Composition for promoting klotho expression and invention related thereto

Abstract

To provide a new composition for increasing expression of the Klotho gene and an application thereof.SOLUTION: The present disclosure provides, in one aspect, the following invention: a composition for enhancing expression of the human Klotho / KL gene, the composition comprising at least a portion of a protein belonging to the TNF superfamily or a nucleic acid encoding at least a portion of the protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present disclosure relates to compositions for promoting the expression of Klotho proteins and inventions related thereto. [Background technology]

[0002] It is generally believed that an organism's lifespan varies greatly depending on two factors: its own genetic factors and the surrounding environment. Organisms grow over the years after birth, and after a certain period of time, they age and die. Aging is generally defined as the gradual decline in the function of organs necessary for survival and reproduction. One example is the decline in cognitive function.

[0003] Numerous studies have been conducted using laboratory animals, and one of the most interesting discoveries is the discovery of a gene called Klotho (Non-Patent Document 1). It has been reported that kl / kl mice (mice with mutations in both Klotho genes on a pair of chromosomes), in which mutations in the Klotho gene have drastically reduced expression, are small in size and have a lifespan of approximately 2 to 5 months. Many studies have concluded that the cause of death is not stunted growth, but premature aging.

[0004] Furthermore, Non-Patent Document 2 shows that the lifespan of mice in which the Klotho gene was introduced and overexpressed was extended by 20%. Another Non-Patent Document 3 shows that the Klotho protein contained in actual human serum decreases with age. These results suggest that the Klotho gene has an important function as an anti-aging gene that suppresses aging, and research into its function is progressing worldwide.

[0005] Numerous studies have suggested that the Klotho gene is associated with various diseases, such as kidney phosphate accumulation, chronic kidney disease, renal fibrosis, osteoporosis, arterial calcification, hypertension, cardiac hypertrophy and fibrosis, ischemic myocardial infarction, chronic obstructive pulmonary disease, decreased insulin production, beta cell loss, neurodegenerative diseases, dementia, and cancer, suggesting that decreased Klotho expression induces these diseases (Non-Patent Document 4). This suggests that Klotho may normally suppress the onset of these diseases.

[0006] Therefore, the idea of ​​administering Klotho protein is a natural one, as supplementing Klotho protein, which decreases with age, is likely to extend lifespan and reduce the risk of developing these diseases. However, the half-life of Klotho protein is extremely short, at 7 hours (Non-Patent Document 5), and it is expected that a large amount of recombinant protein would be required to achieve this. However, Klotho protein is known to undergo numerous glycosylation modifications, making large-scale production using bacteria such as Escherichia coli difficult.

[0007] Therefore, an approach such as that described in Patent Documents 1 and 2, in which expression of the endogenous Klotho gene is promoted so that protein production continues even after it is degraded, is thought to be very effective. [Prior art documents] [Non-patent literature]

[0008] [Non-Patent Document 1] https: / / www.nature.com / articles / 36285 [Non-patent document 2] https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC2536606 / [Non-patent document 3] https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4130489 / [Non-patent document 4] https: / / www.frontiersin.org / articles / 10.3389 / fragi.2022.931331 / full [Non-patent document 5] https: / / www.ncbi.nlm.nih.gov / pmc / articles / PMC4696570 / [Patent documents]

[0009] [Patent Document 1] Patent No. 7412546 [Patent Document 2] Japanese Patent Application Publication No. 2023-175790 Summary of the Invention [Problem to be solved by the invention]

[0010] Increasing the expression of the Klotho / KL gene has the potential to suppress the onset of various diseases, including cancer, and may also have the effect of extending lifespan, which was the original purpose of its discovery. However, due to the extreme instability of the Klotho protein and the difficulty of mass production, administering the protein itself is difficult. Furthermore, although several prior art technologies have been developed and discovered to discover compounds that increase Klotho gene expression, no substance that actually increases Klotho expression in humans has been identified.

[0011] Therefore, there is a great need for a means for increasing the expression of the Klotho gene. Therefore, an object of the present disclosure is to provide a new composition for increasing the expression of the Klotho gene and use thereof. [Means for solving the problem]

[0012] After extensive research, the present inventors focused on culture supernatant obtained from mesenchymal stem cells. After collecting blood samples from 10 healthy volunteers, they administered culture supernatant obtained from mesenchymal stem cells by intravenous infusion. After two weeks, blood samples were taken and the levels of Klotho protein in the blood before and after administration were compared using ELISA. The results showed that the amount of Klotho protein increased statistically significantly after intravenous infusion of culture supernatant obtained from mesenchymal stem cells.

[0013] Further investigation revealed that TWEAK (TNF-like weak inducer of apoptosis) protein in the culture supernatant obtained from mesenchymal stem cells is necessary and sufficient for increasing Klotho expression.

[0014] The invention was completed based on the above findings, and in one aspect, the present disclosure includes the following inventions. (Invention 1) A composition for enhancing expression of the human Klotho / KL gene, the composition comprising at least a portion of a protein belonging to the TNF superfamily, or a nucleic acid encoding at least a portion of the protein. (Invention 2) A composition according to Invention 1, comprising one or more of the following (1) to (3): (1) A protein produced by the gene TNFSF12 (Tumor Necrosis Factor ligand superfamily member 12) / TWEAK (TNF-related weak inducer of apoptosis) that contains the amino acid sequence 97 to 249, including the extracellular domain. (2) A protein containing a sequence having 80% or more sequence similarity to the amino acid sequence of the protein of (1) above. (3) A nucleic acid comprising a sequence encoding any one of the proteins (1) and (2) above. (Invention 3) The composition of Invention 1 or 2, comprising one or more of the following (4) to (6): (4) A protein produced by the gene TNFSF2 (Tumor Necrosis Factor ligand superfamily member 2) / TNF (Tumor Necrosis Factor) / TNFα, which contains the amino acid sequence 77 to 233, including the extracellular domain; (5) A protein containing a sequence having 80% or more sequence similarity to the amino acid sequence of the protein (4) above. (6) A nucleic acid comprising a sequence encoding any one of the proteins (4) and (5) above. (Invention 4) The composition according to any one of inventions 1 to 3, wherein the composition comprises one or more of the following (7) to (9): (7) A protein produced by the gene TNFSF1 (Tumor Necrosis Factor ligand superfamily member 1) / TNFβ (Tumor Necrosis Factor β) / Lymphotoxin α, which contains amino acid sequence 35 to 205; (8) A protein containing a sequence having 80% or more sequence similarity to the amino acid sequence of the protein of (7) above. (9) A nucleic acid comprising a sequence encoding any one of the proteins (7) and (8) above. (Invention 5) The composition according to any one of Inventions 1 to 4, wherein the composition comprises one or more of the following (10) to (12): (10) A protein produced by the gene TNF14 (Tumor Necrosis Factor ligand superfamily member 14) / LIGHT, which contains the amino acid sequence 64 to 240 including the extracellular domain; (11) A protein containing a sequence having 80% or more sequence similarity to the amino acid sequence of the protein of (10) above. (12) A nucleic acid comprising a sequence encoding any one of the proteins (10) and (11) above. (Invention 6) 6. The composition according to any one of inventions 1 to 5, comprising a heterotrimeric protein of Lymphotoxin α and Lymphotoxin β. (Invention 7) A composition according to invention 6, wherein the heterotrimeric protein consists of one Lymphotoxin α and two Lymphotoxin β. (Invention 8) 6. The composition according to any one of Inventions 1 to 5, comprising a culture supernatant obtained by culturing mesenchymal stem cells and / or a secretion product of the mesenchymal stem cells. (Invention 9) A composition according to invention 8, comprising a culture supernatant obtained by serum-free culturing of mesenchymal stem cells and / or a secretion product of the mesenchymal stem cells. (Invention 10) 10. The composition according to any one of Inventions 1 to 9, further comprising vitamin D3. (Invention 11) 11. The composition according to any one of Inventions 1 to 10, which is administered for anti-aging purposes. (Invention 12) 12. The composition according to any one of claims 1 to 11, which is administered to treat one or more of the following conditions: (1) Neurodegenerative disease and / or dementia (2) Cancer growth (3) Hyperphosphatemia (4) Chronic kidney disease (5) Renal fibrosis (6) Osteoporosis (7) Arterial calcification (8) Hypertension (9) Cardiac hypertrophy and / or fibrosis (10) Ischemic myocardial infarction (11) Pulmonary fibrosis (12) Chronic obstructive pulmonary disease (13) Beta cell decrease (14) Decreased insulin (15) Increased diabetic lesions (Invention 13) Use of at least a part of a protein belonging to the TNF superfamily, or a nucleic acid encoding at least a part of said protein, for producing the composition according to any one of Inventions 1 to 12. (Invention 14) A method for treating any one of the symptoms described in invention 12 in a subject in need thereof, the method comprising the step of administering a therapeutically effective amount of a composition described in any one of inventions 1 to 12. (Invention 15) 13. At least a part of a protein belonging to the TNF superfamily, or a nucleic acid encoding at least a part of said protein, for treating any one of the conditions described in invention 12 in a subject in need thereof. [Effects of the Invention]

[0015] The above invention relates to a composition containing a protein belonging to the TNF superfamily (e.g., at least one of TWEAK / TNFSF12, TNFα / TNFSF2, TNFβ / TNFSF1, and LIGHT / TNFSF14). Alternatively, the invention relates to a composition containing a culture supernatant and / or a cell secretion product obtained by culturing mesenchymal stem cells. Administration of these compositions can increase the expression of Klotho. [Brief explanation of the drawings]

[0016] [Figure 1] This graph shows the quantitative comparison of serum Klotho protein levels using ELISA in healthy volunteers before and after intravenous administration of culture supernatant obtained by culturing mesenchymal stem cells. Two weeks after administration, serum Klotho protein levels increased with a statistically significant difference (test method: paired t-test). For subjects A to J, the plots in the POST column are shown in the following order from top to bottom: F, H, G, B, J, A, I, E, D, C. [Figure 2]This figure shows the expression of the Klotho gene in the human renal proximal tubule cell line HK-2. AD-CM represents the culture supernatant obtained by culturing human adipose-derived mesenchymal stem cells, and UC-CM represents the culture supernatant obtained by culturing human umbilical cord-derived mesenchymal stem cells. Medium 1 is sf-DOT medium, Medium 2 is MIFI medium, and Medium 3 is RM medium (all BioMimetics Sympathies, Inc.). Controls were samples supplemented with the medium used to culture mesenchymal stem cells (sf-DOT medium, MIFI medium, or RM medium). Gene abundance (mRNA abundance) was quantified by quantitative PCR (qPCR). Compared to the control, Klotho gene expression levels were significantly increased in HK-2 cells supplemented with AD-CM or UC-CM (test method: one-way ANOVA with Bonferroni post hoc test). [Figure 3] This shows the expression of the Klotho gene in the human renal proximal tubule cell line HK-2. The control was a sample to which an equal volume of PBS(-) was added. When each recombinant protein was added at the concentrations indicated on the graph (unit: ng / mL), Klotho gene expression increased in a concentration-dependent manner. [Figure 4] This figure shows the expression of Klotho in the human renal proximal tubule cell line, HK-2. HK-2 cells were cultured in a medium containing 10% FBS (fetal bovine serum), and then 10 ng / ml TWEAK was added. When FBS (FBS#1) purchased from Sigma-Aldrich (catalog number F7524-500ML) was used, the addition of TWEAK increased Klotho gene expression as before. However, when FBS (FBS#2) purchased from ThermoFisher Scientific (catalog number 10437-028) or FBS (FBS#3) purchased from BIOWEST (catalog number S1560) were used, the addition of TWEAK decreased Klotho gene expression. These results suggest that the type and lot of serum used have a significant impact on TWEAK-induced Klotho gene expression. [Figure 5]This figure shows the expression of the Klotho gene in the human renal proximal tubule cell line HK-2. The control sample was an equal volume of PBS(-). Addition of TWEAK and TNFβ recombinant proteins alone or together significantly increased Klotho gene expression (test method: one-way ANOVA with Bonferroni post hoc test). The values ​​listed next to each protein indicate the final concentration (ng / mL) after addition. For example, "LIGHT 1" indicates that LIGHT protein was added to a final concentration of 1 ng / mL. "LTN" refers to a trimer consisting of one lymphotoxin α and two lymphotoxin β molecules. "L+LTN" refers to the combination of LIGHT and LTN in addition to TWEAK. "L+Tβ" refers to the combination of LIGHT and TNFβ in addition to TWEAK. "L+LTN+Tβ" refers to the combination of TWEAK, LIGHT, LTN, and TNFβ. Each protein was added to "L+LTN," "L+Tβ," and "L+LTN+Tβ" to a final concentration of 1 ng / mL. [Figure 6] This shows the expression of the Klotho gene in the human renal proximal tubule cell line HK-2. The control medium was supplemented with the medium used for culturing mesenchymal stem cells, and CM (Conditioned Medium) represents the culture supernatant obtained by culturing human adipose tissue-derived mesenchymal stem cells (AD-CM) or human umbilical cord-derived mesenchymal stem cells (UD-CM). In the presence of control IgG, the addition of CM certainly increased Klotho gene expression, whereas the increase in Klotho gene expression due to the addition of CM was suppressed in the presence of TWEAK-neutralizing antibody. These results strongly suggest that Klotho gene expression is increased via TWEAK in the CM. [Figure 7]This figure shows the expression of the Klotho gene in the human renal proximal tubule cell line HK-2. The control was a medium supplemented with the medium used for culturing mesenchymal stem cells, and CM was the culture supernatant obtained by culturing human adipose tissue-derived mesenchymal stem cells (AD-CM) or human umbilical cord-derived mesenchymal stem cells (UD-CM). Additionally, active vitamin D3 (calcitriol) was added to examine the effect of combining the culture supernatant with the medium. The culture supernatant alone promoted Klotho gene expression. However, the combination of active vitamin D3 further promoted Klotho gene expression. The increase in expression due to the combination of active vitamin D3 was significantly greater than the increase observed when active vitamin D3 was added without the culture supernatant. [Figure 8] The amino acid sequence of TNFSF12 is shown. [Figure 9] The amino acid sequence of TNFSF2 is shown. [Figure 10] The amino acid sequence of TNFSF1 is shown. [Figure 11] The amino acid sequence of LIGHT is shown. [Figure 12] The amino acid sequence of Lymphotoxin α is shown. [Figure 13] The amino acid sequence of Lymphotoxin β is shown. [Figure 14] The amino acid sequence of a trimer consisting of one Lymphotoxin α and two Lymphotoxin β molecules is shown. The underlined portion indicates the linker sequence. DETAILED DESCRIPTION OF THE INVENTION

[0017] Specific embodiments for carrying out the present invention will be described below. The following description is intended to facilitate understanding of the invention and is not intended to limit the scope of the present invention.

[0018] 1.Definition As used herein, the terms "increase Klotho gene expression," "enhance Klotho gene expression," "enhance Klotho gene expression," "increase Klotho gene expression," and the like are used interchangeably. These terms encompass the purpose of ameliorating specific diseases related to the Klotho gene. Furthermore, the terms are not limited to therapeutic purposes. For example, the terms encompass the purpose of preventing the onset of a specific disease even if the person does not have the disease. Furthermore, the terms encompass the purpose of improving or preventing symptoms such as aging, which are not defined as diseases in current medical practice but which particularly affect the quality of life of elderly people.

[0019] The term "nucleic acid" as used herein includes any one or more of DNA and RNA. The term "nucleic acid" as used herein is not limited to naturally occurring nucleic acids. For example, the term "nucleic acid" as used herein includes any one or more of DNA to which a modified group has been added and RNA to which a modified group has been added.

[0020] The term "anti-aging" as used herein means slowing the progression of aging. Aging encompasses various physical symptoms. For example, aging may encompass any one or more of the following symptoms: the development of skin blemishes, the development of skin wrinkles, the development of tumors, the development of polyps, decreased bone density, muscle weakness, decreased vision, decreased appetite, decreased cognitive function, decreased lung function due to fibrosis, decreased kidney function, decreased cardiac function, decreased liver function, decreased pancreatic function (e.g., insulin production ability), and decreased function due to increased fibrosis in multiple other organs.

[0021] As used herein, the term "having sequence similarity" means that two sequences are similar to each other at a degree of 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more (for example, the similarity may be determined using blastp or the like).

[0022] In a further preferred embodiment, the above-mentioned term "having sequence similarity" may differ only by any one or more of the following substitutions: Group 1: serine, threonine Group 2: Asparagine, Glutamine Group 3: Aspartic acid, glutamic acid Group 4: alanine, valine, leucine, isoleucine (preferably valine, leucine, isoleucine) Group 5: Cysteine, methionine

[0023] In each group, the chemical structure differs only by about 1 to 3 carbon atoms in the side chain. Therefore, even if amino acids within each group are changed, there is a high probability that the function of the protein will be maintained.

[0024] As used herein, the term "therapeutically effective amount" refers to an amount sufficient to cause an improvement in symptoms. In one embodiment, improvement in symptoms means that a statistically significant difference in symptoms is achieved when comparing two groups.

[0025] As used herein, the term "prophylactically effective amount" refers to an amount sufficient to prevent the worsening of symptoms. In one embodiment, preventing the worsening of symptoms means that a statistically significant difference in symptoms is achieved when two groups are compared.

[0026] As used herein, the term "mesenchymal stem cells" (MSCs) refers to a type of stem cell present in adults, and has the ability to differentiate into mesodermally derived tissues such as bone, cartilage, blood vessels, and cardiomyocytes. Mesenchymal stem cells may be derived from one or more of the following tissues: adipose tissue, bone marrow, umbilical cord, umbilical cord blood, amniotic membrane, placenta, dental pulp (derived from permanent teeth), dental pulp (derived from baby teeth), etc.

[0027] As used herein, the term "culture supernatant" refers to a substance obtained directly or indirectly by culturing the above-described mesenchymal stem cells using a liquid medium. An example of a directly obtained substance includes the liquid remaining in the culture vessel immediately after the culture is completed. An example of an indirectly obtained substance refers to a substance obtained by further processing the liquid.

[0028] Examples of further processing may include any one or more of the following: Filtration treatment Centrifugation to collect exosomes Freeze-drying to turn it into a powder After powdering, it is processed into a specific shape (e.g., tablet, pill, etc.) To change into a semi-solid (gel-like) Adding any additional ingredients

[0029] Therefore, the term "culture supernatant" as used herein includes not only liquid forms but also solid and semi-solid forms.

[0030] The liquid medium is used to obtain a culture supernatant and / or to culture mesenchymal stem cells. The liquid medium is not particularly limited, and media known in the art (e.g., DMEM, F12, RPMI, etc.) can be used. Examples of liquid media include one or more of the following: Mesenchymal Stem Cell Basal Medium (Invitrogen Corporation), Mesenchymal Stem Cell Basal Medium (Sanko Junyaku Co., Ltd.), MF Medium (Toyobo Corporation), sf-DOT (registered trademark) (Biomimetics Sympathies Inc.), Nipro / Cell Science Institute (e.g., product numbers A2G00P05C+A2G20P1CC), and Kohjin Bio Co., Ltd. (e.g., product number KBM ADSC-4).

[0031] The liquid medium and the culture supernatant may contain serum and / or may be serum-free. In a preferred embodiment, the liquid medium and the culture supernatant are serum-free. This makes it possible to avoid the influence of components in serum.

[0032] 2. Composition In one embodiment, the composition of the present disclosure relates to a composition for enhancing expression of the human Klotho / KL gene. The composition comprises at least a portion of a protein belonging to the TNF superfamily (and / or a protein having sequence similarity thereto), or a nucleic acid encoding at least a portion of the protein. More specifically, the composition may comprise at least a portion of a protein belonging to the human TNF superfamily (and / or a protein having sequence similarity thereto), or a nucleic acid encoding at least a portion of the protein.

[0033] "At least a portion of a protein" means that the protein has a region of sufficient length to exert its function. For example, at least a portion of a protein belonging to the TNF superfamily means that the protein has sufficient function to bind to a receptor as a ligand. In a more preferred example, at least a portion of a protein belonging to the TNF superfamily may have the function of enhancing expression of the human Klotho / KL gene.

[0034] The nucleic acid encoding at least a portion of the protein may be in the form of DNA or RNA and may be included in the composition. The nucleic acid encoding at least a portion of the protein may be included in the form of a vector (e.g., an expression vector). The nucleic acid encoding at least a portion of the protein may be provided in a form incorporated into a lipid nanoparticle (LNP).

[0035] 2-1.TNF superfamily In one embodiment, the composition of the present disclosure comprises at least a portion of a protein belonging to the TNF superfamily, or a nucleic acid encoding at least a portion of the protein. Proteins belonging to the TNF superfamily include, but are not limited to, any one or more of the following: ·TNFSF12(Tumor Necrosis Factor ligand superfamily member 12) / TWEAK(TNF-related weak inducer of apoptosis) ·TNFSF2(Tumor Necrosis Factor ligand superfamily member 2) / TNF( Tumor Necrosis Factor ) / TNFα ·TNFSF1(Tumor Necrosis Factor ligand superfamily member 1) / TNFβ(Tumor Necrosis Factorβ) / Lymphotoxinα ·TNF14(Tumor Necrosis Factor ligand superfamily member 14) / LIGHT Heterotrimeric protein of lymphotoxin α and lymphotoxin β

[0036] In a preferred embodiment, the composition of the present disclosure comprises at least a portion of a protein produced by TNFSF12 (Tumor Necrosis Factor ligand superfamily member 12) / TWEAK (TNF-related weak inducer of apoptosis), or a nucleic acid encoding at least a portion of the protein.

[0037] 2-1-1.TNF superfamily TNFSF12 TNFSF12 (Tumor Necrosis Factor ligand superfamily member 12) / TWEAK (TNF-related weak inducer of apoptosis) (hereinafter referred to as "TNFSF12" or "TWEAK") is known as a ligand for the FN14 / TWEAKR receptor.

[0038] In a preferred embodiment, the composition of the present disclosure comprises (1) a protein comprising the amino acid sequence of amino acids 97 to 249 (SEQ ID NO: 1), which corresponds to at least a portion of the extracellular domain. More preferably, the amino acid sequence of the protein consists of the sequence shown in SEQ ID NO: 1.

[0039] In another preferred embodiment, the composition of the present disclosure comprises (2) a protein having sequence similarity to the amino acid sequence of positions 97 to 249 of the protein (SEQ ID NO: 1, see FIG. 8).

[0040] In another preferred embodiment, the composition of the present disclosure comprises (3) a nucleic acid comprising a sequence encoding any one of the proteins described in (1) and (2) above.

[0041] Among the proteins belonging to the TNF superfamily, TNFSF12 is particularly effective in enhancing the expression of the human Klotho / KL gene.

[0042] The composition may also include a combination of TNFSF12 (or a protein having sequence similarity to TNFSF12) with another protein (e.g., another protein belonging to the TNF superfamily). The same applies to nucleic acids.

[0043] 2-1-2.TNF Superfamily TNFSF2 TNFSF2 (Tumor Necrosis Factor ligand superfamily member 2) / TNF (Tumor Necrosis Factor) / TNFα (hereinafter referred to as "TNFSF2," "TNFα," or "TNFalpha") is known as a ligand for TNFR1 and TNFR2, and is generally known in the form of transmembrane mTNFα and soluble sTNFα.

[0044] In a preferred embodiment, the composition of the present disclosure comprises (4) a protein comprising the amino acid sequence 77 to 233 of the protein (SEQ ID NO: 2), which sequence corresponds to at least a portion of the extracellular domain. More preferably, the amino acid sequence of the protein consists of the sequence shown in SEQ ID NO: 2.

[0045] In another preferred embodiment, the composition of the present disclosure comprises (5) a protein having sequence similarity to the amino acid sequence 77 to 233 of the protein (SEQ ID NO: 2; see FIG. 9).

[0046] In another preferred embodiment, the composition of the present disclosure comprises (6) a nucleic acid comprising a sequence encoding any one of the proteins (4) and (5) above.

[0047] 2-1-3.TNF Superfamily TNFSF1 TNFSF1 (Tumor Necrosis Factor ligand superfamily member 1) / TNFβ (Tumor Necrosis Factor β) / Lymphotoxin α (hereinafter referred to as "TNFSF1" or "TNFβ" or "TNFbeta") has biological activity similar to that of TNF-α and is known as a ligand for TNFR1 and TNFR2.

[0048] In a preferred embodiment, the composition of the present disclosure comprises (7) a protein comprising the amino acid sequence of amino acids 35 to 205 of the protein (SEQ ID NO: 3, see FIG. 10). More preferably, the amino acid sequence of the protein consists of the sequence shown in SEQ ID NO: 3.

[0049] In another preferred embodiment, the composition of the present disclosure comprises (8) a protein having sequence similarity to the amino acid sequence of positions 35 to 205 of the protein (SEQ ID NO: 3).

[0050] In another preferred embodiment, the composition of the present disclosure comprises (9) a nucleic acid comprising a sequence encoding any one of the proteins (7) and (8) above.

[0051] 2-1-4.TNF superfamily TNF14 Tumor necrosis factor ligand superfamily member 14 (TNF14) / LIGHT (hereinafter referred to as "TNF14" or "LIGHT") is recognized by herpesvirus entry mediator and decoy receptor 3.

[0052] In a preferred embodiment, the composition of the present disclosure comprises (10) a protein comprising the amino acid sequence of amino acids 64 to 240 of the protein (SEQ ID NO: 4, see FIG. 11). More preferably, the amino acid sequence of the protein consists of the sequence shown in SEQ ID NO: 4.

[0053] In another preferred embodiment, the composition of the present disclosure comprises (11) a protein having sequence similarity to the amino acid sequence of amino acids 64 to 240 of the protein (SEQ ID NO: 4).

[0054] In another preferred embodiment, the composition of the present disclosure comprises (12) a nucleic acid comprising a sequence encoding any one of the proteins (10) and (11) above.

[0055] 2-1-5. Heterotrimeric protein of lymphotoxin α and lymphotoxin β In a preferred embodiment, the composition of the present disclosure comprises a heterotrimeric protein of Lymphotoxin α and Lymphotoxin β. In a more preferred embodiment, the heterotrimeric protein consists of one Lymphotoxin α and two Lymphotoxin β. The amino acid sequences of Lymphotoxin α and Lymphotoxin β may be sequences comprising, for example, the sequences represented by SEQ ID NO: 5 (see FIG. 12) and SEQ ID NO: 6 (see FIG. 13). More preferably, the heterotrimeric protein may be a trimeric protein comprising Lymphotoxin α and Lymphotoxin β connected via the linker sequence "GGGGS" (see FIG. 14, SEQ ID NO: 7).

[0056] 2-1-6. Combination In a preferred embodiment, the composition of the present disclosure may contain a combination of multiple proteins of the TNF superfamily. In particular, a combination of TWEAK and a specific protein of the TNF superfamily is particularly preferred from the viewpoint of enhancing the expression of the human Klotho / KL gene. For example, any of the following combinations is preferred: TWEAK+TNFβ TWEAK+TNFβ+LIGHT TWEAK + TNFβ + LIGHT + 1 Lymphotoxinα + 2 Lymphotoxinβ trimers

[0057] 2-2.Other In another embodiment, the composition may further contain vitamin D3 in addition to a protein belonging to the TNF superfamily. For example, vitamin D3 may be mixed with the culture supernatant (in other words, vitamin D3 may not be added to the liquid medium for obtaining the culture supernatant, and the culture supernatant may be collected after the culture is completed, and vitamin D3 may be added to the culture supernatant). By combining vitamin D3 with a protein belonging to the TNF superfamily, Klotho expression can be enhanced.

[0058] Vitamin D3 may be in an active form or an inactive form. Inactive vitamin D3 can typically be converted to an active form by metabolism. Some dosage forms may reach the desired tissue (e.g., kidney, etc.) without going through a metabolic process. In this case, it is preferable to use active vitamin D3.

[0059] Similarly, the amount of vitamin D3 in the composition is not particularly limited. For example, the amount of vitamin D3 in the composition may be 4.16 ng / g (10 picomoles / g) or more (i.e., when the total weight of the composition is 1 g, the composition contains 4.16 ng or more (10 picomoles or more) of vitamin D3). Preferably, the amount of vitamin D3 in the composition may be 41.6 ng / g or more (100 picomoles / g or more), more preferably 416 ng / g or more (1 nanomole / g or more).

[0060] The composition may be an aerosol, liquid, solid, or semi-solid.

[0061] In the case of a liquid, it may be in the form of a syrup, emulsion, suspension, etc. Furthermore, in the case of a liquid, it may be stored in a vial, an intravenous bag, a pre-filled syringe, etc. In the case of a solid, it may be in the form of a tablet, pill, capsule, powder, granule, suppository, etc.

[0062] In a preferred embodiment, when the active ingredient is a solid, it may not be enclosed in a capsule or a coating.

[0063] The composition may include other ingredients. Examples of other ingredients may include any one or more of the following: pH adjuster (e.g., phosphate buffer, Tris buffer, etc.) Inorganic salts (e.g., sodium chloride, potassium chloride, etc.) Sugars (e.g., lactose, sucrose, etc.) Excipients (e.g., water, purified water, alcohol, glycerin, lactose, starch, dextrin, white sugar, precipitated silica, etc.)

[0064] 3. Presence or absence of culture supernatant 3-1. When culture supernatant is included In one embodiment, the composition of the present disclosure comprises a culture supernatant of mesenchymal stem cells and / or a secretion product of the mesenchymal stem cells. Mesenchymal stem cells have the property of secreting proteins belonging to the TNF superfamily described above. The secretion product of the mesenchymal stem cells may be obtained from a fraction in the range of 10 kDa to 30 kDa by fractionation by ultrafiltration, for example. The sizes of many proteins belonging to the TNF superfamily are within this range.

[0065] As described above, the culture supernatant itself may be serum-free. For example, the culture supernatant may be obtained by serum-free culture of mesenchymal stem cells. In this case, the composition containing the culture supernatant itself may also be serum-free.

[0066] 3-2. When culture supernatant is not included In another embodiment, the composition of the present disclosure does not contain a mesenchymal stem cell culture supernatant. Preferably, the composition of the present disclosure does not contain a mesenchymal stem cell culture supernatant and does not contain any secretions of the mesenchymal stem cells. In this case, the protein belonging to the TNF superfamily needs to be obtained from a source other than the mesenchymal stem cell culture supernatant. For example, a protein belonging to the TNF superfamily isolated from a source other than the mesenchymal stem cell culture supernatant (e.g., the protein may be expressed in large quantities by genetic engineering and then purified) may be added to the composition.

[0067] 4. Manufacturing method 4-1. Manufacturing method 1 In one embodiment, the present disclosure relates to a method for producing a composition for enhancing expression of the human Klotho / KL gene, the method comprising the steps of: A process of culturing mesenchymal stem cells in a medium to obtain a culture supernatant Formulating the culture supernatant to obtain a composition suitable for administration.

[0068] The culture conditions are not particularly limited and may be any conditions known in the art. For example, the temperature may be 35°C to 40°C, typically 37°C. The incubator may be appropriately controlled to maintain a CO2 concentration of 5%. The culture period is not particularly limited, but it is preferable to culture for at least 24 hours, preferably 48 hours or more. The upper limit is not particularly limited, but it is 168 hours or less.

[0069] The type of culture medium is not particularly limited, and the above-mentioned culture medium can be used appropriately.

[0070] By culturing mesenchymal stem cells, proteins belonging to the TNF superfamily are secreted from the cells.

[0071] After the culture is completed, the supernatant in the culture vessel may be collected by aspirating. Alternatively, the contents of the culture vessel may be transferred to another vessel and centrifuged, and the supernatant may be collected by aspirating. This supernatant may then be considered the culture supernatant.

[0072] The culture supernatant is then blended to obtain a composition suitable for administration. The treatment required for this is not particularly limited, and may be carried out appropriately depending on the final form of the composition.

[0073] For example, the treatment described above in the explanation of the term "culture supernatant" may be carried out.

[0074] In addition to the above-mentioned processes, for example, a process of forming into a specific shape may be carried out.

[0075] In a preferred embodiment, the method may further comprise the step of formulating with vitamin D3 to obtain a composition suitable for administration.

[0076] For example, if the culture supernatant is liquid, vitamin D3 in powder or liquid form may be mixed with the liquid culture supernatant.For example, if the culture supernatant is solid, it may be dissolved in liquid, and then vitamin D3 in powder or liquid form may be mixed with the liquid culture supernatant.Alternatively, if the culture supernatant is solid, it may be processed into powder form, and the powdered vitamin D3 may be mixed with the powder culture supernatant.

[0077] Other components may be added in a similar manner. Alternatively, in addition to the proteins belonging to the TNF superfamily secreted from mesenchymal stem cells, one or more of the above-mentioned proteins belonging to the TNF superfamily may be added externally to the culture supernatant.

[0078] In another embodiment, the present disclosure relates to use of a protein belonging to the TNF superfamily for producing a composition for enhancing expression of the human Klotho / KL gene. The production method may be the production method described above.

[0079] The culture supernatant may also be used in combination with vitamin D3.

[0080] 4-2. Manufacturing method 2 In another embodiment, the present disclosure relates to a method for producing a composition for enhancing expression of the human Klotho / KL gene. The method is similar to the method described in "Production Method 1," but includes a step of preparing one or more proteins belonging to the TNF superfamily, instead of the "step of obtaining a culture supernatant."

[0081] This step may include, for example, preparing a protein belonging to the TNF superfamily by purchasing it commercially available, or may include expressing the protein belonging to the TNF superfamily by genetic engineering (for example, by using an expression vector encoding the protein belonging to the TNF superfamily) and purifying the protein.

[0082] Furthermore, similar to the method described as "Production Method 1," this method may also include the step of further incorporating vitamin D3 to obtain a composition suitable for administration.

[0083] 5. How to use the composition In one embodiment, the present disclosure relates to use of a composition for enhancing expression of the human Klotho / KL gene. In a more preferred embodiment, the use is in combination with vitamin D3.

[0084] An example of the use of a combination includes separately administering a composition containing a protein belonging to the TNF superfamily (e.g., a composition containing a mesenchymal stem cell culture supernatant) and at least one vitamin D3. The administration may be simultaneous or at different times. However, if the time difference is too large, the combined effect of the composition and the vitamin will be reduced. Therefore, the time difference may preferably be within 1 hour, 30 minutes, or 15 minutes.

[0085] In another embodiment, the present invention relates to a method for enhancing expression of the human Klotho / KL gene. The method comprises the step of administering an effective amount of a composition to a subject in need of enhanced expression of the Klotho / KL gene. Here, the composition may comprise a culture supernatant of mesenchymal stem cells and / or a protein belonging to the TNF superfamily. Furthermore, the term "effective amount" refers to an amount that produces a statistically significant difference in the amount of Klotho in the blood of the administered subject when measured before and after administration. For example, when a composition containing a culture supernatant of mesenchymal stem cells is administered by intravenous infusion, the amount may be 0.1 mL / kg to 10 mL / kg, preferably 2 mL / kg to 10 mL / kg, and preferably 0.5 mL / kg to 5 mL / kg.

[0086] In a preferred embodiment, the administering step includes administering vitamin D3. In this case, the above-mentioned mesenchymal stem cell culture supernatant and vitamin D3 may be contained in the same composition. Alternatively, they may be contained in separate compositions.

[0087] The subject to which the compound is administered is not particularly limited and may be a human or an animal (e.g., a mammal other than a human). Typically, the subject to which the compound is administered is a human. The subject to which the compound is administered may be healthy or suffering from a disease.

[0088] The dose of vitamin D3 should be less than 100 μg per day.

[0089] The above-mentioned dosage refers to the total daily dosage. Therefore, the daily administration may be once or divided into multiple doses. For example, the daily administration may be two, three, four, or more times.

[0090] The administration period is not particularly limited and may be within a range defined by two selected from 1 day, 3 days, 7 days, 30 days, 90 days, 120 days, 180 days, 200 days, and 365 days (e.g., 1 day to 90 days, 90 days to 180 days, etc.).

[0091] The administration route is not particularly limited, and may be systemic administration or local administration. The administration route is not particularly limited, and may be, for example, one or more of the following routes: oral administration, inhalation administration, anal administration, subcutaneous injection, intravenous injection, intramuscular injection, infusion, application to the skin, spraying to the skin, etc. In a preferred embodiment, the administration route is parenteral administration. This is because the active ingredient is a protein belonging to the TNF superfamily, and if administered orally, this protein may be decomposed by digestive juices in the stomach.

[0092] 6. Other uses of the composition, etc. In further embodiments, the compositions of the present disclosure can be used for additional purposes. For example, the compositions of the present disclosure can be used to treat the following conditions: (1) Neurodegenerative disease and / or dementia (2) Cancer growth (3) Hyperphosphatemia (4) Chronic kidney disease (5) Renal fibrosis (6) Osteoporosis (7) Arterial calcification (8) Hypertension (9) Cardiac hypertrophy and / or fibrosis (10) Ischemic myocardial infarction (11) Pulmonary fibrosis (12) Chronic obstructive pulmonary disease (13) Beta cell decrease (14) Decreased insulin (15) Increased diabetic lesions

[0093] Therefore, in one embodiment, the present disclosure relates to a method for treating any one or more of the symptoms (1) to (15) described above, which comprises administering a therapeutically effective amount of a composition containing a protein belonging to the TNF superfamily to a subject who has any one or more of the symptoms (1) to (15) described above and is in need of treatment for the symptoms.

[0094] The above-mentioned symptoms (1) to (15) are all known to be related to increases or decreases in Klotho expression. Therefore, administration of the composition of the present disclosure in one embodiment may increase Klotho expression, thereby improving the above-mentioned symptoms (1) to (15).

[0095] In another embodiment, the present disclosure relates to a method for anti-aging, comprising administering a composition containing a protein belonging to the TNF superfamily in an amount effective for preventing aging. Experiments using aging model mice and the like have shown that increases or decreases in Klotho expression are related to aging phenotypes. Therefore, administration of the composition of the present disclosure in one embodiment may increase Klotho expression, thereby preventing aging.

[0096] In another embodiment, the present disclosure relates to the use of at least a portion of a protein belonging to the TNF superfamily, or a nucleic acid encoding at least a portion of said protein, for the manufacture of a composition, which is then used to treat one or more of the symptoms of (1) to (15) or for anti-aging purposes.

[0097] In another embodiment, the present disclosure relates to the use of at least a portion of a protein belonging to the TNF superfamily, or a nucleic acid encoding at least a portion of said protein, for use in treating any one or more of the symptoms of (1) to (15) or for anti-aging. [Example]

[0098] 1. Preparation of culture supernatant First, we prepared mesenchymal stem cells. Specifically, we used adipose tissue-derived mesenchymal stem cells (AD-CM) and umbilical cord tissue-derived mesenchymal stem cells (UC-CM).

[0099] 1-1. Preparation of adipose tissue-derived mesenchymal stem cells Subcutaneous adipose tissue was isolated from patients scheduled to undergo regenerative medicine using adipose tissue-derived mesenchymal stem cells. This subcutaneous adipose tissue is the raw material necessary for preparing cells for administration. The remaining subcutaneous adipose tissue was used for primary culture. Consent for research use was obtained from the patient in advance.

[0100] Subcutaneous adipose tissue was centrifuged (400 × g for 5 minutes) to separate into three layers. Specifically, the three layers were separated from the top down: lipid fraction, adipose tissue fraction, and aqueous fraction. The middle adipose tissue fraction was retained, and the top and bottom layers were discarded. A 0.15% collagenase enzyme solution was added to the remaining adipose tissue fraction at a volume four times the tissue weight. The adipose tissue was permeated at 37°C for 1 hour for enzyme treatment. After the adipose tissue was dispersed by enzyme treatment, the adipose tissue was centrifuged (400 × g for 5 minutes). The precipitated fraction was suspended in 30 mL of PBS(-) solution to obtain the stromal vascular cell fraction containing mesenchymal stem cells. The suspension was then passed through a cell strainer (mesh size 70 μm), and any tissue debris captured by the cell strainer was discarded. The flow-through fraction was then centrifuged again (400 × g for 5 minutes), and the precipitated fraction was suspended in 6 mL of serum-free culture medium sf-DOT (Biomimetics Sympathies, Inc.). The entire cell suspension was seeded into a T25 flask (CellBIND; Corning, 3289) and placed in an incubator (37°C, 5% CO2) to initiate primary culture.

[0101] The medium was completely replaced every three days. The supernatant was discarded, and cells growing on the flask bottom were selectively expanded. After cells reached semi-confluence in a T-25 flask, 2 mL of enzyme solution (TrypLE Express; Thermo Fisher Scientific, 12604021) was added to detach the cells from the flask bottom (37°C, 5 minutes). The cells were diluted with PBS(-) and centrifuged (400 × g, 5 minutes). The precipitated cells were suspended in sf-DOT culture medium, and an aliquot was taken and counted using trypan blue staining. The cells suspended in sf-DOT were seeded into a new T75 flask (CellBIND; Corning, 3290) and placed in an incubator (37°C, 5% CO2) for subculture (P0 → P1). Subsequent subcultures were repeated in the same manner to obtain the required cell numbers (P1 → P2).

[0102] 1-2. Preparation of mesenchymal stem cells derived from umbilical cord tissue First, consent for research use of the umbilical cord was obtained from the expectant mother. After delivery, the umbilical cord was washed with PBS(-) and cut into approximately 3 mm pieces with a scalpel. A 0.15% collagenase enzyme solution was added at a volume four times the tissue weight. The tissue was then infiltrated at 37°C for 2 hours for enzyme treatment. After the umbilical cord tissue was dispersed by enzyme treatment, the tissue was centrifuged (400 × g for 5 minutes). The precipitated fraction was suspended in 30 mL of PBS(-) as the stromal vascular cell fraction containing mesenchymal stem cells. The suspension was then passed through a cell strainer (70 μm mesh), and any tissue debris trapped by the cell strainer was discarded. The passed fraction was then centrifuged again (400 × g for 5 minutes), and the precipitated fraction was suspended in 12 mL of serum-free culture medium sf-DOT (Biomimetics Sympathies, Inc.). The entire cell suspension was seeded into a T75 flask (CellBIND; Corning, 3290) and placed in an incubator (37°C, 5% CO2) to initiate primary culture.

[0103] The medium was completely replaced every three days. The supernatant was discarded, and cells growing on the flask bottom were selectively expanded. After cells reached semi-confluence in a T-25 flask, 2 mL of enzyme solution (TrypLE Express; Thermo Fisher Scientific, 12604021) was added to detach the cells from the flask bottom (37°C, 5 minutes). The cells were diluted with PBS(-) and centrifuged (400 × g, 5 minutes). The precipitated cells were suspended in sf-DOT culture medium, and an aliquot was taken and counted using trypan blue staining. The cells suspended in sf-DOT were seeded into a new T75 flask (CellBIND; Corning, 3290) and placed in an incubator (37°C, 5% CO2) for subculture (P0 → P1). Subsequent subcultures were repeated in the same manner to obtain the required cell numbers (P1 → P2).

[0104] 1-3. Preparation of culture supernatant First, adipose tissue- or umbilical cord tissue-derived mesenchymal stem cells were cultured in sf-DOT as described above. Adipose tissue- or umbilical cord tissue-derived mesenchymal stem cells were cultured in the same medium at 6000 cells / cm per T75 flask. 2 After collecting the culture supernatant (sf-DOT-derived culture supernatant) on day 4, the cells were washed once with PBS(-). RM medium was then added (12 mL per T75 flask) and cultured for two more days in an incubator (37°C, 5% CO2). The culture supernatant was then collected (RM medium-derived culture supernatant). P1 adipose tissue- or umbilical cord tissue-derived mesenchymal stem cells were similarly cultured in MIFI medium, and MIFI medium-derived culture supernatant was also obtained.

[0105] The collected culture supernatant was filtered through a 0.2 μm PES syringe filter (25 mm GD / X syringe filter (PES 0.2 μm sterilized); 6896-2502; GE Healthcare Japan). The filtered culture supernatant was stored frozen at -20°C or below until use in analysis.

[0106] 1-4. Expression analysis methods Total RNA was extracted from the cells using the ReliaPrep RNA Miniprep system (Promega, Z6012).

[0107] After RNA extraction, cDNA synthesis (PrimeScript RT Master Mix; Takara, RR036A) was performed using 500 ng of RNA, followed by quantitative PCR (Thunderbird Sybr qPCR Mix; TOYOBO, QPS-201X5).

[0108] A mixture for cDNA synthesis was prepared according to the following composition. 5xPrimeScript RT Master Mix 2μl (final concentration 1x) Total RNA 500ng RNase-free H2O, adjust to a total of 10 μl

[0109] The mixture was treated using a Veriti 96-well Thermal Cycler manufactured by Applied Biosystems under the following conditions. 37℃ 15 minutes ↓ 85℃ 5 seconds ↓ 4℃∞

[0110] The synthesized cDNA (10 μl) was diluted 10-fold with 90 μl of TE (10 mM Tris-HCl pH 8.0 + 1 mM EDTA pH 8.0), and the diluted product was subjected to quantitative PCR.

[0111] More specifically, the dilutions were mixed under the following conditions: 2×THUNDERBIRD Probe qPCR Mix 10μl 5mM Forward Primer 0.4μl 5mM Reverse Primer 0.4μl H2O 8.2 μl cDNA (10x diluted) 1 μl

[0112] The PCR cycle conditions were as follows: 1. 95℃ 1 minute (initial denaturation) 2.95℃ 15 seconds (denaturation) 3.60℃ 30 seconds (extension) (Steps 2 and 3 were repeated 40 times, and a fluorescent signal was detected each time step 3 was completed.) 4. The temperature was increased from 65°C to 95°C in 0.5°C increments, and the temperature was held for 5 seconds at each increment before detecting the fluorescent signal.

[0113] Using the above cycles, PCR products were detected, and the uniformity of the PCR products was confirmed by a melting curve.

[0114] As an internal control (a housekeeping gene with equal expression in all cells and conditions), GAPDH ( G lycer aldehyde 3- p phosphate d e h hydroxylase) was used. GAPDH Forward primer: 5'-AGCCACATCGCTCAGACAC-3' (SEQ ID NO: 8) GAPDH Reverse primer: 5'-GCCTAATACGACCAAATCC-3' (SEQ ID NO: 9)

[0115] Different primers were used to detect each gene of interest. Klotho forward primer: 5'-AGCTCTCAAAGCCCACATAC-3' (SEQ ID NO: 10) Klotho reverse primer: 5'-CTGATCTGAGCATAACGATAGA-3' (SEQ ID NO: 11)

[0116] All primers were purchased from FASMAC Co., Ltd. (reverse-phase column purification grade). The expression level of each gene is shown as the normalized expression level obtained by quantitative PCR divided by the expression level of GAPDH. Furthermore, the expression level under control conditions (no supernatant treatment) was normalized to "1." The melting curve confirmed that all PCR products amplified using the above primers were unique (i.e., multiple sequences were not amplified with the same primers).

[0117] 2. Example 1 (Changes in serum klotho levels after administration of culture supernatant) Blood samples were collected from 10 adults, and the amount of Klotho protein in the serum was quantified by ELISA (R&D Systems, catalog number DY5334-05). Next, RM medium-derived culture supernatants of umbilical cord-derived mesenchymal stem cells were prepared using the procedure described above. The culture supernatants were then administered intravenously (1 mL / kg). Two weeks after administration, blood samples were collected again, and the amount of Klotho protein in the serum was quantified by ELISA. The results are shown in Figure 1. The average serum Klotho concentration before administration of the culture supernatant was 320.0 pg / mL. Meanwhile, the average serum Klotho concentration after administration was 427.7 pg / mL. A paired t-test was performed to confirm a significant difference between the two values.

[0118] Therefore, it was demonstrated that administration of the culture supernatant of mesenchymal stem cells increases the amount of Klotho in the serum.

[0119] 3. Example 2 (Changes in Klotho Expression by Administration of Culture Supernatant) Next, we examined the effect of culture supernatant administration on Klotho expression in the kidney-derived cell line HK-2. Klotho is known to be most abundant in kidney tissue compared to other organs.

[0120] The cell line HK-2 was prepared. HK-2 was plated in each well of a 24-well plate (Corning, 3337) at 25,000 cells / cm. 2 The cells were then cultured in serum-free medium, Keratinocyte-SFM (ThermoFisher, catalog number 17005042), for 24 hours (37°C, 5% CO2).

[0121] The medium was then replaced with the aforementioned culture supernatant. Specifically, the medium was replaced with either adipose tissue-derived mesenchymal stem cell culture supernatant (AD-CM) or umbilical cord tissue-derived mesenchymal stem cell culture supernatant (UC-CM). As a control, the medium was replaced with mesenchymal stem cell culture medium. This allowed us to focus solely on factors secreted by adipose tissue- or umbilical cord tissue-derived mesenchymal stem cells. After the medium replacement, the culture was continued for 24 hours. The HK-2 cells were then harvested, and RT-PCR was performed as described above to examine the expression levels of Klotho. The results are shown in Figure 2. Compared to the control untreated with culture supernatant, both the adipose tissue-derived mesenchymal stem cell culture supernatant and the umbilical cord tissue-derived mesenchymal stem cell culture supernatant increased the expression levels of Klotho mRNA. Furthermore, this effect did not vary depending on the type of culture medium (Culture medium 1: sf-DOT medium, Culture medium 2: MIFI medium, Culture medium 3: RM medium (all BioMimetics Sympathies Inc.)), and the effect was present in the secretions of both adipose tissue- and umbilical cord tissue-derived mesenchymal stem cells, suggesting that this may be a universal function of mesenchymal stem cells.

[0122] 4. Example 3 (Changes in Klotho Expression by Administration of TNF Superfamily) Next, the inventors further investigated the components contained in the culture supernatant and came to the hypothesis that proteins of the TNF superfamily contribute to the increase in Klotho mRNA expression. Therefore, instead of treating with the culture supernatant, they tried treating with various proteins of the TNF superfamily.

[0123] The cell line HK-2 was prepared. HK-2 was plated in each well of a 24-well plate (Corning, 3337) at 25,000 cells / cm. 2 The cells were then cultured in serum-free medium, Keratinocyte-SFM (ThermoFisher, catalog number 17005042), for 24 hours (37°C, 5% CO2).

[0124] The medium was then supplemented with varying concentrations of TNF superfamily proteins.

[0125] The following recombinant proteins of the TNF superfamily were used: TWEAK (Peprotech, catalog number AF-310-06) TNFα (Peprotech, catalog number AF-300-01A) TNFβ (Peprotech, Catalog No. 300-01B) LIGHT (Peprotech, Catalog No. 310-09B) Trimer of one Lymphotoxin α and two Lymphotoxin β molecules (R&D Systems, catalog number 8884-LY-025 / CF)

[0126] As a control, an equal volume of PBS(-) containing no TNF superfamily proteins was added. Culture was then continued for 24 hours. HK-2 cells were then harvested, and RT-PCR was performed as described above to examine the expression level of Klotho. The results are shown in Figure 3. Compared to the control containing only PBS(-), it was shown that TNF superfamily proteins increased the expression level of Klotho mRNA.

[0127] 5. Example 4 (Changes in Klotho Expression by Serum) Next, we investigated the effect of serum on the contribution of TNF superfamily proteins to the increase in Klotho mRNA expression, because serum such as FBS is generally known to have different effects on cells depending on the product and lot (https: / / cell.brc.riken.jp / ja / manual / fbs_test).

[0128] The cell line HK-2 was prepared. HK-2 was plated in each well of a 24-well plate (Corning, 3337) at 25,000 cells / cm. 2The cells were seeded in serum-free medium (Keratinocyte-SFM, ThermoFisher, catalog no. 17005042) and cultured in serum-free medium (DMEM, SIGMA, D8900) + 10% FBS (FBS#1: SIGMA-ALDRICH, catalog no. F7524-500ML; FBS#2: ThermoFisher Scientific, catalog no. 10437-028; FBS#3: BIOWEST, catalog no. S1560) for 24 hours at 37°C, 5% CO2. TWEAK and TNFα were then added to a final concentration of 10 ng / ml, and the culture was continued for another 24 hours. HK-2 cells were then harvested and analyzed for Klotho expression by RT-PCR as described above. The results are shown in Figure 4. The type of serum affected Klotho gene expression. These results suggest that the type of serum used has a strong influence on Klotho gene expression induced by TWEAK, TNFα, etc.

[0129] 6. Example 5 (Changes in Klotho Expression by Combined Administration of TNF Superfamily Members) Next, we investigated the effect of combined administration of TNF superfamily members on Klotho expression.

[0130] The cell line HK-2 was prepared. HK-2 was plated in each well of a 24-well plate (Corning, 3337) at 25,000 cells / cm. 2 The cells were then cultured in serum-free medium, Keratinocyte-SFM (ThermoFisher, catalog number 17005042), for 24 hours (37°C, 5% CO2).

[0131] They were then formulated with varying concentrations of TNF superfamily proteins.

[0132] The following recombinant proteins of the TNF superfamily were used alone or in combination: TWEAK (Peprotech, catalog number AF-310-06) TNFα (Peprotech, catalog number AF-300-01A) TNFβ (Peprotech, Catalog No. 300-01B) LIGHT (Peprotech, Catalog No. 310-09B) Trimer of one Lymphotoxin α and two Lymphotoxin β molecules (R&D Systems, catalog number 8884-LY-025 / CF)

[0133] As a control, an equal volume of PBS(-) containing no TNF superfamily proteins was added. After addition, the cells were cultured for 24 hours. After that, HK-2 cells were harvested and RT-PCR was performed as described above to examine the expression level of Klotho. The results are shown in Figure 5. It was demonstrated that the combination of TWEAK and TNFβ further increased the expression level of Klotho.

[0134] 7. Example 6 (Changes in Klotho Expression by Administration of Culture Supernatant and Effects of Antibodies to the TNF Superfamily) The results of Example 2 above showed that the culture supernatant increased the expression level of Klotho. Furthermore, the results of Examples 3 to 5 showed that proteins of the TNF superfamily increased the expression level of Klotho. Therefore, we investigated which components of the culture supernatant contribute to the increase in the expression level of Klotho. Specifically, we tested the hypothesis that the culture supernatant contains proteins of the TNF superfamily, and that these proteins contribute, at least in part, to the increase in the expression level of Klotho.

[0135] The cell line HK-2 was prepared. HK-2 was plated in each well of a 24-well plate (Corning, 3337) at 25,000 cells / cm. 2 The cells were then cultured in serum-free medium, Keratinocyte-SFM (ThermoFisher, catalog number 17005042), for 24 hours (37°C, 5% CO2).

[0136] The medium was then replaced with the aforementioned culture supernatant. Specifically, the medium was replaced with either adipose tissue-derived mesenchymal stem cell culture supernatant AD-CM (Pattern 2) or umbilical cord tissue-derived mesenchymal stem cell culture supernatant UC-CM (Pattern 3). As a control (Pattern 1), the medium was replaced with a medium specifically designed for culturing mesenchymal stem cells. This allowed us to focus exclusively on factors secreted from adipose tissue- or umbilical cord tissue-derived mesenchymal stem cells. Furthermore, each of Patterns 1 to 3 was further divided into two subpatterns. Specifically, a control IgG antibody (BioLegend, catalog number 401302) was added to one of the cultures (Subpattern 1), and an anti-Tweak antibody (BioLegend, catalog number 308302) was added to the other (Subpattern 2). The antibody was added to a final concentration of 20 μg / mL.

[0137] Culture was continued for 24 hours. After that, HK-2 cells were collected, and RT-PCR was performed as described above to examine the expression level of Klotho. The results are shown in Figure 6. When cells were treated with the culture supernatant and an IgG antibody was added as a control, the expression level of Klotho was shown to increase, as in Example 2 above. However, when cells were treated with the culture supernatant and an anti-Tweak antibody was added, the degree of increase in the expression level of Klotho was shown to be suppressed compared to subpattern 1. This indicates that Tweak in the culture supernatant contributes, at least in part, to the increase in the expression level of Klotho.

[0138] 8. Example 7 (Enhancing effect by combination with vitamin D3) Next, we investigated the enhancing effect of combining it with active vitamin D3.

[0139] The cell line HK-2 was prepared. HK-2 was plated in each well of a 24-well plate (Corning, 3336) at 25,000 cells / cm. 2 The cells were then cultured in serum-free medium, Keratinocyte-SFM (ThermoFisher, catalog number 17005042), for 24 hours (37°C, 5% CO2).

[0140] The medium was then replaced with the aforementioned culture supernatant. Specifically, the medium was replaced with either adipose tissue-derived mesenchymal stem cell culture supernatant (AD-CM) or umbilical cord tissue-derived mesenchymal stem cell culture supernatant (UC-CM). As a control, the medium was replaced with a medium specifically designed for culturing mesenchymal stem cells. This allowed us to focus solely on factors secreted from adipose tissue- or umbilical cord tissue-derived mesenchymal stem cells. At the same time, a predetermined amount of active vitamin D3 was added to the culture medium to achieve final concentrations of 0 μM, 0.1 μM, or 1.0 μM. After 24 hours of incubation, the HK-2 cells were harvested and subjected to RT-PCR as described above to examine the expression level of Klotho. The results are shown in Figure 7. Treatment with the culture supernatant demonstrated an increase in Klotho expression, as in Example 2. However, treatment with the culture supernatant and the addition of vitamin D3 (calcitriol) resulted in a more significant increase in Klotho expression. This indicates that the combination with vitamin D3 contributes to the increase in Klotho expression.

[0141] Specific embodiments of the invention have been described above. The above embodiments are merely illustrative examples, and the present invention is not limited to these embodiments. For example, technical features disclosed in one of the above embodiments may be applied to other embodiments. Furthermore, unless otherwise specified, for a particular method, the order of some steps may be interchanged with other steps, and additional steps may be added between two specific steps.

[0142] For example, an embodiment described with respect to a protein of the TNF superfamily may be modified by substituting a protein having sequence similarity in amino acid sequence to a protein of the TNF superfamily. Alternatively, an embodiment described with respect to a protein of the TNF superfamily may be modified by substituting a desired nucleic acid. The desired nucleic acid may be a nucleic acid encoding a protein of the TNF superfamily or a nucleic acid encoding a protein having sequence similarity in amino acid sequence to a protein of the TNF superfamily.

[0143] Also, the use of a composition for a particular purpose may be modified by substituting the use of a protein belonging to the TNF superfamily for a particular purpose.

[0144] The scope of the invention is defined by the claims.

Claims

1. A composition for enhancing expression of the human Klotho / KL gene, the composition comprising at least a portion of a protein belonging to the TNF superfamily, or a nucleic acid encoding at least a portion of the protein.

2. The composition of claim 1, wherein the composition comprises one or more of the following (1) to (3): (1) A protein produced by the gene TNFSF12 (Tumor Necrosis Factor ligand superfamily member 12) / TWEAK (TNF-related weak inducer of apoptosis), which contains the amino acid sequence 97 to 249 including the extracellular domain; (2) A protein containing a sequence having 80% or more sequence similarity to the amino acid sequence of the protein of (1) above. (3) A nucleic acid comprising a sequence encoding any one of the proteins (1) and (2) above.

3. The composition of claim 1, wherein the composition comprises one or more of the following (4) to (6): (4) A protein produced from the gene TNFSF2 (Tumor Necrosis Factor ligand superfamily member 2) / TNF (Tumor Necrosis Factor) / TNFα, which contains the amino acid sequence 77 to 233 including the extracellular domain; (5) A protein containing a sequence having 80% or more sequence similarity to the amino acid sequence of the protein of (4) above. (6) A nucleic acid comprising a sequence encoding any one of the proteins (4) and (5) above.

4. The composition of claim 1, wherein the composition comprises one or more of the following (7) to (9): (7) A protein comprising the amino acid sequence 35 to 205 of the proteins produced from the gene TNFSF1 (Tumor Necrosis Factor ligand superfamily member 1) / TNFβ (Tumor Necrosis Factor β) / Lymphotoxin α; (8) A protein containing a sequence having 80% or more sequence similarity to the amino acid sequence of the protein of (7) above. (9) A nucleic acid comprising a sequence encoding any one of the proteins (7) and (8) above.

5. The composition of claim 1, wherein the composition comprises one or more of the following (10) to (12): (10) A protein produced from the gene TNF14 (Tumor Necrosis Factor ligand superfamily member 14) / LIGHT, comprising an amino acid sequence of 64 to 240 including an extracellular domain; (11) A protein containing a sequence having 80% or more sequence similarity to the amino acid sequence of the protein of (10) above. (12) A nucleic acid comprising a sequence encoding any one of the proteins (10) and (11) above.

6. The composition of claim 1, comprising a heterotrimeric protein of lymphotoxin α and lymphotoxin β.

7. The composition of claim 6, wherein the heterotrimeric protein consists of one lymphotoxin α and two lymphotoxin β.

8. The composition according to claim 1, comprising a culture supernatant obtained by culturing mesenchymal stem cells and / or a secretion product of the mesenchymal stem cells.

9. The composition according to claim 8 , comprising a culture supernatant obtained by serum-free culturing of mesenchymal stem cells and / or a secretion product of the mesenchymal stem cells.

10. 10. The composition of claim 1, further comprising vitamin D3.

11. 10. The composition of claim 1, administered for anti-aging purposes.

12. 10. The composition of claim 1, wherein the composition is administered to treat any one or more of the following conditions: (1) Neurodegenerative disease and / or dementia (2) Cancer growth (3) Hyperphosphatemia (4) Chronic kidney disease (5) Renal fibrosis (6) Osteoporosis (7) Arterial calcification (8) High blood pressure (9) cardiac hypertrophy and / or fibrosis (10) Ischemic myocardial infarction (11) pulmonary fibrosis (12) Chronic obstructive pulmonary disease (13) Beta cell decrease (14) Decreased insulin (15) Increased diabetic lesions

13. 2. Use of at least a portion of a protein belonging to the TNF superfamily, or a nucleic acid encoding at least a portion of said protein, for the manufacture of the composition of claim 1.

14. 13. A method for treating any one of the conditions described in claim 12 in a subject in need thereof, comprising administering a therapeutically effective amount of the composition of claim 1.

15. 13. At least a portion of a protein belonging to the TNF superfamily, or a nucleic acid encoding at least a portion of said protein, for the treatment of any one of the conditions defined in claim 12 in a subject in need thereof.

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