Use of FGF5 transcript variant-2 and its protein for the prevention, suppression, and treatment of liver fibrosis

Abstract

The present invention relates to the use of FGF5-v2 (FGF5 transcript variant 2) gene and its protein for preventing or treating liver fibrosis. The FGF5-v2 fragment gene (transcript and protein expressed therefrom) of the present invention is a mutant gene in which the amino acid sequence of fibroblast growth factor 5 (FGF5) is altered, and has the effect of suppressing the progression and proliferation of liver fibrosis. Therefore, FGF5-v2 is expected to prevent and treat liver fibrosis and block the progression to liver cirrhosis and liver cancer when administered to patients with fatty liver via a vector (e.g., AVV), etc., and is useful as a composition for treating liver fibrosis.

Description

[Technical field]

[0001] The present invention relates to the use of transcript variants of FGF5 (fibroblast growth factor 5) and proteins expressed therefrom for the prevention, suppression, and treatment of liver fibrosis.

[0002] The present invention claims priority based on Korean Patent Application No. 10-2022-0159875 filed on November 25, 2022 and Korean Patent Application No. 10-2023-0156822 filed on November 13, 2023, and all contents disclosed in the specification and drawings of said application are incorporated by reference into this application. [Background technology]

[0003] Liver fibrosis occurs due to the extensive accumulation of extracellular matrix, including collagen, which occurs in most types of chronic liver diseases. Representative cells involved in liver fibrosis include hepatic stellate cells, Kupffer cells, and endothelial cells. Hepatic stellate cells are the main source of extracellular matrix production and are involved in the increased production of various extracellular matrices, including collagen. Kupffer cells exist in the sinusoidal space in the liver and affect the substances produced by activated Kupffer cells on surrounding hepatocytes, endothelial cells, and hepatic stellate cells, promoting liver fibrosis. Endothelial cells play an important role in regulating blood flow in the liver and are also involved in the production of growth factors and extracellular matrix that are involved in the proliferation of hepatic stellate cells due to inflammation and liver fibrosis. Liver fibrosis is accompanied by an exponential expansion of activated hepatic stellate cells, which secrete profibrogenic cytokines and subsequently produce extracellular matrix-associated molecules such as α-SMA, collagen, and tissue inhibitors of metalloproteinases (TIMPs).

[0004] In particular, non-alcoholic fatty liver disease (NAFLD) among liver diseases progresses to non-alcoholic steatohepatitis (NASH) and cirrhosis, and is highly related to metabolic diseases such as obesity, diabetes, and hyperlipidemia that are not caused by alcohol. Non-alcoholic steatohepatitis is recognized as a serious disease worldwide because fat accumulates in liver cells, causing degeneration / necrosis of liver cells, which leads to inflammation and liver fibrosis, which can lead to cirrhosis (or cirrhosis) and liver cancer. NASH occurs due to two separate events that develop from steatosis to fibrosis, which is called the "two-hit process" (Non-Patent Document 1).

[0005] Meanwhile, fibroblast growth factor (FGF) is a type of growth factor, and forms a family consisting of 23 types. It is known that each type has various in vivo functions and activities, and their specific roles have not been fully elucidated. In this situation, FGF5 (fibroblast growth factor 5) was previously known to be involved in the progression of liver fibrosis in non-alcoholic steatohepatitis (NASH) (Non-Patent Document 1). However, the present inventors confirmed that the newly discovered FGF5 mutant can effectively suppress fibrosis in the liver, and focusing on this, they confirmed the potential of the FGF5 mutant as a therapeutic agent for liver diseases based on its excellent anti-fibrotic effect. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent Application Serial No. 08 / 991,601 [Patent Document 2] U.S. Patent Application Serial No. 08 / 409,297 [Patent Document 3] U.S. Pat. No. 5,608,149 [Patent Document 4] U.S. Pat. No. 5,608,144 [Patent Document 5] U.S. Pat. No. 5,604,121 [Patent Document 6] U.S. Pat. No. 5,569,597 [Patent Document 7] U.S. Pat. No. 5,466,785 [Patent Document 8] U.S. Patent No. 5,399,680 [Patent Document 9] U.S. Pat. No. 5,268,463 [Patent Document 10] U.S. Pat. No. 5,608,142 [Patent Document 11] European Patent No. 307,247 [Patent Document 12] International Publication No. 91 / 08291 [Non-patent literature]

[0007] [Non-Patent Document 1] Exp Anim.2014;63(1):85-92. [Non-Patent Document 2] Dayhoff et al., in Atlas of Protein Sequence and Structure, vol 5, supp 3, 1978 Summary of the Invention [Problem to be solved by the invention]

[0008] In response to this, while researching therapeutic strategies that can control liver fibrosis, the present inventors have newly discovered that FGF5 (fibroblast growth factor 5), which is previously known to be involved in the progression of liver fibrosis in nonalcoholic steatohepatitis (NASH), is notably inhibited by FGF-v2 (FGF5 transcript variant 2), a short variant of FGF5 according to the present invention, and thus completed the present invention.

[0009] Therefore, an object of the present invention is to provide an FGF5-v2 transcript mutant and its protein (also referred to as "FGF5-v2 protein") consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing.

[0010] Another object of the present invention is to provide a nucleic acid molecule (ie, the gene for FGF5-v2) encoding the FGF5-v2 protein. Another object of the present invention is to provide a carrier containing the FGF5-v2 gene of the present invention, its transcript, or its protein (or carrying the gene, transcript, or protein); or a composition containing the carrier.

[0011] Another object of the present invention is to provide a pharmaceutical composition for preventing or treating liver fibrosis (or liver fibrosis), comprising as an active ingredient an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence catalog of the present invention; an expression vector containing a nucleic acid molecule encoding the FGF5-v2 protein; or an isolated cell into which the expression vector has been introduced.

[0012] However, the technical problems that the present invention aims to solve are not limited to those mentioned above, and other problems not mentioned will be clearly understood by a person having ordinary skill in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0013] In order to achieve the above-mentioned objects, the present invention provides an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence catalog.

[0014] The present invention also provides a nucleic acid molecule encoding the FGF5-v2 protein. In one embodiment of the present invention, the FGF5-v2 gene may include, but is not limited to, the base sequence of SEQ ID NO: 2 in the sequence listing.

[0015] The present invention also provides an expression vector comprising the nucleic acid molecule. The present invention also provides a cell into which the expression vector has been introduced. Preferably, the cell is an isolated cell.

[0016] The present invention also provides a pharmaceutical composition for preventing or treating liver fibrosis, comprising as an active ingredient an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO:1 in the sequence listing; a nucleic acid molecule encoding the FGF5-v2 protein; an expression vector containing the nucleic acid molecule; or an isolated cell into which the expression vector has been introduced.

[0017] In one embodiment of the present invention, the FGF5-v2 protein, the nucleic acid molecule, the expression vector, or the cell may be carried in a vehicle, but is not limited thereto.

[0018] In another embodiment of the present invention, the composition comprises FGF5-v2 protein, the nucleic acid molecule, or the expression vector as an active ingredient, and the carrier may be one or more selected from the group consisting of virus particles, vesicles, nanoparticles, microparticles, liposomes, transposons, micelles, antibodies, and exosomes, but is not limited thereto.

[0019] In yet another embodiment of the present invention, the liver fibrosis may be, but is not limited to, one or more liver fibrosis selected from the group consisting of alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis C, hepatitis B, autoimmune hepatitis, hepatic encephalopathy, primary biliary sclerosis, liver cancer, and hepatocellular carcinoma.

[0020] In yet another embodiment of the present invention, the non-alcoholic fatty liver disease may be any one or more selected from the group consisting of non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), hepatic steatosis, acute fatty liver of pregnancy (AFLP), NAFLD-related liver failure, NAFLD-related liver fibrosis, NAFLD-related cirrhosis, and NAFLD-related liver cancer, but is not limited thereto.

[0021] In another embodiment of the present invention, the composition may be administered in combination with, but not limited to, an anti-inflammatory agent. In yet another embodiment of the present invention, the anti-inflammatory agent may be, but is not limited to, a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent.

[0022] In addition, the present invention provides a method for preventing or treating liver fibrosis, comprising administering to an individual in need thereof an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing; a nucleic acid molecule encoding the FGF5-v2 protein; an expression vector containing the nucleic acid molecule; or an isolated cell into which the expression vector has been introduced. However, the FGF5-v2 gene (nucleic acid molecule), transcript, and / or protein of the present invention may be administered to an individual using various carriers (transmitters) known in the art other than a vector. As the carrier, for example, an expression vector, a virus, a vesicle, a nanoparticle, a microparticle, a liposome, a transposon, a micelle, an exosome, an antibody, etc. may be used.

[0023] In addition, the present invention provides use of a composition comprising, as an active ingredient, an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO:1; a nucleic acid molecule encoding the FGF5-v2 protein; an expression vector containing the nucleic acid molecule; or an isolated cell into which the expression vector has been introduced, for the prevention or treatment of liver fibrosis.

[0024] In addition, the present invention provides use of a composition comprising, as an active ingredient, an FGF5-v2 protein having the amino acid sequence of SEQ ID NO:1; a nucleic acid molecule encoding the FGF5-v2 protein; an expression vector containing the nucleic acid molecule; or an isolated cell into which the expression vector has been introduced, for producing a formulation for preventing or treating liver fibrosis.

[0025] In addition, the present invention provides use of an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing; a nucleic acid molecule encoding said FGF5-v2 protein; an expression vector comprising said nucleic acid molecule; or an isolated cell into which said expression vector has been introduced, for the prevention or treatment of liver fibrosis.

[0026] In addition, the present invention provides use of an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO: 1 in the sequence listing; a nucleic acid molecule encoding said FGF5-v2 protein; an expression vector containing said nucleic acid molecule; or an isolated cell into which said expression vector has been introduced, for the manufacture of a medicament for the prevention or treatment of liver fibrosis. Effect of the Invention

[0027] The fibroblast growth factor 5 (FGF5) transcript variant FGF5-v2 and its protein according to the present invention were developed by adjusting the amino acid sequence of full-length FGF5. It was confirmed that the transcript variant and its protein have the effect of suppressing the progression of liver fibrosis and improving liver dysfunction associated with liver fibrosis and the like, and various inflammation- and fibrosis-related cytokine increases. Therefore, it is expected that liver fibrosis can be suppressed and preventive, ameliorative and therapeutic effects can be achieved against various diseases including liver cirrhosis and liver cancer by administering to a patient a gene encoding FGF5-v2, its transcript, protein and / or various delivery vehicles carrying these (e.g., AAV). [Brief description of the drawings]

[0028] [Figure 1] 1 shows a recombinant expression vector for transferring and expressing (transcript or protein expression) the gene encoding the FGF5-v2 mutant of the present invention (referred to as "FGF5-v2" or "FGF5-S") into cells. [Figure 2a] FIG. 1 is a schematic diagram showing a series of experimental schedules for creating an in vivo liver fibrosis model (an in vivo NAFLD mouse model induced by a CDAA-HFD diet) and confirming the hepatic fibrosis-suppressing effect of the FGF5-v2 mutant of the present invention (referred to as "FGF5-v2" or "FGF5-S"). [Figure 2b]These results show the change in body weight over time after injection of a carrier containing the gene for full-length FGF5 ("FGF5-F") or the FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into a NAFLD mouse model (Normal, normal mice; Control, untreated control group; the same applies below). [Figure 2c] In normal mice, a comparison group, an FGF5-F-treated group, and an FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S")-treated group, changes in glucose tolerance are shown when glucose is injected after 12 hours of fasting. [Figure 3a] The results are a comparison of NAFLD activity scores (NAS) after injecting genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into a NAFLD mouse model. [Figure 3b] The researchers compared the fibrosis scores after injecting genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into a NAFLD mouse model, and confirmed that injection of the FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") specifically had an inhibitory effect on liver fibrosis. [Figure 4a] The results were obtained by injecting genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into a NAFLD mouse model, and then comparing AST, an indicator of liver function, in the serum of the NAFLD mouse model.The results confirmed that injection of FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") specifically had an AST suppression effect (i.e., an effect of improving liver damage and liver function). [Figure 4b]The results were obtained by injecting genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into a NAFLD mouse model, and then comparing ALT levels during liver function evaluation in the serum of the NAFLD mouse model.The results specifically confirmed the ALT suppression effect (i.e., the effect of improving liver damage and liver function) of injection of FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S"). [Figure 4c] The results show that the serum AST / ALT ratio in a NAFLD mouse model was examined after injecting the genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse model. [Figure 4d] The results show that the levels of triglyceride (TG) in lipid serum of NAFLD mouse models were compared after injecting genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse model. [Figure 4e] 1 is a graph showing a comparison of HDL (high-density lipoprotein) levels in serum lipids in a NAFLD mouse model after injecting the gene for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse model. [Figure 4f] 1 is a graph showing a comparison of the levels of LDL (low-density lipoprotein) in serum lipids in a NAFLD mouse model after injecting the gene for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse model. [Figure 4g] 1 is a graph comparing glucose levels in serum of a NAFLD mouse model after injecting the gene for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse model. [Figure 5a]This is a graph showing the comparison of the levels of α-SMA in fibrosis-related biomarkers in liver tissue mRNA from NAFLD mouse models after injecting genes for FGF5-F or FGF5-v2 variant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse models. The inhibitory effect of α-SMA specifically by injecting FGF5-v2 variant (referred to as "FGF5-v2" or "FGF5-S") was confirmed at the mRNA level. [Figure 5b] This is a graph showing the comparison of the levels of Col1α1 in fibrosis-related biomarkers in liver tissue mRNA from NAFLD mouse models after injecting genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse models. The inhibitory effect of Col1α1 on specific induction by injection of FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") was confirmed. [Figure 5c] This is a graph showing a comparison of the levels of fibronectin, a fibrosis-related biomarker, in liver tissue mRNA from a NAFLD mouse model after injecting the genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse model. [Figure 5d] 1 is a graph showing a comparison of the levels of Vimentin, a fibrosis-related biomarker, in liver tissue mRNA from a NAFLD mouse model after injecting the gene for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse model. [Figure 5e] The results show that the expression levels of FGF5-F and FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") transcripts were measured in NAFLD mice (n=8 per group) injected with the genes for FGF5-F (Figure 5e) or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") (Figure 5f). [Figure 5f] Same as above. [Figure 6a]The results are shown below. After injecting genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into a NAFLD mouse model, the expression of IL-1β protein, an inflammatory marker, was quantitatively analyzed through ELISA experiments using liver tissue from the NAFLD mouse model. [Figure 6b] The results are shown below. After injecting genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into a NAFLD mouse model, the expression of IL-6 protein, an inflammatory marker, was quantitatively analyzed through ELISA experiments using liver tissue from the NAFLD mouse model. [Figure 6c] The results show that after injecting FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") genes into NAFLD mouse models, the expression level of α-SMA protein was analyzed using Western blot analysis of liver tissue from NAFLD mouse models. The inhibitory effect of injection of FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") on the protein level of α-SMA was specifically confirmed. [Figure 7a] The results show a comparison of the expression of various cytokines and chemokines in the serum of NAFLD mouse models after injecting genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse model, confirming the hepatic fibrosis-suppressing effect of FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S"). [Figure 7b] The results show a comparison of the protein level expression of various cytokines and chemokines in the liver tissue of a NAFLD mouse model after injecting genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") into the NAFLD mouse model, and confirmed the inhibitory effect of FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") on cytokines related to liver fibrosis. [Figure 8a]This is an image showing the results of Sirius red staining of liver tissue sections from a NAFLD mouse model injected with the genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S"). The image confirms the inhibitory effect of FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") on liver fibrosis. [Figure 8b] This is an image showing the results of H&E staining of liver tissue sections from a NAFLD mouse model into which the genes for FGF5-F or FGF5-v2 mutant (referred to as "FGF5-v2" or "FGF5-S") were injected. [Figure 9] FIG. 1 shows the full length FGF5 sequence with the regions corresponding to FGF5-v2 mutant (designated "FGF5-v2" or alternatively "FGF5-S") (underlined) and FGF5-v3 (boxed). [Figure 10a] This shows the results of a stimulation experiment using human hepatic stellate cell line (LX2 cells), where the x-axis indicates the time (min) after FGF5 treatment and the y-axis indicates the fold value compared to the control experiment. It was confirmed that the full-length FGF5 protein has a signal transduction effect through Erk phosphorylation. [Figure 10b] This shows the results of a stimulation experiment using a human hepatic stellate cell line (LX2 cells), where the x-axis indicates the time after protein treatment with FGF5-v2 mutant (called "FGF5-v2" or "FGF5-S"), and the y-axis indicates the fold value compared to the control experiment. Through Erk phosphorylation, it was confirmed that the FGF5-v2 mutant (called "FGF5-v2" or "FGF5-S") protein has no signal transduction effect. [Figure 10c]The results of a stimulation experiment using human hepatic stellate cell line (LX2 cells) are shown. The x-axis shows the concentration of FGF5-v2 mutant (called "FGF5-v2" or "FGF5-S") protein pretreated at 1, 3, and 5 times the concentration of FGF5 treatment, and the y-axis shows the fold value compared to the control experiment. It was confirmed that the FGF5-v2 mutant (called "FGF5-v2" or "FGF5-S") protein antagonizes the signaling action of the full-length FGF5 protein. This confirmed the hepatic fibrosis inhibitory effect of the FGF5-v2 mutant (called "FGF5-v2" or "FGF5-S"). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. As used herein, the term "protein" is used interchangeably with "polypeptide" to refer to a polymer of amino acid residues, such as those commonly found in naturally occurring proteins.

[0030] The present invention provides a mutant gene of FGF5 (fibroblast growth factor 5), an FGF5 transcript variant expressed therefrom, and a protein translated therefrom. That is, the present invention aims to provide an FGF5-v2 (FGF5 transcript variant 2) gene, which is an FGF5 fragment newly identified by the present inventors, a transcript expressed therefrom, and a protein translated therefrom.

[0031] The FGF5-v2 variant of the present invention is an FGF5 fragment newly discovered by the present inventors. It may be called "FGF5-v2" or "FGF5-S". The present inventors have newly discovered that the FGF5-v2 protein has different properties from the full-length FGF5 protein and other fragments (FGF5-v1 or FGF5-v3), and is completely different from the parent protein FGF5, and has an improving and treating effect on liver fibrosis. The full-length FGF5 is a known protein, and specific information can be found in public databases such as NCBI (Accession No. NM_004464.4).

[0032] The FGF5-v2 protein of the present invention is characterized in that it contains or consists of the amino acid sequence of SEQ ID NO: 1 in the sequence catalog. In the present invention, the FGF5-v2 protein means its functional equivalent. The functional equivalent refers to a polypeptide having at least 50% or more, 70% or more, preferably 80% or more, and more preferably 90% or more sequence homology (i.e., identity) with the amino acid sequence constituting the FGF5-v2 protein (a preferred example is the amino acid sequence shown in SEQ ID NO: 1 in the sequence catalog). For example, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, It includes a polypeptide having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence homology, and refers to a polypeptide that exhibits substantially the same physiological activity as the parent FGF5-v2 protein (a preferred example is the polypeptide shown in SEQ ID NO: 1 in the sequence catalog). Here, "substantially the same physiological activity" preferably means a preventive, ameliorative or therapeutic effect on liver fibrosis. Preferably, in the present invention, the functional equivalent of the FGF5-v2 protein may be generated as a result of addition, substitution or deletion of a part of the amino acid sequence of SEQ ID NO: 1 in the sequence catalog. In the above, the amino acid substitution is preferably a conservative substitution. Examples of conservative substitutions of naturally occurring amino acids are as follows: aliphatic amino acids (Gly, Ala, Pro), hydrophobic amino acids (Ile, Leu, Val), aromatic amino acids (Phe, Tyr, Trp), acidic amino acids (Asp, Glu), basic amino acids (His, Lys, Arg, Gln, Asn), and sulfur-containing amino acids (Cys, Met).Furthermore, the functional equivalent of the FGF5-v2 protein also includes a variant in which a part of an amino acid is deleted on the amino acid sequence of the FGF5-v2 protein (i.e., a shorter functional fragment).The deletion or substitution of the amino acids is preferably located in a region that is not directly related to the physiological activity of the FGF5-v2 protein. That is, the deletion or substitution is performed within a range that does not affect the physiological activity of the FGF5-v2 protein. In addition, the present invention also includes variants in which several amino acids are added to both ends or within the amino acid sequence of the FGF5-v2 protein. The scope of the functional equivalent of the present invention also includes polypeptide derivatives in which a part of the chemical structure of the polypeptide is modified while maintaining the basic skeleton and physiological activity of the FGF5-v2 protein. For example, structural modifications to change the stability, storage, volatility, or solubility of the protein are included.

[0033] As used herein, sequence homology and identity are defined as the percentage of identical matching residues (amino acid residues or bases) of a candidate sequence to an original sequence (preferably SEQ ID NO:1 in the sequence catalog for amino acid sequences, or preferably SEQ ID NO:2 in the sequence catalog for nucleic acid sequences) after aligning the candidate sequence with the original sequence and introducing gaps. When necessary, conservative substitutions are not considered as part of sequence identity in order to obtain the maximum percentage sequence identity. In addition, in the case of determining homology or identity of protein sequences, N-terminal, C-terminal or internal extensions, deletions or insertions of the FGF5-v2 protein amino acid sequence are not to be construed as sequences that affect sequence identity or homology. In addition, the sequence identity can be determined by standard methods commonly used to compare similar portions of the amino acid sequences of two polypeptides. Computer programs such as BLAST or FASTA align two polypeptides for optimal amino acid matching (along the full length of one or two sequences, or along predicted portions of one or two sequences). The program provides a default opening penalty and a default gap penalty, and provides a scoring matrix such as PAM250 (a standard scoring matrix; Dayhoff et al., in Atlas of Protein Sequence and Structure, vol 5, supp 3, 1978) that can be used in conjunction with the computer program. For example, the percentage identity can be calculated as follows: multiply the total number of identical matches by 100, then divide by the length of the longer sequence in the matched span and the sum of the number of gaps introduced in the longer sequence to align the two sequences.

[0034] The present invention also provides an FGF5-v2 gene encoding the FGF5-v2 protein of the present invention. As used herein, the terms "nucleic acid", "gene sequence", "DNA sequence", "RNA sequence" or "polynucleotide" refer to deoxyribonucleotides or ribonucleotides in single or double stranded form. Unless otherwise specified, known analogs of natural nucleotides that hybridize to nucleic acids in a manner similar to naturally occurring nucleotides are also included.

[0035] In one embodiment of the present invention, the FGF5-v2 gene may be characterized by comprising the base sequence of SEQ ID NO: 2. In another embodiment of the present invention, the FGF5-v2 gene may consist of the base sequence of SEQ ID NO: 2.

[0036] In the present invention, the polynucleotide encoding the target protein may be modified in various ways in the coding region, taking into consideration the degeneracy of codons or the codons preferred by the organism in which the protein is to be expressed, without changing the amino acid sequence of the protein expressed from the coding region, and may be modified in various ways in the portion excluding the coding region, without affecting the expression of the gene, and such modified genes are also within the scope of the present invention, as will be well understood by those skilled in the art. That is, the polynucleotide of the present invention may be modified by substitution, deletion, insertion, or a combination of one or more nucleic acid bases, as long as it encodes a protein having an equivalent activity, and these are also within the scope of the present invention.

[0037] That is, a nucleic acid molecule (gene) represented by a specific sequence in this specification can include not only the sequence but also its biological equivalent. That is, considering a mutation having a biological equivalent activity of a nucleic acid molecule, a nucleic acid molecule in one embodiment is interpreted as including a sequence showing substantial identity with a sequence described in a sequence number in the sequence list. Specifically, a nucleic acid molecule comprising a nucleotide sequence represented by a specific sequence number is not limited to only the nucleotide sequence, and variants of the nucleotide sequence are included within the scope of the present invention. In the sequence list of the present invention, a nucleic acid molecule consisting of a nucleotide sequence represented by a specific sequence number is a concept including functional equivalents of the nucleic acid molecule constituting it, for example, variants in which a part of the nucleotide sequence of the nucleic acid molecule is modified by deletion, substitution, or insertion, but which can function identically to the nucleic acid molecule. Specifically, the nucleic acid molecules disclosed in the present invention may include nucleotide sequences having sequence identity of 70% or more, more preferably 80% or more, even more preferably 90% or more, and most preferably 95% or more with the amino acid sequences shown in the sequence listing by specific SEQ ID NOs. For example, the nucleic acid molecules may include those having sequence identity of 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100%. "Percent sequence identity" for nucleic acid molecules is determined by comparing a comparison region to two optimally aligned sequences, and a portion of the nucleotide sequence in the comparison region can contain additions or deletions (i.e., gaps) compared to the reference sequence (which does not contain additions or deletions) for the optimal alignment of the two sequences.

[0038] The gene (nucleic acid molecule), its transcript, and / or its protein of the FGF5-v2 variant of the present invention can be directly delivered to an individual through various carriers (transmitters) or by itself. The carrier is not limited to a specific type, as long as it delivers the gene, transcript, and / or protein of the FGF5-v2 to a host cell or other target body, and allows the gene to be expressed in the cell or the transcript or protein to exhibit its inherent physiological activity (liver fibrosis suppression activity). For example, as a carrier for delivering the FGF5-v2 variant, an expression vector, a virus (virus particle), vesicles, nanoparticles, microparticles, liposomes, transposons, micelles, antibodies, exosomes, etc. can be used, but are not limited thereto, and gene, transcript, and protein delivery methods known in the art can be applied without limitation. In addition, the protein can be directly delivered together with a storage material.

[0039] For example, the present invention provides an expression vector (recombinant expression vector) comprising the FGF5-v2 gene (i.e., a nucleic acid molecule encoding an FGF5-v2 protein) according to the present invention. That is, the present invention provides an expression vector (recombinant expression vector) comprising a promoter; and an FGF5-v2 gene (FGF5-v2 polynucleotide) operably linked thereto.

[0040] In the present invention, the term "expression" refers to the production of a protein or a nucleic acid in a cell. In the present invention, the term "recombinant vector" refers to a vector capable of expressing a peptide or protein encoded by a heterologous nucleic acid inserted into the vector, and preferably refers to a vector manufactured to be capable of expressing a target protein (FGF5-v2 protein in the present invention). The term "vector" refers to any medium for introducing and / or transferring bases into a host cell in vitro, ex vivo, or in vivo, and may be a replicating unit to which other DNA fragments are bound and which results in replication of the bound fragments, and the "replicating unit" refers to any genetic unit (e.g., plasmid, phage, cosmid, chromosome, virus, etc.) that functions as an independent unit of DNA replication in vivo, i.e., that can replicate under its own control.

[0041] The vector according to the present invention may be, but is not limited to, linear DNA, plasmid DNA or recombinant viral vector. The vector includes, for example, plasmid vector, cosmid vector and viral vector, such as bacteriophage vector, adenovirus vector, lentivirus vector, retrovirus vector and adeno-associated virus vector. Preferably, the vector is an adenovirus vector. The present inventors used pADTrack vector in the specific embodiment.

[0042] The term "promoter" refers to a DNA sequence that regulates the expression of a nucleic acid sequence operably linked to it in a particular host cell. In addition, it may further include an optional operator sequence for regulating transcription, a sequence encoding an appropriate mRNA ribosome binding site, and a sequence regulating the termination of transcription and translation. The promoter may be a constitutive promoter that constantly induces the expression of a target gene at all times, or an inducible promoter that induces the expression of a target gene at a specific position or time, and examples thereof include SV40 promoter, CMV promoter, CAG promoter, CaMV 35S promoter, Rsyn7 promoter (U.S. Patent Application No. 08 / 991,601), rice actin promoter, ubiquitin promoter, ALS promoter (U.S. Patent Application No. 08 / 409,297), etc. In addition, any of the promoters disclosed in U.S. Patent Nos. 5,608,149; 5,608,144, 5,604,121, 5,569,597, 5,466,785, 5,399,680, 5,268,463 and 5,608,142 may be used.

[0043] In addition to the promoter, the recombinant vector of the present invention may include an operator sequence for regulating transcription, a sequence encoding an appropriate mRNA ribosome binding site, a sequence for regulating the termination of transcription and translation, a terminator, etc., and more preferably may further include a polyhistidine tag (an amino acid motif consisting of at least 5 or more histidine residues), a signal peptide gene, an endoplasmic reticulum retention signal peptide, a cloning site, etc., and may further include a selection marker gene such as a tag gene and an antibiotic resistance gene for selecting transformants. In the recombinant vector, the polynucleotide sequence of each gene is operably linked to a promoter. As used herein, the term "operatively linked" refers to a functional bond between a nucleotide expression regulatory sequence such as a promoter sequence and a different nucleotide sequence, whereby the regulatory sequence regulates the transcription and / or translation of the other nucleotide sequence.

[0044] The recombinant vector can be constructed using a prokaryotic or eukaryotic cell as a host. For example, when the vector of the present invention is an expression vector and a prokaryotic cell is used as a host, it generally contains a strong promoter (e.g., pLλ promoter, trp promoter, lac promoter, tac promoter, T7 promoter, etc.) for driving transcription, a ribosome binding site for initiating decoding, and a transcription / decoding termination sequence. When a eukaryotic cell is used as a host, the replication origin that the vector operates in the eukaryotic cell may include, but is not limited to, an f1 replication origin, an SV40 replication origin, a pMB1 replication origin, an adeno replication origin, an AAV replication origin, and a BBV replication origin. In addition, a promoter derived from the genome of a mammalian cell (e.g., a metallothionine promoter) or a promoter derived from a mammalian virus (e.g., an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a cytomegalovirus promoter, and an HSV tk promoter) is used, and generally has a polyadenylation sequence as a transcription termination sequence.

[0045] Representative examples of the tagging gene include Avi tag, Calmodulin tag, polyglutamate tag, E tag, FLAG tag, HA tag, His tag (polyhistidine tag), Myc tag, S tag, SBP tag, IgG-Fc tag, CTB tag, Softag 1 tag, Softag 3 tag, Strep tag, TC tag, V5 tag, VSV tag, Xpress tag, etc. Preferably, the vector according to the present invention may include a myc tag.

[0046] Meanwhile, the expression vector can be introduced into target cells (host cells) as an expression type by methods known in the art, such as infection, transfection, or transduction.

[0047] The gene transfer method using a plasmid expression vector is a method for directly transferring plasmid DNA into mammalian cells, and is a method approved by the FDA for use in humans. Plasmid DNA has the advantage of being homogeneously purified, unlike viral vectors. As the plasmid expression vector that can be used in the present invention, any mammalian expression plasmid known in the art can be used. Representative examples include, but are not limited to, pRK5 (European Patent No. 307,247), pSV16B (International Patent Publication No. 91 / 08291), and pVL1392 (PharMingen). The plasmid expression vector can be introduced into target cells by methods known in the art, including, but not limited to, transient transfection, microinjection, transduction, cell fusion, calcium phosphate precipitation, liposome-mediated transfection, DEAE Dextran-mediated transfection, polybrene-mediated transfection, electroporation, gene gun, and other known methods for injecting DNA into cells.

[0048] In addition, as a method applicable to the present invention, viral expression vectors containing the nucleic acid include, but are not limited to, retroviruses, adenoviruses, herpes viruses, avipox viruses, lenti viruses, etc. The retroviral vector is constructed so that the viral genes are completely deleted or modified so that non-viral proteins are produced in cells infected with the viral vector. The main advantages of retroviral vectors for gene therapy are that they deliver a large amount of genes into replicating cells, accurately integrate the delivered genes into cellular DNA, and do not cause continuous infection after gene transfection. The retroviral vector approved by the FDA is produced using PA317 amphotropic retrovirus packaging cells. An example of a non-retroviral vector is the adenovirus mentioned above. The main advantages of adenovirus are that it can carry a large amount of DNA fragments (36 kb genome) and has the ability to infect non-replicating cells with a very high titer. Herpes viruses may also be usefully employed for human gene therapy.Other than these, any suitable viral vector known in the art may be used in the present invention.

[0049] When the expression vector is a viral recombinant vector, the vector can be transfected into a virus-producing cell, i.e., a packaging cell line. To "transfect" or "transfect", several different techniques that are commonly used to introduce exogenous nucleic acid (DNA or RNA) into prokaryotic or eukaryotic host cells, such as electrophoresis, calcium phosphate precipitation, DEAE-dextran transfection, or lipofection, can be used. The virus containing the target gene (FGF5-v2 gene) according to the present invention is propagated in the packaging cell line and released outside the cell, and the virus can be transduced into a target cell. The nucleic acid of the virus "transduced" into the cell is either inserted into the genome of the cell or is used to produce the target transcript or protein (FGF5-v2 transcript or protein) without being inserted.

[0050] Also, an isolated cell into which the expression vector according to the present invention has been introduced (by transformation, transfection, transduction, etc.) can be provided. The cell refers to a cell for propagating (amplifying) the expression vector. That is, the cell refers to a host cell directly transduced / transformed / transfected with the above-mentioned nucleic acid molecule or expression vector. For example, when the expression vector is a viral vector, the cell may be a packaging cell for producing a virus containing the viral vector. Selection of an appropriate host is believed to be obvious to one of ordinary skill in the art from the teachings of the present application.

[0051] In order to achieve another object of the present invention, the present invention provides a pharmaceutical composition for preventing or treating liver fibrosis (liver fibrosis), comprising an FGF5-v2 transcript, an FGF5-v2 protein, an expression vector comprising a nucleic acid molecule encoding the FGF5-v2 protein according to the present invention; and / or an isolated cell into which the expression vector has been introduced as an active ingredient. The pharmaceutical composition may contain various carriers for delivering the FGF5-v2 gene, transcript, and / or protein of the present invention to a target cell in addition to the above-mentioned components. That is, the pharmaceutical composition may contain the FGF5-v2 gene, transcript, and / or protein supported by a carrier. A detailed description of the carrier is omitted here, as it has been described above.

[0052] In the present invention, "liver fibrosis" or "liver fibrotic disease" refers to a disease in which fibrosis accumulates due to continuous damage to liver tissue caused by various causes, resulting in damage to the liver's morphology and function. The term "liver fibrosis" is used interchangeably with "liver fibrosis." Fibrosis is a phenomenon in which excessive fibrous connective tissue is formed in organs and tissues during the wound recovery process against repeated damage to cells and tissues. Continuous accumulation of liver fibrosis can cause various liver diseases, including liver cancer and cirrhosis, and thus the development of serious diseases can be prevented and liver dysfunction can be treated by suppressing and improving liver fibrosis. The present inventors confirmed that the FGF5-v2 of the present invention effectively suppresses fibrosis in liver tissue in an animal model of liver disease and suppresses the expression of cytokines related to fibrosis and inflammation, thereby improving the disease.

[0053] In one embodiment of the present invention, the liver fibrosis includes, but is not limited to, alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis C, hepatitis B, autoimmune hepatitis, hepatic encephalopathy, primary biliary sclerosis, liver cancer, and hepatocellular carcinoma, as well as other liver fibrosis or chronic liver diseases of unknown or unclear cause, and may include, without limitation, any disease that is directly or indirectly caused by liver fibrosis or that is accompanied by liver fibrosis.

[0054] In one embodiment of the present invention, the non-alcoholic fatty liver disease may be any one or more selected from the group consisting of non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), hepatic steatosis, Acute Fatty Liver of Pregnancy (AFLP), NAFLD-related liver failure, NAFLD-related liver fibrosis, NAFLD-related cirrhosis, and NAFLD-related liver cancer, but is not limited thereto.

[0055] In another embodiment of the present invention, the pharmaceutical composition of the present invention (i.e., FGF5-v2 protein or its expression vector, etc.) is characterized by being administered in combination with an anti-inflammatory agent, but is not limited thereto. That is, FGF5-v2 of the present invention can achieve a more excellent anti-fibrotic effect by being used in combination with an existing anti-inflammatory agent.

[0056] As used herein, the term "combination administration" can be carried out by administering the individual components of the treatment method simultaneously, sequentially, or separately. It is to obtain a combined therapeutic effect by methods such as simultaneously or sequentially administering two or more drugs, or administering them alternately at regular or irregular intervals. The combination therapy is not limited thereto. For example, the efficacy measured through the degree of response, reaction rate, period until disease progression, or survival period is therapeutically superior to the efficacy obtained by administering one or the remaining components of the combination therapy at normal doses, but can provide a synergistic effect.

[0057] The pharmaceutical composition of the present invention can be administered simultaneously, separately, or sequentially with an anti-inflammatory agent. When administered sequentially with an anti-inflammatory agent, the order of administration is not limited, but the administration treatment method can be appropriately adjusted according to the type of anti-inflammatory agent, the patient's condition, the patient's gender, age, etc.

[0058] The present invention also provides a pharmaceutical composition for preventing or treating liver fibrosis, comprising as active ingredients: (i) an expression vector containing the FGF5-v2 protein of the present invention, a nucleic acid molecule encoding the protein, or an isolated cell into which the expression vector has been introduced; and (ii) an anti-inflammatory agent.

[0059] The composition may be in the form of a mixture in which the (i) FGF5-v2 protein and the (ii) anti-inflammatory agent are mixed, and may be in the form for simultaneous administration of the (i) FGF5-v2 protein and the (ii) anti-inflammatory agent. For example, the (i) FGF5-v2 protein and the (ii) anti-inflammatory agent may be in a single dosage form prepared by mixing with a pharma- ceutically acceptable adjuvant, diluent, or carrier. In this case, the (i) FGF5-v2 protein and the (ii) anti-inflammatory agent may each include various components within the category of the drug. In addition, the single dosage form is a dosage form separate from the above dosage forms, and may be administered together with a separate formulation containing another therapeutic agent.

[0060] Alternatively, the composition may be in a form in which (i) FGF5-v2 protein and (ii) anti-inflammatory agent are each formulated and administered simultaneously, separately, or sequentially. In that case, the composition may be a pharmaceutical composition for combined administration for simultaneous or sequential administration, comprising a first pharmaceutical composition containing a pharmacologic effective amount of the (i) FGF5-v2 protein as an active ingredient; and a second pharmaceutical composition containing a pharmacologic effective amount of the (ii) anti-inflammatory agent as an active ingredient.

[0061] The anti-inflammatory agent according to the present invention may be a steroidal anti-inflammatory agent and / or a non-steroidal anti-inflammatory agent, which may be selected from salicylic acid, acetic acid, propionic acid, mefenamic acid, oxicam series or non-acid series, and specific examples thereof include, but are not limited to, aspirin, ibuprofen, naproxen, ketoprofen, and fenoprofen.

[0062] The present invention also provides a kit for preventing or treating liver fibrosis, comprising the pharmaceutical composition according to the present invention. The kit according to the present invention is not limited to a specific form as long as it is for preventing or treating liver fibrosis, and may include any components and devices for producing, storing, expressing, administering, etc., of the FGF5-v2 gene, transcript, protein, and expression vector according to the present invention, without limitation.

[0063] The content of the FGF5-v2 gene, its transcript, its protein, and / or expression vector in the composition of the present invention can be appropriately adjusted depending on the symptoms of the disease, the degree of progression of the symptoms, the condition of the patient, etc., and may be, for example, 0.0001 to 99.9% by weight or 0.001 to 50% by weight based on the weight of the total composition, but is not limited thereto. The content ratio is a value based on the dry amount after removing the solvent.

[0064] The pharmaceutical composition according to the present invention may further comprise suitable carriers, excipients and diluents commonly used in the manufacture of pharmaceutical compositions, such as one or more selected from the group consisting of diluents, binders, disintegrants, lubricants, adsorbents, moisturizers, film coating materials and controlled release additives.

[0065] The pharmaceutical composition according to the present invention can be formulated in the form of a powder, granule, sustained release granule, enteric granule, liquid, eye drops, elixir, emulsion, suspension, spirit, troche, aromatic perfume, lemonade, tablet, sustained release tablet, enteric tablet, sublingual tablet, hard capsule, soft capsule, sustained release capsule, enteric capsule, pill, tincture, soft extract, dry extract, fluid extract, injection, capsule, perfusion solution, plaster, lotion, paste, spray, inhalant, patch, sterile injection solution, or external preparation such as aerosol, according to a conventional method, and the external preparation may have the form of a cream, gel, patch, spray, ointment, plaster, lotion, liniment, paste, or cataplasm.

[0066] Carriers, excipients and diluents that may be included in pharmaceutical compositions according to the invention include lactose, dextrose, sucrose, oligosaccharides, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

[0067] When the formulation is made, it is prepared using a diluent or excipient such as a commonly used filler, extender, binder, wetting agent, disintegrant, surfactant, etc. Additives for the tablets, powders, granules, capsules, pills and lozenges according to the present invention include corn starch, potato starch, wheat starch, lactose, sucrose, glucose, fructose, D-mannitol, precipitated calcium carbonate, synthetic aluminum silicate, calcium hydrogen phosphate, calcium sulfate, sodium chloride, sodium bicarbonate, purified lanolin, microcrystalline cellulose, dextrin, sodium alginate, methylcellulose, sodium carboxymethylcellulose, kaolin, urea, colloidal silica gel, hydroxypropyl starch, hydroxypropylmethylcellulose (HPMC) 1928, HPMC 2208, HPMC 2906, HPMC 2910, propylene glycol, casein, calcium lactate, Primogel and other excipients; gelatin, gum arabic, ethanol, agar powder, cellulose acetate phthalate, carboxymethylcellulose, carboxymethylcellulose calcium, glucose, purified water, sodium caseinate, glycerin, stearic acid, sodium carboxymethylcellulose, sodium methylcellulose, methylcellulose, microcrystalline cellulose, dextrin, hydroxycellulose, hydroxypropyl starch, hydroxymethylcellulose, purified shellac, starch paste, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinyl alcohol, polyvinylpyrrolidone and other binders can be used; hydroxypropylmethylcellulose, corn starch, agar powder, methylcellulose, bentonite, hydroxypropyl starch, sodium carboxymethylcellulose, sodium alginate, carboxymethylcellulose calcium, calcium citrate, sodium lauryl sulfate, anhydrous silicic acid, 1-hydroxypropylcellulose, dextran, ion exchange resin, polyvinyl acetate, formaldehyde-treated casein and gelatin, alginic acid, amylose, guar gum (Guar gum), sodium bicarbonate, polyvinylpyrrolidone, calcium phosphate, gelling starch, gum arabic, amylopectin, pectin, sodium polyphosphate, ethyl cellulose, white sugar, magnesium aluminum silicate, D-sorbitol liquid, light anhydrous silicic acid, and other disintegrants;Lubricants such as calcium stearate, magnesium stearate, stearic acid, hydrogenated vegetable oil, talc, lycopodium, kaolin, petrolatum, sodium stearate, cocoa butter, sodium salicylate, magnesium salicylate, polyethylene glycol (PEG) 4000, PEG 6000, liquid paraffin, hydrogenated soybean oil (Lubri wax), aluminum stearate, zinc stearate, sodium lauryl sulfate, magnesium oxide, macrogol, synthetic aluminum silicate, silicic anhydride, higher fatty acids, higher alcohols, silicone oil, paraffin oil, polyethylene glycol fatty acid ether, starch, sodium chloride, sodium acetate, sodium oleate, dl-leucine, and light silicic anhydride can be used.

[0068] Examples of additives that can be used in the liquid preparation according to the present invention include water, dilute hydrochloric acid, dilute sulfuric acid, sodium citrate, sucrose monostearate, polyoxyethylene sorbitol fatty acid esters (twin esters), polyoxyethylene monoalkyl ethers, lanolin ethers, lanolin esters, acetic acid, hydrochloric acid, aqueous ammonia, ammonium carbonate, potassium hydroxide, sodium hydroxide, prolamine, polyvinylpyrrolidone, ethyl cellulose, and sodium carboxymethylcellulose.

[0069] The syrup according to the present invention may contain a solution of sucrose, other sugars or sweeteners, and may contain, if necessary, flavorings, coloring agents, preservatives, stabilizers, suspending agents, emulsifiers, thickening agents, etc.

[0070] In the emulsion of the present invention, purified water can be used, and emulsifiers, preservatives, stabilizers, fragrances, etc. can be used as necessary. The suspension according to the present invention can contain suspending agents such as acacia, tragacanth, methylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, microcrystalline cellulose, sodium alginate, hydroxypropylmethylcellulose (HPMC), HPMC 1828, HPMC 2906, and HPMC 2910, and can contain surfactants, preservatives, stabilizers, coloring agents, and fragrances, as necessary.

[0071] Injections according to the present invention may contain solvents such as distilled water for injection, 0.9% sodium chloride injection, Ringer's injection, dextrose injection, dextrose + sodium chloride injection, PEG, lactated Ringer's injection, ethanol, propylene glycol, non-volatile oils - sesame oil, cottonseed oil, peanut oil, soybean oil, corn oil, ethyl oleate, isopropyl myristate, benzene benzoate; solubilizing agents such as sodium benzoate, sodium salicylate, sodium acetate, urea, urethane, monoethylacetamide, butazolidine, propylene glycol, teulenes, nicotinamide, hexamine, dimethylacetamide; buffers such as weak acids and their salts (acetic acid and sodium acetate), weak bases and their salts (ammonia and ammonium acetate), organic compounds, proteins, albumin, peptones, gums; isotonicity agents such as sodium chloride; sodium bisulfite (NaHSO 3 ) Carbon dioxide gas, sodium metabisulfite (Na 2 S 2 O 5 ), Sodium sulfite (Na 2 SO 3 ), nitrogen gas (N 2 ), stabilizers such as ethylenediaminetetraacetic acid; sulfating agents such as sodium bisulfite 0.1%, sodium formaldehyde sulfoxylate, thiourea, disodium ethylenediaminetetraacetate, and acetone sodium bisulfite; soothing agents such as benzyl alcohol, chlorobutanol, procaine hydrochloride, glucose, and calcium gluconate; and suspending agents such as sodium CMC, sodium alginate, Tween 80, and aluminum monostearate.

[0072] Suppositories according to the invention may contain any of the following ingredients: cocoa butter, lanolin, witepsol, polyethylene glycol, glycerogelatin, methylcellulose, carboxymethylcellulose, mixture of stearic and oleic acids, Subanal, cottonseed oil, peanut oil, coconut oil, cocoa butter + cholesterol, lecithin, lanet wax, glycerol monostearate, Tween or Span, Imhausen, Monolen (propylene glycol monostearate), glycerin, Adeps solidus, Buytyrum Tego-G, Cebes Pharma 16, Hexalide Base 95, Cotomar, Hydrokote SP, S-70-XXA, S-70-XX75 (S-70-XX95), Hydrokote. Bases such as 25, Hydrocote 711, Idropostal, Massa estrarium (A, AS, B, C, D, E, I, T), Massa-MF, Maspol, Maspol-15, Neospostal-en, Paramound-B, Sposil (OSI, OSIX, A, B, C, D, H, L), Suppository Base Type IV (AB, B, A, BC, BBG, E, BGF, C, D, 299), Spostal (N, Es), Wecoby (W, R, S, M, Fs), and Tegestar triglyceride base (TG-95, MA, 57) can be used.

[0073] Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and such solid preparations are prepared by mixing the extract with at least one or more excipients, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate talc are also used.

[0074] Liquid preparations for oral administration include suspensions, liquid preparations for internal use, emulsions, syrups, etc., and may contain various excipients such as wetting agents, sweeteners, flavorings, preservatives, etc. in addition to water and liquid paraffin, which are frequently used simple diluents. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Non-aqueous solvents and suspensions that can be used include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate.

[0075] The pharmaceutical composition according to the present invention is administered in a pharmacologic effective amount. In the present invention, the term "pharmacologic effective amount" refers to an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to any medical treatment, and the effective dose level can be determined by factors including the type and severity of the patient's disease, the activity of the drug, sensitivity to the drug, administration time, administration route and excretion rate, treatment duration, concurrently used drugs, and other factors well known in the medical field.

[0076] The pharmaceutical composition according to the present invention can be administered as an individual therapeutic agent or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with conventional therapeutic agents, and can be administered singly or in multiple doses. It is important to administer an amount that can obtain maximum effect with minimum amount without side effects, taking into consideration all of the above factors, and this can be easily determined by a person skilled in the art to which the present invention pertains.

[0077] The pharmaceutical compositions of the present invention may be administered to an individual by a variety of routes, including, but not limited to, oral ingestion, subcutaneous injection, intraperitoneal administration, intravenous injection, intramuscular injection, paraspinal space (intradural) injection, sublingual administration, buccal administration, rectal insertion, vaginal insertion, ocular administration, otic administration, nasal administration, inhalation, spraying through the mouth or nose, dermal administration, transdermal administration, and the like, although all manners of administration are foreseeable.

[0078] The pharmaceutical composition of the present invention is determined by the type of drug as an active ingredient, along with various related factors such as the disease to be treated, the administration route, the age, sex, weight, and severity of the disease of the patient. Specifically, the effective amount of the composition according to the present invention varies depending on the age, sex, and weight of the patient, and can generally be administered at 0.001 to 150 mg, preferably 0.01 to 100 mg per kg of body weight every day or every other day, or administered in 1 to 3 divided doses per day. However, since the amount can be increased or decreased depending on the administration route, severity of the disease, sex, weight, age, etc., the above-mentioned dosage does not limit the scope of the present invention in any way.

[0079] In the present invention, the term "individual" refers to a subject in need of treatment for a disease, and more specifically, refers to mammals such as human or non-human primates, mice, rats, dogs, cats, horses, and cows.

[0080] In the present invention, "administration" means providing a given composition of the present invention to an individual by any suitable method. In the present invention, "prevention" means any action to suppress or delay the onset of a target disease, "treatment" means any action to improve or beneficially modify a target disease and its associated metabolic abnormality symptoms by administration of a pharmaceutical composition according to the present invention, and "amelioration" means any action to reduce a parameter related to a target disease, for example, the severity of symptoms, by administration of a composition according to the present invention.

[0081] In the following, preferred examples and experimental examples are presented to aid in understanding the present invention. However, the following examples and experimental examples are merely provided to facilitate understanding of the present invention, and the contents of the present invention are not limited to the following examples and experimental examples.

[0082] Example 1. Production of FGF5-v2 (FGF5 transcript variant 2) and preparation for gene injection The pAdTrack-CMV vector was digested with the restriction enzymes KpnI and HindII, and the gene was synthesized using the PN 5'-AAAGGTACCATGAGCTTGTCCTTCCTCCTC-3' primer (sequence number 19 in the sequence catalog) and 5'-AAAAAGCTTTCACTTATCGTCGTCATCCTTGTAATCTCTGTGAACTTGGCTTAACATATTGGCTTCGT-' primer (sequence number 20 in the sequence catalog), which correspond to FGF5 variant 2, and then ligated after cleavage with the restriction enzymes KpnI and HindII. The recombinant vector (pAdTrack-CMV hFGF5-2) was transformed into E. coli and amplified, and the pAdTrack-CMV hFGF5-2 vector and pAdEASY vector cut with PmeI restriction enzyme were transformed into E. coli BJ5183 cells, and transformed E. coli BJ5183 cells-hFGF5-2 expressing hFGF5-2 were selected. After that, the target gene was isolated and adenovirus hFGF5-2 was produced using 293T cells.

[0083] Example 2. Creation of in vivo diet-induced non-alcoholic fatty liver disease (NAFLD) as a liver fibrosis model and gene injection Specific pathogen-free male C57BL / 6J mice (6 weeks old) were purchased from DBL (Chungcheong-do, Korea) and adapted to the housing environment 1 week before the start of treatment. At 7 weeks of age, mice were randomly divided into two groups. The normal group (n = 8) was fed a commercial standard diet, and the liver fibrosis group (n = 24) was fed a CDAA-HFD (A06071302, Research Diets, NJ, USA) for 6 weeks. CDAA-HFD is a high-fat diet containing L-amino acids and methionine that is deficient in choline, and is used to induce NAFLD (nonalcoholic fatty liver disease; simple fatty liver, steatohepatitis, and liver fibrosis (nonalcoholic fatty liver-associated cirrhosis).

[0084] The liver fibrosis group was further divided into an empty vector administration group, a full-length FGF5 (also called FGF5-F; FGF5 full sequence) administration group, and an FGF5 mutant FGF5-v2 (also called FGF5-S) administration group for the experiment. For this purpose, 1×10 9 Each mouse was injected once through the tail vein of the mouse. The normal mouse group that was not fed a high-fat diet was administered PBS. After 12 days, all mice were subjected to a glucose tolerance test, and after 2 days, the animals were euthanized by CO 2 All mice were weighed weekly. Mice were housed in a temperature (23 ± 3 °C) and relative humidity (40-60%) controlled chamber under specific pathogen-free conditions.

[0085] Example 3. Confirmation of the hepatic fibrosis suppression effect of FGF5-v2 gene injection in an in vivo NAFLD mouse model As described above, normal mice or NAFLD mice were treated with an empty vector (control), full-length FGF5 (FGF5-F), or FGF5-v2 (FGF5-S), and a glucose tolerance test (GTT) was performed. Liver tissues were then collected from each mouse, and fatty liver indicators, NAS score and fibrosis score, were measured. Specifically, the degree of tissue steatosis and fibrosis was evaluated by a pathologist looking at a photograph of stained mouse tissue and scoring it. Steatosis was scored numerically according to quantitative pathological standards (steatosis, inflammation, and hepatocyte swelling). Fibrosis was evaluated by staining mouse liver tissue fragments with Sirius Red, observing them, and scoring them into F0 stage (no fibrosis), F1 stage (mild), F2 stage (moderate), F3 stage (bridging fibrosis), and F4 stage (cirrhosis).

[0086] First, the results of steatosis evaluation are shown in FIG. 3a. NAS score, an index of fatty liver, was significantly increased in NAFLD mice fed CDAA-HFD compared to mice fed normal diet, and no significant difference in NAS score was observed between the untreated control group, the FGF5-F treatment group, and the FGF5-S treatment group among NAFLD mice. Meanwhile, as a result of comparing the degree of fibrosis, as shown in FIG. 3b, the degree of fibrosis significantly increased by CDAA-HFD diet was not significantly different from the untreated control group when treated with full-length FGF5-F, but the degree of fibrosis was greatly reduced when treated with FGF5-v2, the FGF5 transcript variant of the present invention. From the above results, it was confirmed that FGF5-v2 has a specific and significant improving effect on liver fibrosis occurring in fatty liver, rather than on fatty liver itself.

[0087] Example 4. Confirmation of suppression of non-alcoholic fatty liver fibrosis in serum of an in vivo NAFLD mouse model by FGF5-v2 gene injection In this example, the hepatic fibrosis-suppressing effect of the FGF5 transcript mutant of the present invention was confirmed through serum analysis of a mouse model. Specifically, the serum levels of biomarkers indicating the degree of fatty liver fibrosis were quantified.

[0088] Specifically, mice were anesthetized with isoflurane using a respiratory anesthesia machine, then euthanized, and blood was collected by cardiac puncture. The collected blood was centrifuged at 4,000 rpm for 30 minutes at 4°C, and serum was collected and stored at -80°C for further analysis. The levels of all metabolic factors present in serum (e.g., AST, ALT, TG, HDL, LDL, and glucose) were measured in the serum samples obtained above using an Advia 1800 Chemistry System according to the manufacturer's protocol (Siemens Healthcare, Erlangen, Germany).

[0089] As a result, no significant difference was observed in HDL and LDL between the untreated control group and the full-length FGF5 or FGF5-v2 treated groups (Figures 4e and 4f), but the analysis results of AST and ALT, which are used as hepatocyte damage markers in fatty liver disease, showed that the levels of these were significantly reduced in the FGF5-v2 treated group compared to the untreated control group and the full-length FGF5 treated group (Figures 4a to 4c). In addition, it was confirmed that triglyceride (TG) and glucose were significantly reduced in the FGF5-v2 treated group compared to the other groups (Figures 4d and 4g). The above results indicate that the FGF5-v2 of the present invention has a particularly excellent effect on suppressing liver fibrosis in diseases related to non-alcoholic fatty liver.

[0090] Example 5. Confirmation of suppression of liver fibrosis markers in liver tissue mRNA of an in vivo NAFLD mouse model by FGF5-v2 gene injection In this example, the mRNA levels of fibrosis-related factors were analyzed in the liver tissue of each mouse model, and the hepatic fibrosis-suppressing effect of the FGF5 transcript mutant of the present invention was confirmed at the RNA level.

[0091] RNA was isolated using the RNeasy Mini Kit (QIAGEN, Hilden, Germany) according to the manufacturer's protocol. RNA quality and total RNA yield, 260 / 280 and 260 / 230 ratios were measured using a NanoDrop spectrophotometer (Thermo, MA, USA). Total RNA was purified using PrimeScript RT-PCR according to the manufacturer's protocol (Takara, Shiga, Japan). TMThe complementary DNA was reverse transcribed using the 1st strand cDNA Synthesis Kit. Quantification of the complementary DNA template was performed by real-time PCR using SYBR green fluorescence (Takara) on a CFX384 instrument (Bio-Rad, CA, USA). Primer information is as follows: mα-SMA forward 5'-TGC TGA CAG AGG CAC CAC TGA A-3' and reverse 5'-CAG TTG TAC GTC CAG AGG CAT AG-3'; mCol1α1 forward 5'-CCT CAG GGT ATT GCT GGA CAA C-3' and reverse 5'-CAG AAG GAC CTT GTT TGC CAG G-3'; mFibronectin forward 5'-CCC TAT CTC TGA TAC CGT TGT CC-3' and reverse 5'- TGC CGC AAC TAC TGT GAT TCG G-3'; mVimentin forward 5'-CGG AAA GTG GAA TCC TTG CAG G-3' and reverse 5' AGC AGT GAG GTC AGG CTT GGA A-3'; mHPRT forward 5' CTG GTG AAA AGG ACC TCT CGA AG-3' and reverse 5'-CCA GTT TCA CTA ATG ACA CAA ACG-3'.

[0092] As a result, it was shown that α-SMA and Col1α1, which are major markers of fibrosis, were significantly decreased in the FGF5-v2-treated group compared to the control group and the FGF5(Full)-treated group (Figures 5a and 5b). In the case of fibronectin and vimentin, which are also known as EMT markers, the FGF5(Full)-treated group showed increased levels compared to the untreated control group, while the FGF5-v2-treated group showed similar levels to the control group (Figures 5c and 5d). The above results indicate that the FGF5 transcript variant of the present invention can suppress the expression of liver fibrosis-related factors and suppress liver fibrosis in non-alcoholic fatty liver disease. On the other hand, when the expression of full-length FGF5 and FGF5-v2 transcripts was measured in each mouse model into which the full-length FGF5 or FGF5-v2 gene was introduced, it was confirmed that in the full-length FGF5 gene-injected group, no FGF5-v2 transcript was detected and only full-length FGF5 was detected, whereas in the FGF5-v2 gene-injected group, no full-length FGF5 transcript was detected and only FGF5-v2 was detected (Figures 5e and 5f).

[0093] Example 6. Confirmation of suppression of liver fibrosis at the liver tissue protein level by FGF5-v2 gene injection in an in vivo NAFLD mouse model In this example, the protein levels of fibrosis-related factors in the liver tissue of each mouse model were analyzed by ELISA and Western blot, and the hepatic fibrosis-suppressing effect of the FGF5 transcript mutant of the present invention was confirmed at the protein level.

[0094] Specifically, liver tissues isolated from mice were dissolved in T-PER buffer (Thermo, MA, USA) supplemented with PhosSTOP and protease inhibitors (Roche, Basel, Switzerland) and centrifuged at 15,000 rpm for 10 min at 4°C. Protein concentrations were quantified by BCA analysis (Thermo). The amounts of IL-6 and IL-1β (R&D systems, MA, USA) were measured using ELISA experiments against tissue proteins at a fixed concentration, or through immunoblot experiments using SDS-PAGE. Antibodies such as α-SMA (abcam, Cambridge, UK) and β-actin (Cell signaling, MA, USA) were used.

[0095] As a result, the inflammatory markers IL-1β and IL-6 showed no significant difference in the FGF5-v2 treated group compared to the control (see Figures 6a and 6b), but the fibrosis marker α-SMA was significantly reduced in the FGF5-v2 injected mouse model compared to the other groups (Figure 6c). These results indicate that administration of the FGF5 transcript variant of the present invention is particularly effective in reducing liver fibrosis compared to improving inflammation in a mouse model of non-alcoholic fatty liver.

[0096] Example 7. Confirmation of cytokine and chemokine protein expression in serum and liver tissue of an in vivo NAFLD mouse model injected with FGF5-v2 gene In this example, the levels of cytokines and chemokines were analyzed in the serum and liver tissue of each mouse model, and the therapeutic effect of administration of the FGF5 transcript mutant of the present invention was confirmed.

[0097] Specifically, serum was obtained from each mouse as described in Example 4, and liver tissue fractions were obtained in the same manner as in Example 6. Cytokine and chemokine protein expression levels were measured using MILLIPLEX MAP Mouse Cytokine / Chemokine Kit (R&D systems). Fluorescence intensity was read using MAGPIX® (Luminex Corporation, Austin, TX).

[0098] As a result, as shown in Figure 7a, the levels of cytokines and chemokines (CCL2, CCL5, CCL11, and CXCL2) secreted by hepatic stellate cells, the main cells associated with liver fibrosis, were significantly reduced in NAFLD mice administered FGF5-v2 compared to other NAFLD mouse groups. In addition, it was confirmed that CXCL1 and fibroblast growth factor 21 (FGF21), which are involved in fibrosis, were also significantly reduced in the FGF5-v2-administered group. Furthermore, the FGF5-v2-administered group showed higher serum levels of IL-10, an anti-inflammatory cytokine, compared to other groups.

[0099] As shown in Figure 7b, the expression of CCL2, CCL5, CCL11, and CXCL2 was decreased in the FGF5-v2-treated group compared to the other groups at the protein level in liver tissue, and the level of fibroblast growth factor 21 (FGF21) was also significantly decreased.

[0100] The above results indicate that the FGF5 transcript variant of the present invention can improve and treat liver fibrosis by suppressing the production and secretion of cytokines and chemokines that promote fibrosis in non-alcoholic fatty liver disease and increasing the levels of anti-inflammatory cytokines.

[0101] Example 8. Comparison of liver tissue staining (Sirius red staining and H&E staining) of in vivo NAFLD mouse model injected with FGF5-v2 gene In this example, the progression of liver fibrosis was confirmed through Sirius red staining, which is known to be an indicator of fibrosis, in the liver tissue of a NAFLD mouse model, and the progression of fatty liver disease was confirmed through H&E staining.

[0102] Specifically, liver tissue samples were obtained from each mouse in the same manner as in the previous examples, and then fixed in 10% buffered formalin, embedded in paraffin, and cut into 5 μm thick tissue sections. Each tissue section was stained with Sirius Red using a Picrosirius Red staining kit (Abcam) according to the manufacturer's instructions. H&E staining was performed according to standard methods.

[0103] The results of Sirius red staining are shown in FIG. 8a, and the results of H&E staining are shown in FIG. 8b. Comparative analysis of Sirius red stained liver tissues of tissue sections magnified at 100x or 400x showed that the liver tissues of the FGF5 full gene-injected group did not show any significant difference in the degree of Sirius red staining compared to the untreated control group. Meanwhile, it was confirmed that the liver tissues of mice injected with the FGF5-v2 gene showed a significantly reduced degree of Sirius red staining compared to the other groups (FIG. 8a). The above results indicate that FGF5-v2 can effectively suppress fibrosis in the liver.

[0104] Comparative histopathological analysis of H&E stained liver tissues magnified at 100x or 400x magnification confirmed that there was no significant difference in the degree of steatosis between the liver tissues of the FGF5 full gene-injected group, the liver tissues of the FGF5-v2 gene-injected group, and the untreated control group (Figure 8b).The above results indicate that the FGF5-v2 gene has a specific and significant improving effect on liver fibrosis, which is indicated by fatty liver, rather than having an effect on fatty liver itself.

[0105] Example 9. Confirmation of FGF5 antagonistic effect of FGF5-v2 protein through experiments on hepatic stellate cell line In this example, a stimulation experiment was carried out using a hepatic stellate cell line (LX2 cells), and the hepatic fibrosis-suppressing effect of the FGF5 transcript mutant of the present invention was confirmed at the protein level.

[0106] Hepatic stellate cells (LX2 cells) were seeded the day before and then starvated for about an hour the next day, and treated with 100ng / ml of recombinant protein FGF5. The protein levels of total ERK and phospho-ERK were measured at each time point. Hepatoblastoma (HepG2) cells were also used as a control.

[0107] As a result, we confirmed that the total ERK level did not change, but the phospho-ERK level increased in the group treated for only 5 minutes, and gradually decreased from the group treated for 10 minutes onwards (Figure 10a). However, there was not much difference in hepatoblastoma (HepG2).

[0108] In addition, when FGF5-v2 protein alone (FGF5s) was used to examine the effect of FGF5-v2, the hepatic stellate cell stimulation response was quantified using the phospho-ERK reaction under the same conditions as above. The reaction level showed no significant difference compared to the mutant protein (Figure 10b).

[0109] Then, under the same conditions, FGF5 and FGF5-v2 protein (FGF5s) were treated together at 1x, 3x, and 5x concentrations. As a result, when only FGF5 was treated, the phospho-ERK level increased, but when FGF5-v2 was also treated, the phospho-ERK level decreased, and when a larger amount of FGF5-v2 was treated than when the same amount was treated, the phospho-ERK level decreased more, confirming that FGF5-v2 acts as an antagonist of FGF5 (Figure 10c). No change was observed in hepatoblastoma (HepG2) cells treated in the same manner.

[0110] After correcting the experimental results of hepatic stellate cells (LX2 cells) using semi-quantitative values ​​of total ERK, a significant difference was observed when the expression value of phospho-ERK was confirmed. The results are similar to the mechanism that can be inferred from the results of the animal experiments performed above, and indicate that FGF5-v2 protein is effective in suppressing liver fibrosis by antagonizing the action of FGF5 on hepatic stellate cells.

[0111] As can be seen from the above examples, the FGF5 transcript variant, FGF5-v2, according to the present invention can effectively inhibit and improve liver fibrosis by suppressing the levels of fibrosis-promoting factors and increasing the levels of anti-inflammatory cytokines in a non-alcoholic fatty liver animal model. Therefore, the FGF-v2 of the present invention is expected to be useful in preventing and treating various liver fibrosis-related diseases such as liver cirrhosis and liver cancer through its excellent liver fibrosis-inhibiting effect.

[0112] The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified in other specific forms without changing the technical concept or essential features of the present invention. Therefore, it should be understood that the above-described embodiments are merely illustrative and not limiting.

[0113] Sequence information relevant to the present invention is set forth in Table 1 below.

[0114] [Table 1-1]

[0115] [Table 1-2]

[0116] [Table 1-3]

[0117] [Table 1-4] [Industrial Applicability]

[0118] The fibroblast growth factor 5 (FGF5) transcript variant FGF5-v2 and its protein according to the present invention were developed by adjusting the amino acid sequence of full-length FGF5. It was confirmed that the transcript variant and its protein have the effect of suppressing the progression of liver fibrosis and improving liver dysfunction associated with liver fibrosis and the like, and various inflammation- and fibrosis-related cytokine increases. Therefore, it is expected that liver fibrosis can be suppressed and preventive, ameliorative and therapeutic effects can be achieved against various diseases including liver cirrhosis and liver cancer by administering to a patient a gene encoding FGF5-v2, its transcript, protein and / or various delivery vehicles (e.g., AAV) carrying these, and thus the present invention has industrial applicability.

Claims

1. FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO:

1.

2. A nucleic acid molecule encoding the FGF5-v2 protein of claim 1.

3. The nucleic acid molecule according to claim 2, wherein the FGF5-v2 gene comprises the base sequence of SEQ ID NO:

2.

4. An expression vector comprising the nucleic acid molecule of claim 2 or 3.

5. An isolated cell into which the expression vector according to claim 4 has been introduced.

6. A pharmaceutical composition for preventing or treating liver fibrosis, comprising as an active ingredient an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO:1; a nucleic acid molecule encoding said FGF5-v2 protein; an expression vector containing said nucleic acid molecule; or an isolated cell into which said expression vector has been introduced.

7. The pharmaceutical composition according to claim 6, wherein the FGF5-v2 protein, the nucleic acid molecule, the expression vector, or the cell is carried by a vehicle.

8. The pharmaceutical composition according to claim 7, wherein the composition comprises an FGF5-v2 protein, the nucleic acid molecule, or the expression vector as an active ingredient, and the carrier is at least one selected from the group consisting of virus particles, vesicles, nanoparticles, microparticles, liposomes, transposons, micelles, antibodies, and exosomes.

9. The pharmaceutical composition according to claim 6, characterized in that the liver fibrosis is liver fibrosis in one or more selected from the group consisting of alcoholic fatty liver disease, non-alcoholic fatty liver disease, hepatitis C, hepatitis B, autoimmune hepatitis, hepatic encephalopathy, primary biliary sclerosis, liver cancer, and hepatocellular carcinoma.

10. 10. The pharmaceutical composition according to claim 9, wherein the non-alcoholic fatty liver disease is at least one selected from the group consisting of non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver (NAFL), hepatic steatosis, acute fatty liver of pregnancy (AFLP), NAFLD-related liver failure, NAFLD-related liver fibrosis, NAFLD-related cirrhosis, and NAFLD-related liver cancer.

11. The pharmaceutical composition of claim 6, wherein the composition is administered in combination with an anti-inflammatory agent.

12. The pharmaceutical composition according to claim 11, characterized in that the anti-inflammatory agent is a steroidal anti-inflammatory agent or a non-steroidal anti-inflammatory agent.

13. A method for preventing or treating liver fibrosis, comprising administering to an individual in need thereof an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO:1; a nucleic acid molecule encoding said FGF5-v2 protein; an expression vector comprising said nucleic acid molecule; or an isolated cell into which said expression vector has been introduced.

14. Use of a composition comprising, as an active ingredient, an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO:1; a nucleic acid molecule encoding said FGF5-v2 protein; an expression vector containing said nucleic acid molecule; or an isolated cell into which said expression vector has been introduced, for the prevention or treatment of liver fibrosis.

15. Use of an FGF5-v2 protein consisting of the amino acid sequence of SEQ ID NO:1; a nucleic acid molecule encoding said FGF5-v2 protein; an expression vector containing said nucleic acid molecule; or a composition comprising an isolated cell into which said expression vector has been introduced as an active ingredient, in the manufacture of a preparation for the prevention or treatment of liver fibrosis.

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