inventors have succeeded in restoring vision in blind rats by introducing the gene into their retinas.

[0003] However, there is a need for modified channelrhodopsins that have higher ion permeability than those reported so far. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent No. 5322067 Summary of the Invention [Problem to be solved by the invention]

[0005] Therefore, an object of the present invention is to provide a modified channelrhodopsin with high ion permeability. [Means for solving the problem]

[0006] In light of the above, the inventors have conducted extensive research and found that a channelrhodopsin with high ion permeability can be obtained by replacing the third extracellular domain from the N-terminus of the three extracellular domains of Volvox-derived channelrhodopsin with the corresponding extracellular domain of Chlamydomonas reinhardtii-derived channelrhodopsin-2.

[0007] The modified channelrhodopsin of the present invention, which has been made based on the above findings, is a polypeptide in which the third extracellular domain counting from the N-terminus of the three extracellular domains of Volvox-derived channelrhodopsin is substituted with the corresponding extracellular domain of Chlamydomonas reinhardtii-derived channelrhodopsin-2, as described in claim 1, and has the following (a): or (b) It is composed of polypeptides. (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 ( b ) A polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and having channelrhodopsin function. The modified channelrhodopsin according to claim 2 is the modified channelrhodopsin according to claim 1, which is composed of a polypeptide having the amino acid sequence shown in SEQ ID NO:8. The polynucleotide of the present invention is also 3 As described above, claim 1 or 2 The polypeptides that constitute the modified channelrhodopsins described herein are encoded. The expression vector of the present invention also includes the following: 4 Claim operably linked to a promoter as described above. 3 The polynucleotides described herein are included. The cells of the present invention are also 5 As described above, claim 1 or 2 The polypeptides that make up the modified channelrhodopsins described are expressed. Also, claims 6 The cells described are 5 In the described cells, the cells are neuronal cells. The present invention also provides the following claims: 7 1. A method for the manufacture of a medicament for treating a subject suffering from an outer retinal disorder, comprising: or 2 A polypeptide constituting the modified channelrhodopsin of claim 3 The polynucleotides described in claims 4 The use of any of the expression vectors described. Also, claims 8 The described uses are 7 In the described use, the disorder of the outer retina is any one of retinitis pigmentosa, age-related macular degeneration, and retinal detachment. The pharmaceutical composition for treating damage to the outer retina of the present invention is also 9 As described above, claim 1 or 2 The polypeptide constituting the modified channelrhodopsin described above or claims 4 It contains any of the expression vectors described above as an active ingredient. [Effects of the Invention]

[0008] According to the present invention, a modified channelrhodopsin having high ion permeability can be provided. [Brief explanation of the drawings]

[0009] [Figure 1] 1 shows the structure of a plasmid for preparing a p525-expressing adeno-associated virus vector in Example 1. [Figure 2] 1 is a graph showing that p525, p548, and p550 have higher ion permeability than mVChR1 in Test Example 1. [Figure 3] 1 is a graph showing the ion permeability of p578, p579, p580, and p581 in Test Example 4. [Figure 4] 1 is a graph showing that p579 has a shorter τon than p548 in Test Example 5. [Figure 5]10 is a graph showing that p579 has a shorter τoff than p548. [Figure 6] 10 is a graph showing that in Test Example 6, visual evoked potentials can be recorded by introducing the p548 gene into the retina. [Figure 7] 10. The photograph shows that when retinal spread preparations are observed under a fluorescence microscope, p548 expression can be confirmed throughout the neural retina. [Figure 8] 10A and 10B are photographs showing that when retinal slice preparations are observed under a fluorescence microscope, p548 expression can be confirmed mainly in the retinal ganglion cell layer. DETAILED DESCRIPTION OF THE INVENTION

[0010] The modified channelrhodopsin of the present invention is a polypeptide in which the third extracellular domain from the N-terminus of the three extracellular domains of Volvox-derived channelrhodopsin is replaced with the corresponding extracellular domain of Chlamydomonas reinhardtii-derived channelrhodopsin-2.

[0011] A specific example of a Volvox-derived channelrhodopsin is a polypeptide comprising at least the amino acids 67 to 322 of the amino acid sequence shown in SEQ ID NO: 1. The polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1 is a modified channelrhodopsin reported by the present inventors in Patent Document 1 (a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 in Patent Document 1). This modified channelrhodopsin is a polypeptide consisting of 344 amino acids, in which the N-terminal region of Volvox-derived channelrhodopsin is replaced with the N-terminal region of Chlamydomonas reinhardtii-derived channelrhodopsin-1 (a region involved in plasma membrane-localized expression, not including the transmembrane domain) to improve its expression efficiency on the plasma membrane. The amino acids 1 to 66 of the amino acid sequence shown in SEQ ID NO: 1 correspond to the amino acids 1 to 66 of the amino acid sequence of Chlamydomonas reinhardtii-derived channelrhodopsin-1 shown in SEQ ID NO: 2. In the present invention, the modified channelrhodopsin, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 1, is referred to as mVChR1. Furthermore, the matters described in Patent Document 1 are treated as matters described in this specification.

[0012] The amino acid sequence of mVChR1 shown in SEQ ID NO: 1 is as follows: Of the three extracellular domains possessed by mVChR1, the third extracellular domain (EX3), counting from the N-terminus, spans Gly271 to Ser279.

[0013] (amino acid sequence of mVChR1) MSRRPWLLALALAVALAAGSAGASTGSDATVPVATQDGPDYVFHRAHERMLFQTSYTLENNGSVICMPR GQCYCEGWLRSRGT SIEKTIAITLQWVVFALSVACLGWY AYQAWRATCG WEEVYVALIEMMKSIIEAFH TM1 IN1 TM2 EFDSPATLWLSSGNGVV WMRYGEWLLTCPVLLIHL SNLTGLKDDYS KRTMGLLVSDVGCIVWGATSAM C EX1 TM3 IN2 TM4 E T.G. WTKILFFLISLSYGMYTYFHAAKVYIEAFH TVPKG ICRELVRVMAWTFFVAWGMFPVLFLL GTEGFG X2TM5 IN3 TM6 EX3 HIS PYGSAIGHSILDLIAKNMWGVLGNYLRVKIHEHILLY GDIRKKQKITIAGQEMEVETLVAEEEDR TM7 *TM: transmembrane domain IN: intracellular domain EX: extracellular domain

[0014] Taking mVChR1 as an example, the modified channelrhodopsin of the present invention is a polypeptide in which the above-mentioned EX3 is replaced with the corresponding extracellular domain of Chlamydomonas reinhardtii-derived channelrhodopsin-2, i.e., the third extracellular domain counting from the N-terminus of the three extracellular domains possessed by Chlamydomonas reinhardtii-derived channelrhodopsin-2. The amino acid sequence of Chlamydomonas reinhardtii-derived channelrhodopsin-2 is as shown in SEQ ID NO: 3, and the third extracellular domain counting from the N-terminus is Gly233 to Ser241. In conjunction with the substitution of EX3, the transmembrane domains before and after it, i.e., the amino acids on the C-terminus of TM6 adjacent to the N-terminus of EX3 and / or the amino acids on the N-terminus of TM7 adjacent to the C-terminus of EX3, may also be substituted. However, the number of amino acids that may be substituted is preferably a maximum of three. Substitution of more than three amino acids may affect the function of the transmembrane domain.

[0015] The modified channelrhodopsin of the present invention may be a polypeptide in which the third extracellular domain counting from the N-terminus of the three extracellular domains of Volvox-derived channelrhodopsin is replaced with the corresponding extracellular domain of Chlamydomonas reinhardtii-derived channelrhodopsin-2, and in which other domains or regions have been further modified. For example, in the case of mVChR1, the polypeptide may be a polypeptide in which the amino acids 142 to 169 of the amino acid sequence shown in SEQ ID NO: 1 are replaced with the amino acids 143 to 170 of the amino acid sequence of Chlamydomonas reinhardtii-derived channelrhodopsin-1 shown in SEQ ID NO: 2.

[0016] A specific example of such a modified channelrhodopsin is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4. This polypeptide (p525) comprises EX3 of mVChR1 substituted with the corresponding extracellular domain of channelrhodopsin-2 derived from Chlamydomonas reinhardtii, and amino acids 142 to 169 of mVChR1 substituted with amino acids 143 to 170 of channelrhodopsin-1 derived from Chlamydomonas reinhardtii shown in SEQ ID NO: 2 (note that Pro at position 280 of TM7 is substituted with Val in conjunction with the substitution of EX3).

[0017] Furthermore, the modified channelrhodopsin of the present invention may be a polypeptide in which, for example, the sixth transmembrane domain (TM6) counting from the N-terminus of mVChR1 is replaced with the corresponding transmembrane domain of a channelrhodopsin derived from Chloromonas oogama. The amino acid sequence of the channelrhodopsin derived from Chloromonas oogama is as shown in SEQ ID NO: 5, and the sixth transmembrane domain counting from the N-terminus is from Arg187 to Val212.

[0018] Furthermore, the modified channelrhodopsin of the present invention may be a polypeptide in which, for example, in the case of mVChR1, the amino acids from position 323 onwards in the amino acid sequence shown in SEQ ID NO: 1 are replaced with the amino acids from position 265 onwards in the amino acid sequence of the channelrhodopsin derived from Chloromonas grandiflora shown in SEQ ID NO: 5.

[0019] A specific example of such a modified channelrhodopsin is a polypeptide consisting of the amino acid sequence set forth in SEQ ID NO: 6. In this polypeptide (p548), EX3 of mVChR1 is replaced with the corresponding extracellular domain of channelrhodopsin-2 derived from Chlamydomonas reinhardtii, and amino acids 142 to 169 of mVChR1 are replaced with amino acids 143 to 170 of channelrhodopsin-1 derived from Chlamydomonas reinhardtii as set forth in SEQ ID NO: 2. Furthermore, TM6 of mVChR1 is replaced with the corresponding transmembrane domain of channelrhodopsin derived from Chloromonas grandiflora, and amino acids 323 to 344 of mVChR1 are replaced with amino acids 265 to 286 of channelrhodopsin derived from Chloromonas grandiflora as set forth in SEQ ID NO: 5 (note that Pro at position 280 of TM7 is replaced with Val due to the substitution of EX3).

[0020] Another specific example is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7. This polypeptide (p550) has EX3 of mVChR1 substituted with the corresponding extracellular domain of channelrhodopsin-2 derived from Chlamydomonas reinhardtii, and amino acids 142 to 169 of mVChR1 substituted with amino acids 143 to 170 of channelrhodopsin-1 derived from Chlamydomonas reinhardtii shown in SEQ ID NO: 2, and further has TM6 of mVChR1 substituted with the corresponding membrane domain of channelrhodopsin derived from Chlamydomonas gypsum. The amino acids 323 to 344 of mVChR1 have been replaced with amino acids 265 to 286 of the channelrhodopsin derived from Chloromonas grandiflora shown in SEQ ID NO: 5, which is the same as the polypeptide (p548) consisting of the amino acid sequence shown in SEQ ID NO: 6 above, but in addition, the amino acids 28 to 60 of mVChR1 have been deleted (note that in conjunction with the substitution of EX3, Pro at position 280 of TM7 has been replaced with Val).

[0021] The modified channelrhodopsins of the present invention include polypeptides having one or more amino acid deletions, substitutions, additions, or insertions in the amino acid sequences shown in SEQ ID NOs: 4, 6, and 7, respectively, and having channelrhodopsin function. The modified channelrhodopsins of the present invention also include polypeptides consisting of amino acid sequences having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 6, and 7, respectively, and having channelrhodopsin function. Here, "multiple" refers to integers of 50 or less, preferably integers of 30 or less, and more preferably integers of 10 or less, for example, 2 to 9, 2 to 7, or 2 to 5. The sequence identity with the amino acid sequences shown in SEQ ID NOs: 4, 6, and 7, respectively, is preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99%. The percent identity refers to a value calculated using software (e.g., FASTA, DANASYS, BLAST, etc.) that calculates identity between multiple (two) amino acid sequences with default settings. A specific example of such a modified channelrhodopsin is a polypeptide (p528) consisting of the amino acid sequence shown in SEQ ID NO: 8, in which Trp at position 210 of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 is substituted with Tyr and Thr at position 211 is substituted with Val.Further examples include polypeptides in which the His at position 172 of the polypeptides consisting of the amino acid sequences shown in SEQ ID NOs: 4, 6, and 8, or the His at position 139 of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, has been replaced with another amino acid, such as Gly, Ala, Lys, or Arg. Specifically, examples include a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9 (p578) in which the His at position 172 of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 has been replaced with Gly, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10 (p579) in which the His at position 172 has been replaced with Ala, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11 (p580) in which the His at position 172 has been replaced with Lys, and a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12 (p581) in which the His at position 172 has been replaced with Arg. Substitution of His at position 172 of the polypeptides consisting of the amino acid sequences shown in SEQ ID NOs: 4, 6, and 8, or His at position 139 of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7, with another amino acid can change at least one of the time from the start of light irradiation until the channel opens (opening rate: τon) and the time from the cessation of light irradiation until the channel closes (closing rate: τoff). As is well known to those skilled in the art, "having channelrhodopsin function" means having a channel function that controls ion permeability between the outside and inside of a cell by sensing light. It is preferred that at least one of the biological activities evaluated by the degree of photosensitivity, photosensitive wavelength, degree of ion permeability, τon, τoff, etc., is at least equivalent to the biological activity of a polypeptide consisting of the amino acid sequence shown in SEQ ID NOs: 4, 6, 7, and 8.

[0022] The modified channelrhodopsin of the present invention can be produced by genetic engineering techniques. Specifically, first, a polynucleotide encoding the modified channelrhodopsin of the present invention (hereinafter referred to as the "modified channelrhodopsin gene of the present invention") is prepared. The modified channelrhodopsin gene of the present invention can be prepared by techniques known to those skilled in the art. Specifically, for example, the modified channelrhodopsin gene can be prepared by chemical synthesis based on the sequence information of a polynucleotide encoding the modified channelrhodopsin reported by the present inventors in Patent Document 1, a polynucleotide encoding channelrhodopsin-2 derived from Chlamydomonas reinhardtii, and, if necessary, a polynucleotide encoding channelrhodopsin-1 derived from Chlamydomonas reinhardtii or a polynucleotide encoding channelrhodopsin derived from Chlamydomonas gamma. Alternatively, the desired region of each polynucleotide can be amplified based on the sequence information of each polynucleotide using PCR primers that amplify the desired region of each polynucleotide, and then ligated using, for example, the Gibson Assembly system (New England Biolabs). Next, the modified channelrhodopsin gene of the present invention operably linked to a promoter can be incorporated into an expression vector that can maintain replication in host cells, stably express the encoded polypeptide, and stably retain this gene, and the host can be transformed with the resulting recombinant expression vector to produce the modified channelrhodopsin of the present invention in the host. For recombinant techniques, see Proc. Natl. Acad. Sci. USA., 1984 81:5662 and Molecular Cloning: A Laboratory Manual (1989) Second Edition, Cold Spring Harbor Laboratory Press, etc.Examples of expression vectors that can be used include Escherichia coli-derived plasmids (e.g., pET28, pGEX4T, pUC118, pUC119, pUC18, pUC19, and other plasmid DNAs), Bacillus subtilis-derived plasmids (e.g., pUB110, pTP5, and other plasmid DNAs), yeast-derived plasmids (e.g., YEp13, YEp24, YCp50, and other plasmid DNAs), λ phage (λgt11 and λZAP), mammalian plasmids (pCMV and pSV40), viral vectors (e.g., animal virus vectors such as adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, lentivirus vectors, and vaccinia virus vectors, and insect virus vectors such as baculovirus vectors), plant vectors (e.g., binary vectors based on pBI), and cosmid vectors. Here, "operably linked" refers to a functional bond between a promoter sequence and a polynucleotide sequence of interest such that the promoter sequence can initiate transcription of the polynucleotide sequence of interest. The promoter is not particularly limited, and an appropriate promoter may be selected depending on the host. Known constitutive and inducible promoters can be used, but constitutive promoters are preferred. Specific examples include the CMV promoter, SV40 promoter, CAG promoter, synapsin promoter, rhodopsin promoter, CaMV promoter, glycolytic enzyme promoters, lac promoter, trp promoter, tac promoter, GAPDH promoter, GAL1 promoter, PH05 promoter, PGK promoter, thy1 promoter, GRK promoter, and RPEJ promoter.For the purpose of specifically expressing the modified channelrhodopsin of the present invention in a particular cell, the transcriptional regulatory region of a polypeptide gene specifically expressed in that cell (for example, the transcriptional regulatory region of IRBP (Interphotoreceptor retinoid binding protein), which is specifically expressed in photoreceptor cells (Marjorie Nicoud et al., The Journal of Gene Medicine, Volume 9, Issue 12, 1013-1107, December 2007)) may be ligated upstream of these promoters. The modified channelrhodopsin gene of the present invention can be inserted into an expression vector, for example, by creating or linking restriction enzyme sites flanking the modified channelrhodopsin gene of the present invention and inserting it into a restriction enzyme site or multicloning site of an appropriate vector DNA. In addition to the promoter and the modified channelrhodopsin gene of the present invention, the expression vector may optionally contain enhancers and other cis-elements, splicing signals, poly(A) addition signals, selection markers (drug resistance gene markers such as ampicillin resistance markers and tetracycline resistance markers, auxotrophy-complementing gene markers such as LEU1, TRP1, and URA3, dominant selection markers such as APH, DHFR, and TK), ribosome binding sites (RBS), and the like. Transformation of the host can be carried out using the protoplast method, spheroplast method, competent cell method, virus method, calcium phosphate method, lipofection method, microinjection method, gene bombardment method, Agrobacterium method, electroporation, and the like. The transformants thus obtained are cultured under appropriate conditions using a medium containing assimilable carbon sources, nitrogen sources, metal salts, vitamins, and the like. The transformants are typically cultured aerobically, such as by shaking or aeration with stirring, at 25 to 37°C for 3 to 6 hours. During the culture period, the pH is maintained near neutral. The pH is adjusted using inorganic or organic acids, alkaline solutions, etc. During culture, antibiotics such as ampicillin or tetracycline may be added to the medium as needed, depending on the selection marker inserted into the recombinant expression vector.Furthermore, the host used for transformation is not particularly limited as long as it can express the modified channelrhodopsin of the present invention, and examples thereof include bacteria (Escherichia coli and Bacillus subtilis), yeast (Saccharomyces cerevisiae, etc.), animal cells (COS cells, Chinese hamster ovary (CHO) cells, 3T3 cells, BHK cells, HEK293 cells, etc.), and insect cells. The modified channelrhodopsin of the present invention can be obtained in a form that retains its activity by separating and purifying it using a general method from a culture (culture supernatant, cultured cells, cultured bacterial cells, homogenate of cells or bacterial cells, etc.) obtained by culturing a transformant, followed by ultrafiltration concentration, freeze-drying, spray-drying, crystallization, etc. Alternatively, the modified channelrhodopsin of the present invention may be provided in the form of cells that express the modified channelrhodopsin of the present invention without isolation or purification. In this case, the host cells used for transformation are host cells suitable for the subsequent use, such as neurons (photoreceptors, bipolar cells, ganglion cells, etc.), preferably human neurons. Furthermore, when the modified channelrhodopsin of the present invention is used for medical purposes, it may be provided in the form of an expression vector for the modified channelrhodopsin of the present invention. In this case, it is preferable to use an expression vector that is excellent in terms of cellular introduction efficiency, intracellular replication maintenance, stability, expression efficiency, etc. Examples of such vectors include viral vectors such as adeno-associated virus vectors, retrovirus vectors, and lentivirus vectors, (autonomously replicating) plasmids, transposons, etc. Plasmids for constructing expression vectors for the modified channelrhodopsin of the present invention can be prepared, for example, according to the methods described in Tomita H et al., Invest Ophthalmol Vis Sci. 2007 Aug;48(8):3821-6 and Sugano E et al., Invest Ophthalmol Vis Sci. 2005 Sep;46(9):3341-8.

[0023] Examples of the modified channelrhodopsin gene of the present invention include a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 13 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4), a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 14 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6), a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 15 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7), a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 16 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 8), a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 17 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 9), a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 18 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 10), a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 19 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 11), and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 20 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 12). However, the modified channelrhodopsin gene of the present invention is not limited to these polynucleotides and includes polynucleotides that hybridize to the complementary strands of these polynucleotides under stringent conditions and encode a polypeptide having channelrhodopsin function. Also included are polynucleotides that have at least 90%, preferably at least 91%, more preferably at least 92%, more preferably at least 93%, more preferably at least 94%, more preferably at least 95%, more preferably at least 96%, more preferably at least 97%, more preferably at least 98%, and most preferably at least 99% sequence identity to the base sequences shown in each of SEQ ID NOs: 13 to 20, and that encode polypeptides having channelrhodopsin function.Here, "hybridization under stringent conditions" refers to, for example, hybridization for 1 to 24 hours at 30 to 50°C in 3 to 4×SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.2), 0.1 to 0.5% SDS, or preferably for 1 to 24 hours at 40 to 45°C in 3.4×SSC, 0.3% SDS, followed by washing. Examples of washing conditions include sequential washing at room temperature with a solution containing 2×SSC and 0.1% SDS, 1×SSC, and 0.2×SSC. However, the above combinations of conditions are merely examples, and those skilled in the art will be able to achieve similar stringencies by appropriately combining the above factors and other factors that determine hybridization stringency (e.g., concentration, length, and GC content of the hybridization probe, hybridization reaction time, etc.).

[0024] When the modified channelrhodopsin of the present invention is a polypeptide in which the N-terminal region of Volvox-derived channelrhodopsin is replaced with the N-terminal region of Chlamydomonas reinhardtii-derived channelrhodopsin-1 according to Patent Document 1, it retains the high expression efficiency on the cell membrane of the modified channelrhodopsin (e.g., mVChR1) described in Patent Document 1 and further possesses high ion permeability. Therefore, the modified channelrhodopsin of the present invention and an expression vector containing a polynucleotide encoding the same are useful for treating subjects suffering from disorders of the outer retina. Here, "disorder of the outer retina" refers to any disease in which visual dysfunction or visual impairment occurs due to degeneration or loss of photoreceptor cells in the outer retina, while cells other than photoreceptor cells remain normal or retain some of their function. Examples of such diseases include retinitis pigmentosa, age-related macular degeneration, and retinal detachment. The term "subject" refers to a subject who is blind or at risk of blindness due to a disorder of the outer retina. The subject is not limited to humans, but may be other mammals. Examples of other mammals include mice, rats, monkeys, rabbits, dogs, cats, cows, and horses. "Treatment of a subject suffering from a disorder of the outer retina" means restoring visual function to a subject who has become blind or is at risk of becoming blind due to a disorder of the outer retina, compared to before administration of the pharmaceutical agent of the present invention.

[0025] The pharmaceutical composition of the present invention contains the modified channelrhodopsin of the present invention or an expression vector containing a polynucleotide encoding the same as an active ingredient, and is formulated as a medicament for treating a subject suffering from a disorder of the outer retina. The effective amount is an amount that can provide a therapeutic effect for a given symptom and method of use, and is appropriately determined by a person skilled in the art through animal tests and clinical trials, taking into consideration the age, weight, sex, state and severity of the disease, and method of administration of the subject to be administered. In the case of a virus, the viral load is, for example, 10 12 ~10 13 capsids / ml (e.g., approximately 10 13The active ingredient may be formulated with one or more pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers include various buffers, such as saline, phosphate, and acetate buffers. The medicament may contain other therapeutic ingredients. Examples of other therapeutic ingredients include drugs known to treat retinitis pigmentosa, age-related macular degeneration, retinal detachment, and the like. The medicament may be formulated, for example, as an injection for topical administration, eye drops, or eyewash. Injectable preparations may be provided as unit-dose formulations in ampoules or multi-dose containers with the addition of a preservative. The medicament may also be lyophilized for reconstitution with a suitable vehicle, such as pyrogen-free sterile water, prior to use. The medicament is preferably administered by direct injection into the affected area, i.e., the retina, or by direct contact with the vitreous. [Example]

[0026] The present invention will be described in detail below with reference to examples, but the present invention should not be construed as being limited to the following description.

[0027] Example 1: Obtaining cells expressing the modified channelrhodopsin of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 4 (p525) A polynucleotide region encoding amino acids 1 to 141 of the amino acid sequence shown in SEQ ID NO: 1 of mVChR1 described in Patent Document 1, a polynucleotide region encoding amino acids 143 to 170 of the amino acid sequence shown in SEQ ID NO: 2 of Chlamydomonas reinhardtii-derived channelrhodopsin-1, a polynucleotide region encoding amino acids 170 to 270 of the amino acid sequence shown in SEQ ID NO: 1, a polynucleotide region encoding amino acids 233 to 242 of the amino acid sequence shown in SEQ ID NO: 3 of Chlamydomonas reinhardtii-derived channelrhodopsin-2, and a polynucleotide region encoding amino acids 280 to 342 of the amino acid sequence shown in SEQ ID NO: 1 were ligated, and restriction enzyme sequences were added to the 5' and 3' ends of the polynucleotide. This polynucleotide was then chemically synthesized and inserted into the multicloning site of a plasmid for constructing an adeno-associated virus vector. The structure of the resulting plasmid for constructing an adeno-associated virus vector expressing p525 is shown in Figure 1. This plasmid contains a fluorescent protein gene (venus) located in the 3' region of the multicloning site, and the gene of interest is expressed as a fusion protein with the venus gene at the C-terminus. Therefore, this plasmid was transfected into cells using the calcium phosphate method, and cells expressing p525 were identified using the venus gene as an indicator. Specifically, 1.5 mL of 0.3 M CaCl2 was added to a tube containing this plasmid solution (15 μg of plasmid) and mixed by inversion. The contents were then added to a separate tube containing 1.5 mL of 2X HBS (280 mM NaCl, 1.5 mM NaHPO4, 50 mM HEPES, pH 7.1). After mixing by inversion again, the contents were added dropwise to HEK (Human Embryonic Kidney) 293 cells, a human embryonic kidney cell line, cultured in DMEM medium containing 10% FBS. The cells were then cultured at 37°C in 5% CO2. After 6 hours, the medium was replaced with a fresh medium, and after culturing for 2 days, the cells were observed under a fluorescence microscope to confirm the expression of p525 in the cells.

[0028] Example 2: Modified channelrhodopsin of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 6 (obtaining cells expressing p548) a polynucleotide region encoding the 1st to 141st amino acids of the amino acid sequence shown in SEQ ID NO: 1 of mVChR1 described in Patent Document 1; a polynucleotide region encoding the 143rd to 170th amino acids of the amino acid sequence shown in SEQ ID NO: 2 of channelrhodopsin-1 derived from Chlamydomonas reinhardtii; a polynucleotide region encoding the 170th to 244th amino acids of the amino acid sequence shown in SEQ ID NO: 1; a polynucleotide region encoding the 187th to 212th amino acids of the amino acid sequence shown in SEQ ID NO: 5 of channelrhodopsin derived from Chlamydomonas stagnalis; Cells expressing p548 were prepared in the same manner as in Example 1, except that a polynucleotide region encoding amino acids 233 to 242 of the amino acid sequence shown in SEQ ID NO: 3 of channelrhodopsin-2 derived from Taiwan taiwan, a polynucleotide region encoding amino acids 280 to 322 of the amino acid sequence shown in SEQ ID NO: 1, and a polynucleotide region encoding amino acids 265 to 286 of the amino acid sequence shown in SEQ ID NO: 5 were linked together, and restriction enzyme sequences were added to the 5' and 3' ends of the polynucleotide.

[0029] Example 3: Modified channelrhodopsin of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 7 (obtaining cells expressing p550) A polynucleotide region encoding the 1st to 27th amino acids of the amino acid sequence shown in SEQ ID NO: 1 of mVChR1 described in Patent Document 1, a polynucleotide region encoding the 61st to 141st amino acids of the amino acid sequence shown in SEQ ID NO: 1, a polynucleotide region encoding the 143rd to 170th amino acids of the amino acid sequence shown in SEQ ID NO: 2 of channelrhodopsin-1 derived from Chlamydomonas reinhardtii, a polynucleotide region encoding the 170th to 244th amino acids of the amino acid sequence shown in SEQ ID NO: 1, and a polynucleotide region encoding the 187th to 212th amino acids of the amino acid sequence shown in SEQ ID NO: 5 of channelrhodopsin derived from Chlamydomonas gypsum. p550-expressing cells were prepared in the same manner as in Example 1, except that a polynucleotide was chemically synthesized in which a region of the polynucleotide encoding p550, a region of the polynucleotide encoding amino acids 233 to 242 in the amino acid sequence of SEQ ID NO: 3 of channelrhodopsin-2 derived from Chlamydomonas reinhardtii, a region of the polynucleotide encoding amino acids 280 to 322 in the amino acid sequence of SEQ ID NO: 1, and a region of the polynucleotide encoding amino acids 265 to 286 in the amino acid sequence of SEQ ID NO: 5 were ligated, and restriction enzyme sequences were added to the 5' and 3' ends of the polynucleotide, and the polynucleotide was inserted into the multicloning site of a plasmid for preparing an adeno-associated virus vector.

[0030] Test Example 1: Measurement of light-induced currents by patch clamp method in cells expressing p525, p548, and p550 (Part 1) Cells expressing p525, p548, and p550 were examined for venus expression under a microscope and then measured using a patch clamp system (EPC-10, HEKA). The extracellular solution consisted of 138 mM NaCl, 3 mM KCl, 10 mM HEPES, 4 mM NaOH, 1 mM CaCl2, and 2 mM MgCl2, adjusted to pH 7.4 with 1N HCl. The electrode solution consisted of 130 mM CsCl, 1.1 mM EGTA, 2 mM MgCl2, 0.1 mM CaCl2, 10 mM NaCl, 10 mM HEPES, and 2 mM Na2ATP, adjusted to pH 7.2 with 1N CsOH. Light irradiation (light source: LED) was performed for 1 second, with a light intensity of 1 μW / mm. 2 The stimulation interval was 60 seconds, and the clamp potential was -60 mV. The wavelengths were 405, 455, 505, 560, 617, and 656 nm. The results are shown in Figure 2. Figure 2 also shows the measurement results for cells expressing mVChR1 obtained in the same manner as for cells expressing p525, p548, and p550. As is clear from Figure 2, p525, p548, and p550 all had higher ion permeability than mVChR1.

[0031] Test Example 2: Measurement of light-induced currents by patch clamp method in cells expressing p525 (part 2) The light-induced current of p525 was measured in the same manner as in Test Example 1, except that light irradiation was carried out for 10 milliseconds. As a result, the ion permeability of p525 was higher than that of mVChR1.

[0032] Test Example 3: Measurement of light-induced currents using the patch clamp method in cells expressing p528 When the light-induced current of p528 was measured in the same manner as in Test Example 1, it was found that the ion permeability was higher than that of mVChR1.

[0033] Example 4: Obtaining cells expressing the modified channelrhodopsin of the present invention (p578(H172G) consisting of the amino acid sequence shown in SEQ ID NO: 9) The plasmid for constructing the p548-expressing adeno-associated virus vector prepared in Example 2 was subjected to site-directed mutagenesis using a KOD mutagenesis kit (Code No. SMK-101, TOYOBO) according to the manufacturer's instructions, substituting His at position 172 with Gly to prepare a plasmid for constructing the p578-expressing adeno-associated virus vector. Specifically, PCR was performed using 10 pmol / μL of a mutation primer (172G Forward Primer: GGACTGAGCAACCTGACCGGCCTGAA of SEQ ID NO: 21) and 10 pmol / μL of a Reverse Primer (GATCAGGATCACAGGACAGGTCAG of SEQ ID NO: 22) and 50 ng / μL of the plasmid for constructing the p548-expressing adeno-associated virus vector as a template. The PCR reaction cycle consisted of 5 cycles of 94°C for 2 minutes, 98°C for 10 seconds, and 68°C for 7 minutes. The PCR product was treated with the restriction enzyme DpnI to digest the template plasmid for constructing a p548-expressing adeno-associated virus vector, and then T4 Polynucleotide Kinase and Ligase were simultaneously applied to circularize the linear plasmid by self-ligation to obtain a plasmid for constructing a p578-expressing adeno-associated virus vector. Cells expressing p578 were produced in the same manner as in Example 1, except that the thus obtained plasmid for constructing a p578-expressing adeno-associated virus vector was used.

[0034] Example 5: Obtaining cells expressing the modified channelrhodopsin of the present invention (p579(H172A) consisting of the amino acid sequence shown in SEQ ID NO: 10) As a mutation primer, 172A of SEQ ID NO: 23 Forward Primer: GCCCTGAGCAACCTGACCGGCCTGA A A plasmid for constructing a p579-expressing adeno-associated virus vector was prepared and cells expressing p579 were produced in the same manner as in Example 4, except that the plasmid was used.

[0035] Example 6: Obtaining cells expressing the modified channelrhodopsin of the present invention (p580(H172K) consisting of the amino acid sequence shown in SEQ ID NO: 11) As a mutation primer, 172K Forward Primer of SEQ ID NO: 24: AAACTGAGCAACCTGACCGGCCTGA A A plasmid for constructing a p580-expressing adeno-associated virus vector was prepared in the same manner as in Example 4, except that the plasmid was used, and cells expressing p580 were produced.

[0036] Example 7: Obtaining cells expressing the modified channelrhodopsin of the present invention (p581(H172R) consisting of the amino acid sequence shown in SEQ ID NO: 12) As a mutation primer, 172R of SEQ ID NO: 25 Forward Primer: CGCCTGAGCAACCTGACCGGCCTGA A A plasmid for constructing a p581-expressing adeno-associated virus vector was prepared and p581-expressing cells were produced in the same manner as in Example 4, except that the plasmid was used.

[0037] Test Example 4: Measurement of light-induced currents by patch clamp method in cells expressing p578, p579, p580, and p581 The light-induced currents of each were measured in the same manner as in Test Example 1. The results are shown in Figure 3. Figure 3 also shows the measurement results for cells expressing p548. As is clear from Figure 3, the ion permeability of p578, p579, p580, and p581 was lower than that of p548, but higher than that of mVChR1 (see Figure 2 for the ion permeability of mVChR1).

[0038] Test Example 5: Measurement of τ on and τ off by patch clamp method in cells expressing p579 τon and τoff were measured under the same conditions as in the measurement of light-induced current in Test Example 4. The results for τon are shown in Figure 4 and for τoff in Figure 5. Each figure also shows the measurement results for cells expressing p548. As is clear from Figures 4 and 5, p579, in which His at position 172 of p548 is substituted with Ala, had shorter τon and τoff than p548, demonstrating that it controls cells with high temporal resolution.

[0039] Test Example 6: Transfer of p548 gene into retina using adeno-associated virus vector and its effect (Experimental Method) Construction of adeno-associated virus vectors Adeno-associated virus vectors for introducing the p548 gene into the retina were prepared using the AAV helper-free system (Stratagene, La Jalla, CA) according to the manufacturer's instructions. The three plasmids used for constructing the p548-expressing adeno-associated virus vector prepared in Example 2, pAAV-RC, and pHelper, were used to construct the adeno-associated virus vector. Specifically, each plasmid solution (15 μg each) was added to a tube tapped with 1.5 mL of 0.3 M CaCl2, and the tube was then mixed by inversion. The contents were then added to a separate tube containing 1.5 mL of 2X HBS (280 mM NaCl, 1.5 mM NaHPO4, 50 mM HEPES, pH 7.1). After further inversion, the contents were added dropwise to 293T cells cultured in a 15 cm culture dish. The three plasmids were co-transfected by the calcium phosphate method and cultured at 37°C under 5% CO2. After 3 days of culture, the target virus particles were purified from the collected cells. experimental animals Seven-month-old Royal College of Surgeons (RCS: rdy / rdy) rats were used. RCS rats undergo normal retinal development after birth, but photoreceptor cell degeneration begins at 3 weeks of age, and by 3 months of age, photoreceptor cells are almost completely lost, leading to blindness. Therefore, visual evoked potentials cannot be recorded from 7-month-old RCS rats. Introduction of p548 gene into retina Under mixed anesthesia with ketamine (66 mg / kg) and xylazine (3.3 mg / kg), the bulbar conjunctiva of both eyes of RCS rats was incised approximately 1 mm, and 5 μL of virus solution was injected into the vitreous via a 32-gauge microsyringe inserted through the pars plana of the ciliary body. Measurement of visual evoked potentials Two months after intravitreal injection of the virus solution, visual evoked potentials (VEPs) were measured in RCS rats and recorded using a Mayo PuREC evoked potential recording system. After a scalp incision was made to expose the skull, electrodes were placed on the dura 6.8 mm from the midline between bregma and lambda, 3 mm from the center of the evoked potential. A reference electrode was placed on the dura 12 mm from the midline between bregma and lambda. The electrodes were fixed in place with dental cement. VEPs were measured under mixed anesthesia with ketamine (66 mg / kg) and xylazine (3.3 mg / kg) with mydriasis using 1% atropine and 2.5% phenylephrine hydrochloride. Visual evoked potentials were recorded using various LEDs (stimulus wavelengths: 465, 525, and 650 nm) at a 10 ms exposure time and 1 Hz frequency, with 200 repeated stimuli. The recorded data were averaged. Preparation and observation of retinal flat mounts and retinal slices Eight months after intravitreal virus injection in RCS rats, retinal stretch mounts were prepared to confirm p548 expression. The eyes were enucleated and immediately fixed in 4% paraformaldehyde. The anterior segment was removed, and the neural retina was detached from the choroid. The detached neural retina was stretched onto a glass slide and observed under a fluorescence microscope to confirm p548 expression using the venus as an indicator. The retinal stretch mounts were then embedded in an embedding medium for frozen tissue sectioning (OCT compound, Sakura Finetech Japan) to prepare frozen sections (retinal section preparations). P548 expression in the retinal cross-sections was confirmed using the venus as an indicator by observation under a fluorescence microscope.

[0040] (Experimental results) The results of measuring visual evoked potentials are shown in Figure 6. As is clear from Figure 6, by introducing the p548 gene into the retina, visual evoked potentials could be recorded at all stimulus light wavelengths: 465, 525, and 650 nm. The amplitude of the visual evoked potential increased with increasing light intensity. Figure 7 shows a photograph of a retinal spread preparation observed under a fluorescence microscope. As is clear from Figure 7, p548 expression was confirmed throughout the neural retina. Figure 8 shows a photograph of a retinal slice preparation observed under a fluorescence microscope. As is clear from Figure 8, p548 expression was confirmed mainly in the retinal ganglion cell layer (the photograph on the lower right shows stained nuclei). [Industrial Applicability]

[0041] INDUSTRIAL APPLICABILITY The present invention has industrial applicability in that it can provide modified channelrhodopsins with high ion permeability (photoreactivity).

Claims

1. A modified channelrhodopsin is a polypeptide in which the third extracellular domain from the N-terminus of the three extracellular domains of Volvox-derived channelrhodopsin is replaced with the corresponding extracellular domain of Chlamydomonas reinhardtii-derived channelrhodopsin-2, and is composed of the following polypeptide (a) or (b): (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 (b) a polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and having channelrhodopsin function;

2. The modified channelrhodopsin according to claim 1, which is composed of a polypeptide having the amino acid sequence shown in SEQ ID NO:

8.

3. A polynucleotide encoding a polypeptide constituting the modified channelrhodopsin according to claim 1 or 2.

4. An expression vector comprising the polynucleotide of claim 3 operably linked to a promoter.

5. A cell expressing a polypeptide constituting the modified channelrhodopsin according to claim 1 or 2.

6. The cell according to claim 5, wherein the cell is a nerve cell.

7. Use of any of the polypeptides constituting the modified channelrhodopsin described in claim 1 or 2, the polynucleotide described in claim 3, and the expression vector described in claim 4 in the manufacture of a pharmaceutical for treating a subject suffering from a disorder of the outer retina.

8. The use according to claim 7, wherein the damage to the outer retina is any one of retinitis pigmentosa, age-related macular degeneration, and retinal detachment.

9. A pharmaceutical composition for treating damage to the outer layer of the retina, comprising as an active ingredient either a polypeptide constituting the modified channelrhodopsin according to claim 1 or 2 or an expression vector according to claim 4.

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