B-spectrin (sptbn1) deficiency protects mice from high-fat diet-induced liver disease and cancer development

Abstract

To protect mice from high-fat diet-induced liver disease and cancer development through B-spectrin (SPTBN1) deficiency.SOLUTION: This disclosure provides methods of treating obesity, nonalcohol-related fatty liver disease, nonalcoholic steatohepatitis, or hepatocellular cancer in a subject in need thereof, the method comprising administering a therapeutically effective amount of at least one siRNA molecule targeting SPTBN1. At least one of the siRNA molecules that inhibit expression of SPTBN1 comprises 15 to 30 nucleotides. At least one of the siRNA molecules that inhibit expression of SPTBN1 comprises an overhang region of 1 to 6 nucleotides.SELECTED DRAWING: Figure 6B

Description

[Technical field]

[0001] Government support This invention was made with Government support under U01 CA230690 and R01 M023146 awarded by the National Institutes of Health. The Government has certain rights in this invention.

[0002] Reference to Electronically Submitted Sequence Listing The contents of the Sequence Listing, which was submitted electronically with this application in an ASCII text file (Name: 3973_018PC02_Seqlisting_ST25.txt, Size: 37,736 bytes, and Creation Date: November 3, 2021), are hereby incorporated by reference in their entirety.

[0003] The present disclosure provides a method for treating obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of a siRNA molecule targeting SPTBN1. [Background technology]

[0004] Nonalcohol-associated fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are caused by obesity and metabolic disorders and affect one-third of the population worldwide (1, 2). These diseases encompass a spectrum of lipid accumulation in the liver (steatosis), injury, inflammation, hepatocyte ballooning (cell death), and progressive fibrosis (cirrhosis), ultimately leading to carcinogenesis (3, 4). Lipid accumulation in the liver promotes chronic oxidative and endoplasmic reticulum (ER) stress, cell death, immune cell inflammation, fibrosis, and disease progression. These effects are exacerbated by factors such as increased uptake of free fatty acids (FFA), a sedentary lifestyle, and hyperinsulinemia. Depending on whether NASH occurs with or without cirrhosis, liver cancer incidence can vary from 2.4% (without cirrhosis) to 12.8% (with cirrhosis) (1, 3). Obesity increases the risk of liver cancer mortality by twofold and, together with NASH, is responsible for a significant increase in this cancer (5, 6). Despite new therapeutic approaches targeting NASH, few single agents reverse both fibrosis and steatosis, and thus NASH presents a major clinical challenge. (5, 7) Therefore, understanding the molecular mechanisms that converge on abnormal lipid accumulation, fibrosis, and the fatal transformation to hepatocarcinogenesis may provide new approaches to target NASH in unique populations susceptible to disease progression.

[0005] The development of NAFLD is thought to involve de novo lipogenesis along with the abnormal accumulation of free fatty acids, triglycerides, and cholesterol. Activation of hepatocyte death receptor pathways, tumor necrosis factor (TNF), and caspases contribute to the tissue damage and steatohepatitis seen in NASH (8, 9). De novo lipogenesis is stimulated by activation of the transcription factor sterol regulatory element (SRE)-binding protein (SREBP) and inhibition of energy-sensing pathways, such as those involving adenosine monophosphate (AMP)-activated protein kinase (AMPK). SREBP1 is the master lipogenic transcription factor that promotes fatty acid synthesis and contributes to lipogenesis (10). SREBP proteins are maintained in the endoplasmic reticulum by interactions with the proteins INSIG and SCAP. Activation of SREBP in response to sterol depletion or ER stress requires the dissociation of INSIG and SCAP-induced cleavage of SREBP by site-1 protease (S1P) followed by a second cleavage by site-2 protease (S2P) to generate mature forms of SREBP protein that translocate to the nucleus and regulate target gene transcription (11). In stressed cultured cells in the presence of activated caspase-3, SREBP1 and SREBP2 are cleaved and activated by caspase-3, although the physiological context of this is unknown (12). The truncated nuclear forms are termed n-SREBPs, and the full-length ER-localized forms are termed precursor SREBPs.

[0006] The degree of fibrosis is considered the strongest predictor of progression from NALFD to NASH and ultimately to hepatocellular carcinoma (HCC) (1,13,14). Pivotal to liver fibrosis is activation of the transforming growth factor β (TGF-β) pathway (15,16). TGF-β1 is the founding member of this family, and its ligand signals through two serine-threonine kinase receptors (TGFBR2 and TGFBR1) that activate SMAD transcription regulators. The SMAD complex, which contains SMAD3, serves as a central center for fibrotic progression by causing overexpression of extracellular matrix genes, such as those encoding collagens COL1A1, COL1A2, COL3A1, COL5A2, COL6A1, and COL6A3, as well as stimulating genes encoding protease inhibitors, tissue inhibitors of metalloproteinases (TIMPs), and plasminogen activator inhibitor 1 (PAI-1) (16). The SMAD3 complex also represses the gene encoding peroxisome proliferator-activated receptor gamma coactivator 1α (PGC-α) (17).The profibrotic effects of TGF-β1 through SMAD3 involve multiple mechanisms and cell types, including enhancing the invasion or proliferation (or both) of tissue-resident fibroblasts, generating myofibroblasts, inducing epithelial-mesenchymal transition (EMT), and inhibiting collagenolysis (18).

[0007] SPTBN1 (β2-spectrin, also known as β2SP) is a multidomain adaptor protein with functions in the cytoplasm and nucleus (19-21). Specifically, SPTBN1 promotes TGF-β receptor activation of SMAD3 in the cytosol (22) and interacts with SMAD3 in the nucleus to regulate specific target genes (23,24). SPTBN1 is a dynamic tetrameric protein consisting of two antiparallel dimers of alpha and beta subunits. SPTBN1 binding partners include ankyrin, which functions to link proteins in the plasma membrane to the spectrin-containing cytoskeleton, and lamins and the chromatin regulator CTCF (CCCTC-binding factor), which functions to organize chromatin in the nucleus and regulate gene expression (19,23,25,26). SPTBN1 is a substrate for caspases 3 and 7, and the SPTBN1 1454DEVD1457 peptide sequence generates two fragments (160 and 80 kDa) that have distinct and distinct functions in apoptosis and transcription. (27) The importance of SPTBN1 in liver disease comes from the finding that mice treated with shRNA targeting SPTBN1 exhibit less acetaminophen-induced hepatotoxicity. (27) Increased abundance of hepatic SMAD3 and SPTBN1, as well as members of the TGF-β pathway that are associated with profibrotic pathways, is found in approximately 40% of HCC cases, and many HCC cases are associated with NASH (24, 28). [Brief description of the drawings]

[0008] [Figure 1A] none. [Figure 1B] none. [Figure 1C] none. [Figure 1D] none. [Figure 1E] none. [Figure 1F] none. [Figure 1G] none. [Figure 2A] none. [Figure 2B] none. [Figure 2C] none. [Figure 2D] none. [Figure 2E] none. [Figure 2F] none. [Figure 2G] none. [Figure 2H] none. [Figure 3A] none. [Figure 3B] none. [Figure 3C] none. [Figure 3D] none. [Figure 3E] none. [Figure 3F] none. [Figure 4A] none. [Figure 4B] none. [Figure 4C] none. [Figure 4D] none. [Figure 4E] none. [Figure 4F] none. [Figure 4G] none. [Figure 5A] none. [Figure 5B] none. [Figure 5C] none. [Figure 5D] none. [Figure 5E] none. [Figure 5F] none. [Figure 6A] none. [Figure 6B] none. [Figure 6C] none. [Figure 6D] none. [Figure 6E] none. [Figure 6F] none. [Figure 6G] none. [Figure 6H] none. [Figure 6I] none. [Figure 6J] none. [Figure 7A] none. [Figure 7B] none. [Figure 7C] none. [Figure 7D] none. [Figure 7E] none. [Figure 7F] none. [Figure S1A] none. [Figure S1B] none. [Figure S1C] none. [Figure S1D] none. [Figure S1E] none. [Figure S1F] none. [Figure S1G] none. [Fig. S1H] none. [Figure S2A] none. [Figure S2B] none. [Figure S2C] none. [Figure S2D] none. [Figure S2E] none. [Figure S3A] none. [Figure S3B] none. [Figure S3C] none. [Figure S4A] none. [Figure S4B] none. [Figure S4C] none. [Figure S4D] none. [Figure S4E] none. [Figure S5A] none. [Figure S5B] none. Summary of the Invention [Means for solving the problem]

[0009] The present disclosure provides a method for treating a disease, disorder, or condition, such as obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or hepatocellular carcinoma, in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The role of SPTBN1 in liver tumorigenesis was studied by generating liver-specific SPTBN1 conditional knockout (LSKO) mice. LSKO mice, or mice treated with SPTBN1-directed siRNA, were shown to be protected from the adverse effects of a high-fat diet by a mechanism involving reduced expression of profibrotic genes and genes involved in de novo lipogenesis. The mice did not become obese or progress to NASH or HCC. The translational importance of the results was supported by the analysis of SPTBN1 expression in human NASH and HCC, as well as by the finding that siRNA targeting SPTBN1 reversed transcriptional changes in genes involved in fatty acid metabolism and fibrosis induced in a human three-dimensional culture model of NASH. Thus, the results identified a previously unknown role for SPTBN1 in regulating caspase-3-induced SREBP activity in response to stress conditions caused by a high-fat diet (HFD).

[0011] In one embodiment, the present disclosure provides a method for treating obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1.

[0012] In another embodiment, at least one of the siRNA molecules that inhibits expression of SPTBN1 comprises 15-30 nucleotides.

[0013] In another embodiment, at least one of the siRNA molecules that inhibits expression of SPTBN1 comprises 15-20 nucleotides.

[0014] In another embodiment, at least one of the siRNA molecules that inhibits expression of SPTBN1 comprises an overhang region of 1-6 nucleotides.

[0015] In another embodiment, at least one of the siRNA molecules that inhibits expression of SPTBN1 does not comprise an overhang region.

[0016] In another embodiment, at least one of the siRNA molecules that inhibits expression of SPTBN1 is set forth as SEQ ID NO:7.

[0017] In another embodiment, at least one of the siRNA molecules that inhibits expression of SPTBN1 is set forth as SEQ ID NO:8.

[0018] In another embodiment, at least one of the siRNA molecules that inhibits expression of SPTBN1 is set forth as SEQ ID NO:9.

[0019] In another embodiment, at least one of the siRNA molecules that inhibits expression of SPTBN1 is set forth as SEQ ID NO:10.

[0020] In another embodiment, the present disclosure provides a method of treating obesity, non-alcohol-related fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1, wherein at least one of the siRNA molecules that inhibit expression of SPTBN1 is homologous to at least 10 nucleotides of SEQ ID NO: 7. In another embodiment, at least one of the siRNA molecules that inhibit expression of SPTBN1 is homologous to at least 15 nucleotides of SEQ ID NO: 7.

[0021] In another embodiment, the present disclosure provides a method of treating obesity, non-alcohol-related fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1, wherein at least one of the siRNA molecules that inhibit expression of SPTBN1 is homologous to at least 10 nucleotides of SEQ ID NO: 8. In another embodiment, at least one of the siRNA molecules that inhibit expression of SPTBN1 is homologous to at least 15 nucleotides of SEQ ID NO: 8.

[0022] In another embodiment, the present disclosure provides a method of treating obesity, non-alcohol-related fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1, wherein at least one of the siRNA molecules that inhibit expression of SPTBN1 is homologous to at least 10 nucleotides of SEQ ID NO: 9. In another embodiment, at least one of the siRNA molecules that inhibit expression of SPTBN1 is homologous to at least 15 nucleotides of SEQ ID NO: 9.

[0023] In another embodiment, the present disclosure provides a method of treating obesity, non-alcohol-related fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1, wherein at least one of the siRNA molecules that inhibit expression of SPTBN1 is homologous to at least 10 nucleotides of SEQ ID NO: 10. In another embodiment, at least one of the siRNA molecules that inhibit expression of SPTBN1 is homologous to at least 15 nucleotides of SEQ ID NO: 10.

[0024] In another embodiment, the present disclosure provides a method of treating obesity, non-alcohol-related fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1, wherein at least one of the siRNA molecules that inhibit expression of SPTBN1 has at least 90% sequence identity with SEQ ID NO: 7. In another embodiment, at least one of the siRNA molecules that inhibit expression of SPTBN1 has at least 95% sequence identity with SEQ ID NO: 7.

[0025] In another embodiment, the present disclosure provides a method of treating obesity, non-alcohol-related fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1, wherein at least one of the siRNA molecules that inhibit expression of SPTBN1 has at least 90% sequence identity with SEQ ID NO: 8. In another embodiment, at least one of the siRNA molecules that inhibit expression of SPTBN1 has at least 95% sequence identity with SEQ ID NO: 8.

[0026] In another embodiment, the present disclosure provides a method of treating obesity, non-alcohol-related fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1, wherein at least one of the siRNA molecules that inhibit expression of SPTBN1 has at least 90% sequence identity with SEQ ID NO: 9. In another embodiment, at least one of the siRNA molecules that inhibit expression of SPTBN1 has at least 95% sequence identity with SEQ ID NO: 9.

[0027] In another embodiment, the present disclosure provides a method of treating obesity, non-alcohol-related fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1, wherein at least one of the siRNA molecules that inhibit expression of SPTBN1 has at least 90% sequence identity with SEQ ID NO: 10. In another embodiment, at least one of the siRNA molecules that inhibit expression of SPTBN1 has at least 95% sequence identity with SEQ ID NO: 10.

[0028] In another embodiment, the present disclosure provides a method for treating obesity in a subject in need thereof, hi another embodiment, treating obesity comprises reducing the amount of body fat in the subject and / or reducing the subject's body weight.

[0029] In another embodiment, the present disclosure provides a method for treating non-alcohol related fatty liver disease in a subject in need thereof. In another embodiment, treating non-alcohol related fatty liver disease comprises reducing blood triglycerides in the subject.

[0030] In another embodiment, the present disclosure provides a method for treating non-alcoholic steatohepatitis in a subject in need thereof. In another embodiment, treating non-alcoholic steatohepatitis comprises reducing blood triglycerides in the subject.

[0031] In another embodiment, the present disclosure provides a method for treating hepatocellular carcinoma in a subject in need thereof, hi another embodiment, treating hepatocellular carcinoma comprises reducing tumor mass in the subject.

[0032] In another embodiment, 1 to 10 siRNA molecules, such as 1 to 5 siRNA molecules, such as 1 to 3 siRNA molecules, that inhibit the expression of SPTBN1 are administered to the subject. In another embodiment, 1 siRNA molecule that inhibits the expression of SPTBN1 is administered to the subject. In another embodiment, 2 siRNA molecules that inhibit the expression of SPTBN1 are administered to the subject. In another embodiment, 3 siRNA molecules that inhibit the expression of SPTBN1 are administered to the subject. In another embodiment, 4 siRNA molecules that inhibit the expression of SPTBN1 are administered to the subject. In another embodiment, 5 siRNA molecules that inhibit the expression of SPTBN1 are administered to the subject. In another embodiment, 6 siRNA molecules that inhibit the expression of SPTBN1 are administered to the subject. In another embodiment, 7 siRNA molecules that inhibit the expression of SPTBN1 are administered to the subject. In another embodiment, 8 siRNA molecules that inhibit the expression of SPTBN1 are administered to the subject. In another embodiment, 9 siRNA molecules that inhibit the expression of SPTBN1 are administered to the subject. In another embodiment, 10 siRNA molecules that inhibit the expression of SPTBN1 are administered to the subject.

[0033] "Treat," "treating," "treatment," and similar terms are used herein to mean eliminating, alleviating, or ameliorating a disease or condition and / or its associated symptoms. Treating a disease or condition does not require, although it does not exclude, that the disease, condition, or its associated symptoms be completely eliminated.

[0034] The term "therapeutically effective amount," as used herein, refers to that amount of a therapeutic agent, e.g., a siRNA molecule that inhibits expression of SPTBN1, sufficient to result in the amelioration of one or more symptoms of a disorder, or sufficient to prevent the progression, or sufficient to cause regression of a disorder. For example, with respect to the treatment of cancer, in one embodiment a therapeutically effective amount would refer to the amount of a therapeutic agent that results in a therapeutic response, e.g., normalization of blood counts, reduction in the rate of tumor growth, reduction in tumor mass, reduction in the number of metastases, increase in the time it takes for tumor to progress, and / or increases subject survival by at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95% or at least about 100% or more. With respect to the treatment of obesity, in one embodiment, a therapeutically effective amount will refer to the amount of a therapeutic agent that results in at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% or more reduction in body fat or body weight in a subject.With respect to the treatment of NAFLD or NASH, in one embodiment, a therapeutically effective amount will refer to the amount of a therapeutic agent that results in at least about 2%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% or more reduction in blood triglycerides in a subject.

[0035] The terms "G", "C", "A", "T" and "U" each generally refer to naturally occurring nucleotides that contain guanine, cytosine, adenine, thymidine and uracil, respectively, as bases. However, it will be understood that the term "nucleotide" also refers to alternative nucleotides or alternative replacement moieties, which are described in more detail below. Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be replaced with other moieties without substantially altering the base pairing properties of the oligonucleotide containing the nucleotide bearing such replacement moiety. For example, but not limited to, a nucleotide containing inosine as a base can base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine can be replaced with a nucleotide containing, for example, inosine in the nucleotide sequence of a dsRNA as described in the present invention. In another example, adenine and cytosine at any position in an oligonucleotide can be replaced with guanine and uracil, respectively, to form a GU Wobble base pairing with a target mRNA sequence. Sequences containing such replacement moieties are suitable for the compositions and methods featured in this invention.

[0036] The terms "nucleobase" and "base" include the purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds during nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses alternative nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases, such as adenine, guanine, cytosine, thymine, uracil, xanthine and hypoxanthine, as well as alternative nucleobases. Such variants are described, for example, in Hirao et al (2012) Accounts of Chemical Research vol 45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.37 1.4.1.

[0037] The term "nucleoside" refers to a monomeric unit of an oligonucleotide or polynucleotide having a nucleobase and a sugar moiety. Nucleosides can include naturally occurring and alternative nucleosides, such as those described herein. The nucleobase of a nucleoside can be a naturally occurring nucleobase or an alternative nucleobase. Similarly, the sugar moiety of a nucleoside can be a naturally occurring sugar or an alternative sugar.

[0038] The term "alternative nucleoside" refers to a nucleoside having an alternative sugar or alternative nucleobase, such as those described herein.

[0039] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., an "alternative nucleobase" selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uridine, 5-bromouridine, 5-thiazolo-uridine, 2-thio-uridine, pseudouridine, 1-methylpseudouridine, 5-methoxyuridine, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0040] The nucleobase moieties can be represented by the letter symbol for each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter can optionally include alternative nucleobases having equivalent functions.

[0041] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring. Sugar also includes "sugar substitutes," defined as structures that can replace the furanose ring of a nucleoside. In certain embodiments, the sugar substitute is a non-furanose (or 4'-substituted furanose) ring or ring system, or an open ring system. Such structures can include simple changes compared to the natural furanose ring, such as a six-membered ring, or can be more complex, as in the case of the acyclic systems used in peptide nucleic acids. Sugar substitutes can also include sugar substitutes in which the furanose ring is replaced with another ring system, such as a morpholino or hexitol ring system. Sugar moieties useful in preparing oligonucleotides bearing the motif include β-D-ribose, β-D-2' deoxyribose, substituted sugars (e.g., 2', 5' and disubstituted sugars), 4'-S-sugars (e.g., 4'-S-ribose, 4'-S-2'-deoxyribose, and 4'-S-2'-substituted ribose), bicyclic sugar surrogates (e.g., 2'-O-CH 2 -4'-, or 2'-O-(CH 2 ) 2-4'-bridged ribose derived bicyclic sugars, and sugar surrogates (e.g., where the ribose ring is replaced with a morpholino or hexitol ring system). The type of heterocyclic base and internucleoside linkage used at each position is arbitrary and is not a factor in determining the motif. In most nucleosides with surrogate sugar moieties, the heterocyclic nucleobase is generally maintained to allow hybridization.

[0042] "Nucleotide" as used herein refers to a monomeric unit of an oligonucleotide or polynucleotide that includes a nucleoside and an internucleoside linkage. The internucleoside linkage may or may not include a phosphate linkage. Similarly, "linked nucleosides" may or may not be linked by a phosphate linkage. Many "alternative internucleoside linkages" are known in the art, including, but not limited to, phosphate, phosphorothioate, and boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNAs) (e.g., locked nucleosides (LNAs) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNAs), phosphotriesters, phosphorothioates, phosphoramidates, and other variants of the phosphate backbone of natural nucleosides, including those described herein.

[0043] "Alternative nucleotide," as used herein, refers to a nucleotide having an alternative nucleoside or sugar and an internucleoside linkage, which can include an alternative nucleoside linkage.

[0044] The terms "oligonucleotide" and "polynucleotide" as used herein are defined as molecules comprising two or more covalently linked nucleosides as generally understood by those skilled in the art. Such covalently linked nucleosides may also be referred to as nucleic acid molecules or oligomers. Oligonucleotides are often made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, reference is made to the sequence or order of the nucleobase moieties or variants of the covalently linked nucleotides or nucleosides. The oligonucleotides of the present invention may be artificial, chemically synthesized and typically purified or isolated. Oligonucleotides are also intended to include compounds with (i) one or more furanose moieties replaced by furanose derivatives or replaced by any cyclic or acyclic structure that can be used as a covalent attachment point to a base moiety, (ii) one or more phosphorodiester linkages that are either modified, as in the case of phosphoramidate or phosphorothioate linkages, or completely replaced by a suitable linking moiety, as in the case of formacetal or riboacetal linkages, and / or (iii) one or more bonded furanose-phosphodiester linkage moieties that are replaced by any cyclic or acyclic structure that can be used as a covalent attachment point to a base moiety. Oligonucleotides of the present invention may include one or more alternative nucleosides or nucleotides, including, for example, those described herein. It is also understood that oligonucleotides include compounds that lack sugar moieties or nucleobases but can still base pair or hybridize with a target sequence.

[0045] "Oligonucleotide" refers to a short (eg, 100 linked nucleosides or less) polynucleotide.

[0046] As used herein, the term "strand" refers to an oligonucleotide comprising a chain of linked nucleosides. A "strand comprising a nucleobase sequence" refers to an oligonucleotide comprising a chain of linked nucleosides described by a sequence referenced using standard nucleobase nomenclature.

[0047] The term "antisense" as used herein refers to a nucleic acid comprising an oligonucleotide or polynucleotide that is sufficiently complementary to all or a portion of a gene, primary transcript or processed mRNA to interfere with the expression of an endogenous gene (e.g., MLH3). A "complementary" polynucleotide is one that can base-pair according to standard Watson-Crick complementarity rules. Specifically, purines will base-pair with pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired either with thymine (A:T) in the case of DNA or with uracil (A:U) in the case of RNA. It is understood that two polynucleotides can hybridize to each other even if they are not completely complementary to each other, provided that each has at least one region that is substantially complementary to the other.

[0048] The terms "antisense strand" and "guide strand" refer to the strand of a dsRNA that includes a region that is substantially complementary to a target sequence, such as MLH3 mRNA.

[0049] The terms "sense strand" and "passenger strand," as used herein, refer to the strand of a dsRNA that includes a region that is substantially complementary to a region of the antisense strand, as those terms are defined herein.

[0050] The term "dsRNA" refers to an agent that includes a sense strand and an antisense strand that contain linked nucleosides, as that term is defined herein. dsRNA includes, for example, siRNA and shRNA, which mediate targeted cleavage of RNA transcripts via the RNA-induced silencing complex (RISC) pathway. dsRNA induces sequence-specific degradation of mRNA by a process known as RNA interference (RNAi). dsRNA reduces the expression of MLH3 in cells, for example, cells in a subject, such as a mammalian subject. Generally, most of the linked nucleosides in each strand of dsRNA are ribonucleosides, although one or both strands may contain one or more non-ribonucleosides, such as deoxyribonucleosides and / or alternative nucleosides, as described in detail herein.

[0051] The terms "siRNA" and "short interfering RNA" (also known as "small interfering RNA") refer to an RNA agent, preferably a double-stranded agent, of about 10-50 nucleotides in length, optionally where the strand has an overhanging end, e.g., containing one, two or three overhanging linked nucleosides, that are capable of inducing or mediating RNA interference. Naturally occurring siRNAs are generated by the cellular RNAi machinery (e.g., Dicer or its homologs) from longer (e.g., greater than 25 linked nucleosides in length) dsRNA molecules.

[0052] The terms "shRNA" and "short hairpin RNA", as used herein, refer to an RNA agent having a stem-loop structure, comprising first and second regions of complementary sequence, the degree and orientation of complementarity of which is such that base pairing occurs between said regions, and the first and second regions are joined by a loop region, the loop being obtained by the lack of base pairing between nucleobases within the loop region.

[0053] A "chimeric" dsRNA or "chimera," in the context of this invention, is a dsRNA that contains two or more chemically distinct regions, each made up of at least one monomeric unit, i.e., in the case of dsRNA, a nucleoside or nucleotide.

[0054] The duplex region can be of any length that permits specific degradation of the desired target RNA via the RISC pathway, and can be in the range of about 9-36 base pairs in length, e.g., about 10-30 base pairs in length, e.g., about 15-30 base pairs in length, or about 18-20 base pairs in length, e.g., about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairs in length, e.g., about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20 , 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, The length may be 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Ranges and lengths intermediate to the above listed ranges and lengths are also contemplated as part of the invention.

[0055] The two strands forming the duplex structure may be different portions of one longer oligonucleotide molecule, or they may be separate oligonucleotide molecules. When the two strands are portions of one larger molecule, and therefore linked by an uninterrupted chain of linked nucleosides between the 3' end of one strand and the 5' end of each of the other strands forming the duplex structure, the linked strand is referred to as a "hairpin loop." A hairpin loop may contain at least one unpaired nucleobase. In some embodiments, a hairpin loop may contain at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 23, or more unpaired nucleobases. In some embodiments, a hairpin loop may have 10 or fewer linked nucleosides. In some embodiments, a hairpin loop may have 8 or fewer unpaired nucleobases. In some embodiments, a hairpin loop may have 4 to 10 unpaired nucleobases. In some embodiments, the hairpin loop can be 4 to 8 linked nucleosides.

[0056] In one embodiment, each strand of the dsRNA contains 19-23 linked nucleosides that interact with a target RNA sequence, e.g., an MLH3 target mRNA sequence, to induce cleavage of the target RNA. Without wishing to be bound by theory, it is believed that long double-stranded RNA introduced into cells is degraded by a type III endonuclease known as Dicer (Sharp et al., 2002). et al. (2001) Genes Dev. 15:485). Dicer, a ribonuclease III-like enzyme, processes the RNA into 19-23 base pair short interfering RNAs with characteristic di-base 3' overhangs (Bernstein, et al., (2001) Nature 409:363). The dsRNA is then incorporated into the RNA-induced silencing complex (RISC) where one or more helicases unwind the dsRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir, et al., (2001) Genes Dev. 15:188). The two substantially complementary strands of dsRNA are composed of separate RNA molecules, which can be, but are not necessarily, covalently linked. When the two strands are covalently linked by means other than an uninterrupted chain of nucleotides between the 3'-end of one strand and the 5'-end of each of the other strands forming the duplex structure, the linking structure is called a "linker".

[0057] The term "linker" or "linking group" refers to an organic moiety that links two parts of a compound, e.g., is covalently attached to two parts of a compound. The RNA strands can have the same or different number of linked nucleosides. The maximum number of base pairs is the number of linked nucleosides in the shortest strand of the dsRNA minus any overhangs present in the duplex. In addition to the duplex structure, the dsRNA can include one or more nucleoside overhangs. In one embodiment of the dsRNA, at least one strand includes a 3' overhang of at least one nucleoside. In another embodiment, at least one strand includes a 3' overhang of at least two linked nucleosides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 linked nucleosides. In other embodiments, at least one strand of the dsRNA includes a 5' overhang of at least one nucleoside. In certain embodiments, at least one strand comprises a 5' overhang of at least two linked nucleosides, for example, 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or 15 linked nucleosides. In yet other embodiments, both the 3' and 5' ends of one strand of the dsRNA comprise an overhang of at least one nucleoside.

[0058] Linker or linking group also refers to a link between two atoms that connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds. The conjugate moiety can be directly or through a linking moiety (e.g., linker or tether) to the dsRNA. The linker serves to covalently link a third region, e.g., the conjugate moiety, to the dsRNA (e.g., the end A or C of the region). In some embodiments of the present invention, the conjugate or dsRNA conjugate of the present invention can optionally include a linker region disposed between the dsRNA and the conjugate moiety. In some embodiments, the linker between the conjugate and the dsRNA is biologically cleavable. Phosphodiester-containing biodegradable linkers are described in more detail in WO2014 / 076195 (incorporated herein by reference).

[0059] As used herein, the term "nucleoside overhang" refers to at least one unpaired nucleobase that protrudes from the double-stranded structure of dsRNA or siRNA. For example, a nucleoside overhang exists when the 3'-end of one strand of dsRNA extends beyond the 5'-end of the other strand, or vice versa. A dsRNA may contain an overhang of at least one nucleoside, or the overhang may contain at least two nucleosides, at least three nucleosides, at least four nucleosides, at least five nucleosides, or more. A nucleoside overhang comprises or consists of alternative nucleosides, including deoxynucleotides / nucleosides. The overhang(s) may be present on the sense strand, the antisense strand, or any combination thereof. Furthermore, the nucleoside(s) of the overhang may be present on the 5'-end, the 3'-end, or both ends of either the antisense or sense strand of the dsRNA. In certain embodiments, the overhang comprises a self-complementary portion such that the overhang can form a hairpin structure that is stable under physiological conditions.

[0060] The terms "a" and "an" mean one or more. EXAMPLES

[0061] General Methods and Sequence Listing Experimental model and subject details Study population (patients with NASH) The Cambridge cohort consisted of 58 consecutive NAFLD patients (19 NAFL, 24 NASH F0-2, 15 NASH F3-4) recruited at the NASH Unit of Cambridge University Hospitals. All patients had a clinically and biopsy-proven diagnosis of NAFLD (alternative diagnoses and patients with fatty liver due to different aetiology were excluded), histology recorded according to the NASH CRN scoring system (NAS1) by a trained human pathologist, and snap-frozen tissue for research purposes (gene expression by next-generation sequencing, see below). All comparisons were made to the NAFLD group. The study was approved by the local ethical committee and adhered to the principles of the Declaration of Helsinki. All patients gave their informed consent for the use of their clinical / omics data and samples for research purposes. For NASH / HCC patient information from single-cell sequencing from UCLA, detailed patient information is attached in Table 1. [Table 1]

[0062] Mouse strains, HFD feeding and DEN injections All animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals and were approved by the Biomedical Research Ethics Committee for Biomedical Research at George Washington University. C57BL / 6 mice were purchased and operated from Jackson Laboratories. Male and female mice were used in our studies. To generate liver-specific deletion in Sptbn1 mice, Flox sites were inserted on both sides of exon 24 to exon 26 of the Sptbn1 gene, together with a Neo cassette. The Neo cassette was then removed by crossing with Flp mice. Sptbn1-Flox mice were then crossed with Albumin-Cre to generate liver-specific deletion in Sptbn1 mice. For high-fat diet (HFD)-induced hepatic steatosis, 10- to 12-week-old male and female mice were fed a control diet or HFD (ENVIGO, Cat.TD.06414) for 12 to 20 weeks. Blood glucose, TG and cholesterol levels were measured by glucometer, CardioChek PA analyzer and PTS Panels lipid panel test strips. For the DEN-induced liver cancer model, 25 mg / kg DEN was injected into 14-15 day old male and female mice. Tumor progression was analyzed after 6-10 months. Liver and visceral adipose tissue were excised and weighed. Spleen, heart, brain, muscle and brown adipose tissue were also harvested for further histological analysis.

[0063] Generation of cell lines and cell cultures Sptbn1 - / - , Sptbn1 + / -and WT MEFs were generated from Sptbn1 crossbreed mice. Human hepatoma cell lines Hep3B and HepG2, Huh7, and mouse immortalized hepatocyte cell line AML12 were purchased from ATCC and cultured in complete culture medium DMEM / F12 medium (Corning, Cat. 10-090-CV) supplemented with 1% streptomycin-penicillin and 10% FBS (Hyclone, Cat. SH30396.03). The human immortalized hepatocyte cell line THLE2, purchased from ATCC, was cultured in BEGM medium (Lonza / Clonetics Corporation, Cat. CC3170) supplemented with BPE (bovine pituitary extract), hydrocortisone, hEGF, insulin, triiodothyronine, transferrin and retinoic acid from the BEGM culture medium kit (Lonza / Clonetics Corporation, Cat. CC3170), plus 1% streptomycin-penicillin, 10% FBS (Sigma-Aldrich, Cat. F2442), 40 ug fluorophor-ethanolamine (Sigma-Aldrich, Cat. P0503) and 3 ug human recombinant EGF (Corning®, Cat. 354052). THLE2 cells were cultured using flasks or plates pre-coated with 0.01 mg / ml fibronectin, 0.03 mg / ml type I bovine collagen, and 0.01 mg / ml bovine serum albumin. For HepG2 cell lines with stable knockout of Smad3 and / or Sptbn1 by CRISPR / Cas9, HepG2 cells seeded on 6-well plates were transfected with Sptbn1 / HDR knockout CRISPR plasmids (Santa Cruz, sc-401818, sc-401818-HDR) using Lipofectamine LTX (Invitrogen) and Opti-MEM medium (Invitrogen) in triplicate each well according to the manufacturer's instructions. After 48 hours, puromycin (5 μg / ml) was added into the medium for selection of stable knockout cells. The selection medium was changed every 2-3 days.

[0064] Learn how reagent DMEM / F12 medium was obtained from Corning (Cat. 10-090-CV), streptomycin-penicillin (Corning, Cat. 30-002-Cl), fetal bovine serum was purchased from HyClone (Cat. SH30396.03), luminophor-ethanolamine (Cat. P0503), and THEL2 were purchased from ATCC (ATCC Cat. CRL-2706).

[0065] Lipoprotein-deficient serum from fetal bovine was obtained from Sigma-Aldrich (Cat. S5394). BEGM culture medium kit was obtained from Lonza / Clonetics Corporation (Cat. CC3170). Human recombinant EGF was purchased from (Corning, Cat. 354052). Collagen-coated flasks for THLE2 cells were purchased from Thermo Scientific (Cat. 132707).

[0066] Targeting SPTBN1 with siRNA in mouse and human 3D cultures For hydrodynamic injection of siRNA into mouse tails, siSptbn1 and siCtrl were resuspended to 0.25 mM in nuclease-free water (Life Technologies) and incubated with RNAiMAX transfection reagent (Invitrogen) at a 1:1 ratio for 20 minutes at room temperature. These complexes were then diluted to 0.625 uM in TransIT®-QR hydrodynamic delivery solution (Mirus, Madison, WI). Mice were given 2.0 mL of diluted siSptbn1 (1.25 nmol) or siCtrl by hydrodynamic tail vein injection as described previously. After feeding with HFD for 1 week, injections were repeated at 2-week intervals for 6 weeks. For a human 3D perfusion microphysical system consisting of primary human hepatocytes, hepatic Kupffer cells and stellate cells, these cells were co-cultured for 2 weeks in a medium enriched with fatty acids, sugars and insulin as a culture model of human NASH.

[0067] Transfection and cell fractionation Cells seeded in 60 mm plates on day 0 were transfected with 3–5 μg of the indicated plasmid mix on day 1 using Lipofectamine 3000 (Thermo Fisher Scientific, Cat. L3000015) according to the manufacturer's instructions. For TGF-β treatment on day 2, cells were incubated overnight in serum-free DMEM / F12 medium. TGF-β1 (Sigma-Aldrich, Cat. T1654; R&D, Cat. 240-B) was then added at a final concentration of 200 pM for 0.5, 2, 12, 24 or 48 h, after which cells were harvested. For knockdown experiments, siRNA targeting Sptbn1 or Smad3 (Dharmacon) was transfected into HepG2 or Hep3B cells by Lipofectamine RNAiMAX (Thermo Fisher, Cat. 13778150) for 24–48 h.

[0068] For cell fractionation, cells were harvested and lysed with cytoplasmic lysis buffer followed by nuclear lysis buffer according to the manufacturer's instructions for the Nuclear / Cytosolic Fractionation Kit (Biovision, Cat. K266-25).

[0069] Immunoprecipitation and immunoblotting Treated or untreated cells were incubated with freshly made (20 mM Tris-HCl) using a complete protease inhibitor and phosphatase inhibitor cocktail (Sigma-Aldrich, Cat. 4906845001 and Cat. 11836170001). The cells were lysed in 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton®-X100, 1% sodium deoxycholate, 0.1% SDS) or NP-40 lysis buffer (50 mM Tris-HCl, pH 7.5, 0.15 M NaCl, 1% NP-40, 1 mM EDTA) followed by SDS-PAGE and immunoblotting using the following antibodies: anti-SPTBN1 (in-house, rabbit), anti-SREBP1 (Abcam, Cat. Ab191857), anti-vinculin (Proteintech, Cat. 26520-1-AP) and anti-GAPDH (Santa Cruz, Cat. sc-32233), anti-tubulin (Cell Signaling, Cat. 3873), and anti-Smad3 (Cell Signaling, Cat. 9513). For immunoprecipitation, each fraction lysate or whole cell lysate was precleared for 30 min to remove nonspecific binding, and then immunoprecipitation was performed for 3 h or overnight using Protein A / G Mix Magnetic Beads (MilliporeSigma™, Cat. LSKMAGAG02) mixed with anti-SPTBN1 (in-house) (23) or rabbit IgG (Cell Signaling, Cat. 2729) as a control. The immunoprecipitated complexes (magnetic beads-lysate-antibody mix) were washed with washing buffer (10 mM EDTA-Tris, 150 mM NaCl, 20 mM MgCl ). 2 ), eluted with sample buffer, and then subjected to Western blot analysis.

[0070] Luciferase assay For transcriptional reporter assays, HepG2 or Hep3B cells were cultured at 1 × 10 per well. 4 The cells were seeded in 24-well dishes at a density of 1000×. The next day, cells were incubated with Lipofectamine Cells were co-transfected with siRNA (control, or siRNA targeting Sptbn1 and Smad3) and a luciferase reporter (containing the LDLR promoter or SCD promoter) using a 3000 (Thermo-Fisher, Cat. L3000015). - / - ) or Sptbn1(Sptbn1 - / - ), MEF cells lacking Sptbn1 + / - For the wild type, cells were co-transfected with either the wild type LDLR promoter region (pLDLR-luc, Addgene Cat. 14940), the LDLR promoter region harboring an SREBP-unresponsive mutant SRE (pLDLR-luc MUT, Addgene Cat. 14945) or the SCD1 promoter (pGL3-SCD1, a gift from Drs. Giovanni Sorrentino and Giannino Del Sal) with Renilla (Promega) using Lipofectamine 3000 (Thermo Fisher, Cat. L3000015). For rescue experiments, pLDLR-luc or pGL3-SCD1 was co-transfected with the control plasmid or Sptbn1 with V5-Sptbn1. - / - Smad3 in MEF cells or together with Flag-SMAD3 plasmid - / -MEF cells were co-transfected. 24 hours after transfection, cells were treated with 200 pM TGF-β1 (R&D, Cat. 240-B;) for 24 hours and then serum-starved overnight. In all luciferase assays, the expression plasmid Renilla (Promega) served as an internal control to correct for transfection efficiency. Cells were extracted using 100 μl of luciferase cell culture lysis reagent. Ten microliters of cell extract were used for measurement of Renilla enzyme activity. Twenty microliters of cell extract were used for luciferase assay using Dual Luciferase Assay Kit (Promega, Cat. E1980) according to the manufacturer's instructions. Luciferase activity was normalized to Renilla activity (AU) for each sample, and fold change was calculated.

[0071] Immunohistochemistry and immunofluorescence analysis For immunohistochemistry analysis, mouse tissues were fixed in 10% formalin and embedded in paraffin using standard procedures. FFPE sections were stained with hematoxylin and eosin (H&E) to assess gross morphology and ballooning, with Oil Red to determine triglyceride accumulation, and with Sirius Red to determine fibrosis. Different cell types were also determined using Hepatocyte Nuclear Factor 4 (HNF4) (for hepatocytes), Desmin (for hepatic stellate cells), CD31 (for hepatic sinusoidal endothelial cells), F4 / 80 (for macrophages), CD142, ICAM1, and CK19 (for cholangiocytes). Sections were labeled with antibodies specific for SPTBN1, SREBP1, and Smads (Cell Signaling Technology, 2899) antibodies. Diaminobenzidine was used as the chromogen.

[0072] For immunofluorescence analysis, cells were plated at 2 × 10 per dish in DMEM / F12 supplemented with 10% FBS or 1% lipoprotein-deficient serum (LPDS) for 12 h. 4Cells were seeded on glass-bottom dishes at a density of 100x100x100 / mL. Cells treated with oleic acid, staunosporin, or TNF-α with or without TGF-β at different time points were then fixed with 10% formalin for 15 min, followed by permeabilization with 0.1% saponin for 5 min. Cells were then incubated with blocking solution (PBS containing 0.1% saponin and 10% bovine serum albumin) for 1 h at room temperature, followed by incubation with primary antibodies (anti-rabbit SREBP1, anti-rabbit Sptbn1, and anti-rabbit SMAD3 at 1:200) overnight at 4°C. The next day, cells were washed with PBS and incubated with secondary antibodies (anti-rabbit Alexa Fluor Plus 488, goat anti-mouse Alexa Fluor 568, goat anti-rabbit Alexa Fluor 488, Invitrogen) for 1 h at room temperature. Nuclei were counterstained using DAPI. Images were acquired with an IX81 Olympus microscope and a confocal microscope using a ×40 objective.

[0073] RNA extraction and RNA integrity Human RNA was isolated by the following protocol: biopsies were homogenized in STAT-60 (AMS biotechnology, CS-502) (1 ml), mixed (by vortexing), centrifuged at 13,000g / RT for 5 min, the supernatant was mixed with 200 μl chloroform (Sigma, Cat. 650471) and centrifuged (12,000g / 4°C / 15 min), then the supernatant was mixed with 500 ul isopropanol (Sigma, Cat. 33539) and centrifuged (10,000g / 4°C / 10 min) to pellet the RNA, the pellet was washed with ethanol (75%; 1 ml), dried at room temperature and the RNA was resuspended in DEPC-treated water (Quality Biological, Cat. 351-068-131). All reagents, plasticware and equipment used were nuclease-free, sterile and of molecular biology grade. RNA purity (A260 / A280>1.80) and concentration were determined using a Nanodrop (Thermo The RNA integrity was determined using a 2100 Bioanalyzer (RNA 6000 Nano Kit, Agilent, Santa Clara, California, USA); an RNA integrity number (RIN) of 8 or greater was considered as the minimum cutoff for sequencing. For mice, RNA was extracted from either liver tissue or hepatocytes using the RNeasy Plus Mini Kit (Qiagen, Cat. 74134). RNA quality and concentration were assessed using a Thermo Scientific Nanodrop 3300 spectrophotometer, followed by RNA sequencing (Novogene Corporation). For real-time PCR analysis, total RNA was extracted utilizing TRIzol reagent (Invitrogen, Cat. AM9738) according to the manufacturer's instructions. Reverse transcription of 1–2 ug of total RNA was performed by using the Super-Script III First Strand Kit (Invitrogen). Each cDNA was amplified in triplicate over 40 cycles with the iQ™ SYBR Green SuperMix PCR kit (Bio-Rad Laboratories) in a Bio-Rad system (iCycler Thermal Cycler). Primers for real-time PCR are listed in the table below.

[0074] Whole transcriptome amplification and RNA sequencing One microgram of RNA was used to generate a barcoded sequencing library using the Illumina TruSeq® Stranded mRNA Library Preparation Kit (Illumina) according to the manufacturer's instructions. Sequencing libraries were normalized for concentration, pooled together into a 96-plex pool, and sequenced at single end 50 bp (SE50) in three lanes on an Illumina HiSeq 4000 instrument, yielding an average of 15 million reads / sample. Library preparation was performed by the Genomics and Transcriptomic Core at the Institute of Metabolic Sciences, and sequencing was performed at the Genomics Core, Cancer Research UK Cambridge Institute, both at the University of Cambridge.

[0075] NGS data processing and statistical analysis RNA sequencing data were aligned against the human GRCh38 genome and the house mouse (GRCm38 / mm10) using hisat2 V2.1.02 and genes that passed QC were counted using HTSeq2 (v0.11.1)3. The raw gene-level counts were then used for differential gene expression analysis using DESeq24. Log 2 - Transformed copies per million (Log 2 Gene expression was normalized using the CPM (Cross-regression probability distribution), and statistical significance (p<0.05) was assessed using the Wald test in DESeq2 by R. Raw p-values ​​were then adjusted by the Benjamini-Hochberg procedure to suppress a false discovery rate (FDR) of 5.

[0076] Bioinformatics Functional Analysis Whole transcriptome imputation was performed using Ingenuity Pathway Analysis (IPA, Qiagen), followed by filtering statistically significant (p<0.05) hits in IPA with Log2FC below -0.3785 or above 0.3785, Log2CPM>0.5 in human, and / or FPKM>1 in mouse to investigate differentially expressed genes within groups. Upstream regulator networks were generated and ordination ordered by importance (p<0.05 in human) and activation status (Z-score) of the involved genes. We considered only genes that were statistically significant (p<0.05), had Log2FC below -0.3785 or above 0.3785, enriched for "significantly regulated" networks in the comparative analysis, and / or had FDR<0.05.

[0077] Structural modeling of human SPTBN1 and SREBP1c The three-dimensional structures of human SPTBN1 and SREBP1c are still unknown, and therefore in-silico structural modeling was performed using a homology-based fold recognition approach using the web-based Phyre2 (Protein Homology / analogY Recognition Engine V 2.0) server (53). This approach generates three-dimensional models of protein structures using multiple templates covering different query regions (with different identities and therefore varying confidence levels) of the amino acid sequence. Initially, template identification was performed by P-BLAST (Protein Basic Local Alignment Search Tool) against the RCSB Protein Data Bank (PDB), where several templates were found to show favorable sequence identity and overlap with SPTBN1 and SREBP1c to model their structures. Templates from the PDB with the highest sequence identity and overlap, as well as fold structures from the in-house Phyre fold library, were used to generate models of human SPTBN1 and SREBP1c (Table 2). The loop model was optimized using ModLoop (54) followed by model optimization using the Swiss-PDB Viewer (55). [Table 2]

[0078] Model optimization and energy minimization were also performed to find the most stable and lowest energy conformations of SPTBN1 and SREBP1c to avoid any resulting errors and high energy configurations that may lead to physical perturbations and instability of the structures. Swiss-PDB Viewer was used for energy minimization and structure optimization by changing the coordinate geometry in such a way as to release internal strains and reduce the total potential energy of the models.

[0079] Structure-based molecular docking Furthermore, their binding modes were explored by using molecular docking techniques after modeling the SPTBN1 and SREBP1c structures. The structural coordinates of the SPTBN1 fragment encompassing D50-T975, Q1132-T2155, and A2198-K2364, and the SREBP1c fragment encompassing Q295-K374 and P546-S705 were taken from our model structure. Docking was performed using ClusPro 2.0 web server (38). First, the docking results were screened for higher binding affinity, and then the top docked conformations were selected and their possible interactions were further analyzed using PyMOL and LigPlot+.

[0080] statistics Differences between the two groups were assessed using a two-tailed Student's t-test using GraphPad Prism. Pairwise comparisons were performed on weight gain measured weekly. One-way ANOVA with post-hoc Bonferroni's test for multiple comparisons was used. In vitro experiments were performed two to four times. All luciferase experiments were performed at least three times with three replicates. Results are expressed as mean ± standard deviation unless otherwise indicated. In all statistical analyses, p < 0.05 was considered statistically significant.

[0081] Various human and mouse sequence listings Homo sapiens spectrin beta, non-erythrocytic 1 (SPTBN1), transcript variant 1, mRNA NCBI reference sequence: NM_003128.3

[0082] Homo sapiens spectrin beta, non-erythrocytic 1 (SPTBN1), transcript variant 2, mRNA NCBI reference sequence: NM_178313.2

[0083] Mus musculus spectrin beta, non-erythroid type 1 (Sptbn1), transcript variant 1, mRNA NCBI reference sequence: NM_175836.2

[0084] Mus musculus spectrin beta, non-erythroid type (Sptbn1), transcript variant 2, mRNA NCBI reference sequence: NM_009260.2

[0085] Mouse siRNA targeting Sptbn1 Target sequence:

[0086] Sense: CGAUGUUACAAGAACGGUUTT (SEQ ID NO:5).

[0087] Antisense: AACCGUUCUUGUAACAUCGTG (sequence number 6).

[0088] Targeting with human siRNA Target sequence: CCUGAAAGUGAGCGCAUUA (SEQ ID NO:7).

[0089] Target sequence: CCGCAUACGAGGAGCGUGU (SEQ ID NO:8).

[0090] Target sequence: GGACAUGUCUUACGAUGAA (SEQ ID NO:9).

[0091] Target sequence: GUGACAAGGCCGACGAUAU (SEQ ID NO: 10).

[0092] Example 1 Liver-specific low SPTBN1 knockout protects mice from HFD-induced NASH We generated liver-specific conditional SPTBN1 (Sptbn1-flox) knockout mice (LSKO) without hepatocyte SPTBN1 (Supplementary Figure 1A,B). LSKO mice are viable and fertile. Hepatocyte-specific deletion of SPTBN1 was supported by the lack of detectable protein by immunoblot or immunohistochemical staining (Supplementary Figure 1B,C). LSKO mice fed a normal diet were characterized for liver histology and metabolic state (Supplementary Figure 1D-H). On a normal diet, LSKO mice weighed similarly to Floxed control mice (Supplementary Figure 1E), but LSKOs exhibited significantly reduced concentrations of serum triacylglycerol (TG) (Supplementary Figure 1F) and blood glucose (Supplementary Figure 1G), and had slightly elevated insulin sensitivity in an insulin resistance test (Supplementary Figure 1H). Histologically, the livers of LSKO mice had reduced lipid accumulation when compared to Floxed mice (Supplementary Figure 1D). In addition, LSKO and Flox control mice had similar food and water intake, and urine output (Supplementary Figure 2A).

[0093] The impact of HFD was evaluated using LSKO mice. Male and female Flox control and LSKO mice aged 10–12 weeks were placed on a HFD for 12–20 weeks (Figure 1A). The HFD was 60% fat for calories (29–31). After 12 weeks on the HFD, food and water intake, as well as urine output, were comparable between Flox control and LSKO mice (Supplementary Figure 2A). However, after 12 weeks of HFD, LSKO mice gained less weight, had less visceral fat, and were protected from HFD-induced obesity (Figure 1B). Metabolic profile assessment showed that HFD-fed LSKO mice had lower concentrations of serum triglycerides (TG) but serum glucose and total cholesterol concentrations similar to HFD-fed Flox control mice (Figure 1C). The livers of HFD-fed LSKO mice were smaller than those of HFD-fed Flox control mice (Fig. 1D), even though both mouse genotypes had similar levels of Ki67-labeled proliferating cells in their livers (Supplementary Fig. 2B). Analysis of liver function by measuring serum aspartate transaminase (AST) and alanine transaminase (ALT) concentrations showed reduced concentrations in LSKO mice, suggesting better liver function and less liver damage in HFD-fed LSKO mice compared to HFD-fed Flox control mice (Fig. 1D).

[0094] Consistent with less liver damage in HFD-fed LSKO mice, liver histology of these mice revealed normal liver architecture, minimal lipid accumulation, and the absence of possible ballooning or signs of inflammation in the liver (Figure 1E). Electron microscopy revealed fibrogenesis, indicated by collagen deposition in the extracellular matrix, and steatosis, indicated by accumulation of lipid droplets, in the livers of HFD-fed Flox mice, but these changes were absent in the livers of HFD-fed LSKO mice (Figure 1F). Compared with livers from Flox mice fed a HFD for 16 weeks, livers from LSKO mice showed decreased expression of TGF-β / SMAD3-regulated genes associated with liver fibrosis, altered expression of genes associated with inflammation, and decreased expression of genes encoding proteins involved in fatty acid metabolism (Figure 1G).

[0095] Changes in Flox control mice indicated that 16 weeks of HFD caused these mice to develop NAFLD with progression to NASH. Moreover, LSKO mice were protected from this diet-induced liver pathology. LSKO mice fed an HFD were found to be protected from the development of obesity and NAFLD, as well as from the development of NAFLD and HCC.

[0096] Example 2 SPTBN1 regulates the SREBP pathway and fatty acid metabolism To understand how liver-specific loss of SPTBN1 protects mice from liver disease, we conducted targeted studies of certain key regulators in Flox and LSKO mice fed a HFD for 16 weeks and performed comprehensive liver metabolite analysis of RNA sequencing (RNA-seq) data to investigate gene expression differences in the liver of Flox and LSKO mice after 16 weeks of HFD. We examined the transcript and protein abundance of the transcriptional regulator C / EBPα, which regulates adipogenesis (32) and uncoupling protein 2 (UCP2), which reduces mitochondrial ATP production, and has been linked to NASH (33). For none of these, there were differences in transcript or protein abundance levels in the liver of HFD-fed Flox and LSKO mice (Supplementary Figure 2C). Bioinformatics analysis with Ingenuity Pathway Analysis (IPA) showed that some of the pathways with associations with differentially expressed genes were related to stress or infection (Figure 2A). Ingenuity "upstream regulator" analysis of differentially regulated genes in LSKO mice showed an association with decreased or reduced activity of transcription factors controlling lipid and cholesterol metabolism - SREBP, PPARγ (peroxisome proliferator-activated receptor gamma) and PPARα - and the transcription factor XBP1, which is activated by ER stress (Figure 2A). Given the association between loss of SPTBN1 in liver and reduced SREBP and PPARG lipid pathways, we examined the limited mass spectrometry data to set up proteins that co-immunoprecipitated with V5-tagged SPTBN1 from stressed HEK293T cells and relate them to nascent lipogenesis or its regulation (Table 3). Unexpectedly, based on those data, SREBP1 was a possible binding partner of SPTBN1. Therefore, we focused on SREBP1. [Table 3]

[0097] Because SPTBN1 has an anchoring function in both the cytoplasm and nucleus, we assessed whether SREBP1 distribution or abundance in the liver was altered in LSKO mice fed either a normal diet or a HFD. In the livers of Flox mice fed a normal diet, SREBP1 had a focal punctate distribution, with few cells showing nuclear staining (Figure 2B, bottom). In mice fed a HFD for 12–16 weeks, most hepatocytes had nuclear-localized SREBP1 in livers from Flox control mice, consistent with SREBP1 activation. SREBP1 staining was less focal in livers from LSKO mice fed either a normal diet or a HFD, and there were fewer cells with nuclear SREBP1 in livers from HFD-fed LSKO mice, suggesting lower SREBP1 activity (Figure 2B). No differences in SREBP1 transcript abundance were detected in livers from Flox control and LSKO mice fed a normal diet ( Supplementary Figure 2D ), indicating that the reduction in SREBP1 abundance was not mediated at the level of gene expression.

[0098] There are two genes encoding SREBP1 and SREBP2, therefore, we assessed the abundance of pro-SREBP (ER-localized full-length SREBP) and n-SREBP (truncated nuclear SREBP) for both proteins and their ER-localized regulators SCAP and INSIG in the livers of Flox and LSKO mice fed a normal diet or HFD for 12–16 weeks. SCAP and INSIG abundance was similar between Flox and LSKO mice in the normal diet or HFD groups, and SCAP showed a consistent decrease in abundance in HFD-fed mice (Figure 2C). In addition, knockdown of SPTBN1 by siRNA in THLE2 human hepatocytes did not affect INSIG1 abundance (Supplementary Figure 2E). Although varying among individual mice, pro-SREBP1 and n-SREBP1 appeared to be decreased in livers of LSKO mice fed a normal diet or HFD compared with their abundance in livers of Floxed control mice fed a normal diet (Fig. 2C). In HFD-fed mice, the abundance of pro-SREBP2 was similar or slightly lower in livers from Floxed control mice, whereas the abundance of n-SREBP2 appeared to be similar or slightly lower in LSKO mouse livers (Fig. 2C). Thus, the decrease in SREBP1 abundance detected by immunohistochemistry and immunoblot in LSKO mouse livers was not due to altered abundance of SREBP ER-localized regulators or impaired SREBP1 gene expression.

[0099] To determine whether the reduction in SREBP1 in LSKO mouse liver corresponded to a reduction in SREBP1 target gene expression, we analyzed either the transcript and protein abundance of the products of nascent lipogenesis genes or luciferase reporter assays performed with SRE-containing promoters. In the livers of mice fed a normal diet or a HFD for 12–16 weeks, ACC1 (encoding acetyl-CoA carboxylase 1), SCD1 (encoding stearoyl-CoA desaturase-1), and FASN (encoding fatty acid synthase) were less abundant in LSKO tissues, and these changes were also reflected in less abundant proteins (Figure 2D,E). For this reason, we detected a reduction in the expression of SREBP1 target genes in the absence of SREBP1. Consistent with the minimal differences observed in SREBP2 abundance in the livers of Flox and LSKO mice, transcripts of LDLR, a major target of SREBP2 in cholesterol metabolism (34), were found to be comparable in mouse livers, as were the amounts of LDLR protein (Fig. 2D, E). The data suggested that SPTBN1 predominantly regulates SREBP1 but not SREBP2.

[0100] The SREBP-responsive luciferase assay uses luciferase reporters containing a wild-type SRE from the LDLR promoter region (LDLR-luc), an SREBP-unresponsive mutant SRE (mut-LDLR-luc), or an SRE from the SCD1 promoter (SCD-luc) and was assayed in mouse embryonic fibroblast (MEF) cells isolated from wild-type mice and in systemic Sptbn1 cells. - / - Serum starvation, which activates SREBP in cultured cells (35), was found to increase SRE-dependent LDL-luc and SCD-luc activity in wild-type MEFs, and Sptbn1 - / - No or minimal SRE-dependent luciferase activity was detected in cells (Figure 2F), indicating that SREBP1-dependent target gene expression was impaired in the absence of SPTBN1. - / -Re-expression of SPTBN1 in MEF cells restored SRE-dependent luciferase activity (Fig. 2G). We assessed whether TGF-β stimulates SRE-dependent gene expression and found that TGF-β stimulated SRE-dependent luciferase activity in an SPTBN1-dependent manner (Fig. 2F), suggesting that TGF-β signaling involving SPTBN1 may also contribute to de novo lipogenesis.

[0101] WT or SMAD3 - / - Using a luciferase reporter gene expressed in MEFs, we performed experiments to examine whether TGF-β signaling through SMAD3 affects SRE-dependent gene expression. - / - As a positive control, luciferase expression stimulated by TGF-β was observed in MEFs (Fig. 2H, left). - / - from SMAD-dependent promoters in WT MEFs, but not in MEFs ( Fig. 2H , right). These data indicated that TGF-β signaling through SMAD3 was not responsible for TGF-β-mediated enhancement of SRE-dependent gene expression.

[0102] Example 3 SPTBN1 binds to SREBP1 To verify the mass spectrometry data suggesting that SPTBN1 and SREBP1 are part of the same complex (Table 3), SPTBN1 was immunoprecipitated from three HCC cell lines. Pre-SREBP1 and n-SREBP1 were detected in immunoprecipitates from all three cell lines (Fig. 3A, Supplementary Fig. 3A), and the interaction of SPTBN1 with pre-SREBP1 occurred in the cytoplasm (Supplementary Fig. 3B).

[0103] To explore the structural basis of the binding between SPTBN1 and SREBP1 and to map the potential interaction sites, we modeled the three-dimensional structures of SPTBN1 and SREBP1c and then performed structure-based molecular docking simulations. There are two splice variants of SREBP1: SREBP1a and SREBP1c (36). SREBP1c was selected for molecular docking simulations because it is the form that specifically stimulates fatty acid synthesis and is associated with hepatic steatosis (37).

[0104] SPTBN1 is composed of 2,364 amino acid residues and features multiple homologous tandem spectrin repeats, each of which consists of three antiparallel helices flanked by part of a pair of calponin-homology (CH) domains at the N-terminus and a pleckstrin homology (PH) domain at the C-terminus (Figure 3B). SPTBN1 has a caspase-3 cleavage site (1454DEVD1457) in the 11th spectrin repeat. SREBP1c is composed of 1,047 amino acid residues that form an N-terminal DNA-binding domain (bHLH), two transmembrane domains, each with a central cleavage site, and a regulatory domain at the C-terminus. SREBP1c has a caspase-3 cleavage site (433SEPDSP438) before the transmembrane domain (12) (Figure 3B).

[0105] For modeling, SPTBN1 was tested in three fragments encompassing D50-T975, and Q1132-T2155 (Fig. 3C), as well as A2198-K2364 (Supplementary Fig. 3C), and SREBP1c was modeled in two fragments encompassing Q295-K374 (Fig. 3C) and P546-S705. SREBP1c P546-S705 cannot be docked onto SPTBN1. However, the interaction was successful with SREBP1c. was simulated between the Q295–K374 fragment and SPTBN1 ( Figures 3C,D ; Supplementary Figure 3C ; Table 4 ).

[0106] The model predicted that SREBP1c Q295~K374 would conformationally engage with high affinity in the binding cavity of SPTBN1 and that the interaction would be stabilized by multiple hydrogen bonds and van der Waals interactions (Table 4). Based on the lowest interaction energy values ​​(38,39), the predicted models were ranked as having the following affinities (Table 4): SREBP1c Q295~K374 / SPTBN1 Q1132~T2155 (Figure 3D)>SREBP1c Q295~K374 / SPTBN1 D50~T975 (Figure 3C)>>SREBP1c Q295~K374 / SPTBN1 A2198~K2364 (Supplementary Figure 3C). Taken together, the docking analysis predicted SPTBN1 fragments D50-T975 and Q1132-T2155 as preferred SREBP1c Q295-K374 binding sites. [Table 4]

[0107] To test the predictive model, we generated SREBP1 and SPTBN1 fragments to identify the regions required for their interaction. Starting with N-SREBP1 (amino acids M1 to L466), we generated four fragments (Figure 3D). Fragment 2 of SREBP1 corresponds to the modeled portion (Q295 to K374) and coimmunoprecipitated more effectively with full-length V5-tagged SPTBN1 (Figure 3D). Indeed, this region was required for the interaction. Caspase 3 cleaves SPTBN1 into two products, N-SPTBN1 (amino acids M1 to D1454) and C-SPTBN1 (E1455 to K2364), which are detected in acetaminophen-injured liver (27). Although N-SPTBN1 and C-SPTBN1 were generated tagged with V5, only N-SPTBN1 was found to coimmunoprecipitate with Flag-tagged n-SREBP1 (Figure 3E). These data suggest that SPTBN1 and SREBP1 may directly interact. Modeling and fragment interaction analysis led to the hypothesis that conditions that result in caspase-3 activation in cells result in cleavage of SPTBN1, which facilitates the interaction between N-SPTBN1 and n-SREBP1.

[0108] Example 4 Cellular stress induces caspase-3-dependent cleavage of both SREBP1 and SPTBN1 in human hepatocytes and HCC cells Stress-induced caspase activation has been associated with the NASH phenotype (8, 9). RNA-seq data revealed alterations in the ER stress and UPR pathways in the livers of HFD-fed LSKO mice (Figure 2A). Because SREBP1 and SPTBN1 are both substrates of caspase-3 (12, 27) and interaction data suggested that their cleavage products interact (Figure 3E, F), we explored the relationship between caspase-3, SPTBN1, and SREBP1 in immortalized human hepatocyte cell line THLE2 and HCC cell line Huh7. TNFα and cycloheximide or palmitic acid (PA) were used to induce caspase-3 activation. TNFα and PA induce caspase-3 activation in hepatocytes (40, 41) and have been associated with the progression to NASH (42, 43). THLE2 cells exposed to TNFα and cycloheximide exhibited caspase 3 activation (cleaved caspase 3), SREBP1 activation (n-SREBP1), and cleavage of SPTBN1 to generate both N-SPTBN1 and C-SPTBN1 (Figure 4A). Analysis of the cytosolic and nuclear distribution of N-SPTBN1 and C-SPTBN1 expressed as V5-tagged proteins in THLE2 cells showed that N-SPTBN1 was present in both the cytoplasm and nucleus, whereas C-SPTBN1 was only detected in the cytoplasm (Figure 4B). Knockdown of SPTBN1 in Huh7 cells did not attenuate caspase 3 activation induced by TNFα and cycloheximide, or cleavage of SREBP1 in these conditions (Figure 4C). However, expression of SCD1, an SREBP1 target gene, was decreased (Figure 4C). Impaired expression of the SREBP2 target gene LDLR was observed in this HCC cell line exposed to TNFα and cycloheximide (Figure 4D), which contrasts with findings in the liver of chow-fed LSKO mice (Figure 2E) and may be related to the lack of stress in in vivo conditions.

[0109] Exposure of Huh7 cells to PA induced caspase-3 activation, which was reduced by a caspase-3 inhibitor (Z-DEVD-FMK) (Figure 4E). PA also induced cleavage of SPTBN1, which was also reduced by a caspase-3 inhibitor, indicating that STPBN1 cleavage is dependent on caspase-3 activity (Figure 4E). Pro-SREBP1 and SPTBN1, as well as N-SPTBN1, were found to be immunoprecipitated from Huh7 cells exposed to PA, consistent with the interaction between SREBP1 and SPTBN1 mediated by the caspase-3 cleavage product N-SPTBN1 (Figure 4F). In PA-treated cells in which SPTBN1 was knocked down, caspase-3 activation was not affected, but N-SREBP was found to be reduced, suggesting that interaction with SPTBN1 may stabilize n-SREBP1 (Figure 4G).

[0110] In stressed hepatocytes or in hepatocytes cultured with high amounts of PA, caspase-3 cleaved both SREBP1 and SPTBN1, and the cleavage products interacted to stabilize the nuclear form of SREBP1, thereby promoting de novo lipogenesis.

[0111] Biochemical data demonstrated a mechanistic link between nonapoptotic caspase-3 activity and nascent lipogenesis through the formation of nSREBP and stabilization of nSREBP1 by interaction with caspase-cleaved N-SPTBN1, providing a second pathway, other than ER stress-induced activation of SREBP, for the aberrant activation of nascent lipogenesis leading to lipidation and progression to NAFLD. The data suggested that caspase-mediated activation of SREBP1 was independent of changes in SCAP and INSIG, ER-localized regulators of SREBP activation (6, 52), thereby bypassing normal controls that limit nascent lipogenesis under conditions of sufficient or excess lipid.

[0112] Example 5 Human NASH is associated with increased SPTBN1 and CASPASE3 expression, as well as enhanced TGF-β pathway and SREBP1 activity To gain insight into the validity of findings implicating pathways involving SPTBN1 and the caspase 3-mediated interaction pathway between the cleavage products of SPTBN1 and SREBP1 in steatosis, data from four publicly available databases with information on healthy obese subjects and patients with NAFLD, NASH, or HCC were analyzed. Using Ingenuity “upstream regulator” analysis, proteins associated with differential gene expression between early NASH (NASH1 and 2) and NAFLD, and between late NASH (NASH3 and 4) and NAFLD were evaluated. Consistent with the involvement of TGF-β signaling in fibrosis, a major indicator of progression of NASH from NAFLD (14, 44, 45), upstream regulators with increased activity included several proteins in the TGF-β pathway, with the only downregulated protein being SMAD7, an inhibitor of TGF-β signaling (Figure 5A).

[0113] Liver tissue data from healthy obese subjects were compared with those from NASH patients for transcripts of SPTBN1, caspase 3, SREBP1 and SMAD3, as well as SREBP1 target genes involved in lipid biosynthesis. SPTBN1 and caspase 3 transcripts were both increased in NASH patients, while no differences were observed for SREBP1 and SMAD3 (Figure 5B). Significant increases in the expression of FASN, SCD and AACS were found, suggesting increased SREBP1 activity (Figure 5C).

[0114] Because NASH represents a high risk of progression to HCC (1), we performed single-cell RNA-seq for SPTBN1 expression in liver tissue from patients with both NASH and HCC but without cirrhosis. Subsets of cells with high SPTBN1 expression were identified (Figure 5D, Table 1): five subtypes of hepatocytes (Hep-1, Hep-4, Hep-5, Hep-6, and Hep-7), both types of cholangiocytes (Chol-1 and Chol-2), and liver sinusoidal endothelial cells of the portal vein (LSEC-PP). Analysis of single-cell RNA-seq data showed that hepatocytes with high SPTBN1 expression clustered together when plotted on a phenograph (Figure 5E). SPTBN1 abundance was confirmed to be higher in liver tissue from NASH patients compared to liver tissue from healthy controls (Figure 5F).

[0115] Collectively, evaluation of human NASH liver data is consistent with findings from HFD-fed mice implicating SPTBN1 in the progression of this disease. Furthermore, these data support increased activity of TGF-β / SMAD signaling and SREBP1-dependent gene expression in addition to increased SPTBN1 and caspase-3 in NASH.

[0116] Example 6 Targeting SPTBN1 with siRNA in vivo attenuates HFD-induced NAFLD and NASH Liver disease is one of the few with FDA-approved siRNA-based therapeutics (46). Therefore, we performed an experiment to examine whether siRNA-mediated knockdown of SPTBN1 in Flox mice protects them from HFD-induced NAFLD and NASH. Flox mice (10–12 weeks old) were fed a HFD for 12 weeks. One week after the start of the HFD, mice were hydrodynamically injected with either siRNA targeting SPTBN1 (siSptbn1) or an equivalent volume of siRNA negative control (siCtrl) every 2 weeks for a total of three injections (Figure 6A). Mice were followed up weekly for weight gain and survival. Knockdown of SPTBN1 was confirmed by siSptbn1 in the liver of Flox mice at the end of the experiment (Figure 6A, right). In each parameter, siSptbn1-treated mice responded similarly to LSKO mice in response to HFD. Compared to siCtrl-treated mice, siSptbn1-treated mice gained significantly less weight (Figure 6B), accumulated less visceral body fat (Figure 6C), had lower blood TG levels, and had similar blood glucose levels (Figure 6D). Histology, lipid accumulation, and expression of profibrotic and inflammatory genes in the livers of siSptbn1-treated mice were also comparable to LSKO mice, with significantly less expression of profibrotic and inflammatory genes compared to livers from siCtrl-treated mice (Figure 6E), and had normal liver architecture and low lipid accumulation without any signs of NAFLD or NASH (Figure 6F).

[0117] The same benefit of siSPTBN1 was observed in liver-specific SPTBN1 heterozygous mice (LHET) (Supplementary Fig. 4A, B) and female Flox mice (Supplementary Fig. 4C). Thus, siSPTBN1 treatment prevented the development of NAFLD and progression to NASH in mice when started early during the HFD feeding period.

[0118] Hepatocyte-specific absence of SPTBN1 apparently had no adverse effects on liver morphology or function, and siRNA targeting SPTBN1 was an effective therapeutic agent in preclinical mouse NASH models and in attenuating progression to HCC in chemically induced mouse models. In addition, acetaminophen is a commonly used over-the-counter drug that is hepatotoxic (51). Low doses of acetaminophen induce phosphorylation of TGFBR2 and SMAD signaling, and higher toxic doses promote caspase activation, leading to the generation of caspase-cleaved SPTBN1 and severe hepatotoxicity (27). Data suggest that targeting SPTBN1 may be a useful therapy to prevent acetaminophen hepatotoxicity.

[0119] The results place SPTBN1 as a central player in both adipogenesis and fibrosis in response to a HFD: as a participant in TGF-β / SMAD3 signaling, SPTBN1 promotes fibrosis, and as a participant in stress-activated SREBP1 signaling, SPTBN1 promotes de novo lipogenesis and adipogenesis.

[0120] Example 7 Knockdown of SPTBN1 in a human 3D NASH model attenuates NASH-associated transcriptional changes Mouse results showed that SPTBN1 knockdown prevented HFD-induced NAFLD and progression to NASH. This potential therapy was also tested in a newly developed three-dimensional perfusion microphysical system that allows co-culture of primary human hepatocytes, hepatic Kupffer cells, and stellate cells for 2 weeks in medium enriched with fatty acids, sugars, and insulin as a culture model of human NASH (47, 48). siRNA was applied on days 4 and 6, and the three-dimensional cultures were exposed to different concentrations of siSptbn1 and equivalent concentrations of siCtrl (Figure 6G). 25 nM siRNA treatment caused a >90% reduction in SPTBN1 transcripts after 96 hours (Figure 6H, left). No significant differences were observed in lipid accumulation on days 8 or 14 (2 or 7 days after siRNA addition), although there was a trend toward lower amounts in siSptbn1-treated cultures (Figure 6H, right).

[0121] RNA-seq analysis of cultures exposed to siSptbn1 (25 nM or 50 nM) or siCtrl for several days was performed on samples harvested 96 hours after siRNA treatment. Pathway analysis revealed that transcripts encoding proteins involved in fatty acid metabolism, including those involved in lipid transport, triglyceride and glycogen metabolism, and lipoprotein catabolism, were significantly decreased in siSptbn1-treated cultures (Figure 6I). There was a decrease in transcripts encoding proteins involved in fibrosis, and changes in inflammatory gene expression in siSptbn1-treated cultures (Figure 6I). These changes indicated that si SPTBN1 treatment reduced de novo lipogenesis, inflammation, and fibrosis. As expected, siSptbn1-treated cultures showed a striking reduction in gene expression signatures associated with TGF-β signaling (Supplementary Fig. 4E), consistent with mouse models showing that this profile is reduced in LSKO and activated in mouse (HFD-induced NASH) and human NASH (Supplementary Fig. 4E).

[0122] "Upstream regulator" analysis by IPA showed that regulators with higher activity in human NASH compared to NAFLD were uniformly associated with lower activity in siSptbn1-treated cultures compared to siCtrl-treated cultures (Figure 5J). Furthermore, upstream regulators with lower activity in NASH than NAFLD had higher activity in siSPTBN1-treated cultures compared to siCtrl-treated cultures (Figure 5J). Thus, siSptbn1 appears to reverse or counteract regulatory changes associated with NASH, further supporting the concept of siSptbn1 as a protein therapeutic.

[0123] Example 8 LSKO mice are protected from HFD-induced HCC The data showed that complete liver-specific loss of SPTBN1 protected mice from HFD-induced NASH, a well-established risk for HCC (1). Here, we evaluated the involvement of homozygous loss of hepatic SPTBN1 for HCC. First, HCC incidence was examined in LSKO and LHET mice fed a HFD for 20 weeks. We found two tumor nodules in four HFD-fed LHET mice, but no liver tumors in any of the age-matched HFD-fed LSKO mice (Figure S7A). Because progression to HCC did not occur frequently enough in this experimental condition, we switched to a chemically induced HCC model.

[0124] Diethylnitrosamine (DEN) was used to induce HCC. Liver tumor progression was assessed at 24 or 40 weeks after DEN injection. Floxed control, LHET, and LSKO mice all had tumor progression in this model, while LSKO mice were relatively protected (Fig. 7B, C; Fig. S7B). Small nodules were found in the liver at 24 weeks after DEN injection (Fig. 7B), which progressed to tumors by 40 weeks (Fig. 7B, C; Fig. S7B). Most LSKO mice had a lower tumor burden, as indicated by lower tumor numbers, smaller tumor sizes, and improved liver pathology, when compared to Floxed control mice (Fig. 7B, C). Furthermore, fewer proliferating cells, as indicated by Ki67 labeling, were found in LSKO mouse livers compared to Floxed control livers (Fig. 7D). However, no significant differences were observed in labeling with the apoptotic marker caspase-3, suggesting that the reduced tumor burden in LSKO mice was not due to increased apoptosis (Fig. 7E).Consistent with the LSKO HCC-protective phenotype, STAT4, an inhibitor of HCC (49, 50), was identified as an active upstream regulator of genes differentially regulated in the liver of HFD-fed LSKO mice (Fig. 2A).

[0125] To test whether complete loss of SPTBN1 has a protective effect in human HCC, the frequency of SPTBN1 homozygous loss (deep deletion) was analyzed in human HCC. In the data from TCGA, only one patient (0.2%) had homozygous loss of SPTBN1 (1 / 440), whereas heterozygous deletion occurred in 7.8% (30 / 440) of HCC patients (30 / 440). Evaluation of copy number alterations of SPTBN1 in 33 cancer types in the TCGA data also showed that homozygous loss of SPTBN1 was rare, with an overall frequency of 0.1% across 33 cancer types representing 33,039 cancer patients (Figure 7F). Thus, the human cancer results indicate that the findings in LSKO mice are translationally important and that loss of SPTBN1 protects against NASH progression and progression to HCC.

[0126] All of the features described in this specification (including any accompanying claims, abstract and drawings) and / or all of the steps of any method so disclosed may be combined with any of the above aspects in any combination, except combinations in which at least some of such features and / or steps are mutually exclusive.

[0127] It should be understood that the above aspects and examples are not intended to be limiting in any way to the scope of the present disclosure, and that the claims provided herein are intended to encompass all aspects, embodiments, and examples, whether or not expressly provided herein.

[0128] All patents, patent applications and publications cited herein are incorporated by reference in their entirety.

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[0183] 55. N. Guex and MCPeitsch, SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18(15), 2714-2723 (1997). The present invention provides, for example, the following items. (Item 1) A method for treating obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1. (Item 2) 2. The method of claim 1, wherein at least one of the siRNA molecules that inhibits the expression of SPTBN1 comprises 15 to 30 nucleotides. (Item 3) 3. The method according to item 2, wherein at least one of the siRNA molecules that inhibits the expression of SPTBN1 comprises 15 to 20 nucleotides. (Item 4) 4. The method according to any one of items 1 to 3, wherein at least one of the siRNA molecules that inhibits expression of SPTBN1 comprises an overhang region of 1 to 6 nucleotides. (Item 5) 4. The method according to any one of items 1 to 3, wherein at least one of the siRNA molecules that inhibits expression of SPTBN1 does not contain an overhang region. (Item 6) 2. The method of claim 1, wherein at least one of the siRNA molecules that inhibits the expression of SPTBN1 is set forth as SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9 or SEQ ID NO: 10. (Item 7) A method for treating obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1, wherein at least one of the siRNA molecules that inhibit expression of SPTBN1 is homologous to at least 10 nucleotides of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10. (Item 8) A method for treating obesity, non-alcoholic fatty liver disease, non-alcoholic steatohepatitis or hepatocellular carcinoma in a subject in need thereof, comprising administering a therapeutically effective amount of at least one siRNA molecule that inhibits expression of SPTBN1, wherein at least one of the siRNA molecules that inhibit expression of SPTBN1 has at least 90% sequence identity with SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9 or SEQ ID NO:10. (Item 9) 9. The method according to any one of items 1 to 8, for treating obesity in a subject in need thereof. (Item 10) 10. The method of claim 9, wherein treating obesity comprises reducing the amount of body fat in the subject and / or reducing the body weight of the subject. (Item 11) The method according to any one of items 1 to 8, for treating non-alcohol-related fatty liver disease in a subject in need thereof. (Item 12) 12. The method of claim 11, wherein treating non-alcohol related fatty liver disease comprises reducing blood triglycerides in the subject. (Item 13) 9. The method according to any one of items 1 to 8, for treating non-alcoholic steatohepatitis in a subject in need thereof. (Item 14) 14. The method of claim 13, wherein treating nonalcoholic steatohepatitis comprises reducing blood triglycerides in the subject. (Item 15) 9. The method according to any one of items 1 to 8, for treating hepatocellular carcinoma in a subject in need thereof. (Item 16) 16. The method of claim 15, wherein treating hepatocellular carcinoma comprises reducing tumor mass in the subject. (Item 17) 17. The method according to any one of items 1 to 16, comprising administering to the subject one siRNA molecule that inhibits expression of SPTBN1. (Item 18) 17. The method according to any one of items 1 to 16, comprising administering to the subject two siRNA molecules that inhibit the expression of SPTBN1. (Item 19) 17. The method according to any one of items 1 to 16, comprising administering to the subject three siRNA molecules that inhibit the expression of SPTBN1. (Item 20) 17. The method according to any one of items 1 to 16, comprising administering to the subject 4 to 10 siRNA molecules that inhibit expression of SPTBN1.

Claims

[Claim 1] The invention described in the specification.

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