Composition for promoting Klotho expression and related invention

Abstract

The present disclosure aims to provide a novel composition for increasing the expression of the Klotho gene and use thereof. [Solution] In one aspect, the present disclosure provides 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.

Description

[Technical field]

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

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

[0003] Numerous studies have been conducted using experimental 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 of a pair of chromosomes) in which the expression level of the Klotho gene is drastically reduced due to mutations in the gene, are small in body size and die within about 2 to 5 months. Many studies have concluded that the cause of death is not stunted growth, but rapid 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 accumulation of phosphate in the kidney, 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, and that these diseases are induced when Klotho expression is decreased (Non-Patent Document 4). In other words, it has been suggested that Klotho may normally suppress the onset of these diseases.

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

[0007] Therefore, an approach such as that described in Patent Documents 1 and 2, in which the expression of endogenous Klotho genes is promoted to continue production even after the protein is degraded, is considered 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] JP 2023-175790 A 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 is also expected to have the effect of extending lifespan, which was the original background of the discovery. On the other hand, due to the extreme instability of the Klotho protein and the difficulty of mass production, it is difficult to administer the protein itself. Furthermore, although some prior art has been advancing the discovery and development of compounds that increase the expression of the Klotho gene, until now, substances that increase the expression of Klotho in actual humans have not been known.

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

[0012] As a result of intensive research, the inventors focused on culture supernatant obtained from mesenchymal stem cells. After collecting samples from 10 healthy volunteers, they administered culture supernatant obtained from mesenchymal stem cells by drip infusion, and after 2 weeks, they took blood samples again. The amount of Klotho protein in the blood before and after the administration was compared using ELISA. The amount of Klotho protein was statistically significantly increased by the administration of culture supernatant obtained from mesenchymal stem cells by drip infusion.

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

[0014] The invention has been 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 any 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 having 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) A composition according to the first or second aspect of the present invention, comprising any 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 having 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. (Invention 4) A composition according to any one of inventions 1 to 3, comprising any 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, the protein containing amino acid sequence 35 to 205; (8) A protein having 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) A composition according to any one of Inventions 1 to 4, comprising any 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, the protein containing the amino acid sequence 64 to 240 including the extracellular domain; (11) A protein having 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 culture of mesenchymal stem cells and / or a secretion product of the mesenchymal stem cells. (Invention 10) 10. The composition according to any one of claims 1 to 9, further comprising vitamin D3. (Invention 11) A composition according to any one of claims 1 to 10, which is administered for anti-aging purposes. (Invention 12) 12. A 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) Increase in 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 a composition according to any one of Inventions 1 to 12. (Invention 14) A method for treating any one of the conditions 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 1-12. (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 the treatment of any one of the conditions described in invention 12 in a subject in need thereof. Effect of the Invention

[0015] The above invention is 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). Or, the composition contains a culture supernatant obtained by culturing mesenchymal stem cells and / or a cell secretion product. By administering these compositions, the expression of Klotho can be increased. [Brief description of the drawings]

[0016] [Figure 1] This graph shows the quantitative comparison of serum Klotho protein levels by ELISA before and after intravenous administration of culture supernatant obtained by culturing mesenchymal stem cells in healthy subjects. 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. [Diagram 2]The expression of Klotho gene in human renal proximal tubule cell line HK-2 is shown. 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.). The control is a sample to which the medium used for culturing mesenchymal stem cells (sf-DOT medium, MIFI medium, RM medium) was added. The gene amount (mRNA amount) was quantified by quantitative PCR (qPCR). Compared to the control, the expression amount of Klotho gene was significantly increased in HK-2 cells to which AD-CM or UC-CM was added (test method: one-way ANOVA with bonferroni post hoc test). [Diagram 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 amount of PBS(-) was added. When each recombinant protein was added at the concentration (unit: ng / mL) indicated on the graph, the expression of the Klotho gene increased in a concentration-dependent manner. [Figure 4] The expression of Klotho gene in human renal proximal tubule cell line HK-2 is shown. HK-2 was cultured in a culture medium containing 10% FBS (Fetal Bovine Serum) beforehand, and then 10ng / ml TWEAK was added. When FBS (FBS#1) purchased from SIGMA-ALDRICH (catalog number F7524-500ML) was used, the expression of Klotho gene increased by the addition of TWEAK as in the past. However, when FBS (FBS#2) purchased from ThermoFisher Scientific (catalog number 10437-028) and FBS (FBS#3) purchased from BIOWEST (catalog number S1560) were used, the expression of Klotho gene decreased by the addition of TWEAK. This result suggests that the type and lot of serum used have a strong influence on the expression of Klotho gene by TWEAK. [Diagram 5]The expression of Klotho gene in human renal proximal tubule cell line HK-2 is shown. The control is a sample with an equal amount of PBS(-). When TWEAK and TNFβ recombinant proteins were added alone or together, the expression of Klotho gene was significantly increased by adding them together (test method: one-way ANOVA with Bonferroni post hoc test). The value written next to each protein indicates the final concentration (ng / mL) after addition. For example, "LIGHT 1" means that LIGHT protein was added to a final concentration of 1ng / mL. "LTN" means a trimer of one Lymphotoxinα and two Lymphotoxinβ. "L+LTN" means that in addition to TWEAK, LIGHT and LTN were further combined. "L+Tβ" means that in addition to TWEAK, LIGHT and TNFβ were further combined. "L+LTN+Tβ" means that in addition to TWEAK, LIGHT, LTN, and TNFβ were further combined. In "L+LTN", "L+Tβ", and "L+LTN+Tβ", each protein was added to a final concentration of 1 ng / mL. [Figure 6] The figure shows the expression of the Klotho gene in the human renal proximal tubule cell line HK-2. The control is a medium supplemented with the medium used for culturing mesenchymal stem cells, and CM (Conditioned Medium) is 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 the expression of the Klotho gene, whereas in the presence of TWEAK neutralizing antibody, the increase in the Klotho gene due to the addition of CM was suppressed. This result strongly suggests that the expression of the Klotho gene is increased via TWEAK in the CM. [Figure 7]The expression of Klotho gene in human renal proximal tubule cell line HK-2 is shown. The control is a medium added with the medium used for culturing mesenchymal stem cells, and CM is 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 addition, active vitamin D3 (Calcitriol) was added, and the effect of combining with the culture supernatant was examined. The culture supernatant alone promoted the expression of Klotho gene. However, the combination with active vitamin D3 further promoted the expression of Klotho gene. The degree of increase by combining with active vitamin D3 was more remarkable than the increase when active vitamin D3 was added without using 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 is shown. The underlined portion indicates the linker sequence. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Hereinafter, specific embodiments for carrying out the present invention will be described. 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 "increasing the expression of the Klotho gene", "enhancing the expression of the Klotho gene", "enhancing the expression of the Klotho gene", "increasing the expression of the Klotho gene", and the like are used interchangeably. These terms include the purpose of improving a specific disease related to the Klotho gene. Furthermore, the term is not limited to therapeutic purposes. For example, the term includes the purpose of preventing the onset of a specific disease even if the person does not have the disease. Furthermore, the term includes the purpose of improving or preventing symptoms such as aging, which are not defined as diseases in current medical science but which affect the quality of life of the elderly in particular.

[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 natural 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 to slow down the progression of aging. Aging encompasses various physical symptoms. For example, aging may encompass any one or more of the following symptoms: development of skin spots, development of skin wrinkles, development of tumors, development of polyps, loss of bone density, muscle weakness, loss of vision, loss of appetite, loss of cognitive function, loss of lung function due to fibrosis, loss of kidney function, loss of heart function, loss of liver function, loss of pancreatic function (e.g., insulin production ability), loss of function due to increased fibrosis in multiple other organs, etc.

[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 carbons in the side chain. Therefore, even if the amino acids in each group are changed, there is a high probability that the function of the protein can be maintained.

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

[0026] As used herein, the term "mesenchymal stem cell" (MSC) refers to one of the stem cells present in an adult body, and is capable of differentiating into mesodermally derived tissues such as bone, cartilage, blood vessels, and cardiomyocytes. Mesenchymal stem cells may be derived from any 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 milk teeth), and the like.

[0027] As used herein, the term "culture supernatant" refers to a substance obtained directly or indirectly by culturing the above-mentioned 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: By carrying out filtration and Frozen Drying and turning into powder After being converted into powder, it is processed into a specific shape (e.g., tablet, pill, etc.) To change into a semi-solid (gel-like) state Add any additional ingredients

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

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

[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 do not contain serum. 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 the expression of 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 part of a protein means that the protein has a region of sufficient length to exert its function. For example, at least a part of a protein belonging to the TNF superfamily means that the protein has a sufficient function to bind to a receptor as a ligand. In a further preferred example, at least a part of a protein belonging to the TNF superfamily may have a function to enhance the expression of human Klotho / KL gene.

[0034] The nucleic acid encoding at least a part of the protein may be in the form of DNA or may be included in the composition in the form of RNA. The nucleic acid encoding at least a part of the protein may be included in the form of a vector (e.g., an expression vector). The nucleic acid encoding at least a part of the protein may be provided in a form contained in 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 of 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), the sequence corresponding 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 (1) and (2) above.

[0041] Among the proteins belonging to the TNF superfamily, TNFSF12 in particular has a high effect of enhancing the expression of the human Klotho / KL gene.

[0042] Compositions may also include combinations of TNFSF12 (or proteins with sequence similarity to TNFSF12) with other proteins (eg, other proteins that belong to the TNF superfamily). The same is true for 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" or "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 an amino acid sequence of 77 to 233 of the protein (SEQ ID NO: 2), the sequence corresponding 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 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) / Lymphotoxinα (hereinafter referred to as "TNFSF1" or "TNFβ" or "TNFbeta") has biological activity similar to TNF-α and is known as a ligand for TNFR1 and TNFR2.

[0048] In a preferred embodiment, the composition of the present disclosure contains (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 amino acids 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 further preferred embodiment, the heterotrimeric protein comprises one Lymphotoxin α and two Lymphotoxin β. The amino acid sequences of Lymphotoxin α and Lymphotoxin β may be sequences comprising sequences represented by, for example, 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 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 include a combination of multiple proteins of the TNF superfamily. In particular, the combination of TWEAK and a specific protein of the TNF superfamily is particularly preferred from the viewpoint of enhancing the expression of human Klotho / KL gene. For example, any of the following combinations is preferred. TWEAK+TNFβ TWEAK+TNFβ+LIGHT -TWEAK+TNFβ+LIGHT+1 Lymphotoxinα and 2 Lymphotoxinβ trimers

[0057] 2-2.Other In another embodiment, the composition may further contain vitamin D3 in addition to the 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 the protein belonging to the TNF superfamily, the expression of Klotho can be enhanced.

[0058] Vitamin D3 may be active or inactive. Inactive vitamin D3 can typically be converted to active by metabolism. Some forms of administration may reach desired tissue (e.g., kidney, etc.) without 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 4.16ng / g (10 picomoles / g) or more (i.e., when the weight of the entire composition is 1g, the composition contains 4.16ng or more (10 picomoles or more) of vitamin D3).Preferably, the amount of vitamin D3 in the composition may be 41.6ng / g or more (100 picomoles / g or more), more preferably 416ng / 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 liquids, they may be in the form of syrups, emulsions, suspensions, etc. In addition, in the case of liquids, they may be stored in vials, drip bags, pre-filled syringes, etc. In the case of solids, they may be in the form of tablets, pills, capsules, powders, granules, suppositories, etc.

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

[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 includes 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 the above-mentioned proteins belonging to the TNF superfamily. 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 these ranges.

[0065] As mentioned above, the culture supernatant itself may not contain serum. 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 not contain serum.

[0066] 3-2. When culture supernatant is not included In another embodiment, the composition of the present disclosure does not include the culture supernatant of mesenchymal stem cells. Preferably, the composition of the present disclosure does not include the culture supernatant of mesenchymal stem cells, and does not include secretions of the mesenchymal stem cells. In this case, the protein belonging to the TNF superfamily must be obtained from a source other than the culture supernatant of mesenchymal stem cells. For example, the protein belonging to the TNF superfamily isolated from a source other than the culture supernatant of mesenchymal stem cells (for example, the protein may be purified after being expressed in large amounts by genetic engineering) 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 so that CO2 is 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 is 168 hours or less.

[0069] The type of medium is not particularly limited, and the above-mentioned media 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 aspirated and collected. Alternatively, the contents of the culture vessel may be transferred to another vessel and centrifuged, and the supernatant may be aspirated and collected. This supernatant may then be regarded as the culture supernatant.

[0072] The culture supernatant is then mixed to obtain a composition suitable for administration. The procedures required for this purpose are 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 Vitamin D3 to obtain a composition suitable for administration.

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

[0077] Other components may be added in a similar manner. Alternatively, in addition to the protein belonging to the TNF superfamily secreted from mesenchymal stem cells, one or more types of proteins belonging to the TNF superfamily described above may be added from the outside 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 the expression of human Klotho / KL gene. The production method may be the above-mentioned production method.

[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 human Klotho / KL gene, which 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] The step may include, for example, preparing the protein by purchasing a commercially available TNF superfamily protein, or may include expressing the protein by genetic engineering (for example, by using an expression vector encoding the protein of the TNF superfamily) and purifying the protein.

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

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

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

[0085] In another embodiment, the present invention is a method for enhancing the expression of human Klotho / KL gene. The method includes a step of administering an effective amount of a composition to a subject in need of enhanced expression of Klotho / KL gene. Here, the composition may include a culture supernatant of mesenchymal stem cells and / or a protein belonging to the TNF superfamily. In addition, the "effective amount" means 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 drip 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 step of administering 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, both may be contained in separate compositions.

[0087] The subject to which the drug 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 drug is administered is a human. The subject to which the drug is administered may be healthy or may be suffering from a disease.

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

[0089] The above-mentioned dosage refers to the total dosage per day. Therefore, the number of times of administration per day may be one or may be divided into several times. For example, the number of times of administration per day may be two, three, four or more.

[0090] The administration period is not particularly limited. The administration period 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 (for example, 1 day to 90 days, 90 days to 180 days, etc.).

[0091] The administration form 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, drip infusion, application to the skin, spraying to the skin, etc. In a preferred embodiment, the administration route is preferably parenteral administration. This is because the active ingredient is a protein belonging to the TNF superfamily, and if the protein is administered orally, it may be decomposed by the digestive juices of 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 for treating 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) Increase in diabetic lesions

[0093] Thus, in one embodiment, the present disclosure relates to a method for treating any one or more of the above-mentioned symptoms (1) to (15), comprising the step of 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 above-mentioned symptoms (1) to (15) and is in need of treatment for the symptoms.

[0094] It is known that the above-mentioned symptoms (1) to (15) are all related to the increase or decrease in the expression of Klotho. Therefore, by administering the composition of the present disclosure in one embodiment, the expression of Klotho can be increased, and thus the above-mentioned symptoms (1) to (15) can be improved.

[0095] In another embodiment, the present disclosure relates to a method for anti-aging, and the method comprises administering a composition comprising a protein belonging to the TNF superfamily in an amount effective for preventing aging. It is known from experiments using aging model mice and the like that there is a relationship between the increase or decrease in Klotho expression and aging phenotype. Therefore, by administering the composition of the present disclosure in one embodiment, it is possible to 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 used to treat any one or more of the symptoms of (1) to (15) or for anti-aging.

[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 use in anti-aging. EXAMPLES

[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 mesenchymal stem cells derived from adipose tissue Subcutaneous adipose tissue was isolated from patients who were scheduled to undergo regenerative medicine using adipose tissue-derived mesenchymal stem cells. The subcutaneous adipose tissue was used as the raw material for the preparation of cells for administration. The remaining portion of the subcutaneous adipose tissue was used for primary culture. In addition, consent for research use was obtained from the patients in advance.

[0100] The subcutaneous adipose tissue was subjected to centrifugation (400×g for 5 minutes) and separated into three layers. Specifically, the tissue was separated into three layers, namely, a lipid fraction, an adipose tissue fraction, and an aqueous fraction, from the top. The middle layer of the adipose tissue fraction was left, and the top and bottom layers were discarded. A 0.15% collagenase enzyme solution was added to the remaining adipose tissue fraction in an amount of 4 times the tissue weight. The tissue was permeated at 37°C for 1 hour and subjected to enzyme treatment. After the adipose tissue was dispersed by the enzyme treatment, the adipose tissue was subjected to centrifugation (400×g for 5 minutes). The precipitated fraction was suspended in 30 mL of PBS(-) solution as a stromal vascular cell fraction containing mesenchymal stem cells. The suspension was then passed through a cell strainer (mesh size 70 μm diameter), and tissue residues captured by the cell strainer were discarded. The flow-through fraction was then centrifuged again (400×g for 5 min), 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 in a T25 flask (CellBIND; Corning, 3289) and placed in an incubator (37°C, 5% CO2) to initiate primary culture.

[0101] The medium was replaced once every three days. The supernatant was discarded, and the cells growing on the bottom of the flask were selectively grown. 2 mL of enzyme solution (TrypLE Express; Thermo Fisher Scientific, 12604021) was added to the cells in the T-25 flask that had grown to semi-confluence, and the cells were detached from the bottom of the flask (37°C, left to stand for 5 minutes). The cells were diluted with PBS(-) and centrifuged (400×g for 5 minutes). The precipitated cells were suspended in sf-DOT culture medium, and a portion was taken and the cell count was measured using the trypan blue staining method. The cells suspended in sf-DOT were seeded in a new T75 flask (CellBIND; Corning, 3290) and left to stand in an incubator (37°C, 5% CO2) for subculture (P0→P1). Subculture was then performed using the same procedure to obtain the required number of cells (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 in advance. After delivery, the umbilical cord was washed with PBS(-) and cut into pieces of about 3 mm with a scalpel. A 0.15% collagenase enzyme solution was added in an amount four times the tissue weight. The tissue was permeated 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(-) solution as the stromal vascular cell fraction containing mesenchymal stem cells. The suspension was then passed through a cell strainer (mesh size 70 μm diameter), and tissue residues captured by the cell strainer were 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% CO 2 ) to initiate primary culture.

[0103] The medium was replaced once every three days. The supernatant was discarded, and the cells growing on the bottom of the flask were selectively grown. 2 mL of enzyme solution (TrypLE Express; Thermo Fisher Scientific, 12604021) was added to the cells in the T-25 flask that had grown to semi-confluence, and the cells were detached from the bottom of the flask (37°C, left to stand for 5 minutes). The cells were diluted with PBS(-) and centrifuged (400×g for 5 minutes). The precipitated cells were suspended in sf-DOT culture medium, and a portion was taken and the cell count was measured using the trypan blue staining method. The cells suspended in sf-DOT were seeded in a new T75 flask (CellBIND; Corning, 3290) and left to stand in an incubator (37°C, 5% CO2) for subculture (P0→P1). Subculture was then performed using the same procedure to obtain the required number of cells (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 (culture supernatant derived from sf-DOT) on the fourth day, the cells were washed once with PBS(-), and then RM medium was added at 12 mL per T75 flask, and further cultured for 2 days in an incubator (37°C, 5% CO2). The culture supernatant was then collected (culture supernatant derived from RM medium). P1 adipose tissue or umbilical cord tissue-derived mesenchymal stem cells were similarly cultured in MIFI medium, and culture supernatant derived from MIFI medium 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 frozen and stored at -20°C or below until use in analysis.

[0106] 1-4. Expression analysis methods Total RNA was extracted from 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 10μl in total

[0109] The above mixture was treated under the following conditions using a Veriti 96 well Thermal Cycler from Applied Biosystems. 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 solution 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 8.2μl cDNA (10x dilution) 1μl

[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℃ to 95℃ in 0.5℃ increments, and the temperature was held for 5 seconds each time 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 ydrogenase) was used. GAPDH Forward primer: 5'-AGCACATCGCTCAGACAC-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'-AGCTCTCAAGCCACATAC-3' (SEQ ID NO: 10) Klotho reverse primer: 5'-CTGATCTGCAGCTAACGATAGA-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 expression level of each gene obtained by quantitative PCR, which was further normalized by dividing it by the expression level of GAPDH. Furthermore, the expression level under control conditions in which the supernatant was not treated was normalized to "1". It was confirmed by melting curve that all PCR products amplified using the above primers were single (i.e., multiple types of sequences were not amplified with the same primer).

[0117] 2. Example 1 (Changes in serum Klotho levels following administration of culture supernatant) Blood samples were collected from 10 adults in advance, and the amount of Klotho protein in the serum was quantified by ELISA (R&D Systems, catalog number DY5334-05). Next, the culture supernatant from the RM medium of mesenchymal stem cells derived from umbilical cord tissue was prepared by the above-mentioned procedure. The culture supernatant was then administered intravenously (1 mL / kg). Blood samples were collected again two weeks after administration, and the amount of Klotho protein in the serum was quantified by ELISA. The results are shown in Figure 1. The average concentration of Klotho in the serum before administration of the culture supernatant was 320.0 pg / mL. On the other hand, the average concentration of Klotho in the serum after administration of the culture supernatant was 427.7 pg / mL. A paired T test was performed on both values ​​to confirm that there was a significant difference.

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

[0119] 3. Example 2 (Changes in Klotho Expression by Administration of Culture Supernatant) Next, we used the kidney-derived cell line HK-2 to examine the change in Klotho expression by administration of the culture supernatant. 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 cultured 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] Then, the medium was replaced with the above-mentioned culture supernatant. Specifically, the medium was replaced with the culture supernatant AD-CM of adipose tissue-derived mesenchymal stem cells or the culture supernatant UC-CM of umbilical cord tissue-derived mesenchymal stem cells. As a control, the medium was replaced with a medium for culturing mesenchymal stem cells. This makes it possible to focus only on factors secreted from adipose tissue- or umbilical cord tissue-derived mesenchymal stem cells. After the medium replacement, the culture was continued for 24 hours. Then, HK-2 was collected and RT-PCR was performed by the above-mentioned method to examine the expression level of Klotho. The results are shown in Figure 2. Compared to the control that was not treated with the culture supernatant, it was shown that the mRNA expression level of Klotho was increased in both the culture supernatant of adipose tissue-derived mesenchymal stem cells and the culture supernatant of umbilical cord tissue-derived mesenchymal stem cells. Furthermore, this effect did not vary depending on the type of culture medium (Medium 1: sf-DOT medium, Medium 2: MIFI medium, Medium 3: RM medium (all BioMimetics Sympathies, Inc.)), and the effect was present in the secretions of mesenchymal stem cells derived from both adipose tissue and umbilical cord tissue, 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 inventor further investigated the components contained in the culture supernatant and came up with the hypothesis that the TNF superfamily proteins contribute to the increase in the mRNA expression level of Klotho. Therefore, instead of treating with the culture supernatant, treatment was performed with various TNF superfamily proteins.

[0123] The cell line HK-2 was prepared. HK-2 was cultured 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) A trimer of one Lymphotoxin α and two Lymphotoxins β (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. Cultivation was then continued for 24 hours. HK-2 cells were then collected and RT-PCR was carried out as described above to examine the expression level of Klotho. The results are shown in Figure 3. Compared to the control where only PBS(-) was added, it was shown that the expression level of Klotho mRNA was increased by TNF superfamily proteins.

[0127] 5. Example 4 (Changes in Klotho Expression by Serum) Next, we examined 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 cultured in each well of a 24-well plate (Corning, 3337) at 25,000 cells / cm. 2The cells were seeded in a serum-free medium of Keratinocyte-SFM (ThermoFisher, Catalog No. 17005042) and cultured in serum-free medium of DMEM (SIGMA, D8900) + 10% FBS (three different products from different companies were used; 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 (37°C, 5% CO2). Then, TWEAK and TNFα were added to a final concentration of 10ng / ml, and the culture was continued for another 24 hours. Then, HK-2 were collected and RT-PCR was performed by the method described above to examine the expression level of Klotho. The results are shown in Figure 4. The Klotho gene expression was changed by the type of serum. This result suggests that the type of serum used has a strong influence on the 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 cultured 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) A trimer of one Lymphotoxin α and two Lymphotoxins β (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. HK-2 cells were then collected and RT-PCR was performed as described above to examine the expression level of Klotho. The results are shown in Figure 5. It was shown that the expression level of Klotho was further increased by combining TWEAK and TNFβ.

[0134] 7. Example 6 (Changes in Klotho expression by administration of culture supernatant and effects of antibodies against the TNF superfamily) The results of Example 2 above showed that the expression level of Klotho was increased by the culture supernatant. The results of Examples 3 to 5 also showed that the expression level of Klotho was increased by a protein of the TNF superfamily. Therefore, we investigated which component of the culture supernatant contributes to the increase in the expression level of Klotho. Specifically, we verified the hypothesis that the culture supernatant contains a protein of the TNF superfamily and that the protein contributes, at least in part, to the increase in the expression level of Klotho.

[0135] The cell line HK-2 was prepared. HK-2 was cultured 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] Thereafter, the medium was replaced with the above-mentioned culture supernatant. Specifically, the medium was replaced with the culture supernatant AD-CM (pattern 2) of mesenchymal stem cells derived from adipose tissue, or the culture supernatant UC-CM (pattern 3) of mesenchymal stem cells derived from umbilical cord tissue. As a control (pattern 1), the medium was replaced with a medium for culturing mesenchymal stem cells. This makes it possible to focus only on factors secreted from mesenchymal stem cells derived from adipose tissue or umbilical cord tissue. Furthermore, each of patterns 1 to 3 was further divided into two subpatterns. Specifically, an IgG antibody (manufactured by BioLegend, catalog number 401302) was added as a control to one of the patterns (subpattern 1), and an anti-Tweak antibody (manufactured by BioLegend, catalog number 308302) was added to the other (subpattern 2). The antibody was added to a final concentration of 20 μg / mL.

[0137] The culture was continued for 24 hours. Thereafter, HK-2 was collected, and RT-PCR was performed by the above-mentioned method to examine the expression level of Klotho. The results are shown in FIG. 6. When treated with the culture supernatant and IgG antibody was added as a control, the expression level of Klotho was shown to increase, as in Example 2 above. However, when treated with the culture supernatant and 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 partially 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 cultured 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] Then, the medium was replaced with the above-mentioned culture supernatant. Specifically, the medium was replaced with the culture supernatant AD-CM of adipose tissue-derived mesenchymal stem cells or the culture supernatant UC-CM of umbilical cord tissue-derived mesenchymal stem cells. As a control, the medium was replaced with a medium for culturing mesenchymal stem cells. This makes it possible to focus only on factors secreted from adipose tissue-derived or umbilical cord tissue-derived mesenchymal stem cells. At the same time, a predetermined amount of active vitamin D3 was added to a final concentration of 0 μM, 0.1 μM, or 1.0 μM. After continuing the culture for 24 hours, HK-2 was collected and RT-PCR was performed by the above-mentioned method to examine the expression level of Klotho. The results are shown in FIG. 7. When treated with the culture supernatant, it was shown that the expression level of Klotho increased, as in Example 2 above. However, when treated with the culture supernatant and vitamin D3 (Calcitriol) was added, the degree of increase in the expression level of Klotho was even more remarkable. This indicates that the combination with vitamin D3 contributes to the increase in the expression level of Klotho.

[0141] Above, specific embodiments of the invention have been described. The above embodiments are merely specific examples, and the present invention is not limited to the above embodiments. For example, a technical feature disclosed in one of the above embodiments can be applied to other embodiments. In addition, unless otherwise specified, for a particular method, the order of some steps can be replaced with other steps, and an additional step can be added between two specific steps.

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

[0143] The use of a composition for a particular purpose may also 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, The composition comprises any one or more of the following (1) to (9): (1) A protein produced from the gene TNFSF12 (Tumor Necrosis Factor ligand superfamily member 12) / TWEAK (TNF-related weak inducer of apoptosis), which contains an extracellular domain and an amino acid sequence of 97 to 249, and which contains the amino acid sequence represented by SEQ ID NO: 1; (2) A nucleic acid comprising a sequence encoding the protein of (1) above. (3) A protein produced from the gene TNFSF2 (Tumor Necrosis Factor ligand superfamily member 2) / TNF (Tumor Necrosis Factor) / TNFα, which contains an amino acid sequence of 77 to 233 including an extracellular domain, and which contains the amino acid sequence represented by SEQ ID NO: 2; (4) A nucleic acid comprising a sequence encoding the protein of (3) above. (5) A protein produced from the gene TNFSF1 (Tumor Necrosis Factor ligand superfamily member 1) / TNFβ (Tumor Necrosis Factor β) / Lymphotoxin α, which contains the amino acid sequence of 35 to 205, and which contains the amino acid sequence represented by SEQ ID NO: 3; (6) A nucleic acid comprising a sequence encoding the protein of (5) above. (7) A protein produced from the gene TNF14 (Tumor Necrosis Factor ligand superfamily member 14) / LIGHT, which contains an extracellular domain and an amino acid sequence from 64 to 240, and which contains the amino acid sequence represented by SEQ ID NO: 4; (8) A nucleic acid comprising a sequence encoding the protein of (7) above. (9) A heterotrimeric protein consisting of one lymphotoxin α and two lymphotoxin β molecules.

2. 2. The composition of claim 1, comprising a culture supernatant obtained by culturing mesenchymal stem cells, and / or a secretion product of the mesenchymal stem cells corresponding to a fraction in the range of 10 kDa to 30 kDa.

3. 3. The composition of claim 2, comprising a culture supernatant obtained by serum-free culture of mesenchymal stem cells, and / or a secretion product of the mesenchymal stem cells corresponding to a fraction in the range of 10 kDa to 30 kDa.

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

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

6. 10. The composition of claim 1, administered to treat any one or more of the following conditions: (1) Neurodegenerative disease and / or dementia (2) Increase in cancer (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) Increase in diabetic lesions

7. 2. 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 the manufacture of a composition according to claim 1.

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