Composition for use in treating fatty liver disease

Abstract

The present disclosure provides a pharmaceutical composition for use in treating a fatty liver disease. The present disclosure provides a composition (such as pharmaceutical composition) for use in, for example, treating a subject with a fatty liver disease. A composition (such as pharmaceutical composition) according to the present disclosure comprises an insulin-like growth factor binding protein acid-labile subunit (IGFALS) protein or a nucleic acid encoding an IGFALS.

Description

Compositions for use in treating fatty liver disease

[0001] The present disclosure relates to compositions for use in treating fatty liver disease.

[0002] In recent years, the incidence of nonalcoholic steatohepatitis (NASH) has rapidly increased both domestically and internationally, and it has become a major cause of chronic liver failure in Japan. NASH is a pathological condition in which nonalcoholic fatty liver (NAFL) causes liver inflammation and progressive liver fibrosis. Progression of NASH poses a risk of developing liver cirrhosis and liver cancer. NASH is diagnosed based on 1) the absence of a history of alcohol use, as well as liver biopsy findings of (i) macrovesicular and microvesicular fatty deposits, (ii) inflammatory cell infiltration, (iii) hepatocyte ballooning, and (iv) perihepatocyte fibrosis in the centrilobular area, and 3) the absence of other liver diseases such as viral hepatitis (e.g., HBV or HCV) or autoimmune hepatitis.

[0003] Patent Document 1 discloses that blood insulin-like growth factor binding protein acid-labile subunit (IGFALS) levels are reduced in patients with liver fibrosis, and that the degree of liver fibrosis can be predicted based on blood IGFALS levels.

[0004] WO2023 / 013764A

[0005] The present disclosure provides compositions (eg, pharmaceutical compositions) for use in treating a subject having or at risk of developing fatty liver disease.

[0006] According to the present disclosure, for example, the following inventions are provided: (1) A pharmaceutical composition for use in treating hepatitis and / or liver fibrosis in a subject with fatty liver disease or a subject at risk of developing fatty liver disease (e.g., a subject with fatty liver), the pharmaceutical composition comprising an insulin-like growth factor binding protein acid-labile subunit (IGFALS) protein or a nucleic acid encoding IGFALS. (2) The pharmaceutical composition according to (1) above, wherein the subject with fatty liver disease is a subject with non-alcoholic fatty liver disease (NAFLD) or metabolically-associated fatty liver disease (MAFLD). (3) The pharmaceutical composition according to (1) or (2) above, wherein the subject with fatty liver disease is a subject with non-alcoholic steatohepatitis (NASH). (4) The pharmaceutical composition according to any of (1) to (3) above, wherein the subject with fatty liver disease is a subject with a non-alcoholic fatty liver disease (NAFLD) activity score of 4 or higher and a fibrosis grade of 2 or higher. (5) The pharmaceutical composition according to any one of (1) to (4) above, wherein the subject with fatty liver disease has a blood IGFALS level below a reference value. (6) The pharmaceutical composition according to any one of (1) to (5) above, wherein the subject with fatty liver disease is a subject who has been shown to be suitable for administration of an IGFALS protein or a nucleic acid encoding IGFALS by a method for estimating the progression of liver fibrosis in a subject with non-alcoholic fatty liver disease (NAFLD), the method comprising measuring the subject's blood IGFALS level and comparing the blood IGFALS level with a reference value, and wherein a blood IGFALS level lower than the reference value indicates that the subject is suitable for administration of an IGFALS protein or a nucleic acid encoding IGFALS. (7) The pharmaceutical composition according to any one of (1) to (6) above, wherein the nucleic acid encoding IGFALS is messenger RNA. (8) A pharmaceutical composition according to any one of (1) to (7) above, wherein a nucleic acid encoding IGFALS is incorporated into a gene expression cassette of a viral vector, and within the expression cassette, the nucleic acid is operably linked to a control sequence, thereby enabling the IGFALS protein to be expressed in hepatocytes.(9) The pharmaceutical composition according to (8) above, wherein the viral vector is an adeno-associated viral vector (AAV).

[0007] (101) A pharmaceutical composition for use in treating hepatitis and / or liver fibrosis in a subject with NAFLD or MAFLD or a subject at risk of developing NAFLD or MAFLD (e.g., a subject with fatty liver), comprising an insulin-like growth factor binding protein acid-labile subunit (IGFALS) protein or a nucleic acid encoding IGFALS. (102) The pharmaceutical composition according to (101), wherein the subject with NAFLD or MAFLD is a subject with non-alcoholic fatty liver disease (NAFLD) or metabolically-associated fatty liver disease (MAFLD). (103) The pharmaceutical composition according to (101) or (102), wherein the subject with NAFLD or MAFLD is a subject with non-alcoholic steatohepatitis (NASH). (104) The pharmaceutical composition according to any one of (101) to (103) above, wherein the subject with NAFLD or MAFLD has a nonalcoholic fatty liver disease (NAFLD) activity score of 4 or more and a fibrosis grade of 2 or more. (105) The pharmaceutical composition according to any one of (101) to (104) above, wherein the subject with NAFLD or MAFLD has a blood IGFALS level below the reference value. (106) The pharmaceutical composition according to any one of (101) to (105), wherein the subject having NAFLD or MAFLD is a subject who has been shown to be suitable for administration of an IGFALS protein or a nucleic acid encoding IGFALS by a method for estimating the progression of liver fibrosis in a subject, the method comprising measuring the IGFALS level in the subject's blood and comparing the IGFALS level with a reference value, and determining that the IGFALS level in the blood is lower than the reference value, the subject being suitable for administration of the IGFALS protein or a nucleic acid encoding IGFALS. (107) The pharmaceutical composition according to any one of (101) to (106), wherein the nucleic acid encoding IGFALS is messenger RNA.(108) The pharmaceutical composition according to any one of (101) to (107), wherein the nucleic acid encoding IGFALS is incorporated into a gene expression cassette of a viral vector, and within the expression cassette, the nucleic acid is operably linked to a regulatory sequence, thereby enabling the IGFALS protein to be expressed in liver cells. (109) The pharmaceutical composition according to (108), wherein the viral vector is an adeno-associated viral vector (AAV).

[0008] (201) A composition (e.g., a pharmaceutical composition) for use in treating a subject with fatty liver disease or a subject at risk of developing fatty liver disease (e.g., a subject with fatty liver), comprising an insulin-like growth factor binding protein acid-labile subunit (IGFALS) protein or a nucleic acid encoding IGFALS. (202) The composition (e.g., a pharmaceutical composition) according to (201), wherein the subject with fatty liver disease is a subject with non-alcoholic fatty liver disease (NAFLD) or metabolically-associated fatty liver disease (MAFLD). (203) The composition (e.g., a pharmaceutical composition) according to (201) or (202), wherein the subject with fatty liver disease is a subject with non-alcoholic steatohepatitis (NASH). (204) The composition (e.g., pharmaceutical composition) according to any one of (201) to (203), wherein the subject with fatty liver disease has a non-alcoholic fatty liver disease (NAFLD) activity score of 4 or more and a fibrosis grade of 2 or more. (205) The composition (e.g., pharmaceutical composition) according to any one of (201) to (204), wherein the subject with fatty liver disease has a blood IGFALS level below the reference value. (206) The subject having fatty liver disease is a subject who has been shown to be suitable for administration of an IGFALS protein or a nucleic acid encoding IGFALS by a method for estimating the progression of liver fibrosis in a subject having nonalcoholic fatty liver disease (NAFLD), comprising measuring the subject's blood IGFALS level and comparing the blood IGFALS level with a reference value, and a blood IGFALS level lower than the reference value indicates that the subject is suitable for administration of an IGFALS protein or a nucleic acid encoding IGFALS. (207) The composition (e.g., pharmaceutical composition) according to any one of (201) to (206), wherein the nucleic acid encoding IGFALS is messenger RNA.(208) The composition (e.g., pharmaceutical composition) according to any one of (201) to (207), wherein the nucleic acid encoding IGFALS is incorporated into a gene expression cassette of a viral vector, and in the expression cassette, the nucleic acid is operably linked to a control sequence, thereby allowing the IGFALS protein to be expressed in liver cells. (209) The composition (e.g., pharmaceutical composition) according to (208), wherein the viral vector is an adeno-associated viral vector (AAV).

[0009] (301) A pharmaceutical composition for use in treating a subject with NAFLD or MAFLD or a subject at risk of developing NAFLD or MAFLD (e.g., a subject with fatty liver), comprising an insulin-like growth factor binding protein acid-labile subunit (IGFALS) protein or a nucleic acid encoding IGFALS. (302) The pharmaceutical composition according to (301), wherein the subject with NAFLD or MAFLD is a subject with non-alcoholic fatty liver disease (NAFLD) or metabolically-associated fatty liver disease (MAFLD). (303) The pharmaceutical composition according to (301) or (302), wherein the subject with NAFL or MAFL is a subject with non-alcoholic steatohepatitis (NASH). (304) The pharmaceutical composition according to any one of (301) to (303) above, wherein the subject with NAFLD or MAFLD has a nonalcoholic fatty liver disease (NAFLD) activity score of 4 or more and a fibrosis grade of 2 or more. (305) The pharmaceutical composition according to any one of (301) to (304) above, wherein the subject with NAFLD or MAFLD has a blood IGFALS level below the reference value. (306) The pharmaceutical composition according to any one of (301) to (305), wherein the subject having NAFLD or MAFLD is a subject who has been shown to be suitable for administration of an IGFALS protein or a nucleic acid encoding IGFALS by a method for estimating the progression of liver fibrosis in a subject, the method comprising measuring the IGFALS level in the subject's blood and comparing the IGFALS level with a reference value, and determining that the IGFALS level in the blood is lower than the reference value, the subject being suitable for administration of the IGFALS protein or a nucleic acid encoding IGFALS. (307) The pharmaceutical composition according to any one of (301) to (306), wherein the nucleic acid encoding IGFALS is messenger RNA. (308) A pharmaceutical composition according to any one of (301) to (307) above, wherein a nucleic acid encoding IGFALS is incorporated into a gene expression cassette of a viral vector, and within the expression cassette, the nucleic acid is operably linked to a control sequence, thereby allowing the IGFALS protein to be expressed in liver cells.(309) The pharmaceutical composition according to (308) above, wherein the viral vector is an adeno-associated viral vector (AAV).

[0010] Figure 1 shows that blood IGFALS levels in patients with active NASH are lower than those in patients with inactive NASH, and the relationship between blood IGFALS levels and the specificity and sensitivity of diagnosing active NASH. Figure 2 shows a schematic diagram of a NASH model treatment experiment. Figure 3 shows the relative expression levels of each mRNA in a NASH model treated with an adeno-associated virus (AAV) vector containing a nucleic acid encoding IGFALS driven by a liver-specific promoter (human thyroxine-binding globulin (TBG) promoter). The control group (normal) consisted of healthy C57BL / 6J mice of the same age. Figure 4 shows blood IGFALS and IGF1 protein levels in a NASH model treated with an adeno-associated virus (AAV) vector containing a nucleic acid encoding IGFALS driven by a liver-specific promoter or a control vector (EGFP-expressing vector). The control group (normal) consisted of healthy C57BL / 6J mice of the same age. Figure 5 shows lipid droplets (hematoxylin-eosin staining) and liver fibrosis (Sirius Red staining) in the liver tissue of a NASH model treated with an adeno-associated virus (AAV) vector containing a nucleic acid encoding IGFALS driven by a liver-specific promoter or a control vector (EGFP-expressing vector). The bar indicates 100 μm. Figure 6 shows triglyceride, cholesterol, and phospholipid levels in the liver of a NASH model treated with an adeno-associated virus (AAV) vector containing a nucleic acid encoding IGFALS driven by a liver-specific promoter or a control vector (EGFP-expressing vector). Figure 7 shows the effects of liver fibrosis in the liver of a NASH model treated with an adeno-associated virus (AAV) vector containing a nucleic acid encoding IGFALS driven by a liver-specific promoter or a control vector (EGFP-expressing vector). FIG. 8 shows the survival rate of NASH models treated with an adeno-associated virus (AAV) vector containing a nucleic acid encoding IGFALS driven by a liver-specific promoter or a control vector (EGFP-expressing vector).Figure 9 shows the body weight (A), AST level (B), ALT level (C), fasting blood glucose (D), serum NEFA level (E), and serum TG level (F) in a NASH model after treatment with an adeno-associated virus (AAV) vector containing a nucleic acid encoding IGFALS driven by a liver-specific promoter or a control vector (EGFP expression vector). Figure 10 shows the results of alpha-smooth muscle actin (αSMA) and F4 / 80 staining in a NASH model after treatment with an adeno-associated virus (AAV) vector containing a nucleic acid encoding IGFALS driven by a liver-specific promoter or a control vector (EGFP expression vector). The bar indicates 100 μm. Figure 11 shows the preparation scheme of human liver organoids (HLOs) containing multiple cell types. iPS cells (iPSCs) are treated with activin A to obtain definitive endoderm. Foregut spheroids containing both endodermal and mesodermal lineages were obtained by treatment with fibroblast growth factor 4 (FGF4) and glycogen synthase kinase 3 (GSK3) inhibitors. Human liver organoids were obtained through treatment with retinoic acid, hepatocyte growth factor (HGF), dexamethasone (DEX), and oncostatin M (OSM). Figure 12 shows the time course of collagen deposition in the interstitial region of sHLO. Figure 13 shows the accumulation of neutral lipids in HLO treated with oleic acid (OA) or palmitic acid (PA). Neutral lipids were stained with BODIPY 493 / 503. Figure 14 shows a heat map of gene expression in HLO treated with oleic acid (OA) or palmitic acid (PA). The heat map shows relative expression levels. FIG. 15 shows the results of COL1A1 staining and vimentin staining of sections of HLO cultured for 7 days in the presence of OA alone or OA with IGF1 or IGFALS, as well as the area of ​​the COL1A1-positive region.

[0011] As used herein, a "subject" may be a mammal, including, for example, primates such as humans and chimpanzees, laboratory animals such as rats, mice, and rabbits, livestock animals such as pigs, cows, horses, sheep, and goats, and pets such as dogs and cats, and is preferably a human. As used herein, a "patient" refers to a subject having a disease, preferably a human having a disease.

[0012] As used herein, "treatment" refers to therapeutic and / or prophylactic treatment. Therapeutic treatment refers to treating a subject who has developed a condition, and prophylactic treatment refers to treating a subject before the onset of a particular condition, thereby preventing the onset of that condition. Therapeutic treatment can include slowing the rate of progression of a condition, halting the progression of a condition, alleviating a condition, and curing a condition.

[0013] As used herein, "fatty liver disease" refers to a condition in which triglycerides accumulate in the liver. Clinically, patients with triglycerides accumulated in 30% or more of their hepatocytes are diagnosed as having fatty liver disease. Fatty liver disease is also called hepatic steatosis.

[0014] As used herein, "nonalcoholic fatty liver disease" (NAFLD) refers to a condition in which fat accumulates in the liver (fatty liver or hepatic steatosis), occurring in people who have never drunk alcohol or who rarely drink alcohol (less than 30 g / day for men and less than 20 g / day (ethanol equivalent) for women). Alcohol intake can be calculated by multiplying the alcohol content (%) by the amount of alcohol (mL) by the specific gravity of alcohol (0.8 g / mL). NAFLD is a general term for a range of diseases, from nonalcoholic fatty liver to steatohepatitis and liver cirrhosis. Known causes of NAFLD include lifestyle-related diseases such as obesity, diabetes, dyslipidemia, and hypertension, as well as diseases such as sleep apnea syndrome, polycystic ovary syndrome, hypothyroidism, and hypopituitarism, and the administration of total parenteral nutrition and drugs (such as tamoxifen, valproic acid, and amiodarone) after surgery such as pancreaticoduodenectomy and jejunoileal bypass surgery. A diagnosis of NAFLD can be made based on 1) no or little alcohol consumption (less than 30 g / day for men and less than 20 g / day for women), 2) liver biopsy findings showing (i) macrovesicular and microvesicular fatty deposits, (ii) inflammatory cell infiltration, (iii) hepatocyte ballooning, and (iv) perihepatocyte fibrosis in the centrilobular area, and 3) the absence of other liver diseases such as viral hepatitis (viruses include, for example, HBV and HCV) or autoimmune hepatitis.

[0015] As used herein, "metabolic-associated fatty liver disease" (MAFLD) is a disease classification proposed as a new disease classification to replace NAFLD. The diagnostic criteria for MAFLD are, in addition to the presence of hepatic steatosis, 1) a BMI of 25 kg / m 21) Presence of type 2 diabetes; or 2) Presence of two or more of the following seven metabolic at-risk criteria: Waist circumference 94 cm or more (men) or 80 cm or more (women); Blood pressure 130 / 85 mmHg or more; Plasma triglyceride level 150 mg / dL or more; HDL cholesterol level less than 40 mg / dL (men) or less than 50 mg / dL (women); Pre-diabetes; HOMA insulin resistance score 2.5 or more; Plasma hsCRP level greater than 2 mg / dL.

[0016] As used herein, "nonalcoholic fatty liver" (NAFL) refers to fatty liver that shows little progression of the disease. Approximately 80-90% of NAFLD cases fall into the category of NAFL. Treatments for subjects with NAFL include dietary and / or exercise therapy, and weight loss resulting from these. Treatment of the underlying disease (e.g., the diseases listed above) may also be effective.

[0017] As used herein, "nonalcoholic steatohepatitis" (NASH) refers to a condition in which the liver becomes inflamed due to NAFL, leading to the progression of liver fibrosis. As NASH progresses, the subject may develop cirrhosis and liver cancer. It can be diagnosed by liver biopsy, which examines liver tissue. NASH can be diagnosed when a patient has NAFLD and, in addition, 2) liver biopsy findings show either (iii) ballooning of hepatocytes or (iv) perihepatocyte fibrosis in the centrilobular area.

[0018] As used herein, "alcoholic steatohepatitis" (ASH) refers to steatohepatitis caused by habitual and continuous excessive alcohol consumption. ASH can be diagnosed by evaluating liver function abnormalities (e.g., liver tissue lesions are mainly hepatocyte degeneration or necrosis, with 1) significant swelling (ballooning) of hepatocytes mainly in the centrilobular area, 2) various degrees of hepatocyte necrosis, 3) Mallory bodies (alcohol vitreous bodies), or 4) infiltration of polymorphonuclear leukocytes), confirming alcohol drinking history (e.g., drinking an average of 60 g (equivalent to ethanol) or more per day for 5 years or more), and excluding liver damage caused by factors other than alcohol (e.g., negative hepatitis virus markers, negative antimitochondrial antibodies, and negative antinuclear antibodies).

[0019] As used herein, "hepatic fibrosis" refers to a condition in which connective tissue accumulates in the liver. Connective tissue accumulates, for example, due to liver injury repair. In particular, when injury is chronic, repair occurs repeatedly, resulting in accelerated accumulation of connective tissue and fibrosis. Pathologically, fibrosis is initiated by activation of perivascular stellate cells in the liver. These cells contribute to the inflammatory response and may produce fibrosis-inducing factors such as TGF-β while excessively producing extracellular matrix (e.g., collagen) and matrix proteins. Fibrosis grades can be determined, for example, based on Table 1 below (see the NASH clinical research network histological scoring system (Keliner et al., Hepatology, 2005)).

[0020]

[0021] Fibrosis can be improved or prevented from progressing by removing the cause.Medicines for treating fibrosis include PPAR agonists (e.g., thiazolidine derivatives), bile acid nuclear receptor agonists (e.g., bile acid analogs, e.g., obeticholic acid), CCR2 or CCR5 antagonists (e.g., cenicriviroc), kinase inhibitors (e.g., sorafenib), ASK1 antagonists (e.g., selonsertib), and collagen-specific chaperone inhibitors (e.g., siRNA or antisense oligonucleotides against HSP47).

[0022] As used herein, "insulin-like growth factor binding protein acid-labile subunit" (IGFALS) is a blood glycoprotein that binds to insulin-like growth factor 1 (IGF1) and increases its half-life and intravascular localization. IGF1 can bind to six IGF-binding proteins (IGFBP1-6). Most IGF1 circulating in the blood forms a protein complex with IGFALS in addition to either IGFBP3 or IGFBP5 (Baxter, Nat Rev Cancer 2014). Human IGFALS is registered with the National Center for Biotechnology Information (NCBI) under GENE ID: 3483, and its amino acid sequence may be, but is not limited to, the amino acid sequence registered in NCBI Reference Sequence: NP_001139478.1.

[0023] As used herein, a "blood sample" may be whole blood, serum, or plasma. Whole blood may contain an anticoagulant. Serum is a liquid component obtained by collecting blood in a container that does not contain an anticoagulant, allowing the blood to clot, and then centrifuging the collected blood. Plasma is a supernatant obtained by centrifuging blood mixed with an anticoagulant to precipitate blood cell components.

[0024] As used herein, a "protein complex" refers to a complex containing multiple proteins, each of which is associated with at least one other protein. An "antibody-protein complex" refers to a protein complex containing an antibody and a protein, in which the antibody binds to the protein. Protein complexes include protein complexes found in blood samples (protein complexes in blood samples), and are complexes that do not contain antibodies, for example, artificially produced antibodies (e.g., monoclonal antibodies, etc.) (antibody-free protein complexes).

[0025] The term "expression vector" refers to a vector containing a polynucleotide of interest and equipped with a mechanism for expressing the polynucleotide (mRNA) of interest in a cell into which the vector has been introduced. For example, a "polynucleotide expression vector" refers to a vector capable of expressing a polynucleotide in a cell into which the vector has been introduced. In a polynucleotide expression vector, the polynucleotide is operably linked to, for example, a regulatory sequence. The mRNA is translated into a protein in the cell. If the protein is a secretory protein, the protein can be secreted outside the cell.

[0026] <Method of the Present Disclosure> According to the present disclosure, a method of treating a subject is provided. The subject may be a subject with fatty liver disease (e.g., fatty liver). The subject may be a subject with NAFLD or MAFLD. The subject may be a subject with NAFLD or MAFLD, preferably a subject with fibrosis grade 1 or higher, 2 or higher, 3 or higher, or 4 or higher, particularly a subject with fibrosis grade 2 or 3. The method may include administering an IGFALS protein or a nucleic acid encoding an IGFALS protein to the subject. According to the present invention, the subject may be a subject with fatty liver disease (e.g., fatty liver) but without fibrosis.

[0027] According to the present disclosure, there is provided a method of treating a subject, wherein the subject has NAFLD or MAFLD, preferably a subject with a fibrosis grade of 1 or more, 2 or more, 3 or more, or 4 or more, particularly a subject with a fibrosis grade of 2 or 3.

[0028] According to the present disclosure, the subject may have cirrhosis. According to the present disclosure, the subject may have compensated cirrhosis. According to the present disclosure, the subject may have decompensated cirrhosis. According to the present disclosure, the subject may have hepatocellular carcinoma (HCC).

[0029] According to the present disclosure, there is provided a method for preventing hepatitis and / or liver fibrosis in a subject, wherein the subject has NAFL or MAFL, e.g., a subject without hepatitis. According to the present disclosure, there is provided a method for preventing hepatitis and / or liver fibrosis in a subject, wherein the subject has NAFLD or MAFLD, e.g., a subject without liver fibrosis. According to the present disclosure, there is provided a method for preventing NASH in a subject, wherein the subject has NAFLD or MAFLD, e.g., a subject without liver fibrosis.

[0030] The IGFALS protein is isolated or purified. As used herein, "isolated" means separated from one or more components that coexist in the expression environment. As used herein, "purified" means further concentrating the target substance after isolation or improving its purity. The nucleic acid encoding the IGFALS protein is also isolated or purified.

[0031] IGFALS protein is originally a protein secreted into blood.Therefore, IGFALS protein can be administered into blood.Administered into blood can be, for example, by intravenous administration or hepatic artery injection.Alternatively, IGFALS protein can be administered into liver.

[0032] In one aspect, the nucleic acid encoding the IGFALS protein is operably linked to a regulatory sequence. As used herein, a "regulatory sequence" refers to a sequence that drives a gene operably linked to it and has the activity of transcribing RNA from that gene. A regulatory sequence is, for example, a promoter. Examples of promoters include class I promoters (which can be used to transcribe pre-rRNA), class II promoters (which contain a core promoter and upstream promoter elements and can be used to transcribe mRNA), and class III promoters (further classified into types I, II, and III). A regulatory sequence, also known as a regulatory sequence, may be any promoter that can transcribe mRNA in cells such as animal cells and plant cells. For example, various Pol II promoters can be used as the first regulatory sequence. Pol II promoters include, but are not limited to, the CMV promoter, the EF1 promoter (EF1α promoter), the SV40 promoter, the MSCV promoter, the hTERT promoter, the β-actin promoter, the CAG promoter, and the CBh promoter. Promoters can also include promoters that drive bacteriophage-derived RNA polymerases, such as the T7 promoter, T3 promoter, and SP6 promoter, as well as Pol III promoters, such as the U6 promoter. The T7 promoter is preferably used for transcription from circular DNA, while the SP6 promoter is preferably used for transcription from linear DNA. The promoter may also be an inducible promoter. An inducible promoter is a promoter that can induce expression of a polynucleotide operably linked to the promoter only in the presence of an inducer that drives the promoter. Some inducible promoters can induce expression of a polynucleotide operably linked to the promoter only in the absence of an inhibitor that suppresses promoter activity. Inducible promoters include, but are not limited to, promoters that induce gene expression upon heating, such as heat shock promoters. Inducible promoters also include drug-activated promoters.Such drug-inducible promoters include, for example, cumate operator sequences, lambda operator sequences (e.g., 12xlambda Op), tetracycline-inducible promoters, etc. Tetracycline-inducible promoters include, for example, promoters that drive gene expression in the presence of tetracycline or its derivatives (e.g., doxycycline) or reverse tetracycline-controlled transactivator (rtTA). An example of a tetracycline-inducible promoter is the TRE3G promoter.

[0033] In one aspect, the nucleic acid encoding the IGFALS protein operably linked to a regulatory sequence may be incorporated into a protein expression vector. The protein expression vector comprises a gene expression cassette containing the nucleic acid encoding the IGFALS protein operably linked to a regulatory sequence. The protein expression vector may be, but is not limited to, a plasmid vector or a viral vector. Examples of viral vectors include lentiviral vectors, vesicular stomatitis virus (VSV) vectors, adenoviral vectors, adeno-associated virus (AAV) vectors, and Sendai virus vectors. Those skilled in the art can appropriately prepare each vector using standard methods. The AAV vector may be any of AAV1 to AAV10. In a preferred aspect, the AAV vector has liver tropism. In a preferred aspect, the AAV vector may be any of AAV2, AAV3, AAV6, AAV7, AAV8, and AAV9. In a preferred aspect, the protein expression vector may be an AAV8 vector.

[0034] The regulatory sequence may preferably be a liver-specific promoter, which can induce liver-specific expression of a gene operably linked thereto. Examples of liver-specific promoters include the human thyroxine-binding globulin (TBG) promoter, the albumin promoter, and the alpha-1-antitrypsin promoter.

[0035] The gene encoding the IGFALS protein operably linked to the control sequence can be transcribed and translated in mammalian cells to produce the IGFALS protein.The IGFALS protein can be, but is not limited to, a wild-type IGFALS, and can be, for example, an IGFALS having an amino acid sequence registered in the National Center for Biotechnology Information (NCBI) database, for example, NCBI Reference Sequence: NP_001139478.1, or a sequence corresponding thereto (particularly a wild-type IGFALS or a functional IGFALS, particularly a naturally occurring functional IGFALS).The IGFALS can also be a protein having 90% or more sequence identity with the above amino acid sequence and having the function of IGFALS. The function of IGFALS may be, but is not limited to, for example, the ability to bind to IGF1 and IGFBP to form a complex, the ability to dissociate from the complex, or the ability to stabilize IGF1 or IGFBP in the complex.

[0036] In some embodiments, a gene encoding an IGFALS protein operably linked to a regulatory sequence can be carried in a protein expression vector. Thus, the present disclosure provides a protein expression vector carrying a gene encoding an IGFALS protein operably linked to a regulatory sequence in an expressible manner. The protein expression vector can be, for example, a viral vector.

[0037] In some embodiments, the nucleic acid encoding the IGFALS protein may be messenger RNA (mRNA). In some embodiments, at least one uridine in the mRNA may be changed to pseudouridine. The pseudouridine may be 1-methylpseudouridine. The mRNA may be transcribed from cDNA, i.e., it may not have an intron. The mRNA may also have a cap structure at the 5' end (Furuichi Y & Miura K. Nature. 1975;253(5490):374-5). The cap structure can be added to the mRNA by the Anti-Reverse Cap Analogues (ARCA) method using a cap analog (Stepinski J et al. RNA. 2001 Oct;7(10):1486-95). The Cap0 structure of mRNA can be converted to a Cap1 structure by further performing 2'-O methyltransferase treatment. This can be performed by conventional methods, for example, using commercially available kits such as the ScriptCap m7G Capping System and the ScriptCap 2'-O-Methyltransferase Kit, or the T7 mScript Standard mRNA Production System (AR Brown CO., LTD). The mRNA can have a polyA tail. The addition of the polyA tail can be performed by conventional methods, for example, using the A-Plus Poly(A) Polymerase Tailing Kit (AR Brown CO., LTD). Therefore, in one embodiment, the mRNA may be an mRNA having a Cap structure at the 5'-end and a polyA at the 3'-end, and preferably in which at least a portion of the uridines is pseudouridine (preferably 1-methylpseudouridine). The mRNA may be isolated or synthetic.

[0038] mRNA can be encapsulated in lipid nanoparticles (LNPs). This prevents degradation of mRNA in vivo and improves the efficiency of mRNA delivery into cells. Therefore, in one embodiment, the mRNA may have a Cap structure at the 5' end and a polyA at the 3' end, and preferably, at least a portion of the uridines is pseudouridine (preferably 1-methylpseudouridine). Lipid nanoparticles encapsulating such mRNA are also provided. Lipid nanoparticles that can be used include, but are not limited to, lipid nanoparticles described in US Pat. Nos. 9,364,435B, 8,822,668B, 8,802,644B, and 8,058,069B2. Alternatively, mRNA may be encapsulated in a polyion complex micelle or a polyion complex polymersome (Miyata et al., Chem. Soc. Rev., 2012, 41, 2562-2574).

[0039] In one aspect of the present invention, the subject may be a subject whose liver fibrosis level (or the progression of liver fibrosis, liver fibrosis grade, or liver fibrosis score) has been estimated by a method for estimating the liver fibrosis level (or the progression of liver fibrosis, liver fibrosis grade, or liver fibrosis score). In one aspect, the subject may be a subject estimated to have grade 1A, preferably grade 2, or grade 3 fibrosis.

[0040] In one aspect of the present invention, the subject may be a subject at risk of developing fatty liver disease, for example, a subject determined to have low blood IGF1 levels and / or low IGFALS protein levels. Whether or not a subject has low blood IGF1 levels and / or low IGFALS protein levels can be determined by the method described below. A subject at risk of developing fatty liver disease may be, for example, a subject with obesity (e.g., a body mass index (BMI) of 25 or more). BMI is calculated as weight (kg) / (height (m)). 2In one embodiment of the present invention, the treatment enables a subject to exhibit a blood IGF1 protein level equivalent to that of a healthy subject (e.g., within ±30% of that of a healthy subject, preferably within ±20% of that of a healthy subject).

[0041] In one embodiment of the present invention, a subject may be treated to suppress the expansion of the hepatic fibrotic area or the amount of fibrosis accumulation after the treatment. For example, a subject may be treated to suppress the expansion of the hepatic fibrotic area or the amount of fibrosis accumulation after the treatment, compared to a subject who did not receive the treatment or compared to the expansion of the hepatic fibrotic area or the amount of fibrosis accumulation that would be expected if the subject did not receive the treatment.

[0042] In some aspects of the invention, treatment of a subject can improve the subject's survival rate after treatment. For example, treatment of a subject can improve the subject's survival rate after treatment compared to a subject that does not receive the treatment or the survival rate that would be expected if the treatment were not received. In some aspects of the invention, treatment of a subject can reduce or inhibit an increase in the subject's neutral lipid (triglyceride) levels after treatment, for example, lowering neutral lipid levels compared to the neutral lipid levels before administration or compared to a subject that does not receive the treatment or the neutral lipid levels that would be expected if the treatment were not received.

[0043] In some embodiments of the present invention, treatment of a subject can increase IGFALS in the subject after treatment, hi some embodiments, treatment of a subject can increase IGFALS in the subject after treatment by 50% to 300% (e.g., 100% to 200%).

[0044] In one embodiment, a method for estimating liver fibrosis level (or progression of liver fibrosis, liver fibrosis grade, or liver fibrosis score) comprises carrying out the following estimation method. In one embodiment, a method is provided which comprises carrying out the following estimation method and then carrying out the above-mentioned treatment method.

[0045] In some aspects, administration of an IGFALS protein or a nucleic acid encoding an IGFALS protein causes the subject's blood IGFALS protein level to exceed a predetermined cutoff value for blood IGFALS levels.

[0046] <Prediction method of the present disclosure> The method of the present disclosure includes measuring the protein level (i.e., concentration) of IGFALS in a biological sample (e.g., a body fluid sample, preferably a blood sample) obtained from a subject (see WO2023 / 013764A). The biological sample may preferably be a blood sample. Using a blood sample can be advantageous in that it allows for minimally invasive testing and repeated testing, in that it can avoid the liver biopsy that has previously been required for diagnosis. Measurement can be performed using either serum or plasma.

[0047] According to the present disclosure, as liver fibrosis progresses, the blood IGFALS protein level can decrease.In particular, in liver fibrosis grade 2 or higher, the average blood IGFALS protein level can be, for example, half or less of the average of healthy individuals.In some aspects of the present disclosure, the subject can be a NASH patient who does not have fibrosis (has fibrosis grade 0) or has the possibility of having fibrosis.

[0048] In certain aspects of the present disclosure, the patient may be a NASH patient with or at risk of having grade 1 fibrosis (e.g., a grade selected from the group consisting of grades 1A, 1B, and 1C).

[0049] In certain aspects of the present disclosure, the subject may be a NASH patient with or at risk of having grade 2 fibrosis.

[0050] In certain aspects of the present disclosure, the subject may be a NASH patient with or at risk of having grade 3 fibrosis.

[0051] In certain aspects of the present disclosure, the subject may be a NASH patient with or at risk of having grade 4 fibrosis.

[0052] In a preferred embodiment, the subject may be one or more NASH patients selected from the group consisting of NASH patients with no fibrosis (having grade 0 fibrosis) or with the potential for fibrosis, NASH patients with grade 1 fibrosis (e.g., a grade selected from the group consisting of grades 1A, 1B, and 1C) or with the potential for fibrosis, and NASH patients with grade 2 fibrosis or with the potential for fibrosis.

[0053] In a preferred embodiment, the subject with the above-mentioned grade of fibrosis may be a subject diagnosed with NASH or NAFL based on, for example, a drinking history, a liver biopsy, and the exclusion of hepatitis due to other causes, such as viral hepatitis, as described above. In this embodiment, the subject may have a further fibrosis grade determined. Thus, in one embodiment, the liver fibrosis grade can be estimated by a body fluid biopsy (liquid biopsy) for a patient whose presence or absence of fibrosis and liver fibrosis grade have been determined by a liver biopsy or the like.

[0054] In some embodiments, the method of the present disclosure may further include (i) comparing the subject's blood IGFALS protein level (measured value) with (ii) (a) a reference value for healthy individuals (first reference value). If the measured value (the measured value) in (i) is lower than the first reference value, the subject is indicated to have or be likely to have fibrosis of fibrosis grade 2 or higher. Therefore, the method of the present disclosure may further include predicting that a subject having a blood IGFALS protein level lower than the first reference value has or is likely to have fibrosis of fibrosis grade 2 or higher.

[0055] In one aspect, the method of the present disclosure includes (i) comparing the subject's blood IGFALS protein level (measured value) with (ii) (a) a reference value for healthy individuals (first reference value), and may further include inferring that a subject from a blood sample in which the ratio of the subject's blood IGFALS protein level (measured value) to the first reference value (the measured value / first reference value) is equal to or less than a first predetermined cutoff value has or is likely to have liver fibrosis of grade 2 or higher.

[0056] In one aspect, the method of the present disclosure may further include: (i) comparing the blood IGFALS protein level of the subject (measured value) with (ii) (a) a reference value for healthy individuals (first reference value); and inferring that a subject from a blood sample in which the ratio of the measured value to the first reference value (measured value / first reference value) is equal to or greater than a first predetermined cutoff value does not have or is likely to have liver fibrosis of grade 2 or higher (i.e., is likely to not have liver fibrosis of grade 2 or higher).

[0057] The first reference value may be, for example, the mean value, first quartile value, or minimum value of the IGFALS level (preferably, the blood IGFALS level) in a biological sample (e.g., a body fluid sample, preferably a blood sample) of a healthy individual.

[0058] The first predetermined cutoff value may be, for example, less than 1.0, 0.9 or less, 0.8 or less, 0.7 or less, 2 / 3 or less, 0.6 or less, 0.55 or less, 0.5 or less, 0.45 or less, or 0.4 or less. The first predetermined cutoff value may be 0.4 or more, 0.45 or more, 0.5 or more, or 0.55 or more. In a preferred embodiment, the first predetermined cutoff value may be a value in the range of 0.4 to 0.7, preferably 0.5 to 0.6.

[0059] In one aspect, the first reference value may be the average blood IGFALS level of healthy individuals, and the first cutoff value may be a numerical value of 2 / 3 or less, for example, a numerical value in the range of 0.4 to 0.7, preferably a numerical value in the range of 0.5 to 0.6. In one aspect, the first reference value may be the first quartile of blood IGFALS levels of healthy individuals, and the first cutoff value may be a numerical value of 0.9 or less, 0.8 or less, 0.7 or less, or 2 / 3 or less. In one aspect, the first reference value may be the minimum blood IGFALS level of healthy individuals, and the first cutoff value may be a numerical value less than 1.

[0060] In one aspect, the disclosed method may include comparing (i) the blood IGFALS protein level of the subject (measured value) with (ii)(b) a reference value (second reference value) for subjects with liver fibrosis of fibrosis grade 2-3.

[0061] In one aspect, the method of the present disclosure comprises: (i) comparing the blood IGFALS protein level of the subject (measured value) with (ii) (b) a reference value (second reference value) for subjects with liver fibrosis of fibrosis grade 2 to 3; and may further comprise inferring that a subject from a blood sample in which the ratio of the measured value to the second reference value (measured value / second reference value) is less than a second predetermined cutoff value has or is likely to have liver fibrosis of grade 2 or higher.

[0062] In one aspect, the method of the present disclosure may further include: (i) comparing the blood IGFALS protein level of the subject (measured value) with (ii) (b) a reference value (second reference value) for subjects with liver fibrosis of fibrosis grade 2 to 3; and inferring that a subject from a blood sample in which the ratio of the measured value to the second reference value (the measured value / second reference value) is equal to or greater than a second predetermined cutoff value does not have or is likely to have liver fibrosis of grade 2 or higher (i.e., is likely to not have liver fibrosis of grade 2 or higher).

[0063] The second reference value can be the mean, third quartile, or maximum blood IGFALS level of subjects with fibrosis grade 2-3 liver fibrosis.

[0064] The second cutoff value may be a numerical value of 1.8 or less, 1.7 or less, 1.6 or less, 1.5 or less, 1.4 or less, 1.3 or less, 1.2 or less, 1.1 or less, or 1 or less. The second cutoff value may be, for example, a numerical value of 1 or more, 1.1 or more, 1.2 or more, 1.3 or more, or 1.4 or more. The second cutoff value may be, for example, a numerical value in the range of 1 to 1.8, or a numerical value in the range of 1 to 1.5.

[0065] In one aspect, the second reference value is the average blood IGFALS level of subjects with liver fibrosis of fibrosis grade 2 to 3, and the second cutoff value can be a value of 1.8 or less, a value of 1.5 or less, or a value in the range of 1 to 1.8, preferably a value in the range of 1 to 1.5.

[0066] In certain aspects, the methods of the present disclosure may include comparing (i) the subject's blood IGFALS protein level (measured) with (ii)(c) a predetermined cutoff value for blood IGFALS levels.

[0067] In one aspect, the method of the present disclosure may further include: (i) comparing the subject's blood IGFALS protein level (measured value) with (ii)(c) a predetermined cutoff value for blood IGFALS levels; and inferring that a subject from a blood sample in which the measured value is less than the predetermined cutoff value for blood IGFALS levels has or is likely to have liver fibrosis of grade 2 or higher.

[0068] In one aspect, the method of the present disclosure may further include: (i) comparing the subject's blood IGFALS protein level (measured value) with (ii)(c) a predetermined cutoff value for blood IGFALS levels; and inferring that subjects from blood samples in which the measured value is equal to or greater than the predetermined cutoff value for blood IGFALS levels do not have or are likely to have liver fibrosis with a liver fibrosis grade of 2 or higher.

[0069] The predetermined cutoff value for blood IGFALS levels can be, for example, a value of 7 μg / mL or less, a value of 6.9 μg / mL or less, a value of 6.8 μg / mL or less, a value of 6.7 μg / mL or less, a value of 6.6 μg / mL or less, a value of 6.5 μg / mL or less, a value of 6.4 μg / mL or less, a value of 6.3 μg / mL or less, a value of 6.2 μg / mL or less, a value of 6.1 μg / mL or less, a value of 6.0 μg / mL or less, a value of 5.9 μg / mL or less, a value of 5.8 μg / mL or less, a value of 5.7 μg / mL or less, a value of 5.6 μg / mL or less, a value of 5.5 μg / mL or less, a value of 5.4 μg / mL or less, a value of 5.3 μg / mL or less, a value of 5.2 μg / mL or less, a value of 5.1 μg / mL or less, or a value of 5.0 μg / mL or less. The predetermined cutoff value for the blood IGFALS level may be, for example, 5.0 μg / mL or more, 5.1 μg / mL or more, 5.2 μg / mL or more, 5.3 μg / mL or more, 5.4 μg / mL or more, 5.5 μg / mL or more, 5.6 μg / mL or more, 5.7 μg / mL or more, 5.8 μg / mL or more, 5.9 μg / mL or more, or 6.0 μg / mL or more. In a preferred embodiment, the predetermined cutoff value for the blood IGFALS level may be, for example, 4 μg / mL to 7 μg / mL, and more preferably 5 μg / mL to 6 μg / mL.

[0070] According to the present disclosure, there is provided a method as described above, wherein the subject has liver fibrosis of grade 0 or 1 when the method is first performed, and the method of the present disclosure is performed using blood samples obtained from the subject at multiple time points, and determining that the liver fibrosis of the subject estimated to have liver fibrosis of grade 2 or higher in each blood sample obtained after a specific time point has progressed from grade 0 or 1 to grade 2 or higher, and / or estimating the time when the liver fibrosis of the subject estimated to have liver fibrosis of grade 2 or higher progressed from grade 0 or 1 to grade 2 or higher.

[0071] While the subject's fibrosis is grade 0 or 1, the method includes estimating the subject as not having or potentially having fibrosis of liver fibrosis grade 2 or higher. After the subject's fibrosis reaches grade 2 or higher, the method includes estimating the subject as having or potentially having fibrosis of liver fibrosis grade 2 or higher. When the subject's liver fibrosis grade progresses to grade 2 or higher, it can be determined that the subject's fibrosis has progressed to grade 2 or higher. Furthermore, when the subject's liver fibrosis grade progresses to grade 2 or higher, it can be estimated that the liver fibrosis has progressed from grade 0 or 1 to grade 2 or higher between the time of collection of the last blood sample with liver fibrosis grade 0 or 1 and the time of collection of the first blood sample with liver fibrosis grade 2. In this way, the time when the subject's liver fibrosis transitioned from grade 0 or 1 to grade 2 or higher can be estimated. The method of the present disclosure uses a blood sample and is minimally invasive, allowing for repeated testing. Therefore, the method of the present disclosure is suitable for monitoring liver fibrosis grade over time.

[0072] In a preferred embodiment, the blood IGFALS protein level can be estimated by measuring the serum or plasma IGFALS protein level. The serum or plasma IGFALS protein level can be determined by enzyme-linked immunosorbent assay (ELISA). Before measurement, the blood sample may be subjected to gel filtration or other methods to separate IGFALS from other contaminants.

[0073] ELISA includes direct, indirect, sandwich, and competitive methods. In the direct method, IGFALS is immobilized on a support (e.g., a plate surface), and after washing, the immobilized IGFALS is detected with a labeled antibody. In the indirect method, IGFALS is immobilized on a support (e.g., a plate surface), and after washing, the immobilized IGFALS is bound to an antibody, and after further washing, the antibody bound to IGFALS is detected with a labeled secondary antibody. In the sandwich method, a first antibody is immobilized on a support (e.g., a plate surface), and after washing, IGFALS is bound to the immobilized first antibody, and after further washing, IGFALS is detected with a labeled second antibody. In the competitive method, an antibody is immobilized on a support (e.g., a plate surface), washed, and then a sample containing a certain concentration of labeled IGFALS is contacted with the immobilized antibody, and after washing, the amount of label remaining on the support is measured to estimate the IGFALS concentration in the blood or sample. In either case, an antibody that binds to IGFALS can be used.

[0074] In a preferred embodiment, the antibody that binds to IGFALS may be specific for IGFALS. In a preferred embodiment, the antibody that binds to IGFALS may have a specificity of 10 to IGFALS. -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 It can bind with a KD value of M or less.

[0075] In a preferred embodiment, the first antibody and the second antibody can bind to IGFALS simultaneously, and in particular, the first antibody, the second antibody, and IGFALS can form a complex (protein-antibody complex) comprising the first antibody, the second antibody, and IGFALS.

[0076] In some embodiments, IGFALS may form a protein complex with IGF1 and / or IGFBP-3, particularly with IGF1 and IGFBP-3, in blood or a sample. In some embodiments, IGFALS may form a protein complex with IGF1 and / or IGFBP-5, particularly with IGF1 and IGFBP-5, in blood or a sample. Therefore, IGFALS may be measured by detecting the protein complex. In this case, the protein complex can be detected with an antibody that binds to IGFALS and an antibody that binds to IGF1 and / or IGFBP-3 (or IGFBP-5). For example, the protein complex can be adsorbed to a solid surface with an antibody that binds to immobilized IGFALS, and then the protein complex can be detected with an antibody that binds to IGF1 and / or an antibody that binds to IGFBP-3 (or IGFBP-5). Alternatively, the protein complex can be adsorbed to a solid surface using an immobilized antibody that binds to IGF1 and / or an antibody that binds to IGFBP-3 (or IGFBP-5), and then detected using an antibody that binds to IGFALS. Thus, in one aspect, for example, the first antibody may comprise an antibody that binds to IGFALS, and the second antibody may comprise an antibody that binds to IGF1 and / or an antibody that binds to IGFBP-3 (or IGFBP-5), or the first antibody may comprise an antibody that binds to IGF1 and / or an antibody that binds to IGFBP-3 (or IGFBP-5), and the second antibody may comprise an antibody that binds to IGFALS. The protein complex may be detected using the above-mentioned antibodies by a sandwich assay, or by the immunochromatography method described below.

[0077] In some embodiments, IGFALS levels can be measured by chemiluminescent enzyme immunoassay (CLEIA). IGFALS or a protein complex containing IGFALS (e.g., a protein complex with IGF1 and IGF3 (or IGFBP-5)) can be adsorbed (captured) onto a solid surface using an immobilized first antibody. The solid phase can be a plate surface or a bead (e.g., magnetic bead) surface. The solid surface can be washed, and then IGFALS or IGFALS-containing multimers adsorbed to the solid surface can be detected using a second antibody. The magnetic beads can be washed by contacting a magnet with the container from the outside, capturing the magnetic beads on the inner surface of the contact area, and then performing a solution exchange. Various techniques for increasing the sensitivity of CLEIA can be combined with this detection. IGFALS can be captured onto the solid phase directly using a first antibody, or it can be captured using a non-immobilized first antibody in combination with an additional antibody that recognizes the immobilized first antibody. In this case, a complex is formed in the following order: solid phase-additional antibody-first antibody. When the first antibody binds to IGFALS or a protein complex containing IGFALS, the resulting complex can become an antibody complex containing solid phase-additional antibody-first antibody-IGFALS or a protein complex containing IGFALS. After antibody complex formation, washing is performed, and then the bond between the additional antibody and the first antibody is cleaved, thereby releasing an antibody complex containing the first antibody-IGFALS or a protein complex containing the first antibody-IGFALS into the supernatant. This antibody complex can be recovered, captured on another solid surface, and detected with a second antibody. The additional antibody may recognize the first antibody via a labeled molecule bound to the first antibody. The labeled molecule can be dissociated from its bond with the additional antibody, for example, by introducing an excess of free labeled molecule into the system. In this way, the bond between the additional antibody and the first antibody can be cleaved. The labeling molecule is not particularly limited, but may be, for example, 2,4-dinitrophenyl (DNP), in which case the additional antibody may be an antibody that recognizes DNP.

[0078] In a preferred embodiment, the first and second antibodies may be monoclonal antibodies.

[0079] In one aspect, the first antibody and the second antibody may be an antibody having the heavy chain CDR1-3 and light chain CDR1-3 of the anti-human IGFALS antibody produced from clone M6005C04 as the heavy chain CDR1-3 and light chain CDR1-3, respectively, and an antibody having the heavy chain CDR1-3 and light chain CDR1-3 of the anti-human IGFALS antibody produced from clone M6001E07 as the heavy chain CDR1-3 and light chain CDR1-3, respectively. In another embodiment, the first antibody and the second antibody may be an antibody having, as heavy chain CDR1-3 and light chain CDR1-3, respectively, the heavy chain CDR1-3 and light chain CDR1-3 of the anti-human IGFALS antibody produced from clone M6001E07, and an antibody having, as heavy chain CDR1-3 and light chain CDR1-3, respectively, the heavy chain CDR1-3 and light chain CDR1-3 of the anti-human IGFALS antibody produced from clone M6005C04. CDRs can be determined based on the numbering of Kabat et al. (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., 1991, Bethesda: US Dept. of Health and Human Services, PHS, NIH.), Chothia, AbM, contact, IMGT, Aho, or Martin (Enhanced Chothia), etc.

[0080] In one aspect, the first antibody and the second antibody may be, respectively, an antibody having the heavy chain variable region and light chain variable region of the anti-human IGFALS antibody produced from clone M6005C04, and an antibody having the heavy chain variable region and light chain variable region of the anti-human IGFALS antibody produced from clone M6001E07. In one aspect, the first antibody and the second antibody may be, respectively, an antibody having the heavy chain variable region and light chain variable region of the anti-human IGFALS antibody produced from clone M6001E07, and an antibody having the heavy chain variable region and light chain variable region of the anti-human IGFALS antibody produced from clone M6005C04.

[0081] In some embodiments, the first and second antibodies may be anti-human IGFALS antibodies produced from clones M6005C04 and M6001E07, respectively. In some embodiments, the first and second antibodies may be anti-human IGFALS antibodies produced from clones M6001E07 and M6005C04, respectively.

[0082] As a label, an enzyme used in enzyme antibody techniques (e.g., peroxidase, glucose oxidase, and alkaline phosphatase) can be used. As a substrate, a substrate of the above enzyme (a chromogenic substrate, a fluorescent substrate, or a luminescent substrate) can be used. Alternatively, biotin can be used as a label, and an avidin-labeled enzyme can be used to detect a biotinylated antibody. As the avidin, streptavidin, neutravidin, etc. can be used. In either case, the IGFALS level is measured or estimated based on the amount of substrate conversion by the enzyme linked to the antibody bound to IGFALS. As a substrate, a substance that is colored or discolored by the enzyme is preferably used. Calibration methods for estimation and specific estimations can be performed by conventional methods.

[0083] Those skilled in the art can label antibodies using conventional methods. Labeling can be performed, for example, by covalent bonding. In this way, labeled antibodies can be obtained.

[0084] An example of a peroxidase that can be used is horseradish peroxidase (HRP). Horseradish peroxidase generates color, fluorescence, or chemiluminescence when a chromogenic, fluorogenic, or luminescent substrate is added. Therefore, the presence of a labeled capture molecule can be detected using the color, fluorescence, or chemiluminescence as an indicator. Chromogenic substrates for horseradish peroxidase include, for example, tetramethylbenzidine (TMB), o-phenylenediamine (OPD), 2,2-azinobis[3-ethylbenzo-thiazoline-6-sulfonic acid (ABTS)], and Amplex™ Red, which can be used to detect labeled molecules in the presence of hydrogen peroxide.

[0085] Luminescent substrates for alkaline phosphatase include p-nitrophenyl phosphate (pNPP), 4-methylumbelliferyl phosphate (4-MUP), and AttoPhos™, which can be used to detect the captured molecule.

[0086] Glucose oxidase oxidizes glucose to generate gluconic acid and hydrogen peroxide. Hydrogen peroxide can be easily detected using, for example, a colorimetric probe for detecting hydrogen peroxide (e.g., peroxidase). Hydrogen peroxide can be used to generate a color in the presence of peroxidase and its color-developing substrate.

[0087] In the above-described methods of the present disclosure, the sensitivity and specificity may be 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, respectively.

[0088] In the above-described method of the present disclosure, the levels of blood biomarkers other than blood IGFALS can be disregarded, i.e., in the above-described method of the present disclosure, only the blood IGFALS level can be used for evaluation.

[0089] In the above-described methods of the present disclosure, the blood IGFALS level can be estimated by the serum IGFALS level.

[0090] In certain aspects of the methods of the present disclosure, the blood IGFALS level can be determined by mass spectrometry. Mass spectrometry can be performed appropriately by those skilled in the art. Examples of mass spectrometry include matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, electrospray ionization (ESI) mass spectrometry, surface-enhanced laser desorption / ionization time-of-flight (SELDI-TOF) mass spectrometry, quadrupole time-of-flight (Q-TOF) mass spectrometry, atmospheric pressure photoionization mass spectrometry (APPI-MS), Fourier transform mass spectrometry (FTMS), matrix-assisted laser desorption / ionization Fourier transform ion cyclotron resonance mass spectrometry, and secondary ion mass spectrometry (SIMS). Prior to mass spectrometry, the blood sample may be separated, concentrated, or purified by gel filtration or liquid chromatography.

[0091] The method of the present disclosure is an in vitro method. In the method of the present disclosure, the term "method for estimating liver fibrosis level" can be read as "method for estimating liver fibrosis level", "method for determining liver fibrosis level", or "method for obtaining preliminary information for diagnosing liver fibrosis level". In some embodiments, the method of the present disclosure includes a step of diagnosing a human. In some embodiments, the method of the present disclosure does not include a step of diagnosing a human.

[0092] <Composition or pharmaceutical composition of the present disclosure and others> According to the present disclosure, a composition (e.g., supplement) may comprise an effective amount of IGFALS protein or a nucleic acid encoding IGFALS protein is provided. According to the present disclosure, a pharmaceutical composition may comprise a therapeutically effective amount of IGFALS protein or a nucleic acid encoding IGFALS protein is provided. A therapeutically effective amount is the amount of active ingredient that achieves therapeutic benefit and does not cause unacceptable side effects.

[0093] According to the present disclosure, for example, a composition (e.g., a pharmaceutical composition) comprising a gene encoding an IGFALS protein operably linked to such a regulatory sequence is provided. According to the present disclosure, for example, a composition (e.g., a pharmaceutical composition) comprising a protein expression vector carrying a gene encoding an IGFALS protein operably linked to a regulatory sequence is provided.

[0094] The present disclosure provides a composition (e.g., a pharmaceutical composition) containing mRNA encoding an IGFALS protein. The present disclosure also provides nanovesicles encapsulating mRNA encoding an IGFALS protein. The nanovesicles may be vesicles with a submicrometer particle size, preferably vesicles with a diameter of 100 nm or less. The nanovesicles may be lipid nanovesicles. The nanovesicles may be polyion complex micelles.

[0095] According to the present disclosure, there is provided a composition (e.g., a pharmaceutical composition) comprising an IGFALS protein. The IGFALS protein may not have a secretory signal sequence.

[0096] The composition (e.g., pharmaceutical composition) may further contain a pharmaceutically acceptable solvent, excipient, and / or additive. Examples of the solvent include water, preferably water for injection. Examples of the additive include an isotonic agent, a pH adjuster, a dispersant, a surfactant, and a salt. The composition may be a food or beverage composition (e.g., a food composition or a beverage composition). The composition may be, for example, a supplement.

[0097] Also according to the present disclosure, a composition (e.g., a pharmaceutical composition) can be used in the above method. Thus, according to the present disclosure, a composition (e.g., a pharmaceutical composition) for use in the above method is provided.

[0098] The present disclosure provides the use of an IGFALS protein in the manufacture of a composition (e.g., a medicament) for use in the above method. The present disclosure also provides the use of a nucleic acid encoding an IGFALS protein in the manufacture of a composition (e.g., a medicament) for use in the above method.

[0099] According to the present disclosure, there are provided an IGFALS protein for use in the above-mentioned method. Also, according to the present disclosure, there are provided nucleic acids encoding the IGFALS protein for use in the above-mentioned method.

[0100] According to the present disclosure, the methods or compositions (e.g., pharmaceutical compositions) of the present invention can be used in combination with IGF1 or IGFBP or nucleic acids encoding same. According to the present disclosure, the methods or compositions (e.g., pharmaceutical compositions) of the present invention can be used in combination with drugs for treating fibrosis.

[0101] Materials and Methods 1. Animal Experiments C57BL / 6J male mice were purchased from CLEA Japan or Sankyo Lab Services. All mouse experiments were approved by the Animal Experimentation Committee of Tokyo Medical and Dental University and conducted in accordance with the Guidelines for the Care and Use of Laboratory Animals. Mice were housed in a standard environment with a 12-hour light-dark cycle (8:00 AM to 8:00 PM) and provided with food and water ad libitum. Male, 6-week-old STAM mice were purchased from SMC Laboratories. NASH was induced in the STAM mouse model by a single subcutaneous injection of 200 μg of streptozotocin (Sigma-Aldrich, Japan) on day 2 of life and then by feeding High Fat Diet 32 ​​(radiation-sterilized, CLEA Japan) 4 weeks after birth. Survival, clinical symptoms, and behavior were monitored daily, and body weights were recorded weekly and before administration. To measure fasting blood glucose levels, 11-week-old mice were fasted overnight (16 h). Blood glucose levels were measured from tail blood using LAB Gluco (Research & Innovation Japan, JAPAN).

[0102] To induce liver-specific expression of IGFALS and EGFP, AAV8-TGB-IGFALS or AAV8-TGB-EGFP (VectorBuilder, VB210929-1133cnx and VB211004-1032kjx) containing mouse IGFALS ORF (NM_001364896.1) were transfected at 1.2 × 10 per mouse. 11The genome copies were resuspended in 100 μl and injected intravenously into 7-week-old STAM mice via the tail vein. After viral transduction, the mice were euthanized by exsanguination under isoflurane anesthesia at 13 weeks of age.

[0103] 2. Blood Biochemistry Analysis. Mouse serum IGF1 and IGFALS concentrations were measured by ELISA using the IGF1 ELISA Kit Mouse (Proteintech) and Mouse IGFALS ELISA Kit (CUSABIO), respectively. Serum AST and ALT concentrations were measured using a FUJI DRI-CHEM NX500V (Fujifilm Corporation). Blood samples were collected from free-feeding mice to quantify serum triglyceride and nonesterified fatty acid levels. These serum samples were measured using the Lab Assay NEFA and Triglyceride Kit (Fujifilm Wako Shibayagi Co., Ltd.).

[0104] 3. Creation of human iPS cell-derived liver organoids (HLOs). Cell culture was performed in an incubator at 37°C and 95% humidity. The human iPS cell line 1383D6 was provided by the Center for iPS Cell Research and Application, Kyoto University. Human iPS cells were maintained in culture on dishes or plates coated with iMatrix-511 (Nippi) using StemFit AK02N (Ajinomoto) medium.

[0105] Liver organoids were generated from human iPS cells according to the method of Ouchi et al., Cell Metab., 30(2):374-384, 2019. Specifically, human iPS cells were detached with Accutase (Innovative Cell Technologies) and then cultured at a density of 1 × 10 5 cells / cm 2The following day (day 1), the cells were cultured in RPMI 1640 medium (Nacalai Tesque) containing 100 ng / ml Activin A (R&D Systems), 50 ng / ml BMP4 (R&D Systems), and 1x NEAA (ThermoFisher Scientific). On day 2, the cells were cultured in RPMI 1640 medium containing 100 ng / ml Activin A, 0.2% Hyclone dFBS (GE Healthcare), and 1x NEAA. On day 3, the cells were cultured in RPMI 1640 medium containing 100 ng / ml Activin A, 2% Hyclone dFBS, and 1x NEAA to induce definitive endoderm differentiation. On days 4–6, the cells were cultured in Advanced DMEM / F12 medium (ThermoFisher Scientific) containing 500 ng / ml FGF4 (R&D Systems), 3 μM CHIR99021 (R&D Systems), 2% B27 (Life Technologies), 1% N2 (Life Technologies), 10 mM HEPES (Life Technologies), and 1% Glutamax (Life Technologies) to induce differentiation into foregut cells. The resulting foregut cells were embedded in Matrigel (Corning) and cultured in Advanced DMEM / F12 + 2% B27 + 1% N2 + 10 mM HEPES + 1% Glutamax medium containing 2 mM retinoic acid (Merck) for a total of 4 days, with medium changes every 2 days. Subsequently, the cells were cultured in HCM (Lonza, SignleQuots, except for EGF) supplemented with 100 nM dexamethasone (Merck), 20 ng / ml Oncostatin M (PeproTech), and 10 ng / ml HGF (PeproTech) until day 20, with the medium changed every three days, to obtain liver organoids.

[0106] 4. Induction of a steatohepatitis-like state in HLOs. Oleic acid (Sigma-Aldrich) or palmitic acid (Sigma-Aldrich) was added to HLOs cultured up to day 20 at a final concentration of 400 μM. HLOs were harvested 3 days after addition for fat accumulation analysis and 7 days after addition for fibrosis analysis. Lipid accumulation in HLOs was assessed by staining with BODIPY 493 / 503 (ThermoFisher Scientific). After washing with PBS, harvested HLOs were stained with 2 μM BODIPY 493 / 503 and NucBlue Live ReadyProbes Reagent (ThermoFisher Scientific). Stained HLOs were observed using an SP8 confocal laser microscope (Leica). Recombinant IGF1 (R&D Systems) or recombinant IGFALS (R&D Systems) was added together with oleic acid to analyze their effect on fibrosis. These factors were added by changing the medium every three days, and HLOs cultured up to day 7 were subjected to tissue analysis.

[0107] 5. Histological Analysis of Mouse Liver and Organoids. Liver tissue and organoids were fixed overnight in 4% paraformaldehyde and then dehydrated in ethanol. The tissues were then embedded in paraffin, sectioned, and stained with hematoxylin and eosin (H&E) at the TMDU Research Core. Sirius Red staining was performed using the Picro-Sirius Red Stain Kit (ScyTek Laboratories) according to the manufacturer's instructions. Samples were observed under a BZ-X800 microscope (Keyence). The pathological grade and stage of each sample and the NAFLD activity score (NAS) were assessed according to the methods of Brunet et al., J. Gastroenterol., 94: 2467-2474, 1999 and Kleiner et al., Hepatology, 41: 1313-1321, 2005.

[0108] 6. Immunohistochemistry. After deparaffinization, sections were subjected to antigen retrieval using HistoVT One (Nacalai Tesque, Japan) at 90°C for 20 minutes, followed by incubation with 3% hydrogen peroxide for 10 minutes. Sections were blocked in TBS containing 0.05% Tween-20 and 3% BSA for 1 hour at room temperature. Subsequently, sections were incubated overnight at 4°C with primary antibodies (antibodies and dilutions are listed in Table 2). After washing with TBS + 0.1% Tween 20, sections were incubated with Alexa 488-, Alexa 555-, or Alexa 647-conjugated secondary antibodies (Life Technologies) and Hoechst 33342 (DOJINDO) for 1 hour at room temperature. After washing with TBS + 0.1% Tween 20, sections were mounted using PermaFluor Aqueous Mounting Medium (Lab Vision Corporation). Samples were observed using an SP8 confocal laser microscope (Leica). Sirius Red- and COL1A1-positive areas were quantified using Fiji software.

[0109]

[0110] 7. Measurement of Hydroxyproline Hydroxyproline in mouse liver was measured using a Hydroxyproline Assay Kit (QuickZyme Biosciences) according to the attached protocol.

[0111] Lipids were extracted from mouse liver tissue according to the Folch method (J. Biol. Chem. 226(1):497-509, 1957). The amount of lipids (mg) contained in 1 g of tissue was quantified by an enzymatic method at Immuno-Biological Laboratories Co., Ltd.

[0112] 9. RT-qPCR Total RNA was extracted from mouse liver tissue and organoids using the FastGene RNA Basic Kit (Nihon Genetics). Next, genomic DNA was removed and cDNA was synthesized using ReverTra Ace qPCR RT Master Mix with gDNA Remover (Toyobo). Real-time PCR was performed using THUNDERBIRD Next SYBR qPCR Mix (Toyobo) and the primer sets listed in Table 3 on a QuantStudio 3 Real-Time PCR System (ThermoFisher Scientific). Target gene expression levels were normalized to those of B2m (mouse) or ACTB (human organoid).

[0113]

[0114] 10. Statistical Analysis Statistical analysis of two groups was performed using the Student t-test (unpaired). For statistical analysis of three or more groups, significance was tested using one-way ANOVA followed by Tukey's multiple test. Other analyses, such as survival curve analysis, and graph creation were performed using Prism 8 (ver. 8.4.3, GraphPad).

[0115] Results 1. Liver-specific overexpression of IGFALS ameliorates NAFLD pathology in STAM mice. Based on serum analysis of NAFLD patients with liver biopsy data, we previously focused on insulin-like growth factor 1 (IGF1)-binding proteins and found that the acid-labile subunit (ALS or IGFALS), which forms trimers with IGF binding protein (IGFBP) 3 or IGFBP 5 and stabilizes the circulating lifespan of IGF1, is a useful blood marker for identifying early fibrosis or active NASH. Specifically, we demonstrated that blood IGFALS levels are reduced in NASH with fibrosis grade 2 or higher, or in active NASH with fibrosis grade 2 or higher and a NAFLD activity score (NAS) of 4 or higher (see Figure 1).

[0116] To clarify the role of IGFALS in the pathogenesis of NASH, we investigated the effects of hepatic overexpression of IGFALS (expression driven by the TGB promoter) using AAV8, a highly hepatocyte-specific serotype, in STAM mice, a model of NASH. Expression of IGFALS in hepatocytes is thought to induce secretion of IGFALS from the hepatocytes, resulting in its release into surrounding cells or systemically via the bloodstream. As a control, STAM mice were injected with AAV8 expressing EGFP (n = 12 for both AAV8-TGB-EGFP and AAV8-TGB-IGFALS groups). Specifically, mice were injected with AAV8 at 7 weeks of age and observed and sampled until 13 weeks of age (see Figure 2). RT-qPCR analysis of AAV8-induced gene expression in the liver revealed a significant increase in IGFALS mRNA levels in STAM mice treated with AAV8-TGB-IGFALS compared with those in STAM mice treated with AAV8-TGB-EGFP and healthy mice (when the expression level in healthy mice was set at 1.00 (±0.283 SD), the levels were 0.698 ± 0.263 in the AAV8-TGB-EGFP group and 16.22 ± 6.212 in the AAV8-TGB-IGFALS group) (see Figure 3). Furthermore, when the amount of IGFALS in serum was quantified by ELISA, the AAV8-TGB-EGFP-treated group showed lower levels than healthy mice, whereas the AAV8-TGB-IGFALS-treated group showed higher levels of IGFALS than healthy mice (healthy mice: 7.438±1.394 μg / ml, AAV8-TGB-EGFP-treated group: 3.967±1.303 μg / ml, AAV8-TGB-IGFALS-treated group: 10.79±3.137 μg / ml) (see Figure 4). Correlated with this change, serum IGF1 levels were also reduced in the AAV8-TGB-EGFP-treated group, whereas they recovered to levels comparable to those of healthy mice in the AAV8-TGB-IGFALS-treated group (healthy mice: 96.06±9.485 ng / ml, AAV8-TGB-EGFP-treated group: 58.66±19.04 ng / ml, AAV8-TGB-IGFALS-treated group: 107.10±28.61 ng / ml) (see Figure 4).These results suggest that the reduced expression level of IGFALS in STAM mice can be genetically increased using AAV8.

[0117] In the livers of the AAV8-TGB-EGFP-treated group, numerous hepatocytes with accumulated fat were observed, as well as ballooning, a sign of hepatocyte degeneration (Fig. 5, H&E). In contrast, the amount of fatty cells tended to be partially reduced in the AAV8-TGB-IGFALS-treated group. Measurement of triglyceride, cholesterol, and phospholipid levels in liver lipids extracted from the AAV8-TGB-IGFALS-treated group revealed a significant decrease in triglyceride levels (150.4 ± 57.68 mg / g tissue in the AAV8-TGB-EGFP-treated group, 100.9 ± 30.03 mg / g tissue in the AAV8-TGB-IGFALS-treated group) (Fig. 6). Fasting blood glucose levels were elevated in both groups, but the difference was not significant (Fig. 9D). Serum free fatty acid (NEFA) and triglyceride levels were similar between the two groups (Fig. 9E, F). The progression of NAFLD was assessed histopathologically using the NAFLD activity score (NAS). The NAS was significantly lower in the AAV8-TGB-IGFALS-treated group than in the AAV8-TGB-EGFP-treated group (5.333±1.366 vs. 3.111±1.537) (Figure 7). Consistently, F4 / 80-positive macrophage infiltration also tended to be suppressed in the AAV8-TGB-IGFALS-treated group (Figure 10).

[0118] The area of ​​liver fibrosis was assessed by Sirius Red staining. In the AAV8-TGB-EGFP-treated group, periportal and intralobular fibrosis was observed, whereas the extent of fibrosis was reduced in the AAV8-TGB-IGFALS-treated group (1.315±0.5413% in the AAV8-TGB-EGFP-treated group, 0.8485±0.2671% in the AAV8-TGB-IGFALS-treated group) (Fig. 5 and Fig. 7). Furthermore, hydroxyproline accumulation in the tissue was assessed by quantifying the acid hydrolysis product. The amount of hydroxyproline in the AAV8-TGB-IGFALS-treated group was reduced (1.053±0.1968 μmol / g tissue in the AAV8-TGB-EGFP-treated group, 0.8131±0.12084 μmol / g tissue in the AAV8-TGB-IGFALS-treated group) (Fig. 7). Consistent with these results, the proportion of aSMA-positive cells, a marker for activated hepatic stellate cells, also tended to decrease in the AAV8-TGB-IGFALS-administered group (see Figure 10).

[0119] There was no significant difference in body weight at the final age of 13 weeks between the AAV8-TGB-EGFP and AAV8-TGB-IGFALS groups (Fig. 9A). Serum liver enzymes AST and ALT were measured as markers of liver damage. There were no significant differences between the two groups (Fig. 9B and C).

[0120] The survival rate was compared between the AAV8-TGB-EGFP and AAV8-TGB-IGFALS groups. The AAV8-TGB-EGFP group showed a decreased survival rate at 10–11 weeks of age, dropping to 33.3% at 13 weeks of age. The AAV8-TGB-IGFALS group showed a significant improvement in survival rate, remaining at 75.0% (Figure 8).

[0121] 2. Addition of recombinant IGFALS ameliorates fibrosis accumulation in a steatohepatitis-like model of human iPS cell-derived liver organoids. We have previously developed a method to create multicellular liver organoids (human pluripotent stem cell-derived liver organoids, HLOs) from human iPS cells, in which hepatocytes and multiple non-parenchymal cells responsible for inflammation and fibrosis simultaneously develop (see Figure 11). Furthermore, by optimizing the protocol for adding fatty acids to the culture medium, we have constructed a pathological model in vitro that can induce phenotypes equivalent to inflammation and fibrosis from a fatty liver state (Ouchi et al., Cell Metabolism 2019; Shinozawa et al., Gastroenterology 2021; Kimura et al., Cell 2022). Specifically, they found that adding oleic acid, a free fatty acid, led to a fatty liver condition with the accumulation of neutral fat within one day of addition, and further increased expression of inflammatory cytokines (Kimura et al., Cell 2022) and extracellular accumulation of type I collagen within 3-7 days of addition (see Figure 12).

[0122] To explore extracellular signaling factors, such as growth factors and cytokines, associated with inflammation and fibrosis in HLO, we used RT-qPCR to compare the expression levels of various extracellular signaling factors between stimulation with oleic acid (400 mμ), which has high fat accumulation activity and can ultimately induce a fibrotic state, and stimulation with palmitic acid (400 mM), which is highly lipotoxic but does not induce a fibrotic state (see Figure 13). The results showed that IGF1 expression was reduced compared to control on both days 3 and 6 after oleic acid (OA) supplementation (control: 50.90±5.54, OA: 18.89±5.54, PA: 46.03±4.35, P=0.0006, control vs. OA; and control: 100.0±7.32, OA: 24.90±1.16, PA: 73.96±7.22, P<0.0001, control vs. OA) (Fig. 14). These results suggest that IGF1 signaling is suppressed by HLO under conditions that transition from fatty liver to inflammation and fibrosis.

[0123] Next, we analyzed the effects of IGF1 or IGFALS supplementation on steatohepatitis-like HLO. Specifically, we added recombinant IGF1 or IGFALS along with oleic acid and cultured the cells for 7 days. Extracellular COL1A1 accumulation was assessed histologically as a marker of fibrosis. The concentrations of recombinant IGF1 and IGFALS added were based on previous binding experiments using purified proteins (Twigg and Baxter, J. Biol. Chem., 273(11):6074-6079, 1998, and Firth et al., J. Biol. Chem., 273(5):2631-2638, 1998). Specifically, in the presence of IGFBP3 (1.25-3.75 nM), 6.25 nM of IGFALS is estimated to be the concentration required to incorporate 2.0-12 nM of IGF1 into the trimer. Therefore, we evaluated the fibrotic phenotype in cells cultured for 7 days with oleic acid (400 μM) plus IGF1 (12 nM) or IGFALS (6.25 nM), compared with cells cultured with oleic acid (400 μM) alone. Compared to the control group, treatment with oleic acid alone significantly increased the COL1A1-positive fibrotic area, whereas the addition of oleic acid with IGF1 or IGFALS (OA+IGF1, OA+IGFALS) significantly reduced the fibrotic area (COL1A1-positive area: control 1.338±0.304%, OA 7.063±1.728%, OA+IGF1 1.610±0.4024%, OA+IGFALS 3.073±1.152%) (Fig. 15). On the other hand, the abundance of Vimentin-positive mesenchymal cells, corresponding to hepatic stellate cell-like cells, in HLO did not significantly decrease in the OA+IGF1 or OA+IGFALS conditions compared to OA alone (Fig. 15). This suggests that the COL1A1-positive fibrotic changes may be due to the suppression of hepatic stellate cell-like cell activity by IGF1 or IGFALS.

[0124] Discussion: Although active drug development for NASH is currently underway, no highly effective therapeutic agents have yet been commercially available. This study demonstrated that IGFALS, which regulates the formation of IGF1 complexes in the blood, has the ability to improve NASH. Specifically, we demonstrated that IGFALS can improve NASH pathology in both STAM mice, a mouse model of progressive NASH, using gene therapy with AAV8, and in the human liver model HLO, using recombinant IGFALS.

[0125] STAM mice, which have a diabetic background, are known as a model of rapid progression from fatty liver to hepatitis, liver fibrosis, and liver cancer (Pokorny et al., Radil. Diagn., 31(2):145-151, 1990). Steatosis develops at approximately 5-6 weeks of age, followed by hepatitis at 7-8 weeks of age, and fibrosis progresses from 9 weeks onward. In this study, we demonstrated that liver-specific expression of IGFALS by AAV8 administration at 7 weeks of age can reduce fatty liver, hepatitis, liver fibrosis, and improve survival. These preclinical data suggest that replenishment of IGFALS at the hepatitis stage can improve prognosis. Furthermore, when IGFALS expression was restored by AAV8, serum IGF1 levels were elevated to levels comparable to those of normal controls (see Figure 4). Since there was no change in IGF1 gene expression levels in the liver (see Figure 3), it is possible that this increase in blood IGF1 occurred through post-transcriptional regulation, and that the function of IGFALS increased the stability of IGF1 in the blood.

[0126] Drug development for NASH requires validation in models with high clinical relevance to human pathology. Drug discovery research utilizing organoids derived from human stem cells, in particular, is gaining attention. In this study, we demonstrated the anti-fibrotic effect of IGFALS in STAM mice and in human iPS cell-derived liver organoids (HLOs), a model of human NASH we developed. Specifically, we found that the addition of IGF1 or IGFALS as recombinant proteins to the culture medium suppressed the accumulation of type I collagen (COL1A1), a key extracellular matrix protein involved in fibrosis. Studies using mouse models and cell lines have shown that IGF1 acts on hepatic stellate cells to suppress fibrosis (Nishizawa et al., Sci. Rep., 6:34605, 2016, and Sanz et al., Gut., 54(1):134-141, 2005), suggesting that a similar mechanism may be at work in human liver organoids. On the other hand, IGFALS alone does not activate IGF1 signaling, suggesting that the anti-fibrotic effect observed in this study is mediated by endogenous IGF1. In other words, IGF1 and IGFBP3 are secreted from human liver organoids, and the association of recombinant IGFALS with these to form a complex is expected to stabilize the IGF1 ligand activity in the organoid culture environment. The mechanism of IGF1 signaling activation in the liver by IGFALS and its target cells remain to be explored in the future.

[0127] Several studies have demonstrated that IGF1 levels are reduced in NASH. For example, a recent meta-analysis by Yao et al. reported that IGF1 levels are reduced in NAFLD patients compared with healthy controls (Yao et al., Endocrine, 65(2):227-237, 2019). Furthermore, animal studies using mice and rats have shown that continuous administration of recombinant IGF1 reduces hepatic triglyceride accumulation and fibrosis in NAFLD models (Ohkubo et al., Cell Rep., 41(11):111803, 2022; Nishizawa et al., Biochem Biophys Res Commun., 423(2):295-300, 2012; Nishizawa et al., Sci Rep., 6:34605, 2016). This suggests that supplementing IGF1 in NASH may improve the pathology. However, because IGF1 itself exerts physiological effects on cell proliferation and cell survival, theoretical concerns about its carcinogenicity or tumor growth have been raised. Therefore, commercially available IGF1 preparations (e.g., Somazon or Increlex) are contraindicated for patients with malignant tumors. Furthermore, epidemiological studies have shown that high serum IGF1 levels are associated with an increased risk of breast and prostate cancer (Voskuil et al., Cancer Epidemiol Biomarkers Prev.,(1):195-203. 2005). Therefore, approaches other than direct IGF1 supplementation may be safer treatments. However, this study focused on enhancing the stability of endogenous IGF1 by supplementing IGFALS, which may reduce the risk of the aforementioned concerns associated with recombinant IGF1 preparations.

[0128] All documents cited herein are incorporated by reference in their entirety.

Claims

1. A composition for use in treating a subject with non-alcoholic fatty liver disease (NAFLD) activity score of 4 or higher and non-alcoholic steatohepatitis (NASH) with a fibrosis grade of 2 or higher, or a subject at risk of developing said NASH, comprising insulin-like growth factor binding protein acid-labile subunit (IGFALS) protein or a nucleic acid encoding IGFALS, wherein said subject has low blood IGF1 levels and low IGFALS protein levels, and the composition does not contain either insulin-like growth factor 1 (IGF1) protein or a nucleic acid encoding IGF1 protein.

2. 2. The composition of claim 1, wherein the subject with fatty liver disease has a blood IGFALS level below the reference value.

3. The subject having NASH, A method for estimating the progression of liver fibrosis in a subject with NASH, comprising measuring a blood IGFALS level in the subject and comparing the blood IGFALS level with a reference value, wherein a blood IGFALS level lower than the reference value indicates that the subject is suitable for administration of an IGFALS protein or a nucleic acid encoding IGFALS. The composition of claim 1 or 2, wherein the subject is a subject that has been shown by the method to be suitable for administration of an IGFALS protein or a nucleic acid encoding IGFALS.

4. The composition of any one of claims 1 to 3, wherein the nucleic acid encoding IGFALS is messenger RNA.

5. The composition of any one of claims 1 to 4, wherein a nucleic acid encoding IGFALS is incorporated into a gene expression cassette of a viral vector, and within the expression cassette, the nucleic acid is operably linked to a regulatory sequence, thereby allowing the IGFALS protein to be expressed in liver cells.

6. The composition of claim 5 , wherein the viral vector is an adeno-associated viral vector (AAV).