WO2025047991A1

WO2025047991A1 - Myeloid-derived growth factor for use in treating or preventing a liver disorder

Info

Publication number: WO2025047991A1
Application number: PCT/JP2024/080151
Authority: WO
Inventors: Anton PEKCEC; Tomoko Maeda; Masao Honda
Original assignee: Boehringer Ingelheim International Gmbh
Priority date: 2023-09-01
Filing date: 2024-08-30
Publication date: 2025-03-06

Abstract

The present invention relates to the protein myeloid-derived growth factor (MYDGF) or nucleic acids encoding said protein for use in treating or preventing a liver disorder. The present invention also relates to vectors comprising the nucleic acid, host cells expressing the nucleic acid, and methods for use in treating a liver disorder.

Description

DESCRIPTION

TITLE OF INVENTION: MYELOID-DERIVED GROWTH FACTOR FOR USE IN TREATING OR PREVENTING A LIVER DISORDER

TECHNICAL FIELD OF THE INVENTION

[0001]

The present invention relates to the protein myeloid-derived growth factor (MYDGF) or nucleic acids encoding said protein for use in treating or preventing a liver disorder. The present invention also relates to vectors comprising the nucleic acid, host cells expressing the nucleic acid, and methods for use in treating or preventing a liver disorder.

BACKGROUND OF THE INVENTION

[0002]

Myeloid-derived growth factor (MYDGF), also known as Factor 1, is a protein encoded in open reading frame 10 on human chromosome 19 (C19Orf10). The Protein was described in 2007 in a proteome-analysis of the so called fibroblast-like synoviocytes (FLS-cells) as a new secreted Factor in the synovium. A correlation between the secretion of the protein and inflammatory diseases of the joint has been supposed without any experimental or statistical evidence (Weiler et al. , Arthritis Research and Therapy 2007, The identification and characterization of a novel protein, cl9orfl0, in the synovium). A corresponding patent application claims the protein as therapeutic agent for the treatment of joint and for the diagnosis of a tissue undergoing altered growth as well as monitoring changes in a tissue (US 2008/0004232 A1, Characterization of cl9orf10, a novel synovial protein). Another scientific publication describes an enhanced expression of the protein in hepatocellar carcinoma cells (Sunagozaka et al., International Journal of Cancer, 2010, Identification of a secretory protein cl 9orfI0 activated in hepatocellular carcinoma). Recombinant produced protein showed a proliferation enhancing effect on cultured hepatocellar carcinoma cells. It is noted that C19Orf10 has also been referred to as IL-25, IL-27 and IL-27W as it was originally considered an interleukin. However, the terms “IL-25” and “IL-27” have been used inconsistently in the art and have been used to designate a variety of different proteins. For example, US 2004/0185049 refers to a protein as IL-27 and discloses its use in modulating the immune response. This protein is structurally distinct from Factor 1 (compare Factor 1 amino acid sequence according to SEQIDNO: 1 to the amino acid sequence of “IL-27” according to UniProt: Q8NEV9). Similarly, EP 2 130 547 A1 refers to a protein as IL-25 and discloses its use in treating inflammation. This protein has also been referred to in the art as lL-17E and is structurally distinct from Factor 1 (compare the amino acid sequence of Factor 1 according to SEQ ID NO: 1 to the amino acid sequence of “IL-25” according to UniProt: Q9H293).

[0003]

WO 2014/111458 discloses Factor 1 for use in enhancing proliferation and inhibiting apoptosis of non-transformed tissue or non-transformed cells, in particular for use in treating acute myocardial infarction. Further disclosed are inhibitors of Factor 1 for medical use, in particular for use in treating or preventing a disease in which angiogenesis contributes to disease development or progression.

[0004]

Korf-Klingebiel et al. (Nature Medicine, 2015, Vol. 21 (2): 140-149) report C19Orf10 to be secreted by bone marrow cells after myocardial infarction, which protein promotes cardiac myocyte survival and angiogenesis. The authors show that bone marrow-derived monocytes and macrophages produce this protein endogenously to protect and repair the heart after myocardial infarction, and propose the name myeloid-derived growth factor (MYDGF). In particular, treatment with recombinant Mydgf is reported to reduce scar size and contractile dysfunction after myocardial infarction. Although the beneficial effect of MYDGF on MI have been investigated, the effect of MYDGF on a liver disorder including acute liver failure, a chronic liver disease, or a liver transplantation had not been evaluated.

[0005]

Acute liver failure (ALF) is a rare, acute, potentially reversible condition resulting in severe liver impairment and rapid clinical deterioration in patients without preexisting liver disease. Acute liver failure is most commonly caused by viral infections (e.g., hepatitis A, B, and E) and drugs (e.g, acetaminophen, NSAIDs and some antibiotics). Other possible causes of acute liver failure are acute ischemic liver injury, mushroom ingestion, and metabolic diseases such as Wilson's disease, exposure to a toxin etc. The rarity of acute liver failure, along with its severity and heterogeneity, has resulted in a very limited evidence base to guide supportive care (W Bernal et al. NEJM. 2013; 369:2525-2534).

[0006]

Chronic liver disease (CLD) is a progressive deterioration of liver functions for more than six months, which includes synthesis of clotting factors, other proteins, detoxification of harmful products of metabolism, and excretion of bile. CLD is a continuous process of inflammation, destruction, and regeneration of liver parenchyma, which leads to fibrosis and cirrhosis. The spectrum of etiologies is broad for chronic liver disease, which includes toxins, alcohol abuse for a prolonged time, infection, autoimmune diseases, genetic and metabolic disorders (https://www.ncbi.nlm.nih.gov/books/NBK554597/). Chronic liver disease is associated with different degrees of scarring (nonfibrotic, fibrotic, cirrhosis), and compensation (compensated vs. decompensated). Chronic liver disease includes non-alcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH), conditions in which fat accumulates in the liver, chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV) caused by ongoing viral hepatitis, alcoholic liver disease (ALD) caused by long-term alcohol abuse, autoimmune hepatitis, which is a liver disease caused by the body's immune system, primary biliary cholangitis conditions in which the bile ducts are destructed, hardening and scarring of the bile ducts caused by primary sclerosing cholangitis, and metabolic diseases including Wilson's disease, a condition in which copper accumulates in the liver, Alpha- 1 antitrypsin deficiency and hemochromatosis, a condition that causes iron buildup in the body. Chronic liver disease still lacks effective pharmacotherapy.

[0007]

Liver transplantation refers to the treatment of removing a liver whose function has deteriorated due to various causes and transplanting a healthy liver. Liver transplantation includes living-donor liver transplantation and brain-dead liver transplantation. In the Liver transplantation, it is required improving the quality and function of the donor liver prior to transplantation and increasing the likelihood of transplant success and post-transplant recovery in the recipient.

[0008]

There remains a need for means and methods for treating a liver disorder.

SUMMARY OF THE INVENTION

[0009]

The invention provides in a first aspect myeloid-derived growth factor (MYDGF) or a fragment or a variant thereof exhibiting the biological function of MYDGF, for use in treating or preventing a liver disorder.

[0010]

According to one embodiment, the MYDGF protein or a fragment or a variant thereof exhibiting the biological function of MYDGF, is for use in treating or preventing a liver disorder. According to a preferred embodiment, the liver disorder is an acute liver failure, a chronic liver disease, or a 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 with at least 85% amino acid sequence identity to SEQ ID NO: 1.

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

[0013]

According to a further aspect, the present invention provides a nucleic acid encoding the growth factor protein MYDGF or a fragment or a variant thereof exhibiting the biological function of MYDGF, for use in treating or preventing a liver disorder.

[0014]

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

[0015]

According to a further aspect, the present invention provides a vector comprising the nucleic acid of the present invention for use in treating or preventing a liver disorder.

[0016]

According to a further aspect, 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 treating or preventing a liver disorder. Preferably, the host cell expresses the nucleic acid.

[0017]

According to yet another aspect, the present invention provides a pharmaceutical composition comprising the MYDGF protein, the nucleic acid, the vector or the host cell of the present invention and optionally a suitable pharmaceutical excipient, for use in treating or preventing a liver disorder.

[0018]

According to a preferred embodiment, the pharmaceutical composition for use is administered through the oral, intravenous, subcutaneous, intramucosal, intraarterial, intramuscular or intrahepatic route. The administration is preferably through one or more bolus injection(s) and/or infusion(s).

[0019]

According to a further aspect, the present invention provides a method of treating or preventing a liver disorder. The method comprises administering to a patient in need thereof a therapeutically effective amount of MYDGF or fragment or variant thereof exhibiting the biological function of MYDGF. According to a preferred embodiment, the liver disorder is an acute liver failure, a chronic liver disease, or a liver transplantation. According to a further preferred embodiment, the acute liver failure is selected from the group consisting of viral hepatitis, drug- induced liver injury, acute ischemic liver injury and toxic liver damage. According to a further preferred embodiment, the chronic liver diseases is selected from the group consisting of nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection with 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.

[0020]

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

[0021]

According to yet another embodiment, the MYDGF or fragment or variant thereof exhibiting the biological function of MYDGF is administered through one or more bolus injection(s) and/or infusion(s), preferably in a pharmaceutically accepted carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0022]

Figure 1: Preparation of Acute Liver Injury Model (A) Schematic overview of experimental design to induce acute liver injury model by carbon tetrachloride (CCl₄). (B) MYDGF mRNA expression in the liver of mice. (C) and (D) Serum levels of transaminase, ALT/AST, at 24 hrs, 48 hrs and 72 hrs after a single-dose intraperitoneal injection of CCl₄.

Figure 2: Preparation of Chronic Liver Injury Model. (A) Schematic overview of experimental design to induce chronic liver injury model by carbon tetrachloride (CCl₄). (B) MYDGF mRNA expression in the liver of mice at the time points of administration of CCl₄ and 7 days (37d), 14 days (44d) and 28 days (58d) after the end of administration of CCl₄. (C) and (D) The mRNA expression of TNFα and colla2 in the liver at the time points of administration of CCl₄ and 7 days (37d), 14 days (44d) and 28 days (58d) after the end of administration of CCl₄.

Figure 3: MYDGF Protein Therapy in Acute Liver Injury Model. (A) Illustration of a mouse implanted with an osmotic pump. (B) Schematic overview of experimental design in carbon tetrachloride (CCl₄) to induce acute liver injury model. Mice were divided into MYDGF ALZET pump group, and sham-operated control group. (C) Serum MYDGF concentration in the MYDGF ALZET pump group and the sham-operated control group at 24 hrs, 48 hrs and 72 hrs after a single-dose intraperitoneal injection of CCl₄. (D) and (E) Serum levels of transaminase, ALT/AST in the MYDGF ALZET pump group and the sham-operated control group at 24 hrs and 48 hrs after a single-dose intraperitoneal injection of CCl₄. (F) H&E staining of ALI mouse livers at 1 day, 2 days and 3 days after a single-dose intraperitoneal injection of CCl₄. The black line in liver slices indicates the necrotic area. Scale bar: 100 μm. (G) A graph obtained by quantifying using Image J the area ratio of the necrotic area relative to the visual field (damaged area %) in each group stained in (F).

Figure 4: Suppression of hepatic inflammatory response by MYDGF in Acute Liver Injury Model. (A) - (C) The mRNA expression of inflammatory cytokines, TNF-α, IL- 1β, and IL-6 in whole liver tissue in the MYDGF ALZET pump group and the sham-operated control group at 24 hrs, 48 hrs and 72 hrs after a single-dose intraperitoneal injection of CCl₄. (D) The expression of NF-κB, p-NF-κB in liver samples of the MYDGF ALZET pump group and vehicle pump control group at 48 hrs after a single-dose intraperitoneal injection of CCl₄.

Figure 5: MYDGF Protein Therapy in Chronic Liver Injury Model. (A) Schematic overview of experimental design in carbon tetrachloride (CCl₄) induced chronic liver injury model. Mice were divided into sham-operated control group, MYDGF ALZET pump group and control group.(B) Serum MYDGF concentration in sham-operated control group, MYDGF ALZET pump group and control group at 28 days after starting administration of CCl₄. (C) and (D) Serum levels of transaminase, ALT/AST in sham-operated control group, MYDGF ALZET pump group and control group at 28 days after starting administration of CCl₄.

Figure 6: Suppression of liver fibrosis by MYDGF in Chronic Liver Injury Model. (A) Sirius Red staining of livers of sham-operated control group and MYDGF pump group at 28 days after starting administration of CCl₄. H&E staining of livers of control ‘group, sham-operated control group and MYDGF ALZET pump experiment group are also shown. As for Sirius Red staining, positive-staining areas were displayed white in the grayscale image. (B) The positivestaining areas in sham-operated control group and MYDGF ALZET pump group in (A) was quantified by ImageJ. (C) Hydroxyproline content in the liver of sham-operated control group, MYDGF ALZET pump group and control group at 28 days after starting administration of CCl₄. (D) - (G) The mRNA expression of hepatic fibrosis genes TGF-β, ACTA2, COL1A1, and COL1A2 in whole liver tissue of sham-operated control group, MYDGF ALZET pump group and control group at 28 days after starting administration of CCl₄.

Figure 7: Suppression of hepatic inflammatory response by MYDGF in Chronic Liver Injury Model (A) - (C) The mRNA expression of inflammatory cytokines, TNF-α, IL- 1β, and IL-6 in whole liver tissue of sham-operated control group, MYDGF ALZET pump group and control group at 28 days after starting administration of CCl₄. (D) The expression of NF-κB andp-NF-κB in liver samples of sham-operated control group, MYDGF ALZET pump group and control group at 28 days after starting administration of CCl₄.

Figure 8: Suppression of inflammatory response by MYDGF in HepG2 cells. (A) The expression of NF-κB and p-NF-κB in HepG2 cells at 6 hrs after treated with different concentrations of rhMYDGF. (B) The expression of TNF-α mRNA in HepG2 cells at 6 hrs after treating with different concentrations of rhMYDGF.

Figure 9: Induction of MYDGF by inflammatory cytokines in HepG2 cells. (A)-(C) MYDGF mRNA expression in HepG2 cells at 24 hrs after treated with recombinant human inflammatory cytokines TNF-α, IL-1β, and IL-6, respectively. (D) MYDGF mRNA expression in HepG2 cells at 24 hrs after treated with different concentrations of rhIL-6. (E) The expression of MYDGF protein in supernatant and cell lysate in HepG2 cells at 24 hrs after treated with different concentrations of rhIL-6.

DETAILED DESCRIPTION OF THE INVENTION

[0023]

Before the present invention is described in detail below, it is to be understood that this invention is not limited to the particular methodology, protocols and reagents described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. Unless defined otherwise, all technical and scientific tenns used herein have the same meanings as commonly understood by one of ordinary skill in the art.

DEFINITIONS

[0024]

Preferably, the terms used herein are defined as described in "A multilingual glossary of biotechnological terms: (IUPAC Recommendations)", H.G.W. Leuenberger, B. Nagel, and H. Kolbl, Eds., Helvetica Chimica Acta, CH-4010 Basel, Switzerland, (1995).

[0025]

To practice the present invention, unless otherwise indicated, conventional methods of chemistry, biochemistry, cell biology, and recombinant DNA techniques are employed which are explained in the literature in the field (cf, e.g., Molecular Cloning: A Laboratory Manual, 2^nd Edition, J. Sambrook et al. eds., Cold Spring Harbor Laboratory Press, Cold Spring Harbor 1989). Furthermore, conventional methods of clinical cardiology are employed which are also explained in the literature in the field (cf , e.g., Braunwald’s Heart Disease. A Textbook of Cardiovascular Medicine, 9^th Edition, P. Libby et al. eds., Saunders Elsevier Philadelphia, 2011).

[0026]

Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps. As used in this specification and the appended claims, the singular forms “a”, “an”, and “the” include plural referents, unless the content clearly dictates otherwise.

[0027]

Nucleic acid molecules are understood as polymeric macromolecules made from nucleotide monomers. Nucleotide monomers are composed of a nucleobase, a five-carbon sugar (such as but not limited to ribose or 2'-deoxyribose), and one to three phosphate groups. Typically, a polynucleotide is formed through phosphodiester bonds between the individual nucleotide monomers. In the context of the present invention referred to 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]

The term “open reading frame” (ORF) refers to a sequence of nucleotides, that can be translated into amino acids. Typically, such an ORF contains a start codon, a subsequent region usually having a length which is a multiple of 3 nucleotides, but does not contain a stop codon (TAG, TAA, TGA, UAG, UAA, or UGA) in the given reading frame. Typically, ORFs occur naturally or are constructed artificially, i.e. by gene-technological means. An ORF codes for a protein where the amino acids into which it can be translated form a peptide-linked chain.

[0029]

The terms "protein" and "polypeptide" are used interchangeably herein and refer to any peptide-bond-linked chain of amino acids, regardless of length or post-translational modification. Proteins usable in the present invention (including protein derivatives, protein variants, protein fragments, protein segments, protein epitops and protein domains) can be further modified by chemical modification. This means such a chemically modified polypeptide comprises other chemical groups than the 20 naturally occurring amino acids. Examples of such other chemical groups include without limitation glycosylated amino acids and phosphorylated amino acids. Chemical modifications of a polypeptide may provide advantageous properties as compared to the parent polypeptide, e.g. one or more of enhanced stability, increased biological half-life, or increased water solubility. Chemical modifications applicable to the variants usable in the present invention include without limitation: PEGylation, glycosylation of non-glycosylated parent polypeptides, covalent coupling to therapeutic small molecules, like glucagon-like peptide 1 agonists, including exenatide, albiglutide, taspoglutide, DPP4 inhibitors, incretin and liraglutide, or the modification of the glycosylation pattern present in the parent polypeptide. Such chemical modifications applicable to tire variants usable in the present invention may occur co- or post-translational.

[0030]

The term “amino acid” encompasses naturally occurring amino acids as well as amino acid derivatives. A hydrophobic non-aromatic amino acid in the context of the present invention, is preferably any amino acid which has a Kyte-Doolittle hydropathy index of higher than 0.5, more preferably of higher than 1.0, even more preferably of higher than 1.5 and is not aromatic. Preferably, a hydrophobic non-aromatic amino acid in the context of the present invention, is selected from the group consisting of the amino acids alanine (Kyte Doolittle hydropathy index 1.8), methionine (Kyte Doolittle hydropathy index 1.9), isoleucine (Kyte Doolittle hydropathy index 4.5), leucine Kyte Doolittle hydropathy index 3.8), and valine (Kyte Doolittle hydropathy index 4.2), or derivatives thereof having a Kyte Doolittle hydropathy index as defined above. [0031]

The term “variant” is used herein to refer to a polypeptide which differs in comparison to the polypeptide or fragment thereof from which it is derived by one or more changes in the amino acid sequence. The polypeptide from which a protein variant is derived is also known as the parent polypeptide. Likewise, the fragment from which a protein fragment variant is derived from is known as the parent fragment. Typically, a variant is constructed artificially, preferably by gene- technological means. Typically, the parent polypeptide is a wild-type protein or wild-type protein domain. Further, the variants usable in the present invention may also be derived from homologs, orthologs, or paralogs of the parent polypeptide or from artificially constructed variant, provided that the variant exhibits at least one biological activity of the parent polypeptide. Tire changes in the amino acid sequence may be amino acid exchanges, insertions, deletions, N-terminal truncations, or C-tenninal truncations, or any combination of these changes, which may occur at one or several sites. In preferred embodiments, a variant usable in the present invention exhibits a total number 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 in the amino acid sequence (i.e. exchanges, insertions, deletions, N-terminal truncations, and/or C- terminal truncations). The amino acid exchanges may be conservative, and/or semi-conservative, and/or non-conservative. In preferred embodiments, a variant usable in the present invention differs from the protein or domain from which it is derived by 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 acid exchanges, preferably conservative amino acid changes.

[0032] Typical substitutions are among the aliphatic amino acids, among the amino acids having aliphatic hydroxyl side chain, among the amino acids having acidic residues, among the amide derivatives, among the amino acids with basic residues, or the amino acids having aromatic residues. Typical semi-conservative and conservative substitutions are: [0033]

[0034]

Changing from A, F, H, I, L, M, P, V, W or Y to C is semi-conservative if the new cysteine remains as a free thiol. Furthermore, the skilled person will appreciate that glycines at sterically demanding positions should not be substituted and that P should not be introduced into parts of the protein which have an alpha-helical or a beta-sheet structure.

[0035]

Alternatively or additionally, a “variant” as used herein, can be characterized by a certain degree of sequence identity to the parent polypeptide or parent polynucleotide from which it is derived. More precisely, a protein variant in the context of the present invention exhibits at least 85% sequence identity to its parent polypeptide. Preferably, the polypeptide in question and the reference polypeptide exhibit the indicated sequence identity over a continuous stretch 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 continuous stretch 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 the specification with regards to polypeptide and polynucleotide sequence comparisons. This expression preferably refers to a sequence identity of 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% to the respective reference polypeptide or to the respective reference polynucleotide.

[0037]

Fragments of proteins comprise deletions of amino acids, which may be N-terminal truncations, C-terminal truncations or internal deletions or any combination of these. Such variants comprising N-terminal truncations, C-terminal truncations and/or internal deletions are referred to as “fragments” in the context ofthe present application. A fragment may be naturally occurring (e.g. splice variants) or it may be constructed artificially, preferably by gene-technological means. Preferably, a fragment (or deletion variant) has a deletion 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 at its N-terminus and/or at its C-terminus and/or internally as compared to the parent polypeptide, preferably at its N-terminus, at its N- and C-terminus, or at its C-terminus.

[0038]

In case where two sequences are compared and the reference sequence is not specified in comparison to which the sequence identity percentage is to be calculated, the sequence identity is to be calculated with reference to the longer of the two sequences to be compared, if not specifically indicated otherwise.

[0039]

The similarity of nucleotide and amino acid sequences, i.e. the percentage of sequence identity, can be determined via sequence alignments. Such alignments can be carried out with several art-known algorithms, preferably with the mathematical algorithm of Karlin and Altschul (Karlin & Altschul (1993) Proc. Natl. Acad. Sci. USA 90: 5873-5877), with hmmalign (HMMER package, http://hmmer.wustl.edu/) or with the CLUSTAL algorithm (Thompson, J. D., Higgins, D. G. & Gibson, T. J. (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 Clustal X version 2.0. Bioinformatics, 23, 2947-2948.) which are available e.g. on http://npsa-pbil.ibcp.fr/cgi- bin/npsa automat.pl?page=/NPSA/npsa_clustalw.html or on htp://www.ebi.ac.uk/Tools/clustalw2/index.html. Preferably, the CLUSTALW2 algorithm on htp://www.ebi.ac.uk/Tools/clustalw2/index.html is used wherein the parameters used are the default parameters as they are set on htp://www.ebi.ac.uk/Tools/clustalw2/index.html: Alignment type = Slow, protein weight matrix = Gonnet, gap open = 10, gap extension = 0,1 for slow pairwise alignment options and protein weight matrix = Gonnet, gap open = 10, gap extension = 0,20, gap distances = 5, No end gaps = no, Output options: format = Ain w/numbers, Order = aligned. [0040]

The grade of sequence identity (sequence matching) may be calculated using e.g. BLAST, BLAT or BlastZ (or BlastX). A similar algorithm is incorporated into the BLASIN and BLASTP programs of Altschul et al. (1990) J. Mol. Biol. 215: 403-410. BLAST protein searches are performed with the BLASTP program available e.g. on http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&BLAST_PROGRAMS=blastp&PA GE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome. Preferred algorithm parameters used are the default parameters as they are set on htp://blast.ncbi.nhn.nih.gov/Blast.cgi?PROGRAM=blastp&BLAST _PROGRAMS=blastp&PA GE_TYPE=BlastSearch&SHOW_DEFAULTS=on&LINK_LOC=blasthome: Expect threshold = 10, word size = 3, max matches in a query range = 0, matrix = BLOSUM62, gap costs = Existence: 11 Extension: 1, compositional adjustments = conditional compositional score matrix adjustment together with the database of non-redundant protein sequences (nr) to obtain amino acid sequences homologous to the Factor 1 and Factor 2 polypeptides.

[0041]

To obtain gapped alignments for comparative purposes, Gapped BLAST is utilized as described in Altschul et al. (1997) Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs are used. Sequence matching analysis may be supplemented by esta bl ished homology mapping techniques like Shuffle- LAGAN (Brudno M., Bioinformatics 2003b, 19 Suppl 1 :154-I62) or Markov random fields. When percentages of sequence identity are referred to in the present application, these percentages are calculated in relation to the full length of the longer sequence, if not specifically indicated otherwise. [0042]

The term “host cell” as used herein refers to a cell that harbours a nucleic acid of the invention (e.g. in form of a plasmid or virus). Such host cell may either be a prokaryotic (e.g. a bacterial cell) or a eukaryotic cell (e.g. a fungal, plant or animal cell). The cell can be transformed or non-transformed. The cell can be an isolated cell for example in a cell culture or part of a tissue, which itself can be isolated or part of a more complex organization structure such as an organ or an individual.

[0043]

The terms “myeloid-derived growth factor”, “MYDGF”, “Factor 1”, “MYDGF polypeptide or protein” or “Factor 1 polypeptide or protein” are used interchangeably and refer to the protein indicated in NCBI reference sequence NM_019107.3 (human homologue) as well as it mammalian homologues, in particular from mouse or rat. The amino acid sequence of the human homologue is encoded in open reading frame 10 on human chromosome 19 (C19Orf10). Preferably, MYDGF and Factor 1 protein refer to a protein, which comprises, essentially consists or consists of a core segment of human Factor 1 having an amino acid sequence according to SEQ ID NO: 1.

[0044]

Whether or not a protein, variant or fragment exhibits the biological function of MYDGF can be determined by any one of the tests described in the examples below. According to the present invention, a peptide or protein exhibits the biological function of MYDGF if the results obtained with such peptide or protein compared to the results obtained with the MYDGF protein of the present invention shown in at least one of the examples presented herein below 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 over the indicated controls.

[0045]

As used herein, the terms “MYDGF” and “Mydgf ’ both denote myeloid-derived growth factor, wherein ‘ ‘MYDGF ’ is used in the present invention to refer to the human variant and ‘ ‘Mydgf ’ is used to refer to the mouse variant of myeloid-derived growth factor.

[0046]

The term “acute liver failure” as used herein means a rare, acute, potentially reversible condition resulting in severe liver impairment and rapid clinical deterioration in patients without preexisting liver disease (A. Shingina et. al., Am J Gastroenterol. 2023 Jul 1;118(7): 1128-1153). [0047]

The term “chronic liver disease” as used herein means a progressive deterioration of liver functions for more than six months, which includes synthesis of clotting factors, other proteins, detoxification of harmful products of metabolism, and excretion of bile. CLD is a continuous process of inflammation, destruction, and regeneration of liver parenchyma, which leads to fibrosis and cirrhosis. The spectrum of etiologies is broad for chronic liver disease, which includes toxins, alcohol abuse for a prolonged time, infection, autoimmune diseases, genetic and metabolic disorders (https://www.ncbi.nlm.nih.gov/books/NBK554597/). Chronic liver disease is associated with different degrees of scarring (nonfibrotic, fibrotic, cirrhosis), and compensation (compensated vs. decompensated).

[0048]

The term “liver fibrosis” as used herein means the formation of excess fibrous connective tissue in a liver in a reparative or reactive process such as a reactive, benign or pathological state. The trigger is chronic injury, especially if there is an inflammatory component. The connective tissue deposited during fibrosis can interfere with or inhibit the normal architecture and function of the underlying organ ortissue (R. Weiskirchen et. al., Molecular Aspects ofMedicine, Vol 65, 2019, 2-15).

[0049]

The term “cirrhosis” as used herein means a condition in which the liver is scarred and permanently damaged. Scar tissue replaces healthy liver tissue and prevents the liver from working normally. Scar tissue also partly blocks the flow of blood through the liver (https://www.niddk.nih.gov/health-information/liver-disease/cirrhosis/definition-facts). Cirrhosis is generally divided into two stages: compensated and decompensated. [0050]

The term “compensated cirrhosis" is the early stage of liver cirrhosis, where the liver is scarred but still able to function normally or at some level. People with this type of cirrhosis may have mild or no symptoms, but they still need treatment to prevent further damage and complications. [0051]

The term “decompensated cirrhosis” is the advanced stage of cirrhosis, where the liver is so severely scarred that it can’t function properly. People with this type of cirrhosis may have serious complications such as jaundice, ascites, hepatic encephalopathy, hepatorenal syndrome, and variceal hemorrhage.

[0052]

The term “liver transplantation” as used herein means refers to the treatment of removing a liver whose function has deteriorated due to various causes and transplanting a healthy liver. The description of the embodiments comprises further definitions and explanations of terms used throughout the application. These descriptions and definitions are valid for the whole application unless it is otherwise stated.

SEQUENCES

[0053]

Sequences used in the present invention are listed below. SEQ IDNO: 1 (amino acid sequence ofhuman Factor 1, lacking the 31 aa N-terminal signal peptide):

SEQ ID NO: 2 (amino acid sequence of the mouse homologue of Factor 1, lacking the 24 aa N-terminal signal peptide):

SEQ ID NOG (amino acid sequence of human Factor 1, including the N-terminal signal peptide (shown in bold and underlined); UniProtKB - Q969H8):

SEQ ID NO: 4 (amino acid sequence of the mouse homologue of Factor 1, including the N-terminal signal peptide (shown in bold and underlined); UniProtKB - Q9CPT4):

SEQ ID NO: 5 shows the nucleic acid sequence of human Factor 1 encoding MYDGF of SEQ ID NO: 3 (NCBI Gene ID: 56005).

SEQ ID NO: 6 shows the nucleic acid sequence of mouse Factor 1 encoding Mydgf of SEQ ID NO: 4 (NCBI Gene ID: 28106).

EMBODIMENTS

[0054] In the following, the elements of the present invention will be described. These elements are listed with specific embodiments, however, it should be understood that they may be combined in any manner and in any number to create additional embodiments. The variously described examples and preferred embodiments should not be construed to limit the present invention to only the explicitly described embodiments. This description should be understood to support and encompass embodiments which combine the explicitly described embodiments with any number of the disclosed and/or preferred elements. Furthermore, any permutations and combinations of all described elements in this application should be considered disclosed by the description of the present application unless the context indicates otherwise.

[0055]

The present inventors show for the first time the effect of MYDGF on liver injury. The inventors particularly show that administration of MYDGF in a mouse model inhibits acute and chronic liver injury and liver fibrosis. The present inventors also show that MYDGF inhibits hepatic inflammatory response through studies in mouse models and cultured cells. These effects can be used inter alia, for inhibiting tire liver injury and consequent progression of scarring. Therefore, in a first aspect, the invention provides the protein myeloid-derived growth factor (MYDGF) or a fragment or a variant thereof exhibiting the biological function of MYDGF, for use in treating or preventing a liver disorder caused by liver injury.

[0056]

In a second aspect, the invention provides the protein myeloid-derived growth factor (MYDGF) or a fragment or a variant thereof exhibiting the biological function of MYDGF, for use in treating or preventing a liver disorder. According to a preferred embodiment, the liver disorder is an acute liver failure, a chronic liver disease, or a liver transplantation. According to a preferred embodiment, the acute liver failure is selected from the group consisting of viral hepatitis, drug- induced liver injury, acute ischemic liver injury and toxic liver damage. The viral hepatitis is a liver inflammation caused by viral infections, including Hepatitis A, Hepatitis B, Hepatitis C, Hepatitis D and Hepatitis E. The drug-induced liver injury is caused by the toxic effects of certain medications, such as acetaminophen, nonsteroidal anti-inflammatory drugs (NSAIDs), and some antibiotics. The acute ischemic liver injury occurs when there is reduced blood flow to the liver, often due to conditions like liver transplantation, shock, or vascular diseases. The toxic liver damage" is caused by exposure to certain chemicals, toxins, mushroom ingestion or industrial pollutants, such as carbon tetrachloride, aflatoxins, or heavy metals. According to a preferred embodiment, the chronic liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection with 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. The non-alcoholic fatty liver disease (NAFLD) is a condition in which excess fat builds up in the liver for reasons other than excessive alcohol consumption, with fat in the liver but little or no inflammation or liver damage. The non-alcoholic steatohepatitis (NASH) is a condition in which excess fat builds up in the liver for reasons other than excessive alcohol consumption, with inflammation of the liver and liver damage which can cause fibrosis, or scarring, of the liver, in addition to fat in the liver. The chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), can lead to ongoing liver inflammation and fibrosis. The alcoholic liver disease (ALD) is resulting from chronic alcohol consumption, which can lead to inflammation and scarring of the liver tissue. The autoimmune hepatitis is arising from autoimmune hepatitis, a condition in which the immune system mistakenly attacks the liver, resulting in chronic inflammation and fibrosis. The primary biliary cholangitis (PBC) is a chronic liver disease characterized by progressive destruction of the bile ducts, leading to fibrosis and cirrhosis over time. The primary sclerosing cholangitis (PSC) is involving inflammation and scarring of the bile ducts, often associated with inflammatory bowel disease (IBD). The metabolic liver disease is inherited disorders that affect liver function, such as Wilson's disease, hemochromatosis, and alpha- 1 antitrypsin deficiency. The chronic ischemic liver injury occurs when there is reduced blood flow to the liver, often due to conditions like liver transplantation, shock, or vascular diseases.

Also provided is the MYDGF or a fragment or a variant thereof for this use, wherein the liver disorder is a liver transplantation including living-donor liver transplantation and brain-dead liver transplantation. The MYDGF or a fragment or a variant thereof can improve the quality and function of the donor liver prior to transplantation and increasing the likelihood of transplant success and post-transplant recovery in the recipient.

[0057]

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

[0058]

In a preferred embodiment of this aspect of the invention, the protein comprises the amino acid sequence SEQ ID NO: 1, a fragment or a variant thereof exhibiting the biological function of MYDGF, which has at least 85% sequence identity to SEQ ID NO: 1. A person skilled in the art is able to decide without undue burden, which positions in the parental polypeptide can be mutated to which extent and which positions have to be maintained to preserve the functionality of the polypeptide. Such information can, for example, be gained from homologues sequences which can be identified, aligned and analyzed by bioinformatic methods well known in the art. Such analyses are exemplarily described in example 7 and Figures 6 and 7 of WO 2014/111458. Mutations are preferably introduced in those regions of the protein, which are not fully conserved between species, preferably mammals. In a particularly preferred embodiment of the invention, the MYDGF protein comprises, essentially consists or consists of the amino acid sequence 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 to SEQ ID NO: 1.

[0059]

N-terminal deletion variants are also encompassed, which may for example lack one or more amino acids from amino acid position 1 to 24 (based on SEQ ID NO: 1), i.e. from the N- terminally conserved region.

[0060]

C-terminal deletion variants are also encompassed, which may for example lack one or more amino acids from amino acid position 114 to 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 at the N-terminus, at the C-terminus, within the amino acid sequence or combinations thereof. The protein of the first aspect of the present invention may thus further comprise additional amino acid sequences, e.g. for stabilizing or purifying the resulting protein. Examples of such amino acids are 6xHis-tags, myc-tags, or FLAG-tags, which are well known in the art, and which may be present at any position in the protein, preferably at the N-terminus or the C-terminus. A particularly preferred additional sequence is a 6xHis-tag. Preferably, said 6xHis-tag is present on the C-terminus of the MYDGF protein. Depending on the expression system used and, if present, an additional amino acid such as a tag described above, one or more residual amino acids may remain on the N-terminus and/or the C-terminus of the protein. It is emphasized that in the MYDGF protein and Mydgf protein according to the present invention such artefacts may be present, as in shown e.g. in Ebenhoch R. et al., Nat Commun. 2019 Nov 26;10(l):5379, and Polten F. et al., Anal Chem. 2019 Jan 15;91(2): 1302-1308.

[0062]

In some cases it is preferred to mutate protease cleavage sites within the MYDGF protein of the first aspect of the present invention to stabilize the protein (see Segers et al. Circulation 2007, 2011). The skilled person knows how to determine potential proteolytic cleavage sites within a protein. For example, protein sequences can be submitted to websites providing such analysis as, e.g. http://web.expasy.org/peptide_cutter/ or http://pmap.burnham.org/proteases. If the protein sequence according to SEQ ID NO: 1 is submitted to http://web.expasy.org/peptide_cutter/ the following cleavage sites with lower frequency (less then 10) are determined: [0063] Table 1

[0064]

These sites may be altered to remove the recognition/cleavage sequence of the respectively identified protease to increase the serum half-life of the protein.

[0065]

MYDGF has been shown in the present invention to inhibit or prevent acute and chronic liver injury, and liver fibrosis and their associated inflammatory responses. These effects can be used inter alia, for inhibiting the liver injury and consequent progression ofscarring. Accordingly, the present invention provides the MYDGF protein or a fragment or a variant thereof exhibiting the biological function of MYDGF for use in treating or preventing a liver disorder.

[0066]

Without wishing to be bound by any theory, MYDGF suppresses inflammatory responses in the liver through suppression of NF-κB activation, thereby preventing and/or treating a liver disorder.

[0067]

The MYDGF protein may further comprise additional amino acid sequences, e.g. for stabilizing or purifying the resulting protein. For example, it is preferred to mutate protease cleavage sites within the MYDGF protein to stabilize the protein. Suitable proteolytic cleavage sites can be identified as described above.

[0068]

The MYDGF protein or compositions comprising the protein can administered in vivo, ex vivo or in vitro, preferably in vivo.

[0069]

In a further aspect, the present invention provides nucleic acids encoding the MYDGF protein or a fragment or a variant thereof exhibiting the biological function of MYDGF as described herein for use in treating or preventing a liver disorder. The present invention also provides nucleic acids encoding the MYDGF protein or a fragment or a variant thereof exhibiting the biological function of MYDGF as described herein for use in treating or preventing a liver disorder. The nucleic acids for use according to the present invention preferably encode an amino acid sequence having at least 85% sequence identity to SEQ ID NO: 1.

[0070]

Nucleic acid sequences can be optimized in an effort to enhance expression in a host cell. Parameters to be considered include C:G content, preferred codons, and the avoidance of inhibitory secondary structure. These Factors can be combined in different ways in an attempt to obtain nucleic acid sequences having enhanced expression in a particular host (cf. e.g. Donnelly et al., International Publication Number WO 97/47358). The ability of a particular sequence to have enhanced expression in a particular host involves some empirical experimentation. Such experimentation involves measuring expression of a prospective nucleic acid sequence and, if needed, altering the sequence. Starting with a particular amino acid sequence and the known degeneracy of the genetic code, a large number of different encoding nucleic acid sequences can be obtained. The degeneracy of the genetic code arises because almost all amino acids are encoded by different combmations of nucleotide triplets or "codons". The translation of a particular codon into a particular amino acid is well known in the art (see, e.g., Lewin GENES IV, p. 119, Oxford University Press, 1990 ). [0071]

The nucleic acid for use according to the present invention may further comprise a transcriptional control element or expression control sequences positioned to control expression of the protein. Such a nucleic acid together with control elements is often termed as an expression system. The term “expression system” as used herein refers to a system designed to produce one or more gene products of interest. Typically, such system is designed “artificially”, i.e. by gene- technological means usable to produce the gene product of interest in vivo, in vitro or ex vivo. The term “expression system” further encompasses the expression of the gene product of interest comprising the transcription of the polynucleotides, mRNA splicing, translation into a polypeptide, co- and post-translational modification of a polypeptide or protein as well as the targeting of the protein to one or more compartments inside of the cell, the secretion from the cell and the uptake of the protein in the same or another cell. This general description refers to expression systems for the use in eukaryotic cells, tissues or organisms. Expression systems for prokaryotic systems may differ, wherein it is well known in the art, how an expression system for prokaryotic cells is constructed. [0072]

Regulatory elements present in a gene expression cassette generally include: (a) a promoter transcriptionally coupled to a nucleotide sequence encoding the polypeptide, (b) a 5' ribosome binding site functionally coupled to the nucleotide sequence, (c) a terminator joined to the 3' end of the nucleotide sequence, and (d) a 3' polyadenylation signal functionally coupled to the nucleotide sequence. Additional regulatory elements useful for enhancing or regulating gene expression or polypeptide processing may also be present. Promoters are genetic elements that are recognized by an RNA polymerase and mediate transcription of downstream regions. Prefened promoters are strong promoters that provide for increased levels of transcription. Examples of strong promoters are the immediate early human cytomegalovirus promoter (CMV), and CMV with intron A (Chapman et al, Nucl. Acids Res. 19:3979-3986, 1991). Additional examples of promoters include naturally occurring promoters such as the EFl alpha promoter, the murine CMV promoter, Rous sarcoma virus promoter, and SV40 early/late promoters and the [beta]-actin promoter; and artificial promoters such as a synthetic muscle specific promoter and a chimeric muscle-specific/CMV promoter (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 initiation codon. Examples of preferred ribosome binding sites include CCACCAUGG, CCGCC AUGG, and ACCAUGG, where AUG is the initiation codon (Kozak, Cell 44:283-292, 1986). The polyadenylation signal is responsible for cleaving the transcribed RNA and the addition of a poly (A) tail to the RNA. The polyadenylation signal in higher eukaryotes contains an AAUAAA sequence about 11-30 nucleotides from the polyadenylation addition site. The AAUAAA sequence is involved in signalling RNA cleavage (Lewin, Genes IV, Oxford University Press, NY, 1990). The poly (A) tail is important for the processing, export from the nucleus, translation and stability of the mRNA.

[0074]

Polyadenylation signals that can be used as part of a gene expression cassette include the minimal rabbit [beta] -globin polyadenylation signal and the bovine growth hormone polyadenylation (BGH) (Xu et al., Gene 272: 149-156, 2001 , Post et al., U.S. Patent U. S. 5, 122,458). [0075]

Examples of additional regulatory elements useful for enhancing or regulating gene expression or polypeptide processing that may be present include an enhancer, a leader sequence and an operator. An enhancer region increases 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). An enhancer region can be associated with a promoter. [0076]

The expression of the MYDGF protein or variant thereof according to the present invention may be regulated. Such regulation can be accomplished in many steps of the gene expression. Possible regulation steps are, for example but not limited to, initiation of transcription, promoter clearance, elongation of transcription, splicing, export from the nucleus, mRNA stability, initiation of translation, translational efficiency, elongation of translation and protein folding. Other regulation steps, which influence the concentration of a MYDGF polypeptide inside a cell affect the half-life of the protein. Such a regulation step is, for example, the regulated degeneration of proteins. As the proteins of the invention comprise secreted proteins, the protein can be directed to a secretory pathway of the host cell. The efficiency of secretion regulates together with the regulatory steps referring to the expression and protein stability the concentration of the respective protein outside of the cell. Outside of the cell can refer to, for example but not limited to, a culture medium, a tissue, intracellular matrix or space or a body fluid such as blood or lymph.

[0077]

The control of the regulatory steps mentioned above can be, for example, cell-type or tissuetype independent or cell-type or tissue-type specific. In a particularly preferred embodiment of the invention, the control of the regulatory steps is cell-type or tissue-type specific. Such a cell-type or tissue-type specific regulation is preferably accomplished through the regulation steps referring to the transcription of a nucleic acid. This transcriptional regulation can be accomplished through the use of cell-type or tissue-type specific promoter sequences. The result of this cell-type or tissue-type specific regulation can have different grades of specificity. This means, that the expression of a respective polypeptide is enhanced in the respective cell or tissue in comparison to other cell- or tissue-type or that the expression is limited to the respective cell- or tissue-type. Cell- or tissue-type specific promoter sequences are well known in the art and available for a broad range of cell- or tissue-types.

[0078]

The expression is not necessarily cell-type or tissue-type specific but may depend from physiological conditions. Such conditions are for example an inflammation or a wound. Such a physiological condition-specific expression can also be accomplished through regulation at all above mentioned regulation steps. The preferred way of regulation for a physiological conditionspecific expression is the transcriptional regulation. For this purpose a wound or inflammation specific promoter can be used. Respective promoters are, for example, natural occurring sequences, which can be, for example, derived from genes, which are specifically expressed during an immune reaction and/or the regeneration of wounded tissue. Another possibility is the use of artificial promoter sequences, which are, for example constructed through combination of two or more naturally occurring sequences.

[0079]

The regulation can be cell-type or tissue-type specific and physiological condition-specific. Particularly, the expression can be a liver specific expression. Preferably, the expression is liver specific and/or wound specific.

[0080]

Another possibility for a regulation of expression of the MYDGF protein or variant thereof according to the present invention is the conditional regulation of the gene expression. To accomplish conditional regulation, an operator sequence can be used. For example, the Tet operator sequence can be used to repress gene expression. The conditional regulation of gene expression by means of the T et operator together with a Tet repressor is well known in the art and many respective systems have been established for a broad range of prokaryotic and eukaryotic organisms. A person of skill in the art knows how to choose a suitable system and adapt it to the special needs of the respective application.

[0081]

In a particularly preferred embodiment the use of a nucleic acid according to the invention comprises the application to an individual, preferably an individual suffering from a liver disorder. [0082]

According to a further aspect, the present invention provides vectors comprising the nucleic acid or the expression system described herein for use in treating or preventing a liver disorder. [0083]

As used herein, the term "vector" refers to aprotein or a polynucleotide or a mixture thereof which is capable of being introduced or of introducing the proteins and/or nucleic acid comprised therein into a cell. In the context of the present invention it is prefemed that the genes of interest encoded by the introduced polynucleotide are expressed within the host cell upon introduction of the vector or the vectors. Examples of suitable vectors include but are not limited to plasmid vectors, cosmid vectors, phage vectors such as lambda phage, filamentous phage vectors, viral vectors, viral like particles, and bacterial spores.

[0084]

In a preferred embodiment of the invention, the vector is a viral vector. Suitable viral vectors include but are not limited to adenoviral vectors, adeno-associated viral (AAV) vectors, alphaviral vectors, herpes viral vectors, measles viral vectors, pox viral vectors, vesicular stomatitis viral vectors, retroviral vector and lentiviral vectors.

[0085]

In a particularly preferred embodiment of the invention, the vector is an adenoviral or an adeno-associated viral (AAV) vector.

[0086]

Nucleic acids encoding one or more MYDGF proteins or variants thereof according to the invention can be introduced into a host cell, a tissue or an individual using vectors suitable for therapeutic administration. Suitable vectors can preferably deliver nucleic acids into a target cell without causing an unacceptable side effect.

[0087]

In a particularly preferred embodiment the use of a vector according to the invention, comprises the application to an individual in need thereof.

[0088] Vectors comprising nucleic acids encoding the MYDGF protein or fragments or variants thereof exhibiting the biological function of MYDGF described above are preferably for use in treating or preventing a liver disorder.

[0089]

According to a further aspect, the present invention provides a host cell comprising the vector as described herein and expressing the nucleic acid encoding the MYDGF protein or a fragment or a variant thereof exhibiting the biological function of MYDGF for use in treating or preventing a liver disorder.

[0090]

According to a further aspect, the present invention provides pharmaceutical compositions comprising the MYDGF protein or a fragment or a variant thereof exhibiting the biological function of MYDGF and optionally a suitable pharmaceutical excipient, a liver disorder.

[0091]

The term "suitable pharmaceutical excipient", as used herein, refers to a pharmacologically inactive substance such as but not limited to a diluent, excipient, surfactants, stabilizers, physiological buffer solutions or vehicles with which the therapeutically active ingredient is administered. “Pharmaceutical excipients” are also called “pharmaceutical carriers” and can be liquid or solid. Liquid carriers include but are not limited to sterile liquids, such as saline solutions in water and oils, including but not limited to those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. A saline solution is a preferred carrier when the pharmaceutical composition is administered intravenously. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. In a preferred embodiment of the invention, the carrier is a suitable pharmaceutical excipient. Suitable pharmaceutical excipients comprise starch, glucose, lactose, sucrose, gelatine, malt, rice, flour, chalk, sitica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the tike. Such suitable pharmaceutical excipients are preferably pharmaceutically acceptable.

[0092]

“PharmaceuticaUy acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generatiy recognized pharmacopeia for use in animals, and more particularly in humans.

[0093]

The term "composition" is intended to include the formulation of the active compound with encapsulating material as a carrier providing a capsule in which the active component with or without other carriers, is surrounded by a carrier, which is thus in association with the active compound.

[0094]

The term "active ingredient" refers to the substance in a pharmaceutical composition or formulation that is biologically active, i.e. that provides pharmaceutical value. In the context of the invention, the active ingredient is the MYDGF protein or the fragment or variant thereof exhibiting the biological function of MYDGF. A pharmaceutical composition may comprise one or more active ingredients which may act in conjunction with or independently of each other. The active ingredient can be formulated as neutral or salt forms. The salt form is preferably a pharmaceutically acceptable salt.

[0095]

The term "pharmaceutically acceptable salt" refers to, for example but not limited to, a salt of the MYDGF polypeptides of the present invention including the fragments and variants thereof described herein. Suitable pharmaceutically acceptable salts include acid addition salts which may, for example, be 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, where the peptide carries an acidic moiety, suitable pharmaceutically acceptable salts thereof 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 halide, hydroxide, carboxylate, sulfate, phosphate, nitrate, alkyl sulfonate and aryl sulfonate). Illustrative examples of pharmaceutically acceptable salts include, but are not limited to, acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bicarbonate, bisulfate, bitartrate, borate, bromide, butyrate, calcium edetate, camphorate, camphorsulfonate, camsylate, carbonate, chloride, citrate, clavulanate, cyclopentanepropionate, digluconate, dihydrochloride, dodecylsulfate, edetate, edisylate, estolate, esylate, ethanesulfonate, formate, fumarate, gluceptate, glucoheptonate, gluconate, glutamate, glycerophosphate, glycolylarsanilate, hemisulfate, heptanoate, hexanoate, hexylresorcinate, hydrabamine, hydrobromide, hydrochloride, hydroiodide, 2-hydroxy- ethanesulfonate, hydroxynaphthoate, iodide, isothionate, lactate, lactobionate, laurate, lauryl sulfate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, methylsulfate, mucate, 2- naphthalenesulfonate, napsylate, nicotinate, nitrate, N-methylglucamine ammonium salt, oleate, oxalate, pamoate (embonate), palmitate, pantothenate, pectinate, persulfate, 3-phenylpropionate, phosphate/diphosphate, picrate, pivalate, polygalacturonate, propionate, salicylate, stearate, sulfate, subacetate, succinate, tannate, tartrate, teoclate, tosylate, triethiodide, undecanoate, valerate, and the like (cf. e.g. S. M. Berge et al., "Pharmaceutical Salts", J. Pharm. Sci., 66, pp. 1-19 (1977)). [0096]

According to one embodiment, the active ingredient is administered to a cell, a tissue or an individual in an effective amount. An “effective amount” is an amount of an active ingredient sufficient to achieve the intended purpose. The active ingredient may be a therapeutic agent. The effective amount of a given active ingredient will vary with parameters such as the nature of the ingredient, the route of administration, the size and species of the individual to receive the active ingredient, and the purpose of the administration. The effective amount in each individual case may be determined empirically by a skilled artisan according to established methods in the art. As used in the context of the invention, ''administering'' includes in vivo administration to an individual as wed as administration direcdy to cells or tissue in vitro or ex vivo.

[0097]

In a preferred embodiment of the invention, the pharmaceutical compositions are customized for the treatment of a disease or disorder. As used herein, “treat”, “treating” or “treatment” of a disease or disorder means accomplishing one or more of the following: (a) reducing the severity of the disorder; (b) limiting or preventing development of symptoms characteristic of the disorders) being treated; (c) inhibiting worsening of symptoms characteristic of the disorders) being treated; (d) limiting or preventing recurrence of the disorders) in patients that have previously had the disorders); (e) limiting or preventing recurrence of symptoms in patients that were previously symptomatic for the disorders); (f) reduction of mortality after occurrence of a disease or a disorder; (g) healing; and (h) prophylaxis of a disease. The term “'ameliorating” is also encompassed by the term “treating”. As used herein, “prevent”, “preventing”, “prevention”, or “prophylaxis” of a disease or disorder means preventing that such disease or disorder occurs in patient.

[0098]

In a particularly preferred embodiment of the invention, a treatment with a pharmaceutical composition according to the invention comprises the treatment of an individual in need of such treatment.

[0099]

The pharmaceutical composition contemplated by the present invention may be formulated in various ways well known to one of skill in the art. For example, the pharmaceutical composition of the present invention may be in liquid form such as in the form of solutions, emulsions, or suspensions. Preferably, the pharmaceutical composition of the present invention is formulated for parenteral administration, preferably for intravenous, intra-arterial, intramuscular, subcutaneous, transdermal, intrapulmonaiy, intraperitoneal, intrahepatic administration, or administration via mucous membranes, preferably for intravenous, subcutaneous, or intraperitoneal administration. A preparation for oral or anal administration is also possible. Preferably, the pharmaceutical composition of tire present invention is in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood. The aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9, more preferably to a pH of from 5 to 7), if necessary. The pharmaceutical composition is preferably in unit dosage form. In such form the pharmaceutical composition is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of pharmaceutical composition such as vials or ampoules. [0100]

The pharmaceutical composition is preferably administered through the intravenous, intraarterial, intramusculuar, subcutaneous, transdermal, intrapulmonary, intraperitoneal, or intrahepatic route, wherein other routes of administration known in the art are also comprised.

[0101]

If the pharmaceutical composition is used as a treatment for an individual, the use of the pharmaceutical composition can replace the standard treatment for the respective disease or condition or can be administered additionally to the standard treatment. In the case of an additional use of the pharmaceutical composition, the pharmaceutical composition can be administered before, simultaneously or after a standard therapy.

[0102]

It is further preferred that the pharmaceutical composition is administered once or more than once. This comprises 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 time span for the administration of the pharmaceutical is not limited. Preferably, the administration does not exceed 1, 2, 3, 4, 5, 6, 7 or 8 weeks.

[0103]

A single dose of the pharmaceutical composition, can independently form the overall amount of administered doses, or the respective time span of administration can include administration as one or more bolus injection(s) and/or infusion(s).

[0104]

According to a further aspect, the present invention provides a method of treating a liver disorder, comprising administering to a patient in need thereof a therapeutically effective amount of MYDGF or fragment or variant thereof exhibiting the biological function of MYDGF. In said method, the 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 respect, the fragment or variant comprises an amino acid sequence with at least 85% amino acid sequence identity to SEQ ID NO:1.

[0105]

In a preferred embodiment of the method of the present invention, the liver disorder is an acute liver failure, a chronic liver disease, or a liver transplantation. According to a further preferred embodiment, the acute liver failure is selected from the group consisting of viral hepatitis, drug- induced liver injury, acute ischemic liver injury and toxic liver damage. According to a further preferred embodiment, the chronic liver diseases is selected from the group consisting of nonalcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection with 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.

[0106]

According to a preferred embodiment of this method of the present invention, the MYDGF protein or fragment or variant thereof exhibiting the biological function of MYDGF is administered through one or more bolus injection(s) and/or infusion(s), preferably in a pharmaceutically accepted carrier.

[0107]

EXAMPLES

The Examples are designed to further illustrate the present invention and serve a better understanding. They are not to be construed as limiting the scope of the invention in any way. [0108]

Materials and methods used in the Examples:

Unless stated otherwise, the following materials and methods were used in the examples.

[0109]

Blood was collected in EP tubes, kept at 4°C for 30 minutes, and centrifuged at 15,000 rpm for 10 minutes at 4°C.Liver tissues were collected and snapfrozen in liquid nitrogen for proteins and in RNAlater (Invitrogen) for RNA extraction and analysis.

A portion of the liver was fixed in 10% phosphate-buffered formalin, embedded in paraffin blocks, processed histologically, stained with hematoxylin and eosin (H&E) and Sirius Red. Analysis and quantification of positive-staining areas measured by Image J software.

To analysis serum MYDGF concentration, we coat Nunc MaxiSorp™ flat-bottom (Thermo Fisher Scientific) with 5ug/ml anti-MYDGF capture antibody (PPB-32537, provided by Boehringer Ingelheim), bind sample with 3ug/ml Biotin HumanSF20/MYDGF Antibody (PPB- 50118, provided by Boehringer Ingelheim), develop with Streptavidin-HRP (Southern Biotech) and Substrate Reagent (R&D Systems). Finally, the absorbance of sample was measured at 450nm.

Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) concentrations were measured with Transaminase Cll Test Wako kits (Wako) according to the manufacturer’s instructions. Hydroxyproline content in the whole liver tissue were measured with Hydroxyproline Assay Kit ™ (Cell Biolabs Catalog Number: STA-675) according to the manufacturer’ s instructions. [0110]

The human hepatocellular carcinoma cell line HepG2 was seeded onto collagen-coated plates and maintained 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 in a humidified atmosphere at 37°C and 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) and subsequently incubated in serum-free DMEM for 6 hours before exposure to recombinant MYDGF protein.

[0111]

Whole-cell lysates from mouse liver and HepG2 cells were prepared and lysed in a 1 x RIPA Lysis Buffer (EMD Milfipore) containing complete Protease Inhibitor Cocktail and PhosSTOP (Roche Applied Science). The following primary antibodies were used: NF-κB, p-NF- κB(Ser536), GAPDH (all Cell Signaling Technology); SF20/MYDGF (R&D Systems). [0112]

Total RNA was isolated from frozen liver tissue samples in RNAlater using a RNeasy Mini Kit(QIAGEN), and cell samples using a NIPPON RNA Kit (Nippon Gene), according to the manufacturer's protocol. cDNA was synthesized from 100 ng total RNA using a High-capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific). RTD-PCR was conducted using TaqMan Gene Expression Assay Identification. The following TaqMan probes(Thermo Fisher Scientific) were used: MYDGF (Hs00384077_ml, Mm00840829_ml ), TNF-α (Hs00174128_ml, Mm00443258_ml), IL-1β (Mm00434228_ml), IL-6 (Mm00446190jml), Collal (Mm00801666_gl), Colla2 (Mm00483888_ml), Acta2 (Mm01546133 ml). Quantitative gene expression data were normalized to the expression levels of the housekeeping gene GAPDH. [0113] <Recombinant Proteins and Chemicals>

Recombinant human MYDGF (PROTEOS-1066-Batch2, PPB-11924): The nucleic acid sequence encoding human Factor 1 is available under NCBI Gene ID: 56005 (SEQ ID NO: 6). The amino acid sequence of human Factor 1 including the N-terminal signal peptide is detailed in SEQ ID NO: 3. In the examples, human MYDGF without the signal peptide (SEQ ID NO: 1) was used and expressed as detailed in Ebenhoch R. et al, Crystal structure and receptor-interacting residues of MYDGF - a protein mediating ischemic tissue repair (Nat Commun. 2019 Nov 26;10(l):5379 and Polten et al. Plasma Concentrations of Myeloid-Derived Growth Factor in Healthy Individuals and Patients with Acute Myocardial Infarction as Assessed by Multiple Reaction Monitoring-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 were presented as means ± standard deviation (SD) and analyzed using GraphPad Prism 9.4.1 software. Experiments were repeated at least 3 times. Two-tailed unpaired Student t test or one-way analysis of variance was used to evaluate the data. Pearson’s correlation coefficients were used to assess relationship. A value of p < 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 carbon tetrachloride (CCl₄) induced liver injury models.

Ex.1 - 1 : Preparation of Acute liver injury model

C57BL/6J mice (malelOw) were divided into 6 groups (each n=5). The mice were intraperitoneally injected withl0% CCl₄ (Com Oil 180 μL +CCl₄20 μL/mice). Liver and serum were collected at 6 hrs, 12 hrs, 24 hrs, 48 hrs and 72 hrs after injection of CCl₄ (Fig. 1 A).

The expression of MYDGF mRNA in the liver was evaluated at each time (Fig.1B ). The serum levels of transaminases, AST and ALT were evaluated at 24 hrs, 48 hrs and 72 hrs (Figs. 1C and 1D ).The expression of MYDGF mRNA in the liver increased from 6hrs and peaked at 12 hrs after CCl₄ injection. The levels of MYDGF mRNA were maintained from 12 hrs to 48 hrs and slightly decreased at 72 hrs (Fig.1B). The serum levels of transaminases AST and ALT elevated from 24 hrs and peaked at 48 hrs and returned to basal at 72 hrs (Figs. 1C and 1D).

[0116]

Ex. 1-2: Preparation of Chronic liver injury model

C57BL/6J mice (malelOw) were intraperitoneally administered with 10% CCl₄ (Com Oil 180 μL +CCl₄20 μL/mice) every three times, 10 times in total for 30 days. After cessation of CCl₄, livers and serum were collected at 7 days, 14 days and 28 days (Fig. 2A). The mRNA expression of MYDGF, TNFα and colla2 in the liver were evaluated at each time (Figs. 2B, 2C and 2D ).

The expression of MYDGF mRNA in the liver was continuously induced by the long-term administration of CCl₄ (Fig. 2B). The expression of mRNA of TNFα and colla2 in liver were upregulated by CCl₄. Interestingly, the expression of colla2 mRNA were significantly inversely correlated with the expression of MYDGF mRNA (Figs. 2C and 2D).

[0117] Example 2: The Effect of MYDGF on acute liver injury

Ex.2-1: Protocol

Male C57BL/6 mice (aged 8-10 weeks, weighing 20-25 g) were maintained in a temperature-controlled (22 ± 2°C) pathogen-free animal facility under a standard 12 h light/dark cycle. To induce acute liver injury model, mice were intraperitoneally injected with either a single dose of com oil (vehicle control) or CCl4 (1 μL/g intraperitoneally), diluted 1:9 in com oil. In MYDGF experiment group, an osmotic pump (Alzet model 1007D, 20 μg/d, 7 days, filled with 20 μg of recombinant human MYDGF per 12 μl diluted in PBS) was implanted in the subcutaneous interscapular pocket (Fig. 3A). In sham-operated control group, a skin incision was made between the shoulder blades, and the wound was closed with surgical staples.

The above-mentioned experiment group (MYDGF ALZET pump group), and a sham- operated control group were intraperitoneally injected with a single-dose of CCl4. After injection, mice were euthanized at 24 hrs, 48 hrs and 72 hrs and their serum and liver tissue samples were collected for evaluation (n=5 per time point per group) (Fig.3B).

For histological examination and NF-kB activation in liver, the control group was a vehicle pump control group instead of the sham operation control group. In the vehicle pump control group, an osmotic pump (Alzet model 1007D, 12 μl/d of PBS, 7 days) was implanted in the subcutaneous interscapular pocket. PBS (vehicle) was administered at a flow rate of 12 μl/d.

Serum MYDGF concentration in the MYDGF ALZET pump group and the sham- operated control group was analyzed by ELISA (Fig.3 C) . The high levels of serum MYDGF were maintained throughout the study in the MYDGF ALZET pump group.

[0118]

Ex.2-2: Serum levels of transaminase

Serum levels of transaminase, AST/ALT were evaluated at 24 hrs, 48 hrs and 72 hrs in the MYDGF ALZET pump group and the sham-operated control group. Serum ALT and AST levels in the two groups are shown as international units per liter. Serum levels of ALT and AST were significantly lower in mice with MYDGF ALZET pump group than sham-operated control group at 24 hrs and 48 hrs (Figs.3D and 3E ).

[0119]

Ex.2-3: Histological examination

The liver samples of the MYDGF ALZET pump group and vehicle pump control group at different time points after a single-dose intraperitoneal injection of CCl₄ was stained with hematoxylin and eosin (H&E) (Fig. 3F).

Histological examination of collected livers showed the CCl₄ induced necrosis area around central vein (Fig.3F, zone3). The black line in liver slices indicates the necrotic area. Necrosis area was most extensive at 24 hrs and gradually decreased over the period. Necrosis area tends to be smaller in MYDGF ALZET pump group compared to vehicle pump control group (Fig.3F). The percent necrosis area in each group was quantified by Image J. Semi quantification of necrosis area showed the significant lower necrosis in MYDGF ALZET pump group compared to control group at 24 hrs and 48 hrs (Figs.3F and 3G).

[0120]

Ex.2-4: Suppression of hepatic inflammatory response

(1) mRNA expression of inflammatory cytokines

The 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-operated control group. The mRNA expression of TNFα and IL6 at 24 hrs and 48 hrs were significantly down regulated in MYDGF ALZET pump group than the control group (Figs.4A and 4C). The mRNA expression of IL 1β at 24 hrs, 48 hrs and 72 hrs were significantly down regulated in MYDGF ALZET pump group than sham-operated control group (Fig.4B ).

(2) NF-kB activation in liver

The expression of NF-κB, p-NF-κB in liver samples of the MYDGF ALZET pump group and vehicle pump control group at 48 hrs after a single-dose intraperitoneal injection of CCl4 was analysed by western blot.

NF-kB activation in liver was repressed in MYDGF ALZET pump group compared to vehicle pump control group (Fig.4D ).

[0121]

Example 3: The Effect of MYDGF on chronic liver injury

Ex.3-1 : Protocol

Male C57BL/6 mice (aged 8-10 weeks, weighing 20-25 g) were maintained in a temperature-controlled (22 ± 2°C) pathogen-free animal facility under a standard 12 h light/dark cycle. To induce chronic liver injury model, mice were intraperitoneally administered with either a com oil (vehicle control) or 10% CCl₄ (Com Oil 180 μL +CCl₄20 μL/mice) every three days, 10 times in total for 27 days. In experiment group, an osmotic pump (Alzet model 1007D, 20 μg/d, 7 days, filled with 20 μg of recombinant human MYDGF per 12 μl diluted in PBS) was implanted in the subcutaneous interscapular pocket. In sham-operated control group, a skin incision was made between the shoulder blades, and the wound was closed with surgical staples.

Mice were divided into sham-operated group (n=6), pump experiment group (MYDGF ALZET pump group) (n=6) and control group (n=3). The sham-operated group were treated with CCl4 intraperitoneal injection + sham-operation. The pump experiment group were treated with CCl4 intraperitoneal injection + rhMYDGF pump-implantation. The control group were received vehicle intraperitoneal injection without pump implantation.

Mice were euthanized at 28 days after starting administration of CCl4 and their serum and liver tissue samples were collected for evaluation (Fig.5A).

Serum MYDGF concentration in sham-operated control group, MYDGF ALZET pump group and control group at 28 days after starting administration of CCl₄ was analyzed by ELISA (Fig.5B).

[0122]

Ex.3-2: Serum levels of transaminase

Serum transaminase, AST/ALT in the three groups were measured for the evaluation of liver injury. Serum levels of ALT and AST were significantly lower inmice with MYDGF ALZET pump group than sham-operated control group (Figs.5C and 5D).

[0123]

Ex.3-3: Suppression of liver fibrosis

(1) Histological examination

The livers of sham-operated group and pump experiment group were harvested at 30 28 days after starting administration of CCl4 and stained using Sirius Red for fibrosis analysis and quantification of positive-staining areas as liver fibrosis area measured by Image J software. Semi quantification of fibrosis area showed the significant lower fibrosis in MYDGF ALZET pump group compared to sham-operated control group (Figs.6A and 6B).

(2) Hydroxyproline content

Hydroxyproline content in the liver of the three groups was measured for the evaluation of liver fibrosis. Hydroxyproline content in the liver are shown as microgram per gram of whole liver tissue (Fig. 6C). The hydroxyproline content was significantly lower in MYDGF ALZET pump group than sham-operated control group.

(3) mRNA expression of hepatic fibrosis genes

The mRNA expression ofhepatic fibrosis genes TGF-P, ACTA2, COL1 A1, and COL1 A2 in whole liver tissue of the three groups were analyzed by RTD-PCR in the three groups (Figs. 6D- 6G). The mRNA expressions of TGF-β, ACTA2, COL1A1, COL1A2 were significantly down regulated in MYDGF ALZET pump group than sham-operated control group.

[0124]

Ex.3-5: Suppression ofhepatic inflammatory response

(1) mRNA expressions of inflammatory cytokines

The mRNA expressions of inflammatory cytokines TNF-α, IL-1β, and IL-6 in whole liver tissue were analyzed by RTD-PCR (Figs. 7A-7C). The mRNA expression of TNF-α, IL- 1β, and IL-6 were significantly down regulated in MYDGF ALZET pump group than sham-operated control group.

(2) NF-kB activation in liver

The total and phosphorylated levels of NF-κB in whole liver tissue from mice were analyzed by western blotting (Fig. 7D). NF-kB activation in liver was repressed in MYDGF ALZET pump group compared to sham-operated control group.

RhMYDGF suppresses hepatic inflammatory response in a mouse model of chronic hepatitis-induced liver fibrosis.

[0125]

Example 4: Inhibition of NF-κB activation and expression of cytokines in HepG2 cells

Ex. 4-1 : MYDGF suppressed NF-κB activation in HepG2 cells.

The ant-inflammatory effect of MYDGF in HepG2 cells was evaluated in vitro. HepG2 cells were treated with different concentrations of rhMYDGF (100 ng/mL and 500 ng/mL), and NF-κB phosphorylation activity were analyzed by western blotting at 6 hrs (n=3). MYDGF dose dependently repressed the expression of phosphorylated NF-kB (Fig. 8 A).

[0126]

Ex. 4-2: MYDGF decreased TNF-α expression in HepG2 cells.

The ant-inflammatory effect of MYDGF in HepG2 cells was evaluated in vitro. HepG2 cells were treated with different concentrations of rhMYDGF (100 ng/mL and 500 ng/mL), and the expression of TNF-α mRNA were analyzed by RTD-PCR at 6 hrs (n=3) (Fig. 8B).

MYDGF significantly decreased the expression of TNF-α mRNA expression.

It was shown that decrease of expression of inflammatory cytokine and suppression ofNF- κB activation by MYDGF were recognized.

[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, respectively, was analyzed by RTD-PCR at 24 hrs (n=3) (Figs. 9A-9C). Dose-dependent effects of rhIL-6 on mRNA expression (Fig.9D) and supernatant protein (Fig.9E) of MYDGF in HepG2 cells were analyzed by RTD-PCR and western blotting, respectively at 24 hrs (n=3).

TNFα and IL 1β did not induce MYDGF, however, IL6 significantly induced MYDGF in mRNA and protein levels.

Claims

1. Myeloid-derived growth factor (MYDGF) or a fragment or a variant thereof exhibiting the biological function of MYDGF, for use in treating or preventing a liver disorder.

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

3. The MYDGF or a fragment or a variant thereof according to claim 1 or 2, wherein the acute liver failure is selected from the group consisting of viral hepatitis, drag-induced liver injury, acute ischemic liver injury and toxic liver damage, wherein the chronic liver diseases is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection with 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. The MYDGF or a fragment or a variant thereof according to any one of claim 1 to 3, wherein the MYDGF comprises:

(i) SEQ ID NO: 1; or

(ii) a fragment or variant of SEQ ID NO: 1 exhibiting the biological function of MYDGF, and wherein the variant comprises an amino acid sequence with at least 85% amino acid sequence identity to SEQ ID NO: 1.

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

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

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

8. The nucleic acid according to claim 6 or 7, wherein the acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury and toxic liver damage, wherein the chronic liver disease is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), alcohohc 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 claim 6 to 8, wherein the MYDGF comprises:

(i) SEQ ID NO: 1; or

10. A vector comprising the nucleic acid of any one of claims 6 to 9, for use in t in treating or preventing a liver disorder.

11. A host ceh comprising the nucleic acid of any one of claims 6 to 7 or the vector according to claim 9, for use in treating or preventing a liver disorder.

12. A pharmaceutical composition comprising Myeloid-derived growth factor (MYDGF) or a fragment or a variant thereof exhibiting the biological function of MYDGF, or a nucleic acid encoding the MYDGF or a fragment or a variant thereof, or a vector comprising the nucleic acid, or a host cell comprising the nucleic acid or the vector, for use in treating or preventing a liver disorder.

13. The pharmaceutical composition according to claim 12, wherein the liver disorder is an acute liver failure, a chronic liver disease, or a liver transplantation.

14. The pharmaceutical composition according to claim 12 or 13, wherein the acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury and toxic liver damage, wherein the chronic liver diseases is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection with hepatitis B virus (HBV) or hepatitis C virus (HCV), alcohohc 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 the MYDGF comprises:

(i) SEQ ID NO: 1; or

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

17. The pharmaceutical composition according to any one of claims 12 to 16, wherein said pharmaceutical composition is administered through the oral, intravenous, subcutaneous, intramucosal, intraarterial, intramuscular or intrahepatic route.

18. The pharmaceutical composition according to claim 17, wherein the administration is through one or more bolus injection(s) and/or infusion(s).

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

20. The method according to claim 19, wherein the liver disorder is an acute liver failure, a chronic liver disease, or a liver transpl an tation .

21. The method according to claim 19 or 20, wherein the acute liver failure is selected from the group consisting of viral hepatitis, drug-induced liver injury, acute ischemic liver injury and toxic liver damage, wherein the chronic liver diseases is selected from the group consisting of non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), chronic infection with 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 the MYDGF comprises:

(i) SEQ ID NO: 1; or

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

24. The method according to any one of claims 19 to 23, wherein said MYDGF is administered through the oral, intravenous, subcutaneous, intramucosal, intraarterial, intramuscular or intrahepatic route.

25. The method according to claim 24, wherein the administration is through one or more bolus injection(s) and/or infusion(s).