Combination for treating and / or preventing liver disease

Abstract

One embodiment of the present invention relates to a combination for treating and / or preventing liver disease, the combination comprising: an HNF1 gene or HNF1 protein; a FOXA gene or FOXA protein; and an HNF6 gene or HNF6 protein.

Description

Combinations for treating and / or preventing liver disease

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

[0002] There are no effective treatments for liver diseases such as cirrhosis, liver fibrosis, and NASH. Furthermore, when medical treatment reaches its limits for severe liver disease, liver transplantation is the only option, due to the challenges of securing donors and finding a suitable match. Meanwhile, artificial livers, which are being developed around the world, are unable to adequately replace liver function and have yet to be put to practical use.

[0003] In one embodiment, the present invention aims to provide an agent or method for treating and / or preventing liver disease.

[0004] The present inventors have found that liver disease can be treated and / or prevented by using a specific combination of reprogramming factors. The present invention encompasses the following embodiments: (1) A combination for treating and / or preventing liver disease, comprising an HNF1 gene or HNF1 protein, a FOXA gene or FOXA protein, and an HNF6 gene or HNF6 protein. (2) The combination according to (1), wherein the liver disease is selected from the group consisting of cirrhosis, liver fibrosis, and non-alcoholic steatohepatitis (NASH). (3) The combination according to (1), wherein the liver disease is cirrhosis or liver fibrosis. (4) The combination according to any of (1) to (3), wherein HNF1 is HNF1A. (5) The combination according to any of (1) to (4), wherein FOXA is FOXA3. (6) The combination according to any of (1) to (5), comprising an HNF1 gene, a FOXA gene, and an HNF6 gene. (7) The combination according to (6), wherein the HNF1 gene, the FOXA gene, and the HNF6 gene are contained in a vector. (8) The combination according to (7), wherein the HNF1 gene, the FOXA gene, and the HNF6 gene are contained in separate vectors. (9) The combination according to (7) or (8), wherein the vector is an adeno-associated virus (AAV). (10) The combination according to (9), wherein the vector is AAV8. (11) The combination according to any of (7) to (10), wherein the HNF1 gene, the FOXA gene, and the HNF6 gene are operably linked to a hepatocyte-specific promoter in a vector. (12) The combination according to any of (1) to (6), wherein the HNF1 gene, the FOXA gene, and the HNF6 gene are contained in lipid nanoparticles. (13) The combination according to any of (1) to (12), wherein the combination is a pharmaceutical composition. (14) A method for treating and / or preventing liver disease, comprising administering to a subject an HNF1 gene or HNF1 protein, a FOXA gene or FOXA protein, and an HNF6 gene or HNF6 protein. (15) The method according to (14), wherein the liver disease is selected from the group consisting of cirrhosis, hepatic fibrosis, and nonalcoholic steatohepatitis (NASH). (16) The method according to (14), wherein the disease is cirrhosis or hepatic fibrosis.(17) The method according to any one of (14) to (16), wherein HNF1 is HNF1A. (18) The method according to any one of (14) to (17), wherein FOXA is FOXA3. (19) The method according to any one of (14) to (18), comprising administering the HNF1 gene, the FOXA gene, and the HNF6 gene. (20) The method according to (19), wherein the HNF1 gene, the FOXA gene, and the HNF6 gene are contained in a vector. (21) The method according to (20), wherein the HNF1 gene, the FOXA gene, and the HNF6 gene are each contained in a separate vector. (22) The method according to (20) or (21), wherein the vector is an adeno-associated virus (AAV). (23) The method according to (22), wherein the vector is AAV8. (24) The method according to any one of (21) to (23), wherein the HNF1 gene, the FOXA gene, and the HNF6 gene are operably linked to a hepatocyte-specific promoter in a vector. (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 a lipid nanoparticle. (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 contained in a pharmaceutical composition.

[0005] The present invention provides agents or methods for treating and / or preventing liver diseases.

[0006] Figure 1A shows the results of gene expression analysis of AFP and EpCAM after AAV8-mediated transduction of human iHepPC inducers into liver fibrosis model mice. Figure 1B shows the results of EpCAM immunostaining after AAV8-mediated transduction of human iHepPC inducers into liver fibrosis model mice. Figure 2 shows the results of fibrotic tissue staining (A), relative fibrotic tissue expression (B), Col1a1 gene expression (C), and serum alanine aminotransferase (ALT) (D) and aspartate aminotransferase (AST) (E) after AAV8-mediated transduction of human iHepPC inducers into liver fibrosis model mice. Figure 3 shows the results of fat deposition measurement after AAV8-mediated transduction of human iHepPC inducers into liver fibrosis model mice. Figure 4 shows the results of histopathological analysis after AAV8-mediated transduction of human iHepPC inducers into liver fibrosis model mice. A shows the results of hematoxylin-eosin (HE) staining, and B shows the results of NAFLD activity score (NAS). Figure 5 shows the results of fibrotic tissue staining (A), the percentage of fibrotic tissue (B), Col1a1 gene expression level (C), and serum alanine aminotransferase (ALT) (D) and aspartate aminotransferase (AST) (E) levels after LNP-mediated introduction of human iHepPC inducers into liver fibrosis model mice. Figure 6 shows the results of fat deposition measurements after LNP-mediated introduction of human iHepPC inducers into liver fibrosis model mice. Figure 7 shows the results of histopathological analysis after LNP-mediated introduction of human iHepPC inducers into liver fibrosis model mice. A shows the results of hematoxylin-eosin (HE) staining, and B shows the results of NAFLD activity score (NAS).

[0007] 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 ("combination" is also referred to as "combined product" in this specification).

[0008] As used herein, examples of liver diseases include simple fatty liver, nonalcoholic fatty liver disease (NAFLD) (also known as "metabolic dysfunction-associated fatty liver disease (MASLD)"), nonalcoholic 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 disease (e.g., hepatitis A, hepatitis B, hepatitis C), hepatic ischemia, and liver cancer. Further examples of liver diseases include congenital liver disease (e.g., congenital metabolic disorders, congenital hepatic fibrosis, Alagille syndrome), autoimmune liver disease, biliary atresia, acute liver failure, and drug-induced liver injury. The liver disease may be selected from the group consisting of cirrhosis, hepatic fibrosis, and nonalcoholic steatohepatitis (NASH), and may be, for example, cirrhosis or hepatic fibrosis.

[0009] As used herein, "treatment" includes alleviation of symptoms associated with the disease, as well as continuation of improved symptoms, suppression of recurrence, and all treatments associated with the disease. As used herein, "prevention" includes preventing the onset of symptoms associated with the disease or reducing the risk thereof.

[0010] The combinations described herein may be combinations of reprogramming factors. As used herein, "reprogramming" refers to a process of changing the differentiated state of a cell to a differentiated state different from that of the cell or to an undifferentiated state. Factors used for such reprogramming (reprogramming factors) include combinations comprising or consisting of the HNF1 gene or HNF1 protein, the FOXA gene or FOXA protein, and the HNF6 gene or HNF6 protein. Specific combinations include any of the following combinations 1) to 8), for example, combination 1) or 8): 1) a combination of the HNF1 gene, FOXA gene, and HNF6 gene, 2) a combination of the HNF1 gene, FOXA protein, and HNF6 gene, 3) a combination of the HNF1 gene, FOXA gene, and HNF6 protein, 4) a combination of the HNF1 gene, FOXA protein, and HNF6 protein, 5) a combination of the HNF1 protein, FOXA gene, and HNF6 gene, 6) a combination of the HNF1 protein, FOXA protein, and HNF6 gene, 7) a combination of the HNF1 protein, FOXA gene, and HNF6 protein, 8) a combination of the HNF1 protein, FOXA protein, and HNF6 protein.

[0011] In one embodiment, the combination described herein may be a combination of the HNF1 gene, the FOXA gene, and the HNF6 gene, for example, a combination of HNF1 mRNA, FOXA mRNA, and HNF6 mRNA.

[0012] Hepatocyte Nuclear Factor 1 (HNF1) is a homeodomain protein with two isoforms, HNF1A and HNF1B.

[0013] Hepatocyte Nuclear Factor 6 (HNF6) is a homeodomain transcription factor involved in human tissue development. It regulates the development of various tissues, including the pancreas and liver, as well as the expression of various hepatic genes.

[0014] FOXA is a hepatocyte nuclear factor (transcription factor) required for the earliest stage of liver tissue formation, and includes FOXA1, FOXA2, and FOXA3. These FOXA transcription factors share over 90% amino acid homology in the common forkhead / winged helix domain, suggesting that they have functional complementarity with each other.

[0015] The combinations described herein may contain other factors. When other factors are included, it is preferable that the factors do not substantially reduce the efficacy of treating and / or preventing a disease. Factors that can be added to the combination include, for example, the MYC gene or protein. MYC family genes are known as transcription factors that act by binding to nuclear DNA, and in humans, they include c-MYC, L-MYC, and N-MYC.

[0016] The amino acid sequences of the above reprogramming factors and the nucleotide sequences of the genes encoding these factors are shown in Table 1.

[0017]

[0018] Genes encoding these factors or portions thereof can be cloned with reference to "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)) or the like, or can be obtained from addgene or the like.

[0019] Herein, 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 also be designated in the same manner as above.

[0020] The reprogramming factors (genes or proteins) used herein are not limited to nucleic acid sequences having the nucleotide sequences represented by the SEQ ID NOs in Table 1 or amino acid sequences having the amino acid sequences represented by the SEQ ID NOs in Table 1. The following mutants can also be used as long as they have the function of a reprogramming factor: (a) A protein consisting of an amino acid sequence in which one or several (e.g., 10 or less, 5 or less, 4 or less, 3 or less, or 2) amino acids have been deleted, substituted, or added in the amino acid sequence shown in Table 1 (the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, or 10), and which has the function of a reprogramming factor. (b) A protein consisting of an amino acid sequence that has 80% or more, 85% or more, 90% or more, 95% or more, 98% or more, or 99% or more (e.g., 90% or more) sequence identity to the amino acid sequence shown in Table 1 (the amino acid sequence shown in SEQ ID NOs: 2, 4, 6, 8, or 10), and which has the function of a reprogramming factor. (c) A nucleic acid encoding the protein of (a) above. (d) A nucleic acid encoding the protein of (b) above. (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 (amino acid sequence shown in SEQ ID NO: 1, 3, 5, 7, or 9) and encodes a protein that functions as a reprogramming factor.

[0021] As used herein, "stringent conditions" may refer to, for example, 5x SSC, 5x Denhardt's solution, 0.5% SDS, 50% formamide, and 50°C. Under these conditions, it is expected that DNA and / or RNA with higher homology can be obtained more efficiently as the temperature is increased. However, several factors are thought to affect the stringency of hybridization, including temperature, DNA and / or RNA concentration, DNA and / or RNA length, ionic strength, time, and salt concentration, and one skilled in the art can achieve similar stringency by appropriately selecting these factors.

[0022] Whether a particular protein functions as a reprogramming factor can be determined by well-known methods. For example, a combination described herein containing a particular protein can be introduced into cells that are not hepatic stem cells or hepatic progenitor cells, cultured for a predetermined period of time, and then examined by determining whether the expression of markers of hepatic stem cells or hepatic progenitor cells, such as albumin, α-fetoprotein, and E-cadherin, is increased in the cells.

[0023] The combinations described herein may be, but are not limited to, the following combinations (1) to (3), such as the combination (2) or (3): (1) a combination of (a) the HNF1A gene or HNF1A protein, (b) the FOXA3 gene or FOXA3 protein, and (c) the HNF6 gene or HNF6 protein; (2) a combination of (a) the HNF1A gene, (b) the FOXA3 gene, and (c) the HNF6 gene; (3) a combination of (a) the HNF1A protein, (b) the FOXA3 protein, and (c) the HNF6 protein.

[0024] The combination may comprise, consist of, or consist essentially of the above factors.

[0025] Reprogramming factors can be produced by genetic engineering (see, for example, "Molecular Cloning, A Laboratory Manual (4th edition)" (Cold Spring Harbor Laboratory Press (2012)).

[0026] When the reprogramming factor is a gene, the gene may be, for example, DNA or RNA (e.g., mRNA). The reprogramming factor may also be contained in a vector. In one embodiment, the present invention relates to a vector comprising the combination described herein. The vector may be an expression vector in which the reprogramming factor and / or other DNA sequence is operably linked to one or more expression control sequences. Examples of expression control sequences include promoters, enhancers, and transcription termination regions. As used herein, the term "operably linked" refers to a gene being linked to an expression control sequence and being expressible in cells, etc. By operably linking the reprogramming factor to a hepatocyte-specific promoter in the vector, the specificity to hepatocytes may be enhanced, and efficacy and / or side effects may be improved. Examples of hepatocyte-specific promoters include the thyroxine-binding globulin (TBG) gene promoter, the albumin gene promoter, the transthyretin (TTR) gene promoter, the apolipoprotein gene promoter, and the Pr1 promoter.

[0027] The genes encoding the reprogramming factors may all be contained in one vector, or each gene may be contained in a separate vector, or some genes may be contained in one vector and the remaining genes may be contained in another vector.

[0028] Expression vectors that can be used herein include plasmids, bacteriophages, and viral vectors derived from retroviruses, vaccinia viruses, adenoviruses, lentiviruses, and adeno-associated viruses, such as AAV8 and Sendai virus. When AAV8, which has high affinity for hepatocytes, is used, specificity to hepatocytes can be enhanced, which can improve efficacy and / or reduce side effects.

[0029] In one embodiment, the combination described herein is comprised in a lipid nanoparticle (LNP). In one embodiment, the present invention relates to an LNP comprising a combination described herein.

[0030] When the reprogramming factor is a protein, the efficiency of introduction into cells can be increased by linking the protein to a membrane-permeable peptide or by using a cationic lipid.

[0031] The combinations described herein can be administered to a subject, for example, by injection into the skin tissue, blood vessels (eg, intravenous), or intraperitoneal cavity of an animal.

[0032] In one embodiment, the combination described herein is contained in one or more pharmaceutical compositions.

[0033] The pharmaceutical compositions described herein may contain, in addition to the combinations described herein, pharmaceutically acceptable carriers (excipients, fillers, binders, lubricants, etc.) and / or known additives (buffers, isotonicity agents, chelating agents, colorants, preservatives, fragrances, flavoring agents, sweeteners, etc.). For example, lipid nanoparticles (LNPs) can be included as carriers to facilitate gene delivery.

[0034] The pharmaceutical compositions described herein can generally be administered systemically or locally, orally or parenterally. The pharmaceutical compositions described herein can be administered to a living body, for example, by intravenous injection, intramuscular injection, intraperitoneal injection, subcutaneous injection, or suppository, and can be administered, for example, by intravenous injection. The dosage of the pharmaceuticals described herein can be appropriately determined depending on age, body weight, symptoms, therapeutic effect, administration method, treatment time, etc. For example, the pharmaceutical composition or active ingredient can be administered to an individual in an amount of 0.0001 to 100 mg / kg per day, preferably 0.01 to 100 mg / kg per day.

[0035] The pharmaceutical compositions described herein can be prepared in the form of pharmaceutically acceptable preparations, which can be prepared in the form of injections such as sterile solutions, suspensions, and freeze-dried preparations, or tablets, granules, powders, capsules, emulsions, suspensions, syrups, etc., according to conventional procedures.

[0036] A "subject" to which the combinations or pharmaceutical compositions described herein are administered includes human and non-human organisms. Examples of non-human organisms include birds and non-human mammals (e.g., cows, monkeys, cats, mice, rats, guinea pigs, hamsters, pigs, dogs, rabbits, sheep, and horses).

[0037] In one embodiment, the present invention relates to a method for treating and / or preventing liver disease, comprising administering to a subject a combination described herein, or an HNF1 gene or protein, a FOXA gene or protein, and an HNF6 gene or protein, wherein the combination, HNF1 gene or protein, FOXA gene or protein, HNF6 gene or protein, and liver disease in this embodiment are as described herein.

[0038] Example 1: Hepatocyte regeneration via in vivo reprogramming. In this example, a liver fibrosis model (liver cirrhosis model) was created by subcutaneously injecting carbon tetrachloride (Fujifilm Wako Pure Chemical Industries, Ltd.) mixed with olive oil (Nacalai Tesque) at a dose of 5 μl per gram of mouse body weight into C57BL / 6J mice (KBT Oriental) twice a week for 8 weeks. Human iHepPC inducers (FOXA3, HNF1A, and HNF6: designated h3F) were introduced using AAV8, an adeno-associated virus with high affinity for hepatocytes. Specifically, vectors containing each of the above inducers were generated by recombining the Cre sequence of AAV.TBG.PI.Cre.rBG (Plasmid #107787) (addgene) with the respective inducer sequences (the FOXA3, HNF1A, and HNF6 sequences shown in Table 1) according to standard methods. These vectors were transfected into HEK293 cells using polyethyleneimine (PEI Max) (Polysciences) according to the accompanying protocol to generate AAV8. AAV8 was injected into the tail vein once, 4 weeks after the start of carbon tetrachloride administration. While carbon tetrachloride administration continued, gene expression analysis of alpha-fetoprotein (AFP) and epithelial cell adhesion molecule (EpCAM) was performed using livers 2, 4, 7, 14, and 28 days after AAV8 administration, using qPCR and immunohistochemistry. Gene expression analysis and immunostaining were performed according to the method described by Hiroki Inada et al., Nature Communications, Vol. 11, Article number: 5292, 2020. AFP is a hepatic progenitor cell marker, and EpCAM is expressed in hepatic progenitor cells and bile duct epithelial cells. In AAV8, expression of human iHepPC-inducing factors is controlled by the hepatocyte-specific thyroxine-binding globulin (TBG) gene promoter, indicating that human iHepPC-inducing factors are primarily expressed in hepatocytes.

[0039] The results are shown in Figure 1. It was found that the gene expression levels of AFP and EpCAM peaked 7 and 14 days, respectively, after the expression of human iHepPC inducers (A). The values ​​shown in the graph are relative to the gene expression levels in the livers of mice treated with carbon tetrachloride for 4 weeks, immediately before AAV8 administration.

[0040] Furthermore, immunostaining revealed many more EpCAM-positive cells in the livers of mice expressing human iHepPC-inducing factors than in the livers of mock-control mice administered an empty vector (AAV8-TBG-Null) that did not contain the human iHepPC-inducing factors (B). Cell DNA was stained with DAPI (blue).

[0041] Example 2: Effect of in vivo reprogramming on hepatic fibrosis improvement by hepatocyte blastogenesis. In this example, a mouse model of liver cirrhosis prepared as in Example 1 was used. Four weeks after the start of carbon tetrachloride administration, AAV8 containing a human iHepPC inducer was transduced as in Example 1. Carbon tetrachloride administration was continued, and after another four weeks, liver tissue fibrosis and Col1a1 gene expression were analyzed, and serum ALT and AST levels were measured. Specifically, liver tissue was fixed in formalin, paraffin sections were prepared, and the area of ​​fibrotic tissue stained with Sirius Red was quantified using a KEYENCE BZ-X800. Furthermore, cDNA synthesized from mRNA extracted from liver tissue using ISOGEN II (NIPPON GENE) was used to synthesize cDNA using a ReverTra qPCR RT kit (TOYOBO). qPCR was performed using specific primers (TCCTCCAGGGATCCAACGA (SEQ ID NO: 11) and GGCAGGCGGGAGGTCTT (SEQ ID NO: 12)) to analyze Col1a1 gene expression. Serum ALT and AST levels were measured using Transaminase CII-Test Wako (Fujifilm Wako Pure Chemical Industries, Ltd.), and an empty vector (AAV8-TBG-Null) containing no human iHepPC inducer was used as a mock control.

[0042] The results are shown in Figure 2. Staining of fibrotic tissue with Sirius Red revealed that the amount of red-stained fibrotic tissue was reduced by the expression of human iHepPC inducer (A, B). Furthermore, gene expression of collagen (Col1a1), the main component of fibrotic tissue, was also reduced (C). Furthermore, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), liver injury markers, were also reduced by the expression of human iHepPC inducer (D, E). The values ​​in Figures 2B–E represent the endpoint value at 8 weeks divided by the value at 4 weeks, immediately before AAV8 administration.

[0043] Example 3: Fatty Liver Improvement by Hepatocyte Blastogenesis via In Vivo Reprogramming In this example, a cirrhosis model mouse prepared as in Example 1 was used. Four weeks after the start of carbon tetrachloride administration, AAV8 containing human iHepPC inducers was introduced as in Example 1. Carbon tetrachloride administration was continued, and fat deposition in liver tissue was measured after another four weeks. Specifically, frozen sections of liver tissue were prepared, and the area of ​​fat deposition stained with Oil Red O was quantified using a KEYENCE BZ-X800. An empty vector (AAV8-TBG-Null) not containing human iHepPC inducers was used as a mock control.

[0044] The results are shown in Figure 3. When lipid droplets were stained with Oil Red O, the area of ​​red-stained fat deposits was reduced by the expression of human iHepPC inducers (A, B).

[0045] Example 4: Improved Liver Histology by Hepatocyte Blastogenesis via In Vivo Reprogramming In this example, a cirrhosis model mouse prepared as in Example 1 was used. Four weeks after the start of carbon tetrachloride administration, AAV8 containing human iHepPC inducers was introduced as in Example 1. Carbon tetrachloride administration was continued, and liver histology was analyzed after another four weeks. Specifically, liver tissue was fixed in formalin, paraffin sections were prepared, and the degree of hepatocyte ballooning, steatosis, and inflammatory cell infiltration was scored after staining with hematoxylin and eosin (HE). The specific scoring method followed David E. Kleiner et al., Hepatology, 2005 Jun, 41(6):1313-21. An empty vector (AAV8-TBG-Null) lacking human iHepPC inducers was used as a mock control.

[0046] The results are shown in Figure 4. Histopathological analysis using hematoxylin and eosin (HE) staining showed that expression of human iHepPC inducer improved ballooning hepatocytes, steatosis, and inflammatory cell infiltration, and reduced NAFLD activity score (NAS) (A, B).

[0047] Example 5: Improvement of Liver Fibrosis by Expression of Human iHepPC Inducing Factors Using LNPs In this example, a cirrhosis model mouse prepared as in Example 1 was used. Four weeks after the start of carbon tetrachloride administration, lipid nanoparticles (LNPs) containing messenger RNA (mRNA) for human induced hepatic progenitor cell (human iHepPC) inducing factors (FOXA3, HNF1A, HNF6: designated h3F) were administered at a dose of 2 μg / g (mouse body weight) of mRNA once every five days for a total of six doses. The production of LNPs containing mRNA was outsourced to GenScript. The FOXA3 mRNA sequence used in this example is shown in SEQ ID NO: 13, the HNF1A mRNA sequence in SEQ ID NO: 14, and the HNF6 mRNA sequence in SEQ ID NO: 15. Carbon tetrachloride was administered continuously during LNP administration, and 4 weeks after the start of LNP administration, liver tissue fibrosis and Col1a1 gene expression were analyzed, and serum ALT and AST levels were measured as in Example 2. LNP containing green fluorescent protein (GFP) mRNA (LNP-GFP) was used as a control.

[0048] The results are shown in Figure 5. Staining of fibrotic tissue with Sirius Red revealed that the amount of red-stained fibrotic tissue was reduced by the expression of human iHepPC inducer (A, B). Furthermore, gene expression of collagen (Col1a1), a major component of fibrotic tissue, was also reduced (C). Furthermore, serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST), which are liver damage markers, were also reduced by the expression of human iHepPC inducer (D, E).

[0049] Example 6: Improvement of fatty liver by expression of human iHepPC inducer using LNP In this example, a cirrhosis model mouse prepared in the same manner as in Example 1 was used. Four weeks after the start of carbon tetrachloride administration, lipid nanoparticles (LNP) containing messenger RNA (mRNA) for human induced hepatic progenitor cells (human iHepPC) inducers (FOXA3, HNF1A, HNF6: designated h3F) were administered once every five days, a total of six times, as in Example 5. Carbon tetrachloride administration continued during this time, and fat deposition in liver tissue was measured four weeks after the start of LNP administration, as in Example 3. LNP containing green fluorescent protein (GFP) mRNA (LNP-GFP) was also used as a control.

[0050] The results are shown in Figure 6. When lipid droplets were stained with Oil Red O, the area of ​​red-stained fat deposits was reduced by the expression of human iHepPC inducer (A, B).

[0051] Example 7: Improvement of Liver Histology by Expression of Human iHepPC Inducing Factors Using LNP In this example, a cirrhosis model mouse prepared in the same manner as in Example 1 was used. Four weeks after the start of carbon tetrachloride administration, lipid nanoparticles (LNP) containing messenger RNA (mRNA) for human induced hepatic progenitor cells (human iHepPC) inducing factors (FOXA3, HNF1A, HNF6: designated as h3F) were administered once every five days, a total of six times, as in Example 5. Carbon tetrachloride was administered continuously during this period, and liver histology analysis was performed in the same manner as in Example 4 four weeks after the start of LNP administration. In addition, LNP containing green fluorescent protein (GFP) mRNA (LNP-GFP) was used as a control.

[0052] The results are shown in Figure 7. Histopathological analysis using hematoxylin and eosin (HE) staining showed that expression of human iHepPC inducer improved ballooning hepatocytes, steatosis, and inflammatory cell infiltration, and reduced NAFLD activity score (NAS) (A, B).

Claims

1. A combination for treating and / or preventing liver diseases, 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 disease is 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 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, comprising the HNF1 gene, the FOXA gene, and the HNF6 gene in a vector.

8. The combination according to claim 7, comprising the HNF1 gene, the FOXA gene, and the HNF6 gene in separate vectors.

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

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

11. The combination according to claim 8, wherein the HNF1 gene, the FOXA gene, and the HNF6 gene are operably linked to a hepatocyte-specific promoter in the vector.

12. The combination according to claim 1, comprising the HNF1 gene, the FOXA gene, and the HNF6 gene in lipid nanoparticles.

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

Images