Modified channelrhodopsin

Abstract

Provided is a modified channelrhodopsin having high ion permeability (photoreactivity). The solution is to replace the third extracellular domain from the N-terminal side of the three extracellular domains possessed by channelrhodopsin derived from Volvox with the corresponding extracellular domain of channelrhodopsin-2 derived from Chlamydomonas reinhardtii.

Description

[Technical Field]

[0001] This invention relates to modified channel rhodopsin. More specifically, it relates to modified channel rhodopsin with high ion permeability (photoreactivity). [Background Technology]

[0002] It is known that research worldwide is being conducted on optogenetics, a method for reconstructing visual function through gene introduction and expression, where nerve cells control cellular responses via light-responsive proteins (channel rhodopsin). In Patent Document 1, the inventors also reported a modified channel rhodopsin whose expression efficiency on the cell membrane was improved by replacing the N-terminal region of channel rhodopsin derived from *Volvox* with the N-terminal region of channel rhodopsin-1 derived from *Chlamydomonas reinhardtii*. This modified channel rhodopsin can be induced to excite by opening ion channels upon light exposure, and the inventors successfully restored vision by introducing its gene into the retina of blind rats.

[0003] However, there is a demand for modified channel rhodopsin with higher ion permeability than the modified channel rhodopsin reported to date.

[0004] [Existing Technical Documents]

[0005] [Patent Documents]

[0006] Patent Document 1: Patent No. 5322067

[0007] [Summary of the Invention]

[0008] [The problem the invention aims to solve]

[0009] Therefore, the present invention aims to provide modified channel rhodopsin with high ion permeability.

[0010] [Methods used to solve problems]

[0011] Based on the above points, the inventors conducted in-depth research and discovered that by replacing the third extracellular domain from the N-terminus of the three extracellular domains of channel rhodopsin derived from Volvox with the corresponding extracellular domain of channel rhodopsin-2 derived from Chlamydomonas reinhardtii, a channel rhodopsin with high ion permeability was obtained.

[0012] Based on the above insights, the modified channelo-rhodopsin of the present invention, as described in Embodiment 1, is a polypeptide in which the third extracellular domain from the N-terminus of the three extracellular domains of channelo-rhodopsin derived from Volvox is replaced with the corresponding extracellular domain of channelo-rhodopsin-2 derived from Chlamydomonas reinhardtii.

[0013] In addition, the modified channel rhodopsin described in Embodiment 2 is formed by the modified channel rhodopsin described in Embodiment 1, wherein the channel rhodopsin derived from Volvox contains at least amino acids from positions 67 to 322 of the amino acid sequence shown in SEQ ID NO: 1.

[0014] In addition, the modified channel rhodopsin described in Embodiment 3 is formed by replacing amino acids at positions 142 to 169 of the amino acid sequence shown in SEQ ID NO: 1 with amino acids at positions 143 to 170 of the amino acid sequence of channel rhodopsin-1 derived from Chlamydomonas reinhardtii, as shown in SEQ ID NO: 2.

[0015] In addition, the modified channel rhodopsin described in Embodiment 4 is any one of (a) to (c) of the modified channel rhodopsin described in Embodiment 3.

[0016] (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4

[0017] (b) A polypeptide consisting of an amino acid sequence containing one or more amino acid deletions, substitutions, additions, or insertions in the amino acid sequence shown in SEQ ID NO: 4, and possessing channel rhodopsin function.

[0018] (c) A polypeptide consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 4 and having channel rhodopsin function.

[0019] In addition, the modified channel rhodopsin described in Embodiment 5 is a polypeptide composed of the amino acid sequence shown in SEQ ID NO: 8 in the modified channel rhodopsin described in Embodiment 4.

[0020] In addition, the modified channel rhodopsin described in Embodiment 6 is formed by replacing the sixth transmembrane domain from the N-terminus of the seven transmembrane domains of channel rhodopsin derived from Volvox with the corresponding transmembrane domain of channel rhodopsin derived from Chloromonasoogama in the modified channel rhodopsin described in any one of Embodiments 1 to 3.

[0021] In addition, the modified channel rhodopsin described in Embodiment 7 is formed by replacing the amino acid from position 323 onwards in the modified channel rhodopsin described in Embodiment 2 or 3 with the amino acid from position 265 onwards in the amino acid sequence of the channel rhodopsin derived from Chloromonas oogama shown in SEQ ID NO: 5.

[0022] In addition, the modified channel rhodopsin described in Embodiment 8 is any one of (a) to (c) below in the modified channel rhodopsin described in Embodiment 6 or 7.

[0023] (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6

[0024] (b) A polypeptide consisting of an amino acid sequence containing one or more amino acid deletions, substitutions, additions, or insertions in the amino acid sequence shown in SEQ ID NO: 6, and possessing channel rhodopsin function.

[0025] (c) A polypeptide consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 6 and having channel rhodopsin function.

[0026] In addition, the modified channel rhodopsin described in Embodiment 9 is a polypeptide formed by replacing His at position 172 of the polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 with other amino acids in the modified channel rhodopsin described in Embodiment 8.

[0027] In addition, the modified channel rhodopsin described in Embodiment 10 is a polypeptide composed of the amino acid sequence shown in any one of SEQ ID NO: 9 to 12 in the modified channel rhodopsin described in Embodiment 9.

[0028] In addition, the modified channel rhodopsin described in Embodiment 11 is any one of (a) to (c) below in the modified channel rhodopsin described in Embodiment 6 or 7.

[0029] (a) A polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7

[0030] (b) A polypeptide consisting of an amino acid sequence containing one or more amino acid deletions, substitutions, additions, or insertions in the amino acid sequence shown in SEQ ID NO: 7, and possessing channel rhodopsin function.

[0031] (c) A polypeptide consisting of an amino acid sequence having at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 7 and having channel rhodopsin function.

[0032] In addition, the polynucleotide of the present invention, as described in Embodiment 12, encodes a polypeptide as described in any one of Embodiments 1 to 11.

[0033] In addition, the expression vector of the present invention, as described in Embodiment 13, contains the polynucleotide described in Embodiment 12 that is functionally linked to the promoter.

[0034] In addition, the cells of the present invention, as described in Embodiment 14, express the polypeptides described in any one of Embodiments 1 to 11.

[0035] Furthermore, in the cell described in Embodiment 15, the cell described in Embodiment 14 is a nerve cell.

[0036] In addition, the present invention, as described in Embodiment 16, is used in the manufacture of a medicine for treating a test subject with an outer retinal defect by any of the polypeptides described in Embodiments 1 to 11, the polynucleotides described in Embodiment 12, and the expression vectors described in Embodiment 13.

[0037] In addition, in the use described in Embodiment 17, the outer retinal layer defect is any one of retinitis pigmentosa, age-related macular degeneration, or retinal detachment, as described in Embodiment 16.

[0038] In addition, the pharmaceutical composition of the present invention for treating outer retinal defects, as described in Embodiment 18, contains any one of the polypeptides described in Embodiments 1 to 11 or any one of the expression vectors described in Embodiment 13 as an active ingredient.

[0039] [The effects of the invention]

[0040] The present invention provides modified channel rhodopsin with high ion permeability.

[0041] [Brief explanation of the attached image]

[0042]

【 Figure 1 [This refers to the composition of the plasmid used to create the adeno-associated virus vector expressing p525 in Example 1.]

[0043]

【 Figure 2 The graph shows that p525, p548, and p550 have higher permeability than mVChR1 ions in Experiment Example 1.

[0044]

【 Figure 3 The graph above is a coordinate graph showing the ion permeability of p578, p579, p580, and p581 in Experimental Example 4.

[0045]

【 Figure 4 The graph shows that in Experiment 5, p579 is shorter than p548 and τon is shorter.

[0046]

【 Figure 5 The graph shows that in Experiment 5, p579 is shorter than p548 and τoff is shorter.

[0047]

【 Figure 6 The image shows that in Experiment 6, the p548 gene was introduced into the retina, and a coordinate graph of visual evoked potentials could be recorded.

[0048]

【 Figure 7 The image shown is a photograph from Experiment 6, which demonstrates the expression of p548 in the entire neuroretina when the retinal extension specimen is observed under a fluorescence microscope.

[0049]

【 Figure 8 The image shown is from Experiment 6, where p548 expression was dominant in retinal ganglion cell layers when retinal slice specimens were observed under a fluorescence microscope.

Detailed Implementation Methods

[0050] The modified channel rhodopsin of the present invention is a polypeptide in which the third extracellular domain from the N-terminus of the three extracellular domains of channel rhodopsin from Volvox is replaced with the corresponding extracellular domain of channel rhodopsin-2 from Chlamydomonas reinhardtii.

[0051] As a specific example of channel rhodopsin derived from the genus *Volvox*, a polypeptide containing at least amino acids from positions 67 to 322 of the amino acid sequence shown in SEQ ID NO: 1 can be cited. The polypeptide composed of the amino acid sequence shown in SEQ ID NO: 1 is the modified channel rhodopsin reported by the inventors in Patent Document 1 (a polypeptide composed of the amino acid sequence shown in SEQ ID NO: 10 in Patent Document 1). This modified channel rhodopsin is a 344-amino acid polypeptide whose expression efficiency on the cell membrane is improved by replacing the N-terminal region of channel rhodopsin derived from the genus *Volvox* with the N-terminal region of channel rhodopsin-1 derived from *Chlamydomonas reinhardtii* (a region related to cell membrane localization and expression and lacking a transmembrane domain). The amino acids at positions 1 to 66 of the amino acid sequence shown in SEQ ID NO: 1 are the same as the amino acids at positions 1 to 66 of the channel rhodopsin-1 derived from *Chlamydomonas reinhardtii* shown in SEQ ID NO: 2. In this invention, the modified channel rhodopsin, which is a polypeptide composed of the amino acid sequence shown in SEQ ID NO: 1, is referred to as mVChR1. Furthermore, matters described in Patent Document 1 are treated as matters described in this specification.

[0052] The amino acid sequence of mVChR1 shown in SEQ ID NO: 1 is as follows. The third extracellular domain (EX3) of the three extracellular domains of mVChR1 from the N-terminus is Gly271 to Ser279.

[0053] [Amino acid sequence of mVChR1]

[0054]

[0055] ※TM: Transmembrane domain

[0056] IN: Intracellular domain

[0057] EX: Extracellular domain

[0058] The modified channel rhodopsin of the present invention, when taking mVChR1 as an example, replaces the aforementioned EX3 with the corresponding extracellular domain of channel rhodopsin-2 derived from Chlamydomonas reinhardtii, that is, a polypeptide formed by the third extracellular domain from the N-terminus of the three extracellular domains possessed by channel rhodopsin-2 from Chlamydomonas reinhardtii. The amino acid sequence of channel rhodopsin-2 derived from Chlamydomonas reinhardtii is as shown in SEQ ID NO: 3, and its third extracellular domain from the N-terminus is Gly233~Ser241. The substitution of EX3 may also involve the substitution of the transmembrane domains before and after it, namely, the amino acids on the C-terminal side of TM6 adjacent to the N-terminus of EX3, and / or the amino acids on the N-terminal side of TM7 adjacent to the C-terminus of EX3. The number of amino acids optionally substituted is preferably a maximum of three. Substitution of more than three amino acids raises concerns about affecting the function of the transmembrane domains.

[0059] The modified channelomorphin of the present invention, besides having the third extracellular domain from the N-terminus of the three extracellular domains of channelomorphin derived from Volvox replaced by the corresponding extracellular domain of channelomorphin-2 derived from Chlamydomonas reinhardtii, can also be a polypeptide formed by further alteration of other domains or regions. For example, when taking mVChR1 as an example, it can also be a polypeptide formed by replacing amino acids 142 to 169 of the amino acid sequence shown in SEQ ID NO: 1 with amino acids 143 to 170 of the amino acid sequence of channelomorphin-1 derived from Chlamydomonas reinhardtii shown in SEQ ID NO: 2.

[0060] As a specific example of such modified channel rhodopsin, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4 can be cited. This polypeptide (p525) is formed by replacing EX3 of mVChR1 with the corresponding extracellular domain of channel rhodopsin-2 from Chlamydomonas reinhardtii, and replacing amino acids 142-169 of mVChR1 with amino acids 143-170 of channel rhodopsin-1 from Chlamydomonas reinhardtii as shown in SEQ ID NO: 2 (furthermore, along with the replacement of EX3, Pro at position 280 of TM7 is replaced with Val).

[0061] Furthermore, the modified channel rhodopsin of the present invention, for example, when mVChR1 is used, can also be a polypeptide in which the sixth transmembrane domain (TM6) from its N-terminus is replaced with the corresponding transmembrane domain of channel rhodopsin derived from *Chloromonas oogama*. The amino acid sequence of channel rhodopsin derived from *Chloromonas oogama*, as shown in SEQ ID NO: 5, has the sixth transmembrane domain from its N-terminus as Arg187–Val212.

[0062] In addition, the modified channel rhodopsin of the present invention, for example, when mVChR1 is taken as an example, can also be a polypeptide formed by replacing the amino acid from position 323 onwards in the amino acid sequence shown in SEQ ID NO: 1 with the amino acid from position 265 onwards in the amino acid sequence of the channel rhodopsin derived from Chloromonas oogama shown in SEQ ID NO: 5.

[0063] As a specific example of such modified channel rhodopsin, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 6 can be cited. This polypeptide (p548) is formed by replacing EX3 of mVChR1 with the corresponding extracellular domain of channel rhodopsin-2 from Chlamydomonas reinhardtii, and replacing amino acids 142-169 of mVChR1 with amino acids 143-170 of channel rhodopsin-1 from Chlamydomonas reinhardtii as shown in SEQ ID NO: 2. Furthermore, TM6 of mVChR1 is replaced with the corresponding transmembrane domain of channel rhodopsin from Chloromonas oogama, and amino acids 323-344 of mVChR1 are replaced with amino acids 265-286 of channel rhodopsin from Chloromonas oogama as shown in SEQ ID NO: 5. (Furthermore, along with the replacement of EX3, Pro at position 280 of TM7 is replaced with Val.)

[0064] As another specific example, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 7 can be cited. This polypeptide (p550) is formed by replacing EX3 of mVChR1 with the corresponding extracellular domain of channel rhodopsin-2 from Chlamydomonas reinhardtii, and replacing amino acids 142-169 of mVChR1 with amino acids 143-170 of channel rhodopsin-1 from Chlamydomonas reinhardtii as shown in SEQ ID NO: 2. Furthermore, TM6 of mVChR1 is replaced with the corresponding transmembrane domain of channel rhodopsin from Chloromonas oogama, and amino acids 323-344 of mVChR1 are replaced with amino acids 265-286 of channel rhodopsin from Chloromonas oogama as shown in SEQ ID NO: 5. The amino acid sequence shown in NO:6 is the same as that of the polypeptide (p548), and it is formed by deleting amino acids 28 to 60 of mVChR1 (in addition, with the substitution of EX3, Pro at position 280 of TM7 is replaced with Val).

[0065] The modified channel rhodopsin of the present invention comprises a polypeptide having one or more amino acid deletions, substitutions, additions, or insertions in the amino acid sequences shown in SEQ ID NO: 4, 6, and 7, and having channel rhodopsin function. Additionally, it comprises a polypeptide having at least 90% sequence identity with the amino acid sequences shown in SEQ ID NO: 4, 6, and 7, and having channel rhodopsin function. Herein, "multiple" refers to an integer of 50 or less, preferably an integer of 30 or less, more preferably an integer 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 NO: 4, 6, and 7 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%. Furthermore, the % of similarity refers to the value calculated using software (e.g., FASTA, DANASYS, BLAST, etc.) that calculates the similarity between multiple (2) amino acid sequences with default settings. As a specific example of such modified channel rhodopsin, a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4, where Trp at position 210 is replaced with Tyr and Thr at position 211 is replaced with Val, is an example (p528). In addition, examples include polypeptides in which the His at position 172 of the amino acid sequence shown in SEQ ID NO: 4, 6, and 8, or the His at position 139 of the amino acid sequence shown in SEQ ID NO: 7, is replaced with other amino acids, such as Gly, Ala, Lys, and Arg. Specifically, examples include polypeptides in which the His at position 172 of the amino acid sequence shown in SEQ ID NO: 6 is replaced with Gly (p578), polypeptides in which the His at position 172 of the amino acid sequence shown in SEQ ID NO: 10 is replaced with Ala (p579), polypeptides in which the His at position 172 of the amino acid sequence shown in SEQ ID NO: 11 is replaced with Lys (p580), and polypeptides in which the His at position 172 of the amino acid sequence shown in SEQ ID NO: 12 is replaced with Arg (p581), etc. By replacing His at position 172 of the polypeptide consisting of the amino acid sequences shown in SEQ ID NO: 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 other amino acids, at least one of the time from the start of light irradiation to the opening of the channel (opening rate: τon) and the time from the stop of light irradiation to the closing of the channel (closing rate: τoff) can be changed.Furthermore, "having channel rhodopsin function" refers to having a channel function that controls ion permeability between the outer and inner sides of a cell by sensing light, as is known to those skilled in the art. Preferably, at least one of the biological activities evaluated by the degree of photosensitivity or the wavelength of light sensing, the degree of ion permeability, τon or τoff, etc., is at least equivalent to the biological activity of the polypeptide composed of the amino acid sequences shown in SEQ ID NO: 4, 6, 7, 8, respectively.

[0066] The modified channel rhodopsin of the present invention can be manufactured by genetic engineering methods. Specifically, firstly, the polynucleotide encoding the modified channel rhodopsin of the present invention (hereinafter referred to as the "modified channel rhodopsin gene of the present invention") is modulated. The modified channel rhodopsin gene of the present invention can be modulated by methods known to those skilled in the art. Specifically, for example, the inventors can chemically synthesize the modified channel rhodopsin based on the sequence information of the polynucleotide encoding the modified channel rhodopsin reported in Patent Document 1 and the polynucleotide encoding channel rhodopsin-2 from Chlamydomonas reinhardtii, and furthermore, if necessary, the polynucleotide encoding channel rhodopsin-1 from Chlamydomonas reinhardtii or the polynucleotide encoding channel rhodopsin from Chloromonas oogama. Alternatively, the desired regions of the respective polynucleotides can be amplified using PCR primers that amplify the desired regions of the respective polynucleotides based on their sequence information, and ligation can be performed using a system such as the Gibson Assembly system (New England Biolabs). Next, the modified channel rhodopsin gene of the present invention, functionally linked to the promoter, is replicated and maintained in a host bacterium, stably expressing the encoded polypeptide. This is then integrated into an expression vector that stably maintains the gene. The resulting recombinant expression vector is used to transform the host, allowing the production of the modified channel rhodopsin of the present invention in the host. For recombinant techniques, see Proc. Natl. Acad. Sci. USA., 1984 81:5662 or Molecular Cloning: A Laboratory Manual (1989) Second edition, Cold Spring Harbor Laboratory Press, etc.As expression vectors, plasmids derived from *Escherichia coli* (e.g., pET28, pGEX4T, pUC118, pUC119, pUC18, pUC19, and other plasmid DNAs), plasmids derived from *Bacillus subtilis* (e.g., pUB110, pTP5, and other plasmid DNAs), plasmids derived from yeast (e.g., YEp13, YEp24, YCp50, and other plasmid DNAs), λ phage (λgt11 or λZAP), mammalian plasmids (pCMV or pSV40), viral vectors (e.g., animal viral vectors such as adenovirus vectors, adeno-associated virus vectors, retroviral vectors, lentiviral vectors, vaccinia virus vectors, etc., insect viral vectors such as baculovirus vectors), plant vectors (e.g., binary vector pBI line), and granular vectors can be used. "Functionally linked" refers to the functional binding between a promoter sequence and a target polynucleotide sequence in a manner that allows transcription of the target polynucleotide sequence to be initiated by the promoter sequence. The promoter is not particularly limited; a suitable promoter can be selected according to the host. Well-known constitutive or inducible promoters can be used, with constitutive promoters being preferred. Specific examples include the CMV promoter, SV40 promoter, CAG promoter, synaptic protein promoter, rhodopsin promoter, CaMV promoter, glycolytic enzyme promoter, lac promoter, trp promoter, tac promoter, GAPDH promoter, GAL1 promoter, PH05 promoter, PGK promoter, thy1 promoter, GRK promoter, and RPEJ promoter. The modified channelo-rhodopsin of the present invention can also be specifically expressed in specific cells, such that the upstream of these promoters binds to 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) specifically expressed in photoreceptor cells (Marjorie Nicoud et al., The Journal of Gene Medicine, Volume 9, Issue 12, 1013-1107, December 2007)). Insertion of the modified channelo-rhodopsin gene of the present invention into the expression vector is performed, for example, by creating or ligating a restriction endonuclease site side-attached to the modified channelo-rhodopsin gene of the present invention, and inserting it into the appropriate restriction endonuclease site or multiple cloning site of the vector DNA.In addition to the promoter and the modified channelomorphosin gene of this invention, the expression vector may also contain, as needed, enhancers and other cis-elements, splicing signals, poly-A additional signals, selection markers (drug resistance gene markers such as ampicillin resistance markers and tetracycline resistance markers, nutritional requirement complementation gene markers such as LEU1, TRP1, and URA3, and preferential selection markers such as APH, DHFR, and TK), ribosome binding sites (RBS), etc. Host transformation can be performed using protoplast methods, protoplastid methods, competent cell methods, viral methods, calcium phosphate methods, lipid transfection methods, microinjection methods, gene bombardment methods, Agrobacterium methods, electroporation, etc. The resulting transformants are cultured in a medium containing available carbon sources, nitrogen sources, metal salts, vitamins, etc., under appropriate conditions. Transformant culture is typically carried out under aerobic conditions such as shaking culture or aerated and stirred culture at 25–37°C for 3–6 hours. During cultivation, the pH is maintained near neutral. pH adjustments are made using inorganic or organic acids, alkaline solutions, etc. During cultivation, antibiotics such as ampicillin or tetracycline may be added to the culture medium as needed, corresponding to the selection marker inserted into the recombinant expression vector. Furthermore, the host used in transformation is not particularly limited as long as it can express the modified channel rhodopsin of this invention; examples include bacteria (Escherichia coli or Bacillus subtilis), yeast (Saccharomyces cerevisiae, etc.), animal cells (COS cells, Chinese hamster ovary (CHO) cells, 3T3 cells, BHK cells, HEK293 cells, etc.), insect cells, etc. The modified channel rhodopsin of this invention can be separated or purified from cultures obtained from the transformation organism (culture supernatant, cultured cells, cultured bacterial cells, cell or bacterial cell homogenates, etc.) using conventional methods, and obtained in a form that retains its activity through ultrafiltration concentration, freeze-drying, spray drying, crystallization, etc. Alternatively, the modified channelo-rhodopsin of the present invention may be provided in the cellular form expressing the modified channelo-rhodopsin of the present invention without isolation or purification. In this case, the host cell used in transformation is a host cell suitable for subsequent use, such as nerve cells (receptor cells, bipolar cells, ganglion cells, etc.), preferably human nerve cells. Furthermore, when using the modified channelo-rhodopsin of the present invention for medical purposes, it may also be provided in the form of an expression vector of the modified channelo-rhodopsin of the present invention. In this case, it is preferable to use an expression vector with excellent cell introduction efficiency, intracellular replication maintenance, stability, and expression efficiency. Examples of such vectors include viral vectors such as adenovirus vectors, retroviral vectors, and lentiviral vectors, as well as (autoreplicating) plasmids and transposons.The plasmids for preparing the modified channel rhodopsin expression vector of the present invention can be modulated according to, for example, the methods described in Tomita H et al., Invest Ophthalmol Vis Sci. 2007 Aug; 48(8): 3821-6 or Sugano E et al., Invest Ophthalmol Vis Sci. 2005 Sep; 46(9): 3341-8.

[0067] Examples of the modified channel rhodopsin gene of the present invention include, for example, a polynucleotide consisting of the base 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 base 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 base 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 base 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 base 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 base 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 base 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 base sequence shown in SEQ ID NO: 20 (encoding a polypeptide consisting of the amino acid sequence shown in SEQ ID NO: 4). (The polypeptide constituted by the amino acid sequence shown in NO: 12). However, the modified channel rhodopsin gene of the present invention is not limited to these polynucleotides, but includes polynucleotides that hybridize under strict conditions with complementary strands of these polynucleotides, and polynucleotides encoding polypeptides having channel rhodopsin function. In addition, polynucleotides containing 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 with the respective base sequences shown in SEQ ID NO: 13 to 20, and encoding polypeptides having channel rhodopsin function. The term "hybridization under stringent conditions" includes, for example, hybridization at 30–50°C, in 3–4 × SSC (150 mM sodium chloride, 15 mM sodium citrate, pH 7.2), and in 0.1–0.5% SDS for 1–24 hours, preferably hybridization at 40–45°C, in 3.4 × SSC, and in 0.3% SDS for 1–24 hours, followed by washing. Examples of washing conditions include, for instance, a solution containing 2 × SSC and 0.1% SDS, and continuous washing at room temperature using 1 × SSC solution and 0.2 × SSC solution.However, the combination of the above conditions is illustrative. Those skilled in the art can achieve the same level of stringency by appropriately combining the above-mentioned elements or other elements (e.g., concentration, length and GC content of the hybridization probe, reaction time of hybridization, etc.) that determine the stringency of hybridization.

[0068] The modified channelomorphosin of the present invention is based on Patent Document 1. When a polypeptide is formed by replacing the N-terminal region of channelomorphosin derived from *Volvox* with the N-terminal region of channelomorphosin-1 derived from *Chlamydomonas reinhardtii*, it retains the high expression efficiency on the cell membrane characteristic of the modified channelomorphosin (e.g., mVChR1) described in Patent Document 1, and further possesses high ion permeability. Therefore, the modified channelomorphosin of the present invention, or an expression vector containing a polynucleotide encoding it, is useful for the treatment of subjects suffering from outer retinal layer disorders. Here, "outer retinal layer disorders" refers to any disease in which visual function is impaired or absent due to degeneration or disappearance of photoreceptor cells in the outer layer of the retina, but cells other than photoreceptor cells remain normal or partially functional. Examples of such diseases include retinitis pigmentosa, age-related macular degeneration, and retinal detachment. "Test subject" refers to a subject who is blind due to an outer retinal barrier or is at risk of blindness. The test subject is not limited to humans and may also be other mammals. Examples of other mammals include mice, rats, monkeys, rabbits, dogs, cats, cattle, and horses. "Treatment of test subjects with outer retinal barriers" refers to the restoration of visual function in test subjects who are blind due to an outer retinal barrier or are at risk of blindness, compared to before the application of the medicine of this invention.

[0069] The pharmaceutical composition of the present invention uses the modified channel rhodopsin of the present invention or an expression vector containing a polynucleotide encoding the same as an active ingredient, and is formulated as a pharmaceutical preparation for treating a subject with an outer retinal defect. The effective amount is the amount that provides a therapeutic effect for a given symptom or method of administration, and is suitably determined by those skilled in the art through animal testing or clinical trials, taking into account factors such as the age, weight, sex, disease state or severity, and method of administration of the subject. In the case of a virus, the viral load is, for example, 10. 12 ~10 13 Capsules / ml (e.g., about 10) 13(Each capillary / ml). When formulated as a pharmaceutical, the active ingredient may be formulated together with one or more pharmaceutically permissible carriers. Examples of pharmaceutically permissible carriers include various buffer solutions, such as physiological saline, phosphate, and acetate buffers. The pharmaceutical may also contain additional therapeutic ingredients. Examples of additional therapeutic ingredients include drugs known as therapeutic agents for retinitis pigmentosa, age-related macular degeneration, and retinal detachment. The pharmaceutical can be formulated as, for example, injections, eye drops, and eye washes for topical application. Injectable formulations may include preservatives and be provided as unit dosage forms, such as ampoules or multi-dose containers. Additionally, the pharmaceutical can be a lyophilized agent prepared by recombining with a suitable medium, such as sterile water without pyrogenic substances, before use. The administration of the pharmaceutical is preferably by direct injection into the affected area of ​​the test subject, i.e., into the retina, or by direct contact with the vitreous humor.

[0070]

Example

[0071] The present invention will now be described in detail with reference to embodiments. However, the present invention is not limited to the following description.

[0072] [Example 1: Modified channel rhodopsin of the present invention, consisting of the amino acid sequence shown in SEQ ID NO: 4 (obtained from cells expressing p525)]

[0073] A polynucleotide region encoding amino acids 1-141 of the amino acid sequence shown in SEQ ID NO: 1 (encoding mVChR1) as described in Patent Document 1, a polynucleotide region encoding amino acids 143-170 of the channel rhodopsin-1 from Chlamydomonas reinhardtii shown in SEQ ID NO: 2, a polynucleotide region encoding amino acids 170-270 of the amino acid sequence shown in SEQ ID NO: 1, a polynucleotide region encoding amino acids 233-242 of the channel rhodopsin-2 from Chlamydomonas reinhardtii shown in SEQ ID NO: 3, and a polynucleotide region encoding amino acids 280-342 of the amino acid sequence shown in SEQ ID NO: 1, along with polynucleotides containing restriction endonuclease sequences appended to their 5' and 3' ends, was chemically synthesized and inserted into the multiple cloning site of a plasmid for preparing an adeno-associated virus vector. The structure of the adeno-associated virus vector preparing a p525-expressing plasmid thus modulated is shown in [illustration missing]. Figure 1This plasmid contains a fluorescent protein gene (venus) in the 3' region of the multiple cloning site, and the target gene is expressed as a fusion protein with venus appended to the C-terminal region. This plasmid is then transfected into cells using the calcium phosphate method, and cells expressing p525 are identified using venus as an indicator. Specifically, after adding 1.5 mL of 0.3 M CaCl2 to a tube containing the plasmid solution (15 μg) and inverting the mixture, 1.5 mL of 2× HBS (280 mM NaCl, 1.5 mM Na2HPO4, 50 mM HEPES, pH 7.1) prepared in a separate tube is added. After inverting and stirring again, the contents are added dropwise to HEK (Human Embryonic Kidney) 293 cells, a cell line derived from human fetal kidney, cultured in DMEM medium containing 10% FBS. The cells are then cultured at 37°C with 5% CO2. Six hours later, the culture medium was replaced with fresh culture medium, and after two days of culture, the cells were observed under a fluorescence microscope to confirm the expression of p525 in the cells.

[0074] [Example 2: Modified channel rhodopsin of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 6 (obtained from cells expressing p548)]

[0075] In addition to the chemically synthesized polynucleotide region encoding amino acids 1-141 of the amino acid sequence shown in SEQ ID NO: 1 (encoding mVChR1 as described in Patent Document 1), the polynucleotide region encoding amino acids 143-170 of the channel rhodopsin-1 from Chlamydomonas reinhardtii from SEQ ID NO: 2, the polynucleotide region encoding amino acids 170-244 of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide region encoding amino acids 187-212 of the channel rhodopsin from Chloromonas oogama from SEQ ID NO: 5, the polynucleotide region encoding amino acids 233-242 of the channel rhodopsin-2 from Chlamydomonas reinhardtii from SEQ ID NO: 3, the polynucleotide region encoding amino acids 280-322 of the amino acid sequence shown in SEQ ID NO: 1, and the polynucleotide region encoding SEQ ID NO: 1, the polynucleotide region encoding amino acids 143-170 of the channel rhodopsin-1 from Chlamydomonas reinhardtii from SEQ ID NO: 1, the polynucleotide region encoding amino acids 170-244 of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide region encoding amino acids 187-212 of the channel rhodopsin from Chloromonas oogama from SEQ ID NO: 5, the polynucleotide region encoding amino acids 233-242 of the channel rhodopsin-2 from Chlamydomonas reinhardtii from SEQ ID NO: 1, and the polynucleotide region encoding amino acids 280-322 of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide region encoding SEQ ID NO: 1, the polynucleotide region encoding amino acids 143-170 of the channel rhodopsin-1 from Chlamydomonas reinhardtii from SEQ ID NO: 1, and the polynucleotide region The polynucleotide region of amino acids 265-286 of the amino acid sequence shown in NO:5, and the polynucleotide with the restriction endonuclease sequence appended to its 5' and 3' ends, are inserted outside the multiple cloning site of the plasmid used to prepare adeno-associated virus vector, and cells expressing p548 are prepared in the same manner as in Example 1.

[0076] [Example 3: Modified channel rhodopsin of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 7 (obtained from cells expressing p550)]

[0077] In addition to the chemically synthesized polynucleotide region encoding amino acids 1-27 of the amino acid sequence shown in SEQ ID NO: 1 encoding mVChR1 as described in Patent Document 1, the polynucleotide region encoding amino acids 61-141 of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide region encoding amino acids 143-170 of the amino acid sequence shown in SEQ ID NO: 2 of channel rhodopsin-1 derived from Chlamydomonas reinhardtii, the polynucleotide region encoding amino acids 170-244 of the amino acid sequence shown in SEQ ID NO: 1, the polynucleotide region encoding amino acids 187-212 of the amino acid sequence shown in SEQ ID NO: 5 of channel rhodopsin derived from Chloromonas oogama, and the polynucleotide region encoding amino acids 233-242 of the amino acid sequence shown in SEQ ID NO: 3 of channel rhodopsin-2 derived from Chlamydomonas reinhardtii, the polynucleotide region encoding SEQ ID NO: 1, and the polynucleotide region encoding amino acids 143-170 of the amino acid sequence shown in SEQ ID NO: 2 of channel rhodopsin derived from Chlamydomonas reinhardtii, the polynucleotide region encoding amino acids 143-170 of the amino acid sequence shown in SEQ ID NO: 2 of channel rhodopsin derived from Chlamydomonas reinhardtii, the polynucleotide region encoding amino acids 143-244 of channel rhodopsin-1 ... The polynucleotide region of amino acids 280-322 of the amino acid sequence shown in NO:1 and the polynucleotide region of amino acids 265-286 of the amino acid sequence shown in SEQ ID NO:5, along with the polynucleotides with restriction endonuclease sequences appended to their 5' and 3' ends, were inserted outside the multiple cloning site of the plasmid used to prepare adeno-associated virus vector, and cells expressing p550 were prepared in the same manner as in Example 1.

[0078] [Experimental Example 1: Measurement of photoinduced currents in cells expressing p525, p548, and p550 using the patch-clamp method (Part 1)]

[0079] For cells expressing p525, p548, and p550, venus expression was confirmed under a microscope, and measurements were performed 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 1 N 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 1 N CsOH. Light irradiation (LED light source) was set to 1 second, and light intensity was set to 1 μW / mm². 2The stimulation interval was set to 60 seconds, and the fixed potential was set to -60mV. Wavelengths were set to 405, 455, 505, 560, 617, and 656 nm respectively. The results are shown below. Figure 2 The results of assays on cells expressing mVChR1 obtained in the same manner as those expressing p525, p548, and p550 are also presented together. Figure 2 .from Figure 2 It was found that p525, p548, and p550 all have higher permeability than mVChR1 ions.

[0080] [Experimental Example 2: Measurement of photoinduced current in cells expressing p525 using the patch-clamp method (Part 2)]

[0081] Except for irradiation with light for 10 milliseconds, when the photoinduced current of p525 was measured in the same manner as in Example 1, it showed higher permeability than mVChR1 ions.

[0082] [Experimental Example 3: Measurement of photoinduced current in cells expressing p528 using the patch-clamp method]

[0083] When the photoinduced current of p528 was measured in the same manner as in Experimental Example 1, it showed higher permeability than mVChR1 ions.

[0084] [Example 4: Modified channel rhodopsin of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 9 (obtained from cells expressing p578(H172G))]

[0085] Using the adeno-associated virus vector preparation plasmid expressing p548 modulated in Example 2, site-specific mutations were introduced using the KOD mutagenesis kit (Code No. SMK-101, TOYOBO) according to its manual, replacing His at position 172 with Gly to modulate the adeno-associated virus vector preparation plasmid expressing p578. Specifically, PCR was performed using 10 pmol / μL of the mutation primer (SEQ ID NO: 21, 172G forward primer: GGACTGAGCA ACCTGACCGG CCTGAA) and 10 pmol / μL of the reverse primer (SEQ ID NO: 22, GATCAGGATC ACAGGACAGG TCAG), with 50 ng / μL of the adeno-associated virus vector preparation plasmid expressing p548 as a template. The PCR reaction cycle consisted of 5 cycles: 94°C for 2 min → 98°C for 10 sec → 68°C for 7 min. The PCR product was treated with the restriction endonuclease DpnI to digest the adeno-associated virus vector preparation plasmid expressing p548, which served as a template. Subsequently, the linear plasmid was self-ligated into a circular form by the simultaneous action of T4 polynucleotide kinase and ligase, yielding the adeno-associated virus vector preparation plasmid expressing p578. Cells expressing p578 were prepared in the same manner as in Example 1, except that the adeno-associated virus vector preparation plasmid expressing p578 was used.

[0086] [Example 5: Modified channel rhodopsin of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 10 (obtained from cells expressing p579(H172A))]

[0087] Except for using the 172A forward primer of SEQ ID NO: 23: GCCCTGAGCA ACCTGACCGGCCTGAA as a variant primer, the plasmid for preparing the adeno-associated virus vector expressing p579 was modulated in the same manner as in Example 4 to prepare cells expressing p579.

[0088] [Example 6: Modified channel rhodopsin of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 11 (obtained from cells expressing p580(H172K))]

[0089] Except for using the 172K forward primer of SEQ ID NO: 24: AAACTGAGCA ACCTGACCGGCCTGAA as a variant primer, the plasmid for preparing the adeno-associated virus vector expressing p580 was modulated in the same manner as in Example 4 to prepare cells expressing p580.

[0090] [Example 7: Modified channel rhodopsin of the present invention consisting of the amino acid sequence shown in SEQ ID NO: 12 (obtained from cells expressing p581(H172R))]

[0091] Except for using the 172R forward primer of SEQ ID NO: 25: CGCCTGAGCA ACCTGACCGGCCTGAA as a variant primer, the plasmid for preparing the adeno-associated virus vector expressing p581 was modulated in the same manner as in Example 4 to prepare cells expressing p581.

[0092] [Experimental Example 4: Measurement of photoinduced currents in cells expressing p578, p579, p580, and p581 using the patch-clamp method]

[0093] The photoinduced currents were measured in the same manner as in Experimental Example 1. The results are shown in... Figure 3 The results of assays for cells expressing p548 are also shown in [the table / image / etc.]. Figure 3 For example from Figure 3 Clearly, the ion permeability of p578, p579, p580, and p581 is lower than that of p548, but higher than that of mVChR1 (the ion permeability of mVChR1 is referenced). Figure 2 ).

[0094] [Experimental Example 5: Determination of τon and τoff in p579-expressing cells using the patch-clamp method]

[0095] τon and τoff were measured under the same conditions as the photoinduced current measurement in Example 4. For τon, the results are shown in... Figure 4 For τoff, the results are shown in Figure 5 The assay results for cells expressing p548 are shown together in their respective figures. From Figure 4 and Figure 5 It was found that when His at position 172 of p548 was replaced with p579 of Ala, both τon and τoff were shorter than those of p548, and the cell degradation was controlled by a higher time.

[0096] [Experimental Example 6: Introduction of the p548 gene into the retina using an adeno-associated virus vector and its effects]

[0097]

Experimental Methods

[0098] [Preparation of adeno-associated virus vectors]

[0099] Using an AAV-free auxiliary system (Stratagene, La Jalla, CA), and according to its manual, an adeno-associated virus vector for introducing the p548 gene into the retina was prepared using three plasmids: pAAV-RC, pHelper, and a plasmid for preparing the p548-expressing adeno-associated virus vector modulated in Example 2. Specifically, after adding 1.5 mL of 0.3 M CaCl2 to a tapped tube containing the respective plasmid solutions (15 μg each) and inverting the tube, the contents were added to 1.5 mL of 2×HBS (280 mM NaCl, 1.5 mM Na2HPO4, 50 mM HEPES, pH 7.1) prepared in a separate tube. After inverting and stirring again, the contents were added dropwise to 293T cells cultured in 15 cm culture dishes. The three plasmids were co-transfected using the calcium phosphate method, and the cells were cultured at 37°C with 5% CO2. After 3 days of culture, the viral particles were purified from the recovered cells for the intended purpose.

[0100] Laboratory animals

[0101] Seven-month-old Royal College of Surgeons (RCS:rdy / rdy) rats were used. Although RCS rats develop a retina normally after birth, photoreceptor cell degeneration begins from 3 weeks of age, and by 3 months of age, the photoreceptor cells have largely disappeared, leading to blindness. Therefore, no visual evoked potentials were recorded in 7-month-old RCS rats.

[0102] [Introduction of the p548 gene into the retina]

[0103] Under mixed anesthesia with ketamine (66 mg / kg) and xylazine (3.3 mg / kg), the conjunctiva of both eyes of RCS rats was cut by about 1 mm, and 5 μL of virus solution was injected into the vitreous body by inserting a 32-gauge microinjector through the flat part of the piloid body.

[0104] Measurement of visual evoked potentials

[0105] Two months after intravitreal injection of a viral solution into RCS rats, visual evoked potentials (VAPs) were measured using a PuREC evoked response recording device (Mayo). The electrodes used for VEP measurement were positioned on the dura mater approximately 6.8 mm from the anterior fontanelle and 3 mm from its center, with the skull exposed by scalp incision. The reference electrode was positioned on the dura mater 12 mm from the anterior fontanelle. The electrodes were fixed with dental cement. VEP measurements were performed under mixed anesthesia with ketamine (66 mg / kg) and xylazine (3.3 mg / kg), with mydriasis caused by 1% atropine and 2.5% phenylephrine hydrochloride. Visual stimulation was performed using various LEDs (stimulation wavelengths: 465, 525, 650 nm) as light sources, repeated 200 times at a stimulation frequency of 1 Hz for 10 ms, and the results were recorded by summation and averaging.

[0106] Preparation and observation of retinal stretch specimens and retinal section specimens.

[0107] Eight months after intravitreal injection of a viral solution into RCS rats, retinal stretch specimens were prepared to confirm p548 expression. The eyeballs were enucleated, immediately fixed with 4% paraformaldehyde solution, and the anterior ocular portion was removed. The neuroretina was then detached from the choroid. p548 expression was confirmed by stretching the detached neuroretina onto a glass slide and observing it under a fluorescence microscope, using venus as an indicator. Next, frozen sections (retinal sections) were prepared by embedding the prepared retinal stretch specimens in a freezing tissue sectioning medium (OCT compound from Sakura Finetek, Japan). These sections were then observed under a fluorescence microscope, using venus as an indicator to confirm p548 expression in the retinal cross-section.

[0108]

Experimental Results

[0109] The results of visual evoked potential measurements are shown in Figure 6 For example from Figure 6 It is clear that by introducing the p548 gene into the retina, visual evoked potentials can be recorded at any of the stimulation wavelengths of 465, 525, and 650 nm. The stronger the light intensity, the larger the amplitude of the visual evoked potential. A photograph of a retinal stretching specimen observed under a fluorescence microscope is shown below. Figure 7 For example from Figure 7 Clearly, p548 expression can be confirmed throughout the neuroretina. A photograph of a retinal section specimen observed under a fluorescence microscope is shown. Figure 8 For example from Figure 8 It is clear that p548 is the predominant expression level, which can be confirmed in the retinal ganglion cell layer (the lower right photo shows the stained nucleus).

[0110] [Potential for Industrial Applications]

[0111] The present invention has industrial potential at the point where modified channels of rhodopsin can provide high ion permeability (photoreactivity).

Claims

1. Modified channel rhodopsin, which is a polypeptide consisting of an amino acid sequence represented by any one of SEQ ID NO: 4, 6-12.

2. A polynucleotide encoding the polypeptide of claim 1.

3. An expression vector containing the polynucleotide of claim 2, which is functionally linked to a promoter.

4. Cultured cells expressing the polypeptide of claim 1.

5. The cell of claim 4, wherein the cell is a nerve cell.

6. Use of any one of the polypeptide of claim 1, the polynucleotide of claim 2, or the expression vector of claim 3 in the manufacture of a medicament for treating a subject with an outer retinal defect.

7. The use as claimed in claim 6, wherein the outer retinal layer defect is any of the following: retinitis pigmentosa, age-related macular degeneration, or retinal detachment.

8. A pharmaceutical composition for treating outer retinal defects, comprising either the polypeptide of claim 1 or the expression vector of claim 3 as an active ingredient.

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