Bone marrow-derived growth factors for use in the treatment or prevention of liver disease

Abstract

This provides means and methods for treating liver disease. [Solution] The present invention relates to a myeloid-derived growth factor (MYDGF) protein, or a nucleic acid encoding the protein, for use in the treatment or prevention of liver disease. The present invention also relates to a vector containing the nucleic acid, a host cell expressing the nucleic acid, and a method for use in the treatment of liver disease.

Description

[Technical Field]

[0001] The present invention relates to a myeloid-derived growth factor (MYDGF) protein or a nucleic acid encoding the same, for use in the treatment or prevention of liver diseases. The present invention also relates to a vector comprising the nucleic acid, a host cell expressing the nucleic acid, and a method for use in the treatment or prevention of liver diseases. [Background Art]

[0002] Myelin-derived growth factor (MYDGF), also known as Factor 1, is a protein encoded in open reading frame 10 (C19Orf10) of human chromosome 19. This protein was described in 2007 as a novel secretory factor in the synovial membrane in proteomic analysis of fibroblast-like synovial cells (FLS cells). The correlation between the secretion of this protein and inflammatory joint diseases has been suggested without experimental or statistical evidence (Weiler et al., Arthritis Research and Therapy 2007, "Identification and Characterization of a Novel Synovial Protein c19orf10"). A corresponding patent application claims this protein as a therapeutic agent for the treatment of joints, the diagnosis of growth abnormalities in tissues, and the monitoring of tissue changes (US2008 / 0004232A1, "Characterization of a Novel Synovial Protein c19orf10"). Another scientific paper reports enhanced expression of this protein in hepatocellular carcinoma cells (Sunagozaka et al., International Journal of Cancer, 2010, Identification of secreted protein c19orf10 activated in hepatocellular carcinoma cells). Recombinantly produced protein showed a growth-promoting effect in cultured hepatocellular carcinoma cells. Originally thought to be an interleukin, C19Orf10 is also called IL-25, IL-27, or IL-27W. However, the terms "IL-25" and "IL-27" have been used inconsistently in the art and have been used to refer to various different proteins. For example, US2004 / 0185049 refers to the protein as IL-27 and discloses its use in regulating immune responses. This protein is structurally different from factor I (compare the amino acid sequence of factor I shown in SEQ ID NO: 1 with the amino acid sequence of "IL-27" shown in UniProt:Q8NEV9). Similarly, in EP2130547A1, the protein is called IL-25, and its use in the treatment of inflammation is disclosed.This protein is also known as IL-17E in this technical field and is structurally different from factor I (compare the amino acid sequence of factor I shown in SEQ ID NO: 1 with the amino acid sequence of "IL-25" shown in UniProt:Q9H293).

[0003] WO2014 / 111458 discloses Factor1 for promoting the proliferation of non-transformed tissues or non-transformed cells and inhibiting apoptosis, particularly for use in the treatment of acute myocardial infarction. Furthermore, Factor1 inhibitors for medical use, particularly for the treatment or prevention of diseases in which angiogenesis contributes to the onset or progression of the disease, are also disclosed.

[0004] Korf-Klingebiel et al. (Nature Medicine, 2015, Vol.21(2):140-149) reported on C19Orf10, a protein secreted by bone marrow cells after myocardial infarction that promotes cardiomyocyte survival and angiogenesis. The authors showed that bone marrow-derived monocytes and macrophages endogenously produce this protein, protecting and repairing the heart after myocardial infarction, and proposed naming it myelo-derived growth factor (MYDGF). In particular, treatment with recombinant MyDGF has been reported to reduce scar size and systolic dysfunction after myocardial infarction. While the beneficial effects of MYDGF on myocardial infarction have been studied, the effects of MYDGF on liver diseases, including acute liver failure, chronic liver disease, or liver transplantation, have not been evaluated.

[0005] Acute liver failure (ALF) is a rare, acute, and potentially reversible condition that causes severe liver dysfunction and rapid clinical deterioration in patients without pre-existing liver disease. Acute liver failure is most commonly caused by viral infections (e.g., hepatitis A, B, E) or medications (e.g., acetaminophen, NSAIDs, and certain antibiotics). Other causes of acute liver failure include acute ischemic liver injury, mushroom ingestion, metabolic disorders such as Wilson's disease, and exposure to toxins. Because acute liver failure is rare and its severity and diversity, there is very limited evidence to guide supportive care (W Bernal et al. NEJM. 2013; 369:2525-2534).

[0006] Chronic liver disease (CLD) is a condition characterized by a progressive decline in liver function over a period of six months or more, including the synthesis of coagulation factors and other proteins, detoxification of harmful metabolites, and bile excretion. CLD is a continuous process of inflammation, destruction, and regeneration of the liver parenchyma, leading to fibrosis and cirrhosis. The etiologies of chronic liver disease are diverse and include toxins, long-term alcohol abuse, infections, autoimmune diseases, genetic disorders, and metabolic disorders (https: / / www.ncbi.nlm.nih.gov / books / NBK554597 / ). Chronic liver disease is associated with varying degrees of scarring (non-fibrosis, fibrosis, cirrhosis) and compensation (compensated cirrhosis and decompensated cirrhosis). Chronic liver diseases include non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH), chronic infections of hepatitis B virus (HBV) and hepatitis C virus (HCV) due to the progression of viral hepatitis, alcoholic liver disease (ALD) due to long-term alcohol abuse, autoimmune hepatitis, a liver disease caused by the body's immune system, primary biliary cholangitis, primary sclerosing cholangitis causing hardening and scarring of the bile ducts, Wilson's disease, where copper accumulates in the liver, alpha-1 antitrypsin deficiency, and metabolic diseases such as hemochromatosis, where iron accumulates in the body. Effective drug therapies for chronic liver diseases have not yet been established.

[0007] Liver transplantation is a treatment that involves removing a liver whose function has deteriorated due to various causes and transplanting a healthy liver. There are two types of liver transplants: living donor liver transplantation and brain-dead donor liver transplantation. In liver transplantation, it is necessary to improve the quality and function of the donor liver before transplantation to increase the success rate of the transplant and the recovery rate of the recipient after transplantation.

[0008] Means and methods for treating liver disease are still needed. [Overview of the Initiative]

[0009] In a first embodiment, the present invention provides myeloid-derived growth factor (MYDGF), or a fragment or variant thereof exhibiting a biological function, for use in the treatment or prevention of liver disease.

[0010] According to one embodiment, the MYDGF protein, or a fragment or variant thereof exhibiting the biological function of MYDGF, is used for the treatment or prevention of liver disease. According to a preferred embodiment, the liver disease is acute liver failure, chronic liver disease, or liver transplantation.

[0011] According to a preferred embodiment, the MYDGF protein comprises SEQ ID NO: 1. Alternatively, the MYDGF protein comprises a fragment or variant of SEQ ID NO: 1, which exhibits the biological function of MYDGF, wherein the variant comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1.

[0012] In a preferred embodiment, the liver disease is acute liver failure, chronic liver disease, or liver transplantation. In a further preferred embodiment, acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury, and toxic liver injury. In a further preferred embodiment, chronic liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection due to hepatitis B virus (HBV) or hepatitis C virus (HCV), alcoholic liver disease (ALD), autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerotic cholangitis (PSC), metabolic liver disease, and chronic ischemic liver injury.

[0013] In a further embodiment, the present invention provides nucleic acids encoding the growth factor protein MYDGF, or fragments or variants thereof exhibiting the biological function of MYDGF, for use in the treatment or prevention of liver disease.

[0014] According to one embodiment, the nucleic acid encodes an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1.

[0015] In a further embodiment, the present invention provides a vector comprising the nucleic acid of the present invention for use in the treatment or prevention of liver disease.

[0016] In a further embodiment, the present invention provides a host cell comprising the nucleic acid of the present invention or the vector of the present invention for use in the treatment or prevention of liver disease. Preferably, the host cell expresses the nucleic acid of the present invention.

[0017] In yet another aspect, the present invention provides a pharmaceutical composition for use in the treatment or prevention of liver disease, comprising the MYDGF protein, nucleic acid, vector or host cell of the present invention and optionally a suitable pharmaceutical excipient.

[0018] According to preferred embodiments, the pharmaceutical composition of the present invention is administered orally, intravenously, subcutaneously, intramucosally, intra-arterially, intramuscularly, or intrahepatically. Administration is preferably carried out by one or more bolus injections and / or intravenous infusions.

[0019] In a further embodiment, the present invention provides a method for treating or preventing liver disease. This method comprises administering a therapeutically effective amount of MYDGF, or a fragment or variant thereof exhibiting the biological function of MYDGF, to a patient in need. In a preferred embodiment, the liver disease is acute liver failure, chronic liver disease, or liver transplantation. In a further preferred embodiment, acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury, and toxic liver injury. In a further preferred embodiment, chronic liver disease is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection by hepatitis B virus (HBV) or hepatitis C virus (HCV), alcoholic liver disease (ALD), autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerotic cholangitis (PSC), metabolic liver disease, and chronic ischemic liver injury.

[0020] According to one embodiment, MYDGF comprises SEQ ID NO: 1, or a fragment or variant of SEQ ID NO: 1 exhibiting the biological function of MYDGF. The variant comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1.

[0021] In yet another embodiment, MYDGF, or a fragment or variant thereof exhibiting the biological function of MYDGF, is administered by one or more bolus injections and / or infusions, preferably in a pharmaceutically acceptable carrier. [Brief explanation of the drawing]

[0022] [Figure 1] (A) Schematic diagram of the experimental design for inducing an acute liver injury model using carbon tetrachloride (CCl4). (B) Expression of MYDGF mRNA in mouse liver. (C) and (D) Serum transaminase (ALT / AST) concentrations at 24, 48, and 72 hours after a single intraperitoneal administration of CCl4. [Figure 2]Construction of a chronic liver injury model. (A) Schematic diagram of the experimental design for inducing a chronic liver injury model with carbon tetrachloride (CCl4). (B) MYDGF mRNA expression in mouse liver at the time of CCl4 administration and 7 (37), 14 (44), and 28 (58) days after the end of CCl4 administration. (C) and (D) TNFα and col1a2 mRNA expression in the liver at the time of CCl4 administration and 7 (37), 14 (44), and 28 (58) days after the end of CCl4 administration. [Figure 3] MYDGF protein therapy in an acute liver injury model. (A) Figure of a mouse with an implanted osmotic pump. (B) Schematic diagram of the experimental design using carbon tetrachloride (CCl4) to induce an acute liver injury model. Mice were divided into a MYDGF ALZET pump group and a sham surgery control group. (C) Serum MYDGF concentrations in the MYDGF ALZET pump group and sham surgery control group at 24, 48, and 72 hours after a single intraperitoneal administration of CCl4. (D) and (E) Serum transaminases and ALT / AST concentrations in the MYDGF ALZET pump group and sham surgery control group at 24 and 48 hours after a single intraperitoneal administration of CCl4. (F) H&E staining of the liver of ALI mice after a single intraperitoneal administration of CCl4, at 1, 2, and 3 days. Black lines in liver sections indicate necrotic areas. Scale bar: 100 μm. A graph showing the ratio of the necrotic area to the visual field (damage area %) in each group stained with (G) and (F), quantified using Image J. [Figure 4] Suppression of hepatic inflammatory response by MYDGF in an acute liver injury model. (A)-(C) mRNA expression of inflammatory cytokines, TNF-α, IL-1β, and IL-6 in all liver tissues of the MYDGF ALZET pump group and sham surgery control group at 24, 48, and 72 hours after single intraperitoneal administration of CCl4. (D) Expression of NF-κB and p-NF-κB in liver samples of the MYDGF ALZET pump group and solvent pump control group at 48 hours after single intraperitoneal administration of CCl4. [Figure 5]MYDGF protein therapy in a chronic liver injury model. (A) Schematic diagram of the experimental design in a carbon tetrachloride (CCl4)-induced chronic liver injury model. Mice were divided into a sham surgery control group, a MYDGF ALZET pump group, and a control group. (B) Serum MYDGF concentrations in the sham surgery control group, MYDGF ALZET pump group, and control group 28 days after the start of CCl4 administration. (C) and (D) Serum transaminase and ALT / AST concentrations in the sham surgery control group, MYDGF ALZET pump group, and control group 28 days after the start of CCl4 administration. [Figure 6] Suppression of liver fibrosis by MYDGF in a model of suppressive chronic liver injury. (A) Sirius red staining of livers of the control group that underwent sham surgery and the pump group 28 days after the start of MYDGFCCl4 administration. Staining results for the control group, sham surgery control group, and MYDGFALZET pump experimental group are also shown. For Sirius red staining, positive stained areas are shown in white in the grayscale image. (B) The sham surgery control group and the MYDGF(A)ALZET pump group were quantified in ImageJ. (C) Hydroxyproline content in the liver of the sham surgery control group, ALZET pump group and control group 28 days after the start of MYDGFCCl4 administration. (D)-(G) mRNA expression of liver fibrosis genes TGF-β, ACTA2, COL1A1, COL1A2 in all liver tissue of the sham surgery control group. ALZET pump group and control group 28 days after the start of MYDGFCCl4 administration. [Figure 7] Suppression of hepatic inflammatory response by MYDGF in a chronic liver injury model: (A)-(C) mRNA expression of inflammatory cytokines, TNF-α, IL-1β, and IL-6 in all liver tissues of the sham surgery control group, MYDGF ALZET pump group, and control group 28 days after the start of CCl4 administration. (D) Expression of NF-κB and p-NF-κB in liver samples of the sham surgery control group, MYDGF ALZET pump group, and control group 28 days after the start of CCl4 administration. [Figure 8]Inhibition of inflammatory response by MYDGF in HepG2 cells. (A) Expression of NF-κB and p-NF-κB in HepG2 cells 6 hours after treatment with different concentrations of rhMYDGF. (B) Expression of TNF-α mRNA in HepG2 cells 6 hours after treatment with different concentrations of rhMYDGF. [Figure 9] Induction of MYDGF by inflammatory cytokines in HepG2 cells. (A)-(C) Expression of MYDGF mRNA in HepG2 cells 24 hours after treatment with recombinant human inflammatory cytokines TNF-α, IL-1β and IL-6, respectively. (D) Expression of MYDGF mRNA in HepG2 cells 24 hours after treatment with different concentrations of rhIL-6. (E) Expression of MYDGF protein in supernatant and cell lysate of HepG2 cells 24 hours after treatment with different concentrations of rhIL-6.

[0023] [Detailed Description of the Invention] Before describing the present invention in detail below, it should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described herein. This is because these methods are modifiable. It should also be understood that the terms used herein are for the purpose of describing specific embodiments only, and are not intended to limit the scope of the present invention. The scope of the present invention is limited only by the appended claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art.

[0024] [Definitions] Preferably, the terms used herein are defined as described in "Multilingual Glossary of Biotechnology Terms: (IUPAC Recommendations)", edited by HGW Leuenberger, B. Nagel, and H. Koelbl, Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0025] To carry out the present invention, conventional methods of chemistry, biochemistry, cell biology, and recombinant DNA technology described in the literature of the art are used unless otherwise specified (see, for example, Molecular Cloning: A Laboratory Manual, 2nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Furthermore, conventional methods of clinical cardiology described in the literature of the art are also used (see, for example, Braunwald's Heart Disease: A Textbook of Cardiovascular Medicine, 9th Edition, P. Libby et al. eds., Saunders Elsevier Philadelphia, 2011).

[0026] Throughout this specification and the subsequent claims, unless otherwise specified in the context, the word “comprises” and variations such as “comprising” shall be understood to include, but not to exclude, any other integers, steps, or groups of integers or steps. As used in this specification and the appended claims, the singular forms “a,” “an,” and “the” shall also include the plural forms unless otherwise explicitly stated.

[0027] Nucleic acid molecules are understood as macromolecules composed of nucleotide monomers. Nucleic acid monomers consist of a nucleic acid base, a five-carbon sugar (e.g., ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, polynucleotides are formed by phosphate diester bonds between individual nucleotide monomers. In this invention, nucleic acid molecules include, but are not limited to, ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The terms "polynucleotide" and "nucleic acid" are used interchangeably herein.

[0028] An "open reading frame" (ORF) refers to a nucleotide sequence that can be translated into amino acids. Typically, such an ORF contains a start codon and a region that is usually a multiple of three nucleotides in length, but does not contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA). ORFs usually exist in nature or are artificially constructed using genetic engineering techniques. ORFs encode proteins in which the translatable amino acids form peptide chains.

[0029] The terms “protein” and “polypeptide” are used interchangeably herein and refer to amino acid chains linked by peptide bonds, regardless of length or whether or not they have post-translational modifications. Proteins usable in this invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitopes, and protein domains) can be further modified by chemical modification. This means that such chemically modified polypeptides may contain chemical groups other than the 20 naturally occurring amino acids. Examples of such other chemical groups include, but are not limited to, glycosylated amino acids and phosphorylated amino acids. Chemical modification of polypeptides may result in advantageous properties compared to the parent polypeptide, such as one or more of the following: improved stability, extended biological half-life, or improved water solubility. Chemical modifications applicable to variants usable in this invention include, but are not limited to, PEGylation, glycosylation of non-glycosylated parent polypeptides, covalent bonding with therapeutic small molecules such as exenatide, albiglutide, taspoglutide, DPP4 inhibitors, incretins, glucagon-like peptide 1 agonists such as liraglutide, or modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications, applicable to mutants usable in this invention, can occur during or after translation.

[0030] The term "amino acid" includes not only natural amino acids but also amino acid derivatives. In this invention, a hydrophobic non-aromatic amino acid refers to an amino acid that is not aromatic and preferably has a Kyte Doolittle hydroxyl index of 0.5 or higher, more preferably 1.0 or higher, and even more preferably 1.5 or higher. Preferably, the hydrophobic non-aromatic amino acid in the context of this invention is selected from the group consisting of alanine (Kyte Doolittle hydroxyl index 1.8), methionine (Kyte Doolittle hydroxyl index 1.9), isoleucine (Kyte Doolittle hydroxyl index 4.5), leucine (Kyte Doolittle hydroxyl index 3.8), and valine (Kyte Doolittle hydroxyl index 4.2), or their derivatives having the Kyte Doolittle hydroxyl index as defined above.

[0031] In this specification, the term “mutant” is used to refer to a polypeptide that differs from the polypeptide or fragment from which it is derived due to one or more changes in its amino acid sequence. The polypeptide from which a protein mutant is derived is also called the parent polypeptide. Similarly, the fragment from which a protein fragment mutant is derived is also called the parent fragment. Typically, mutants are constructed artificially, preferably by genetic engineering means. Typically, the parent polypeptide is the wild-type protein or wild-type protein domain. Furthermore, mutants usable in this invention may also be derived from homologs, orthologs, or paralogs of the parent polypeptide, or from artificially constructed mutants, in which case the mutant shall exhibit at least one biological activity of the parent polypeptide. Changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-terminal cleavages, C-terminal cleavages, or any combination of these changes, and may occur at one or more sites. In a preferred embodiment, the mutants usable in the present invention exhibit a total of up to 23 (up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23) changes (i.e., exchanges, insertions, deletions, N-terminal cleavage, and / or C-terminal cleavage) in the amino acid sequence. The amino acid exchanges may be conservative, semi-conservative, and / or non-conservative. In a preferred embodiment, the mutants usable in the present invention differ from the protein or domain from which they are derived by exchanges of up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids, preferably conservative amino acid changes.

[0032] Typical substitutions are between aliphatic amino acids, between amino acids with aliphatic hydroxyl side chains, between amino acids with acidic residues, between amide derivatives, between amino acids with basic residues, or between amino acids with aromatic residues. Typical semi-conservative and conservative substitutions are as follows:

[0033] JPEG2026530536000002.jpg128150

[0034] Substitutions of A, F, H, I, L, M, P, V, W, or Y to C are semi-conservative if the new cysteine ​​remains as a free thiol. Furthermore, those skilled in the art will understand that glycine in sterically demanding positions should not be substituted, and that P should not be introduced into regions of proteins that have α-helix or β-sheet structures.

[0035] Alternatively or additionally, the “mutants” as used herein may be characterized by a degree of sequence identity with the parent polypeptide or parent polynucleotide from which they are derived. More precisely, a protein mutant in the context of the present invention exhibits at least 85% sequence identity with its parent polypeptide. Preferably, the polypeptide in question and the reference polypeptide exhibit the indicated sequence identity over a contiguous range of 20, 30, 40, 45, 50, 60, 70, 80, 90, 100 or more amino acids, or over the entire length of the reference polypeptide. Preferably, the polynucleotide in question and the reference polynucleotide exhibit the indicated sequence identity over a contiguous range of 60, 90, 120, 135, 150, 180, 210, 240, 270, 300 or more nucleotides, or over the entire length of the reference polypeptide.

[0036] The term “at least 85% sequence identity” is used throughout this specification in reference to sequence comparisons of polypeptides and polynucleotides. This expression preferably refers to at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with respect to each reference polypeptide or each reference polynucleotide.

[0037] Protein fragments consist of amino acid deletions and may include N-terminal cleavage, C-terminal cleavage, internal deletion, or any combination thereof. Such variants, including N-terminal cleavage, C-terminal cleavage, and / or internal deletion, are referred to as “fragments” in the context of this application. Fragments may be naturally occurring (e.g., splice variants) or may be artificially constructed, preferably by genetic engineering means. Preferably, a fragment (or deletion variant) has up to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, or 23 amino acids deleted at its N-terminus and / or C-terminus and / or internally, preferably at its N-terminus, its N-terminus and C-terminus, or its C-terminus, compared to the parent polypeptide.

[0038] When comparing two sequences, and no reference sequence is specified as the one to be compared when calculating the sequence identity percentage, the sequence identity is calculated based on the longer of the two sequences being compared, unless otherwise specified.

[0039] The percentage of similarity between nucleotide sequences and amino acid sequences, i.e., sequence identity, can be determined by sequence alignment. Such alignments can be performed using several algorithms known in the art, preferably the mathematical algorithms of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), hmmalign (HMMER package, http: / / hmmer.wustl.edu / ), or the CLUSTAL algorithm (Thompson, JD, Higgins, DG & Gibson, TJ (1994) Nucleic Acids Res. 22, 4673-80), or the CLUSTALW2 algorithm (Larkin MA, Blackshields G, Brown NP, Chenna R, McGettigan PA, McWilliam H, Valentin F, Wallace IM, Wilm A, Lopez R, Thompson JD, Gibson TJ, Higgins DG. (2007)). Clustal W and ClustalX versions 2.0. Bioinformatics, 23, 2947-2948. These are available, for example, at http: / / npsa-pbil.ibcp.fr / cgi-bin / npsa_automat.pl?page= / NPSA / npsa_clustalw.html or http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html. Preferably, the CLUSTALW2 algorithm from http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html is used.The parameters used here are the default parameters set at http: / / www.ebi.ac.uk / Tools / clustalw2 / index.html: For the slow pairwise alignment option, Alignment type = slow, Protein weight matrix = Gonnet, Gap open = 10, Gap expansion for slow pairwise alignment = 0,1, Protein weight matrix = Gonnet, Gap open = 10, Gap expansion = 0,20, Gap distance = 5, No end gap = none, Output options: Format = Aln w / numbers, Order = Aligned.

[0040] The degree of sequence identity (sequence matching) can be calculated using, for example, BLAST, BLAT, BlastZ (or BlastX). Similar algorithms are incorporated into the BLASTN and BLASTP programs in Altschul et al. (1990) J.Mol.Biol.215:403-410. BLAST protein searches are performed using the BLASTP program, which is available, for example, as follows. http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome. The recommended algorithm parameters are the following default parameters. http: / / blast.ncbi.nlm.nih.gov / Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PAGE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome:Expected threshold=10, word size=3, maximum matches in query range=0, matrix=BLOSUM62, gap cost=present:11, extension:1, configuration adjustment=conditional configuration score matrix adjustment and a database of non-redundant protein sequences (nr) for obtaining amino acid sequences homologous to Factor1 and Factor2 polypeptides.

[0041] To obtain gap alignment for comparative purposes, Gapped BLAST is used, as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When using the BLAST and Gapped BLAST programs, the default parameters of each program are used. Sequence matching analysis can be supplemented by established homology mapping methods such as Shuffle-LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1: I54-I62) or Markov random fields. Where sequence identity percentages are mentioned in this application, unless otherwise specified, these percentages are calculated as a ratio to the total length of the longer sequence.

[0042] In this specification, the term “host cell” means a cell that harbors the nucleic acid of the present invention (e.g., in the form of a plasmid or virus). Such a host cell may be either a prokaryotic cell (e.g., a bacterial cell) or a eukaryotic cell (e.g., a fungal, plant, or animal cell). The cell may be transformed or untransformed. The cell may be, for example, an isolated cell in a cell culture or part of a tissue, the tissue itself may be isolated or part of a more complex tissue structure such as an organ or an individual.

[0043] The terms “myelin-derived growth factor,” “MYDGF,” “factor I,” “MYDGF polypeptide or protein,” or “factor I polypeptide or protein” are used interchangeably and refer to the protein shown in the NCBI reference sequence NM_019107.3 (human homolog), as well as its mammalian homologs, particularly those derived from mice or rats. The amino acid sequence of the human homolog is encoded in open reading frame 10 (C19Orf10) on human chromosome 19. Preferably, MYDGF and factor I protein refer to proteins that essentially consist of, or consist of, the core segment of human factor I having the amino acid sequence described in Sequence ID No. 1.

[0044] Whether a protein, variant, or fragment exerts the biological function of MYDGF can be determined by any of the tests described in the following examples. According to the present invention, a peptide or protein exerts the biological function of MYDGF if the results obtained using the peptide or protein achieve at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95%, or 100% of the effect reported for MYDGF against a given control group, compared to the results obtained using the MYDGF protein of the present invention shown in at least one example described herein.

[0045] As used herein, the terms "MYDGF" and "Mydgf" both refer to bone marrow-derived growth factor. In this invention, "MYDGF" is used to refer to a human variant, and "Mydgf" is used to refer to a mouse variant of bone marrow-derived growth factor.

[0046] As used herein, the term “acute hepatic failure” means a rare, acute, potentially reversible condition that results in severe hepatic impairment and rapid clinical deterioration in patients without pre-existing liver disease (A. Shingina et.al., Am J Gastroenterol. 2023 Jul 1;118(7):1128-1153).

[0047] As used herein, “chronic liver disease” refers to a condition in which liver function, including the synthesis of coagulation factors and other proteins, detoxification of harmful metabolites, and bile excretion, progressively declines over a period of six months or more. Chronic liver disease (CLD) is a continuous process of repeated inflammation, destruction, and regeneration of the liver parenchyma, leading to fibrosis and cirrhosis. The etiologies of chronic liver disease are diverse and include toxins, long-term alcohol abuse, infections, autoimmune diseases, genetic disorders, and metabolic disorders (https: / / www.ncbi.nlm.nih.gov / books / NBK554597 / ). Chronic liver disease differs in the degree of scarring (non-fibrotic, fibrotic, cirrhotic) and compensation (compensated vs. decompensated).

[0048] In this specification, the term "hepatic fibrosis" means the formation of excessive fibrous connective tissue in the liver during a repair or reaction process, such as a reactive, benign, or pathological condition. The precipitating factor is chronic injury, particularly when there is an inflammatory component. The connective tissue deposited during fibrosis may disrupt or impair the normal structure and function of the underlying organ or tissue (R. Weiskirchen et.al., Molecular Aspects of Medicine, Vol 65, 2019, 2-15).

[0049] As used herein, the term “cirrhosis” refers to a condition in which the liver becomes scarred and permanently damaged. Scarred tissue replaces healthy liver tissue and impairs normal liver function. Scarred tissue also partially blocks blood flow through the liver (https: / / www.niddk.nih.gov / health-information / liver-disease / cirrhosis / definition-facts). Cirrhosis is generally divided into two stages: compensated cirrhosis and decompensated cirrhosis.

[0050] "Compensated cirrhosis" refers to the early stage of cirrhosis. The liver is scarred, but its function is normal or partially maintained. In this type of cirrhosis, symptoms may be mild or absent, but treatment is necessary to prevent further damage and complications.

[0051] "Decompensated cirrhosis" refers to a progressive stage of cirrhosis in which the liver undergoes severe scarring and ceases to function normally. Patients with this type of cirrhosis may present with serious complications such as jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, and variceal bleeding.

[0052] In this specification, “liver transplantation” refers to a treatment that involves removing a liver whose function has deteriorated due to various causes and transplanting a healthy liver. The description of embodiments includes further definitions and explanations of terms used throughout this application. These explanations and definitions are effective throughout this application unless otherwise noted.

[0053] [array] The sequence used in this invention is shown below. Sequence ID 1 (amino acid sequence of human Factor1 lacking the 31-amino acid N-terminal signal peptide): VSEPTTVAFDVRPGGVVHSFSHNVGPGDKYTCMFTYASQGGTNEQWQMSLGTSEDHQHFTCTIWRPQGKSYLYFTQFKAEVRGAEIEYAMAYSKAAFERESDVPLKTEEFEVTKTAVAHRPGAFKAELSKLVIVAKASRTEL Sequence ID 2 (amino acid sequence of the mouse homolog of Factor1 lacking the 24-amino acid N-terminal signal peptide): VSEPTTVPFDVRPGGVVHSFSQDVGPGNKFTCTFTYASQGGTNEQWQMSLGTSEDSQHFTCTIWRPQGKSYLYFTQFKAELRGAEIEYAMAYSKAAFERESDVPLKSEEFEVTKTAVSHRPGAFKAELSKLVIVAKAARSEL SEQ ID NO:3 (Amino acid sequence of human Factor1, including the N-terminal signal peptide (shown in bold and underlined); UniProtKB - Q969H8): JPEG2026530536000003.jpg30150SEQ ID NO: 4 (Amino acid sequence of the mouse homolog of Factor1, including the N-terminal signal peptide (shown in bold and underlined); UniProtKB - Q9CPT4): JPEG2026530536000004.jpg31150 Sequence ID 5 shows the nucleic acid sequence of human Factor1 encoding MYDGF of Sequence ID 3 (NCBI gene ID: 56005). Sequence ID 6 shows the nucleic acid sequence of mouse Factor1 (NCBI gene ID: 28106) encoding Mydgf in Sequence ID 4. [Modes for carrying out the invention]

[0054] [Embodiment] The elements of the present invention are described below. These elements are listed along with specific embodiments, but it should be understood that they can be combined in any way and in any number to create further embodiments. The various examples and preferred embodiments described should not be construed as limiting the invention to only the embodiments explicitly described. This description should be understood as supporting and encompassing the embodiments explicitly described and embodiments that combine any number of the disclosed elements and / or preferred elements. Furthermore, any permutation and combination of all elements described in this application should be considered disclosed by this description unless otherwise stated in the context.

[0055] The inventors have for the first time demonstrated the effects of MYDGF on liver damage. In particular, they have shown that administration of MYDGF in a mouse model suppresses acute and chronic liver damage and liver fibrosis. Furthermore, studies using mouse models and cultured cells have shown that MYDGF also suppresses liver inflammatory responses. These effects can be used, in particular, to suppress the progression of liver damage and associated scarring. Accordingly, in a first embodiment, the present invention provides the protein myelo-derived growth factor (MYDGF) or a fragment or variant thereof exhibiting the biological function of MYDGF for use in the treatment or prevention of liver disease caused by liver damage.

[0056] In a second embodiment, the present invention provides myeloid-derived growth factor (MYDGF) proteins, or fragments or variants thereof exhibiting biological functions of MYDGF, for use in the treatment or prevention of liver disease. According to a preferred embodiment, the liver disease is acute liver failure, chronic liver disease, or liver transplantation. According to a preferred embodiment, acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury, and toxic liver injury. Viral hepatitis is inflammation of the liver caused by viral infections, including hepatitis A, B, C, D, and E. Drug-induced liver injury is caused by the toxic effects of certain drugs, such as acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), and certain antibiotics. Acute ischemic liver injury occurs when blood flow to the liver is reduced due to conditions such as liver transplantation, shock, or vascular disease. Toxic liver injury is caused by ingestion of certain chemicals, toxins, mushrooms, or exposure to industrial pollutants such as carbon tetrachloride, aflatoxins, or heavy metals. In preferred embodiments, chronic liver diseases include non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infections caused by hepatitis B virus (HBV) or hepatitis C virus (HCV), alcoholic liver disease (ALD), autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), metabolic liver disease, and chronic ischemic liver injury. Non-alcoholic fatty liver disease (NAFLD) is a disease in which excessive fat accumulates in the liver due to causes other than excessive alcohol consumption, and although fat accumulates in the liver, there is little to no inflammation or liver damage. Non-alcoholic steatohepatitis (NASH) is a disease in which excessive fat accumulates in the liver due to reasons other than excessive alcohol consumption, and is accompanied by liver inflammation and liver damage, and can cause not only fat accumulation in the liver but also liver fibrosis and scarring. Chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) can cause persistent inflammation and fibrosis of the liver. Alcoholic liver disease (ALD), caused by chronic alcohol consumption, can lead to inflammation and scarring of liver tissue. Autoimmune hepatitis is a disease in which the immune system mistakenly attacks the liver, resulting in chronic inflammation and fibrosis.Primary biliary cholangitis (PBC) is a chronic liver disease characterized by progressive destruction of the bile ducts, leading to fibrosis and cirrhosis over time. Primary sclerosing cholangitis (PSC) is a disease characterized by inflammation and scarring of the bile ducts, and is often associated with inflammatory bowel disease (IBD). Metabolic liver diseases include Wilson's disease, hemochromatosis, and alpha-1 antitrypsin deficiency, which are genetic disorders that affect liver function. Chronic ischemic liver injury develops when blood flow to the liver is reduced due to liver transplantation, shock, vascular disease, etc. Furthermore, MYDGF or its fragments or variants are also provided for this purpose. Treatments for liver disease include liver transplantation, including living donor liver transplantation and brain-dead donor liver transplantation. MYDGF or its fragments or variants can improve the quality and function of the donor liver before transplantation, thereby increasing the success rate of transplantation and the recovery rate of recipients after transplantation.

[0057] In a particularly preferred embodiment of the present invention, the protein comprises the amino acid sequence or a fragment thereof of SEQ ID NO: 1. Preferably, the protein has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with SEQ ID NO: 1.

[0058] In a preferred embodiment of this aspect of the present invention, the protein comprises the amino acid sequence of SEQ ID NO: 1, a fragment or variant thereof exhibiting the biological function of MYDGF, and having at least 85% sequence identity with SEQ ID NO: 1. Those skilled in the art can determine, without undue burden, which positions of the parent polypeptide can be mutated and to what extent, and which positions need to be preserved to maintain the functionality of the polypeptide. Such information can be obtained, for example, from homologous sequences that can be identified, aligned, and analyzed by bioinformatics techniques well known in the art. Such analyses are illustrated in Example 7 of WO 2014 / 111458 and in Figures 6 and 7. The mutations are preferably introduced into regions of the protein that are not fully conserved between species, preferably between mammals. In a particularly preferred embodiment of the present invention, the MYDGF protein comprises, essentially consists of, or consists of, the amino acid sequence of SEQ ID NO: 1, or a fragment or variant thereof exhibiting the biological function of MYDGF. Preferably, the protein has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1.

[0059] N-terminal deletion mutants are also included, which may, for example, lack one or more amino acids from amino acid positions 1 through 24 (based on SEQ ID NO:1), i.e., from the N-terminal conserved region.

[0060] C-terminal deletion mutants are also included, which may, for example, lack one or more amino acids between amino acid positions 114 and 142 (based on SEQ ID NO:1).

[0061] On the other hand, amino acids can be added to the MYDGF protein. Such additions include additions to the N-terminus, C-terminus, within the amino acid sequence, or a combination thereof. Therefore, the protein of the first embodiment of the present invention may further include additional amino acid sequences, for example, to stabilize or purify the resulting protein. Examples of such amino acids include 6xHis tags, myc tags, or FLAG tags. These are well known in the art and can be located at any position in the protein, but are preferably located at the N-terminus or C-terminus. A particularly preferred addition sequence is the 6xHis tag. Preferably, the 6xHis tag is located at the C-terminus of the MYDGF protein. Depending on the expression system used and any additional amino acids, such as the tags mentioned above, one or more residual amino acids may remain at the N-terminus and / or C-terminus of the protein. It is emphasized that the MYDGF protein and Mydgf protein according to the present invention may contain artifacts, such as those shown in Ebenhoch R. et al., Nat Commun. 2019 Nov 26;10(1):5379 and Polten F. et al., Anal Chem. 2019 Jan 15;91(2):1302-1308.

[0062] In some cases, it is preferable to stabilize the protein by mutating the protease cleavage sites within the MYDGF protein of the first embodiment of the present invention (see Segers et al. Circulation 2007, 2011). Those skilled in the art know how to identify potential proteolytic cleavage sites within a protein. For example, a protein sequence can be submitted to a website that provides analysis, such as http: / / web.expasy.org / peptide_cutter / or http: / / pmap.burnham.org / proteases. If the protein sequence with SEQ ID NO:1 is submitted to http: / / web.expasy.org / peptide_cutter / , the following low-frequency (less than 10) cleavage sites will be identified.

[0063] [Table 1]

[0064] These sites are modified to remove the recognition / cleavage sequences of their respective identified proteases, potentially extending the serum half-life of the protein.

[0065] In this invention, MYDGF has been shown to inhibit or prevent acute and chronic liver injury, as well as hepatic fibrosis and associated inflammatory responses. These effects can be used, in particular, to inhibit the progression of liver injury and associated scarring. Accordingly, this invention provides MYDGF protein, or fragments or variants thereof exhibiting the biological function of MYDGF, for use in the treatment or prevention of liver disease.

[0066] While not intending to be bound by any particular theory, MYDGF suppresses the inflammatory response in the liver by inhibiting NF-κB activation, thereby preventing and / or treating liver disease.

[0067] The MYDGF protein may further contain additional amino acid sequences, for example, to stabilize or purify the resulting protein. For example, it is preferable to mutate the protease cleavage sites within the MYDGF protein to stabilize the protein. Appropriate proteolytic cleavage sites can be identified as described above.

[0068] MYDGF protein or compositions containing the protein can be administered in vivo, in vitro, or in vitro, and is preferably administered in vivo.

[0069] In a further embodiment, the present invention provides nucleic acids encoding the MYDGF protein described herein, or a fragment or variant thereof exhibiting the biological function of MYDGF, for use in the treatment or prevention of liver disease. The present invention also provides nucleic acids encoding the MYDGF protein described herein, or a fragment or variant thereof exhibiting the biological function of MYDGF, for use in the treatment or prevention of liver disease. The nucleic acids used in accordance with the present invention preferably encode an amino acid sequence having at least 85% sequence identity with SEQ ID NO: 1.

[0070] Nucleic acid sequences can be optimized to enhance expression in host cells. Parameters to consider include C:G content, preferred codons, and avoidance of inhibitory secondary structures. These factors can be combined in various ways to obtain nucleic acid sequences with enhanced expression in a particular host (see, e.g., Donnelly et al., international publication number WO 97 / 47358). Whether a particular sequence can enhance expression in a particular host is determined by several empirical experiments. Such experiments include measuring the expression of the target nucleic acid sequence and modifying the sequence as needed. Starting from a specific amino acid sequence and the degeneracy of the known genetic code, numerous different coding nucleic acid sequences can be obtained. The degeneracy of the genetic code arises because almost all amino acids are encoded by different combinations of nucleotide triplets, i.e., "codons." Translation from a specific codon to a specific amino acid is well known in the art (see, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990).

[0071] Nucleic acids used in accordance with the present invention may further include transcriptional regulatory elements or expression regulatory sequences arranged to control protein expression. Such nucleic acids, together with the regulatory elements, are often referred to as expression systems. As used herein, the term “expression system” refers to a system designed to produce one or more target gene products. Typically, such systems are designed “artificially,” i.e., by genetic technology means available for producing the target gene product in vivo, in vitro, or ex vivo. The term “expression system” further encompasses the expression of the target gene product, including the transcription of polynucleotides, mRNA splicing, translation into polypeptides, translational and posttranslational modifications of polypeptides or proteins, as well as the targeting of the protein to one or more compartments within a cell, secretion from the cell, and uptake of the protein into the same or other cells. This general description refers to expression systems for use in eukaryotic cells, tissues, or organisms. Expression systems in prokaryotic systems may differ, and how expression systems in prokaryotic cells are constructed is well known in the art.

[0072] The regulatory elements included in a gene expression cassette generally include (a) a promoter transcriptionally bound to the nucleotide sequence encoding a polypeptide, (b) a 5' ribosome binding site functionally bound to the nucleotide sequence, (c) a terminator bound to the 3' end of the nucleotide sequence, and (d) a 3' polyadenylation signal functionally bound to the nucleotide sequence. Additional regulatory elements useful for promoting or controlling gene expression or polypeptide processing may also exist. A promoter is a genetic element recognized by RNA polymerase that mediates the transcription of downstream regions. A preferred promoter is a potent promoter that increases transcription levels. Examples of potent promoters include the human cytomegalovirus pre-early promoter (CMV) and CMV containing intron A (Chapman et al, Nucl. Acids Res. 19:3979-3986, 1991). Further examples of promoters include native promoters such as the EF1 alpha promoter, mouse CMV promoter, Roussarcoma virus promoter, SV40 early / late promoter, and [β]-actin promoter, as well as artificial promoters such as synthetic muscle-specific promoters and chimeric muscle-specific / CMV promoters (Li et al., Nat. Biotechnol. 17:241-245, 1999; Hagstrom et al., Blood 95:2536-2542, 2000).

[0073] The ribosome binding site is located at or near the start codon. Examples of preferred ribosome binding sites include CCACCAUGG, CCGCCAUGG, and ACCAUGG, where AUG is the start codon (Kozak, Cell 44:283-292, 1986). Polyadenylation signals are responsible for the cleavage of transcribed RNA and the addition of a poly(A) tail to the RNA. In higher eukaryotes, polyadenylation signals include an AAUAAA sequence located approximately 11–30 nucleotides away from the polyadenylation site. The AAUAAA sequence is involved in the signaling of RNA cleavage (Lewin, Genes IV, Oxford University Press, NY, 1990). The poly(A) tail is important for mRNA processing, nuclear transport, translation, and stability.

[0074] Polyadenylation signals that can be used as part of a gene expression cassette include minimal rabbit [β]globin polyadenylation signals and bovine growth hormone polyadenylation (BGH) signals (Xu et al., Gene 272:149-156, 2001; Post et al., U.S. Patent US 5,122,458).

[0075] Examples of additional regulatory elements useful for promoting or controlling gene expression or polypeptide processing include enhancers, leader sequences, and operators. Enhancer regions promote transcription. Examples of enhancer regions include the CMV enhancer and the SV40 enhancer (Hitt et al., Methods in Molecular Genetics 7:13-30, 1995; Xu, et al., Gene 272:149-156, 2001). Enhancer regions may be associated with promoters.

[0076] The expression of the MYDGF protein or its variants according to the present invention can be regulated. Such regulation can be achieved at many stages of gene expression. Possible regulatory stages include, but are not limited to, transcription initiation, promoter clearance, transcription elongation, splicing, transport from the nucleus, mRNA stability, translation initiation, translation efficiency, translation elongation, and protein folding. Other regulatory stages that affect the concentration of the MYDGF polypeptide in the cell also affect the protein's half-life. Such regulatory stages include, for example, regulated denaturation of the protein. Since the proteins of the present invention include secretory proteins, the proteins can be induced into the secretory pathway of the host cell. Secretory efficiency, along with regulatory stages related to expression and protein stability, regulates the extracellular concentration of the protein. Extracellular refers to, but is not limited to, culture media, tissues, intracellular matrix or intercellular spaces, or bodily fluids such as blood or lymph.

[0077] The control of the regulatory steps described above may be, for example, independent of cell type or tissue type, or specific to cell type or tissue type. In a particularly preferred embodiment of the present invention, the control of the regulatory steps is specific to cell type or tissue type. Such cell type or tissue type-specific regulation is preferably achieved through a regulatory step relating to nucleic acid transcription. This transcriptional regulation can be achieved by using a cell type or tissue type-specific promoter sequence. The results of this cell type or tissue type-specific regulation may have different levels of specificity. This means that the expression of each polypeptide is enhanced in each cell or tissue compared to other cell types or tissue types, or that the expression is limited to each cell type or tissue type. Cell type or tissue type-specific promoter sequences are well known in the art and are available for a wide range of cell types or tissue types.

[0078] Expression is not necessarily cell-type or tissue-type specific and may depend on physiological conditions. Such conditions include, for example, inflammation and wounds. Such physiological condition-specific expression can also be achieved through regulation in all the regulatory steps described above. A preferred method for regulating physiological condition-specific expression is transcriptional regulation. For this purpose, wound or inflammation-specific promoters can be used. Each promoter is, for example, a native sequence derived from a gene specifically expressed during an immune response and / or wound tissue regeneration. Another possibility is the use of artificial promoter sequences constructed from, for example, a combination of two or more native sequences.

[0079] Regulation may be cell type or tissue type specific, and physiological condition specific. In particular, expression may be liver-specific. Preferably, expression is liver-specific and / or wound-specific.

[0080] Another possibility for controlling the expression of the MYDGF protein or its variants according to the present invention is conditional control of gene expression. Operator sequences can be used to achieve conditional control. For example, Tet operator sequences can be used to repress gene expression. Conditional control of gene expression using Tet operators and Tet repressors is well known in the art, and many systems have been established in a wide range of prokaryotes and eukaryotes. Those skilled in the art know how to select an appropriate system and adapt it to the specific needs of each application.

[0081] In a particularly preferred embodiment, the use of nucleic acids according to the present invention includes application to individuals, preferably individuals suffering from liver disease.

[0082] In a further embodiment, the present invention provides a vector comprising a nucleic acid or expression system described herein for use in the treatment or prevention of liver disease.

[0083] In this specification, the term “vector” refers to a protein, polynucleotide, or mixture thereof that can be introduced into a cell or into a cell containing proteins and / or nucleic acids. In the present invention, it is preferable that the target gene encoded by the introduced polynucleotide is expressed in the host cell upon introduction of the vector(s). Suitable vectors include, but are not limited to, plasmid vectors, cosmid vectors, phage vectors such as lambda phages, filamentous phage vectors, viral vectors, virus-like particles, and bacterial spores.

[0084] In a preferred embodiment of the present invention, the vector is a viral vector. Suitable viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus (AAV) vectors, alphavirus vectors, herpesvirus vectors, measles virus vectors, poxvirus vectors, vesicular stomatitis virus vectors, retrovirus vectors, and lentivirus vectors.

[0085] In a particularly preferred embodiment of the present invention, the vector is an adenovirus or an adeno-associated virus (AAV) vector.

[0086] Nucleic acids encoding one or more MYDGF proteins or their variants according to the present invention can be introduced into host cells, tissues, or organisms using a vector suitable for therapeutic administration. A suitable vector is preferably capable of delivering the nucleic acid to target cells without causing unacceptable side effects.

[0087] In a particularly preferred embodiment, the use of the vector according to the present invention includes application to an individual that requires it.

[0088] A vector containing a nucleic acid encoding the MYDGF protein, or a fragment or variant thereof exhibiting the aforementioned biological function of MYDGF, is preferably used for the treatment or prevention of liver disease.

[0089] In a further embodiment, the present invention provides host cells expressing nucleic acids encoding the MYDGF protein or a fragment or variant thereof exhibiting the biological function of MYDGF, for use in the treatment or prevention of liver damage, comprising the vector described herein.

[0090] In a further embodiment, the present invention provides a pharmaceutical composition for liver disease comprising a MYDGF protein or a fragment or variant thereof exhibiting the biological function of MYDGF, and optionally a suitable pharmaceutical excipient.

[0091] As used herein, the term “suitable pharmaceutical excipient” refers to a diluent, excipient, surfactant, stabilizer, physiological buffer, or medium used when administering a therapeutic active ingredient. “Pharmaceutical excipient” is also called “pharmaceutical carrier” and may be liquid or solid. Examples of liquid carriers include, but are not limited to, sterile solutions such as saline dissolved in water, and oils such as peanut oil, soybean oil, mineral oil, and sesame oil, which are of petroleum, animal, plant, or synthetic origin. Saline, aqueous dextrose, and glycerol solutions can also be used as liquid carriers, particularly for injections. Saline is a preferred carrier when administering pharmaceutical compositions intravenously. Examples of suitable pharmaceutical carriers are described in E.W. Martin's “Remington's Pharmaceutical Sciences.” In preferred embodiments of the present invention, the carrier is a suitable pharmaceutical excipient. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, skim milk powder, glycerol, propylene glycol, water, and ethanol. Such suitable pharmaceutical excipients are preferably pharmaceutically acceptable.

[0092] "Pharmacologically acceptable" means that it is approved by a federal or state regulatory authority, or that its use in animals, particularly humans, is listed in the United States Pharmacopeia or other generally accepted pharmacopoeias.

[0093] The term “composition” is intended to include a combination of an active compound and an encapsulating material that provides a capsule in which the active component is surrounded by a carrier, with or without other carriers, thereby associating with the active compound.

[0094] The term "active ingredient" refers to a substance that is biologically active, i.e., has medicinal value, in a pharmaceutical composition or formulation. In this invention, the active ingredient is the MYDGF protein, or a fragment or variant thereof that exhibits the biological function of MYDGF. A pharmaceutical composition may contain one or more active ingredients that may act in conjunction with each other or independently. The active ingredient may be formulated in neutral or salt form. The salt form is preferably a pharmaceutically acceptable salt.

[0095] The term “pharmaceutically acceptable salt” refers to, but is not limited to, salts of the MYDGF polypeptide of the present invention, including, for example, the fragments and variants thereof described herein. Suitable pharmaceutically acceptable salts include, for example, acid addition salts formed by mixing a solution of the polypeptide of the present invention with a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, citric acid, tartaric acid, carbonic acid, or phosphoric acid. Furthermore, if the peptide has an acidic moiety, suitable pharmaceutically acceptable salts may include alkali metal salts (e.g., sodium or potassium salts), alkaline earth metal salts (e.g., calcium or magnesium salts), and salts formed with suitable organic ligands (e.g., ammonium, quaternary ammonium, and amine cations formed using counter anions such as halides, hydroxides, carboxylates, sulfates, phosphates, nitrates, alkyl sulfons, and aryl sulfons). Specific examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipine, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, tartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphor sulfonate, cansylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecyl sulfate, edetate, edisylate, estrate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolyl arsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydravamin, hydrobromide, hydrochloride, and hydroiodide.2-Hydroxyethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methyl sulfate, mucinate, 2-naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate / diphosphate, picrate, pivaphosphate, polygalacturonate, propionate, salicylate, stearate, sulfate, acetate, succinate, tannate, tartrate, theocrine, tosylate, triethiodide, undecanoate, valerate, etc. (Example: SM Berge et al., "Pharmaceutical Includes "Salts," J. Pharm. Sci. 66, pp. 1-19 (1977).

[0096] According to one embodiment, an active ingredient is administered to cells, tissues, or an organism in an effective amount. “Effective amount” means an amount of the active ingredient sufficient to achieve the intended purpose. The active ingredient may be a therapeutic agent. The effective amount of a particular active ingredient varies depending on parameters such as the properties of the ingredient, the route of administration, the size and species of the organism to which the active ingredient is administered, and the purpose of administration. The effective amount in each case can be determined empirically by those skilled in the art according to methods established in the art. “Administration” as used in this invention includes not only intra vivo administration to an organism, but also in vitro or direct administration to cells or tissues outside the body.

[0097] In preferred embodiments of the present invention, the pharmaceutical composition is customized for the treatment of a disease or disorder. In this specification, “to treat,” “to cure,” or “to treat” a disease or disorder means achieving one or more of the following: (a) reducing the severity of the disorder; (b) suppressing or preventing the onset of symptoms characteristic of the disorder being treated; (c) suppressing the exacerbation of symptoms characteristic of the disorder being treated; (d) suppressing or preventing recurrence of the disorder in a patient who has previously suffered from the disorder; (e) suppressing or preventing recurrence of symptoms in a patient who has previously shown symptoms of the disorder; (f) reducing the mortality rate after the onset of the disease or disorder; (g) curing; and (h) preventing the disease. The term “to improve” is also included in the term “to treat.” In this specification, “to prevent,” “prevent,” or “preventive method” of a disease or disorder means preventing the onset of such disease or disorder in a patient.

[0098] In a particularly preferred embodiment of the present invention, treatment with the pharmaceutical composition according to the present invention includes treatment of an individual in need of such treatment.

[0099] The pharmaceutical compositions intended by the present invention can be formulated in various ways well known to those skilled in the art. For example, the pharmaceutical compositions of the present invention may be in liquid form, such as solutions, emulsions, or suspensions. Preferably, the pharmaceutical compositions of the present invention are formulated for parenteral administration, preferably for intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intrapulmonary, or intraperitoneal administration. The route of administration is intrahepatic or mucosal, preferably intravenous, subcutaneous, or intraperitoneal. Formulations for oral or rectal administration are also possible. Preferably, the pharmaceutical compositions of the present invention are in the form of a sterile aqueous solution, which may contain, for example, a salt or other substance such as glucose in sufficient quantity to make the solution isotonic with blood. The aqueous solution should be appropriately buffered as needed (preferably pH 3 to 9, more preferably pH 5 to 7). The pharmaceutical compositions are preferably in unit dosage forms. In such forms, the pharmaceutical composition is divided into unit doses containing an appropriate amount of the active ingredient. The unit dosage forms can be formulations in which individual amounts of the pharmaceutical composition are packaged, such as vials or ampoules.

[0100] The pharmaceutical composition is preferably administered via intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intrapulmonary, intraperitoneal, or intrahepatic routes, but other routes of administration known in the art are also included.

[0101] When a pharmaceutical composition is used as a therapeutic agent for an individual, it may be administered as a substitute for or in addition to standard treatment for the respective disease or condition. When used in addition to standard treatment, the pharmaceutical composition may be administered before, concurrently with, or after standard treatment.

[0102] Furthermore, it is preferable to administer the pharmaceutical composition once or more times. This includes 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 times. The duration of administration of the pharmaceutical is not limited. Preferably, administration does not exceed 1, 2, 3, 4, 5, 6, 7, or 8 weeks.

[0103] The single dose of a pharmaceutical composition can independently form the total amount of dose administered, or each time range of administration may include administration as one or more bolus injections and / or infusions.

[0104] In a further embodiment, the present invention relates to a method for treating a liver disease, comprising administering to a patient in need of treatment a therapeutically effective amount of MYDGF, or a fragment or variant thereof exhibiting the biological function of MYDGF. In the method, MYDGF preferably comprises SEQ ID NO: 1, or a fragment or variant thereof exhibiting the biological function of MYDGF of SEQ ID NO: 1. In this regard, the fragment or variant comprises an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO: 1.

[0105] In preferred embodiments of the method of the present invention, the liver disease is acute liver failure, chronic liver disease, or liver transplantation. In more preferred embodiments, acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury, and toxic liver injury. In even more preferred embodiments, the chronic liver disease is non-alcoholic fatty liver disease (NAFLD), and the chronic liver disease is selected from the group consisting of non-alcoholic steatohepatitis (NASH), chronic infection due to hepatitis B virus (HBV) or hepatitis C virus (HCV), alcoholic liver disease (ALD), autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerotic cholangitis (PSC), metabolic liver disease, and chronic ischemic liver injury.

[0106] According to a preferred embodiment of this method of the present invention, the MYDGF protein, or a fragment or variant thereof exhibiting the biological function of MYDGF, is preferably administered by one or more bolus injections and / or infusions in a pharmaceutically acceptable carrier.

[0107] [Examples] The following examples are provided to further illustrate and enhance understanding of the present invention and do not limit its scope.

[0108] Materials and methods used in the examples: Unless otherwise specified, the following materials and methods were used in the examples.

[0109] <Sample Processing and Analysis> Blood was collected in EP tubes, stored at 4°C for 30 minutes, and centrifuged at 15,000 rpm for 10 minutes at 4°C. Liver tissue was collected, rapidly frozen in liquid nitrogen for protein analysis, and rapidly frozen in RNAlater (Invitrogen) for RNA extraction and analysis. A portion of the liver was fixed with 10% phosphate-buffered formalin, embedded in a paraffin block, and then histologically processed. Hematoxylin-eosin (H&E) staining and Sirius Red staining were performed. Analysis and quantification of positively stained areas were performed using ImageJ software. To analyze MYDGF concentration in serum, Nunc MaxiSorp TM Flat-bottomed filters (Thermo Fisher Scientific) were coated with 5 ug / ml anti-MYDGF capture antibody (PPB-32537, provided by Boehringer Ingelheim). Samples were then conjugated with 3 ug / ml biotin human SF20 / MYDGF antibody (PPB-50118, provided by Boehringer Ingelheim), and colored with streptavidin-HRP (provided by Southern Biotech) and substrate reagent (provided by R&D Systems). Finally, the absorbance of the samples was measured at 450 nm. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were measured using the Transaminase CII Test Wako Kit (Wako) according to the manufacturer's instructions. The hydroxyproline content in all liver tissue was measured using the Hydroxyproline Assay Kit™ (Cell Biolabs catalog number: STA-675) according to the manufacturer's instructions.

[0110] <Cell culture> Human hepatocellular carcinoma cell line HepG2 was seeded on collagen-coated plates, and cultured in Dulbecco's Modified Eagle Medium (DMEM, Life Technologies) supplemented with 10% fetal bovine serum (FBS), 100 U / mL penicillin and 100 mg / mL streptomycin at 37°C under a humidified atmosphere with 5% CO2. To minimize the potential influence of growth factors present in FBS on cell behavior, cells were washed twice with phosphate-buffered saline (PBS), then cultured in serum-free DMEM for 6 hours, and subsequently exposed to recombinant MYDGF protein.

[0111] <Western Blotting> Whole cell lysates were prepared from mouse liver cells and HepG2 cells, and lysed in 1×RIPA Lysis Buffer (EMD Millipore) containing a protease inhibitor cocktail and PhosSTOP (Roche Applied Science). The following primary antibodies were used: NF-κB, p-NF-κB (Ser536), GAPDH (all from Cell Signaling Technology), and SF20 / MYDGF (R&D Systems).

[0112] <RNA Extraction and RTD-PCR Analysis> Total RNA was isolated from frozen liver tissue samples using the RNeasy Mini Kit (QIAGEN) via RNAlater, and from cell samples using the NIPPON RNA Kit (Nippon Gene), following the manufacturer's protocol. cDNA was synthesized from 100 ng of total RNA using the High-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RTD-PCR was performed using TaqMan Gene Expression Assay Identification. The following TaqMan probes (Thermo Fisher Scientific) were used: MYDGF (Hs00384077_m1, Mm00840829_m1), TNF-α (Hs00174128_m1, Mm00443258_m1), IL-1β (Mm00434228_m1), IL-6 (Mm00446190_m1), Col1a1 (Mm00801666_g1), Col1a2 (Mm00483888_m1), and Acta2 (Mm01546133_m1). Quantitative gene expression data were standardized to the expression level of the housekeeping gene GAPDH.

[0113] <Recombinant proteins and chemicals> Recombinant human MYDGF (PROTEOS-1066-Batch2, PPB-11924): The nucleic acid sequence encoding human Factor1 is available under NCBI gene ID: 56005 (SEQ ID NO: 6). The amino acid sequence of human Factor1, including the N-terminal signal peptide, is described in detail in SEQ ID NO: 3. In the examples, signal peptide-free human MYDGF (SEQ ID NO:1) was used and evaluated as detailed in Ebenhoch R. et al. "Crystal structure and receptor interaction residues of MYDGF - a protein that mediates ischemic tissue repair" (Nat Commun. 2019 Nov 26;10(1):5379) and Polten et al. "Multiple reaction monitoring - Plasma concentrations of bone marrow-derived growth factor in healthy individuals and patients with acute myocardial infarction by evaluation by mass spectrometry. Anal Chem. 2019 Jan 15;91(2):1302-1308". Recombinant human TNF-α, IL-1β, and IL-6 were purchased from R&D Systems.

[0114] <Statistical analysis> Data are presented as mean ± standard deviation (SD) and analyzed using GraphPad Prism 9.4.1 software. The experiment was repeated at least three times. Two-tailed unpaired Student's t-test or one-way ANOVA was used to evaluate the data. Pearson's correlation coefficient was used to evaluate relationships. A p-value < 0.05 was considered statistically significant. Data are presented as mean ± SD. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0115] Example 1: Analysis of MYDGF expression in a carbon tetrachloride (CCl4)-induced liver injury model. Example 1-1: Creation of an acute liver injury model C57BL / 6J mice (male, 10 weeks old) were divided into 6 groups (5 mice per group). 10% CCl4 (180 μL corn oil + 20 μL CCl4 / mouse) was administered intraperitoneally to the mice. Liver and serum samples were collected at 6, 12, 24, 48, and 72 hours after CCl4 administration (Figure 1A). MYDGF mRNA expression in the liver was evaluated at each time point (Figure 1B). Serum transaminases, AST, and ALT levels were assessed at 24, 48, and 72 hours (Figures 1C and 1D). MYDGF mRNA expression in the liver increased from 6 hours after CCl4 infusion, peaking at 12 hours. MYDGF mRNA levels were maintained from 12 to 48 hours and slightly decreased at 72 hours (Figure 1B). Serum transaminases AST and ALT levels increased from 24 hours, peaking at 48 hours and returning to baseline levels at 72 hours (Figures 1C and 1D).

[0116] Examples 1-2: Creation of a chronic liver injury model C57BL / 6J mice (10 weeks old, male) were intraperitoneally administered 10% carbon tetrachloride (180 μL corn oil + 20 μL carbon tetrachloride per mouse) in 10 doses every 3 times for a total of 10 doses over 30 days. Liver and serum samples were collected on days 7, 14, and 28 after discontinuation of carbon tetrachloride administration (Figure 2A). mRNA expression of MYDGF, TNFα, and col1a2 in the liver was evaluated at each time point (Figures 2B, 2C, 2D). MYDGF mRNA expression in the liver was sustainably induced by long-term administration of CCl4 (Figure 2B). TNFα and col1a2 mRNA expression in the liver were elevated by CCl4. Interestingly, col1a2 mRNA expression was significantly inversely correlated with MYDGF mRNA expression (Figures 2C and 2D).

[0117] Example 2: Effects of MYDGF on acute liver injury Example 2-1: Protocol Male C57BL / 6 mice (8-10 weeks old, 20-25g body weight) were housed in a temperature-controlled (22±2°C) pathogen-free animal facility under a standard 12-hour light-dark cycle. To induce an acute liver injury model, mice were given a single intraperitoneal dose of corn oil (solvent control) or CCl4 diluted 1:9 with corn oil (1 μL / g intraperitoneally). In the MYDGF experimental group, an osmotic pump (Alzet model 1007D, 20 μg / day, 7 days, filled with 20 μg of recombinant human MYDGF per 12 μl diluted in PBS) was implanted in the subcutaneous interscapular pocket (Figure 3A). In the sham surgery control group, a skin incision was made between the scapulae and the wound was closed with surgical staples. The experimental group (MYDGF ALZET pump group) and the sham surgery control group were administered CCl4 as a single intraperitoneal dose. Mice were euthanized 24, 48, and 72 hours after administration, and serum and liver tissue samples were collected and evaluated (n=5 per group and per time point) (Figure 3B). For histological examination of the liver and testing of NF-κB activation, a solvent pump group was established as the control group instead of a sham surgery control group. In the solvent pump group, an osmotic pump (Alzet model 1007D, PBS 12 μl / day, 7 days) was implanted in the subcutaneous interscapular pocket. PBS (solvent) was administered at a flow rate of 12 μl / day. Serum MYDGF concentrations were analyzed by ELISA in the MYDGF ALZET pump group and the sham surgery control group (Figure 3C). Serum MYDGF concentrations remained high throughout the study period in the MYDGF ALZET pump group.

[0118] Example 2-2: Serum transaminase levels Serum transaminase levels and AST / ALT levels were evaluated at 24, 48, and 72 hours in both the MYDGF ALZET pump group and the sham surgery control group. Serum ALT and AST levels in both groups are expressed in international units per liter. In mice in the MYDGF ALZET pump group, serum ALT and AST levels were significantly lower at 24 and 48 hours compared to the sham surgery control group (Figures 3D and 3E).

[0119] Examples 2-3: Histological examination Liver samples from the MYDGF ALZET pump group and the media pump control group were stained with hematoxylin and eosin (H&E) at various time points after a single intraperitoneal injection of CCl4 (Figure 3F). Histological examination of the collected liver tissue revealed a CCl4-induced necrotic area around the central vein (Figure 3F, Zone 3). The black lines in the liver sections indicate the necrotic area. The necrotic area was widest at 24 hours and then gradually decreased. The MYDGF ALZET pump group tended to have a smaller necrotic area compared to the control group (Figure 3F). The proportion of necrotic area in each group was quantified using ImageJ. Semi-quantification of the necrotic area showed that necrosis was significantly lower in the MYDGF ALZET pump group compared to the control group at 24 and 48 hours (Figures 3F and 3G).

[0120] Examples 2-4: Suppression of inflammatory response in the liver (1) mRNA expression of inflammatory cytokines mRNA expression of inflammatory cytokines TNF-α, IL-1β, and IL-6 in whole liver tissue was analyzed by RTD-PCR in the MYDGF ALZET pump group and the sham surgery control group. In the MYDGF ALZET pump group, TNF-α and IL-6 mRNA expression was significantly lower at 24 and 48 hours compared to the control group (Figures 4A and 4C). IL-1β mRNA expression was also significantly lower in the MYDGF ALZET pump group compared to the sham surgery control group at 24, 48, and 72 hours (Figure 4B). (2) Activation of NF-κB in the liver NF-κB and p-NF-κB expression in liver samples from the MYDGF ALZET pump group and the media pump control group 48 hours after a single intraperitoneal injection of CCl4 was analyzed by Western blotting. NF-κB activation in the liver was suppressed in the MYDGF ALZET pump group compared to the control group (Figure 4D).

[0121] Example 3: Effect of MYDGF on chronic liver injury Example 3-1: Protocol Male C57BL / 6 mice (8-10 weeks old, 20-25g body weight) were housed in a temperature-controlled (22±2°C) pathogen-free animal facility under a standard 12-hour light-dark cycle. To create a chronic liver injury model, mice were intraperitoneally administered either corn oil (control group) or 10% CCl4 (180 μL corn oil + 20 μL CCl4 / mouse) every 3 days for 10 doses over 27 days. In the experimental group, an osmotic pump (Alzet model 1007D, 20 μg / day for 7 days, filled with recombinant human MYDGF 20 μg / 12 μl diluted in PBS) was implanted in the subcutaneous interscapular pocket. In the control group, which underwent sham surgery, a skin incision was made between the scapulae and the wound was closed with surgical staples. Mice were divided into three groups: a sham surgery group (n=6), a pump experiment group (MYDGF ALZET pump group) (n=6), and a control group (n=3). The sham surgery group received intraperitoneal injection of CCl4 and a sham surgery. The pump experiment group received intraperitoneal injection of CCl4 and implantation of an rhMYDGF pump. The control group received intraperitoneal injection of a solvent without pump implantation. Mice were euthanized 28 days after the start of CCl4 administration, and serum and liver tissue samples were collected for evaluation (Figure 5A). Serum MYDGF concentrations in the sham surgery control group, MYDGF ALZET pump group, and control group were analyzed by ELISA 28 days after the start of CCl4 administration (Figure 5B).

[0122] Example 3-2: Serum transaminase levels To evaluate liver damage, serum transaminase and AST / ALT levels were measured in three groups. Mice in the MYDGF ALZET pump group had significantly lower serum ALT and AST levels compared to the sham surgery group (Figures 5C and 5D).

[0123] Example 3-3: Suppression of liver fibrosis (1) Histological examination The livers of the sham surgery group and the pump experiment group were removed 30 to 28 days after the start of carbon tetrachloride administration. Fibrosis analysis was performed using Sirius Red staining, and positively stained areas were quantified for measurement of liver fibrosis area using ImageJ software. Semi-quantification of fibrosis area showed that the MYDGF ALZET pump group had significantly lower fibrosis compared to the sham surgery control group (Figures 6A and 6B). (2) Hydroxyproline content To evaluate liver fibrosis, hydroxyproline content in the livers of three groups was measured. Hydroxyproline content in the liver is expressed in micrograms per gram of total liver tissue (Figure 6C). The MYDGF ALZET pump group had significantly lower hydroxyproline content compared to the sham surgery group. (3) mRNA expression of liver fibrosis genes mRNA expression of liver fibrosis genes TGF-β, ACTA2, COL1A1, and COL1A2 was analyzed in all liver tissues of the three groups using RTD-PCR (Figure 6D-6G). In the MYDGF ALZET pump group, mRNA expression of TGF-β, ACTA2, COL1A1, and COL1A2 was significantly reduced compared to the sham surgery group.

[0124] Examples 3-5: Suppression of inflammatory response in the liver (1) mRNA expression of inflammatory cytokines mRNA expression of inflammatory cytokines TNF-α, IL-1β, and IL-6 in whole liver tissue was analyzed by RTD-PCR (Figure 7A-7C). In the MYDGF ALZET pump group, mRNA expression of TNF-α, IL-1β, and IL-6 was significantly reduced compared to the sham surgery group. (2) Activation of NF-κB in the liver The total amount and phosphorylation levels of NF-κB in whole mouse liver tissue were analyzed by Western blotting (Figure 7D). In the MYDGF ALZET pump group, NF-κB activation in the liver was suppressed compared to the sham surgery group. RhMYDGF suppresses the inflammatory response in the liver in a mouse model of chronic hepatitis-induced hepatic fibrosis.

[0125] Example 4: Inhibition of NF-κB activation and cytokine expression in HepG2 cells Example 4-1: MYDGF suppressed NF-κB activation in HepG2 cells. The anti-inflammatory effects of MYDGF in HepG2 cells were evaluated in vitro. HepG2 cells were treated with different concentrations of rhMYDGF (100 ng / mL and 500 ng / mL), and NF-κB phosphorylation activity was analyzed by Western blotting after 6 hours (n=3). MYDGF suppressed phosphorylated NF-κB expression in a dose-dependent manner (Figure 8A).

[0126] Example 4-2: MYDGF reduced TNF-α expression in HepG2 cells. The anti-inflammatory effects of MYDGF in HepG2 cells were evaluated in vitro. HepG2 cells were treated with different concentrations of rhMYDGF (100 ng / mL and 500 ng / mL), and TNF-α mRNA expression was analyzed using RTD-PCR after 6 hours (n=3) (Figure 8B). MYDGF significantly reduced TNF-α mRNA expression. MYDGF was shown to reduce the expression of inflammatory cytokines and suppress NF-κB activation.

[0127] Example 5: Induction of MYDGF by inflammatory cytokines in HepG2 cells. MYDGF mRNA expression in HepG2 cells treated with recombinant human inflammatory cytokines TNF-α, IL-1β, and IL-6 was analyzed by RTD-PCR 24 hours later (n=3) (Figure 9A-9C). The dose-dependent effects of rhIL-6 on MYDGF mRNA expression (Figure 9D) and supernatant protein (Figure 9E) in HepG2 cells were analyzed by RTD-PCR and Western blotting 24 hours later (n=3). TNFα and IL1β did not induce MYDGF, but IL6 significantly induced MYDGF at both the mRNA and protein levels.

Claims

1. Myelo-derived growth factor (MYDGF), or fragments or variants thereof exhibiting the biological function of MYDGF, for use in the treatment or prevention of liver disease.

2. The MYDGF or a fragment or variant thereof according to claim 1, wherein the liver disease is acute liver failure, chronic liver disease, or liver transplantation.

3. MYDGF or a fragment or variant thereof according to claim 1 or 2, wherein acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury, and toxic liver injury, and chronic liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection due to hepatitis B virus (HBV) or hepatitis C virus (HCV), alcoholic liver disease (ALD), autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), metabolic liver disease, and chronic ischemic liver injury.

4. MYDGF or a fragment or variant thereof according to any one of claims 1 to 3, wherein MYDGF comprises the following: (i) Sequence ID 1; or (ii) A fragment or variant of SEQ ID NO: 1 that exhibits the biological function of MYDGF and contains an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO:

1.

5. The MYDGF protein comprises SEQ ID NO: 1, and has one additional amino acid at the N-terminus and / or C-terminus of the protein of SEQ ID NO: 1, according to any one of claims 1 to 4, or a fragment or variant thereof.

6. A nucleic acid encoding MYDGF or a fragment or variant thereof according to any one of claims 1 to 5, for use in the treatment or prevention of liver disease.

7. The nucleic acid according to claim 6, wherein the liver disease is acute liver failure, chronic liver disease, or liver transplantation.

8. The nucleic acid according to claim 6 or 7, wherein acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury, and toxic liver injury, and chronic liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection due to hepatitis B virus (HBV) or hepatitis C virus (HCV), alcoholic liver disease (ALD), autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), metabolic liver disease, and chronic ischemic liver injury.

9. The nucleic acid according to any one of claims 6 to 8, wherein MYDGF comprises the following: (i) Sequence ID 1; or (ii) A fragment or variant of SEQ ID NO: 1 that exhibits the biological function of MYDGF and contains an amino acid sequence having at least 85% amino acid sequence identity with SEQ ID NO:

1.

10. A vector comprising the nucleic acid according to any one of claims 6 to 9 for use in the treatment or prevention of liver disease.

11. A host cell comprising the nucleic acid according to any one of claims 6 to 7 or the vector according to claim 9, for use in the treatment or prevention of liver disease.

12. A pharmaceutical composition comprising bone marrow-derived growth factor (MYDGF) or a fragment or variant thereof exhibiting the biological function of MYDGF, or a nucleic acid encoding MYDGF or a fragment or variant thereof, or a vector containing said nucleic acid, or a host cell containing said nucleic acid or vector, for use in the treatment or prevention of liver disease.

13. A pharmaceutical composition according to claim 12, wherein the liver disease is acute liver failure, chronic liver disease, or liver transplantation.

14. The pharmaceutical composition according to claim 12 or 13, wherein acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury, and toxic liver injury, and chronic liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection due to hepatitis B virus (HBV) or hepatitis C virus (HCV), alcoholic liver disease (ALD), autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), metabolic liver disease, and chronic ischemic liver injury.

15. The pharmaceutical composition according to any one of claims 12 to 14, wherein MYDGF comprises the following: (i) Sequence ID 1; or (ii) Fragments or mutants of SEQ ID NO: 1 that exhibit the biological function of MYDGF, and Here, the mutant contains an amino acid sequence that has at least 85% amino acid sequence identity with SEQ ID NO:

1.

16. The pharmaceutical composition according to any one of claims 12 to 15, wherein the MYDGF protein comprises SEQ ID NO: 1, and the protein of SEQ ID NO: 1 has one additional amino acid at its N-terminus and / or C-terminus.

17. A pharmaceutical composition according to any one of claims 12 to 16, administered by oral, intravenous, subcutaneous, intramucosal, intraarterial, intramuscular, or intrahepatic route.

18. The pharmaceutical composition according to claim 17, wherein administration is by one or more bolus injections and / or intravenous infusions.

19. A method for treating or preventing liver disease, comprising administering to a patient in need of the treatment of myelo-derived growth factor (MYDGF), or a fragment or variant thereof exhibiting the biological function of MYDGF, or a nucleic acid encoding MYDGF, a fragment or variant thereof, or a vector containing the nucleic acid, or a host cell containing the nucleic acid or the vector.

20. The method according to claim 19, wherein the liver disease is acute liver failure, chronic liver disease, or liver transplantation.

21. The method according to claim 19 or 20, wherein acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury, and toxic liver injury, and chronic liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection due to hepatitis B virus (HBV) or hepatitis C virus (HCV), alcoholic liver disease (ALD), autoimmune hepatitis, primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC), metabolic liver disease, and chronic ischemic liver injury.

22. The method according to any one of claims 19 to 21, wherein MYDGF includes the following: (i) Sequence ID 1; or (ii) Fragments or mutants of SEQ ID NO: 1 that exhibit the biological function of MYDGF, and Here, the mutant contains an amino acid sequence that has at least 85% amino acid sequence identity with SEQ ID NO:

1.

23. The method according to any one of claims 19 to 22, wherein the MYDGF protein comprises SEQ ID NO: 1, and the protein of SEQ ID NO: 1 has one additional amino acid at its N-terminus and / or C-terminus.

24. The method according to any one of claims 19 to 23, wherein MYDGF is administered by oral, intravenous, subcutaneous, mucosal, arterial, intramuscular or hepatic route.

25. The method according to claim 24, wherein administration is by one or more bolus injections and / or intravenous infusions.

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