Functionally enhanced stem cells primed with PRL-1 variant and therapeutic composition comprising same

Abstract

The present invention relates to functionally enhanced stem cells and a therapeutic composition comprising same and, more specifically, to functionally enhanced stem cells primed with a PRL-1 variant and a composition for treating metabolic diseases, comprising the functionally enhanced stem cells. According to the present invention, functionally enhanced mesenchymal stem cells can be obtained without artificial gene introduction, and the mesenchymal stem cells functionally enhanced according to the present invention exhibit increased cell growth and suppressed cellular senescence, thereby enabling the preparation of more effective stem cell therapeutics.

Description

Function-enhanced stem cells primed with a PRL-1 variant and a therapeutic composition containing the same

[0001] Cross-reference of related applications

[0002] This application claims priority to Korean Patent Application No. 10-2024-0191746 filed on December 19, 2024, the disclosure of which is incorporated herein by reference in its entirety.

[0003] Sequence list

[0004] The present application includes a sequence listing, which has been submitted electronically in XML format and the entirety thereof is incorporated by reference into the present application. A copy of the said sequence listing, created on December 19, 2025, is named KC25182_SEQUENCE LISTING.xml and has a size of 2.76 kilobytes.

[0005] The present invention relates to function-enhanced stem cells and therapeutic compositions containing the same, and more specifically, to function-enhanced stem cells primed with a PRL-1 variant and compositions for treating metabolic diseases containing function-enhanced stem cells.

[0006] Mesenchymal stem cells (MSCs) are cells that retain stemness and self-renewal capabilities and have the ability to differentiate into various mesenchymal tissues; they can be extracted from tissues such as bone marrow, adipose tissue, umbilical cord blood, synovium, bone tissue, subpatellar fat, and placenta.

[0007] Mesenchymal stem cells possess immunomodulatory capabilities that inhibit the activation and proliferation of T and B lymphocytes, suppress the activity of natural killer (NK) cells, and regulate the functions of dendritic cells and macrophages, making them capable of allotransplantation and xenotransplantation. Recognizing this potential of mesenchymal stem cells, numerous studies are currently underway to utilize them as cell therapies.

[0008] Recently, among studies on mesenchymal stem cells, research is being conducted to overexpress specific factors in mesenchymal stem cells and to use mesenchymal stem cells with overexpressed specific factors as cell therapies, and there are attempts to treat diseases by overexpressing PEDF in mesenchymal stem cells (Korean Registered Patent No. 10-2198942) and by overexpressing PRL-1 in mesenchymal stem cells (Korean Registered Patent No. 10-2216646; Korean Registered Patent No. 10-2314158; Korean Registered Patent No. 10-2159630).

[0009] Meanwhile, PTP4A1 (protein tyrosine phosphatase type ₃), also known as PRL-1 (phosphatase of regenerating liver-1), belongs to a group of 109 mammalian protein tyrosine phosphatases capable of removing phosphorylation of tyrosine residues and serine / threonine residues, and is known to affect cell growth and liver regeneration.

[0010] There is a method of delivering a plasmid containing the PRL-1 gene into cells using an electroporation method called AMAXA to induce overexpression of PRL-1 in mesenchymal stem cells (Korean Registered Patent No. 10-2216646; Korean Registered Patent No. 10-2314158; Korean Registered Patent No. 10-2159630). When genes are delivered directly into cells using such electroporation methods, safety issues may arise, which may become an obstacle to their future use as cell therapy.

[0011] Accordingly, the inventors of the present invention, through diligent efforts to develop a stem cell therapeutic agent that is safer and has higher therapeutic efficacy, confirmed that when a PRL-1 variant is applied to mesenchymal stem cells, cell growth is promoted and mesenchymal stem cells with inhibited cellular senescence and enhanced stem cell capabilities can be obtained, thereby completing the present invention.

[0012] [Prior Art Literature]

[0013] [Patent Literature]

[0014] Korean Registered Patent No. 10-2198942

[0015] Korean Registered Patent No. 10-2216646

[0016] Korean Registered Patent No. 10-2314158

[0017] Korean Registered Patent No. 10-2159630

[0018] The present invention aims to solve one or more or all of the problems of the aforementioned prior art.

[0019] The objective of the present invention is to provide a PRL-1 variant with enhanced cell permeability.

[0020] Another objective of the present invention is to provide a method for producing function-enhanced stem cells using a PRL-1 variant.

[0021] Another objective of the present invention is to provide functionally enhanced stem cells.

[0022] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of metabolic diseases containing functionally enhanced stem cells as an active ingredient.

[0023] Another objective of the present invention is to provide a method for the prevention or treatment of metabolic diseases, comprising administering function-enhancing stem cells to a subject in need thereof.

[0024] The objectives of the present invention are not limited to those mentioned above. The objectives of the present invention will become more apparent from the following description and will be realized by the means and combinations thereof described in the claims.

[0025] A representative configuration of the present invention for achieving the above objective is as follows.

[0026] In one aspect of the present invention, a PRL-1 variant is provided comprising a peptide represented by the amino acid sequence of SEQ ID NO. 2 and a PRL-1 peptide, wherein the cell-permeating peptide is fused to the PRL-1 peptide.

[0027] In some embodiments, the PRL-1 variant may be represented by the amino acid sequence of SEQ ID NO. 1.

[0028] In another aspect of the present invention, a method for producing functionally enhanced stem cells is provided, comprising the following steps:

[0029] (a) a step of enhancing the function of stem cells by treating mesenchymal stem cells with the PRL-1 variant of claim 1; and

[0030] (b) A step of obtaining stem cells with enhanced functions.

[0031] In some embodiments, treatment with the PRL-1 variant may be performed before, during, or after culture of mesenchymal stem cells.

[0032] In some embodiments, mesenchymal stem cells may be derived from tissues selected from the group consisting of placenta, amniotic membrane, umbilical cord blood, umbilical cord, fat, muscle, nerve, and amniotic membrane.

[0033] In another embodiment, the mesenchymal stem cells may be placental-derived stem cells.

[0034] In some embodiments, the placenta-derived stem cells may be placental chorionic valve-derived mesenchymal stem cells obtained by a method comprising the following steps:

[0035] (i) A step of obtaining a chorionic plate membrane from the placenta separated from the mother's body after childbirth;

[0036] (ii) a step of collecting mesenchymal stem cells by scraping the cells inside the chorionic valve obtained in step (i);

[0037] (iii) a step of adding a solution containing trypsin and ethylenediaminetetraacetate to the cells obtained in step (ii) to perform an enzymatic reaction at 20 to 30°C, and adding fetal bovine serum to stop the enzymatic reaction; and

[0038] (iv) A step of centrifuging the reaction solution obtained in step (iii) and culturing the recovered cells in a medium supplemented with fetal bovine serum and antibiotics.

[0039] In some embodiments, the treatment concentration of the PRL-1 variant may be 1 to 100,000 ng / mL.

[0040] In another aspect of the present invention, a functionally enhanced stem cell is provided that is prepared by the method for preparing a functionally enhanced stem cell as described herein and has (i) upregulation of one or more stem cell markers selected from the group consisting of Oct 4, Nanog and Sox2; (ii) downregulation of beta-galactosidase expression; or the gene expression characteristics of (i) and (ii) compared to a mesenchymal stem cell not treated with a PRL-1 variant.

[0041] In another aspect of the present invention, a pharmaceutical composition for the prevention or treatment of metabolic diseases is provided, containing functionally enhanced stem cells as described in this specification as an active ingredient.

[0042] In some embodiments, metabolic diseases may be selected from the group consisting of liver cirrhosis, liver fibrosis, alcoholic liver disease, fatty liver, liver cancer, polycystic ovary syndrome, ovarian failure, obesity, diabetes, dyslipidemia, hypertension, thyroid eye disease, macular degeneration, and diabetic retinopathy.

[0043] In another aspect of the present invention, a PRL-1 variant or a nucleic acid encoding the amino acid sequence of SEQ ID NO. 1 is provided.

[0044] In another aspect of the present invention, a nucleic acid delivery vehicle comprising nucleic acid as described herein is provided.

[0045] In some embodiments, the nucleic acid delivery vehicle may be selected from the group consisting of recombinant vectors, lipid nanoparticles (LNPs), polymer nanoparticles (PNPs), liposomes, exosomes, lipids, colloids, macromolecular complexes, microspheres, nanoparticles, microspheres, beads, oil-in-water emulsions, micelles, and viral vectors.

[0046] In another aspect of the present invention, a cell or microorganism transformed into a nucleic acid or nucleic acid carrier as described herein is provided.

[0047] In another aspect of the present invention, a cell culture composition comprising a PRL-1 variant or a PRL-1 variant represented by the amino acid sequence of SEQ ID NO. 1 is provided.

[0048] In some embodiments, the PRL-1 variant may be present at a concentration of 1 to 100,000 ng / mL.

[0049] In another aspect of the present invention, a method for preventing or treating metabolic diseases is provided, comprising administering functionally enhanced stem cells as described herein to a subject in need thereof.

[0050] In some embodiments, metabolic diseases may be selected from the group consisting of liver cirrhosis, liver fibrosis, alcoholic liver disease, fatty liver, liver cancer, polycystic ovary syndrome, ovarian failure, obesity, diabetes, dyslipidemia, hypertension, thyroid eye disease, macular degeneration, and diabetic retinopathy.

[0051] In another aspect of the present invention, a use for the prevention or treatment of metabolic diseases of functionally enhanced stem cells as described herein is provided.

[0052] The present invention provides a PRL-1 variant with improved cell permeability.

[0053] The PRL-1 variant of the present invention can be treated to mesenchymal stem cells to achieve functional enhancement of mesenchymal stem cells without the introduction of artificial genes into the cells.

[0054] The PRL-1 variant of the present invention has enhanced cell permeability, which can enhance the functional enhancement effect of mesenchymal stem cells.

[0055] The functionally enhanced mesenchymal stem cells of the present invention exhibit increased cell growth and inhibition of cellular aging, and can be utilized as a more effective stem cell therapeutic agent.

[0056] The functionally enhanced mesenchymal stem cells of the present invention exhibit an excellent inhibitory effect on fat production, making them useful for the development of treatments for metabolic diseases.

[0057] Figure 1 illustrates the morphology of mesenchymal stem cells following the introduction of aPRL-1 (a recombinant protein sold by Abcam) or CPP (CPP conjugate recombinant PRL-1) peptides.

[0058] Figure 2 is a graph showing the change in growth rate of mesenchymal stem cells following the introduction of aPRL-1 or CPP peptide.

[0059] Figure 3a shows the staining results of β-galactosidase, an aging marker of mesenchymal stem cells, following the introduction of aPRL-1 or CPP peptide. Figure 3b is a graph showing the β-galactosidase positive signal (multiple) following the introduction of aPRL-1 or CPP peptide.

[0060] Figure 4 shows the flow cytometry (FACS) results of marker factors in mesenchymal stem cells following the introduction of aPRL-1 or CPP peptides.

[0061] Figures 5a to 5d illustrate the results of confirming the expression levels of OCT4, a mesenchymal stem cell capability marker, by RT-PCR following the introduction of aPRL-1 or CPP peptides. Figure 5a is a graph showing the relative OCT4 mRNA expression levels of passage 6 following the introduction of aPRL-1 or CPP peptides. Figure 5b illustrates the RT-PCR results of passage 6 following the introduction of aPRL-1 or CPP peptides. Figure 5c is a graph showing the relative OCT4 mRNA expression levels of passage 7 following the introduction of aPRL-1 or CPP peptides. Figure 5d illustrates the RT-PCR results of passage 7 following the introduction of aPRL-1 or CPP peptides.

[0062] Figures 6a and 6b illustrate the results of confirming the inhibitory effect on lipid production following co-culture with aPRL-1 or CPP peptide-enhanced stem cells after induced oxidative stress with H2O2 in retinal pigment epithelial cell lines. Figure 6a shows the results of fixing cells and BODIPY (Lipid droplet) fluorescence staining after co-culture with retinal pigment epithelial cell lines treated with H2O2. Figure 6b is a graph showing lipid droplets per cell, confirming that the inhibitory effect on lipid synthesis by CPP-primed mesenchymal stem cells was superior compared to the aPRL-1 treatment group.

[0063] Figures 7a and 7b illustrate the results of confirming the inhibitory effect on adipogenesis following co-culture with aPRL-1 or CPP peptide-enhanced stem cells after induced fibrosis with TAA (Thioacetamide) in ovarian granulosa cell lines. Figure 7a shows the results of fixing cells and staining them with BODIPY (Lipid droplet) fluorescence after co-culture with ovarian granulosa cell lines treated with TAA. Figure 7b is a graph showing lipid droplets per cell, confirming that the inhibitory effect on adipogenesis by CPP-primed mesenchymal stem cells was superior compared to the aPRL-1 treatment group.

[0064] Figures 8a and 8b illustrate the results of confirming the inhibitory effect on lipid production following co-culture with aPRL-1 or CPP peptide-enhanced stem cells after induced fibrosis with TAA (Thioacetamide) in liver epithelial cell lines. Figure 8a shows the results of fixing and BODIPY (Lipid droplet) fluorescence staining of liver epithelial cell lines after co-culture with TAA. Figure 8b is a graph showing lipid droplets per cell, confirming that the inhibitory effect on lipid synthesis by CPP-primed mesenchymal stem cells was superior compared to the aPRL-1 treatment group.

[0065] The following detailed description of the present invention will be described with reference to specific drawings (where drawings are available) regarding specific embodiments in which the present invention may be practiced, but the present invention is not limited thereto and is limited only by the appended claims to all scopes identical or equivalent to those described in the claims. It should be understood that various embodiments of the present invention are different but need not be mutually exclusive. For example, specific shapes, structures, and characteristics described in this specification may be modified from some embodiments to others or realized by combining multiple embodiments without departing from the technical spirit and scope of the present invention. Technical and academic terms used in this specification have the same meaning as commonly used in the field to which the present invention belongs, unless otherwise defined. For the purpose of interpreting this specification, the definitions set forth below shall apply, and terms expressed in the singular form shall be interpreted as also referring to the plural form (i.e., at least one) unless inappropriate in the context, and vice versa.

[0066] In this specification, the term “about” refers to the usual margin of error for each value known to a person skilled in the art. Furthermore, unless otherwise specified, all numbers, values, and / or expressions representing ingredients, conditions, compositions, amounts, etc. used in this specification should be understood as being modified by the term “about”, as these numbers are essentially approximations that reflect the various uncertainties of measurement that occur in obtaining these values ​​among other things. According to one example, cases may include ±10% or less, ±9% or less, ±8% or less, ±7% or less, ±6% or less, ±5% or less, ±4% or less, ±3% or less, ±2% or less, ±1% or less, or ±0.5% or less from a given numeric value.

[0067] In this specification, when a configuration is described as "containing" or "comprising" a component, this means that, unless specifically stated otherwise, it does not exclude other components but may include additional components.

[0068] In this specification, the terms “as described herein,” “as described herein,” “as described herein,” or “as described herein” should be understood to refer to the contents disclosed throughout this specification, including the detailed description of the invention, claims, drawings, and embodiments. When used in relation to a specific term or component, this expression is intended to encompass all definitions, embodiments, preferred embodiments, and numerical ranges provided for such term in any part of this specification.

[0069] The present invention is based on the discovery that a PRL-1 variant obtained by fusing at least partially PRL-1 with a specific cell-permeating peptide can enhance the function of mesenchymal stem cells. Accordingly, in one aspect of the present invention, a PRL-1 variant (PRL-1 fusion polypeptide) comprising a cell-permeating peptide represented by the amino acid sequence (RRQRRTSKLMKR) of SEQ ID NO. 2 and a PRL-1 peptide is provided.

[0070] In the PRL-1 variant of the present invention, a cell-permeating peptide can be fused to the PRL-1 peptide. For example, the cell-permeating peptide can be fused to the N-terminus or C-terminus of the PRL-1 peptide, preferably to the N-terminus. The fusion is performed in the direction from the N-terminus to the C-terminus.

[0071] In one embodiment, the PRL-1 variant of the present invention may include cysteine ​​residues at both ends of the cell-permeating peptide sequence (i.e., Cys-CPP-Cys). A disulfide bond is formed between the two cysteine ​​residues, connecting the two ends of the cell-permeating peptide to form a ring structure, which can further improve cell permeability.

[0072] In another aspect of the present invention, a PRL-1 variant represented by the amino acid sequence of SEQ ID NO. 1 is provided. The amino acid sequence of SEQ ID NO. 1 is shown in Table 1.

[0073] In the present invention, PRL-1 (phosphatase of regenerating liver-1) may include PRL-1 derived from vertebrates including humans, such as mammals, fish, amphibians, birds, or reptiles. Additionally, PRL-1 may also include precursors of PRL-1. Through previous research, the inventors confirmed that overexpression of PRL-1 can be induced without transduction by treating mesenchymal stem cells with PRL-1, and filed related documents in Korean Patent Application No. 2023-0078281 and International Patent Application No. PCT / KR2023 / 016955, the entire contents of which are incorporated herein by reference.

[0074] In the present invention, the PRL-1 peptide or PRL-1 variant affects cells, for example, due to its presence in a culture medium. The PRL-1 peptide or PRL-1 variant can affect the interior of cells, for example, mesenchymal stem cells, to overexpress the PRL-1 gene. Overexpression means that the expression of the PRL-1 gene is increased compared to the case where the cells are not treated with the PRL-1 peptide or PRL-1 variant. In this specification, "through the PRL-1 peptide" or "through the PRL-1 variant" refers to the cells being affected due to the presence of the PRL-1 peptide. The process by which the cells are affected may be the PRL-1 peptide affecting the interior of the cells.

[0075] In one embodiment of the present invention, it was confirmed that when a variant of PRL-1 with enhanced cell permeability is produced and mesenchymal stem cells are cultured by adding the PRL-1 variant to a culture medium, functionally enhanced stem cells with improved cell proliferation ability, inhibition of cellular senescence, and / or cell therapeutic ability can be produced.

[0076] The PRL-1 gene in mesenchymal stem cells can be overexpressed through a PRL-1 variant. Overexpression means that the expression of the PRL-1 gene is increased compared to the case without PRL-1 peptide treatment.

[0077] Mesenchymal stem cells can express one or more surface antigens selected from the group consisting of CD44, CD73, CD90, CD105, HLA-DR, and HLA-G. Even when mesenchymal stem cells are cultured by adding a PRL-1 variant to the culture medium, the surface antigen expression characteristics of the mesenchymal stem cells can be maintained. Mesenchymal stem cells exhibit reduced expression of Col1. The cell therapeutic ability of mesenchymal stem cells is enhanced through the PRL-1 peptide, which can increase the efficacy of reducing Col1 expression. Mesenchymal stem cells exhibit increased expression of one or more genes selected from the group consisting of albumin, Cyclin D1, and HNF1a. The cell therapeutic ability of mesenchymal stem cells is enhanced through the PRL-1 peptide, which can increase the efficacy of increasing the expression of one or more genes selected from the group consisting of albumin, Cyclin D1, and HNF1a.

[0078] In some embodiments, the PRL-1 variant may be a fusion protein that further comprises another protein or peptide. In a method for preparing a fusion protein, a polynucleotide encoding PRL-1 or a PRL-1 variant may be ligated to a frame with a polynucleotide encoding another protein or peptide.

[0079] Technologies known in the art can be used for this purpose. For peptides fused with PRL-1 or PRL-1 variants, known peptides such as FLAG (Hopp, TP et al., BioTechnology 6:1204-1210, 1988), 6x His residues consisting of 6 histidines (His), 10x His, influenza hemagglutinin (HA), human c-myc fragment, VSV-GP fragment, p18HIV fragments, T7-tag, HSV-tag, E-tag, SV40T antigen fragment, lck-tag, alpha-tubulin fragment, B-tag, and Protein C fragment may be used.

[0080] In addition, it is possible to link PRL-1 or a PRL-1 variant with glutathione-S-transferase (GST), influenza hemagglutinin (HA), immunoglobulin constant regions, β-galactosidase, or maltose-binding protein (MBP) to create a fusion protein.

[0081] In another aspect of the present invention, a method for producing functionally enhanced stem cells is provided, comprising the following steps:

[0082] (a) a step of enhancing the function of stem cells by treating mesenchymal stem cells with the PRL-1 variant described herein; and

[0083] (b) A step of obtaining stem cells with enhanced function. Preferably, the stem cells may be mesenchymal stem cells.

[0084] In the present invention, mesenchymal stem cells (MSCs) may refer to cells capable of maintaining self-renewal and stemness maintenance and differentiating into various mesenchymal tissues, and may include mesenchymal stem cells of mammals, such as animals including humans.

[0085] Mesenchymal stem cells may be derived from the placenta, amniotic membrane, umbilical cord blood, umbilical cord, fat, muscle, nerve, or amniotic membrane, and preferably may be umbilical cord-derived, umbilical cord blood-derived, bone marrow-derived, placenta-derived, or fat-derived mesenchymal stem cells, and more preferably may be placenta-derived mesenchymal stem cells.

[0086] Placenta-derived mesenchymal stem cells can be derived from various tissues constituting the placenta, such as amniotic epithelial cells, the amniotic membrane, the trophoblast, and the chorionic membrane. Preferably, placenta-derived mesenchymal stem cells may be mesenchymal stem cells derived from the chorionic plate of the placenta, and more preferably, may be mesenchymal stem cells derived from the chorionic plate membrane.

[0087] The isolation of mesenchymal stem cells can be performed using conventional methods known in the art.

[0088] In some embodiments, the placenta-derived stem cells may be characterized as placenta-chorionic valve-derived mesenchymal stem cells obtained by a method comprising the following steps:

[0089] (i) A step of obtaining a chorionic plate membrane from the placenta separated from the mother's body after childbirth;

[0090] (ii) a step of collecting mesenchymal stem cells by scraping the cells inside the chorionic valve obtained in step (i);

[0091] (iii) a step of adding a solution containing trypsin and ethylenediaminetetraacetate to the cells obtained in step (ii) to perform an enzymatic reaction at 20 to 30°C, and adding fetal bovine serum to stop the enzymatic reaction; and

[0092] (iv) A step of centrifuging the reaction solution obtained in step (iii) and culturing the recovered cells in a medium supplemented with fetal bovine serum and antibiotics.

[0093] In some embodiments, treatment with the PRL-1 variant may be performed before, during, or after culture of mesenchymal stem cells.

[0094] In some embodiments, the treatment concentration of the PRL-1 variant may be 0.001 ng / mL to 100,000 ng / mL. Specifically, the treatment concentration of the PRL-1 variant may be 0.005 ng / mL to 50,000 ng / mL, 0.01 ng / mL to 10,000 ng / mL, 0.05 ng / mL to 5,000 ng / mL, 0.1 ng / mL to 1,000 ng / mL, or 0.5 ng / mL to 500 ng / mL. Preferably, the treatment concentration of the PRL-1 variant may be 0.5 ng / mL to 200 ng / mL, 0.5 ng / mL to 100 ng / mL, 0.5 ng / mL to 50 ng / mL, or 0.8 ng / mL to 20 ng / mL, and more preferably 1 ng / mL to 15 ng / mL, 1 ng / mL to 12 ng / mL, or 1 ng / mL to 10 ng / mL.

[0095] In another aspect of the present invention, a functionally enhanced stem cell produced by the method described herein is provided.

[0096] In some embodiments, the enhanced stem cells may exhibit (i) upregulation of one or more stem cell markers selected from the group consisting of Oct 4, Nanog and Sox2 compared to mesenchymal stem cells not treated with the PRL-1 variant; (ii) downregulation of β-galactosidase expression; or gene expression characteristics of both (i) and (ii).

[0097] In some embodiments, the function-enhanced stem cells may be negative for hematopoietic markers (CD31, CD34, and CD45) and positive for non-hematopoietic markers (CD44, CD90, and CD105).

[0098] In another aspect of the present invention, a composition is provided comprising the function-enhancing stem cells described herein as an active ingredient. In some embodiments, a pharmaceutical composition for the prevention or treatment of metabolic diseases is provided, comprising the function-enhancing stem cells described herein as an active ingredient. The pharmaceutical composition of the present invention may be mixed with one or more pharmaceutically acceptable carriers and / or excipients. Pharmaceutically acceptable carriers and / or excipients are well known to be commonly used in the art and may be appropriately selected from these.

[0099] In another aspect of the present invention, a method for preventing or treating metabolic diseases is provided, comprising administering an effective amount of the function-enhancing stem cells described herein to a subject in need thereof.

[0100] In another aspect of the present invention, the use of the function-enhancing stem cells described herein for the prevention or treatment of metabolic diseases is provided.

[0101] In this specification, the term “treatment” generally means obtaining desired pharmacological and / or physiological effects. These effects are therapeutic in that they partially or completely cure a disease and / or harmful effects resulting from such disease. Desired therapeutic effects include, but are not limited to, prevention of the onset or recurrence of the disease, improvement of symptoms, reduction of any direct or indirect pathological consequences of the disease, reduction of the rate of disease progression, improvement or alleviation of the disease state, and remission or improved prognosis. Preferably, “treatment” may mean medical intervention for an already manifested disease or disorder.

[0102] The term "prevention" means obtaining the desired preventive pharmacological and / or physiological effects in the sense of partially or completely preventing a disease or its symptoms.

[0103] The terms "effective amount" or "pharmaceuticalally effective amount" refer to an amount sufficient to achieve a desired therapeutic or preventive effect. Specifically, it includes the amount necessary to alleviate, relieve, stabilize, reverse, or stop the symptoms of a disease, to suppress the progression of the disease, or to prevent the onset of the disease. The effective amount may vary depending on various factors, such as the individual's age, weight, health status, gender, route of administration, severity of the disease, and the type of specific composition administered, and can be appropriately determined by a person skilled in the art.

[0104] The term “subject” is used interchangeably with “individual” or “patient” and may be a mammal requiring the recovery or enhancement of mitochondrial function, e.g., primates (e.g., humans), companion animals (e.g., dogs, cats, etc.), livestock animals (e.g., cattle, pigs, horses, sheep, goats, etc.), and laboratory animals (e.g., rats, mice, guinea pigs, etc.). In some embodiments, the subject is a human.

[0105] The term "metabolic disease" may refer to a disease resulting from abnormal lipid production and metabolic disorders caused by abnormalities in lipid metabolism. Specifically, metabolic diseases may be selected from a group consisting of liver cirrhosis, liver fibrosis, alcoholic liver disease, fatty liver, liver cancer, polycystic ovary syndrome, ovarian failure, obesity, diabetes, dyslipidemia, hypertension, thyroid eye disease, macular degeneration, and diabetic retinopathy. Examples of abnormal lipid production and metabolic disorders may include obesity, diabetes, dyslipidemia, metabolic diseases, hypertension, thyroid eye disease, or degenerative diseases related to abnormal lipid production and metabolic disorders, and may include metabolic disease-based menopausal disorders, ovarian dysfunction (ovarian failure, polycystic ovary syndrome), and metabolic osteoporosis.

[0106] Liver disease may include liver dysfunction, liver disorder, hepatitis, hepatotoxicity, cholestasis, fatty liver, cirrhosis, hepatic ischemia, alcoholic liver disease, liver abscess, hepatic coma, hepatic atrophy, or liver cancer. A state in which the above liver disease persists chronically and fails to perform normal liver functions may be referred to as "hepatic dysfunction." Patients in a state of hepatic dysfunction may have significantly increased expression of fibrosis-related genes compared to normal individuals; and / or significantly decreased concentrations of Cyclin D1, HNF1α, and albumin (ALB) compared to normal individuals.

[0107] In another aspect of the present invention, a nucleic acid encoding the PRL-1 variant described herein or a nucleic acid encoding the amino acid sequence of SEQ ID NO. 1 is provided.

[0108] In another aspect of the present invention, a nucleic acid delivery vehicle is provided comprising a nucleic acid encoding the PRL-1 variant described herein or a nucleic acid encoding the amino acid sequence of SEQ ID NO. 1.

[0109] In some embodiments, the nucleic acid delivery vehicle may be a recombinant vector, lipid nanoparticle (LNP), polymer nanoparticle (PNP), liposome, exosome, lipid, colloid, macromolecular complex, microsphere, nanoparticle, microsphere, bead, oil-in-water emulsion, micelle, or viral vector.

[0110] In another aspect of the present invention, a cell or microorganism comprising the nucleic acid or nucleic acid carrier described herein is provided.

[0111] In another aspect of the present invention, a culture composition comprising the PRL-1 variant described herein is provided. The culture composition may be a culture medium. The culture medium may be a culture medium known in the art that is conventionally used for the culture of mesenchymal stem cells. The PRL-1 variant may be present in the culture medium at a concentration of 0.001 ng / mL to 100,000 ng / mL. Specifically, the concentration of the PRL-1 variant may be 0.005 ng / mL to 50,000 ng / mL, 0.01 ng / mL to 10,000 ng / mL, 0.05 ng / mL to 5,000 ng / mL, 0.1 ng / mL to 1,000 ng / mL, or 0.5 ng / mL to 500 ng / mL. Preferably, the concentration of the PRL-1 variant may be 0.5 ng / mL to 200 ng / mL, 0.5 ng / mL to 100 ng / mL, 0.5 ng / mL to 50 ng / mL, or 0.8 ng / mL to 20 ng / mL, and more preferably 1 ng / mL to 15 ng / mL, 1 ng / mL to 12 ng / mL, or 1 ng / mL to 10 ng / mL.

[0112] The culture medium may include α-MEM medium and DMEM / F12 medium. The culture medium may include additional substances. The additional substances may be penicillin-streptomycin, heparin, and fibroblast growth factor.

[0113] The present invention will be explained in more detail below through the following examples. However, the examples are merely illustrative of the present invention, and the scope of the present invention is not limited thereto.

[0114] Examples

[0115] Example 1. Preparation of PRL-1 variant

[0116] Method for screening and constructing PRL-1 variants from a peptide database

[0117] 159 peptide sequences in the Cargo database were identified in the peptide database CPPsite 2.0: A Database of Cell Penetrating Peptides (http: / crdd.osdd.net / raghava / cppsite / ), and the cell-permeable peptide RQIKIWFQNRRMKWKK was selected among the peptide sequences based on prior art.

[0118] It is known that when cysteine ​​amino acid sequences are included at both ends of a cell-permeable peptide, the cell permeability is increased by the formation of a cyclic structure of the cell-permeable peptide [Patel et al., Sci Rep, 2019 PMID: 31000738]. Based on this, amino acid sequences expected to have excellent cell permeability were conjugated to the N-terminal of the existing PRL-1 sequence, and 6X Histamine was conjugated to the C-terminal to construct a final variant. The final sequence of the constructed PRL-1 variant is shown in Table 1 (amino acid sequence of PRL-1 variant (CPP)), and it was named CPP.

[0119] The isolation and purification of the recombinant protein were entrusted to GeneScript (South Korea). A recombinant vector was constructed, introduced into TurboCHO cells, and expression was confirmed, and the protein was isolated and purified to obtain the recombinant protein.

[0120] [Table 1]

[0121]

[0122] Example 2. Isolation of placenta-derived mesenchymal stem cells (UNIPla cells)

[0123] UNIPla cells were isolated according to the following method for isolating and culturing placental-derived mesenchymal stem cells (see Korean Registered Patent No. 10-0900309).

[0124] Informed consent was obtained from healthy mothers who had delivered normally at 37 weeks or more, without medical, obstetric, or surgical issues, fetal malformations, or multiple births. The normal placenta obtained from the mother was rapidly transported in a sterile container on ice, and its morphological and structural characteristics were recorded visually. The chorionic valve covering the fetal side of the placenta was pulled off, and the chorionic valve was washed five times with phosphate-buffered saline containing antibiotics (1% penicillin-streptomycin) to remove contaminants present in the tissue that may have occurred during collection and transport.

[0125] The washed chorionic valve was spread with the amniotic cell side down in a sterile 150 mm glass dish, and the cells of the mesenchymal stem cell layer inside the chorionic valve were scraped and collected using a sterile slide glass. The collected cells were washed three times with sterile HBSS solution and centrifuged at 1,000 rpm for 5 minutes. After removing the supernatant, 10 mL of 0.25% trypsin / EDTA solution was added to the remaining cells, and the mixture was incubated for 30 minutes at room temperature while mixing slowly. After adding 1 mL of fetal bovine serum to inhibit the enzymatic reaction, the supernatant (primary enzymatic reaction solution) was separated and transferred to a 50 mL conical tube. 10 mL of 0.25% trypsin / EDTA solution was added to the remaining cells, and the mixture was reacted for 30 minutes at room temperature while mixing slowly. The supernatant was separated and combined with the first enzyme reaction solution. 2.5 mL of fetal bovine serum was added again to stop the enzyme reaction, and the mixture was centrifuged at 1,000 rpm for 5 minutes. The supernatant was then removed, and the cells were harvested.

[0126] The obtained cells were added to 3 mL of culture medium (DMEM / F12 supplemented with 10% fetal bovine serum, 1% penicillin-streptomycin, 1 μg / mL heparin, and 25 ng / mL FGF-4), mixed well, placed in a T25 flask, and cultured in a CO2 incubator at 37°C. Subculture was performed approximately every 5 days, taking into account the cell growth rate, and cells cultured up to the 10th passage were used as UNIPla cells.

[0127] Example 3. Confirmation of cell morphology after culture of PRL-1 or PRL-1 variant

[0128] UNIPla cells obtained by the method of Example 2 were cultured in alpha-MEM medium supplemented with 10% fetal bovine serum, 1% gentamycin, 1 µg / mL heparin, and 25 ng / mL fibroblast growth factor-4 (FGF-4) in an incubator at 37°C under conditions of 20% O2 and 5% CO2.

[0129] Solutions were prepared by adding aPRL-1 (a recombinant protein sold by Abcam) and CPP (a PRL-1 variant) to the culture medium of UNIPla cells at concentrations of 1 ng / mL or 10 ng / mL, respectively. The cells were cultured in the prepared medium for 6 days and observed under an inverted microscope.

[0130] As a result, referring to Figure 1, it was confirmed that the cell morphology did not change even when aPRL-1 or CPP was cultured in a culture medium at a concentration of 1 ng / mL or 10 ng / mL for 6 days.

[0131] Example 4. Confirmation of cell growth after culture of PRL-1 or PRL-1 variant

[0132] A culture medium solution was prepared by adding aPRL-1 or CPP to the culture medium of UNIPla cells at a concentration of 1 ng / mL or 10 ng / mL, respectively. UNIPla cells were cultured in the prepared culture medium for 6 days, treated with Trypsin to harvest the cells, and some cells were stained with trypan blue to measure the total number of cells using a hemocytometer.

[0133] As a result, referring to Figure 2, it was confirmed that the total number of cells increased under conditions where aPRL-1 and CPP were cultured for 6 days in culture medium at concentrations of 1 ng / mL or 10 ng / mL compared to UNIPla grown in culture medium.

[0134] Example 5. Confirmation of cell senescence after culture of PRL-1 or PRL-1 variant

[0135] A culture medium solution was prepared by adding aPRL-1 or CPP to the culture medium of UNIPla cells at a concentration of 1 ng / mL or 10 ng / mL, respectively. After culturing UNIPla cells in the prepared culture medium for 6 days (6th passage, passage 6), the cells were subcultured once more and cultured in the culture medium for another 6 days (7th passage, passage 7).

[0136] Staining of cultured cells for Senescence beta-galactosidase was performed according to the manufacturer's protocol (Senescence Cells Histochemical Staining Kit (Sigma-Aldrich, CS0030)). The staining method is as follows. Cultured cells were washed with PBS, fixed with Fixation buffer (2% formaldehyde, 0.2% glutaraldehyde, 7.04 mM Na2HPO4, 1.47 mM KH2PO4, 0.137 M NaCl, and 2.68 mM KCl) at room temperature for 5 minutes, and washed three times with PBS. The cells were incubated with the staining reagents (Staining Solution 1X, 5 mM Potassium Ferricyanide, 5 mM Potassium Ferrocyanide, X-gal Solution 1 mg / mL) at 37°C for 24 hours. After staining, the solution was replaced with 1X PBS, and the cells were observed and analyzed under an inverted microscope.

[0137] As a result, referring to Figures 3a and 3b, a large number of aged cells were observed under conditions of UNIPla and aPRL-1 concentration of 1 ng / mL grown in the culture medium, and it was confirmed that the number of aged cells decreased under conditions of aPRL-1 concentration of 10 ng / mL and CPP concentrations of 1 ng / mL or 10 ng / mL.

[0138] It was confirmed that cellular senescence was significantly more inhibited in the group treated with CPP (1 ng / mL) compared to the group treated with aPRL-1 (1 ng / mL).

[0139] Example 6. Confirmation of stem cell marker factors in cells after culture of PRL-1 or PRL-1 variants

[0140] A culture medium solution was prepared by adding aPRL-1 or CPP to the culture medium of UNIPla cells at a concentration of 1 ng / mL or 10 ng / mL, respectively. After culturing UNIPla cells in the prepared culture medium for 6 days (6th passage, passage 6), the cells were subcultured once more and cultured in the culture medium for another 6 days (7th passage, passage 7).

[0141] Cultured cells were treated with Trypsin to harvest them, and some of the cells were 5 x 10 based on living cells. 6 Cells were prepared and suspended in 1 mL of FACS buffer. Antibodies against hematopoietic markers (CD31, CD34, and CD45) and non-hematopoietic markers (CD44, CD90, and CD105) were added and reacted for 30 minutes, after which the cells were analyzed using a flow cytometer (FACS).

[0142] As a result, referring to Figure 4, it was confirmed that the hematopoietic markers (CD31, CD34, and CD45) were negative and the non-hematopoietic markers (CD44, CD90, and CD105) were positive.

[0143] Example 7. Confirmation of stem cell potential of cells after culture of PRL-1 and PRL-1 variants

[0144] aPRL-1 or CPP was added to the culture medium of UNIPla cells to a concentration of 1 ng / mL or 10 ng / mL, respectively. Cells cultured in the prepared culture medium for 6 days (passage 6) and cells cultured in the culture medium after one more passage and for 6 days (passage 7) were recovered, and RT-PCR (reverse transcript polymerase chain reaction) for OCT4 was performed.

[0145] Cultured cells were treated with Trypsin to harvest them, and Trizol was added to the isolated cells to lyse them, after which they were placed on ice for 10 minutes. Subsequently, 200 µl of Chloroform was added, the mixture was inverted, and incubated at room temperature for 5 minutes. The resulting solution was then centrifuged at 12,000 RCF at 4°C for 15 minutes. 400 µl of the supernatant was collected, an equal amount of isopropanol was added, and the mixture was reacted at room temperature for 10 minutes followed by centrifugation at 12,000 RCF at 4°C for 15 minutes. After washing with 75% EtOH and drying at room temperature for 30 minutes, DEPC water was added and the mixture was incubated at 55°C for 10 minutes. Finally, cDNA was synthesized according to the manufacturer's protocol (PrimeScript RT reagent Kit (Perfect Real Time)). The synthesized cDNA was used as a template, and PCR was performed using primers for OCT4 and Taq polymerase. After the PCR reaction, DNA was electrophoresed on a 1.5% agarose gel, and bands were detected using a gel doc.

[0146] As a result, referring to Figures 5a to 5d, it was confirmed that the expression level of OCT4 was similar to that of UNIPla under cell conditions in which aPRL-1 or CPP was cultured in culture medium at a concentration of 1 ng / mL or 10 ng / mL for 6 days (6th passage, passage 6) or under cell conditions in which the cell was cultured one more time and cultured in culture medium for 6 days (7th passage, passage 7).

[0147] Example 8. Confirmation of inhibitory effect on cellular adipogenesis after co-culture with PRL-1 and PRL-1 variant-enhanced stem cells

[0148] aPRL-1 or CPP was added to the culture medium of UNIPla cells to a concentration of 1 ng / mL or 10 ng / mL, respectively. Cells cultured in the prepared medium for 6 days were harvested and co-cultured with a retinal pigment epithelial cell line while being treated with 200 µM H2O2. After 2 hours of co-culture, the cells were fixed and fluorescently stained with BODIPY (Lipid droplet) and DAPI (nucleus) for observation under a fluorescence microscope.

[0149] As a result, referring to Figures 6a and 6b, it was confirmed that the formation of lipid droplets increased in the group treated with H2O2 compared to the control group, and in the group co-cultured with UNIPla cells, it was confirmed that the number of lipid droplets decreased under conditions of UNIPla, aPRL-1 concentration of 1 ng / mL or 10 ng / mL, and CPP concentration of 1 ng / mL or 10 ng / mL.

[0150] aPRL-1 or CPP was added to the culture medium of UNIPla cells to a concentration of 1 ng / mL or 10 ng / mL, respectively. Cells cultured in the prepared medium for 6 days were harvested and co-cultured with ovarian granulosa cell lines treated with 70 mM TAA (Thioacetamide). After 24 hours of co-culture, the cells were fixed and fluorescently stained with BODIPY (Lipid droplet) and DAPI (nucleus) for fluorescence microscopy observation.

[0151] As a result, referring to Figures 7a and 7b, it was confirmed that the formation of lipid droplets increased in the group treated with TAA compared to the control group, and in the group co-cultured with UNIPla cells, it was confirmed that the number of lipid droplets decreased under conditions of UNIPla, aPRL-1 concentration of 1 ng / mL or 10 ng / mL, and CPP concentration of 1 ng / mL or 10 ng / mL.

[0152] aPRL-1 or CPP was added to the culture medium of UNIPla cells to a concentration of 1 ng / mL or 10 ng / mL, respectively. Cells cultured in the prepared medium for 6 days were harvested and co-cultured with liver epithelial cell lines treated with 70 mM TAA (Thioacetamide). After 24 hours of co-culture, the cells were fixed and fluorescently stained with BODIPY (Lipid droplet) and DAPI (nucleus) for observation under a fluorescence microscope.

[0153] As a result, referring to Figures 8a and 8b, it was confirmed that the formation of lipid droplets increased in the group treated with TAA compared to the control group, and in the group co-cultured with UNIPla cells, it was confirmed that the number of lipid droplets decreased under conditions of UNIPla, aPRL-1 concentration of 1 ng / mL or 10 ng / mL, and CPP concentration of 1 ng / mL or 10 ng / mL.

[0154] Foregoing, specific parts of the present invention have been described in detail. It will be apparent to those skilled in the art that such specific descriptions are merely preferred embodiments and do not limit the scope of the invention. Accordingly, the actual scope of the invention is defined by the appended claims and their equivalents.

[0155] Statistical analysis

[0156] All experimental results obtained from the embodiments of the present invention were subjected to multi-group comparison using the non-parametric Kruskal-Wallis test. Post-hoc analysis was performed using the Conover-Iman method, and significance levels were adjusted using Benjamini-Hochberg correction. Statistical significance levels were indicated as *p<0.05, **p<0.01, and ***p<0.001.

Claims

1. A PRL-1 variant comprising a peptide represented by the amino acid sequence of SEQ ID NO. 2 and a PRL-1 peptide, wherein the cell-permeating peptide is fused to the PRL-1 peptide.

2. In Paragraph 1, The above PRL-1 variant is represented by the amino acid sequence of SEQ ID NO. 1 PRL-1 variant.

3. A method for manufacturing function-enhanced stem cells comprising the following steps: (a) a step of treating mesenchymal stem cells with the PRL-1 variant of claim 1 to enhance the function of the stem cells; and (b) a step of obtaining the stem cells with enhanced function.

4. In Paragraph 3, A method for producing functionally enhanced stem cells, characterized by performing the above-mentioned PRL-1 variant treatment before, during, or after the culture of mesenchymal stem cells.

5. In Paragraph 3, A method for producing functionally enhanced stem cells, characterized in that the above mesenchymal stem cells are derived from a tissue selected from the group consisting of placenta, amniotic membrane, umbilical cord blood, umbilical cord, fat, muscle, nerve, and amniotic membrane.

6. In Paragraph 5, A method for producing a functionally enhanced stem cell characterized in that the above mesenchymal stem cell is a placenta-derived stem cell.

7. In Paragraph 6, A method for producing function-enhancing stem cells, characterized in that the placenta-derived stem cells are placental chorionic valve-derived mesenchymal stem cells obtained by a method comprising the following steps: (i) A step of obtaining a chorionic plate membrane from the placenta separated from the mother's body after childbirth; (ii) a step of collecting mesenchymal stem cells by scraping the cells inside the chorionic valve obtained in step (i); (iii) a step of adding a solution containing trypsin and ethylenediaminetetraacetate to the cells obtained in step (ii) to perform an enzymatic reaction at 20 to 30°C, and adding fetal bovine serum to stop the enzymatic reaction; and (iv) A step of centrifuging the reaction solution obtained in step (iii) and culturing the recovered cells in a medium supplemented with fetal bovine serum and antibiotics.

8. In Paragraph 3, A method for producing function-enhanced stem cells, characterized in that the treatment concentration of the above PRL-1 variant is 1 to 100,000 ng / mL.

9. A function-enhanced stem cell prepared by the method of claim 3 and having (i) upregulation of one or more stem cell markers selected from the group consisting of Oct 4, Nanog and Sox2 compared to mesenchymal stem cells not treated with a PRL-1 variant; (ii) downregulation of beta-galactosidase expression; or the gene expression characteristics of (i) and (ii).

10. A pharmaceutical composition for the prevention or treatment of metabolic diseases containing the function-enhancing stem cells of claim 9 as an active ingredient.

11. In Paragraph 10, A pharmaceutical composition characterized in that the above metabolic disease is selected from the group consisting of liver cirrhosis, liver fibrosis, alcoholic liver disease, fatty liver, liver cancer, polycystic ovary syndrome, ovarian insufficiency, obesity, diabetes, dyslipidemia, hypertension, thyroid eye disease, macular degeneration, and diabetic retinopathy.

12. A PRL-1 variant of claim 1 or a nucleic acid encoding the amino acid sequence of SEQ ID NO.

1.

13. A nucleic acid carrier comprising the nucleic acid of paragraph 12.

14. In Paragraph 13, The nucleic acid delivery vehicle is characterized by being selected from the group consisting of recombinant vectors, lipid nanoparticles (LNPs), polymer nanoparticles (PNPs), liposomes, exosomes, lipids, colloids, macromolecular complexes, microspheres, nanoparticles, microspheres, beads, oil-in-water emulsions, micelles, and viral vectors.

15. A cell or microorganism transformed with the nucleic acid of paragraph 12 or the nucleic acid carrier of paragraph 13.

16. A cell culture composition comprising the PRL-1 variant of claim 1 or a PRL-1 variant represented by the amino acid sequence of SEQ ID NO.

1.

17. In Paragraph 16, A cell culture composition characterized by the above PRL-1 variant being present at a concentration of 1 to 100,000 ng / mL.

18. A method for the prevention or treatment of metabolic diseases comprising administering the function-enhancing stem cells of paragraph 9 to a subject in need thereof.

19. In Paragraph 18, A method for prevention or treatment characterized in that the above metabolic disease is selected from the group consisting of liver cirrhosis, liver fibrosis, alcoholic liver disease, fatty liver, liver cancer, polycystic ovary syndrome, ovarian failure, obesity, diabetes, dyslipidemia, hypertension, thyroid eye disease, macular degeneration, and diabetic retinopathy.

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