Modified photoreceptor chloride channel

Abstract

The present invention addresses the issue of providing a modified photoreceptive chloride channel that has a narrow wavelength sensitivity range, has both short τon and short τoff, and has excellent photoreactivity. The solution is a polypeptide in which a region from the fourth transmembrane domain to the sixth transmembrane domain, counted from an N terminal-side of a Guillardia theta-derived photoreceptive chloride channel-1 (GtACR1), is replaced by a region corresponding to a Guillardia theta-derived photoreceptive chloride channel-2 (GtACR2).

Description

[Technical Field]

[0001] The present invention relates to a modified photoreceptor chloride channel, and more specifically, to a modified photoreceptor chloride channel with excellent photoresponsive properties, including a narrow wavelength sensitivity range, a short time from the start of light irradiation until the channel opens (open rate: τon), and a short time from the end of light irradiation until the channel closes (close rate: τoff). [Background technology]

[0002] Optogenetics, a technique that involves transfecting neurons with a light-responsive protein (channelrhodopsin) to express light, is widely used to restore visual function. Optogenetics, a method for controlling cellular responses by irradiating light onto the cells, is widely used. Light-activated channels include cation channels, which transport cations between cells, and anion channels, which transport anions. However, light-activated anion channels have only recently been discovered, and reports on their modified forms are scarce compared to those of light-activated cation channels. In this context, Kato et al. focused on photoreceptor chloride channel-1 (GtACR1) isolated from the green alga Guillardia theta (Non-Patent Document 1) and reported that a modified photoreceptor chloride channel (FLASH), in which Arg83 and Asn239 were replaced with Glu, exhibited a shorter τoff than GtACR1 (Non-Patent Document 2). However, there has been no report to date on a modified photoreceptor chloride channel that has excellent light response properties, such as a narrow wavelength sensitivity range and short τ on and τ off . [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Govorunova, EGet al., Natural light-gated anion channels: a family of microbial rhodopsins for advanced optogenetics.,Science,349,647-650(2015) [Non-patent document 2] Hideaki E. Kato et al.,Structural mechanisms of selectivity and gating in anion channel rhodopsins.,Nature,561,349-354(2018) Summary of the Invention [Problem to be solved by the invention]

[0004] Therefore, an object of the present invention is to provide a modified photoreceptor chloride channel that has excellent photoresponsive properties, including a narrow wavelength sensitivity range and short τon and τoff. [Means for solving the problem]

[0005] In light of the above, the inventors have conducted extensive research and found that by replacing the region from the fourth to sixth transmembrane domains counting from the N-terminus of GtACR1, a seven-transmembrane protein, with the corresponding region (i.e., the region from the fourth to sixth transmembrane domains counting from the N-terminus) of photoreceptor chloride channel-2 (GtACR2), which is the same seven-transmembrane protein as GtACR1 and was isolated from Guillardia theta along with GtACR1, it is possible to narrow the wavelength sensitivity range compared to GtACR1 and GtACR2, and also to shorten both τon and τoff compared to GtACR1 and GtACR2.

[0006] The modified photoreceptor chloride channel of the present invention, which has been made based on the above findings, is a polypeptide in which the region from the fourth transmembrane domain to the sixth transmembrane domain counting from the N-terminus of GtACR1 is substituted with the corresponding region of GtACR2, 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: 3 ( b ) A polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and having photoreceptor chloride channel function. Furthermore, as described in claim 2, the modified photoreceptor chloride channel of the present invention is a polypeptide in which the region from the fourth transmembrane domain to the sixth transmembrane domain counting from the N-terminus of GtACR1 is substituted with the corresponding region of GtACR2, and the modified photoreceptor chloride channel comprises the following (a): or (b) It is composed of polypeptides. (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 ( b ) A polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 5 and having photoreceptor chloride channel function. Furthermore, as set forth in claim 3, the polynucleotide of the present invention encodes a polypeptide constituting the modified photoreceptor chloride channel set forth in claim 1 or 2. Furthermore, as set forth in claim 4, the expression vector of the present invention comprises the polynucleotide set forth in claim 3 operably linked to a promoter. Furthermore, as set forth in claim 5, the cell of the present invention expresses a polypeptide constituting the modified photoreceptor chloride channel set forth in claim 1 or 2. Furthermore, the cells described in claim 6 are cells according to claim 5, which are cells that constitute a retina. Furthermore, as set forth in claim 7, the present invention relates to the use of any one of the polypeptide constituting the modified photoreceptor chloride channel of claim 1 or 2, the polynucleotide of claim 3, or the expression vector of claim 4 in the manufacture of a pharmaceutical for treating a subject suffering from a disorder of the outer retina. The use according to claim 8 is the use according to claim 7, wherein the damage to the outer layer of the retina is any one of retinitis pigmentosa, age-related macular degeneration, and retinal detachment. Furthermore, as described in claim 9, the pharmaceutical composition of the present invention for treating disorders of the outer retina contains, as an active ingredient, either a polypeptide constituting the modified photoreceptor chloride channel described in claim 1 or 2 or an expression vector described in claim 4. [Effects of the Invention]

[0007] According to the present invention, it is possible to provide a modified photoreceptor chloride channel that has a narrow wavelength sensitivity range, as well as short τ on and τ off, and has excellent photoresponsive properties. [Brief explanation of the drawings]

[0008] [Figure 1] 1 shows the structure of a plasmid for preparing a ChimGt12-expressing adeno-associated virus vector in Example 1. [Figure 2] 1 is a graph showing that ChimGt12 has a narrower wavelength sensitivity range than GtACR1 and GtACR2 in Test Example 1. [Figure 3] 10 is a graph showing that ChimGt12 has a shorter τon than GtACR1 and GtACR2. [Figure 4] 10 is a graph showing that ChimGt12 has a shorter τoff than GtACR1 and GtACR2. [Figure 5] 1 is a graph showing that in Test Example 3, the reduction in retinal thickness can be significantly suppressed by introducing the ChimGt12 gene into the retina. [Figure 6] 10 is a graph showing that the hyperpolarization response of photoreceptor cells is increased by introducing the ChimGt12 gene into the retina. DETAILED DESCRIPTION OF THE INVENTION

[0009] The modified photoreceptor chloride channel of the present invention is a polypeptide in which the region from the fourth to sixth transmembrane domains counting from the N-terminus of GtACR1 isolated from Guillardia theta, as reported in Non-Patent Document 1, is replaced with the corresponding region of GtACR2. GtACR1 is a polypeptide consisting of the following 295 amino acids (SEQ ID NO: 1), and the region from the fourth to sixth transmembrane domains counting from the N-terminus extends from Asn123 to Phe213. GtACR2 is a polypeptide consisting of the following 291 amino acids (SEQ ID NO: 2), and the region from the fourth to sixth transmembrane domains counting from the N-terminus extends from Asn119 to Ile209 (see, for example, Non-Patent Document 1 for the amino acid sequences of GtACR1 and GtACR2, if necessary).

[0010] (Amino acid sequence of GtACR1) MSSITCDPAIYGEWSRENQFCVEKSLITL DGIKYVQLVMAVVSACQVFFMVT RAPKVPW EAIYLPTTEM TM1 TM2 ITYSLAF TGNGYIRVANGKYLP WARMASWLCTCPIMLGLVS NMALVKYKSIPL NPMMIAASSICTVFGI TM3 TM4 TA SVVLD PLHVWLYCFISSIFFIFEMVVAFAIFAITIHDFQT IGS PMSLKVVERLKLMRIVFYVSWMAY TM5 TM6 PILWSF SSTGACIMS ENTSSVLYLLGDALCKNTYGILLWATT WGLLNGKWDRDYVKGRNVDGTLMPEYE TM7 QDLEKGNTERYEDARAGET *TM: transmembrane domain

[0011] (Amino acid sequence of GtACR2) MASQVVYGEWASTHTECYNMSRIDS TFVSLLQLVWAVVSGCQTIFMIS RAPKVPW ESVYLPFVESITYA TM1 TM2 LAS TGNGTLQMRDGRFFP WSRMASWLCTCPIMLGQIS NMALVKYKSIPL NPIAQAASIIRVVMGITA TI TM3 TM4 SPA EYMKWLFFFFGATCLVFEYSVVFTIFQVGLYGFES VGT PLAQKVVVRIKMLRLIFFIAWTMFPIVW TM5 TM6 LI SPTGVCVIH ENVSAILYLLADGLCKNTYGVILWSTA WGVLEGKWDPACLPGQEKPEADDPFGLNHEK TM7 NAPPNDEVNIRMFGR *TM: transmembrane domain

[0012] The modified photoreceptor chloride channel of the present invention may be a polypeptide in which the region from the fourth transmembrane domain counting from the N-terminus of GtACR1 to the sixth transmembrane domain is substituted with the corresponding region of GtACR2, and in which other domains or regions are further modified. For example, the modified photoreceptor chloride channel may be a polypeptide in which the intracellular domain between the third transmembrane domain and the fourth transmembrane domain counting from the N-terminus of GtACR1, or the extracellular domain between the sixth transmembrane domain and the seventh transmembrane domain, is further substituted with the corresponding domain of GtACR2. The intracellular domain between the third transmembrane domain and the fourth transmembrane domain counting from the N-terminus of GtACR1 and the extracellular domain between the sixth transmembrane domain and the seventh transmembrane domain, respectively, consists of the amino acids between TM3 and TM4 and the amino acids between TM6 and TM7, as described above.

[0013] A specific example of the modified photoreceptor chloride channel of the present invention is a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3. In this polypeptide (ChimGt12), the region from the fourth to sixth transmembrane domains counting from the N-terminus of GtACR1 is replaced with the corresponding region of GtACR2, and further, the extracellular domain between the sixth and seventh transmembrane domains is replaced with the corresponding domain of GtACR2.

[0014] The modified photoreceptor chloride channel of the present invention may also be a polypeptide comprising the N-terminal region of channelrhodopsin-1 derived from Chlamydomonas reinhardtii, for example, all or part of amino acids 1 to 71 of the amino acid sequence of ChR1 shown in SEQ ID NO: 4, added to its N-terminus. A specific example thereof is a polypeptide comprising the amino acid sequence shown in SEQ ID NO: 5. This polypeptide (mV2Gt12) comprises amino acids 1 to 24 of the amino acid sequence of ChR1 shown in SEQ ID NO: 4 added to the N-terminus of ChemGt12.

[0015] The modified photoreceptor chloride channels 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: 3 and 5, respectively, and having photoreceptor chloride channel function. The modified photoreceptor chloride channels 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: 3 and 5, respectively, and having photoreceptor chloride channel function. Here, "multiple" refers to integers of 50 or less, preferably integers of 30 or less, 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: 3 and 5, 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 the identity between multiple (two) amino acid sequences with default settings. Furthermore, as is well known to those skilled in the art, "having a photoreceptor chloride channel function" means having a channel function that controls ion permeability between the outside and inside of a cell by sensing light, and it is preferable 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 set forth in SEQ ID NO: 3 or 5, respectively.

[0016] The modified photoreceptor chloride channel of the present invention can be produced by genetic engineering techniques. Specifically, first, a polynucleotide encoding the modified photoreceptor chloride channel of the present invention (hereinafter referred to as the "modified photoreceptor chloride channel gene of the present invention") is prepared. Modified photoreceptor chloride channel of the present invention genecan be prepared by techniques known to those skilled in the art. Specifically, for example, they can be prepared by chemical synthesis based on the sequence information of polynucleotides encoding GtACR1 and GtACR2. Alternatively, they can be prepared by amplifying desired regions of each polynucleotide using PCR primers that amplify the desired regions of each polynucleotide based on the sequence information of each polynucleotide, and then ligating them using, for example, the Gibson Assembly system (New England Biolabs). Next, the modified photoreceptor chloride channel gene of the present invention operably linked to a promoter is incorporated into an expression vector that can replicate and maintain in host cells, stably express the encoded polypeptide, and stably retain this gene. The resulting recombinant expression vector can then be used to transform a host, thereby producing the modified photoreceptor chloride channel of the present invention in the host. For details of 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 photoreceptor chloride channel of the present invention in a particular cell, the transcriptional regulatory region of a polypeptide gene specifically expressed in that cell (e.g., 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, pp. 1013-1107, December 2007)) may be ligated upstream of these promoters. The modified photoreceptor chloride channel gene of the present invention can be inserted into an expression vector, for example, by creating or linking restriction enzyme sites flanking the modified photoreceptor chloride channel gene of the present invention and inserting the gene into a restriction enzyme site or multicloning site of an appropriate vector DNA.In addition to the promoter and the modified photoreceptor chloride channel 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 complementation gene markers such as LEU1, TRP1, and URA3, dominant selection markers such as APH, DHFR, and TK), ribosome binding sites (RBS), etc. 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, etc. The transformant thus obtained is cultured under appropriate conditions using a medium containing assimilable carbon sources, nitrogen sources, metal salts, vitamins, etc. The transformant is typically cultured aerobicly, such as by shaking or aeration with agitation, at 25 to 37°C for 3 to 6 hours. The pH is maintained near neutral during the culture period. The pH is adjusted using inorganic or organic acids, alkaline solutions, or the like. During culture, antibiotics such as ampicillin or tetracycline may be added to the medium, if necessary, depending on the selection marker inserted into the recombinant expression vector. The host used for transformation is not particularly limited as long as it can express the modified photoreceptor chloride channel 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 photoreceptor chloride channel of the present invention can be isolated and purified by conventional methods from the culture obtained by culturing the transformant (culture supernatant, cultured cells, cultured bacterial cells, cell or bacterial homogenate, etc.), and then obtained in a form that retains its activity by ultrafiltration concentration, freeze-drying, spray-drying, crystallization, or the like. Alternatively, the modified photoreceptor chloride channel of the present invention may be provided in the form of cells expressing the modified photoreceptor chloride channel 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.) or retinal pigment epithelial cells, which are cells that constitute the retina, preferably cells that constitute the human retina, but other cells may also be used. Furthermore, when the modified photoreceptor chloride channel of the present invention is used for medical purposes, it may be provided in the form of an expression vector for the modified photoreceptor chloride channel of the present invention. In this case, it is preferable to use an expression vector that has excellent efficiency in introducing into cells, maintenance of intracellular replication, 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, and transposons. Plasmids for constructing expression vectors for the modified photoreceptor chloride channel 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.

[0017] Examples of the modified photoreceptor chloride channel gene of the present invention include a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 6 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3) and a polynucleotide consisting of the nucleotide sequence shown in SEQ ID NO: 7 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5). However, the modified photoreceptor chloride channel 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 photoreceptor chloride channel function. Furthermore, the modified photoreceptor chloride channel gene of the present invention also includes 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 nucleotide sequences shown in SEQ ID NOs: 6 and 7, respectively, and encode a polypeptide having photoreceptor chloride channel 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.).

[0018] The modified photoreceptor chloride channel of the present invention has a narrower wavelength sensitivity range than GtACR1 and GtACR2, and both τon and τoff are shorter than those of GtACR1 and GtACR2, resulting in excellent photoresponsive properties. A narrow wavelength sensitivity range is advantageous in that it facilitates the design of wavelength selectivity for controlling neuronal excitation and inhibition, while short τon and τoff are advantageous in that it enables neuronal control with high temporal resolution. Therefore, the modified photoreceptor chloride channel of the present invention and an expression vector containing a polynucleotide encoding the channel are useful for treating subjects suffering from damage to the outer retina by suppressing visual dysfunction or impairment caused by degeneration or loss of photoreceptor cells, and by improving existing visual dysfunction or impairment. Here, "damage to the outer retina" refers to any disease in which visual dysfunction or 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 has lost his or her sight due to a disorder in the outer retina or who is at risk of losing his or her sight. 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 in the outer retina" refers to restoring visual function to a level before administration of the pharmaceutical agent of the present invention in a subject who has lost his or her sight due to a disorder in the outer retina or who is at risk of losing his or her sight. The modified photoreceptor chloride channel of the present invention is also useful for various disorders involving light responses, such as disorders of the brain or central or peripheral nervous system, spinal cord injury, and autoimmune diseases.

[0019] The pharmaceutical composition of the present invention contains as an active ingredient the modified photoreceptor chloride channel of the present invention or an expression vector containing a polynucleotide encoding the channel, 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 13 The 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]

[0020] 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.

[0021] Example 1: Obtaining cells expressing the modified photoreceptor chloride channel of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 3 (ChimGt12) This was obtained in accordance with the method described by the present inventors in WO2011 / 019081 as follows. A polynucleotide encoding the amino acids from the third transmembrane domain counting from the N-terminus of GtACR1 to the intracellular domain between the fourth transmembrane domain, a polynucleotide encoding the amino acids from the fourth transmembrane domain counting from the N-terminus of GtACR2 to the extracellular domain between the sixth transmembrane domain and the seventh transmembrane domain, and a polynucleotide encoding the amino acids from the seventh transmembrane domain counting from the N-terminus of GtACR1 to the C-terminus were ligated to form a polynucleotide with restriction enzyme sequences added to its 5' and 3' ends, and inserted into the multicloning site of a plasmid for producing an adeno-associated virus vector. The structure of the resulting plasmid for producing an adeno-associated virus vector expressing ChimGt12 is shown in Figure 1. This plasmid contains a fluorescent protein gene (venus) in the 3' region of the multicloning site, and the gene of interest is expressed as a fusion protein with venus added to the C-terminal region. Therefore, this plasmid was transfected into cells using the calcium phosphate method, and cells expressing ChimGt12 were identified using venus 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 at 37°C under 5% CO2. After 6 hours, the medium was replaced with fresh medium. After 2 days of culture, the cells were observed under a fluorescent microscope to confirm ChimGt12 expression in the cells.

[0022] Example 2: Obtaining cells expressing the modified photoreceptor chloride channel of the present invention (mV2Gt12) consisting of the amino acid sequence shown in SEQ ID NO: 5 Cells expressing mV2Gt12 were prepared in the same manner as in Example 1, except that a polynucleotide encoding the amino acids of ChimGt12 was linked to the 3' end of a polynucleotide encoding amino acids 1 to 24 of the amino acid sequence of ChR1 shown in SEQ ID NO: 4, and restriction enzyme sequences were added to the 5' and 3' ends of the polynucleotide.

[0023] Test Example 1: Measurement of light-induced current, τ on and τ off by patch clamp method in cells expressing ChimGt12 (Measurement method) After confirming the expression of venus under a microscope, cells expressing ChimGt12 were 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 interstimulus interval was 60 seconds, and the clamp potential was set to 0 mV. The wavelengths were 405, 455, 505, 560, 617, and 656 nm.

[0024] (Measurement results) The results of light-induced current measurements are shown in Figure 2, and the results of τon and τoff measurements are shown in Figures 3 and 4, respectively (n>11). Each figure also shows the results of measurements on GtACR1-expressing cells (n=7) and GtACR2-expressing cells (n=8), obtained in the same manner as for ChimGt12-expressing cells. As can be seen from Figure 2, the wavelength sensitivity ranges of ChimGt12 and GtACR1 were comparable on the short wavelength side, but were shorter on the long wavelength side in ChimGt12 than in GtACR1. The wavelength sensitivity ranges of ChimGt12 and GtACR2 were comparable on the long wavelength side, but were shorter on the short wavelength side in ChimGt12 than in GtACR2. GtACR2 was highly responsive to light at 400 nm, which is the shorter wavelength with higher light energy, raising concerns about photodamage due to cellular hyperpolarization. As can be seen from Figures 3 and 4, the τon and τoff of ChimGt12 were shorter than those of GtACR1 and GtACR2 across the entire wavelength range, with some exceptions. Furthermore, when comparing the fluorescence images of cells expressing ChimGt12, GtACR1, and GtACR2, the cells expressing GtACR1 and GtACR2 were significantly shorter than those of GtACR1. GtACR2 Cells expressing GtACR1 and GtACR2 showed strong fluorescence, which may be due to the inability of GtACR1 and GtACR2 to maintain the correct conformation within the cells, raising concerns about cytotoxicity. However, such fluorescence was hardly observed in cells expressing ChimGt12.

[0025] Test Example 2: Measurement of light-induced current, τ on and τ off by patch clamp method in cells expressing mV2Gt12 Using the same measurement method as in Test Example 1, the same measurement results as in the cells expressing ChimGt12 were obtained.

[0026] Test Example 3: Introduction of the ChimGt12 gene into the retina using an adeno-associated virus vector and its effect (Experimental Method) Construction of adeno-associated virus vectors Adeno-associated virus vectors for introducing the ChimGt12 gene into the retina were prepared using the AAV helper-free system (Stratagene, La Jalla, CA) according to the manufacturer's instructions. Each plasmid solution (15 μg each) was added to a tapped tube, and 1.5 mL of 0.3 M CaCl2 was added. After inverting, the contents were transferred 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 inverting again, 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 in 5% CO2. After 3 days of culture, the cells were harvested and the desired virus particles were purified. The plasmid for constructing the ChimGt12-expressing adeno-associated virus vector was prepared using the GRK promoter instead of the CAG promoter used in the plasmid for constructing the ChimGt12-expressing adeno-associated virus vector prepared in Example 1, or the RPEJ promoter linked upstream to 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, pp. 1013-1107, December 2007), in order to express ChimGt12 specifically in photoreceptor cells. As a control, a plasmid for constructing the adeno-associated virus vector for expressing only venus was prepared in the same manner as the plasmid for constructing the ChimGt12-expressing adeno-associated virus vector prepared in Example 1.The adeno-associated virus serotypes used were M8 (a mutant in which Tyr at position 733 of the capsid protein of type 8 was replaced with Phe according to Hilda Petrs-Silva et al., Molecular Therapy, Vol. 17, No. 3, 463-471, March 2009) or DJ (Funakoshi). experimental animals P23H (strain 2) rats aged 16 or 24 weeks were used. P23H rats initially develop normal retinal formation after birth, but then undergo a gradual progression of photoreceptor cell degeneration, resulting in the loss of approximately half of the photoreceptors by 4 months of age. The degeneration continues to progress slowly thereafter, eventually resulting in the loss of most of the photoreceptors and blindness. Introduction of ChimGt12 gene into retina Under mixed anesthesia with ketamine (66 mg / kg) and xylazine (3.3 mg / kg), a 1 mm incision was made in the bulbar conjunctiva of both eyes of P23H rats, and a 32-gauge microsyringe was inserted through the pars plana of the ciliary body to inject 5 μL of virus solution into the vitreous. Alternatively, an incision was made above the bulbar conjunctiva, and a 30-gauge needle was used to incise the sclera approximately 1 mm away from the optic nerve, and 3 μL of virus solution was administered subretinal- ly through this site using a 32-gauge microsyringe. Measurement of retinal thickness Before and one month after virus administration, retinal optical coherence tomography (OCT) was performed under mixed anesthesia with ketamine (66 mg / kg) and xylazine (3.3 mg / kg) and with the pupils dilated with 1% atropine and 2.5% phenylephrine hydrochloride using a NIDEK RS-3000. Electroretinogram measurement Before and every month after virus administration, electroretinograms were measured under mixed anesthesia with ketamine (66 mg / kg) and xylazine (3.3 mg / kg) and with the pupils dilated with 1% atropine and 2.5% phenylephrine hydrochloride. The electroretinograms were recorded using a Mayo PuREC recording device. The light stimulus intensities were 0.01, 3.0, and 10.0 cd·s / m. 2 The three stages are:

[0027] (Experimental results) The results of retinal thickness measurements are shown in Figure 5. As can be seen from Figure 5, when the virus was administered intravitreally to express only the venus (CAG-Venus-M8 iv), the thicknesses of the entire retina (ILM-RPE), photoreceptor layer (ONL-RPE), and outer nuclear layer (ONL) decreased to approximately 75, 70, and 65, respectively, one month after administration, with the pre-administration thickness set at 100, and continued to decrease thereafter. In contrast, when the virus was administered intravitreally (iv) or subretina (subretina) to express ChimGt12, the decrease in each thickness was significantly suppressed. The results of electroretinogram measurements are shown in Figure 6. As can be seen from Figure 6, when the virus was administered intravitreally to express only the venus, the photoreceptor hyperpolarization response was significantly reduced compared to pre-administration. In contrast, when the virus was administered intravitreally or subretinaly to express ChimGt12, the hyperpolarization response of photoreceptors increased one month after administration, with some exceptions (at a light stimulus intensity of 0.01 cd·s / m 2 (The a-wave could not be measured in this study.) This suggests that ChimGt12 not only inhibits the degeneration of photoreceptor cells, but also improves their function. [Industrial Applicability]

[0028] The present invention has industrial applicability in that it can provide a modified photoreceptor chloride channel having excellent photoresponsive properties, including a narrow wavelength sensitivity range and short τon and τoff.

Claims

1. A modified photoreceptor chloride channel is a polypeptide in which the region from the fourth transmembrane domain to the sixth transmembrane domain counting from the N-terminus of Guillardia theta-derived photoreceptor chloride channel-1 (GtACR1) is replaced with the corresponding region of Guillardia theta-derived photoreceptor chloride channel-2 (GtACR2), and is composed of the following polypeptide (a) or (b): (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 3 (b) a polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 3 and having photoreceptor chloride channel function;

2. A modified photoreceptor chloride channel is a polypeptide in which the region from the fourth transmembrane domain to the sixth transmembrane domain counting from the N-terminus of Guillardia theta-derived photoreceptor chloride channel-1 (GtACR1) is replaced with the corresponding region of Guillardia theta-derived photoreceptor chloride channel-2 (GtACR2), and is composed of the following polypeptide (a) or (b): (a) a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 5 (b) a polypeptide having an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 5 and having photoreceptor chloride channel function;

3. A polynucleotide encoding a polypeptide constituting the modified photoreceptor chloride channel of 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 photoreceptor chloride channel according to claim 1 or 2.

6. The cell according to claim 5, which is a cell that constitutes the retina.

7. Use of a polypeptide constituting the modified photoreceptor chloride channel described in claim 1 or 2, a polynucleotide described in claim 3, or an 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 either the polypeptide constituting the modified photoreceptor chloride channel of claim 1 or 2 or the expression vector of claim 4 as an active ingredient.

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