Combination for the treatment and / or prevention of liver disease

Abstract

In one embodiment, the present invention relates to a combination for treating and / or preventing liver disease, comprising the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and the HNF6 gene or HNF6 protein.

Description

Technical Field

[0001] In one embodiment, the present invention relates to a combination for treating and / or preventing liver disease. Background Technology

[0002] There are no effective treatments for liver diseases such as cirrhosis, liver fibrosis, and NASH. Furthermore, regarding the treatment of severe liver diseases, if medical treatment reaches its limits, liver transplantation remains the only option, but challenges exist in securing donors and matching donors. On the other hand, artificial livers, which are being developed worldwide, cannot fully replace the functions of the liver and have not yet been put into practical use. Summary of the Invention

[0003] In one embodiment, the subject of the present invention is to provide a medicament or method for treating and / or preventing liver diseases.

[0004] The inventors have discovered that liver diseases can be treated and / or prevented by using a specific combination of reprogramming factors. The present invention includes the following embodiments.

[0005] (1) A combination for treating and / or preventing liver disease, comprising the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and the HNF6 gene or HNF6 protein.

[0006] (2) According to the combination described in (1), wherein the aforementioned liver disease is selected from the group consisting of cirrhosis, liver fibrosis and non-alcoholic steatohepatitis (NASH).

[0007] (3) According to the combination described in (1), wherein the aforementioned liver disease is cirrhosis or liver fibrosis.

[0008] (4) The combination according to any one of (1) to (3), wherein HNF1 is HNF1A.

[0009] (5) The combination according to any one of (1) to (4), wherein FOXA is FOXA3.

[0010] (6) The combination according to any one of (1) to (5) comprises the HNF1 gene, the FOXA gene and the HNF6 gene.

[0011] (7) The combination according to (6), wherein the HNF1 gene, FOXA gene and HNF6 gene are contained in the vector.

[0012] (8) According to the combination described in (7), wherein the HNF1 gene, FOXA gene and HNF6 gene are contained in different vectors.

[0013] (9) The combination according to (7) or (8), wherein the vector is adeno-associated virus (AAV).

[0014] (10) The combination according to (9), wherein the carrier is AAV8.

[0015] (11) The combination according to any one of (7) to (10), wherein the HNF1 gene, the FOXA gene and the HNF6 gene are operatively linked to a hepatocyte-specific promoter in the vector.

[0016] (12) The combination according to any one of (1) to (6), wherein the HNF1 gene, the FOXA gene and the HNF6 gene are contained in the lipid nanoparticles.

[0017] (13) The combination according to any one of (1) to (12) is a pharmaceutical composition.

[0018] (14) A method for treating and / or preventing liver disease, comprising administering the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and the HNF6 gene or HNF6 protein to a subject.

[0019] (15) According to the method of (14), wherein the aforementioned liver disease is selected from the group consisting of cirrhosis, liver fibrosis and non-alcoholic steatohepatitis (NASH).

[0020] (16) The method according to (14), wherein the aforementioned disease is cirrhosis or liver fibrosis.

[0021] (17) The method according to any one of (14) to (16), wherein HNF1 is HNF1A.

[0022] (18) The method according to any one of (14) to (17), wherein FOXA is FOXA3.

[0023] (19) The method according to any one of (14) to (18) includes administering the HNF1 gene, the FOXA gene and the HNF6 gene.

[0024] (20) According to the method of (19), wherein the HNF1 gene, FOXA gene and HNF6 gene are contained in the vector.

[0025] (21) According to the method described in (20), wherein the HNF1 gene, FOXA gene and HNF6 gene are contained in different vectors.

[0026] (22) The method according to (20) or (21), wherein the vector is adeno-associated virus (AAV).

[0027] (23) According to the method described in (22), wherein the carrier is AAV8.

[0028] (24) The method according to any one of (21) to (23), wherein the HNF1 gene, the FOXA gene and the HNF6 gene are operatively linked to a hepatocyte-specific promoter in the vector.

[0029] (25) The method according to any one of (14) to (19), wherein the HNF1 gene, the FOXA gene and the HNF6 gene are contained in the lipid nanoparticles.

[0030] (26) The method according to any one of (14) to (25), wherein the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and the HNF6 gene or HNF6 protein are included in the pharmaceutical composition.

[0031] According to the present invention, pharmaceutical agents or methods for treating and / or preventing liver diseases are provided. Attached Figure Description

[0032] Figure 1 Figure A shows the results of gene expression analysis of AFP and EpCAM when human iHepPC inducing factors were introduced into a mouse model of liver fibrosis using AAV8. Figure 1 Figure B shows the results of EpCAM immunostaining when human iHepPC inducing factors were introduced into a liver fibrosis model using AAV8.

[0033] Figure 2 The results show the staining of fibrotic tissue (A), relative values ​​of fibrotic tissue (B), gene expression level of Col1a1 (C), serum alanine aminotransferase (ALT) level (D), and serum aspartate aminotransferase (AST) level (E) when human iHepPC inducing factor is introduced into a mouse model of liver fibrosis using AAV8.

[0034] Figure 3 The results show the measurement of fat deposition when human iHepPC inducing factors are introduced into a mouse model of liver fibrosis using AAV8.

[0035] Figure 4 The results of histopathological analysis are shown when human iHepPC inducing factors are introduced into a mouse model of liver fibrosis using AAV8. A shows the results of hematoxylin-eosin (HE) staining, and B shows the results of NAFLD activity score (NAS).

[0036] Figure 5The results show the staining of fibrotic tissue (A), the proportion of fibrotic tissue (B), the gene expression level of Col1a1 (C), and the levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum when human iHepPC inducing factor was introduced into a mouse model of liver fibrosis using LNP.

[0037] Figure 6 The results show the measurement of fat deposition when human iHepPC inducing factors are introduced into a mouse model of liver fibrosis using LNP.

[0038] Figure 7 The results of histopathological analysis are shown when human iHepPC inducing factors are introduced into a mouse model of liver fibrosis using LNP. A shows the results of hematoxylin-eosin (HE) staining, and B shows the results of NAFLD activity score (NAS). Detailed Implementation

[0039] In one embodiment, the present invention relates to a combination for treating and / or preventing liver disease, comprising the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and the HNF6 gene or HNF6 protein (the term "combination" is also referred to as "composition" in this specification).

[0040] In this specification, examples of liver diseases may include simple fatty liver, non-alcoholic fatty liver disease (NAFLD) (also known as metabolic dysfunction-associated fatty liver disease (MASLD)), non-alcoholic steatohepatitis (NASH) (also known as metabolic dysfunction-associated steatohepatitis (MASH)), alcoholic liver disease (ALD), alcohol-related liver disease, cholestatic liver disease, liver fibrosis, cirrhosis, liver failure, hepatitis, steatohepatitis, viral liver diseases (e.g., hepatitis A, hepatitis B, hepatitis C), hepatic ischemia, and liver cancer. Further examples of liver diseases may include congenital liver diseases (e.g., congenital metabolic disorders, congenital liver fibrosis, Alagille syndrome), autoimmune liver diseases, biliary atresia, acute liver failure, and drug-induced liver injury. Liver diseases may be selected from the group consisting of, for example, cirrhosis, liver fibrosis, and non-alcoholic steatohepatitis (NASH), and may include either cirrhosis or liver fibrosis.

[0041] In this instruction manual, "treatment" includes not only improving symptoms related to the aforementioned diseases, but also maintaining improved symptoms, suppressing recurrence, and all other treatments related to the aforementioned diseases. In this instruction manual, "prevention" includes preventing the occurrence of symptoms related to the aforementioned diseases or reducing their risk.

[0042] The combinations described in this specification can be combinations of reprogramming factors. In this specification, "reprogramming" refers to the process of changing the differentiation state of a cell to a different differentiation state or an undifferentiated state. As factors used for such reprogramming (reprogramming factors), combinations comprising or consisting of the following can be listed: the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and the HNF6 gene or HNF6 protein. Specific combinations can be any combination of 1) to 8) below, such as combinations of 1) or 8):

[0043] 1) A combination of the HNF1 gene, FOXA gene, and HNF6 gene;

[0044] 2) A combination of the HNF1 gene, FOXA protein, and HNF6 gene;

[0045] 3) A combination of the HNF1 gene, the FOXA gene, and the HNF6 protein;

[0046] 4) A combination of HNF1 gene, FOXA protein, and HNF6 protein;

[0047] 5) A combination of HNF1 protein, FOXA gene, and HNF6 gene;

[0048] 6) A combination of HNF1 protein, FOXA protein, and HNF6 gene;

[0049] 7) A combination of HNF1 protein, FOXA gene, and HNF6 protein;

[0050] 8) A combination of HNF1, FOXA and HNF6 proteins.

[0051] In one embodiment, the combination described in this specification may be a combination of the HNF1 gene, the FOXA gene, and the HNF6 gene, such as a combination of HNF1 mRNA, FOXA mRNA, and HNF6 mRNA.

[0052] HNF1 (Hepatocyte Nuclear Factor 1) is a protein with a homologous domain, and there are two isoforms: HNF1A and HNF1B.

[0053] HNF6 (Hepatocyte Nuclear Factor 6) is a homologous domain transcription factor involved in human tissue development. It controls the development of various tissues, including the pancreas and liver, and regulates the expression of various liver genes.

[0054] FOXA is a nuclear transcription factor (transcription factor) required in the earliest stages of liver tissue formation, including FOXA1, FOXA2, and FOXA3. These FOXA transcription factors share more than 90% homology at the amino acid level in a common forkhead / winged helix domain, and are therefore considered to be functionally complementary to each other.

[0055] The combinations described in this specification may also include other factors. When other factors are included, they preferably do not substantially reduce the therapeutic and / or preventative effects on the disease. Examples of factors that can be added to the combination include, for instance, MYC genes or proteins. The MYC family of genes are known as transcription factors that bind to nuclear DNA and function as transcription factors; in humans, there are c-MYC, L-MYC, and N-MYC.

[0056] The amino acid sequences of the above reprogramming factors and the base sequences of the genes encoding these factors are shown in Table 1.

[0057] [Table 1]

[0058]

[0059] The genes encoding these factors, or a portion thereof, can be cloned with reference to "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)) or obtained from addgene, etc.

[0060] In this specification, the gene encoding HNF1A is referred to as the "HNF1A gene," and the gene encoding HNF6 is referred to as the "HNF6 gene." Genes encoding other factors can be referred to in the same way.

[0061] The reprogramming factors (genes or proteins) used in this specification are not limited to nucleic acid sequences having the base sequences represented by the sequence numbers shown in Table 1 or amino acid sequences having the amino acid sequences represented by the sequence numbers shown in Table 1. As long as they have the function of reprogramming factors, the mutants shown below can also be used:

[0062] (a) A protein consisting of an amino acid sequence in which one or more amino acids (e.g., less than 10, less than 5, less than 4, less than 3, or 2) are missing, substituted, or added in the amino acid sequence shown in Table 1 (amino acid sequences shown in sequence numbers 2, 4, 6, 8, or 10), and having the function of a reprogramming factor.

[0063] (b) A protein comprising an amino acid sequence having 80%, 85%, 90%, 95%, 98%, or 99% (e.g., 90%) sequence identity with the amino acid sequence shown in Table 1 (the amino acid sequence shown in sequence number 2, 4, 6, 8, or 10), and having the function of a reprogramming factor.

[0064] (c) A nucleic acid that encodes the protein described in (a) above;

[0065] (d) A nucleic acid that encodes the protein described in (b) above;

[0066] (e) A nucleic acid that hybridizes under stringent conditions with a nucleic acid consisting of a base sequence complementary to the base sequence shown in Table 1 (the amino acid sequence shown in sequence number 1, 3, 5, 7 or 9) and encodes a protein that functions as a reprogramming factor.

[0067] In this specification, "strict conditions" can be, for example, 5×SSC, 5×Denhardt solution, 0.5% SDS, 50% formamide, and 50°C. Under these conditions, it can be expected that higher temperatures will result in more efficient acquisition of DNA and / or RNA with high homology. However, factors affecting the strictness of hybridization include temperature, DNA and / or RNA concentration, DNA and / or RNA length, ionic strength, time, salt concentration, etc., and those skilled in the art can achieve the same strictness by appropriately selecting these factors.

[0068] Whether a specific protein functions as a reprogramming factor can be investigated using known methods. For example, a combination of proteins described in this specification containing the specific protein can be introduced into cells that are not liver stem cells or liver progenitor cells and cultured for a specified period. The investigation can then be conducted based on whether the expression of markers of liver stem cells or liver progenitor cells, such as albumin, alpha-fetoprotein, and E-cadherin, increases in the cells.

[0069] The combinations described in this specification are not limited, and for example, they can be combinations of (1) to (3), or combinations of (2) or (3):

[0070] (1)(a) a combination of HNF1A gene or HNF1A protein, (b) FOXA3 gene or FOXA3 protein, and (c) HNF6 gene or HNF6 protein;

[0071] (2)(a) A combination of HNF1A gene, (b) FOXA3 gene and (c) HNF6 gene;

[0072] (3)(a) A combination of HNF1A protein, (b) FOXA3 protein and (c) HNF6 protein.

[0073] The combination may contain the above factors, may be composed of the above factors, or may be essentially composed of the above factors.

[0074] Reprogramming factors can be manufactured through genetic engineering (see, for example, “Molecular Cloning, A Laboratory Manual (4th edition)” (Cold Spring Harbor Laboratory Press (2012)).

[0075] When the reprogramming factor is a gene, the gene can be, for example, DNA or RNA (e.g., mRNA). Alternatively, the reprogramming factor can also be contained in a vector. In one embodiment, the present invention relates to a vector comprising the combinations described herein. The vector can also be an expression vector that operatively links the reprogramming factor and / or other DNA sequences to one or more expression regulatory sequences. Examples of expression regulatory sequences include promoters, enhancers, and transcription termination regions. In this specification, the state in which a gene is linked to an expression regulatory sequence and can be expressed in cells, etc., can be described as "operatively linked." By operatively linking the reprogramming factor to a hepatocyte-specific promoter in a vector, hepatocyte specificity can be improved, efficacy enhanced, and / or side effects reduced. Examples of hepatocyte-specific promoters include the promoters of the thyroxine-binding globulin (TBG) gene, the albumin gene, the transthyretin (TTR) gene, the apolipoprotein gene, and the Pr1 promoter.

[0076] Genes encoding reprogramming factors can be contained entirely in a single vector, or each gene can be contained in a separate vector. Alternatively, a subset of genes can be contained in one vector, while the remainder can be contained in other vectors.

[0077] In addition to plasmids and bacteriophages, other expression vectors that can be used in this specification include viral vectors derived from retroviruses, vaccinia viruses, adenoviruses, lentiviruses, adeno-associated viruses, such as AAV8 and Sendai virus. Using AAV8, which has a high affinity for hepatocytes, can improve the specificity to hepatocytes, enhance efficacy, and / or reduce side effects.

[0078] In one embodiment, the combination described herein is contained in lipid nanoparticles (LNPs). In one embodiment, the present invention relates to LNPs comprising the combination described herein.

[0079] When the reprogramming factor is a protein, the efficiency of its delivery into the cell can be improved by linking the protein to membrane-permeable peptides or by using cationic lipids.

[0080] The combinations described in this instruction manual can be administered to the subject, for example, by injection into the skin tissue, intravascular (e.g., intravenous), or intraperitoneal cavity of an animal.

[0081] In one embodiment, the combination described herein is contained in one or more pharmaceutical compositions.

[0082] In addition to the combinations described herein, the pharmaceutical compositions described in this specification may also contain pharmaceutically acceptable carriers (excipients, extenders, binders, lubricants, etc.) and / or known additives (buffers, isotonic agents, chelating agents, colorants, preservatives, flavorings, tasters, sweeteners, etc.). For example, by incorporating lipid nanoparticles (LNPs) as carriers, gene delivery can be facilitated.

[0083] The pharmaceutical compositions described in this specification can generally be administered systemically or locally, orally or non-orally. These compositions can be administered to the body via intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or suppositories, for example, via intravenous injection. The dosage of the drugs described in this specification can be appropriately set according to age, weight, symptoms, therapeutic effect, method of administration, and treatment time. For example, for an individual, the pharmaceutical composition or active ingredient can be administered at a dose of 0.0001–100 mg / kg daily, preferably 0.01–100 mg / kg.

[0084] The pharmaceutical compositions described in this specification can be formulated into pharmaceutically acceptable preparations. These preparations can be formulated by conventional means into sterile solutions, suspensions, lyophilized formulations, injections, tablets, granules, powders, capsules, emulsions, suspensions, syrups, etc.

[0085] The “test subject” given to the combination or pharmaceutical composition described in this specification includes humans or other organisms. Examples of organisms other than humans include birds and non-human mammals (e.g., cattle, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses).

[0086] In one embodiment, the present invention relates to a method for treating and / or preventing liver disease, comprising administering to a subject a combination of the ingredients described herein, or the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and the HNF6 gene or HNF6 protein. The combination of the ingredients, the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, the HNF6 gene or HNF6 protein, and the liver disease described herein are as described in this specification.

[0087] Example

[0088] <Example 1: In vivo reprogramming-mediated hepatocyte immaturity>

[0089] In this embodiment, a liver fibrosis model (cirrhosis model) was prepared as follows: carbon tetrachloride (FUJIFILM Wako Pure Chemical Corporation) and olive oil (NACALAI TESQUE) were mixed and injected subcutaneously into C57BL / 6J mice (KBT Oriental) at a dose of 5 μl per 1 g mouse body weight, twice a week for 8 weeks. AAV8, an adeno-associated virus (AAV) with high affinity for hepatocytes, was used to introduce the inducing factors (FOXA3, HNF1A, HNF6: denoted as h3F) of human inducible hepatic progenitor cells (human iHepPC). Specifically, a vector containing the aforementioned inducing factors was prepared by recombining the Cre sequence of AAV.TBG.PI.Cre.rBG (Plasmid #107787) (addgene) with the sequences of each inducing factor (the base sequences of FOXA3, HNF1A, and HNF6 shown in Table 1) using conventional methods. AAV8 was created by transfecting HEK293 cells with polyethyleneimine (PEI Max) (Poly Sciences) according to the accompanying protocol. AAV8 was administered via a single tail vein injection at a time point 4 weeks after the start of carbon tetrachloride administration. Then, while continuing carbon tetrachloride administration, liver samples were used at 2, 4, 7, 14, and 28 days after AAV8 administration for qPCR-based gene expression analysis and immunostaining-based protein expression analysis of alpha-fetoprotein (AFP) and epithelial cell adhesion molecule (EpCAM). Gene expression analysis and immunostaining were performed according to the methods described in Hiroki Inada et al., Nature Communications, Vol. 11, Article number: 5292, 2020. AFP is a marker of hepatic progenitor cells, and EpCAM is expressed in hepatic progenitor cells and bile duct epithelial cells. In AAV8, human iHepPC inducible factor is mainly expressed in hepatocytes because the expression of human iHepPC inducible factor is controlled by the promoter of the hepatocyte-specific thyroxine-binding globulin (TBG) gene.

[0090] The results are shown in Figure 1 It was clarified that the gene expression levels of AFP and EpCAM peaked on days 7 and 14, respectively, after human iHepPC inducible factor expression (A). The values ​​in the graph show relative values ​​based on gene expression levels in mouse livers 4 weeks after carbon tetrachloride administration and before AAV8 administration.

[0091] In addition, compared with the livers of Mock control mice given an empty vector (AAV8-TBG-Null) that did not contain human iHepPC inducible factor, a large number of EpCAM-positive cells (B) were detected by immunostaining in the livers expressing human iHepPC inducible factor. The DNA of the cells was stained with DAPI (blue).

[0092] <Example 2: Improvement of Liver Fibrosis Brought About by Liver Cell Rejuvenation Mediated by In Vivo Reprogramming>

[0093] In this embodiment, a cirrhotic mouse model was created in the same manner as in Example 1. Four weeks after the start of carbon tetrachloride administration, AAV8 containing the inducing factor for human iHepPC was introduced, similar to Example 1. Then, after another four weeks of continued carbon tetrachloride administration, liver fibrosis and Col1a1 gene expression, as well as serum ALT and AST levels, were measured. Specifically, formalin-fixed liver tissue was prepared into paraffin sections, and the area of ​​fibrotic tissue stained with Sirius red was quantified using a KEYENCE BZ-X800. In addition, mRNA was extracted from liver tissue using ISOGEN II (NIPPON GENE), and cDNA was synthesized from the mRNA using the Rever Tra qPCRRT kit (TOYOBO). The cDNA was then used for qPCR analysis of Col1a1 gene expression using specific primers (TCCTCCAGGGATCCAACGA (SEQ ID NO. 11) and GGCAGGCGGGAGGTCTT (SEQ ID NO. 12)). Serum ALT and AST levels were also measured using Transaminase CII-Test Wako (FUJIFILM Wako PureChemical Corporation). An empty vector (AAV8-TBG-Null) without human iHepPC inducing factor was used as a mock control.

[0094] The results are shown in Figure 2 In staining of fibrotic tissue using Sirius red, the amount of fibrotic tissue stained red was reduced due to the expression of human iHepPC inducible factor (A, B), which in turn reduced the gene expression of collagen (Col1a1), a major component of fibrotic tissue (C). Furthermore, the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), markers of liver injury, were also reduced due to the expression of human iHepPC inducible factor (D, E). Figure 2The values ​​in the B-E chart represent the values ​​obtained by dividing the 8-week time point that will be the endpoint by the values ​​of the 4-week time points that will be given to AAV8.

[0095] <Example 3: Improvement of fatty liver induced by hepatocyte immaturity mediated by in vivo reprogramming>

[0096] In this embodiment, a cirrhotic mouse model was created in the same manner as in Example 1. Four weeks after the start of carbon tetrachloride administration, AAV8 containing the human iHepPC inducing factor was introduced, similar to Example 1. Then, after another four weeks of continued carbon tetrachloride administration, fat deposition in the liver tissue was measured. Specifically, frozen sections of liver tissue were prepared, and the area of ​​fat deposition regions stained with Oil Red O was quantified using a KEYENCE BZ-X800. Additionally, an empty vector (AAV8-TBG-Null) without the human iHepPC inducing factor was used as a mock control.

[0097] The results are shown in Figure 3 In the staining of lipid droplets using Oil Red O, the area of ​​the lipid deposition region stained red was reduced due to the expression of human iHepPC inducible factor (A, B).

[0098] <Example 4: Improved Liver Tissue Imaging Effects Caused by Liver Cell Rejuvenation Mediated by In Vivo Reprogramming>

[0099] In this embodiment, a cirrhotic mouse model was created in the same manner as in Example 1. Four weeks after the start of carbon tetrachloride administration, AAV8 containing the human iHepPC inducing factor was introduced, similar to Example 1. Then, after another four weeks of continued carbon tetrachloride administration, liver tissue images were analyzed. Specifically, liver tissue was formalin-fixed, paraffin sections were prepared, and hematoxylin-eosin (HE) staining was performed. The degree of ballooning hepatocyte degeneration, fatty degeneration, and inflammatory cell infiltration was scored. The scoring method was based on David E Kleiner et al., Hepatology, 2005 Jun, 41(6):1313-21. Additionally, an empty vector (AAV8-TBG-Null) without the human iHepPC inducing factor was used as a mock control.

[0100] The results are shown in Figure 4Histopathological analysis using hematoxylin-eosin (HE) staining showed that expression of human iHepPC inducing factor improved ballooning hepatocyte degeneration, fatty degeneration, and inflammatory cell infiltration in hepatocytes, and reduced NAFLD activity scores (NAS) (A, B).

[0101] <Example 5: Improvement of liver fibrosis by using LNP-mediated expression of human iHepPC inducible factor>

[0102] In this embodiment, cirrhotic mice were prepared in the same manner as in Example 1. Four weeks after the start of carbon tetrachloride administration, lipid nanoparticles (LNPs) containing messenger RNA (mRNA) of human inducible hepatic progenitor cells (human iHepPC) inducing factors (FOXA3, HNF1A, HNF6: denoted as h3F) were administered at a dose of 2 μg / g (mice body weight) once every 5 days for a total of 6 administrations. The fabrication of the mRNA-containing LNPs was commissioned to GenScript. The mRNA sequences of FOXA3 used in this embodiment are indicated by sequence number 13, HNF1A by sequence number 14, and HNF6 by sequence number 15. Carbon tetrachloride was also administered during the LNP administration period, and liver fibrosis and Col1a1 gene expression were analyzed, as well as serum ALT and AST levels, in the same manner as in Example 2, four weeks after the start of LNP administration. In addition, LNP (LNP-GFP) containing mRNA of green fluorescent protein (GFP) was used as a control.

[0103] The results are shown in Figure 5 In staining of fibrotic tissue using Sirius red, the amount of fibrotic tissue stained red was reduced due to the expression of human iHepPC inducible factor (A, B), which in turn reduced the gene expression of collagen (Col1a1), a major component of fibrotic tissue (C). Furthermore, the levels of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST), markers of liver injury, were also reduced due to the expression of human iHepPC inducible factor (D, E).

[0104] <Example 6: Improvement of fatty liver induced by using LNP expression of human iHepPC inducing factor>

[0105] In this embodiment, using a cirrhotic mouse model prepared in the same manner as in Example 1, lipid nanoparticles (LNPs) containing messenger RNA (mRNA) of human inducible hepatic progenitor cells (human iHepPC) (FOXA3, HNF1A, HNF6: denoted as h3F) were administered 6 times every 5 days, 4 weeks after the start of carbon tetrachloride administration, as in Example 5. Carbon tetrachloride administration continued during this period, and fat deposition in the liver tissue was measured 4 weeks after the start of LNP administration, as in Example 3. Additionally, LNPs containing mRNA of green fluorescent protein (GFP) (LNP-GFP) were used as a control.

[0106] The results are shown in Figure 6 In the staining of lipid droplets using Oil Red O, the area of ​​the lipid deposition region stained red was reduced due to the expression of human iHepPC inducible factor (A, B).

[0107] <Example 7: Improvement of liver tissue images by using LNP-mediated expression of human iHepPC inducible factor>

[0108] In this embodiment, using a cirrhotic mouse model prepared in the same manner as in Example 1, lipid nanoparticles (LNPs) containing messenger RNA (mRNA) of human inducible hepatic progenitor cells (human iHepPC) (FOXA3, HNF1A, HNF6: denoted as h3F) were administered 6 times every 5 days, 4 weeks after the start of carbon tetrachloride administration, as in Example 5. Carbon tetrachloride administration continued during this period, and liver tissue images were analyzed 4 weeks after the start of LNP administration, as in Example 4. Additionally, LNPs containing green fluorescent protein (GFP) mRNA (LNP-GFP) were used as a control.

[0109] The results are shown in Figure 7 Histopathological analysis using hematoxylin-eosin (HE) staining showed that expression of human iHepPC inducing factor improved ballooning hepatocyte degeneration, fatty degeneration, and inflammatory cell infiltration in hepatocytes, and reduced NAFLD activity scores (NAS) (A, B).

Claims

1. A combination for treating and / or preventing liver disease, comprising the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and the HNF6 gene or HNF6 protein.

2. The combination according to claim 1, wherein, The liver diseases were selected from the group consisting of cirrhosis, liver fibrosis, and non-alcoholic steatohepatitis (NASH).

3. The combination according to claim 1, wherein, The liver disease mentioned is cirrhosis or liver fibrosis.

4. The combination according to claim 1, wherein, HNF1 is HNF1A.

5. The combination according to claim 1, wherein, FOXA is FOXA3.

6. The combination according to claim 1, comprising the HNF1 gene, the FOXA gene, and the HNF6 gene.

7. The combination according to claim 6, wherein, The HNF1, FOXA, and HNF6 genes are contained in the vector.

8. The combination according to claim 7, wherein, The HNF1, FOXA, and HNF6 genes are contained in different vectors.

9. The combination according to claim 7, wherein, The vector is adeno-associated virus (AAV).

10. The combination according to claim 9, wherein, The carrier is AAV8.

11. The combination according to claim 8, wherein, The HNF1, FOXA, and HNF6 genes are operatively linked to hepatocyte-specific promoters in the vector.

12. The combination according to claim 1, wherein, The HNF1, FOXA, and HNF6 genes are contained in lipid nanoparticles.

13. The combination according to claim 1, wherein it is a pharmaceutical composition.

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