Gene expression regulator, prophylactic drug or therapeutic drug for alzheimer's disease, and method for improving dementia

Abstract

To provide a novel gene expression regulator that can regulate (e.g., inhibit or suppress) the expression of protein involved in amyloid β-associated or tau protein-associated dementias, e.g., Alzheimer's disease, and / or in cerebral inflammation.SOLUTION: Provided are: a gene expression regulator which is a microparticle containing miRNA for which a target gene is a gene related to an expression of at least one protein from amyloid precursor protein (APP), β-secretase (BACE1), NMDA-activating protein, glycogen synthetase kinase-3β (GSK-3β), and polyglutamine-binding protein-1 (PQBP1); a prophylactic drug or a therapeutic drug for Alzheimer's disease; and a method for improving dementia.SELECTED DRAWING: Figure 19

Description

[Technical Field]

[0001] The present invention relates to a gene expression regulator, a preventive or therapeutic agent for Alzheimer's disease, and a method for ameliorating dementia. [Background technology]

[0002] Alzheimer's disease is a neurodegenerative disease that currently accounts for more than half of all cases of dementia, and further development of treatments and prevention methods is desired. Research to date has made progress in elucidating the mechanisms underlying Alzheimer's disease. It is believed that one of the key points for preventing, slowing the progression of, and ameliorating Alzheimer's disease is to inhibit the increase, aggregation, accumulation, and deposition of amyloid beta protein and tau protein, a microtubule-associated protein. Furthermore, although details will be discussed later, therapeutic drugs for Alzheimer's disease and their mechanisms of action are becoming increasingly known (see, for example, Non-Patent Document 1).

[0003] On the other hand, it has been suggested that neuron-derived exosomes are involved in the metabolism of amyloid β protein in the brain and the onset and progression of Alzheimer's disease (see, for example, Patent Document 1). Patent Document 1 focuses on the fact that drugs capable of promoting exosome production have not yet been fully investigated, and describes an exosome production promoter that can easily promote exosome production in the body, and contains as an active ingredient a substance that can inhibit the function and / or suppress the expression of acid ceramidase. [Prior art documents] [Patent documents]

[0004] [Patent Document 1] Patent Publication No. 2021-083415 [Non-patent literature]

[0005] [Non-Patent Document 1] Clinical Neurology 54(12), 1178-1180, 2014 Summary of the Invention [Problem to be solved by the invention]

[0006] However, as is clear from the method for solving the problem, Patent Document 1 focuses on human neurons, where amyloid beta accumulation actually occurs, and does not focus on exosomes derived from other cells. For example, Patent Document 1 only describes an example of promoting exosome production in human neuroblastoma-derived SH-SY5Y cells. Furthermore, the literature cited in Patent Document 1 includes a non-patent document suggesting that neuron-derived exosomes highly express amyloid beta-binding glycolipids, have the ability to capture amyloid beta protein, and work with microglia, phagocytes in the brain, to remove amyloid beta protein, and another non-patent document suggesting that neuron-derived exosomes capture amyloid beta on their surface and transport it to microglia, thereby increasing the efficiency of amyloid beta protein degradation.

[0007] The problem that the present invention aims to solve is to provide a novel gene expression regulator that can control (suppress, inhibit, etc.) the expression of proteins associated with amyloid beta-related or tau protein-related dementia such as Alzheimer's disease and brain inflammation. [Means for solving the problem]

[0008] The inventors have found that microparticles containing specific microRNAs can regulate (e.g., suppress or inhibit) the expression of proteins associated with amyloid-beta- or tau-protein-related dementia and brain inflammation.

[0009] Specifically, the present invention and preferred configurations thereof are as follows.

[0010] [1] A gene expression regulator, which is a microparticle containing miRNA that targets genes involved in the expression of at least one of the following proteins: amyloid precursor protein (APP), beta-secretase (BACE1), NMDA-activating protein, glycogen synthase kinase-3β (GSK-3β), and polyglutamine-binding protein-1 (PQBP1). [2] The gene expression regulator according to [1], wherein the microparticles are exosomes. [3] A gene expression regulator according to [1] or [2], wherein the microparticles contain miRNA that targets a gene involved in the expression of at least one of the proteins APP, BACE1, NMDA-activating protein, GSK-3β, and PQBP1 at a concentration higher than that of the culture supernatant of dental pulp-derived stem cells. [4] An inhibitor of the expression of genes involved in APP expression. The gene expression regulator according to any one of [1] to [3], wherein the microparticles contain miRNA that targets a gene involved in the expression of APP. [5] The gene expression regulator according to [4], wherein the microparticles contain at least one of the following APP suppression-related miRNAs: APP suppression-related miRNAs: hsa-miR-101-3p, hsa-miR-106b-5p, hsa-miR-1229-5p, hsa-miR-1238-3p, hsa-miR-1260b, hsa-miR-1276, hsa-miR-128-3p, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-144-3p, hsa-miR-151a-3p, hsa-miR-153-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-185-5p, hsa-miR-186-5p, hsa-miR-194-5p, hsa-miR-195-5p, hsa-miR-196a-5p, hsa-miR-203a-3p, hsa-miR-20a-5p, hsa-miR-222-3p, hsa-miR-298, hsa-miR-31-5p, hsa-miR-3120-3p, hsa-miR-3132, hsa-miR-323a-3p, hsa-miR-324-5p, hsa-miR-328-3p, hsa-miR-3620-3p, hsa-miR-3646, hsa-miR-369-3p, hsa-miR-373-3p, hsa-miR-374c-5p, hsa-miR-381-3p, hsa-miR-382-5p, hsa-miR-383-5p, hsa-miR-411-3p, hsa-miR-423-3p, hsa-miR-424-3p, hsa-miR-424-5p, hsa-miR-4484, hsa-miR-455-3p, hsa-miR-4786-5p, hsa-miR-484, hsa-miR-490-5p, hsa-miR-497-5p, hsa-miR-500a-5p, hsa-miR-5093, hsa-miR-532-3p, hsa-miR-532-5p, hsa-miR-539-3p, hsa-miR-548f-3p, hsa-miR-551b-5p, hsa-miR-5584-5p, hsa-miR-567, hsa-miR-5696, hsa-miR-582-5p, hsa-miR-6073, hsa-miR-873-5p, hsa-miR-93-5p. [6] It is an inhibitor of the gene involved in the expression of BACE1. The gene expression regulator according to any one of [1] to [5], wherein the microparticles contain miRNA that targets a gene involved in the expression of BACE1. [7] The gene expression regulator according to [6], wherein the microparticles contain at least one of the following BACE1 inhibition-related miRNAs: BACE1 inhibition-related miRNAs: hsa-miR-107, hsa-miR-124-3p, hsa-miR-1267, hsa-miR-128-3p, hsa-miR-129-2-3p, hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141-3p, hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-195-5p, hsa-miR-200a-3p, hsa-miR-203a-3p, hsa-miR-212-3p, hsa-miR-212-5p, hsa-miR-24-3p, hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-298, hsa-miR-299-3p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-328-3p, hsa-miR-339-5p, hsa-miR-340-5p, hsa-miR-369-3p, hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-374c-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-424-5p, hsa-miR-455-3p, hsa-miR-497-5p, hsa-miR-505-3p, hsa-miR-532-5p, hsa-miR-7-5p, hsa-miR-874-3p, hsa-miR-9-5p. [8] It is an inhibitor of the gene involved in the expression of GSK-3β. The gene expression regulator according to any one of [1] to [7], wherein the microparticles contain miRNA that targets a gene involved in the expression of GSK-3β. [9] The gene expression regulator according to [8], wherein the microparticles contain at least one of the following GSK-3β inhibition-related miRNAs: GSK-3β inhibition-related miRNAs: hsa-let-7a-3p、hsa-let-7b-3p、hsa-let-7f-1-3p、hsa-let-7f-2-3p、hsa-miR-101-3p、 hsa-miR-1185-1-3p, hsa-miR-1185-2-3p, hsa-miR-124-3p, hsa-miR-128-3p, hsa-miR-129-5p hsa-miR-132-3p, hsa-miR-137, hsa-miR-140-5p, hsa-miR-142-5p, hsa-miR-144-3p hsa-miR-150-5p、hsa-miR-155-5p、hsa-miR-15a-5p、hsa-miR-15b-5p、hsa-miR-16-5p、 hsa-miR-1910-5p, hsa-miR-195-5p, hsa-miR-199a-5p, hsa-miR-212-3p, hsa-miR-218-5p hsa-miR-219a-5p、hsa-miR-23a-3p、hsa-miR-24-3p、hsa-miR-26a-5p、hsa-miR-26b-5p、 hsa-miR-27a-3p, hsa-miR-28-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-346 hsa-miR-369-3p、hsa-miR-374a-5p、hsa-miR-374b-5p、hsa-miR-374c-5p、hsa-miR-377-3p、 hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410-3p, hsa-miR-424-5p, hsa-miR-425-5p hsa-miR-4465, hsa-miR-497-5p, hsa-miR-582-5p, hsa-miR-6083, hsa-miR-708-5p hsa-miR-9-5p, hsa-miR-92a-3p, hsa-miR-96-5p, hsa-miR-98-3p.

[10] A gene expression regulator according to any one of [1] to [9], wherein the microparticles are at least one of an inhibitor of gene expression related to APP expression, an inhibitor of gene expression related to BACE1 expression, and an inhibitor of gene expression related to GSK-3β expression, and contain hsa-miR-16-5p.

[11] It is an inhibitor of NMDA-activated gene expression. the NMDA-activated protein is at least one protein selected from the group consisting of DLG1, CAMK2D, CAMK2A, and CAPN1; The gene expression regulator according to any one of [1] to

[10] , wherein the microparticles contain at least one miRNA group selected from the group consisting of the following DLG1 suppression-related miRNA group, the group consisting of the following CAMK2D suppression-related miRNA group, the group consisting of the following CAMK2A suppression-related miRNA group, and the group consisting of the following CAPN1 suppression-related miRNA group, whose target gene is a gene involved in the protein expression of at least one of DLG1, CAMK2D, CAMK2A, and CAPN1; DLG1 suppression-related miRNAs: hsa-miR-1-3p, hsa-miR-142-5p, hsa-miR-204-5p, hsa-miR-206, hsa-miR-21-5p, hsa-miR-218-5p, hsa-miR-340-5p, hsa-miR-613. CAMK2D suppression-related miRNAs: hsa-let-7a-5p, hsa-miR-101-3p, hsa-miR-129-5p, hsa-miR-139-5p, hsa-miR-144-3p, hsa-miR-145-5p, hsa-miR-185-5p, hsa-miR-203a-3p, hsa-miR-204-5p, hsa-miR-211-5p, hsa-miR-24-3p, hsa-miR-27a-3p, hsa-miR-30a-3p, hsa-miR-31-5p, hsa-miR-361-5p, hsa-miR-421, hsa-miR-484, hsa-miR-494-3p, hsa-miR-505-3p, hsa-miR-7-5p. CAMK2A suppression-related miRNAs: hsa-miR-129-5p, hsa-miR-137, hsa-miR-142-5p, hsa-miR-148a-3p, hsa-miR-149-3p, hsa-miR-152-3p, hsa-miR-25-3p, hsa-miR-27a-3p, hsa-miR-32-5p, hsa-miR-338-3p, hsa-miR-340-5p, hsa-miR-363-3p, hsa-miR-3665, hsa-miR-4534, hsa-miR-4665-5p, hsa-miR-4688, hsa-miR-485-5p, hsa-miR-5010-5p, hsa-miR-505-5p, hsa-miR-5698, hsa-miR-625-5p, hsa-miR-92a-3p. CAPN1 suppression-related miRNAs: hsa-miR-1-3p, hsa-miR-124-3p, hsa-miR-140-5p, hsa-miR-17-3p, hsa-miR-22-3p, hsa-miR-34a-5p, hsa-miR-6511b-5p.

[12] Microparticles, Among the DLG1 suppression-related miRNAs, hsa-miR-21-5p, Among the CAMK2D suppression-related miRNAs, hsa-let-7a-5p, Among the miRNAs related to CAMK2A suppression, hsa-miR-92a-3p, A gene expression regulator according to

[11] , which contains hsa-miR-22-3p from the CAPN1 repression-related miRNA group.

[13] It is an inhibitor of PQBP1 expression. The gene expression regulator according to any one of [1] to

[12] , wherein the microparticles contain miRNA that targets a gene involved in the expression of PQBP1.

[14] The gene expression regulator according to

[13] , wherein the microparticles contain at least one of the following PQBP1 suppression-related miRNAs: PQBP1 suppression-related miRNAs: hsa-miR-6727-3p, hsa-miR-6727-5p.

[15] The microparticles are purified and isolated from the culture supernatant of dental pulp-derived stem cells; The gene expression regulator according to any one of [1] to

[14] , wherein the gene expression regulator does not contain components excluding exosomes from the culture supernatant of dental pulp-derived stem cells.

[16] A preventive or therapeutic drug for Alzheimer's disease, comprising the gene expression regulator according to any one of [1] to

[15] .

[17] A method for improving dementia, comprising administering an effective amount of a gene expression regulator described in any one of [1] to

[15] , or an effective amount of a preventive or therapeutic drug for Alzheimer's disease described in

[16] , to a subject who has developed amyloid beta-related or tau protein-related dementia. [Effects of the Invention]

[0011] According to the present invention, it is possible to provide a novel gene expression regulator that can regulate the expression of proteins associated with amyloid beta-related or tau protein-related dementia such as Alzheimer's disease and brain inflammation. [Brief explanation of the drawings]

[0012] [Figure 1] FIG. 1 is a flow chart showing the pathogenesis of Alzheimer's disease. [Figure 2] FIG. 2 is a schematic diagram of the non-amyloidogenic pathway of APP processing. [Figure 3] FIG. 3 is a schematic representation of APP processing in the amyloidogenic pathway. [Figure 4] Figure 4(A) is a schematic diagram of the induction of inflammatory gene expression by AIDS virus cDNA via the PQBP1-cGAS-STING pathway. Figure 4(B) is a schematic diagram of the induction of inflammatory gene expression by tau protein via the PQBP1-cGAS-STING pathway. [Figure 5]Figure 5 is a heat map showing the expression levels of miRNAs expressed in exosomes (expression regulator of the gene in Example 1; SGF) purified from the culture supernatant of dental pulp-derived stem cells targeting APP, compared with the expression levels of miRNAs in the cerebral cortex. [Figure 6] Figure 6 is a heat map showing the expression levels of miRNAs expressed in exosomes (expression regulator of the gene in Example 1; SGF) purified from the culture supernatant of dental pulp-derived stem cells that target beta-secretase (BASE1), compared with the expression levels of miRNAs in the cerebral cortex. [Figure 7] Figure 7 is a heat map showing the expression levels of miRNAs expressed in exosomes (expression regulator of the gene in Example 1; SGF) purified from the culture supernatant of dental pulp-derived stem cells targeting DLG1, compared with the expression levels of miRNAs in the cerebral cortex. [Figure 8] Figure 8 is a heat map showing the expression levels of miRNAs expressed in exosomes (expression regulator of the gene in Example 1; SGF) purified from the culture supernatant of dental pulp-derived stem cells targeting CAMK2D, compared with the expression levels of miRNAs in the cerebral cortex. [Figure 9] Figure 9 is a heat map showing the expression levels of miRNAs expressed in exosomes (expression regulator of the gene in Example 1; SGF) purified from the culture supernatant of dental pulp-derived stem cells targeting CAMK2A, compared with the expression levels of miRNAs in the cerebral cortex. [Figure 10] Figure 10 is a heat map showing the expression levels of miRNAs expressed in exosomes (expression regulator of the gene in Example 1; SGF) purified from the culture supernatant of dental pulp-derived stem cells targeting CAPN1, compared with the expression levels of miRNAs in the cerebral cortex. [Figure 11]Figure 11 is a heat map showing the expression levels of miRNAs expressed in exosomes (expression regulator of the gene in Example 1; SGF) purified from the culture supernatant of dental pulp-derived stem cells that target GSK-3β, compared with the expression levels of miRNAs in the cerebral cortex. [Figure 12] Figure 12 is a heat map showing the expression levels of miRNAs expressed in exosomes (expression regulator of the gene in Example 1; SGF) purified from the culture supernatant of dental pulp-derived stem cells that target PQBP1, compared with the expression levels of miRNAs in the cerebral cortex. [Figure 13] Figure 13 is a heat map (page 1) showing the expression levels of amyloid-β or tau protein-related miRNAs in exosomes (the gene expression regulator of Example 1; SGF) purified from the culture supernatant of dental pulp-derived stem cells, compared with the expression levels of miRNAs in the cerebral cortex. Figure 13-2 is a continuation (page 2) of the heat map in Figure 13. [Figure 14] FIG. 14 is a bar graph showing the expression level of the APP gene in Test Example 4. [Figure 15] FIG. 15 is a bar graph showing the expression level of the BASE1 gene in Test Example 4. [Figure 16] FIG. 16 is a bar graph showing the expression level of the CAMK2D gene in Test Example 4. [Figure 17] FIG. 17 is a bar graph showing the expression level of the GSK-3β gene in Test Example 4. [Figure 18] FIG. 18 is a bar graph showing the expression level of the PQBP1 gene in Test Example 4. [Figure 19] FIG. 19 is a bar graph showing the expression level of each miRNA in Test Example 5. DETAILED DESCRIPTION OF THE INVENTION

[0013] The present invention will be described in detail below. The following description of the constituent elements may be based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. In this specification, a numerical range expressed using "to" means a range that includes the numerical values ​​before and after "to" as the lower and upper limits.

[0014] [Gene expression regulators] The gene expression regulator of the present invention is a microparticle containing miRNA whose target gene (also referred to as "target") is a gene involved in the expression of at least one of the following proteins: amyloid precursor protein (APP), β-secretase (BACE1), NMDA-activating protein, and glycogen synthase kinase-3β (GSK-3β). The gene expression regulator of the present invention can regulate the expression of genes related to proteins associated with amyloid-β- or tau-protein-related dementia and brain inflammation. As a result, the gene expression regulator of the present invention can preferably ameliorate, and more preferably prevent or treat, amyloid-β- or tau-protein-related dementia and brain inflammation. Treatment refers to alleviating the symptoms of amyloid-β- or tau-protein-related dementia and brain inflammation, and does not necessarily mean achieving a complete cure. Treatment of amyloid-β- or tau-protein-related dementia preferably involves alleviating symptoms of cognitive function and behavior. Preferred embodiments of the gene expression regulator of the present invention will be described below.

[0015] <Definition of dementia> In the present invention, dementia generally refers to a state in which cognitive function is acquiredly impaired. Examples of types of dementia include Alzheimer's disease (AD), vascular dementia (VaD), dementia with Lewy bodies (DLB, including Parkinson's disease with dementia (PDD)), frontotemporal lobar degeneration (including frontotemporal dementia (FTD), semantic dementia (SD), and progressive non-fluent aphasia (PNFA)), alcoholic dementia, mixed type, mild cognitive impairment (MCI), and early-onset dementia. The dementia for which the gene expression regulator of the present invention is administered is preferably amyloid-β-related or tau protein-related dementia. There are no particular limitations on the amyloid-β-related or tau protein-related dementia, and examples include a group of diseases in which tau accumulation occurs, known as tau pacing. Specifically, the dementia for which the gene expression regulator of the present invention is administered is preferably Alzheimer's disease, frontotemporal dementia (FTD or bvFTD), semantic dementia (SD), or progressive non-fluent aphasia (PNFA). FTD includes Pick's disease, etc. The dementia to which the gene expression regulator of the present invention is administered is more preferably Alzheimer's dementia.

[0016] <Mechanism of Alzheimer's disease> A flow chart showing the pathogenesis of Alzheimer's disease is shown in Figure 1.

[0017] First, the flowchart shown by the solid arrows in FIG. 1 will be explained. (1) Production of amyloid precursor protein (APP). APP production is further enhanced by mutations in the causative genes (APP, PS1, PS2) and ApoE4 (a risk factor). (2) Production of amyloid beta protein (Aβ). Furthermore, the production of Aβ is enhanced by the inhibition of Aβ-degrading enzymes (neprilysin, insulin-degrading enzyme, etc.). (3) Aggregation, accumulation, and deposition of amyloid beta protein. Furthermore, abnormalities in intracellular calcium homeostasis, reactive oxygen species, and endoplasmic reticulum stress accelerate the aggregation, accumulation, and deposition of Aβ. (21) Neurofibrillary tangles, cell death, and reactive glial proliferation. (22) Incidence of Alzheimer's disease.

[0018] Next, the flowchart shown by the unpainted arrows in FIG. 1 will be described. (11) Induction of abnormal phosphorylation of tau protein progresses through (3) aggregation, accumulation, and deposition of amyloid beta protein. (12) Soluble monomeric tau protein is released from microtubules. (13) Monomeric tau protein migrates from the axon to the soma-dendrites of neurons, where it interacts with the Src tyrosine kinase fyn. (14) fyn is localized and activated in dendrites. (15) Phosphorylation of excitatory NMDA receptor GluN2B. Amplification of glutamate signaling promotes phosphorylation of excitatory NMDA receptor GluN2B. (16) Ca in the cell 2+ Increased inflow. (17) Increased toxicity of Aβ. (21) Neurofibrillary tangles, cell death, and reactive glial proliferation. (22) Incidence of Alzheimer's disease.

[0019] (APP processing) In relation to the production of amyloid β protein (Aβ) (2) in Figure 1, the processing of amyloid precursor protein (APP) will be explained. FIG. 2 is a schematic diagram of the non-amyloidogenic pathway of APP processing. First, when APP is cleaved by α-secretase, C83 (a fragment consisting of 83 amino acids on the C-terminal side) remains in the cell membrane, and sAPPα (a fragment consisting of the extracellular domain on the N-terminal side) is released outside the cell. Next, C83 is cleaved by γ-secretase to produce P3 peptide and CTF, resulting in no Aβ production.

[0020] Meanwhile, Figure 3 is a schematic diagram of APP processing in the amyloidogenic pathway. First, APP is cleaved by β-secretase (BACE1), leaving C99 (a 99 amino acid fragment on the C-terminal side) in the cell membrane, and sAPPβ (an extracellular domain on the N-terminal side) is released outside the cell. C99 is then cleaved by γ-secretase to form Aβ peptides (Aβ1-40 or Aβ1-42). γ-secretase is composed of presenilin 1 (PS1) or presenilin 2 (PS2), nicastrin, APH-1, and PEN2.

[0021] <Alzheimer's disease treatment drugs and their mechanisms of action> The gene expression regulator of the present invention is preferably capable of ameliorating dementia, particularly Alzheimer's disease. Below, the therapeutic drug for Alzheimer's disease and its mechanism of action will be explained together with the mechanism of action of the gene expression regulator of the present invention. Alzheimer's disease treatment drugs and their mechanisms of action include the following:

[0022] (a) Inhibitors of the enzyme that breaks down acetylcholine Acetylcholine is a substance released at the synapse at the tip of a nerve cell's projection and transmits information to the next nerve cell, and the number of cells that produce acetylcholine decreases in the brains of people with Alzheimer's disease. Inhibitors of the enzyme that breaks down acetylcholine (such as donepezil hydrochloride) suppress the breakdown and loss of acetylcholine at the synapse, thereby increasing the amount of acetylcholine. In addition, inhibitors of acetylcholine decomposition enzymes are less related to the gene expression regulator of the present invention.

[0023] (b) Stimulants for nicotinic receptors Acetylcholine signaling is triggered by binding to nicotinic receptors. There are two types of acetylcholine receptors: nicotinic receptors (also called nicotinic acetylcholine receptors) and muscarinic receptors. Nicotinic receptors are pentamer structures made up of subunits, and function as ion channels when acetylcholine binds to them. The number of nicotinic receptors is reduced in the brains of patients with Alzheimer's disease. Drugs such as galantamine hydrobromide bind to a different site from acetylcholine, enhancing the function of nicotinic receptors (APL action). It should be noted that stimulants for nicotine receptors are less related to the gene expression regulator of the present invention.

[0024] (c) NMDA receptor inhibitors By suppressing the activation of NMDA receptors induced by increased Aβ and tau, it prevents cell death due to excessive neuronal excitation. NMDA is involved in memory, learning, and cerebral ischemia. Proteins that regulate NMDA include DLG1, CAMK2D, CAMK2A, CAPN1, and EPHB2. When the microparticles of the gene expression regulator of the present invention contain miRNAs targeting genes involved in the expression of NMDA-activating proteins, they function as NMDA receptor inhibitors. In particular, the gene expression regulator of the present invention functions as at least one NMDA receptor inhibitor selected from the group consisting of DLG1 expression inhibitors, CAMK2D expression inhibitors, CAMK2A expression inhibitors, and CAPN1 expression inhibitors.

[0025] (d) BACE1 inhibitor It inhibits BACE1, the enzyme that produces Aβ, thereby suppressing the production of Aβ. In the gene expression regulator of the present invention, when the microparticles contain miRNA that targets a gene involved in the expression of β-secretase (BACE1), they function as BACE1 inhibitors.

[0026] (e) GSK-3β inhibitors Glycogen synthase kinase-3β (GSK-3β) is an enzyme that phosphorylates multichain proteins and plays a variety of important roles in maintaining and regulating normal cell life. Pathological overexpression of GSK-3β promotes the deposition of Aβ in the brain and the accumulation of tau protein in neurons. GSK-3β inhibitors suppress Aβ deposition and the abnormal phosphorylation of tau protein (see Table 1 below; Clinical Neurology 54(12), 1178-1180, 2014). In the gene expression regulator of the present invention, when the microparticles contain miRNA that targets a gene involved in the expression of glycogen synthase kinase-3β (GSK-3β), they function as GSK-3β inhibitors.

[0027] [Table 1]

[0028] (f) APP inhibitor APP inhibitors inhibit the production of amyloid precursor protein (APP) and suppress the production of APP. In the gene expression regulator of the present invention, when the microparticles contain miRNA that targets a gene involved in the expression of amyloid precursor protein (APP), they function as APP inhibitors.

[0029] <Brain inflammation> The gene expression regulator of the present invention is preferably capable of ameliorating brain inflammation. The mechanism of action of brain inflammation will be explained below together with the mechanism of action of the gene expression regulator of the present invention. Tau protein, which is involved in the pathology of several neurodegenerative diseases, including Alzheimer's disease, frontotemporal lobar degeneration, Parkinson's disease, and Huntington's disease, is recognized by PQBP1, a known intracellular receptor for the AIDS virus, in brain microglia, inducing brain inflammation. Specifically, tau activates microglia via the PQBP1-cGAS-STING pathway, promoting brain inflammation (Nature Communications volume 12, Article number: 6565 (2021)). Figure 4(A) is a schematic diagram of the induction of inflammatory gene expression via the PQBP1-cGAS-STING pathway by AIDS virus cDNA. Figure 4(B) is a schematic diagram of the induction of inflammatory gene expression via the PQBP1-cGAS-STING pathway by tau protein. Therefore, when the microparticles of the gene expression regulator of the present invention contain miRNA that targets the gene involved in the expression of polyglutamine-binding protein-1 (PQBP1), they function as PQBP1 inhibitors, and as a result, the gene expression regulator of the present invention can improve tau protein-associated dementia (particularly Alzheimer's disease) and brain inflammation.

[0030] <Small particles> The miRNA, which is the active ingredient of the gene expression regulator of the present invention, is contained in the microparticles. The microparticles used as gene expression regulators of the present invention are derived from dental pulp-derived stem cells, for example, by secretion, budding, or dispersion from mesenchymal stem cells, such as dental pulp-derived stem cells, and are exuded, released, or shed into cell culture media. The microparticles are preferably contained in the culture supernatant of dental pulp-derived stem cells, and more preferably, are microparticles derived from the culture supernatant of dental pulp-derived stem cells. However, microparticles derived from the culture supernatant of dental pulp-derived stem cells do not necessarily have to be obtained from the culture supernatant of dental pulp-derived stem cells. For example, even if microparticles isolated from the interior of dental pulp-derived stem cells by any method are the same as microparticles that can be isolated from the culture supernatant of dental pulp-derived stem cells, they can still be considered microparticles derived from the culture supernatant of dental pulp-derived stem cells. The microparticles derived from the culture supernatant of dental pulp-derived stem cells or the like may be used in a state contained in the culture supernatant or in a state purified from the culture supernatant. The microparticles are preferably microparticles purified from the culture supernatant. The origin of the microparticles can be determined by known methods. For example, the method described in J Stem Cell Res Ther (2018) 8:2 can be used to determine whether the microparticles are derived from dental pulp-derived stem cells, adipose-derived stem cells, bone marrow-derived stem cells, umbilical cord-derived stem cells, or other stem cells. Specifically, the origin of each microparticle can be determined based on the miRNA pattern of the microparticles.

[0031] (miRNA) In the present invention, the microparticles contain miRNAs that target genes involved in the expression of at least one of the following proteins: amyloid precursor protein (APP), β-secretase (BACE1), NMDA-activating protein, glycogen synthase kinase-3β (GSK-3β), and polyglutamine-binding protein-1 (PQBP1). In the present invention, miRNA (MicroRNAs) are RNA molecules of, for example, 21 to 25 bases (nucleotides). miRNA can regulate gene expression by degrading target gene (target) mRNA or suppressing it at the decoding stage. In the present invention, miRNA may be single-stranded (monomer) or double-stranded (dimer). Furthermore, in the present invention, miRNA is preferably mature miRNA cleaved by ribonuclease such as Dicer.

[0032] The sequences of the miRNAs described herein, such as hsa-miR-16-5p, are registered in publicly known databases (e.g., the miRBase database) with associated accession numbers, allowing those skilled in the art to uniquely determine the sequences. For example, the accession number for hsa-miR-16-5p is MIMAT0000069, and the sequence is registered in the miRBase database. Hereinafter, the accession numbers for each miRNA will be omitted. However, the miRNA in this specification also includes variants that differ by about 1 to 5 bases from the mature miRNA, such as hsa-miR-16-5p. Furthermore, each miRNA in this specification includes a polynucleotide consisting of a base sequence that has identity with the base sequence of each miRNA (e.g., hsa-miR-16-5p), or a polynucleotide consisting of a base sequence complementary to the base sequence, and has the function of the miRNA in this invention. "Identity" refers to the degree of identity when the sequences to be compared are appropriately aligned. The term "identity" refers to the percentage of exact amino acid matches between the sequences. Alignment can be performed using any algorithm, such as BLAST. The identity is, for example, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or approximately 99%. A polynucleotide consisting of an identical nucleotide sequence may have, for example, point mutations, deletions, and / or additions in the nucleotide sequence of an miRNA. The number of nucleotides such as point mutations is, for example, 1 to 5, 1 to 3, 1 to 2, or 1. A polynucleotide consisting of a complementary nucleotide sequence is, for example, a polynucleotide that hybridizes with a polynucleotide consisting of an miRNA nucleotide sequence under stringent conditions and includes a polynucleotide having the function of the miRNA of the present invention. Stringent conditions are not particularly limited, but include, for example, the conditions described in

[0028] of JP 2017-184642 A, the contents of which are incorporated herein by reference.

[0033] In the present invention, it is preferable that the microparticles contain miRNAs that target genes involved in the expression of at least one of the proteins APP, BACE1, NMDA-activating protein, GSK-3β, and PQBP1 at a concentration higher than that of the culture supernatant of dental pulp-derived stem cells. Preferred embodiments of the miRNA contained in the microparticles will be described below.

[0034] (1) APP expression inhibitors The gene expression regulator of the present invention is preferably an inhibitor of APP expression, in which case the microparticles preferably contain miRNA that targets a gene involved in APP expression. In the present invention, it is more preferable that the microparticles contain at least one of the following APP suppression-related miRNAs (63 types): APP suppression-related miRNAs: hsa-miR-101-3p, hsa-miR-106b-5p, hsa-miR-1229-5p, hsa-miR-1238-3p, hsa-miR-1260b, hsa-miR-1276, hsa-miR-128-3p, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-144-3p hsa-miR-151a-3p、hsa-miR-153-3p、hsa-miR-15a-5p、hsa-miR-15b-5p、hsa-miR-16-5p、 hsa-miR-17-5p, hsa-miR-185-5p, hsa-miR-186-5p, hsa-miR-194-5p, hsa-miR-195-5p hsa-miR-196a-5p, hsa-miR-203a-3p, hsa-miR-20a-5p, hsa-miR-222-3p, hsa-miR-298 hsa-miR-31-5p, hsa-miR-3120-3p, hsa-miR-3132, hsa-miR-323a-3p, hsa-miR-324-5p hsa-miR-328-3p, hsa-miR-3620-3p, hsa-miR-3646, hsa-miR-369-3p, hsa-miR-373-3p hsa-miR-374c-5p, hsa-miR-381-3p, hsa-miR-382-5p, hsa-miR-383-5p, hsa-miR-411-3p hsa-miR-423-3p, hsa-miR-424-3p, hsa-miR-424-5p, hsa-miR-4484, hsa-miR-455-3p hsa-miR-4786-5p, hsa-miR-484, hsa-miR-490-5p, hsa-miR-497-5p, hsa-miR-500a-5p hsa-miR-5093, hsa-miR-532-3p, hsa-miR-532-5p, hsa-miR-539-3p, hsa-miR-548f-3p hsa-miR-551b-5p, hsa-miR-5584-5p, hsa-miR-567, hsa-miR-5696, hsa-miR-582-5p hsa-miR-6073, hsa-miR-873-5p, hsa-miR-93-5p.

[0035] The microparticles preferably contain 5 or more types of APP suppression-related miRNAs, more preferably 10 or more types, particularly preferably 20 or more types, and even more particularly preferably 30 or more types.

[0036] It is preferable that the microparticles contain any one of the APP suppression-related miRNAs hsa-miR-101-3p, hsa-miR-153-3p, hsa-miR-16-5p, hsa-miR-222-3p and hsa-miR-93-5p, and it is particularly preferable that they contain at least hsa-miR-16-5p. The microparticles preferably contain at least one type of APP suppression-related miRNA group, with a Log2Ratio of the read count obtained by analysis using IMOTA of 4.0 or more, more preferably 10.0 or more, and particularly preferably 15.0 or more. The microparticles preferably contain hsa-miR-101-3p, hsa-miR-153-3p, hsa-miR-16-5p, hsa-miR-222-3p, and hsa-miR-93-5p, with a Log2Ratio of the read count obtained by analysis using IMOTA of 4.0 or more, more preferably 10.0 or more, and particularly preferably 15.0 or more.

[0037] Note that hsa-miR-16-5p, which is included in the APP suppression-related miRNA group, is also an inhibitor of a gene involved in BACE1 expression and a gene involved in GSK-3β expression. That is, in the present invention, the microparticles are preferably at least one of an inhibitor of a gene involved in APP expression, an inhibitor of a gene involved in BACE1 expression, and an inhibitor of a gene involved in GSK-3β expression, and also contain hsa-miR-16-5p. Furthermore, it is more preferable that the microparticles are an inhibitor of the expression of a gene involved in the expression of APP, an inhibitor of the gene involved in the expression of BACE1, and an inhibitor of the gene involved in the expression of GSK-3β, and contain hsa-miR-16-5p.

[0038] The gene expression regulator of the present invention preferably has an expression level of hsa-miR-16-5p that is 1.1 times or more, more preferably 1.5 times or more, and particularly preferably 2 times or more, higher than that of exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells.

[0039] When the gene expression control agent of the present invention is an agent for inhibiting the expression of the APP gene, it is preferable that the expression of the APP gene in any cell can be inhibited to 0.8 times or less of the normal level (in untreated cells), more preferably to 0.6 times or less, and particularly preferably to 0.4 times or less.

[0040] (2) BACE1 inhibitors The gene expression regulator of the present invention is preferably an inhibitor of BACE 1. In this case, the microparticles preferably contain miRNA that targets a gene involved in the expression of BACE 1. In the present invention, it is more preferable that the microparticles contain at least one type of miRNA among the following group of BACE1 inhibition-related miRNAs (50 types). BACE1 inhibition-related miRNAs: hsa-miR-107, hsa-miR-124-3p, hsa-miR-1267, hsa-miR-128-3p, hsa-miR-129-2-3p, hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141-3p, hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-195-5p, hsa-miR-200a-3p, hsa-miR-203a-3p, hsa-miR-212-3p, hsa-miR-212-5p, hsa-miR-24-3p, hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-298, hsa-miR-299-3p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-328-3p, hsa-miR-339-5p, hsa-miR-340-5p, hsa-miR-369-3p, hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-374c-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-424-5p, hsa-miR-455-3p, hsa-miR-497-5p, hsa-miR-505-3p, hsa-miR-532-5p, hsa-miR-7-5p, hsa-miR-874-3p, hsa-miR-9-5p.

[0041] The microparticles preferably contain 5 or more types of BACE1 inhibition-related miRNAs, more preferably 10 or more types, particularly preferably 20 or more types, and even more particularly preferably 30 or more types.

[0042] The microparticles preferably contain any one of hsa-miR-101-3p, hsa-miR-16-5p, and hsa-miR-29a-3p from the BACE1 inhibition-related miRNA group, and it is particularly preferable that they contain at least hsa-miR-16-5p. The microparticles preferably contain at least one type of BACE1 inhibition-related miRNA, with a Log2Ratio of the read count obtained by analysis using IMOTA of 4.0 or more, more preferably 10.0 or more, and particularly preferably 15.0 or more. The microparticles preferably contain hsa-miR-101-3p, hsa-miR-16-5p, and hsa-miR-29a-3p, with a Log2Ratio of the read count obtained by analysis using IMOTA of 4.0 or more, more preferably 10.0 or more, and particularly preferably 15.0 or more.

[0043] When the gene expression control agent of the present invention is an inhibitor of BACE1 gene expression, it is preferable that the expression of BACE1 gene in any cell can be suppressed to 0.8 times or less of the normal level (in untreated cells), more preferably to 0.75 times or less, and particularly preferably to 0.7 times or less.

[0044] (3) NMDA-activated gene expression inhibitors The gene expression regulator of the present invention is preferably an agent for suppressing the expression of an NMDA-activated gene. In this case, the NMDA-activated protein is preferably at least one of DLG1, CAMK2D, CAMK2A, and CAPN1, and the microparticles preferably contain miRNAs that target genes involved in the expression of at least one of DLG1, CAMK2D, CAMK2A, and CAPN1. In the present invention, it is more preferable that the microparticles contain at least one type of miRNA group selected from the following group of DLG1 suppression-related miRNAs, the following group of CAMK2D suppression-related miRNAs, the following group of CAMK2A suppression-related miRNAs, and the following group of CAPN1 suppression-related miRNAs (51 types in total). DLG1 suppression-related miRNAs: hsa-miR-1-3p, hsa-miR-142-5p, hsa-miR-204-5p, hsa-miR-206, hsa-miR-21-5p, hsa-miR-218-5p, hsa-miR-340-5p, hsa-miR-613. miRNA group related to CAMK2D inhibition: hsa-let-7a-5p, hsa-miR-101-3p, hsa-miR-129-5p, hsa-miR-139-5p, hsa-miR-144-3p, hsa-miR-145-5p, hsa-miR-185-5p, hsa-miR-203a-3p, hsa-miR-204-5p, hsa-miR-211-5p, hsa-miR-24-3p, hsa-miR-27a-3p, hsa-miR-30a-3p, hsa-miR-31-5p, hsa-miR-361-5p, hsa-miR-421, hsa-miR-484, hsa-miR-494-3p, hsa-miR-505-3p, hsa-miR-7-5p. miRNA group related to CAMK2A inhibition: hsa-miR-129-5p, hsa-miR-137, hsa-miR-142-5p, hsa-miR-148a-3p, hsa-miR-149-3p, hsa-miR-152-3p, hsa-miR-25-3p, hsa-miR-27a-3p, hsa-miR-32-5p, hsa-miR-338-3p, hsa-miR-340-5p, hsa-miR-363-3p, hsa-miR-3665, hsa-miR-4534, hsa-miR-4665-5p, hsa-miR-4688, hsa-miR-485-5p, hsa-miR-5010-5p, hsa-miR-505-5p, hsa-miR-5698, hsa-miR-625-5p, hsa-miR-92a-3p. miRNA group related to CAPN1 inhibition: hsa-miR-1-3p, hsa-miR-124-3p, hsa-miR-140-5p, hsa-miR-17-3p, hsa-miR-22-3p, hsa-miR-34a-5p, hsa-miR-6511b-5p.

[0045] It is preferable that the microparticles contain five or more types of miRNAs selected from the group of miRNAs associated with DLG1 inhibition, the group of miRNAs associated with CAMK2D inhibition, the group of miRNAs associated with CAMK2A inhibition, and the group of miRNAs associated with CAPN1 inhibition, more preferably ten or more types, particularly preferably twenty or more types, and even more particularly preferably thirty or more types.

[0046] The tiny particles Among the DLG1 suppression-related miRNAs, hsa-miR-21-5p, Among the CAMK2D suppression-related miRNAs, hsa-let-7a-5p, Among the miRNAs related to CAMK2A suppression, hsa-miR-92a-3p, It is particularly preferable that the miRNAs contain hsa-miR-22-3p among the group of miRNAs associated with CAPN1 repression. The microparticles preferably contain at least one of the DLG1 suppression-related miRNA group, the CAMK2D suppression-related miRNA group, the CAMK2A suppression-related miRNA group, and the CAPN1 suppression-related miRNA group, with a Log2Ratio of the read count obtained by analysis using IMOTA of 4.0 or more, more preferably 10.0 or more, and particularly preferably 15.0 or more. It is preferable that the microparticles contain hsa-miR-21-5p, hsa-let-7a-5p, hsa-miR-92a-3p, and hsa-miR-22-3p in a Log2Ratio of 4.0 or more of the read count obtained by analysis using IMOTA, more preferably 10.0 or more of hsa-miR-21-5p, hsa-let-7a-5p, hsa-miR-92a-3p, and hsa-miR-22-3p, and particularly preferably 15.0 or more of hsa-miR-21-5p, hsa-let-7a-5p, and hsa-miR-92a-3p.

[0047] The gene expression regulator of the present invention preferably has an expression level of hsa-miR-21-5p that is 1.1 times or more, more preferably 1.5 times or more, particularly preferably 2 times or more, and even particularly preferably 5 times or more, compared to exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. The gene expression regulator of the present invention preferably expresses hsa-let-7a-5p at a level 1.1-fold or more, and more preferably at a level 1.5-fold or more, compared to exosomes obtained from the culture supernatant of adipose-derived stem cells.Furthermore, the gene expression regulator of the present invention preferably expresses hsa-let-7a-5p at a level greater than 1.0-fold, and more preferably at a level 1.05-fold or more, compared to exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. The gene expression regulator of the present invention preferably has an expression level of hsa-miR-92a-3p that is 1.1 times or more, more preferably 1.5 times or more, particularly preferably 2 times or more, and even particularly preferably 4 times or more, compared to exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells. The gene expression regulator of the present invention preferably has an expression level of hsa-miR-22-3p that is 1.1 times or more, more preferably 1.5 times or more, and particularly preferably 2 times or more, compared to exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells.

[0048] When the gene expression regulator of the present invention is an inhibitor of the expression of an NMDA-activated gene (preferably the CAMK2D gene), it is preferable that the expression of the NMDA-activated gene in any cell can be suppressed to 0.8 times or less of the normal level (in untreated cells), more preferably to 0.5 times or less, and particularly preferably to 0.3 times or less.

[0049] (4) GSK-3β inhibitors The gene expression regulator of the present invention is preferably an inhibitor of GSK-3β, in which case the microparticles preferably contain miRNA that targets a gene involved in the expression of GSK-3β. It is more preferable that the microparticles contain at least one type of miRNA from the group of GSK-3β inhibition-related miRNAs (54 types) described below. GSK-3β inhibition-related miRNAs: hsa-let-7a-3p, hsa-let-7b-3p, hsa-let-7f-1-3p, hsa-let-7f-2-3p, hsa-miR-101-3p, hsa-miR-1185-1-3p, hsa-miR-1185-2-3p, hsa-miR-124-3p, hsa-miR-128-3p, hsa-miR-129-5p, hsa-miR-132-3p, hsa-miR-137, hsa-miR-140-5p, hsa-miR-142-5p, hsa-miR-144-3p, hsa-miR-150-5p, hsa-miR-155-5p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-1910-5p, hsa-miR-195-5p, hsa-miR-199a-5p, hsa-miR-212-3p, hsa-miR-218-5p, hsa-miR-219a-5p, hsa-miR-23a-3p, hsa-miR-24-3p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-28-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-346, hsa-miR-369-3p, hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-374c-5p, hsa-miR-377-3p, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410-3p, hsa-miR-424-5p, hsa-miR-425-5p, hsa-miR-4465, hsa-miR-497-5p, hsa-miR-582-5p, hsa-miR-6083, hsa-miR-708-5p, hsa-miR-9-5p, hsa-miR-92a-3p, hsa-miR-96-5p, hsa-miR-98-3p.

[0050] The microparticles preferably contain 5 or more types of GSK-3β inhibition-related miRNAs, more preferably 10 or more types, particularly preferably 20 or more types, and even more particularly preferably 30 or more types.

[0051] The microparticles preferably contain one of hsa-miR-16-5p and hsa-miR-29a-3p from the group of miRNAs associated with GSK-3β inhibition, and it is particularly preferable that they contain at least hsa-miR-16-5p. The microparticles preferably contain at least one type of GSK-3β inhibition-related miRNA, with a Log2Ratio of the read count obtained by analysis using IMOTA of 4.0 or more, more preferably 10.0 or more, and particularly preferably 15.0 or more. The microparticles preferably contain hsa-miR-16-5p and hsa-miR-29a-3p, with a Log2Ratio of the read count obtained by analysis using IMOTA of 4.0 or more, more preferably 10.0 or more, and particularly preferably 15.0 or more.

[0052] When the gene expression regulator of the present invention is an inhibitor of the GSK-3β gene, it is preferable that the expression of the GSK-3β gene in any cell can be suppressed to 0.8 times or less of the normal level (in untreated cells), more preferably to 0.75 times or less, and particularly preferably to 0.7 times or less.

[0053] (5) PQBP1 expression inhibitor The gene expression regulator of the present invention is preferably an inhibitor of PQBP1 expression. In this case, it is more preferable that the microparticles contain miRNA that targets a gene involved in PQBP1 expression. As shown in Nature Communications volume 12, Article number: 6565 (2021), miRNA that inhibits PQBP1, which is involved in tau activation, may be useful in the treatment of tau diseases (Alzheimer's disease and brain inflammation).

[0054] It is particularly preferred that the microparticles contain at least one type of miRNAs associated with PQBP1 repression described below, and it is even more preferred that they contain both types. PQBP1 suppression-related miRNAs: hsa-miR-6727-3p, hsa-miR-6727-5p. It is more preferable that the microparticles contain at least hsa-miR-6727-3p from the group of miRNAs associated with PQBP1 repression, and it is particularly preferable that the microparticles contain both hsa-miR-6727-3p and hsa-miR-6727-5p. The microparticles preferably contain at least one type of miRNA from the PQBP1 repression-related miRNA group, with a Log2Ratio of the read count obtained by analysis using IMOTA of 1.0 or more, more preferably 2.0 or more, and particularly preferably 3.0 or more. The microparticles preferably contain hsa-miR-6727-3p and hsa-miR-6727-5p, with a Log2Ratio of the read count obtained by analysis using IMOTA of 2.0 or more, more preferably 3.0 or more, and particularly preferably 4.0 or more.

[0055] The gene expression regulator of the present invention preferably has an expression level of hsa-miR-6727-3p that is 1.1 times or more, more preferably 1.5 times or more, and particularly preferably 2 times or more, compared to exosomes obtained from the culture supernatant of adipose-derived stem cells or exosomes obtained from the culture supernatant of umbilical cord-derived stem cells.

[0056] When the gene expression control agent of the present invention is an inhibitor of the expression of the PQBP1 gene, it is preferable that the expression of the PQBP1 gene in any cell can be suppressed to 0.8 times or less of the normal level (in untreated cells), more preferably to 0.6 times or less, and particularly preferably to 0.55 times or less.

[0057] (Type of miRNA) Here, microparticles derived from the culture supernatant of dental pulp-derived stem cells contain approximately 2,600 types of small RNA. Of these, approximately 1,800 types are miRNA. Of these miRNAs, 180 to 200 types are abundant. The miRNAs abundant in microparticles derived from dental pulp-derived stem cells are characterized by the fact that they contain many microRNAs related to the treatment of cranial nerve diseases and eye diseases, a finding that was previously unknown and newly discovered by the present inventors. This characteristic is significantly different from the types of miRNAs abundant in other microparticles of mesenchymal stem cells. For example, the miRNAs abundant in microparticles of adipose-derived stem cells and microparticles of umbilical cord-derived stem cells contain almost no microRNAs related to the treatment of cranial nerve diseases and eye diseases.

[0058] The microparticles derived from the culture supernatant of dental pulp-derived stem cells preferably contain five or more types of microRNAs (hereinafter also referred to as Alzheimer's-related microRNAs) that can control the expression of proteins associated with amyloid beta-related or tau protein-related dementia such as Alzheimer's disease and brain inflammation, more preferably ten or more types, particularly preferably twenty or more types, even more particularly preferably thirty or more types, and most preferably 100 or more types.

[0059] (Types of microparticles) The microparticles are preferably at least one type selected from the group consisting of exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, and exovesicles, or microvesicles, and are more preferably exosomes. The diameter of the microparticles is preferably 10 to 1000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm. Furthermore, it is desirable that the surface of the microparticles contains tetraspanin molecules such as CD9, CD63, and CD81, and this may be CD9 alone, CD63 alone, or CD81 alone, or any combination of two or three of these. Hereinafter, a preferred embodiment in which exosomes are used as microparticles will be described, but the microparticles used in the present invention are not limited to exosomes.

[0060] Preferably, exosomes are extracellular vesicles that are released from cells upon fusion of multivesicular bodies with the plasma membrane. The surface of the exosome preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells. The exosomes preferably contain intracellular substances of dental pulp-derived stem cells, such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins. Exosomes are known to be used for cell-to-cell communication by transporting genetic information from one cell to another, and they are easily traceable and can be targeted to specific regions.

[0061] (Fine particle content) There is no particular limitation on the amount of microparticles contained in the gene expression regulator of the present invention. 8 It is preferable to contain more than 1.0 × 10 8It is more preferable to include 2.0 × 10 8 It is particularly preferable that the number of atoms contained is 2.5 × 10 or more. 8 It is more particularly preferred that the number of atoms contained is 1.0 × 10 or more. 9 It is even more particularly preferred that the number of the hydroxyl groups contained is 1 or more. Furthermore, there is no particular limitation on the concentration of microparticles contained in the gene expression regulator of the present invention. 8 It is preferable to contain more than 2.0 × 10 8 It is more preferable to have more than 4.0 × 10 8 It is particularly preferable that the content is 5.0 × 10 8 It is more particularly preferable that the content is 2.0 × 10 9 It is even more particularly preferred that the concentration is 1 / mL or more. A preferred embodiment of the gene expression regulator of the present invention contains such a large amount or high concentration of microparticles, thereby maintaining high levels of miRNA and the like, and controlling (particularly suppressing) the expression of proteins associated with amyloid beta-related or tau protein-related dementia and brain inflammation.

[0062] <Other ingredients> In addition to the microparticles, the gene expression regulator of the present invention may contain other components depending on the type of animal to which it is administered and the purpose of administration, as long as the effects of the present invention are not impaired. Examples of other components include nutritional components, antibiotics, cytokines, protective agents, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, soothing agents, stabilizers, preservatives, antiseptics, etc. Examples of nutritional components include fatty acids and vitamins. Examples of antibiotics include penicillin, streptomycin, and gentamicin. Carriers include materials known as pharmaceutically acceptable carriers. The gene expression regulator of the present invention may be the dental pulp-derived stem cell culture supernatant itself or the microparticles themselves, or may be a pharmaceutical composition further containing a pharmaceutically acceptable carrier, excipient, etc. The purpose of the pharmaceutical composition is to facilitate the administration of the dental pulp-derived stem cell culture supernatant or the microparticles to a recipient.

[0063] The pharmaceutically acceptable carrier is preferably a carrier (including a diluent) that does not cause significant irritation to the subject to be administered and does not suppress the biological activity and properties of the administered compound. Examples of the carrier include propylene glycol; (physiological) saline; emulsion; buffer solution; culture medium, such as DMEM or RPMI; and cryopreservation medium containing components that scavenge free radicals.

[0064] The gene expression regulator of the present invention may contain an active ingredient of a conventionally known gene expression regulator, and those skilled in the art can appropriately modify it depending on the intended use, the subject of administration, etc.

[0065] On the other hand, it is preferable that the gene expression regulator of the present invention does not contain any specific substances. For example, it is preferable that the gene expression regulator of the present invention does not contain dental pulp-derived stem cells. Furthermore, the gene expression regulator of the present invention preferably does not contain MCP-1. However, it may contain cytokines other than MCP-1. Examples of other cytokines include those described in

[0014] to

[0020] of JP 2018-023343 A. Furthermore, the gene expression regulator of the present invention preferably does not contain Siglec 9. However, it may contain other sialic acid-binding immunoglobulin-like lectins other than Siglec 9. Preferably, the gene expression regulator of the present invention is substantially free of serum (fetal bovine serum, human serum, sheep serum, etc.) and is also substantially free of conventional serum substitutes such as knockout serum replacement (KSR). The gene expression regulator of the present invention preferably contains the above-mentioned other components in an amount (solid content) of 1% by mass or less, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

[0066] <Method of manufacturing a gene expression regulator> There are no particular limitations on the method for producing the gene expression regulator of the present invention. The expression regulator of the gene of the present invention may be prepared by preparing a culture supernatant of dental pulp-derived stem cells or the like, followed by purifying microparticles from the culture supernatant of the dental pulp-derived stem cells. Alternatively, the expression regulator of the gene of the present invention may be prepared by purifying microparticles from the culture supernatant of commercially purchased dental pulp-derived stem cells. Furthermore, the expression regulator of the gene of the present invention may be prepared by obtaining a composition containing the culture supernatant of dental pulp-derived stem cells that had been discarded (or by appropriately purifying the composition), purifying microparticles from it.

[0067] (Method for preparing culture supernatant of dental pulp-derived stem cells, etc.) The culture supernatant of dental pulp-derived stem cells and the like is not particularly limited. The culture supernatant of dental pulp-derived stem cells, etc. is preferably substantially free of serum. For example, the serum content of the culture supernatant of dental pulp-derived stem cells, etc. is preferably 1% by mass or less, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

[0068] Dental pulp-derived stem cells may be derived from humans or non-human animals, including the same animals (species) as those to which the gene expression regulator of the present invention is administered, as described below, and preferably mammals.

[0069] There are no particular limitations on the dental pulp-derived stem cells used in the culture supernatant. Stem cells from exfoliated deciduous teeth, stem cells from deciduous teeth obtained by other methods, and stem cells from permanent teeth (DPSCs) can be used. In addition to human deciduous tooth pulp stem cells and human permanent tooth pulp stem cells, stem cells derived from dental pulp of animals other than humans, such as porcine deciduous tooth pulp stem cells, can also be used. In addition to exosomes, dental pulp-derived stem cells can produce various cytokines, such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-beta (TGF-β)-1 and -3, TGF-α, KGF, HBEGF, SPARC, other growth factors, and chemokines, as well as many other physiologically active substances. In the present invention, it is particularly preferred that the dental pulp-derived stem cells used in the culture supernatant of dental pulp-derived stem cells are stem cells derived from dental pulp that contain many proteins, and it is preferred to use deciduous dental pulp stem cells. That is, in the present invention, it is preferred to use the culture supernatant of deciduous dental pulp stem cells.

[0070] The dental pulp-derived stem cells used in the present invention may be natural or genetically modified, as long as they can achieve the intended treatment. In particular, the present invention can use immortalized stem cells derived from dental pulp. By using immortalized stem cells that can proliferate virtually indefinitely, the amount and composition of biological factors contained in the stem cell culture supernatant can be stabilized over a long period of time. There are no particular limitations on the immortalized stem cells derived from dental pulp. The immortalized stem cells are preferably non-cancerous immortalized stem cells. Immortalized stem cells derived from dental pulp can be prepared by adding the following low molecular weight compounds (inhibitors) alone or in combination to dental pulp-derived stem cells and culturing them. The TGFβ receptor inhibitor is not particularly limited as long as it has an effect of inhibiting the function of transforming growth factor (TGF) β receptor, and examples thereof include 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyridine, and the like. Examples of suitable pyrazoles include 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridin-2-yl)-4-(4-quinonyl)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (all manufactured by Merck), and SB431542 (Sigma-Aldrich). A-83-01 is preferred. The ROCK inhibitor is not particularly limited as long as it has the effect of inhibiting the function of Rho-associated kinase. Examples of ROCK inhibitors include GSK269962A (Axonmedchem), Fasudil hydrochloride (Tocris Bioscience), Y-27632, and H-1152 (all Fujifilm Wako Pure Chemical Industries, Ltd.). Y-27632 is preferred. The GSK3 inhibitor is not particularly limited as long as it inhibits GSK-3 (Glycogen synthase kinase 3), and examples include A 1070722, BIO, and BIO-acetoxime (all manufactured by TOCRIS). MEK inhibitors are not particularly limited as long as they have the effect of inhibiting the function of MEK (MAP kinase-ERK kinase), and examples include AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86-9766), SL327, U0126-EtOH (all from Selleck), PD98059, U0124, U0125 (all from Cosmo Bio Co., Ltd.), etc.

[0071] When the gene expression regulator of the present invention is used in regenerative medicine, in accordance with the requirements of the Act on Safety of Regenerative Medicine, etc., the culture supernatant of dental pulp-derived stem cells or these immortalized stem cells, or the composition containing microparticles derived therefrom, does not contain somatic stem cells other than dental pulp-derived stem cells, etc. The gene expression regulator of the present invention may contain mesenchymal stem cells or other somatic stem cells other than dental pulp-derived stem cells, etc., but preferably does not contain them. Examples of somatic stem cells other than mesenchymal stem cells include, but are not limited to, stem cells derived from the dermal system, digestive system, bone marrow system, nervous system, etc. Examples of somatic stem cells from the dermal system include epithelial stem cells, hair follicle stem cells, etc. Examples of somatic stem cells from the digestive system include pancreatic (general) stem cells, hepatic stem cells, etc. Examples of somatic stem cells from the bone marrow system (other than mesenchymal stem cells) include hematopoietic stem cells, etc. Examples of somatic stem cells from the nervous system include neural stem cells, retinal stem cells, etc. The gene expression regulator of the present invention may contain, but preferably does not contain, stem cells other than somatic stem cells, including embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).

[0072] There are no particular limitations on the method for preparing the culture supernatant of dental pulp-derived stem cells or immortalized stem cells, and conventional methods can be used. The culture supernatant of dental pulp-derived stem cells, etc., is a culture medium obtained by culturing dental pulp-derived stem cells. For example, a culture supernatant usable in the present invention can be obtained by separating and removing cellular components after culturing dental pulp-derived stem cells. Culture supernatants that have been appropriately subjected to various treatments (e.g., centrifugation, concentration, solvent substitution, dialysis, freezing, drying, lyophilization, dilution, desalting, storage, etc.) may also be used.

[0073] Dental pulp-derived stem cells for obtaining the conditioned medium can be selected by conventional methods based on cell size or morphology, or as adhesive cells. Adhesive cells or their subcultured cells can be selected from dental pulp cells collected from shed deciduous or permanent teeth. The conditioned medium for dental pulp-derived stem cells can be obtained by culturing selected stem cells.

[0074] It is preferable that the "culture supernatant of dental pulp-derived stem cells, etc." is a culture medium that does not contain the cells themselves obtained by culturing dental pulp-derived stem cells, etc. In one embodiment, the culture supernatant of dental pulp-derived stem cells used in the present invention preferably does not contain cells (regardless of cell type) as a whole. This characteristic clearly distinguishes the composition of this embodiment from various compositions that contain dental pulp-derived stem cells, as well as dental pulp-derived stem cells themselves. A typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and is composed only of the culture supernatant of dental pulp-derived stem cells. The dental pulp-derived stem cell culture supernatant used in the present invention may contain the culture supernatant of both deciduous dental pulp-derived stem cells and adult dental pulp-derived stem cells. The dental pulp-derived stem cell culture supernatant used in the present invention preferably contains the deciduous dental pulp-derived stem cell culture supernatant as an active ingredient, more preferably 50% by mass or more, and preferably 90% by mass or more. It is particularly preferable that the dental pulp-derived stem cell culture supernatant used in the present invention is a composition composed solely of the deciduous dental pulp-derived stem cell culture supernatant.

[0075] The culture medium for dental pulp-derived stem cells to obtain the culture supernatant can be a basal medium or a basal medium supplemented with serum, etc. Examples of basal media that can be used include Dulbecco's Modified Eagle's Medium (DMEM), Iscove's Modified Dulbecco's Medium (IMDM) (GIBCO, etc.), Ham's F12 Medium (HamF12) (Sigma, GIBCO, etc.), and RPMI 1640 medium. Examples of ingredients that can be added to the medium include serum (fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (knockout serum replacement (KSR), etc.), bovine serum albumin (BSA), antibiotics, various vitamins, and various minerals. However, to prepare serum-free "dental pulp-derived stem cell culture supernatant," it is recommended to use serum-free medium throughout the entire process or for the final or penultimate few subcultures. For example, serum-free dental pulp-derived stem cell culture supernatant can be prepared by culturing dental pulp-derived stem cells in serum-free medium. Serum-free dental pulp-derived stem cell culture supernatant can also be obtained by performing one or more subcultures and culturing the final or penultimate few subcultures in serum-free medium. Alternatively, serum-free dental pulp-derived stem cell culture supernatant can also be obtained by removing serum from the collected culture supernatant using dialysis, solvent replacement using a column, or other methods.

[0076] The conditions commonly used for culturing dental pulp-derived stem cells to obtain a culture supernatant can be applied as is. The method for preparing the culture supernatant of dental pulp-derived stem cells may be the same as the cell culture method described below, except that the steps of isolating and selecting stem cells are appropriately adjusted depending on the type of stem cells. Those skilled in the art can appropriately isolate and select dental pulp-derived stem cells depending on the type of stem cells. In addition, special conditions may be applied to the culture of dental pulp-derived stem cells to produce large amounts of microparticles such as exosomes, such as low temperature, low oxygen, and microgravity conditions, or co-culture with some kind of stimuli.

[0077] The culture supernatant of dental pulp-derived stem cells used in the present invention for preparing microparticles such as exosomes may contain other components in addition to the culture supernatant of dental pulp-derived stem cells, but it is preferable that it is substantially free of other components. However, each type of additive used in preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells and then stored.

[0078] (Method for preparing microparticles) The microparticles can be prepared by purifying them from the culture supernatant of dental pulp-derived stem cells or the like.

[0079] Purification of microparticles is preferably separation of a fraction containing microparticles from the culture supernatant of dental pulp-derived stem cells, and more preferably isolation of microparticles. Microparticles can be isolated by separating them from non-associated components based on a property of the microparticle, for example, they can be isolated based on molecular weight, size, morphology, composition, or biological activity. In the present invention, microparticles can be purified by separating a specific fraction (e.g., precipitate) containing a large amount of microparticles obtained by centrifuging the culture supernatant of dental pulp-derived stem cells. Unnecessary components (insoluble components) from fractions other than the specified fraction may be removed. The solvent, dispersion medium, and unnecessary components do not necessarily need to be completely removed from the gene expression regulator of the present invention. Centrifugation conditions include 100 to 20,000 g for 1 to 30 minutes. In the present invention, microparticles can be purified by filtering the culture supernatant of dental pulp-derived stem cells or a centrifuged product thereof. Unnecessary components can be removed by filtration. Furthermore, by using a filtration membrane with an appropriate pore size, removal of unnecessary components and sterilization can be performed simultaneously. The material and pore size of the filtration membrane used for filtration are not particularly limited. Filtration can be performed using a filtration membrane with an appropriate molecular weight or size cutoff using a known method. From the viewpoint of facilitating the separation of exosomes, the pore size of the filtration membrane is preferably 10 to 1,000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm. In the present invention, the culture supernatant of dental pulp-derived stem cells, its centrifuged product, or its filtered product can be further separated using a separation method such as column chromatography. For example, high-performance liquid chromatography (HPLC) using various columns can be used. The column can be a size exclusion column or a binding column. One or more properties or biological activities of the microparticles can be used to track the microparticles (or their activity) in each fraction at each processing stage. For example, light scattering, refractive index, dynamic light scattering, or UV-visible light detectors can be used to track the microparticles. Alternatively, specific enzyme activity, etc. can be used to track activity in each fraction. As a method for purifying microparticles, the method described in

[0034] to

[0064] of JP-A No. 2019-524824 may be used, the contents of which are incorporated herein by reference.

[0080] The final form of the gene expression regulator of the present invention is not particularly limited. For example, the gene expression regulator of the present invention may be in the form of microparticles packed in a container together with a solvent or dispersion medium; microparticles gelled with a gel and packed in a container; or microparticles solidified by freezing and / or drying, formulated, or packed in a container. Examples of containers include tubes, centrifuge tubes, bags, and the like suitable for cryopreservation. The freezing temperature can be, for example, -20°C to -196°C.

[0081] Compared to compositions that can be used as conventional gene expression regulators, the gene expression regulator of the present invention has advantages such as ease of mass production, the ability to utilize stem cell culture medium that was previously discarded as industrial waste, and reduced disposal costs for stem cell culture medium. In particular, when the dental pulp-derived stem cell culture supernatant is a culture supernatant from human dental pulp-derived stem cells, the gene expression regulator of the present invention has the advantage of being highly safe from immunological and other standpoints and posing fewer ethical concerns when applied to humans. When the dental pulp-derived stem cell culture supernatant is a culture supernatant from dental pulp-derived stem cells from a patient with dementia, the gene expression regulator of the present invention will be safer and pose fewer ethical concerns when applied to that patient. When the gene expression regulator of the present invention is derived from the conditioned medium of dental pulp-derived stem cells, it can also be used in restorative medicine. In particular, compositions containing microparticles derived from the conditioned medium of dental pulp-derived stem cells are preferred for restorative medicine. It is known that in regenerative medicine based on stem cell transplantation, stem cells are not the main players in regeneration, but rather the liquid components produced by stem cells, together with the patient's own stem cells, repair organs. This solves the difficult issues associated with conventional stem cell transplantation, such as carcinogenesis, standardization, administration method, storage, and culture method, and makes restorative medicine possible using compositions containing conditioned medium of dental pulp-derived stem cells or microparticles derived therefrom. Compared to stem cell transplantation, the use of the gene expression regulator of the present invention is safer, as it is less likely to cause tumorigenesis because cells are not transplanted. Furthermore, the gene expression regulator of the present invention has the advantage of being of consistently standardized quality. It can be mass-produced and efficiently administered, allowing for low-cost use.

[0082] [How to improve dementia] The method of the present invention for ameliorating dementia comprises administering an effective amount of the gene expression regulator of the present invention to a subject who has developed dementia.

[0083] There are no particular limitations on the step of administering the gene expression regulator of the present invention to a subject who has developed dementia. Examples of administration methods include spraying or inhalation into the oral cavity, nasal cavity, or respiratory tract, infusion, topical administration, and nasal drops, with minimal invasive methods being preferred. A preferred method of local administration is injection. Electroporation is also preferred, which applies a voltage (electric pulse) to the skin surface to temporarily create minute holes in the cell membrane, allowing the active ingredient to penetrate into the dermis layer, which is difficult to reach with conventional care. Examples of local administration include intravenous administration, intraarterial administration, intraportal vein administration, intradermal administration, subcutaneous administration, intramuscular administration, and intraperitoneal administration, with intraarterial administration, intravenous administration, subcutaneous administration, and intraperitoneal administration being more preferred. In addition, various formulation techniques can be used to alter the in vivo distribution of gene expression regulators. Many methods for altering in vivo distribution are known to those skilled in the art. Examples of such methods include, for example, protecting exosomes in vesicles composed of substances such as proteins, lipids (e.g., liposomes), carbohydrates, or synthetic polymers. The gene expression regulator of the present invention administered to a subject with dementia may circulate within the subject's body and reach a specific tissue. There are no particular limitations on the number of doses and the interval between doses. The number of doses can be at least once a week, preferably at least five times, more preferably at least six times, and particularly preferably at least seven times. The interval between doses is preferably one hour to one week, more preferably half a day to one week, and particularly preferably one day (once a day). However, this can be adjusted appropriately depending on the target organism species and symptoms of the target. The gene expression regulator of the present invention is preferably used to administer the gene expression regulator to a subject who has developed dementia at least once a week over the effective therapeutic period. When the subject is a human, the more frequent the administration per week, the more preferable, and administration is preferably at least five times a week over the effective therapeutic period, with daily administration being preferred. 2.0×10 9 When using a culture supernatant of dental pulp-derived stem cells at a concentration of 1000 cells / ml, in a mouse model, the amount is preferably 0.1 to 5 ml per mouse (approximately 25 g), more preferably 0.3 to 3 ml, and even more preferably 0.5 to 1 ml. 0.1×10 8 When microparticles at a concentration of particles / μg are used, in a mouse model, the amount is preferably 1 to 50 μg per mouse (approximately 25 g), more preferably 3 to 30 μg, and even more preferably 5 to 25 μg. The preferred range of the dose per body weight for other animals can be calculated proportionally from the dose per body weight (approximately 25 g) for the model mouse, but can be adjusted appropriately depending on the symptoms of the subject.

[0084] There are no particular limitations on the animal (biological species) to which the gene expression regulator of the present invention is administered. The target animal to which the gene expression regulator of the present invention is administered is preferably a mammal, bird (chicken, quail, duck, etc.), or fish (salmon, trout, tuna, bonito, etc.). The mammal may be either a human or a non-human mammal, with humans being particularly preferred. More preferred non-human mammals are cows, pigs, horses, goats, sheep, monkeys, dogs, cats, mice, rats, guinea pigs, and hamsters.

[0085] The gene expression regulator of the present invention may be used in combination with a conventionally known gene expression regulator, and may also be used in combination with a conventionally known therapeutic or preventive agent for amyloid-β- or tau protein-related dementia or brain inflammation. [Example]

[0086] The features of the present invention will be explained in more detail below with reference to examples, comparative examples, and reference examples. The materials, amounts used, ratios, treatment contents, treatment procedures, etc. shown in the following examples can be changed as appropriate without departing from the spirit of the present invention. Therefore, the scope of the present invention should not be construed as being limited by the specific examples shown below.

[0087] [Example 1] <Preparation of culture supernatant of dental pulp-derived stem cells> Culture supernatant of human deciduous dental pulp stem cells was prepared and collected according to the method described in Example 6 of Patent No. 6296622, except that DMEM medium was used instead of the DMEM / HamF12 mixed medium. Primary culture was performed with the addition of fetal bovine serum (FBS), and subculture was performed using the primary culture medium. The supernatant of the subculture medium was then collected so that it did not contain FBS, and the culture supernatant of deciduous dental pulp stem cells was prepared. Note that DMEM is Dulbecco's modified Eagle's medium, and F12 is Ham's F12 medium.

[0088] <Exosome preparation> Exosomes from dental pulp-derived stem cells were purified and collected from the culture supernatant of the resulting dental pulp-derived stem cells using the following ultracentrifugation method. The culture supernatant (100 mL) of primary dental pulp stem cells was filtered through a 0.22-micrometer pore size filter, and the solution was centrifuged at 100,000 × g for 60 minutes at 4°C. The supernatant was decanted, and the exosome-enriched pellet was resuspended in phosphate-buffered saline (PBS). The resuspended sample was centrifuged at 100,000 × g for 60 minutes. The pellet was again collected from the bottom of the centrifuge tube (approximately 100 μl) as the concentrated sample. Protein concentration was determined using a microBSA protein assay kit (Pierce, Rockford, IL). The exosome-containing composition (concentrated solution) was stored at -80°C. A composition containing exosomes purified from the culture supernatant of dental pulp-derived stem cells was used as the gene expression regulator sample of Example 1.

[0089] The average particle size and concentration of the microparticles contained in the gene expression regulator of Example 1 were evaluated. The average particle size of the microparticles contained in the gene expression regulator of Example 1 was 50 to 150 nm. The gene expression regulator of Example 1 was 1.0 × 10 9 It is a highly concentrated exosome solution of 2.0 x 10 9 It was a highly concentrated exosome solution with 100 cells / ml. Furthermore, the components of the gene expression regulator obtained in Example 1 were analyzed by known methods. As a result, it was found that the gene expression regulator in Example 1 does not contain dental pulp-derived stem cells, MCP-1, or Siglec 9. Therefore, it was found that the active ingredient of the gene expression regulator in Example 1 is an active ingredient different from MCP-1 and Siglec 9, which are active ingredients in the mesenchymal stem cell culture supernatant, and their analogs.

[0090] [Comparative Example 1] <Preparation of adipose-derived stem cell culture supernatant> A culture supernatant of adipose-derived stem cells was prepared in the same manner as in Example 1, except that adipose-derived stem cells were used. Except for using the culture supernatant of adipose-derived stem cells, exosomes from adipose-derived stem cells were purified in the same manner as in Example 1. The obtained exosomes from adipose-derived stem cells were used as the gene expression inhibitor of Comparative Example 1.

[0091] Comparative Example 2 <Preparation of umbilical cord-derived stem cell culture supernatant> A culture supernatant of umbilical cord-derived stem cells was prepared in the same manner as in Example 1, except that umbilical cord-derived stem cells were used. Except for using the culture supernatant of umbilical cord-derived stem cells, exosomes from umbilical cord-derived stem cells were purified in the same manner as in Example 1. The obtained exosomes from umbilical cord-derived stem cells were used as the gene expression inhibitor of Comparative Example 2.

[0092] [Test Example 1]: MicroRNA expressed in exosomes The small RNAs contained in the gene expression inhibitor of Example 1 were analyzed by next-generation sequencing (NGS). NGS analysis identified 1,787 miRNAs contained in the gene expression inhibitor of Example 1 (exosomes from dental pulp-derived stem cells). The results are shown in Table 2 below.

[0093] [Table 2]

[0094] [Test Example 2]: Comparison with miRNA in the cerebral cortex MicroRNA analysis was performed using IMOTA (Interactive Multi-Omics-Tissue Atlas). We searched for protein-regulating microRNAs in the cerebral cortex using IMOTA. IMOTA is an interactive multi-omics atlas that allows researchers to investigate the interactions and expression levels of miRNAs, mRNAs, and proteins in various tissues and cells (Nucleic Acids Research, Volume 46, Issue D1, 4 January 2018, Pages D770-D775, "IMOTA: an interactive multi-omics tissue atlas for the analysis of human miRNA-target interactions"). In the cerebral cortex, the Omic counts were 3,071 proteins, 14,182 mRNAs, and 1,124 miRNAs. Furthermore, the Omic counts with a high expression rate in the cerebral cortex were 1,119 proteins, 1,110 mRNAs, and 145 miRNAs.

[0095] Of the 1,124 miRNAs expressed in the cerebral cortex and the 1,787 miRNAs expressed in exosomes purified from the culture supernatant of dental pulp-derived stem cells (the gene expression inhibitor of Example 1; SGF exosome), 859 miRNAs were expressed in common. In other words, of the miRNAs expressed in the gene expression inhibitor of Example 1 (exosomes from dental pulp-derived stem cells), the proportion of miRNAs also expressed in the cerebral cortex was 48.1%, an extremely high figure.

[0096] Furthermore, the 122 miRNAs listed in Tables 3 and 4 below were expressed by the gene expression inhibitor of Example 1 (exosomes from dental pulp-derived stem cells) and had high expression in the cerebral cortex. [Table 3] [Table 4]

[0097] [Test Example 3]: Search for disease-related microRNAs Based on the analysis results obtained, we searched for microRNAs related to disease. Disease-related miRNAs were extracted using IMOTA. Here, we searched for microRNAs that suppress proteins related to Alzheimer's disease or that regulate proteins that are targets of therapeutic drugs. In this Test Example 3, we searched for microRNAs that suppress β-secretase (BACE1) and APP, which are proteins related to Alzheimer's disease. We searched for microRNAs that suppress NMDA, inhibit GSK-3β, or suppress PQBP1, which are proteins that are targets of therapeutic drugs. The results obtained are shown in FIGS. 5 to 13-2, comparing the expression levels of each miRNA in the cerebral cortex and the gene expression regulator of Example 1 (SGF).

[0098] The miRNAs expressed in exosomes purified from the culture supernatant of dental pulp-derived stem cells that target APP were the following APP-suppressing miRNA group (63 types). hsa-miR-101-3p, hsa-miR-106b-5p, hsa-miR-1229-5p, hsa-miR-1238-3p, hsa-miR-1260b, hsa-miR-1276, hsa-miR-128-3p, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-144-3p, hsa-miR-151a-3p, hsa-miR-153-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-185-5p, hsa-miR-186-5p, hsa-miR-194-5p, hsa-miR-195-5p, hsa-miR-196a-5p, hsa-miR-203a-3p, hsa-miR-20a-5p, hsa-miR-222-3p, hsa-miR-298, hsa-miR-31-5p, hsa-miR-3120-3p, hsa-miR-3132, hsa-miR-323a-3p, hsa-miR-324-5p, hsa-miR-328-3p, hsa-miR-3620-3p, hsa-miR-3646, hsa-miR-369-3p, hsa-miR-373-3p, hsa-miR-374c-5p, hsa-miR-381-3p, hsa-miR-382-5p, hsa-miR-383-5p, hsa-miR-411-3p, hsa-miR-423-3p, hsa-miR-424-3p, hsa-miR-424-5p, hsa-miR-4484, hsa-miR-455-3p, hsa-miR-4786-5p, hsa-miR-484, hsa-miR-490-5p, hsa-miR-497-5p, hsa-miR-500a-5p, hsa-miR-5093, hsa-miR-532-3p, hsa-miR-532-5p, hsa-miR-539-3p, hsa-miR-548f-3p, hsa-miR-551b-5p, hsa-miR-5584-5p, hsa-miR-567, hsa-miR-5696, hsa-miR-582-5p, hsa-miR-6073, hsa-miR-873-5p, hsa-miR-93-5p.

[0099] The miRNAs expressed in exosomes purified from the culture supernatant of dental pulp-derived stem cells that target β-secretase (BASE1) were the following BACE1 inhibition-related miRNA group (41 types). hsa-miR-107, hsa-miR-124-3p, hsa-miR-1267, hsa-miR-128-3p, hsa-miR-129-2-3p, hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141-3p, hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-195-5p, hsa-miR-200a-3p, hsa-miR-203a-3p, hsa-miR-212-3p, hsa-miR-212-5p, hsa-miR-24-3p, hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-298, hsa-miR-299-3p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-328-3p, hsa-miR-339-5p, hsa-miR-340-5p, hsa-miR-369-3p, hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-374c-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-424-5p, hsa-miR-455-3p, hsa-miR-497-5p, hsa-miR-505-3p, hsa-miR-532-5p, hsa-miR-7-5p, hsa-miR-874-3p, hsa-miR-9-5p.

[0100] The miRNAs expressed in exosomes purified from the culture supernatant of dental pulp-derived stem cells that target proteins involved in NMDA activation were the following DLG1 suppression-related miRNA group, the following CAMK2D suppression-related miRNA group, the following CAMK2A suppression-related miRNA group, and the following CAPN1 suppression-related miRNA group (51 types in total). DLG1 suppression-related miRNAs: hsa-miR-1-3p, hsa-miR-142-5p, hsa-miR-204-5p, hsa-miR-206, hsa-miR-21-5p, hsa-miR-218-5p, hsa-miR-340-5p, hsa-miR-613. CAMK2D suppression-related miRNAs: hsa-let-7a-5p, hsa-miR-101-3p, hsa-miR-129-5p, hsa-miR-139-5p, hsa-miR-144-3p, hsa-miR-145-5p, hsa-miR-185-5p, hsa-miR-203a-3p, hsa-miR-204-5p, hsa-miR-211-5p, hsa-miR-24-3p, hsa-miR-27a-3p, hsa-miR-30a-3p, hsa-miR-31-5p, hsa-miR-361-5p, hsa-miR-421, hsa-miR-484, hsa-miR-494-3p, hsa-miR-505-3p, hsa-miR-7-5p. CAMK2A inhibition-related miRNA group: hsa-miR-129-5p, hsa-miR-137, hsa-miR-142-5p, hsa-miR-148a-3p, hsa-miR-149-3p, hsa-miR-152-3p, hsa-miR-25-3p, hsa-miR-27a-3p, hsa-miR-32-5p, hsa-miR-338-3p, hsa-miR-340-5p, hsa-miR-363-3p, hsa-miR-3665, hsa-miR-4534, hsa-miR-4665-5p, <​​​​​​​​​​​​​​The miRNAs expressed in exosomes purified from the culture supernatant of dental pulp-derived stem cells that target GSK-3β were the following GSK-3β inhibition-related miRNA group (54 types). hsa-let-7a-3p, hsa-let-7b-3p, hsa-let-7f-1-3p, hsa-let-7f-2-3p, hsa-miR-101-3p, hsa-miR-1185-1-3p, hsa-miR-1185-2-3p, hsa-miR-124-3p, hsa-miR-128-3p, hsa-miR-129-5p, hsa-miR-132-3p, hsa-miR-137, hsa-miR-140-5p, hsa-miR-142-5p, hsa-miR-144-3p, hsa-miR-150-5p, hsa-miR-155-5p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-1910-5p, hsa-miR-195-5p, hsa-miR-199a-5p, hsa-miR-212-3p, hsa-miR-218-5p, hsa-miR-219a-5p, hsa-miR-23a-3p, hsa-miR-24-3p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-28-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-346, hsa-miR-369-3p, hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-374c-5p, hsa-miR-377-3p, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410-3p, hsa-miR-424-5p, hsa-miR-425-5p, hsa-miR-4465, hsa-miR-497-5p, hsa-miR-582-5p, hsa-miR-6083, hsa-miR-708-5p, hsa-miR-9-5p, hsa-miR-92a-3p, hsa-miR-96-5p, hsa-miR-98-3p.

[0102] The miRNAs expressed in exosomes purified from the culture supernatant of dental pulp-derived stem cells that target PQBP1 were the following PQBP1 suppression-related miRNA group (2 types). hsa-miR-6727-3p, hsa-miR-6727-5p.

[0103] There are 153 miRNAs expressed in exosomes from dental pulp-derived stem cells that are expected to be useful in treating Alzheimer's disease, and the results of comparing their expression levels are shown in the heat map in Figure 13. The density in the heat map is shown as the log ratio of the read count values.

[0104] In the above Test Example 3, microRNAs were discovered that suppress beta-secretase (BACE1) or APP, proteins involved in Alzheimer's disease, as well as microRNAs that suppress NMDA, a protein targeted by therapeutic drugs, and that inhibit GSK-3β or suppress PQBP1. In particular, Figures 13 and 13-2 show that of the 859 miRNAs whose expression was confirmed in the cerebral cortex and also confirmed by the gene expression inhibitor of Example 1, 153 miRNAs have the effect of targeting and suppressing genes related to amyloid beta or tau protein (such as the genes that cause Alzheimer's disease).

[0105] [Test Example 4]: Regulation of gene expression Exosomes from dental pulp-derived stem cells obtained in Example 1 or exosomes from adipose-derived stem cells obtained in Comparative Example 1 were added to cerebral cortical neuron cells (neuronal cells derived from Alzheimer's disease patients). 1,000 exosomes were added per cerebral cortical neuron cell. After 72 hours, the cerebral cortical neuron cells were collected, and mRNA was purified. The purified mRNA was used to quantify the expression of target genes for expression analysis that are associated with Alzheimer's disease (expression is increased). On the other hand, cerebral cortical neuron cells to which exosomes had not been added were used as a control (Reference Example 1), and mRNA from the recovered cerebral cortical neuron cells was purified, and the expression of the target genes for expression analysis was quantified using the same method as in Example 1 and Comparative Example 1. The expression levels of the target genes for expression analysis in Example 1 (cells treated with exosomes from dental pulp-derived stem cells) and Comparative Example 1 (cells treated with exosomes from adipose-derived stem cells) were calculated as relative values, with the expression level of the target genes for expression analysis in Reference Example 1 (control; untreated cells) set at 1.0. The target genes for expression analysis were five types: amyloid precursor protein (APP) gene, β-secretase (BACE1) gene, NMDA-activating protein (CAMK2D) gene, glycogen synthase kinase-3β (GSK-3β) gene, and polyglutamine-binding protein-1 (PQBP1) gene. The results obtained for the expression levels of each target gene for expression analysis are shown in Figures 14 to 18.

[0106] 14 to 18, it was found that the gene expression regulator of the present invention (Example 1) can regulate the expression of five target genes for expression analysis (genes related to the expression of APP, BACE1, NMDA-activating protein CAMK2D, GSK-3β, and PQBP1) that are related to Alzheimer's dementia (expression of which increases in the control). In particular, it was found that the gene expression regulator of Example 1 can more significantly suppress the expression of five target genes for expression analysis that are related to Alzheimer's dementia compared to the gene expression regulator of Comparative Example 1.

[0107] [Test Example 5]: miRNA types and expression levels MiRNA analysis of the gene expression inhibitor of Example 1 (exosomes from dental pulp-derived stem cells), the gene expression inhibitor of Comparative Example 1 (exosomes derived from adipose tissue stem cells; ADSCs), and the gene expression inhibitor of Comparative Example 2 (exosomes derived from umbilical cord stem cells; Placenta-MSCs) was carried out using qPCR in the same manner as in Test Example 4. Of the obtained analysis results, the expression levels of the following miRNAs, which are target genes for Alzheimer's disease, were compared. hsa-miR-16-5p (APP suppression-related miRNA, BACE1 inhibition-related miRNA, GSK-3β inhibition-related miRNA), hsa-miR-21-5p (DLG1 suppression-related miRNA), hsa-let7-a-5p (CAMK2D suppression-related miRNA) hsa-miR-92a-3p (CAMK2A suppression-related miRNA), hsa-miR-22-3p (CAPN1 suppression-related miRNA). The results are shown in Figure 19. In Figure 19, the expression level of miRNA in the gene expression inhibitor of Example 1 (exosomes from dental pulp-derived stem cells) is shown as a relative value, assuming it is 100.

[0108] Figure 19 shows that the expression level of miRNA (microRNA predicted to have a therapeutic effect) that suppresses a group of genes whose expression is increased in association with Alzheimer's disease in the gene expression inhibitor of Example 1 (exosomes from dental pulp-derived stem cells) was significantly higher than that of exosomes derived from other mesenchymal stem cells (exosomes derived from adipose stem cells in Comparative Example 1 and exosomes derived from umbilical cord stem cells in Comparative Example 2).

Claims

1. A gene expression regulator, which is a microparticle containing miRNA that targets a gene involved in the expression of at least one protein selected from amyloid precursor protein (APP), β-secretase (BACE1), NMDA-activating protein, glycogen synthase kinase-3β (GSK-3β), and polyglutamine-binding protein-1 (PQBP1).

2. The gene expression regulator according to claim 1 , wherein the microparticles are exosomes.

3. The gene expression control agent according to claim 1 or 2, wherein the microparticles contain miRNA that targets a gene involved in the expression of at least one of the proteins APP, BACE1, NMDA-activating protein, GSK-3β, and PQBP1 at a concentration higher than that of the culture supernatant of dental pulp-derived stem cells.

4. an expression inhibitor of a gene involved in the expression of the APP; The gene expression regulator according to any one of claims 1 to 3, wherein the microparticles contain miRNA that targets a gene involved in the expression of the APP.

5. The gene expression regulator according to claim 4, wherein the microparticles contain at least one of the following miRNAs related to APP suppression: APP suppression-related miRNA group: hsa-miR-101-3p, hsa-miR-106b-5p, hsa-miR-1229-5p, hsa-miR-1238-3p, hsa-miR-1260b, hsa-miR-1276, hsa-miR-128-3p, hsa-miR-142-3p, hsa-miR-142-5p, hsa-miR-144-3p, hsa-miR-151a-3p, hsa-miR-153-3p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-185-5p, hsa-miR-186-5p, hsa-miR-194-5p, hsa-miR-195-5p, hsa-miR-196a-5p, hsa-miR-203a-3p, hsa-miR-20a-5p, hsa-miR-222-3p, hsa-miR-298, hsa-miR-31-5p, hsa-miR-3120-3p, hsa-miR-3132, hsa-miR-323a-3p, hsa-miR-324-5p, hsa-miR-328-3p, hsa-miR-3620-3p, hsa-miR-3646, hsa-miR-369-3p, hsa-miR-373-3p, hsa-miR-374c-5p, hsa-miR-381-3p, hsa-miR-382-5p, hsa-miR-383-5p, hsa-miR-411-3p, hsa-miR-423-3p, hsa-miR-424-3p, hsa-miR-424-5p, hsa-miR-4484, hsa-miR-455-3p, hsa-miR-4786-5p, hsa-miR-484, hsa-miR-490-5p, hsa-miR-497-5p, hsa-miR-500a-5p, hsa-miR-5093, hsa-miR-532-3p, hsa-miR-532-5p, hsa-miR-539-3p, hsa-miR-548f-3p, hsa-miR-551b-5p, hsa-miR-5584-5p, hsa-miR-567, hsa-miR-5696, hsa-miR-582-5p, hsa-miR-6073, hsa-miR-873-5p, hsa-miR-93-5p.

6. an inhibitor of a gene involved in the expression of BACE1; The gene expression regulator according to any one of claims 1 to 5, wherein the microparticles contain miRNA that targets a gene involved in the expression of BACE1.

7. The gene expression regulator according to claim 6, wherein the microparticles contain at least one of the following BACE1 inhibition-related miRNAs: BACE1 inhibition-related miRNA group: hsa-miR-107, hsa-miR-124-3p, hsa-miR-1267, hsa-miR-128-3p, hsa-miR-129-2-3p, hsa-miR-140-3p, hsa-miR-140-5p, hsa-miR-141-3p, hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-17-5p, hsa-miR-195-5p, hsa-miR-200a-3p, hsa-miR-203a-3p, hsa-miR-212-3p, hsa-miR-212-5p, hsa-miR-24-3p, hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-298, hsa-miR-299-3p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-29c-3p, hsa-miR-328-3p, hsa-miR-339-5p, hsa-miR-340-5p, hsa-miR-369-3p, hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-374c-5p, hsa-miR-382-5p, hsa-miR-421, hsa-miR-424-5p, hsa-miR-455-3p, hsa-miR-497-5p, hsa-miR-505-3p, hsa-miR-532-5p, hsa-miR-7-5p, hsa-miR-874-3p, hsa-miR-9-5p.

8. an inhibitor of a gene involved in the expression of GSK-3β; The gene expression regulator according to any one of claims 1 to 7, wherein the microparticles contain miRNA that targets a gene involved in the expression of GSK-3β.

9. The gene expression regulator according to claim 8, wherein the microparticles contain at least one of the following GSK-3β inhibition-related miRNAs: GSK-3β inhibition-related miRNA group: hsa-let-7a-3p, hsa-let-7b-3p, hsa-let-7f-1-3p, hsa-let-7f-2-3p, hsa-miR-101-3p, hsa-miR-1185-1-3p, hsa-miR-1185-2-3p, hsa-miR-124-3p, hsa-miR-128-3p, hsa-miR-129-5p, hsa-miR-132-3p, hsa-miR-137, hsa-miR-140-5p, hsa-miR-142-5p, hsa-miR-144-3p, hsa-miR-150-5p, hsa-miR-155-5p, hsa-miR-15a-5p, hsa-miR-15b-5p, hsa-miR-16-5p, hsa-miR-1910-5p, hsa-miR-195-5p, hsa-miR-199a-5p, hsa-miR-212-3p, hsa-miR-218-5p, hsa-miR-219a-5p, hsa-miR-23a-3p, hsa-miR-24-3p, hsa-miR-26a-5p, hsa-miR-26b-5p, hsa-miR-27a-3p, hsa-miR-28-5p, hsa-miR-29a-3p, hsa-miR-29b-3p, hsa-miR-346, hsa-miR-369-3p, hsa-miR-374a-5p, hsa-miR-374b-5p, hsa-miR-374c-5p, hsa-miR-377-3p, hsa-miR-409-3p, hsa-miR-409-5p, hsa-miR-410-3p, hsa-miR-424-5p, hsa-miR-425-5p, hsa-miR-4465, hsa-miR-497-5p, hsa-miR-582-5p, hsa-miR-6083, hsa-miR-708-5p, hsa-miR-9-5p, hsa-miR-92a-3p, hsa-miR-96-5p, hsa-miR-98-3p.

10. The gene expression control agent according to any one of claims 1 to 9, wherein the microparticles are at least one of an expression inhibitor of a gene involved in the expression of the APP, an inhibitor of a gene involved in the expression of the BACE1, and an inhibitor of a gene involved in the expression of the GSK-3β, and contain hsa-miR-16-5p.

11. an expression inhibitor of the NMDA-activating gene, the NMDA-activating protein is at least one protein selected from the group consisting of DLG1, CAMK2D, CAMK2A, and CAPN1; The gene expression regulator according to any one of claims 1 to 10, wherein the microparticles contain at least one miRNA group selected from the group consisting of the following DLG1 suppression-related miRNA group, the following CAMK2D suppression-related miRNA group, the following CAMK2A suppression-related miRNA group, and the following CAPN1 suppression-related miRNA group, which target genes involved in the expression of at least one type of protein selected from the group consisting of DLG1, CAMK2D, CAMK2A, and CAPN1; DLG1 suppression-related miRNA group: hsa-miR-1-3p, hsa-miR-142-5p, hsa-miR-204-5p, hsa-miR-206, hsa-miR-21-5p, hsa-miR-218-5p, hsa-miR-340-5p, hsa-miR-613. CAMK2D suppression-related miRNA group: hsa-let-7a-5p, hsa-miR-101-3p, hsa-miR-129-5p, hsa-miR-139-5p, hsa-miR-144-3p, hsa-miR-145-5p, hsa-miR-185-5p, hsa-miR-203a-3p, hsa-miR-204-5p, hsa-miR-211-5p, hsa-miR-24-3p, hsa-miR-27a-3p, hsa-miR-30a-3p, hsa-miR-31-5p, hsa-miR-361-5p, hsa-miR-421, hsa-miR-484, hsa-miR-494-3p, hsa-miR-505-3p, hsa-miR-7-5p. CAMK2A suppression-related miRNA group: hsa-miR-129-5p, hsa-miR-137, hsa-miR-142-5p, hsa-miR-148a-3p, hsa-miR-149-3p, hsa-miR-152-3p, hsa-miR-25-3p, hsa-miR-27a-3p, hsa-miR-32-5p, hsa-miR-338-3p, hsa-miR-340-5p, hsa-miR-363-3p, hsa-miR-3665, hsa-miR-4534, hsa-miR-4665-5p, hsa-miR-4688, hsa-miR-485-5p, hsa-miR-5010-5p, hsa-miR-505-5p, hsa-miR-5698, hsa-miR-625-5p, hsa-miR-92a-3p. CAPN1 suppression-related miRNA group: hsa-miR-1-3p, hsa-miR-124-3p, hsa-miR-140-5p, hsa-miR-17-3p, hsa-miR-22-3p, hsa-miR-34a-5p, hsa-miR-6511b-5p.

12. The microparticles are Among the DLG1 suppression-related miRNA group, hsa-miR-21-5p, Among the CAMK2D suppression-related miRNA group, hsa-let-7a-5p, Among the CAMK2A suppression-related miRNA group, hsa-miR-92a-3p, The gene expression control agent according to claim 11, which contains hsa-miR-22-3p among the group of miRNAs related to CAPN1 repression.

13. The PQBP1 expression inhibitor, The gene expression regulator according to any one of claims 1 to 12, wherein the microparticles contain miRNA that targets a gene involved in the expression of PQBP1.

14. The gene expression regulator according to claim 13, wherein the microparticles contain at least one of the following miRNAs related to PQBP1 repression: PQBP1 suppression-related miRNA group: hsa-miR-6727-3p, hsa-miR-6727-5p.

15. The microparticles are purified and isolated from a culture supernatant of dental pulp-derived stem cells, The gene expression regulator according to any one of claims 1 to 14, wherein the gene expression regulator does not contain components other than the exosomes from the culture supernatant of the dental pulp-derived stem cells.

16. A preventive or therapeutic agent for Alzheimer's disease, comprising the gene expression regulator according to any one of claims 1 to 15.

17. A method for improving dementia, comprising administering an effective amount of a gene expression regulator according to any one of claims 1 to 15, or an effective amount of a preventive or therapeutic drug for Alzheimer's disease according to claim 16, to a subject who has developed amyloid beta-related or tau protein-related dementia.

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