WO2022131322A1 - 神経細胞賦活剤 - Google Patents

神経細胞賦活剤 Download PDF

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WO2022131322A1
WO2022131322A1 PCT/JP2021/046462 JP2021046462W WO2022131322A1 WO 2022131322 A1 WO2022131322 A1 WO 2022131322A1 JP 2021046462 W JP2021046462 W JP 2021046462W WO 2022131322 A1 WO2022131322 A1 WO 2022131322A1
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protein
cell
cells
nerve
seq
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French (fr)
Japanese (ja)
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淳 高橋
文平 佐俣
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Kyoto University NUC
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Kyoto University NUC
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Priority to EP21906683.4A priority Critical patent/EP4265270A4/en
Priority to US18/257,143 priority patent/US20240033332A1/en
Priority to JP2022570052A priority patent/JPWO2022131322A1/ja
Publication of WO2022131322A1 publication Critical patent/WO2022131322A1/ja
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Definitions

  • the present invention relates to a nerve cell activator, a cell transplantation aid, a therapeutic or preventive agent for a neurological disorder, a composition for treating a neurological disorder, and the like.
  • the engraftment rate of transplanted cells in the transplantation treatment of neurological diseases is low, and the proportion of cells that differentiate into nerve cells and fully mature is very low, which has been a major obstacle to the therapeutic effect. ..
  • An object of the present invention is to develop and provide a nerve cell activator that activates brain nerve cells. Further, a further subject of the present invention is an agent that promotes at least one or more of engraftment of transplanted cells, differentiation of the engrafted cells into nerve cells, and maturation of the nerve cells in cell transplantation of a neurological disease. To provide.
  • Cst7, Spp1, A2m, Apoc1, Ccl12, Lyz2, Klk6, Gm11428, Cd52, Lgals3bp, Mpeg1, Rac2, Serpina3n, Gm14295, etc. are nerve cells. It has been found that it has a nerve cell activating effect such as an effect of promoting the elongation of the axon, an effect of increasing the side branch of the nerve axon, an effect of suppressing cell death on nerve cells, and an effect of increasing the number of nerve cells.
  • Lyz2 significantly increases the density of dopaminergic neurons derived from transplanted cells in cell transplantation into the striatum of the ventral tissue of the midbrain using mice.
  • the present invention has been completed. The present invention is based on this finding and provides the following.
  • LYZ lysoteam
  • SPP1 secretory phosphoprotein 1
  • PGrn progranulin
  • Ctsd cathepsin S (Ctss), apolypoprotein D (Apod), Sparc, CD52, macrophage expression gene 1 (MPEG1), CC motif chemokine ligand 2 (CCL2), cystatin F (CST7), Rac family low molecular weight GTP Aze 2 (RAC2), Serpin Family A Member 3 (SERPINA3), Anosmin 1 (ANOS1), Cathepsin Deptidase 6 (KLK6), ⁇ 2-Macroglobulin (A2M), GM14295, Galectin 3 Binding Protein (LGALS3BP), Apolypoprotein C1 The agent selected from the group consisting of (APOC1) and their orthologs.
  • the agent according to (1), wherein the nerve cell is a brain nerve cell.
  • the agent according to (1) or (2), wherein the activation of nerve cells is promotion of elongation of axons, axon side branches, and / or dendrites.
  • the agent according to (1) or (2), wherein the activation of nerve cells is suppression of nerve cell degeneration and / or nerve cell death.
  • a cell transplantation aid comprising the agent according to any one of (1) to (4).
  • a therapeutic or prophylactic agent for a neurological disease which comprises the agent according to any one of (1) to (4).
  • the agent according to (6) wherein the disease is selected from the group consisting of neurodegenerative diseases, brain tumors, multiple sclerosis, epilepsy, head trauma, cerebral infarction, stroke, and ischemia.
  • the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis, Lewy body dementias, spinocerebellar degeneration, progressive supranuclear palsy, and cortical basal nucleus degeneration.
  • the agent according to (7) which is selected from the group consisting of.
  • a composition for treating or preventing a neurological disorder which comprises the agent according to any one of (6) to (8).
  • a method for activating brain nerve cells which comprises a step of administering the agent according to any one of (1) to (4) to a subject.
  • (11) A method for treating or preventing a neurological disease in a subject, wherein the agent according to any one of (6) to (8) or the composition according to (9) is administered to the subject. Including, method.
  • the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis, Lewy body dementia, spinocerebellar degeneration, progressive supranuclear palsy, and cortical basal nucleus degeneration.
  • the agent according to (14), wherein the disease is selected from the group consisting of neurodegenerative diseases, brain tumors, multiple sclerosis, epilepsy, head trauma, cerebral infarction, stroke, and ischemia.
  • the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis, Lewy body dementia, spinocerebellar degeneration, progressive supranuclear palsy, and cortical basal nucleus degeneration.
  • the use according to (17), wherein the disease is selected from the group consisting of neurodegenerative diseases, brain tumors, multiple sclerosis, epilepsy, head trauma, cerebral infarction, stroke, and ischemia.
  • the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis, Lewy body dementia, spinocerebellar degeneration, progressive supranuclear palsy, and cortical basal nucleus degeneration.
  • a cell transplantation aid containing the following protein or a nucleic acid encoding the protein, wherein the protein is lysoteam (LYZ), secretory phosphoprotein 1 (SPP1), progranulin (PGrn), cathepsin.
  • LYZ lysoteam
  • SPP1 secretory phosphoprotein 1
  • PGrn progranulin
  • Ctsd cathepsin S (Ctss), apolypoprotein D (Apod), Sparc, CD52, macrophage expression gene 1 (MPEG1), CC motif chemokine ligand 2 (CCL2), cystatin F (CST7), Rac family low molecular weight GTP Aze 2 (RAC2), Serpin Family A Member 3 (SERPINA3), Anosmin 1 (ANOS1), Cathepsin Deptidase 6 (KLK6), ⁇ 2-Macroglobulin (A2M), GM14295, Galectin 3 Binding Protein (LGALS3BP), Apolypoprotein C1 A cell transplant aid selected from the group consisting of (APOC1) and their orthologs.
  • the cell transplantation aid according to (1'), wherein the cell is one or more cells selected from the group consisting of neural stem cells, neural progenitor cells, and nerve cells.
  • the cell transplantation aid according to any one of (1') to (4') which is used in combination with transplanted cells.
  • the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis, Lewy body dementias, spinal cord cerebral degeneration, progressive supranuclear palsy, and cortical basal nucleus degeneration.
  • the cell transplantation aid according to (7') which is selected from the group consisting of diseases.
  • the composition for treating a neurological disease which comprises the cell transplantation aid according to any one of (1') to (8') and cells for transplantation.
  • (10') A method of transplanting cells to a recipient, the cell transplantation aid administration step of administering the cell transplantation aid according to (1') to the recipient, and the cell transplantation of transplanting the cells to the recipient.
  • a method including steps.
  • (11') The method according to (10'), wherein the cell is one or more cells selected from the group consisting of neural stem cells, neural progenitor cells, and neural cells.
  • (12') The method according to (11'), wherein the neural stem cell, neural progenitor cell, and neural cell are cells induced to differentiate from pluripotent stem cells.
  • 13') The method according to any one of (10') to (12'), wherein the cell transplantation aid administration step is performed simultaneously with the cell transplantation step or after the cell transplantation step.
  • the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis, Lewy body dementias, spinocerebellar degeneration, progressive supranuclear palsy, and cortical basal nucleus degeneration.
  • the method according to (16') which is selected from the group consisting of diseases.
  • the neurological disorder is selected from the group consisting of neurodegenerative diseases, brain tumors, multiple sclerosis, epilepsy, head trauma, cerebral infarction, stroke, and ischemia. Transplant aid.
  • the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis, Lewy body dementias, spinal cord cerebral degeneration, progressive supranuclear palsy, and cortical basal nucleus degeneration.
  • the cell transplantation aid according to (19') which is selected from the group consisting of diseases.
  • the cell transplantation aid according to any one of (1') to (5') in the manufacture of a pharmaceutical for treating a neurological disorder is selected from the group consisting of neurodegenerative diseases, brain tumors, multiple sclerosis, epilepsy, head trauma, cerebral infarction, stroke, and ischemia. ..
  • the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis, Lewy body dementias, spinocerebellar degeneration, progressive supranuclear palsy, and cortical basal nucleus degeneration.
  • This specification includes the disclosure contents of Japanese Patent Application No. 2020-209492 and Japanese Patent Application No. 2020-209568, which are the basis of the priority of the present application.
  • brain nerve cells can be activated.
  • a promoting effect can be obtained for at least one of the number, density, and maturity of nerve cells derived from the transplanted cells. This is expected to improve the transplantation effect.
  • FIG. 13.5 It is a figure which shows the cell aggregate produced from the cerebral cortex tissue of the mouse of the embryonic day 13.5. It is a figure which shows the result of transplanting a cell aggregate into a cortical injury model.
  • Figure 2A shows the results of transplantation of cell aggregates immediately after cortical injury.
  • Figure 2B shows the results of transplantation of cell aggregates 7 days after cortical injury. Only donor-derived cells have been stained with anti-GFP antibody.
  • FIG. 4A and 4B show enlarged views of the area shown in the frame in the upper panel. Arrowheads indicate GFP-positive and Fastblue-positive cell bodies in the implant (ie, neuronal cell bodies derived from the implant and projecting axons to the spinal cord). It is a figure which shows the magnification change of the gene expression 7 days after a cortical injury, compared with the time immediately after a cortical injury (day 0) for each factor extracted as a gene whose gene expression increased or decreased 7 days after a cortical injury.
  • FIG. 6A shows neurons cultured in the presence of recombinant proteins of each factor.
  • the arrowhead indicates the position of the cell body.
  • FIG. 6B shows the quantitative results of TAU-positive nerve axon length. Error bars indicate standard deviation, * indicates P ⁇ 0.05, ** indicates P ⁇ 0.01, *** indicates P ⁇ 0.001 (one-way ANOVA).
  • FIG. 10A shows neurons cultured in the presence of each concentration of mLYZ2 recombinant protein.
  • FIG. 10B shows the quantitative results of TAU-positive nerve axon length. It is a figure which shows the result that the number of the nerve axon side branch of a TAU positive nerve axon was increased by the commercially available mLYZ2 recombinant protein.
  • FIG. 12A shows neurons cultured in the presence of each concentration of hLYZ.
  • FIG. 12B shows the quantitative results of TAU-positive nerve axon length. It is a figure which shows the result that the elongation of the TAU positive nerve axon of the human ES cell-derived dopaminergic nerve cell was promoted by hLYZ.
  • FIG. 13A shows neurons cultured in the presence of each concentration of hLYZ.
  • FIG. 13B shows the quantitative results of TAU-positive nerve axon length.
  • FIG. 14A shows neurons cultured in the absence or presence of mLYZ2.
  • FIG. 14B shows the quantitative results of TAU-positive nerve axon length. It is a figure which shows the result that the total number of cells of the TAU positive neuron derived from the human iPS cell-derived cerebral cortex organoid was increased by the commercially available hLYZ recombinant protein.
  • FIG. 17A the results of the group in which the human Lyz recombinant protein was added to the culture medium (Lyz) and the group in which the human Lyz recombinant protein was not added (Ctrl) during the culture before cell transplantation are shown.
  • FIG. 17A the grafts of the Ctrl group and the Lyso group are indicated by arrows.
  • FIG. 17B shows the size of the graft.
  • the horizontal line in each group indicates the average value. * Indicates P ⁇ 0.05 (Student's t-test).
  • FIG. 18A shows the state of axons in the absence of LYZ (Ctrl) and in the presence of LYZ (Lyz 500 ng / mL).
  • the scale bar at the bottom left of the photo shows 200 ⁇ m.
  • FIG. 18B shows the time course of axon length from day 0 to day 7 (0 to 168 hr) of culture.
  • the axon length shown on the vertical axis indicates the total value (mm) of the nerve axon length included in the unit area (mm 2 ) of the acquired image.
  • the quantitative results of axon length on the 7th day of culture in the presence of each factor are shown.
  • CathD indicates cathepsin D (Ctsd)
  • CathS indicates cathepsin S (Ctss).
  • the amount of each factor shown in the figure indicates the amount in 1 mL of the medium.
  • the error bar indicates the standard deviation
  • * indicates P ⁇ 0.05 (One-way ANOVA with Holm-Sidak's post hoc analysis).
  • the results of evaluating the cell viability on the 7th day of culture by Alamar Blue are shown.
  • CathD indicates cathepsin D (Ctsd)
  • CathS indicates cathepsin S (Ctss).
  • the amount of each factor shown in the figure indicates the amount in 1 mL of the medium.
  • Ctrl mean indicates cells to which no candidate factor was added
  • 1% Triton mean indicates cells artificially injured by treatment with a surfactant (1% Triton). Error bars indicate standard deviation.
  • the results of evaluating the cell viability under cytotoxicity (H 2 O 2 ) conditions by Alamar Blue are shown.
  • CathD indicates cathepsin D (Ctsd)
  • CathS indicates cathepsin S (Ctss).
  • the amount of each factor shown in the figure indicates the amount in 1 mL of the medium.
  • the error bar indicates the standard deviation
  • * indicates P ⁇ 0.05 (One-way ANOVA with Holm-Sidak's post hoc analysis).
  • the first aspect of the present invention is a nerve cell activator.
  • the neuronal activator of this embodiment is a protein such as LYZ, CD52, MPEG1, CCL2, CST7, RAC2, SERPINA3, ANOS1, KLK6, A2M, GM14295, SPP1, LGALS3BP, APOC1, PGrn, Ctsd, Ctss, Apod, and Sparc.
  • an axon elongation promoting effect and a cell death suppressing effect on brain nerve cells can be obtained.
  • nerve cell activation means promotion of differentiation of nerve stem cells or neural precursor cells into nerve cells, as well as survival, differentiation, maturation of nerve cells, and with other nerve cells. It means the effect of promoting connection, circuit formation, or function, or the effect of protecting nerve cell survival, differentiation, maturation, connection with other nerve cells, circuit formation, or function against damage to nerve cells. .. Neuronal activation includes, but is not limited to, growth and regeneration promotion of nerve processes (eg, axons, axon side branches, and / or dendrites), spine formation and elongation, suppression of nerve cell degeneration, and Examples include suppression of cell death of nerve cells.
  • the promotion of elongation of the axon side branch or dendrite includes, for example, an increase in neurite length, promotion of branching, and the like.
  • the "nerve cell” is not particularly limited as long as it is a cell of the nervous lineage.
  • the nervous system is divided into a central nervous system (brain or spinal cord) and a peripheral nervous system, but unless otherwise specified, the nervous cells may be the central nervous system or the peripheral nervous system.
  • the cells of the nervous lineage also include cells that do not generate action potentials but are connected to and input to the nervous system (for example, photoreceptor cells).
  • the stage of cell differentiation is not limited, and the stage of maturation of the differentiated nerve cell is not limited.
  • the cells may be neural stem cells, neural progenitor cells, immature neurons, or mature neurons.
  • the cell type of a nerve cell is not limited, and for example, a dopaminergic nerve cell, a noradrenalinergic nerve cell, a serotoninergic nerve cell, a glutamate-working nerve cell, a GABA-working nerve cell, a cholinergic nerve cell, or It may be a histaminergic nerve cell or the like.
  • the nerve cell may be a nerve cell derived from a living body (for example, the brain) or an artificially produced nerve cell. Examples of the nerve cell derived from a living body include a nerve cell existing or functioning in an arbitrary region of the living body (for example, the brain), a nerve cell isolated from an arbitrary region of the living body (for example, the brain), and the like.
  • artificially produced neurons examples include neurons obtained by inducing differentiation from (pluripotent) stem cells such as ES cells and iPS cells. Further, in this embodiment, the nerve cells are present in the living body (for example, in the brain or transplanted in the brain) or cultured in vitro unless otherwise specified. It doesn't matter if it is done.
  • brain nerve cell is a nerve cell that can exist or function in any region of the brain among the above-mentioned nerve cells.
  • the regions in which brain nerve cells can exist or function in the brain are not limited, for example, the cerebrum (cerebral cortex, cerebral white matter, cerebral basal nucleus), cerebellum (cerebellar cortex, cerebellum nucleus), and / or brain stem (mesencephalon, middle brain). , Bridge, cerebrum).
  • Examples of brain nerve cells include nerve cells in the cerebral cortex, nerve cells in the cerebral cortex, nerve cells in the nerve nucleus, and the nerve nucleus also includes a nerve nucleus including a plurality of nerve nuclei such as the basal nucleus of the cerebral brain.
  • suitable neurons are brain neurons, more preferably neurons in the cerebral cortex and / or neurons in the midbrain.
  • the nerve cell of the cerebral cortex the nerve cell of the primary motor cortex is preferable, and the nerve cell of the primary motor cortex V layer is particularly preferable.
  • nerve cells in the midbrain nerve cells in the ventral tegmental area are preferable, and dopaminergic nerve cells present in the substantia nigra are particularly preferable.
  • plural means an integer of 2 or more, for example, an integer of 2 to 10, 2 to 7, 2 to 5, 2 to 4, or 2 to 3.
  • mutant protein refers to any protein derived from the amino acid sequence of a wild-type protein and having an amino acid sequence different from that of the wild-type protein.
  • mutant proteins include splicing variants, mutants based on SNPs and the like, active fragments of proteins and the like.
  • the mutant protein may also include an artificial protein having an amino acid sequence different from that of the wild protein, which is prepared based on the amino acid sequence of the wild protein.
  • the term "active fragment” means a partial region of a protein, and the fragment contains 50% or more, 60% or more, 70% or more, 80% or more, 90% or more, 95% of the activity of the full-length protein.
  • the above refers to a polypeptide fragment that retains 98% or more, or equivalent or more.
  • the amino acid length of the active fragment is not particularly limited as long as the activity of the protein is maintained.
  • amino acid identity means that in the amino acid sequences of the two polypeptides to be compared, a gap is appropriately inserted in one or both of them so as to maximize the number of matching amino acid residues.
  • substitution refers to a group of conservative amino acids having similar properties such as charge, side chain, polarity, and aromaticity among the 20 amino acids constituting a natural protein. Refers to replacement. For example, uncharged polar amino acid group (Gly, Asn, Gln, Ser, Thr, Cys, Tyr), branched amino acid group (Leu, Val, Ile), neutral amino acid group (Gly, Ile) having a low polar side chain.
  • the neuronal cell activators of the present invention are LYZ, CD52, MPEG1, CCL2, CST7, RAC2, SERPINA3, ANOS1, KLK6, A2M, GM14295, SPP1, LGALS3BP, APOC1, listed in Table 1 and Table 1'below.
  • neuronal activator Indicated herein as “neuronal activator”; the neuronal activator may also be referred to as a “transplant cofactor” as described in the second aspect below), or the protein, the active fragment, or
  • the nucleic acid encoding the ortholog (referred to as “nucleic acid encoding a neuronal activator” in the present specification) is included as an essential active ingredient.
  • the neuronal activator of the present invention comprises the LYZ, CST7, CCL12, CD52, MPEG1, RAC2, SPP1, PGrn, Ctsd, Ctss, Apod, and Sparc proteins, active fragments thereof, and any of them.
  • a protein selected from the group consisting of orthologs, or the protein, the active fragment, or the nucleic acid encoding the ortholog is included as an essential active ingredient.
  • the neuronal activator of the invention comprises a group consisting of the above-mentioned LYZ, SPP1, PGrn, Ctsd, Ctss, Apod, and Sparc proteins, active fragments thereof, and orthologs thereof.
  • a protein selected from, or a nucleic acid encoding the protein, the active fragment, or the ortholog is included as an essential active ingredient.
  • the neuronal activator of the invention is a protein selected from the group consisting of the above-mentioned LYZ, SPP1, PGrn and Sparc proteins, active fragments thereof, and orthologs thereof.
  • the protein, the active fragment, or the nucleic acid encoding the ortholog is included as an essential active ingredient.
  • the neuronal activator of the invention is a protein selected from the group consisting of the above-mentioned LYZ, SPP1, and PGrn proteins, active fragments thereof, and orthologs thereof.
  • the protein, the active fragment, or the nucleic acid encoding the ortholog is included as an essential active ingredient.
  • the neuronal activator of the present invention is a protein selected from the group consisting of any of the above-mentioned LYZ and PGrn proteins, an active fragment thereof, and an ortholog thereof, or the protein, the active fragment thereof.
  • the nucleic acid encoding the ortholog is included as an essential active ingredient.
  • the neuronal activator of the present invention is a protein selected from the group consisting of the above-mentioned LYZ, an active fragment thereof, and an ortholog thereof, or a nucleic acid encoding the LYZ, the active fragment, or the ortholog. Is included as an essential active ingredient. Each active ingredient will be specifically described below.
  • LYZ and LYZ ortholog Lysozyme is a human wild-type LYZ protein consisting of the amino acid sequence shown in SEQ ID NO: 2. It has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 2. However, a mutant LYZ protein having an activity equal to or higher than that of the human wild LYZ protein; or 80% or more, 85% or more, preferably 90% or more, 95% or more, more preferably 96 with the amino acid sequence shown in SEQ ID NO: 2. It may be a mutant LYZ protein having an amino acid sequence having an identity of% or more, 97% or more, 98% or more, or 99% or more and having an activity equal to or higher than that of a human wild LYZ protein.
  • the LYZ ortholog has an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in SEQ ID NO: 2.
  • Orthologs of non-human species eg, mouse LYZ2 protein consisting of the amino acid sequence set forth in SEQ ID NO: 1).
  • CD52 and CD52 ortholog CD52 is a human wild-type CD52 protein consisting of the amino acid sequence shown in SEQ ID NO: 4. It has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 4, and is a human wild-type. Variant CD52 protein with activity equal to or greater than that of the CD52 protein; or 80% or more, 85% or more, preferably 90% or more, 95% or more, more preferably 96% or more, 97% with the amino acid sequence shown in SEQ ID NO: 4. As described above, it may be a mutant CD52 protein having an amino acid sequence having 98% or more, or 99% or more identity, and having an activity equal to or higher than that of the human wild-type CD52 protein.
  • the CD52 ortholog has an amino acid sequence of 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identical to the amino acid sequence shown in SEQ ID NO: 4. It may be an ortholog of a species other than human (for example, a mouse CD52 protein consisting of the amino acid sequence shown in SEQ ID NO: 3).
  • Macrophage expressed gene 1 is a human wild-type MPEG1 protein consisting of the amino acid sequence shown in SEQ ID NO: 7. One or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 7. A mutant MPEG1 protein having the same or higher activity as the human wild-type MPEG1 protein; or 80% or more, 85% or more, preferably 90% or more, 95% with the amino acid sequence shown in SEQ ID NO: 7. The above is more preferably a mutant MPEG1 protein having an amino acid sequence having 96% or more, 97% or more, 98% or more, or 99% or more identity and having an activity equal to or higher than that of the human wild type MPEG1 protein. May be.
  • the MPEG1 ortholog has an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in SEQ ID NO: 7.
  • Orthologs of non-human species eg, mouse MPEG1 protein consisting of the amino acid sequence set forth in SEQ ID NO: 5 or 6).
  • CCL2 and CCL2 ortholog CC motif chemokine ligand 2 is a human wild-type CCL2 protein consisting of the amino acid sequence shown in SEQ ID NO: 9.
  • One or more amino acids are deleted or substituted in the amino acid sequence shown in SEQ ID NO: 9.
  • a mutant CCL2 protein having an added amino acid sequence and having an activity equal to or higher than that of the human wild-type CCL2 protein; or 80% or more, 85% or more, preferably 90% or more, the amino acid sequence shown in SEQ ID NO: 9.
  • the CCL2 ortholog has an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in SEQ ID NO: 9.
  • Orthologs of non-human species eg, mouse CCL12 protein consisting of the amino acid sequence set forth in SEQ ID NO: 8).
  • Cystatin F is a human wild-type CST7 protein consisting of the amino acid sequence shown in SEQ ID NO: 11.
  • the CST7 ortholog has an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in SEQ ID NO: 11.
  • Orthologs of non-human species eg, mouse CST7 protein consisting of the amino acid sequence set forth in SEQ ID NO: 10).
  • Rhone family small GTPase 2 (RAC2 and RAC2 ortholog)
  • RAC2 is a human wild-type RAC2 protein consisting of the amino acid sequence shown in SEQ ID NO: 13; one or more amino acids are deleted in the amino acid sequence shown in SEQ ID NO: 13. , 80% or more, 85% or more, preferably 90% with the amino acid sequence set forth in SEQ ID NO: 13; A variant having an amino acid sequence having 95% or more, more preferably 96% or more, 97% or more, 98% or more, or 99% or more identity, and having an activity equal to or higher than that of the human wild-type RAC2 protein. It may be a RAC2 protein.
  • the RAC2 ortholog has an amino acid sequence of 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in SEQ ID NO: 13.
  • Orthologs of non-human species eg, mouse RAC2 protein consisting of the amino acid sequence set forth in SEQ ID NO: 12).
  • Serpin family A member 3 is a human wild-type SERPINA3 protein consisting of the amino acid sequence shown in SEQ ID NO: 15. One or more amino acids are deleted or substituted in the amino acid sequence shown in SEQ ID NO: 15. Alternatively, a mutant SERPINA3 protein having an added amino acid sequence and having an activity equal to or higher than that of the human wild SERPINA3 protein; or 80% or more, 85% or more, preferably 90% or more, the amino acid sequence shown in SEQ ID NO: 15.
  • a mutant SERPINA3 protein having an amino acid sequence having 95% or more, more preferably 96% or more, 97% or more, 98% or more, or 99% or more identity and having an activity equal to or higher than that of the human wild SERPINA3 protein. May be.
  • the SERPINA3 ortholog has an amino acid sequence of 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identical to the amino acid sequence shown in SEQ ID NO: 15.
  • Orthologs of non-human species eg, mouse SERPINA3N protein consisting of the amino acid sequence set forth in SEQ ID NO: 14).
  • Anosmin 1 is a human wild-type ANOS1 protein consisting of the amino acid sequence shown in SEQ ID NO: 17; an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 17. And 80% or more, 85% or more, preferably 90% or more, 95% or more, more preferably with the amino acid sequence shown in SEQ ID NO: 17; May be a mutant ANOS1 protein having an amino acid sequence having 96% or more, 97% or more, 98% or more, or 99% or more identity and having an activity equal to or higher than that of the human wild ANOS1 protein.
  • the ANOS1 ortholog has an amino acid sequence of 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identical to the amino acid sequence shown in SEQ ID NO: 17.
  • Orthologs of non-human species eg, mouse GM11428 protein consisting of the amino acid sequence set forth in SEQ ID NO: 16).
  • Kallikrein related-peptidase 6 is a human wild-type KLK6 protein consisting of the amino acid sequence shown by any of SEQ ID NOs: 19 to 23; 1 or 1 in the amino acid sequence shown by any of SEQ ID NOs: 19-23.
  • a mutant KLK6 protein having an amino acid sequence in which a plurality of amino acids have been deleted, substituted or added and having an activity equal to or higher than that of the human wild KLK6 protein; or the amino acid sequence shown in any of SEQ ID NOs: 19 to 23.
  • the KLK6 ortholog has 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in any of SEQ ID NOs: 19-23. It may be an ortholog of a species other than human having an amino acid sequence having (for example, a mouse KLK6 protein consisting of the amino acid sequence shown in SEQ ID NO: 18).
  • A2M and A2M orthologs ⁇ 2-macroglobulin (A2M) is a human wild-type A2M protein consisting of the amino acid sequence shown by any of SEQ ID NOs: 25 to 28; 1 or 1 in the amino acid sequence shown by any of SEQ ID NOs: 25 to 28.
  • a mutant A2M protein having an amino acid sequence in which a plurality of amino acids have been deleted, substituted or added and having an activity equal to or higher than that of a human wild-type A2M protein; or the amino acid sequence shown by any of SEQ ID NOs: 25 to 28.
  • It has an amino acid sequence having 80% or more, 85% or more, preferably 90% or more, 95% or more, more preferably 96% or more, 97% or more, 98% or more, or 99% or more identity, and is human wild. It may be a mutant A2M protein having an activity equal to or higher than that of the type A2M protein.
  • the A2M ortholog has 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in any of SEQ ID NOs: 25 to 28. It may be an ortholog of a species other than human having an amino acid sequence having (for example, a mouse A2M protein consisting of the amino acid sequence shown in SEQ ID NO: 24).
  • GM14295 and GM14295 Ortholog are mouse wild-type GM14295 protein consisting of the amino acid sequence shown in SEQ ID NO: 29; it has an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 29.
  • It may be a mutant GM14295 protein having an amino acid sequence having an identity of% or more, 97% or more, 98% or more, or 99% or more, and having an activity equal to or higher than that of the wild mouse GM14295 protein.
  • the GM14295 ortholog has an amino acid sequence of 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in SEQ ID NO: 29.
  • Orthologs of species other than mice may be used.
  • the GM14295 ortholog is not known in humans.
  • SPP1 and SPP1 ortholog Secreted phosphoprotein 1
  • SPP1 also called osteopontin
  • SPP1 is a human wild-type SPP1 protein consisting of the amino acid sequence represented by SEQ ID NO: 35 or 36; one or more in the amino acid sequence represented by SEQ ID NO: 35 or 36.
  • the SPP1 ortholog is an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown by SEQ ID NO: 35 or 36. It may be an ortholog of a non-human species having
  • LGALS3BP and LGALS3BP ortholog Galectin 3 binding protein is a human wild-type LGALS3BP protein consisting of the amino acid sequence shown in SEQ ID NO: 38; one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 38.
  • Variant LGALS3BP protein having the same or higher activity as the human wild-type LGALS3BP protein; or 80% or more, 85% or more, preferably 90% or more, 95% with the amino acid sequence shown in SEQ ID NO: 38.
  • the above is more preferably a mutant LGALS3BP protein having an amino acid sequence having 96% or more, 97% or more, 98% or more, or 99% or more identity and having an activity equal to or higher than that of the human wild-type LGALS3BP protein. You may.
  • the LGALS3BP ortholog has an amino acid sequence of 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identical to the amino acid sequence shown in SEQ ID NO: 38.
  • Orthologs of non-human species eg, mouse LGALS3BP protein consisting of the amino acid sequence set forth in SEQ ID NO: 37).
  • Apolipoprotein C1 is a human wild-type APOC1 protein consisting of the amino acid sequence shown by any of SEQ ID NOs: 41 to 44; one or more amino acids in the amino acid sequence shown by any of SEQ ID NOs: 41 to 44.
  • a variant APOC1 protein that has an amino acid sequence deleted, substituted or added and has activity equal to or greater than that of the human wild APOC1 protein; or 80% or more of the amino acid sequence set forth in any of SEQ ID NOs: 41-44.
  • amino acid sequence having 85% or more, preferably 90% or more, 95% or more, more preferably 96% or more, 97% or more, 98% or more, or 99% or more identity, with the human wild APOC1 protein. It may be a mutant APOC1 protein having equivalent or higher activity.
  • the APOC1 ortholog has 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in any of SEQ ID NOs: 41 to 44. It may be an ortholog of a non-human species having an amino acid sequence (eg, a mouse APOC1 protein consisting of the amino acid sequence set forth in SEQ ID NO: 39 or 40).
  • Progranulin is a human wild-type PGrn protein consisting of the amino acid sequence shown in SEQ ID NO: 90; one or more amino acids have been deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 90.
  • a mutant PGrn protein having an amino acid sequence and having an activity equal to or higher than that of the human wild PGrn protein; or 80% or more, 85% or more, preferably 90% or more, 95% or more, the amino acid sequence shown by SEQ ID NO: 90.
  • mutant PGrn protein having an amino acid sequence having 96% or more, 97% or more, 98% or more, or 99% or more identity and having an activity equal to or higher than that of a human wild-type PGrn protein. good.
  • the PGrn ortholog has an amino acid sequence of 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identical to the amino acid sequence shown by SEQ ID NO: 90.
  • Orthologs of non-human species eg, mouse PGrn protein consisting of the amino acid sequence set forth in SEQ ID NO: 89).
  • Cathepsin D is a human wild-type Ctsd protein consisting of the amino acid sequence shown in SEQ ID NO: 92; an amino acid sequence in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 92.
  • Cathepsin D protein having activity equal to or higher than that of human wild-type Ctsd protein; or 80% or more, 85% or more, preferably 90% or more, 95% or more, more preferably with the amino acid sequence shown in SEQ ID NO: 92.
  • the Ctsd ortholog has an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in SEQ ID NO: 92.
  • Orthologs of non-human species eg, mouse Ctsd protein consisting of the amino acid sequence set forth in SEQ ID NO: 91).
  • Cathepsin S is a human wild-type Ctss protein consisting of the amino acid sequence represented by SEQ ID NO: 95 or 96; one or more amino acids are deleted, substituted or added in the amino acid sequence represented by SEQ ID NO: 95 or 96.
  • a mutant Ctss protein having the same or higher activity as the human wild-type Ctss protein; or 80% or more, 85% or more, preferably 90% or more, the amino acid sequence represented by SEQ ID NO: 95 or 96.
  • the Ctss ortholog is an amino acid sequence having 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown by SEQ ID NO: 95 or 96. It may be an ortholog of a non-human species (eg, a mouse Ctss protein consisting of the amino acid sequence set forth in SEQ ID NO: 93 or 94).
  • a non-human species eg, a mouse Ctss protein consisting of the amino acid sequence set forth in SEQ ID NO: 93 or 94.
  • Apolipoprotein D is a human wild-type Apod protein consisting of the amino acid sequence shown in SEQ ID NO: 100; an amino acid in which one or more amino acids are deleted, substituted or added in the amino acid sequence shown in SEQ ID NO: 100.
  • a mutant Apod protein having a sequence and having an activity equal to or higher than that of the human wild Apod protein; or 80% or more, 85% or more, preferably 90% or more, 95% or more, more than the amino acid sequence shown in SEQ ID NO: 100. It may be a mutant Apod protein having an amino acid sequence having 96% or more, 97% or more, 98% or more, or 99% or more identity, and having an activity equal to or higher than that of the human wild Apod protein. ..
  • the Apod ortholog has an amino acid sequence of 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown by SEQ ID NO: 100.
  • Orthologs of non-human species eg, mouse Apod protein consisting of the amino acid sequence set forth in any of SEQ ID NOs: 97-99).
  • the Sparc protein is a human wild-type Sparc protein consisting of the amino acid sequence shown by any of SEQ ID NOs: 103 to 105; one or more amino acids are deleted, substituted or added in the amino acid sequence shown by any of SEQ ID NOs: 103 to 105.
  • a mutant Sparc protein having an amino acid sequence that is equal to or higher than that of the human wild-type Sparc protein; or 80% or more, 85% or more, preferably 90 with the amino acid sequence shown by any of SEQ ID NOs: 103 to 105.
  • % Or more 95% or more, more preferably 96% or more, 97% or more, 98% or more, or a mutation having an amino acid sequence having 99% or more identity and having an activity equal to or higher than that of the human wild-type Sparc protein. It may be a type Sparc protein.
  • the Sparc ortholog has 60% or more, 70% or more, 80% or more, 90% or more, 95% or more, or 99% or more and less than 100% identity with the amino acid sequence shown in any of SEQ ID NOs: 103 to 105. It may be an ortholog of a non-human species having an amino acid sequence (eg, a mouse Sparc protein consisting of the amino acid sequence set forth in SEQ ID NO: 101 or 102).
  • Nerve cell activators their corresponding NCBI accession numbers, and SEQ ID NOs are illustrated in Tables 1 and 1'below. As described in the second aspect described later, the nerve cell activating factors exemplified in Table 1 and Table 1'can also be referred to as transplantation cofactors.
  • neuronal activators are fused with, for example, lipofection, cell membrane permeability peptides (eg, HIV-derived TAT, polyarginine, BP100, Penetratin, pVEC, pAntp, VP22, Transportan, R7, MPG, and Pep-1). , May be introduced into cells by a method such as microinjection.
  • cell membrane permeability peptides eg, HIV-derived TAT, polyarginine, BP100, Penetratin, pVEC, pAntp, VP22, Transportan, R7, MPG, and Pep-1).
  • nucleic acid encoding the protein, the active fragment, or the nucleic acid encoding the ortholog is a nucleic acid encoding any of the above nerve cell activators.
  • Nucleic acids encoding neuronal activators are LYZ, CD52, MPEG1, CCL2, CST7, RAC2, SERPINA3, ANOS1, KLK6, A2M, GM14295, SPP1, LGALS3BP, APOC1, PGrn, Ctsd, Ctss, Apod, or Sparc.
  • the DNA encoding the nerve cell activator may be a cDNA prepared by using a gene encoding the nerve cell activator or a transcript thereof as a template.
  • RNA encoding a neuronal activator includes both pre-mRNA and mature mRNA, but substantially means mature mRNA.
  • DNA encoding the nerve cell activator As a specific example of the DNA encoding the nerve cell activator (cDNA encoding the nerve cell activator), the DNA consisting of the base sequence shown by SEQ ID NO: 46, which encodes the human wild-type LYZ protein; the mouse wild-type LYZ2 protein is encoded.
  • DNA DNA; DNA consisting of the base sequence shown by SEQ ID NO: 83 or 84, which encodes the mouse wild APOC1 protein; DNA consisting of the base sequence shown by SEQ ID NO: 107, which encodes the human wild PGrn protein; The DNA consisting of the base sequence shown in SEQ ID NO: 106; the DNA consisting of the base sequence shown in SEQ ID NO: 109; the DNA consisting of the base sequence shown in SEQ ID NO: 109; the base sequence shown in SEQ ID NO: 108 encoding the mouse wild type Ctsd protein.
  • DNA consisting of DNA DNA consisting of the base sequence represented by SEQ ID NO: 112 or 113, which encodes the human wild-type Ctss protein; DNA consisting of the base sequence represented by SEQ ID NO: 110 or 111, which encodes the mouse wild-type Ctss protein; DNA consisting of the base sequence shown by SEQ ID NO: 117, which encodes the type Apod protein; DNA consisting of the base sequence shown by any of SEQ ID NOs: 114 to 116, which encodes the mouse wild type Apod protein; encodes the human wild type Sparc protein.
  • the mRNA encoding the nerve cell activating factor includes an mRNA containing a base sequence in which t (thymine) is replaced with u (uracil) as a coding region (translation region) in any of the above base sequences.
  • the mRNA encoding the nerve cell activator includes a cap structure at the 5'end, a poly A chain at the 3'end, a 5'untranslated region (5'UTR) upstream of the start codon, and / or. It may include the 3'untranslated region (3'UTR) downstream of the stop codon.
  • the 5'UTR and / or the 3'UTR may contain a sequence for adjusting the amount of translation from mRNA, for example, the 3'UTR is a sequence that increases the amount of translation from mRNA (for example,). Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), etc.) may be included.
  • Nucleic acids encoding neuronal activators, their corresponding NCBI accession numbers, and SEQ ID NOs are illustrated in Tables 2 and 2'below. As described in the second aspect described later, the nucleic acid encoding the nerve cell activating factor exemplified in Table 2 and Table 2'can also be said to be the nucleic acid encoding the transplantation cofactor.
  • the nucleic acid encoding the nerve cell activator may be DNA or RNA. If the nucleic acid is in the form of DNA, it may be included in a gene expression vector. When the nucleic acid is in the form of DNA, it may be introduced into cells by methods such as electroporation, lipofection, liposomes, and microinjection.
  • the "gene expression vector” refers to an expression unit that contains a gene or a gene fragment in an expressible state and can control the expression of the gene or the like.
  • the "expressible state” means that a gene or the like encoding a nerve cell activating factor is arranged in the downstream region under the control of a promoter. Therefore, in the cells into which the vector has been introduced (for example, brain nerve cells), the expression of the nerve cell activator is induced by the promoter.
  • the gene expression vector various expression vectors capable of replicating and expressing in the body of the subject (for example, brain nerve cells) can be used.
  • viral vectors, plasmid vectors, and artificial chromosome vectors are exemplified.
  • the viral vector is not particularly limited as long as it can infect cells (for example, brain cells) and can express a nerve cell activator in the cells.
  • an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a lentivirus vector, a Sendai virus vector and the like are exemplified.
  • the plasmid vector is not particularly limited as long as it is a vector that can be replicated in mammalian cells.
  • the plasmid vector examples include pCMV6-XL3, pEGFP-C1, pGBT-9, pcDNA, pcDM8, pREP4 and the like.
  • the artificial chromosome vector examples include a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), and a bacterial artificial chromosome (BAC, PAC).
  • a promoter is a promoter having an activity of inducing gene expression in a cell (for example, a brain nerve cell).
  • CMV promoter CMV-IE promoter
  • LTR promoter LTR promoter
  • SV40 promoter SV40 promoter
  • RSV promoter RSV promoter
  • HSV-TK promoter EF1 ⁇ promoter
  • Ub promoter Ub promoter
  • metallothioneine promoter SR ⁇ promoter
  • CAG promoter CAG promoter and the like
  • the gene expression vector may contain a control sequence other than the promoter and / or a reporter gene and the like.
  • the control sequence includes an expression control sequence (eg, enhancer, ribosome binding sequence, terminator, poly A addition signal, etc.), intron sequence, nuclease recognition sequence (eg, restriction enzyme recognition sequence, loxP sequence recognized by Cre recombinant enzyme, etc.). Examples include sequences targeted by artificial nucleases such as ZFN and TALEN, sequences targeted by the CRISPR / Cas9 system, etc.), replication origin sequences (SV40 replication origin sequences, etc.), and the like.
  • the reporter gene include a gene encoding a fluorescent protein such as GFP and RFP, a luciferase gene, and the like.
  • a gene expression vector contains an expression cassette (promoter, gene sequence, and terminator) for gene expression to insert the nucleic acid encoding the gene into the chromosome or to excise the nucleic acid inserted into the chromosome as needed. It may have a transposon sequence before and after the unit).
  • the transposon sequence is not particularly limited, but piggyBac is exemplified. In order to introduce an expression cassette into a chromosome using a transposon, it is desirable to introduce the transposase into the cell together with a vector having the expression cassette.
  • the above-mentioned vector may contain a nucleic acid encoding the transposase, or another vector may contain a nucleic acid encoding the transposase, and the nucleic acid may be introduced into a cell at the same time. May be. Furthermore, the gene product encoding the transposase may be directly introduced.
  • the preferred transposase is the transposase corresponding to the transposon sequence described above, preferably the piggyBac transposase.
  • the nucleic acid is in the form of RNA, it may be introduced into cells by techniques such as electroporation, lipofection, and microinjection.
  • the active ingredient in the nerve cell activator may be contained in a plurality of types or in a plurality of forms.
  • the neuronal activator used in the neuronal activator of the present invention is from the proteins shown in Tables 3, 5, 7, and 9 below, active fragments thereof, and orthologs thereof. It may be a protein selected from the group.
  • the nucleic acids encoding the proteins shown in Tables 3, 5, and 7 and their corresponding NCBI accession numbers are illustrated in Tables 4, 6 and 8 below.
  • the nerve cell activating factors exemplified in Tables 3, 5, 7, and 9 can also be referred to as transplantation cofactors.
  • the nucleic acid encoding the nerve cell activator exemplified in Tables 4, 6 and 8 can also be said to be the nucleic acid encoding the transplantation cofactor.
  • the content of the active ingredient contained in the nerve cell activator varies depending on the type and form of the active ingredient (nucleic acid such as protein, DNA or RNA), the dosage form of the nerve cell activator, and the type of solvent or carrier described later. Therefore, it may be determined as appropriate in consideration of each condition.
  • a single dose of the nerve cell activator is adjusted to contain an effective amount of the active ingredient.
  • the amount of the active ingredient may be a total amount including the effective amount.
  • the "effective amount” means an amount necessary for exerting a function as an active ingredient and which gives little or no adverse side effects to a subject to which the active ingredient is applied. This effective amount may vary depending on various conditions such as subject information, application route, and number of applications.
  • the nerve cell activator of the present invention may be used as a medicine, the content of the active ingredient is finally determined by the judgment of a doctor, a pharmacist, or the like.
  • the nerve cell activator of the present invention may be used in an amount of 0.01 ng to 10,000 ng, 0.1 ng to 1,000 ng, or 1 ng to 100 ng, for example, 1 ng, 10 ng, 20 ng, 50 ng, or 100 ng. ..
  • the "subject” is a living body to which the nerve cell activator of the present invention is applied.
  • humans domestic animals (cattle, horses, sheep, goats, pigs, chickens, ostriches, etc.), race horses, pet animals (dogs, cats, rabbits, etc.), experimental animals (mouses, rats, guinea pigs, monkeys, marmosets, etc.) Etc. are applicable.
  • It is preferably a human (in this case, particularly referred to as a "subject"), such as a human who has or is likely to develop a neurological disorder.
  • subject information is various information regarding the characteristics and conditions of the subject. For example, age, weight, gender, general health, presence or absence of disease, disease progression or severity, drug sensitivity, presence or absence of concomitant drugs, resistance to treatment, and the like can be mentioned.
  • neuronal activation is the promotion of axon, axon side branch, and / or dendrite elongation.
  • nerve cell activation is suppression of nerve cell degeneration and / or suppression of nerve cell death.
  • the dosage form of the nerve cell activator of the present invention is not particularly limited. Any form may be used as long as it can be delivered to the target site without inactivating the active ingredient in the body of the subject.
  • the specific dosage form differs depending on the application method described later. Since the application method can be roughly divided into parenteral administration and oral administration, a dosage form suitable for each administration method may be used.
  • the preferred dosage form is a liquid preparation that can be directly administered to the target site or systemically administered via the circulatory system.
  • liquid preparations include injections.
  • the injection is prepared by appropriately combining it with the above-mentioned excipients, emulsifiers, suspensions, surfactants, stabilizers, pH adjusters, etc., and mixing them in a generally accepted unit dose form required for pharmaceutical practice. Can be transformed into.
  • it may be formulated as an artificial extracellular matrix that can be implanted in a living body for the purpose of sustained release of the active ingredient or local action.
  • preferred dosage forms include solids (including tablets, capsules, drops, troches), granules, powders, powders, liquids (drinks for internal use, emulsions, syrups). ). If it is a solid agent, it may be made into a dosage form with a skin known in the art, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets, if necessary. be able to.
  • each of the above dosage forms may be within the range of the dosage form known in the art in each dosage form, and is not particularly limited.
  • the method for producing the nerve cell activator of the present invention may be formulated according to a conventional method in the art.
  • the application route of the nerve cell activator of the present invention may be oral administration or parenteral administration.
  • the oral administration method is generally systemic administration, but the parenteral administration method can be further subdivided into local administration and systemic administration.
  • Local administration corresponds to, for example, intracerebral administration, intracerebral administration, etc.
  • systemic administration corresponds to, for example, intravenous administration (intravenous injection) and intraarterial administration.
  • intravenous administration intravenous injection
  • intraarterial administration intraarterial administration.
  • the nerve cell activator of the present invention When the nerve cell activator of the present invention is locally administered, it should be directly administered to the brain, for example, the cerebrum (for example, frontal lobe or temporal lobe), brain stem, midbrain, or substantia nigra, or the retina or eyeball by injection or the like.
  • Or may be administered intraventricularly.
  • in the case of systemic administration it may be administered intravenously.
  • the dose
  • the effect of activating nerve cells for example, brain nerve cells
  • nerve cells for example, brain nerve cells
  • the effect of promoting axonal elongation and the effect of axillary cord It provides an effect of increasing the number of side branches) and a protective effect against damage to nerve cells (for example, an effect of suppressing nerve cell death and an effect of suppressing nerve cell degeneration).
  • the nerve cell activator of the present invention includes a nerve cell maturation promoting agent, a nerve cell differentiation promoting agent, a nerve protrusion elongation promoting agent (axon, axon side branch, and / or a dendritic protrusion elongation promoting agent), and a nerve. It can also be used as a cell protective agent, a neuronal cell death inhibitor, and / or a neuronal cell degeneration inhibitor.
  • the second aspect of the present invention is a cell transplantation aid.
  • the cell transplantation aid of this embodiment includes the nerve cell activator according to the first aspect. More specifically, the cell transplantation aids of this embodiment include LYZ, CD52, MPEG1, CCL2, CST7, RAC2, SERPINA3, ANOS1, KLK6, A2M, GM14295, SPP1, LGALS3BP, APOC1, PGrn, Ctsd, Ctss, Apod. , And proteins such as Sparc, active fragments thereof, and proteins selected from the group consisting of their orthologs, or nucleic acids encoding the proteins, and are used for the purpose of assisting cell transplantation.
  • cell transplantation aid means at least one of the general evaluation criteria for cell transplantation, such as engraftment of cells transplanted in vivo, differentiation into desired cells, and the level of differentiation. It means promoting the above and assisting cell transplantation. More specifically, it may assist in cell transplantation by promoting the number, density, or maturity of the desired cells derived from the transplanted cells.
  • transplanted cells (cells for transplantation)
  • nerve cells and cells that can differentiate into nerve cells.
  • cells that can differentiate into nerve cells include neural stem cells and neural progenitor cells.
  • the transplanted cell may be a cell derived from a living body (cell collected from the living tissue of the target animal) or an artificially produced cell.
  • the transplanted cells are preferably matched or within the acceptable range for the recipient and the tissue-compatible type, for example, the cells of the recipient, the cells derived from the donor for which the tissue-compatible type is acceptable, and the tissue-compatible type are not within the acceptable range. It may be any cell produced from a donor cell which is a gene editing technique.
  • neural stem cell refers to a stem cell having pluripotency capable of differentiating into nerve cells and glial cells and having self-renewal ability.
  • the "neural progenitor cell” refers to an undifferentiated cell having an ability to differentiate only into a nerve cell.
  • the type of neural stem cell or neural progenitor cell is not particularly limited, and any neural stem cell or neural progenitor cell can be used as long as it can differentiate into a target neural cell.
  • pluripotent stem cell refers to a stem cell having pluripotency capable of differentiating into various cells existing in a living body and also having proliferative ability.
  • embryonic stem (ES) cells embryonic stem (ntES) cells derived from cloned embryos obtained by nuclear transplantation, sperm stem cells (GS cells), embryonic germ cells (EG cells), artificial pluripotency.
  • GS cells sperm stem cells
  • EG cells embryonic germ cells
  • artificial pluripotency examples thereof include stem (iPS) cells, cultured fibroblasts and pluripotent cells (Muse cells) derived from bone marrow stem cells.
  • the "artificially produced cell” is, for example, a cell obtained by inducing differentiation from a pluripotent stem cell or a somatic cell without passing through a pluripotent stem cell by a direct reprogramming method. Directly induced cells.
  • the cell obtained by inducing differentiation from a pluripotent stem cell may be, for example, an organoid cell produced from a human pluripotent stem cell.
  • “maturation of nerve cells” is not limited to, for example, extension of neurites (eg, axons, axon side branches, and / or dendrites), formation and elongation of spines, and other nerves. Synaptogenesis and circuit formation with cells or target cells can be mentioned. Promotion of elongation of axon side branches or dendrites includes, for example, increase in neurite length, promotion of branching, and the like.
  • the cell transplantation aid of the present invention comprises the nerve cell activator according to the first aspect. More specifically, the cell transplantation aid of the present invention includes LYZ, CD52, MPEG1, CCL2, CST7, RAC2, SERPINA3, ANOS1, KLK6, A2M, GM14295, SPP1, and LGALS3BP shown in Tables 1 and 1'above. , APOC1, PGrn, Ctsd, Ctss, Apod, and Sparc proteins, each protein listed in Tables 3, 5, 7, and 9 above, any active fragment thereof, and any ortholog thereof.
  • the protein of choice (referred to herein as "transplant cofactor”; the transplant cofactor can also be referred to as a nerve cell activator as described in the first aspect above, and these terms are compatible herein.
  • the protein, the active fragment, or the nucleic acid encoding the ortholog (referred to herein as "nucleic acid encoding a transplantation cofactor”; eg, Table 2, Table 2'. , Table 4, Table 6, and Table 8.
  • the nucleic acid encoding the transplantation cofactor as described in the first aspect described above can also be said to be the nucleic acid encoding the nerve cell activator. In the specification, these terms can be used interchangeably) as an essential active ingredient.
  • the contents described as “neuronal cell activating factor” or “nucleic acid encoding a neuron activating agent” in the first embodiment are applied.
  • the cell transplantation aids of the present invention are LYZ, CST7, CCL12, CD52, MPEG1, RAC2, SPP1, PGrn, Ctsd, Ctss, Apod, and Sparc proteins, active fragments thereof, and any of them.
  • a protein selected from the group consisting of orthologs, or the protein, the active fragment, or the nucleic acid encoding the ortholog is included as an essential active ingredient.
  • the cell transplantation aid of the invention comprises a group consisting of the LYZ, SPP1, PGrn, Ctsd, Ctss, Apod, and Sparc proteins described above, active fragments thereof, and orthologs thereof.
  • a protein selected from, or a nucleic acid encoding the protein, the active fragment, or the ortholog is included as an essential active ingredient.
  • the cell transplantation aid of the present invention is a protein selected from the group consisting of the above-mentioned LYZ, SPP1, PGrn and Sparc proteins, active fragments thereof, and orthologs thereof.
  • the protein, the active fragment, or the nucleic acid encoding the ortholog is included as an essential active ingredient.
  • the cell transplantation aid of the invention is a protein selected from the group consisting of the above-mentioned LYZ, SPP1, and PGrn proteins, active fragments thereof, and orthologs thereof.
  • the protein, the active fragment, or the nucleic acid encoding the ortholog is included as an essential active ingredient.
  • the cell transplantation aid of the present invention is a protein selected from the group consisting of any of the above-mentioned LYZ and PGrn proteins, an active fragment thereof, and an ortholog thereof, or the protein, the active fragment thereof.
  • the nucleic acid encoding the ortholog is included as an essential active ingredient.
  • the cell transplantation aid of the present invention is a protein selected from the group consisting of the above-mentioned LYZ, an active fragment thereof, and an ortholog thereof, or a nucleic acid encoding the LYZ, the active fragment, or the ortholog. Is included as an essential active ingredient. Since each active ingredient conforms to the description of the first aspect, further description thereof is omitted here.
  • transplantation cofactors corresponding NCBI accession numbers, and SEQ ID NOs are based on the nerve cell activators exemplified in Tables 1 and 1'above, and the description thereof is omitted here.
  • Transplantation aids include, for example, lipofection, fusion with cell membrane permeability peptides (eg, HIV-derived TAT, polyarginine, BP100, Penetratin, pVEC, pAntp, VP22, Transportan, R7, MPG, and Pep-1), microinjection. It may be introduced into a transplanted cell or a cell differentiated from the transplanted cell by a method such as.
  • cell membrane permeability peptides eg, HIV-derived TAT, polyarginine, BP100, Penetratin, pVEC, pAntp, VP22, Transportan, R7, MPG, and Pep-1
  • nucleic acid encoding the protein, the active fragment, or the ortholog is a nucleic acid encoding any of the above transplantation cofactors.
  • Nucleic acids encoding transplant cofactors are LYZ, CD52, MPEG1, CCL2, CST7, RAC2, SERPINA3, ANOS1, KLK6, A2M, GM14295, SPP1, LGALS3BP, APOC1, PGrn, Ctsd, Ctss, Apod, or Sparc.
  • RNA encoding a transplantation cofactor includes both pre-mRNA and mature mRNA, but substantially means mature mRNA.
  • DNA encoding a transplantation cofactor a DNA consisting of the base sequence shown in SEQ ID NO: 46, which encodes a human wild-type LYZ protein; and a mouse wild-type LYZ2 protein.
  • DNA consisting of the base sequence shown by SEQ ID NO: 51 which encodes the human wild-type MPEG1 protein
  • DNA consisting of the base sequence shown by SEQ ID NO: 49 or 50, which encodes the mouse wild-type MPEG1 protein encoding the human wild-type CCL2 protein.
  • DNA SEQ ID NO: 79, which encodes the human wild SPP1 protein Or DNA consisting of the base sequence shown by 80; DNA consisting of the base sequence shown by any of SEQ ID NOs: 74 to 78, which encodes the mouse wild-type SPP1 protein; the base shown by SEQ ID NO: 82, which encodes the human wild-type LGALS3BP protein.
  • DNA consisting of a sequence DNA consisting of the base sequence shown by SEQ ID NO: 81, which encodes the mouse wild-type LGALS3BP protein; DNA consisting of the base sequence shown by any of SEQ ID NOs: 85-88, which encodes the human wild-type APOC1 protein; DNA consisting of the base sequence represented by SEQ ID NO: 83 or 84, which encodes the mouse wild APOC1 protein; DNA consisting of the base sequence shown by SEQ ID NO: 107, which encodes the human wild PGrn protein; encodes the mouse wild PGrn protein.
  • the mRNA encoding the transplantation cofactor includes an mRNA containing a base sequence in which t (thymine) is replaced with u (uracil) as a coding region (translation region) in any of the above base sequences.
  • the mRNA encoding the transplantation cofactor includes a cap structure at the 5'end, a poly A chain at the 3'end, a 5'untranslated region (5'UTR) upstream of the start codon, and / or termination. It may include the 3'untranslated region (3'UTR) downstream of the codon.
  • the 5'UTR and / or the 3'UTR may contain a sequence for adjusting the amount of translation from mRNA, for example, the 3'UTR is a sequence that increases the amount of translation from mRNA (for example,). Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), etc.) may be included.
  • WPRE Woodchuck hepatitis virus post-transcriptional regulatory element
  • Examples of the nucleic acid encoding the transplantation cofactor, the corresponding NCBI accession number, and the SEQ ID NO: are based on the nucleic acid encoding the neuronal activator exemplified in Tables 2 and 2'above, and are described herein. The explanation is omitted.
  • the nucleic acid encoding the transplantation aid may be DNA or RNA (for example, mRNA). If the nucleic acid is in the form of DNA, it may be included in a gene expression vector. Nucleic acid may be introduced into transplanted cells or cells differentiated from transplanted cells by techniques such as electroporation, lipofection, liposomes, and microinjection.
  • the "expressible state” means that a gene or the like encoding a transplantation cofactor is arranged in the downstream region under the control of the promoter. Therefore, in the cells into which the vector has been introduced (for example, transplanted cells), the promoter induces the expression of the transplantation cofactor.
  • the gene expression vector various expression vectors capable of replicating and expressing in a transplanted cell or a cell differentiated from the transplanted cell (for example, a nerve cell) can be used.
  • viral vectors, plasmid vectors, and artificial chromosome vectors are exemplified.
  • the viral vector is not particularly limited as long as it can infect cells (for example, brain cells) and can express a transplantation cofactor in the cells.
  • an adenovirus vector, an adeno-associated virus (AAV) vector, a retrovirus vector, a lentivirus vector, a Sendai virus vector and the like are exemplified.
  • the plasmid vector is not particularly limited as long as it is a vector that can be replicated in mammalian cells.
  • Specific examples of the plasmid vector include pCMV6-XL3, pEGFP-C1, pGBT-9, pcDNA, pcDM8, pREP4 and the like.
  • Examples of the artificial chromosome vector include a human artificial chromosome (HAC), a yeast artificial chromosome (YAC), and a bacterial artificial chromosome (BAC, PAC).
  • the promoter is a promoter having an activity of inducing gene expression in a transplanted cell or a cell differentiated from the transplanted cell (for example, a nerve cell).
  • CMV promoter CMV-IE promoter
  • LTR promoter LTR promoter
  • SV40 promoter SV40 promoter
  • RSV promoter HSV-TK promoter
  • EF1 ⁇ promoter Ub promoter
  • metallothioneine promoter SR ⁇ promoter
  • CAG promoter CAG promoter and the like are exemplified.
  • the nucleic acid When the nucleic acid is in the form of RNA, it may be introduced into cells by techniques such as electroporation, lipofection, and microinjection.
  • the active ingredient may be contained in a plurality of kinds or a plurality of forms in the cell transplantation aid.
  • the transplantation aids used in the cell transplantation aids of the present invention are any of the nerve cell activators shown in Tables 3, 5, 7, and 9 above, any active fragment thereof, and any of the active fragments thereof. It may be a protein selected from the group consisting of any ortholog.
  • nucleic acids encoding transplantation cofactors corresponding to the nerve cell activators shown in Tables 3, 5, and 7 above, and NCBI accession numbers corresponding thereto are exemplified in Tables 4, 6, and 8 above. It is based on the nucleic acid encoding the nerve cell activating factor, and the description thereof is omitted here.
  • the content of the active ingredient contained in the cell transplantation aid varies depending on the type and form of the active ingredient (nucleic acid such as protein, DNA or RNA), the dosage form of the cell transplantation aid, and the type of solvent or carrier described later. Therefore, it may be determined as appropriate in consideration of each condition.
  • a single dose of the cell transplantation aid is adjusted to contain an effective amount of the active ingredient.
  • the amount of the active ingredient may be a total amount including the effective amount.
  • the "effective amount” means an amount necessary for exerting a function as an active ingredient and which gives little or no adverse side effects to a subject to which the active ingredient is applied. This effective amount may vary depending on various conditions such as subject information, application route, and number of applications.
  • the content of the active ingredient is finally determined by the judgment of a doctor, a pharmacist, or the like.
  • the cell transplantation aid of the present invention is 0.01 ng to 10,000 ng, 0.1 ng to 1,000 ng, or 1 ng to 100 ng, for example, 1 ng, 10 ng, 20 ng, 50 ng. Or you may use it at 100 ng.
  • the "subject” is a living body to which the cell transplantation aid of the present invention is applied.
  • humans domestic animals (cattle, horses, sheep, goats, pigs, chickens, ostriches, etc.), race horses, pet animals (dogs, cats, rabbits, etc.), experimental animals (mouses, rats, guinea pigs, monkeys, marmosets, etc.) Etc. are applicable.
  • It is preferably a human (in this case, particularly referred to as a "subject"), such as a human who has or is likely to develop a neurological disorder.
  • a subject to be transplanted is called a "recipient".
  • the dosage form of the cell transplantation aid of the present invention is not particularly limited. Any form may be used as long as it can be delivered to a target site such as a transplantation site or a nerve injury site without inactivating the active ingredient in the body of the subject.
  • the specific dosage form differs depending on the application method described later. Since the application method can be roughly divided into parenteral administration and oral administration, a dosage form suitable for each administration method may be used.
  • the preferred dosage form is a liquid preparation that can be directly administered to a target site such as a transplant site or a nerve injury site or systemically administered via the circulatory system.
  • liquid preparations include injections.
  • the injection is prepared by appropriately combining it with the above-mentioned excipients, emulsifiers, suspensions, surfactants, stabilizers, pH adjusters, etc., and mixing them in a generally accepted unit dose form required for pharmaceutical practice. Can be transformed into.
  • it may be formulated as an artificial extracellular matrix that can be implanted in a living body for the purpose of sustained release of the active ingredient or local action.
  • preferred dosage forms include solids (including tablets, capsules, drops, troches), granules, powders, powders, liquids (drinks for internal use, emulsions, syrups). ). If it is a solid agent, it may be made into a dosage form with a skin known in the art, for example, sugar-coated tablets, gelatin-encapsulated tablets, enteric-coated tablets, film-coated tablets, double tablets, and multi-layer tablets, if necessary. be able to.
  • each of the above dosage forms may be within the range of the dosage form known in the art in each dosage form, and is not particularly limited.
  • the method for producing the cell transplantation aid of the present invention may be formulated according to a conventional method in the art.
  • the application route of the cell transplantation aid of the present invention may be oral administration or parenteral administration.
  • the oral administration method is generally systemic administration, but the parenteral administration method can be further subdivided into local administration and systemic administration.
  • Local administration includes, for example, intracerebral administration, intracerebroventricular administration, administration of transplanted cells to or near the transplantation site, administration to a site where a cell body of a nerve cell projecting to the transplantation site is present, and the like.
  • As the transplantation site the site exemplified in the section of "brain nerve cells" is exemplified.
  • Systemic administration includes, for example, intravenous administration (intravenous injection) and intraarterial administration.
  • the dose may be any amount as long as it is effective for the active ingredient to respond. The effective amount is appropriately selected according to the subject information as described above.
  • the administration time of the cell transplantation aid of the present invention is not limited, and may be before cell transplantation, at the same time as cell transplantation, after cell transplantation, or a combination thereof.
  • “cell transplantation” may refer to “the act of introducing cells for transplantation into the recipient's body”.
  • “simultaneously with cell transplantation” means “the same day as the day when cell transplantation is performed", and is preferably “from the start to the end of the process for cell transplantation”.
  • the cell transplantation aid of the present invention may be administered to a recipient within 6 hours, 4 hours, 2 hours, and 1 hour before and after cell transplantation.
  • the cells for transplantation may be treated with the cell transplantation aid of the present invention prior to cell transplantation.
  • the cells for transplantation may be cultured for a certain period of time in the presence of the cell transplantation aid of the present invention prior to cell transplantation.
  • the period of culturing in the presence of the cell transplantation aid of the present invention is not limited, for example, 2 hours or more, 12 hours or more, 1 day or more, 2 days or more, 3 days or more, 4 days or more, 1 week or more, or 2 It may be more than a week.
  • the culture conditions before cell transplantation are not limited, for example, 0.01 ng to 10,000 ng, 0.1 ng to 1,000 ng, or 1 ng to 100 ng, for example, 1 ng, 10 ng, 20 ng for 100,000 cells for transplantation. , 50 ng, or 100 ng of cells for transplantation may be cultured in a culture medium containing a cell transplantation aid.
  • the cells for transplantation treated with the cell transplantation aid of the present invention may be transplanted to the recipient prior to cell transplantation, and at the same time, the cell transplantation aid of the present invention may be administered to the recipient.
  • the number of administrations of the cell transplantation aid of the present invention is not limited, and it may be either a single administration or a plurality of administrations.
  • a single dose or a plurality of doses may be administered at any time point after transplantation.
  • the cell transplantation aid of the present invention may preferably be continuously administered.
  • the recipient of the cell transplantation aid of the present invention is at least once every two weeks, at least once a week, for example, at least once every two days, at least once a day, or at least twice a day. May be administered to.
  • the cell transplantation aid of the present invention can be used in combination with transplanted cells.
  • the cell transplantation aid of the present invention may be mixed as a protein or nucleic acid in a cell suspension or cell mass containing transplanted cells.
  • the protein may be added directly to a cell suspension or a cell mass, or may be added as a sustained-release matrix in which the protein is embedded.
  • the sustained release matrix is not limited, and examples thereof include naturally derived materials such as extracellular matrix (for example, collagen) and biocompatible synthetic materials.
  • the mixing ratio of the cell transplantation aid of the present invention to the transplanted cells is not limited as long as the effect on the transplanted cells is maintained.
  • the cell transplantation aid of the present invention is 0.01 ng to 10,000 ng, 0.1 ng to 1,000 ng, or 1 ng to 100 ng, for example, 1 ng, 10 ng, 20 ng, 50 ng. , Or can be mixed at a ratio of 100 ng.
  • the cell transplantation aid of the present invention may be introduced into a transplanted cell before cell transplantation so that the transplanted cell itself can express the cell transplantation aid transiently or continuously.
  • the transplantation cofactor may be introduced into the transplanted cell by a method such as lipofection, fusion with a cell membrane-permeable peptide, or microinjection.
  • the transplantation cofactor may be introduced into the transplanted cell as a nucleic acid by a method such as electroporation, lipofection, liposome, or microinjection.
  • a third aspect of the present invention is a therapeutic or prophylactic agent for a neurological disorder (hereinafter referred to as “therapeutic / prophylactic agent”).
  • the therapeutic / prophylactic agent of this embodiment contains the nerve cell activator of the first aspect, and can treat or prevent neurological diseases such as neurodegenerative diseases, head trauma, and cerebral infarction.
  • the therapeutic / prophylactic agents of the invention may include the active ingredient as an essential ingredient and further include solvents, pharmaceutically acceptable carriers, or other agents as selective ingredients.
  • the therapeutic / prophylactic agent of the present invention may be composed of only the active ingredient. However, in order to facilitate the formation of a dosage form and maintain the pharmacological effect and / or the dosage form of the active ingredient, it is preferable that the drug is configured to include a pharmaceutically acceptable carrier described later.
  • the active ingredient in the therapeutic / prophylactic agent of the present invention is the nerve cell activator of the present invention, and its composition has already been described in detail in the first aspect. Therefore, only the selective components will be described below.
  • the therapeutic / prophylactic agents of the present invention can be dissolved in pharmaceutically acceptable solvents as needed.
  • “Pharmaceutically acceptable solvent” means a solvent usually used in the field of pharmaceutical technology.
  • water or an aqueous solution, or an organic solvent can be mentioned.
  • the aqueous solution include saline solution, isotonic solution containing glucose or other auxiliary agents, phosphate buffer solution, and sodium acetate buffer solution.
  • the auxiliary agent include D-sorbitol, D-mannose, D-mannitol, sodium chloride, low-concentration nonionic surfactants, polyoxyethylene sorbitan fatty acid esters, and the like.
  • the organic solvent include ethanol.
  • the therapeutic / prophylactic agents of the present invention may optionally include a pharmaceutically acceptable carrier.
  • “Pharmaceutically acceptable carrier” refers to an additive commonly used in the field of pharmaceutical technology. For example, excipients, binders, disintegrants, fillers, emulsifiers, fluidity modifiers, lubricants, human serum albumin and the like can be mentioned.
  • Excipients include, for example, sugars such as monosaccharides, disaccharides, cyclodextrins and polysaccharides, metal salts, citric acid, tartrate acid, glycine, polyethylene glycol, pluronic, kaolin, silicic acid, or combinations thereof. Be done.
  • Binders include, for example, starch paste using vegetable starch, pectin, xanthan gum, simple syrup, glucose solution, gelatin, tragacant, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose, cellac, paraffin, polyvinylpyrrolidone or a combination thereof. Can be mentioned.
  • disintegrant examples include the starch, lactose, carboxymethyl starch, crosslinked polyvinylpyrrolidone, agar, laminaran powder, sodium hydrogencarbonate, calcium carbonate, alginic acid or sodium alginate, polyoxyethylene sorbitan fatty acid ester, sodium lauryl sulfate, and stearic acid.
  • examples include acid monoglycerides or salts thereof.
  • examples of the filler include petrolatum, the sugar and / or calcium phosphate.
  • emulsifier examples include sorbitan fatty acid ester, glycerin fatty acid ester, sucrose fatty acid ester, and propylene glycol fatty acid ester.
  • flow addition regulator and lubricant examples include silicates, talc, stearate or polyethylene glycol.
  • solubilizers In addition to the above, if necessary, solubilizers, suspending agents, diluents, dispersants, surfactants, painkillers, stabilizers, absorption promoters, bulking agents, etc., which are usually used in pharmaceutical compositions, etc.
  • the carrier is used to avoid or suppress the decomposition of the active ingredient by an enzyme or the like in the subject body, facilitate the formulation and administration method, and maintain the dosage form and drug efficacy, if necessary. It may be used as appropriate. Since the dosage form and application method of the therapeutic / preventive agent of the present invention are in accordance with the description of the first aspect, the description thereof is omitted here.
  • the neurological disease targeted by the therapeutic / prophylactic agent of the present invention is not particularly limited as long as it is a disease accompanied by degeneration, shedding, or cell death of nerve cells.
  • a disease accompanied by degeneration, shedding, or cell death of nerve cells For example, neurodegenerative diseases, brain tumors, head injuries, cerebral contusions, ischemia, cerebrovascular accidents, epilepsy, multiple sclerosis and the like can be mentioned.
  • Neurodegenerative disease is a disease in which the structure and function of nerve cells are damaged and progressively lack.
  • the neurodegenerative disease may be a disease associated with the accumulation of abnormal proteins in the brain or nerve cell death.
  • Specific neurodegenerative diseases include, but are not limited to, muscle atrophic lateral sclerosis, primary lateral sclerosis, Parkinson's disease, FTDP-17, progressive supranuclear palsy, corticobasal degeneration, and Huntington.
  • Parkinson's disease dystonia, neurodegenerative disease with intracerebral iron deposition, frontotemporal corticobasal degeneration, idiopathic corticobasal degeneration, essential tremor, Perry syndrome, mild cognitive impairment (MCI), Alzheimer's disease, silver granules Diseases, Levy body dementia, Down's syndrome, multilineage atrophy, corticobasal degeneration, prion disease, pantothenic acid kinase-related neurodegenerative disease, fist fighter dementia, chronic traumatic encephalopathy and the like.
  • MCI mild cognitive impairment
  • Alzheimer's disease silver granules Diseases
  • Levy body dementia Down's syndrome
  • multilineage atrophy corticobasal degeneration
  • prion disease pantothenic acid kinase-related neurodegenerative disease
  • fist fighter dementia chronic traumatic encephalopathy and the like.
  • the diseases targeted by the therapeutic / prophylactic agents of the present invention are preferably Parkinson's disease, Alzheimer's disease, Huntington's disease, muscle atrophic lateral sclerosis, Lewy body dementia, spinocerebellar degeneration, and progressive supranuclear disease. Paralysis and cortical basal nucleus degeneration.
  • Ischemia includes cerebrovascular dementia, cerebrovascular Parkinson syndrome, cerebral amyloid angiopathy, ischemic optic neuropathy, cerebrovascular accident described below, and other diseases associated with ischemic pathology (eg, ischemic disease). Meningitis, encephalitis, cerebral abscess, etc.).
  • Cerebrovascular disorders include, for example, stroke, cerebral infarction, spinal cord infarction, cerebrovascular sinus thrombosis, cerebrovascular spasm syndrome, cerebrovascular inflammation, cerebral bleeding, submucosal bleeding, haze disease, cerebral arteriovenous fistula, cerebral arteriovenous malformation, Examples include cerebral aneurysm, cervical / cerebral artery dissection, and the like.
  • the neurological diseases targeted by the therapeutic / prophylactic agent of the present invention are preferably neurodegenerative diseases, brain tumors, multiple sclerosis, epilepsy, head trauma, cerebral infarction, stroke, or ischemia.
  • a nerve disease is treated by activating nerve cells such as brain nerve cells and promoting their survival, differentiation, maturation, etc., and / or by protecting the nerve cells. Or it can be prevented.
  • a fourth aspect of the present invention is a therapeutic composition for neurological disorders (hereinafter, simply referred to as “therapeutic composition”).
  • the therapeutic composition of this embodiment contains the cell transplantation aid of the second aspect and the cells for transplantation, and can be used for the purpose of treating a neurological disease.
  • the therapeutic compositions of the present invention may include the active ingredient as an essential ingredient and further include solvents, pharmaceutically acceptable carriers, or other agents as selective ingredients.
  • the therapeutic composition of the present invention may be composed of only the active ingredient. However, in order to facilitate the formation of a dosage form and maintain the pharmacological effect and / or the dosage form of the active ingredient, it is preferable that the drug is configured to include a pharmaceutically acceptable carrier described later.
  • the active ingredient in the therapeutic composition of the present invention is the cell transplantation aid of the present invention and cells for transplantation (transplanted cells).
  • the cell transplantation aid and the composition of the transplanted cells have already been described in detail in the second aspect. Therefore, only the selective components will be described below.
  • composition of the present invention can be dissolved in a pharmaceutically acceptable solvent, if necessary.
  • pharmaceutically acceptable solvent shall be in accordance with the description of the third aspect.
  • compositions of the present invention may optionally include a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier shall be in accordance with the description of the third aspect.
  • the dosage form and application method of the therapeutic composition of the present invention are in accordance with the description of the second aspect, and thus the description thereof is omitted here.
  • the neurological disease targeted by the therapeutic composition of the present invention is not particularly limited as long as it is a disease accompanied by degeneration, shedding, or cell death of nerve cells.
  • the neurological disease targeted by the therapeutic composition of the present invention is based on the target disease described in the third aspect, and the description thereof is omitted here.
  • a kit containing the above-mentioned nerve cell activator is provided. Further, according to the present invention, a kit containing the cell transplantation aid of the present invention and cells for transplantation is provided.
  • INDUSTRIAL APPLICABILITY According to the present invention, there is provided a method for treating or preventing a neurological disease, which comprises a step of administering the above-mentioned nerve cell activator to a subject. Further, according to the present invention, there is also a method of transplanting cells to a recipient, which comprises a cell transplantation aid administration step of administering the above-mentioned cell transplantation aid to the recipient, and a cell transplantation step of transplanting the cells to the recipient. Provided.
  • a method for treating or preventing a neurological disease which comprises a step of administering the above-mentioned nerve cell activator to a subject.
  • the present invention also provides the use of the aforementioned cell transplantation aids in the manufacture of pharmaceuticals for treating neurological disorders.
  • Example 1 Cell transplantation into a cerebral cortex injury model> (Purpose) Cell transplantation is performed in the brain immediately after or 7 days after cortical injury, and the efficiency of engraftment and axonal elongation of the transplanted cells is compared. We will examine how the transplantation results change depending on the host brain environment.
  • a cortical injury model was prepared using 11-week-old immunodeficient mice (CB-17 / Icr-scid / scidJcl, Claire Japan). A biopsy trepan and an aspirator were used at a position 2.0 mm rostrally and laterally from Bregma to injure a depth of 1 mm from the brain surface.
  • CPM count-per-million
  • Example 3 Gene expression analysis of cortical injury model 2> (Purpose) Further candidate factors are identified based on the gene expression analysis results of Example 2. (Method) The following databases: The human secretome and membrane proteome (https://www.proteinatlas.org/humanproteome/tissue/secretome) were used to classify genes / proteins. As a result, membrane proteins & secretory factors (116 genes), secretory factors (772 genes), and membrane proteins (3,422 genes) were extracted. The "membrane protein & secretory factor” corresponds to a gene encoding a plurality of protein isoforms (splice variants) including a secretory isoform and a membrane-bound isoform.
  • the factors with high gene expression 7 days after cortical injury were screened from the gene expression analysis results of Example 2. Specifically, from the results of gene expression immediately after cortical injury (day 0) and 7 days later, the candidate factors with high gene expression 7 days after cortical injury were identified as the following criteria: (a1) and (b), or Extracted using (a2) and (b).
  • p value ⁇ 0.05 and the CPM value on the 7th day is 10 or more.
  • Example 4 Nerve axon elongation promoting effect by each factor> (Purpose) Recombinant proteins of each factor are prepared using cultured cells, and the effect of promoting axon elongation on cortical neurons is verified in vitro.
  • Method 1 Preparation of culture supernatant containing recombinant protein
  • a recombinant protein was prepared by the following method. The mouse-derived full-length sequence of each factor was cloned and integrated into an expression vector, and after plasmid extraction, transfection into HEK293T cells was performed. Then, after performing recovery culture for 1 to 2 days, the transfection medium and DMEM / F12 medium (DMEM / F12 with L-Glutamine, Penicillin / Streptomycin, N2-supplement, B27-supplement) were mixed 1: 1.
  • DMEM / F12 medium DMEM / F12 with L-Glutamine, Penicillin / Streptomycin, N2-supplement, B27-supplement
  • the cells were cultured in medium for 1 day, and then in DMEM / F12 medium (DMEM / F12 with L-Glutamine, Penicillin / Streptomycin, N2-supplement, B27-supplement medium) for 1 day.
  • DMEM / F12 medium DMEM / F12 with L-Glutamine, Penicillin / Streptomycin, N2-supplement, B27-supplement medium
  • the culture supernatant was filtered through a 0.22 ⁇ m filter and then stored frozen.
  • LYZ2 was selected as an example from the above factors, and further functional analysis was performed. It is considered that the effects found for LYZ2 in the following examples can be obtained for other factors as well.
  • LYZ2 Effect of promoting neural axon elongation by mouse LYZ2 recombinant protein> (Purpose)
  • LYZ2 is selected as an example from the above factors, and functional analysis of mLYZ2 is performed using a mouse LYZ2 (mLYZ2) recombinant protein expressed and purified using the Brevibacillus expression system, and a commercially available mLYZ2 recombinant protein. ..
  • mLYZ2 recombinant protein added at 0 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL (DMEM / F12 supplemented with L-Glutamine (2 mM), B27-supplement (1 ⁇ )) , N2-supplement (1 ⁇ ), Penicyllin / Streptomycin (1 ⁇ )), and immunostaining and evaluation with anti-TAU antibody were performed after 6 days of culture. As a result, it was shown that the nerve axons were elongated (Fig. 10) and the nerve axon side branches were increased (Fig. 11) even when the commercially available mLYZ2 was used.
  • Example 6 Nerve axon elongation effect by human LYZ recombinant protein> (Purpose) To verify the effect of human LYZ (hLYZ) recombinant protein on human neurons (human cerebral cortical neurons and human dopamine neurons).
  • TAU-positive nerve axons were visualized by immunostaining using an anti-TAU antibody, and the nerve axon length was quantified. As a result, it was shown that the TAU-positive nerve axons of nerve cells derived from human ES cell-derived cerebral cortical organoids are elongated by hLYZ (Fig. 12).
  • Dopaminergic neurons derived from human ES cells According to the method described in the literature (Kriks S., et al., Nature, 2011, 480 (7378): 547-51.), Differentiation was induced for 37 days and treated with enzymes. Dispersed human ES cell-derived dopaminergic neurons were added with hLYZ of 0 ng / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, and 1,000 ng / mL (Neurobasal medium supplemented with GlutaMAX-I (Neurobasal medium supplemented with GlutaMAX-I).
  • TAU-positive nerve axons were visualized by immunostaining using an anti-TAU antibody, and the nerve axon length was quantified. As a result, it was shown that the TAU-positive nerve axons of human ES cell-derived dopamine neurons are elongated by hLYZ (Fig. 13).
  • Example 7 Effect on the number of nerve cells> (Purpose) The effect of the commercially available mouse LYZ2 recombinant protein (mLYZ2) and the human LYZ recombinant protein (hLYZ) on the total number of TAU-positive neurons will be investigated.
  • mLYZ2 mouse LYZ2 recombinant protein
  • hLYZ human LYZ recombinant protein
  • the nerve axon length and the total number of TAU-positive neurons were quantified by immunostaining using an anti-TAU antibody. As a result, it was shown that the total number of TAU-positive neurons increased (Fig. 14). From this result, it was shown that mLYZ2 has a nerve cell death inhibitory effect.
  • mice LYZ2 recombinant protein Effect of mouse LYZ2 recombinant protein on transplanted cells> (Purpose) When a population of cells isolated from the brain of a fetal mouse is transplanted into the brain of an adult mouse, the mouse LYZ2 recombinant protein (mLYZ2) is administered at the same time.
  • mLYZ2 mouse LYZ2 recombinant protein
  • Midbrain ventral tissue containing dopamine neural progenitor cells was removed from the brain of a fetal mouse (C57BL / 6-Tg (CAG-EGFP), Shimizu laboratory supplies) on the 11th day of pregnancy. After enzymatic treatment, a cell suspension containing 100,000 cells was prepared with PBS containing a final concentration of 10 ⁇ M Y-27632. MLYZ2 (20 ng) was added to this cell suspension and transplanted into the striatum of a 12-week-old Black 6 / N mouse (male). Mice transplanted without adding mLYZ2 to the cell suspension were used as a control group.
  • Example 9 Effect of human Lyz recombinant protein on transplanted cells> (Purpose) Dopamine neural progenitor cells derived from human ES cells are cultured for a certain period of time in the presence of human Lyz recombinant protein, and then transplanted into the brain of an adult mouse. To investigate the effect of human Lyz on the engraftment of transplanted cells.
  • Dopamine neural progenitor cells were prepared from human ES cells (Kh-ES1) by the method described in the literature (Doi D, et al., Stem Cell Reports, 2014, 6; 2 (3): 337-50.). From the 18th day of culture, 100 ng / mL of human Lysozyme protein (abcam / ab158839) was added to the culture medium for cell culture. After enzymatic treatment of dopamine neural progenitor cells on the 29th day of culture, a cell suspension containing 200,000 cells was prepared with PBS containing a final concentration of 10 ⁇ M Y-27632. In this example, Lyz was not additionally added to the cell suspension.
  • This cell suspension was transplanted into the striatum of 16-week-old immunodeficient mice (SCID mice; Shimizu Laboratory Materials Co., Ltd., CB-17 / lcrHsd-Prkdcscid) (Lyz group). Mice transplanted with dopamine neural progenitor cells cultured without adding Lyz to the culture medium were used as a control group (Ctrl).
  • FIG. 17 shows the results of comparing the size of the grafts between the Lyz group and the control group.
  • dopamine neural progenitor cells (Lyz group) cultured in the presence of Lyz had significantly increased graft size compared to cells cultured in the absence of Lyso (control group). rice field. From this result, it was shown that transplantation of cells pretreated with Lyz significantly promoted cell engraftment.
  • Example 10 Evaluation of nerve axon elongation promoting effect and cell death suppressing effect by further candidate factors> (Purpose) 7 candidate factors: lysozyme (LYZ), secretory phosphoprotein 1 (SPP1 or Osteopontin), progranulin (PGrn), cathepsin D (Cathepsin D; Ctsd), cathepsin S (Cathepsin S; Ctss), apolypoprotein D For (Apod) and Sparc, the effect of promoting cathepsin extension and the effect of suppressing cell death are evaluated.
  • LYZ lysozyme
  • SPP1 or Osteopontin secretory phosphoprotein 1
  • PGrn progranulin
  • cathepsin D Cathepsin D; Ctsd
  • cathepsin S Cathepsin S; Ctss
  • apolypoprotein D For (Apod) and Sparc the effect of promoting cathep
  • Human recombinant proteins were used for each candidate factor.
  • the human LYZ recombinant protein is Recombinant Human Lysozyme protein (abcam, ab158839)
  • the human SPP1 recombinant protein is Recombinant Human Osteopontin (OPN) Protein (R & D Systems, 1433-OP-050 / CF), human PGrn group.
  • OPN Human Osteopontin
  • the replacement protein is Recombinant Human Progranulin Protein (R & D Systems, 2420-PG-050), the human Ctsd recombinant protein is Recombinant Human Cathepsin D Protein (R & D Systems, 1014-AS-010), and the human Ctss recombinant protein is Recombinant Human Cathepsin S. Protein (R & D Systems, 1183-CY-010), Human Apod recombinant protein is Recombinant Human Apolipoprotein D Protein (Novus Biologicals, NBP1-99548), Human Sparc recombinant protein is Recombinant Human SPARC Protein (R & D Systems, 941-SP-) 050) was used.
  • each candidate factor of (1) above was added to the medium, and the culture was continued until the 7th day in the presence of each candidate factor.
  • Each factor was diluted with a medium so that the final concentration of 1 well (200 ⁇ l) was the concentration shown in FIGS. 18 to 21.
  • live cell imaging was performed using IncuCyte to measure changes in the axon length of each cell over time.
  • FIG. 18 shows the change over time in axon length when Lyz was added to the medium.
  • FIG. 19 shows the measurement results of axon length on the 7th day of culture in the presence of each factor. It was shown that axon length increased in the presence of Lyz. In addition, the axon length tended to increase even in the presence of PGrn, Spp1, or Sparc.
  • the cell viability was evaluated by the Alamar Blue assay after the completion of live cell imaging on the 7th day of culture in (2) above.
  • cerebral cortical organoids on days 47 to 70 of induction of differentiation were dispersed and seeded on a dish coated with laminin (LM511-E8) at a density of 40,000 cells / cm 2 .
  • the medium composition at this time was DMEM / F12 supplemented with L-Glutamine (2 mM), B27-supplement (1 ⁇ ), N2-supplement (1 ⁇ ), Penicillin / Streptomycin (1 ⁇ ), Y-27632 (10 ⁇ M). ).
  • the medium composition at this time was DMEM / F12 supplemented with L-Glutamine (2 mM), B27-supplement (1 ⁇ ), N2-supplement (1 ⁇ ), and Penicillin / Streptomycin (1 ⁇ ).
  • the Alamar Blue assay was performed by the reagent-attached method using alamarBlue® Cell Viability Reagent (Thermo Fisher Scientific, DAL1025).
  • As a control group cells to which no candidate factor was added (Fig. 20, Ctrl mean) and cells artificially injured by treatment with a detergent (1% Triton) (Fig. 20, 1% Triton mean) were also treated. Evaluation was performed.
  • Figure 20 shows the evaluation results of cell viability. All of the candidate factors used tended to have higher cell viability compared to controls. This tendency was especially high in Lyso and PGrn.

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