Visual function regeneration agent or visual function decline prevention agent

Abstract

To provide a visual function regeneration agent or a visual function decline prevention agent that has excellent regenerative ability for visual function. [Solution] The visual function regeneration agent or visual function decline prevention agent of the present invention contains a chimeric protein as an active ingredient, which has the amino acid sequence of an ion-transporting receptor rhodopsin derived from microorganisms and the amino acid sequence of a G protein-coupled receptor rhodopsin derived from animals. Preferably, the chimeric protein is such that the amino acid sequences of the cytoplasmic second loop and / or cytoplasmic third loop of the ion-transporting receptor rhodopsin derived from microorganisms are replaced with the amino acid sequences of the cytoplasmic second loop and / or cytoplasmic third loop of a G protein-coupled receptor rhodopsin.

Description

Technical Field

[0001] The present invention relates to a visual function regenerating agent or a preventive agent for visual function decline.

Background Art

[0002] Rhodopsin is a photosensitive receptor having a seven transmembrane structure in the retina of humans and animals, but ion channel type and ion pump type rhodopsins derived from microorganisms are also known.

[0003] For example, Non-Patent Document 1 discloses channelrhodopsin 2 (ChR2), which is an ion channel type rhodopsin. Further, in Non-Patent Document 2, it has been reported that a certain visual function is regenerated in mice and rats by introducing a mutant channelrhodopsin into retinal ganglion cells.

Prior Art Documents

Non-Patent Documents

[0004]

Non-Patent Document 1

Non-Patent Document 2

Summary of the Invention

Problems to be Solved by the Invention

[0005] However, ion channel rhodopsins have not yet shown sufficient regenerative effects on visual function, and there is room for improvement.

[0006] This invention has been made in view of the above circumstances, and aims to provide a visual function regeneration agent or a visual function decline prevention agent having excellent visual function regeneration capabilities. [Means for solving the problem]

[0007] The inventors of this invention have discovered that a chimeric protein created by fusing two completely different types of rhodopsins—an ion-transporting rhodopsin derived from microorganisms and a G protein-coupled receptor rhodopsin derived from animals—actually possesses excellent regenerative capacity for visual function, and have completed the present invention. More specifically, the present invention consists of the following components.

[0008] (1) A visual function regeneration agent or visual function decline prevention agent containing as an active ingredient a chimeric protein having the amino acid sequence of an ion transport receptor rhodopsin derived from microorganisms and the amino acid sequence of a G protein-coupled receptor rhodopsin derived from animals.

[0009] (2) The chimeric protein is such that the amino acid sequence of the cytoplasmic second loop and / or cytoplasmic third loop of the amino acid sequence of the ion transport receptor rhodopsin derived from microorganisms is the same as the amino acid sequence of the cytoplasmic second loop and / or cytoplasmic second loop of the G protein-coupled receptor rhodopsin. A visual function regeneration agent or visual function decline prevention agent as described in (1), wherein the amino acid sequence of the third loop has been replaced.

[0010] (3) The visual function regeneration agent or visual function decline prevention agent according to (1) or (2), wherein the ion transport receptor rhodopsin derived from the microorganism is a rhodopsin derived from a microorganism belonging to the genus Gloeobacter, and the G protein-coupled receptor rhodopsin is a rhodopsin derived from a bovine or human.

[0011] (4) The visual function regeneration agent or visual function decline prevention agent according to any of (1) to (3), wherein the chimeric protein has an amino acid sequence encoded by the DNA described in any of (a) to (d) below. (a) DNA having a base sequence that encodes the amino acid sequence described in any of Sequence IDs 1-4 (b) DNA having a base sequence that can hybridize under stringent conditions with a base sequence complementary to the base sequence encoding the amino acid sequence described in any of Sequence IDs 1-4. (c) DNA having a base sequence that encodes an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in any of the amino acid sequences described in SEQ ID NOs: 1 to 4, and which has the ability to regenerate visual function or prevent visual function decline. (d) DNA comprising a base sequence encoding an amino acid sequence having 90% or more homology to any of the amino acid sequences described in Sequence ID No. 1 to 4, and having the ability to regenerate visual function or prevent visual function decline.

[0012] (5) A visual function regeneration agent or visual function decline prevention agent containing as an active ingredient an expression vector into which DNA encoding the amino acid sequence of a chimeric protein described in any of (1) to (4) is incorporated.

[0013] (6) A visual function regeneration agent or visual function decline prevention agent described in any of (1) to (5), used for the treatment or prevention of retinitis pigmentosa.

[0014] (7) An adeno-associated virus (AAV) vector or lentiviral vector into which a sequence of a chimeric protein having the amino acid sequence of an ion-transporting receptor rhodopsin derived from a microorganism and the amino acid sequence of a G protein-coupled receptor rhodopsin derived from an animal is inserted.

[0015] (8) Use of an adeno-associated virus (AAV) vector or a lentiviral vector into which a sequence of a chimeric protein having an amino acid sequence of a microbial ion transport type receptor rhodopsin and an amino acid sequence of an animal-derived G protein-coupled receptor rhodopsin is inserted for producing a medicament for regenerating visual function or preventing visual function decline.

Advantages of the Invention

[0016] According to the present invention, excellent visual function regeneration ability can be achieved.

Brief Description of the Drawings

[0017] [Figure 1] The image obtained by observing the retina of a wild-type mouse injected with AAV2-CAGGS-EGFP-WPRE-pA into the vitreous body under a fluorescence microscope. [Figure 2] (a) A graph showing the result of extracellular potential recording of retinal ganglion cells by a multi-electrode array (MEA) for a control retinitis pigmentosa model (rd1) mouse. (b) A graph showing the result of extracellular potential recording of retinal ganglion cells by a multi-electrode array (MEA) for a retinitis pigmentosa model (rd1) mouse injected with AAV2-CAGGS-GR / BvRh-WPRE-pA. [Figure 3] (a) A figure showing the results of extracellular potential recording of retinal ganglion cells by a multi-electrode array (MEA) for a control retinitis pigmentosa model (rd1) mouse and (b) a retinitis pigmentosa model (rd1) mouse injected with AAV2-CAGGS-GR / BvRh-WPRE-pA. The upper part of FIG. 3 is a raster plot showing the firing of retinal ganglion cells for 10 times, and the lower part of FIG. 3 is a histogram with the firing frequency per second on the vertical axis.

Embodiments for Carrying out the Invention

[0018] Hereinafter, specific embodiments of the present invention will be described in detail. However, the present invention is not limited to the following embodiments, and can be implemented with appropriate modifications within the scope of the object of the present invention. In addition, regarding the overlapping parts of the description, the description may be omitted as appropriate, but it does not limit the gist of the invention.

[0019] <Visual function regenerating agent or visual function decline preventive agent> The visual function regenerating agent or visual function decline preventive agent of the present invention contains, as an active ingredient, a chimeric protein having the amino acid sequence of an ion transport type receptor rhodopsin derived from a microorganism and the amino acid sequence of a G protein-coupled receptor rhodopsin derived from an animal.

[0020] Rhodopsin has a pigment called retinal inside, and when this receives light, it is activated and the visual signal is transmitted to the brain. Since the ion transport type receptor rhodopsin derived from a microorganism does not release retinal even when irradiated with light, it can be repeatedly activated by absorbing light, but it cannot activate G protein like the G protein-coupled receptor rhodopsin derived from an animal. In contrast, according to the present invention, by fusing the animal-derived G protein-coupled receptor rhodopsin to the ion transport type receptor rhodopsin derived from a microorganism that can be repeatedly used, while maintaining the function of repeatedly activating the ion channel type receptor rhodopsin of the ion transport type receptor rhodopsin derived from a microorganism, it is speculated that high activity via the endogenous G protein by the G protein-coupled receptor can be obtained, and an excellent visual regeneration effect can be obtained. Thus, the rhodopsin derived from a microorganism and the G protein-coupled receptor derived from an animal are completely different types of receptors, and the present inventors have actually found that a chimeric protein combining such two types of receptors has the ability to regenerate excellent visual function. In addition, as described above, since it can be repeatedly activated while obtaining high activity, a preventive effect on the decline of visual function (for example, suppression of the progression of retinal diseases such as retinitis pigmentosa) can also be expected.

[0021] Examples of ion transport receptor rhodopsins include ion pump receptor rhodopsins and ion channel receptor rhodopsins.

[0022] The chimeric protein of the present invention is a chimeric protein of microbial-derived ion-transport receptor rhodopsin and G protein-coupled receptor rhodopsin, and has a seven-transmembrane structure. In the present invention, it is preferable that the chimeric protein of microbial-derived ion-transport receptor rhodopsin and G protein-coupled receptor rhodopsin be designed to have high levels of both the function of repeatedly activating microbial-derived ion-transport receptor rhodopsin and the G protein activity of G protein-coupled receptor rhodopsin. In this respect, it is preferable that the chimeric protein of the present invention maintains high levels of activity of both and exhibits particularly high visual function regeneration ability, in which the amino acid sequences of the cytoplasmic second loop and / or cytoplasmic third loop of the amino acid sequence of microbial-derived ion-transport receptor rhodopsin are replaced with the amino acid sequences of the cytoplasmic second loop and / or cytoplasmic third loop of G protein-coupled receptor rhodopsin. The terms "cytoplasmic second loop" and "cytoplasmic third loop" refer to the loops located second and third from the N-terminus, respectively, out of the seven loops.

[0023] Examples of ion-transporting receptor rhodopsins derived from microorganisms include those from eubacteria such as the genus Gloeobacter, and eukaryotes such as the genera Volvox, Chlamydomonas, and Guillardia. Examples of Gloeobacter include Gloeobacter violaceus. Examples of Volvox include Volvox carteri. Examples of Chlamydomonas include Chlamydomonas reinhardtii. Examples of Guillardia include Guillardia theta. To obtain higher visual regeneration and preventive effects, structural compatibility with the G protein activation loop and membrane translocation efficiency are considered important. Microbial ion transport receptor rhodopsins are particularly favored to belong to the genus Gloeobacter because they exhibit good structural compatibility with the G protein activation loop and membrane translocation efficiency. In particular, Gloeobacter violaceus is preferred among microorganisms belonging to the genus Gloeobacter. Furthermore, it is preferable to fuse microorganisms belonging to the genus Gloeobacter with bovine or human G protein-coupled receptor rhodopsins from animal-derived sources. The genus Gloeobacter is also favored because it has the important property of being well expressed in both the bacterium Escherichia coli and the eukaryote human cell.

[0024] Examples of animal-derived G protein-coupled receptor rhodopsins include those derived from cattle, humans, mice, rats, cats, dogs, pigs, sheep, and horses. Of these, rhodopsins derived from cattle or humans are particularly preferred.

[0025] More specifically, the chimeric protein is preferably one that has an amino acid sequence encoded by the DNA described in any of (a) to (d) below. (a) DNA having a base sequence that encodes the amino acid sequence described in any of Sequence IDs 1-4 (b) DNA having a base sequence that can hybridize under stringent conditions with a base sequence complementary to the base sequence encoding the amino acid sequence described in any of Sequence IDs 1-4. (c) DNA having a base sequence that encodes an amino acid sequence in which one or more amino acids are substituted, deleted and / or added in any of the amino acid sequences described in SEQ ID NOs: 1 to 4, and which has the ability to regenerate visual function or prevent visual function decline. (d) DNA comprising a base sequence encoding an amino acid sequence having 90% or more homology to any of the amino acid sequences described in Sequence ID No. 1 to 4, and having the ability to regenerate visual function or prevent visual function decline.

[0026] The cytoplasmic second loop of the G protein-coupled receptor rhodopsin described above is preferably one having the amino acid sequence encoded by the DNA described in (e) to (h) below. (e) DNA having a base sequence encoding the amino acid sequence described in Sequence ID No. 5 or 6 (f) DNA having a base sequence that can hybridize under stringent conditions with a base sequence complementary to the base sequence encoding the amino acid sequence described in Sequence ID No. 5 or 6. (g) DNA having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence described in SEQ ID NO: 5 or 6. (h) DNA consisting of a base sequence encoding an amino acid sequence having 90% or more homology to the amino acid sequence described in Sequence ID No. 5 or 6.

[0027] The third cytoplasmic loop of the G protein-coupled receptor rhodopsin described above is as follows: DNA having the amino acid sequence encoded by the DNA described in (i) to (l) is preferred. (i) DNA having a base sequence that encodes the amino acid sequence described in Sequence ID No. 7 (j) DNA having a base sequence that is complementary to the base sequence encoding the amino acid sequence described in Sequence ID No. 7 and a base sequence that can hybridize under stringent conditions. (k) DNA having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence described in Sequence ID No. 7. (l) DNA consisting of a base sequence encoding an amino acid sequence having 90% or more homology to the amino acid sequence described in Sequence ID No. 7.

[0028] The nucleotide sequence encoding the amino acid sequence described in any of Sequence IDs 1 to 4 is a preferred sequence of the nucleotide sequence encoding the chimeric protein of the present invention. The nucleotide sequence encoding the amino acid sequence described in any of Sequence IDs 1 to 4 has the ability to regenerate visual function or prevent visual function decline. In this specification, "the nucleotide sequence has the ability to regenerate visual function or prevent visual function decline" means that the polypeptide encoded by the nucleotide sequence has the ability to regenerate visual function or prevent visual function decline. Furthermore, DNA having the nucleotide sequence encoding the amino acid sequence described in any of Sequence IDs 1 to 4 further includes various variants and homologs that have the ability to regenerate visual function or prevent visual function decline. Variants and homologs of DNA having the nucleotide sequence encoding the amino acid sequence described in any of Sequence IDs 1 to 4 include, for example, DNA having a nucleotide sequence that can hybridize with the nucleotide sequence encoding the amino acid sequence described in any of Sequence IDs 1 to 4 under stringent conditions. Furthermore, variants and homologs of DNA having the nucleotide sequence encoding the amino acid sequence described in any of Sequence IDs 5 to 7 include DNA having a nucleotide sequence that can hybridize with the nucleotide sequence encoding the amino acid sequence described in any of Sequence IDs 5 to 7 under stringent conditions. Examples of "stringent conditions" include carrying out the reaction in a normal hybridization buffer at 40-70°C (preferably 50-67°C, more preferably 60-65°C) and washing in a washing solution with a salt concentration of 15-300 mM (preferably 15-150 mM, more preferably 15-60 mM, even more preferably 30-50 mM).

[0029] Any of Sequence IDs 1 to 4 can be used as the amino acid sequence of the chimeric protein of the present invention. The DNA encoding the amino acid sequence of the chimeric protein of the present invention also includes DNA having a base sequence encoding an amino acid sequence in which one or more amino acids are substituted, deleted, and / or added in the amino acid sequence described in any of Sequence IDs 1 to 4. Here, in any of Sequence IDs 1 to 4, "one or more" usually means within 50 amino acids, preferably within 30 amino acids, and more preferably within 10 amino acids (e.g., within 5 amino acids, within 3 amino acids, and 1 amino acid). Also, in any of Sequence IDs 5 to 7, "one or more" usually means within 6 amino acids, preferably within 5 amino acids, and more preferably within 4 amino acids (e.g., within 3 amino acids, within 2 amino acids, and 1 amino acid). When maintaining the visual function regeneration ability or visual function decline prevention ability of the chimeric protein, it is desirable that the mutated amino acid residues be mutated to other amino acids in which the properties of the amino acid side chain are conserved. For example, the properties of amino acid side chains include hydrophobic amino acids (A, I, L, M, F, P, W, Y, V), hydrophilic amino acids (R, D, N, C, E, Q, G, H, K, S, T), amino acids with aliphatic side chains (G, A, V, L, I, P), amino acids with hydroxyl group-containing side chains (S, T, Y), amino acids with sulfur atom-containing side chains (C, M), amino acids with carboxylic acid and amide-containing side chains (D, N, E, Q), amino acids with base-containing side chains (R, K, H), and amino acids with aromatic-containing side chains (H, F, Y, W) (all in parentheses are single-letter abbreviations for amino acids). It is also well known that proteins with amino acid sequences modified by the deletion, addition, and / or substitution of one or more amino acid residues with other amino acids maintain their biological activity (Mark, DF et al., Proc.). Natl.Acad.Sci.USA(1984)81,5662-5666、Zoller,M.J.& Smith,M.Nucleic Acids Research(1982)10,6487-6500、Wang,A.et al.,Science 224,1431-1433、Dalbadie-McFarland,G.et al.,Proc.Natl.Acad.Sci.USA(1982)79,6409-6413)。

[0030] Mutants and homologs of DNA having a base sequence encoding the amino acid sequence described in any of SEQ ID NOs: 1 to 4 include DNA consisting of a base sequence that has high homology to the base sequence encoding the amino acid sequence described in any of SEQ ID NOs: 1 to 4. Such DNA preferably has 90% or more homology, more preferably 95% or more (96% or more, 97% or more, 98% or more, 99% or more) to the base sequence encoding the amino acid sequence described in any of SEQ ID NOs: 1 to 4. Mutants and homologs of DNA having a base sequence encoding the amino acid sequence described in any of SEQ ID NOs: 5 to 7 include DNA consisting of a base sequence that has high homology to the base sequence encoding the amino acid sequence described in any of SEQ ID NOs: 5 to 7. Such DNA preferably has 90% or more homology, more preferably 95% or more (96% or more, 97% or more, 98% or more, 99% or more) to the base sequence encoding the amino acid sequence described in any of SEQ ID NOs: 5 to 7. The homology of amino acid sequences and nucleotide sequences can be determined by the BLAST algorithm developed by Karlin and Altschul (Proc.Natl.Acad.Sci.USA 90:5873-5877, 1993). Based on this algorithm, programs called BLASTN and BLASTX have been developed (Altschul et al. J.Mol.Biol. 215:403-410, 1990). When analyzing nucleotide sequences using BLASTN based on BLAST, parameters such as score=100 and wordlength=12 are used. Similarly, when analyzing amino acid sequences using BLASTX based on BLAST, parameters such as score=50 and wordlength=3 are used. When using the BLAST and Gapped BLAST programs, the default parameters for each program are used. Specific methods for these analyses are publicly known (http: / / www.ncbi.nlm.nih.gov.).

[0031] In this invention, "DNA" may be either a sense strand or an antisense strand (for example, one that can be used as a probe), and its shape may be single-stranded or double-stranded. It may also be genomic DNA, cDNA, or synthetic DNA.

[0032] The method for obtaining the DNA of the present invention is not particularly limited, but includes known methods such as obtaining cDNA by reverse transcription from mRNA (e.g., RT-PCR), preparing it from genomic DNA, synthesizing it by chemical synthesis, and isolating it from a genomic DNA library or cDNA library (see, for example, Japanese Patent Application Publication No. 11-29599).

[0033] The chimeric protein used in the visual function regeneration agent or visual function decline prevention agent of the present invention can be prepared, for example, by using a transformant into which an expression vector containing DNA encoding the aforementioned chimeric protein has been introduced. For example, first, this transformant is cultured under appropriate conditions to synthesize the chimeric protein encoded by this DNA. Then, the chimeric protein of the present invention can be obtained by recovering the synthesized protein from the transformant or culture medium.

[0034] More specifically, it can be produced by inserting the DNA encoding the aforementioned chimeric protein into a suitable expression vector. "Suitable vector" refers to prokaryotes and / or The vector can be any vector capable of replicating, maintaining, or self-replicating within various eukaryotic hosts, and can be appropriately selected according to the purpose of use. For example, a high-copy vector can be selected when a large amount of DNA is to be obtained, and an expression vector can be selected when a polypeptide (chimeric protein) is to be obtained. Specific examples are not limited to those described above, but include, for example, known vectors described in Japanese Patent Publication No. 11-29599.

[0035] Furthermore, the expression vector can be used not only for synthesizing chimeric proteins, but also in the visual function regeneration agent or visual function decline prevention agent of the present invention. That is, the visual function regeneration agent or visual function decline prevention agent of the present invention may contain as an active ingredient an expression vector into which DNA encoding the amino acid sequence of the above-mentioned chimeric protein is incorporated. By directly introducing such an expression vector into humans, it can be used for visual function regeneration and prevention of visual function decline. In this case, a vector that can be introduced into human cells is used. Suitable vectors include, for example, adeno-associated virus vectors (AAV vectors) and lentiviral vectors.

[0036] The method of introducing the vector can be appropriately selected depending on the type of vector and host. Specific examples are not limited to these, but for instance, when bacteria are used as the host, known methods such as the protoplast method and the competent method (see, for example, Japanese Patent Publication No. 11-29599) can be used. Furthermore, when the expression vector is used as the active ingredient of the visual function regeneration agent or visual function decline prevention agent of the present invention, it can be introduced, for example, by injecting the above-mentioned AAV vector into the eye.

[0037] The host into which the expression vector is introduced can be any host that is compatible with the expression vector and can be transformed. Specific examples, though not particularly limited, include known natural cells or artificially established cells such as bacteria, yeast, animal cells, and insect cells (see Japanese Patent Publication No. 11-29599), or animals such as humans and mice. The culture of the transformants can be carried out by appropriately selecting a known nutrient medium according to the type of transformant, etc., and adjusting the temperature, pH of the nutrient medium, culture time, etc., as appropriate, so that a large amount of chimeric protein can be easily obtained (see, for example, Japanese Patent Publication No. 11-29599).

[0038] The methods for isolating and purifying chimeric proteins are not particularly limited and include known methods such as those utilizing solubility, differences in molecular weight, and charges (see, for example, Japanese Patent Publication No. 11-29599).

[0039] In this specification, "active ingredient" refers to an ingredient contained in an amount necessary to obtain a visual function regeneration effect or a visual function decline prevention effect, and other ingredients may be included as long as the effect is not impaired to a level below the desired level. Furthermore, the visual function regeneration agent or visual function decline prevention agent of the present invention may be formulated. Furthermore, the route of administration of the visual function regeneration agent or visual function decline prevention agent of the present invention may be either oral or parenteral, and can be appropriately set depending on the form of the formulation, etc.

[0040] For oral administration, the formulation may be used in various forms such as tablets, granules, fine granules, powders, and capsules, and the formulation may contain additives such as commonly used binders, encapsulants, excipients, lubricants, disintegrants, and wetting agents. In addition, for oral administration, the formulation may be formulated in liquid form such as oral aqueous solutions, suspensions, emulsions, and syrups, or in a dry state that is redissolved at the time of use.

[0041] For parenteral administration, the formulation may be contained in unit dose ampoules, multi-dose containers, or tubes, and may also contain additives such as stabilizers, buffers, preservatives, and isotonic agents. Furthermore, for parenteral administration, the formulation may be placed in an appropriate carrier (sterilized) at the time of use. It may also be formulated as a powder that can be redissolved in water, etc. Parenteral administration methods include intravitreous administration, subconjunctival administration, anterior chamber administration, and ophthalmic administration, with intravitreous administration being preferred.

[0042] The visual function regeneration agent or visual function decline prevention agent of the present invention described above can be used for visual function regeneration or prevention of visual function decline by administering it to humans in the manner described above.

[0043] In this invention, "visual function regeneration" refers to the improvement of impaired visual function, which may be partial or complete regeneration. "Prevention of visual function decline" refers to preventing the decline of visual function or suppressing the progression of visual function decline. Examples of such visual functions include visual acuity, contrast sensitivity, light and dark adaptation, and color vision.

[0044] The visual function regeneration agent or visual function decline prevention agent of the present invention may be used for applications expected from visual function regeneration or prevention of visual function decline, for example, for the treatment or prevention of diseases related to visual function decline. Examples of diseases related to visual function decline include retinitis pigmentosa, age-related macular degeneration, myopic macular degeneration, macular dystrophy, diabetic retinopathy, uveitis, and retinal detachment.

[0045] <Vector> The present invention includes adeno-associated virus (AAV) vectors or lentiviral vectors in which a chimeric protein sequence having the amino acid sequence of a microbial ion-transporting receptor rhodopsin and the amino acid sequence of an animal-derived G protein-coupled receptor rhodopsin is inserted.

[0046] Furthermore, the present invention includes the use of an adeno-associated virus (AAV) vector or lentiviral vector into which a chimeric protein sequence having the amino acid sequence of a microbial ion-transporting receptor rhodopsin and the amino acid sequence of an animal-derived G protein-coupled receptor rhodopsin is inserted, for the production of a pharmaceutical product that restores visual function or prevents visual function decline.

[0047] The same type of chimeric protein as described above can be used. [Examples]

[0048] The following experiments on visual function were conducted using mice.

[0049] (Experimental animals) The experiment used wild-type mice (C57BL / 6J, CLEA Nippon Co., Ltd.) and retinitis pigmentosa model (rd1) mice (C3H / HeJ Jcls, CLEA Nippon Co., Ltd.), both of which were 3-week-old males.

[0050] (Production of DNA encoding the chimeric protein (GR / BvRh)) A chimeric protein is encoded by substituting the amino acid sequence corresponding to positions 137-145 from the N-terminus, which corresponds to the second cytoplasmic loop of Gloeobacter violaceus rhodopsin (GR, SEQ ID NO: 8), with the amino acid sequence corresponding to positions 137-145 of bovine rhodopsin (BvRh) (SEQ ID NO: 9), and substituting the amino acid sequence corresponding to positions 198-206 from the N-terminus, which corresponds to the third cytoplasmic loop of Gloeobacter violaceus rhodopsin, with the amino acid sequence corresponding to positions 225-252 of bovine rhodopsin, and further substituting glutamic acid, the 132nd amino acid of Gloeobacter violaceus rhodopsin, with glutamine. The DNA was inserted into the pCDNA3.1 vector. Mutants were generated using the quick-change method.

[0051] (Construction of adeno-associated virus (AAV) vectors with inserted chimeric protein sequences) EGFP or GR / BvRh genes were subcloned into the AAV2 shuttle plasmid to create two viral expression constructs: AAV2-CAGGS-EGFP-WPRE-pA (vector for EGFP expression) and AAV2-CAGGS-GR / BvRh-WPRE-pA (vector for chimeric protein expression). Viral vector packaging was performed by transfecting HEK293 cells with three plasmids: the vector plasmid, the AAV vector plasmid, and the adenovirus helper plasmid. The viral vectors were purified using the cesium chloride method. In the vectors, "ITR" stands for "Inverted Terminal Repeat". "CAGGS" is the sequence of the CAG promoter region. "WPRE" stands for "woodchuck hepatitis virus post-transcriptional regulatory element". "pA" stands for peptide tag. "EGFP" stands for "enhanced green fluorescent protein".

[0052] (Intravitreal injection) In wild-type mice or retinitis pigmentosa model (rd1) mice, a mixture of medetomidine hydrochloride (0.75 mg / kg), midazolam (4 mg / kg), and butorphanol tartrate (5 mg / kg) was administered intraperitoneally. Under general anesthesia, the aforementioned AAV vectors ("AAV2-CAGGS-EGFP-WPRE-pA" or "AAV2-CAGGS-GR / BvRh-WPRE-pA") were injected into the vitreous humor from near the serrated edge using a microsyringe fitted with a 32-gauge needle, at a rate of 1 × 10⁻¹⁶. 12 A 1 μl injection of vg / ml was administered.

[0053] (Reporter observation) Seven weeks after injection of AAV2-CAGGS-EGFP-WPRE-pA into wild-type mice, the retinas were extracted, fixed with 4% paraformaldehyde for 1 hour, and the whole-mounted retinas were observed using a fluorescence microscope. The results are shown in Figure 1. In Figure 1, GCL: ganglion cell layer, INL: inner granular layer, and ONL: outer granular layer. The observation revealed green fluorescence (e.g., the arrow in Figure 1) in the retina, confirming that the vector was introduced and the target gene was expressed normally.

[0054] (Multielectrode Array Recording (MEA): Measurement using a multi-electrode array) Seven weeks after injecting AAV2-CAGGS-GR / BvRh-WPRE-pA into retinitis pigmentosa model (rd1) mice, the eyeballs were extracted under general anesthesia. The retina was then extracted by placing it in Ames medium (Sigma-Aldrich, St. Louis, MO; A1420) bubbling with 95% O2 and 5% CO2. The retina was mounted so that the ganglion cell layer was facing downwards and in contact with the electrode, and light stimulation was performed (white light, 1000 cd / m²). 2 Extracellular potential recordings of retinal ganglion cells were performed (for 1 second). Extracellular potential recordings of retinal ganglion cells were also performed using the same method on a control mouse (rd1) of retinitis pigmentosa that had not been injected with AAV2-CAGGS-GR / BvRh-WPRE-pA. The MEA2100-Lite system (Multi-Channel Systems, Reutlingen, Germany) was used for multi-electrode array measurements. The results are shown in Figure 2. In Figure 2, (a) shows the graph for control mice, and (b) shows the graph for mice injected with AAV2-CAGGS-GR / BvRh-WPRE-pA. In the graphs in Figure 2, the horizontal axis represents elapsed time. The area indicated by the arrow shows the area where light stimulation was applied.

[0055] As shown in Figure 2, no changes were observed in the light-stimulated area in the control group, but an increase in potential was observed in mice injected with AAV2-CAGGS-GR / BvRh-WPRE-pA. This result indicates that GR / BvRh has a visual function regeneration effect in retinitis pigmentosa.

[0056] Furthermore, multi-electrode array measurements were performed using the same method as described above, and a raster plot of 10 retinal ganglion cell firings (upper panel of Figure 3) and a histogram with the firing frequency per second on the vertical axis (lower panel of Figure 3) were obtained. Light stimulation was performed for 0 to 1 second. In Figure 3, (a) shows the graph for control mice, and in Figure 3, (b) shows the graph for mice injected with AAV2-CAGGS-GR / BvRh-WPRE-pA.

[0057] As shown in Figure 3, no light response was observed in control mice, whereas in mice injected with AAV2-CAGGS-GR / BvRh-WPRE-pA, ganglion cell firing was observed, demonstrating a visual regeneration effect.

Claims

[Claim 1] The invention described in the detailed description of the invention.

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