Compositions and methods for treatment of liver disease

Abstract

To provide compositions and methods for treating or preventing liver disease.SOLUTION: Aspects of the invention relate to methods including administering to a subject an agent that inhibits WISP1. Other aspects of the invention relate to methods including administering to a subject an HSC that expresses an agent that inhibits WISP1.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application is a continuation of U.S. Provisional Patent Application No. 62 / 747,900, filed October 19, 2018. 3, the contents of which are incorporated herein by reference in their entirety. No. 6,399,433, which is incorporated herein by reference in its entirety.

[0002] The field of the invention relates to the treatment of liver disease. [Background technology]

[0003] The liver is a very important organ that filters metabolic products and synthesizes proteins, which are necessary for digestion. The liver is able to produce the necessary biochemicals. Specifically, it produces bile and breaks down fats. Chronic and progressive liver disease can lead to accumulation of bile, severe inflammation, scarring, This can result in progressive destruction of the bile ducts in the liver, which can lead to inflammatory bowel disease and fibrosis. As liver tissue is replaced by scar tissue, liver function becomes increasingly impaired. For example, in the case of primary biliary cholangitis (PBC), drugs that can improve survival rates include Only ursodeoxycholic acid (UDCA) is available. Unfortunately, most UDCA-treated patients 40% of patients do not respond adequately to this therapy. More effective therapeutic agents are needed for the treatment of Summary of the Invention

[0004] The invention described herein is based in part on the discovery of miRNA-15a, miRNA-412, and and inhibition of WISP1 by anti-WISP1 antibody demonstrated a central role in the progression of liver fibrogenesis. The finding that induced quiescence of activated hepatic stellate cells (HSCs), a cell type that plays a key role in miR-15a and WISP1 IgG directly target WISP1 and We found that we could inhibit the profibrogenic function of this protein in activated HSCs. As further shown herein.

[0005] Thus, one aspect described herein is a method for treating or preventing liver disease. Thus, an antibody or antibody reagent that inhibits WISP1 can be administered to a subject in need thereof. The method includes:

[0006] In one embodiment of any aspect, the liver disease is selected from the group consisting of Alagille syndrome; alcohol-related liver disease; Alpha 1-antitrypsin deficiency; autoimmune hepatitis; benign liver tumors; biliary atresia; liver cirrhosis Crigler-Najjar syndrome; Galactosemia; Gilbert syndrome; Hemochromatosis hepatic encephalopathy; hepatitis A; hepatitis B; hepatitis C; hepatorenal syndrome; intrahepatic cholestasis of pregnancy Intracellular cholangiocarcinoma (ICP); Lysosomal acid lipase deficiency (LAL-D); Liver cysts; Liver cancer; Neonatal Jaundice; Nonalcoholic fatty liver disease; Nonalcoholic steatohepatitis; Primary biliary cholangitis ( PBC); Primary Sclerosing Cholangitis (PSC); Progressive Familial Intrahepatic Cholestasis (PFIC) Reye's syndrome; glycogen storage disease type I; scleroderma; and Wilson's disease.

[0007] In one embodiment of any aspect, WISP1 is selected from the group consisting of WISP1v, WISP1vx, and WISP1 delta is a splice variant selected from the group consisting of exons 3-4 .

[0008] In one embodiment of any aspect, the antibody or antibody reagent that inhibits WISP1 is mab1 680, AF1680, SAB2501114, ab60114, and ab65943 is selected from the group consisting of:

[0009] In one embodiment of any aspect, the amino acid sequence of the antibody or antibody reagent is selected from the group consisting of SEQ ID NOs: 1-4. , 6, or 12-120.

[0010] In one embodiment of any aspect, the subject is a mammal.

[0011] In one embodiment of any aspect, WISP1 is inhibited in the target cell. In one embodiment of any aspect, the target cell is a mammalian cell. The target cell is a hepatic stellate cell, a fibroblast, or a myofibroblast. In this state, hepatic stellate cells are in a quiescent state.

[0012] In one embodiment of any aspect, the antibody or antibody reagent is administered by direct injection, subcutaneous injection, intramuscular injection, or the like. , orally, transdermally, or intranasally.

[0013] In one embodiment of any aspect, inhibiting WISP1 comprises inhibiting WISP1 activity; or reducing WISP1 protein levels. , the activity of WISP1 is at least 50%, at least 60%, At least 70%, at least 80%, at least 90%, or greater inhibition In one embodiment of any aspect, the level of WISP1 is compared to a suitable control, At least 50%, at least 60%, at least 70%, at least 80%, at least is reduced by 90% or more.

[0014] Another aspect described herein is an antibody or antibody reagent and pharmaceutical agent that inhibits WISP1. The composition includes a carrier acceptable to the subject.

[0015] In one embodiment of any aspect, the composition is formulated to treat or prevent liver disease. It is being done.

[0016] Another aspect described herein is a method for treating liver disease in a subject, comprising: (a) administering to a subject a therapeutically effective amount of Levels of WISP1 and / or Yap, Col1a1, and Acta2 in elephant biological samples (b) comparing the measurement in (a) with a reference level; (c) detecting the reference level. Compared with Bell, (a) WISP1 and / or Yap, Col1a1, and Acta2 (d) identifying the increased subjects as having liver disease; and and administering an antibody or antibody reagent that inhibits WISP1.

[0017] In one embodiment of any aspect, the method further comprises, prior to (a), obtaining a biological sample from the subject. It further includes:

[0018] In one embodiment of any aspect, the biological sample is a blood sample, tissue, buffy coat, blood Qing, or organization.

[0019] Yet another aspect described herein is a method for treating or preventing liver disease, comprising administering to a subject a therapeutically effective amount of In one embodiment, the method includes administering to a subject in need thereof an agent that inhibits WISP1. Provide.

[0020] In one embodiment of any aspect, the agent that inhibits WISP1 is a small molecule, an antibody, or an anti- Somatic reagents, peptides, genome editing systems, viral vectors, miRNA, lncRNA, mRNA In one embodiment of any aspect, the siRNA is selected from the group consisting of siRNA, siRNA, and siRNA. The cloRNA is miRNA15a or miRNA412.

[0021] In one embodiment of any aspect, the agent is administered by direct injection, subcutaneous injection, intramuscular injection, oral injection, transdermal injection, or the like. , or by intranasal administration.

[0022] Another aspect described herein is a method for treating a subject with a medicament for inhibiting WISP1 and a pharma- ceutically acceptable A composition is provided that includes a carrier.

[0023] Another aspect described herein is a method for treating liver disease in a subject, comprising: (a) administering to a subject a therapeutically effective amount of To detect the levels of WISP1 and / or Acta2 in elephant biological samples; b) comparing the measurement of (a) with a reference level; (c) comparing the measurement of (a) with the reference level. Identifying subjects with increased WISP1 and / or Acta2 as having liver disease and (d) administering to a subject having liver disease an agent that inhibits WISP1. The present invention provides a method comprising:

[0024] Yet another aspect described herein is a genetically engineered vector expressing an agent that inhibits WISP1. The present invention relates to a method for producing an engineered hepatic stellate cell or a population thereof, the method comprising: contacting the inhibitor with an inhibitory agent for a period of time sufficient to allow expression of the agent; and culturing the cells at said temperature.

[0025] In one embodiment of any aspect, the cells are in a quiescent state.

[0026] In one embodiment of any aspect, the contacting causes the cell to express an agent or a vector encoding an agent. In one embodiment of any aspect, the contacting includes transduction, nuclease transfection, electroporation, direct injection, and / or transfection Includes options.

[0027] Another aspect described herein is a method for producing a hepatic stellate molecule according to any of the methods described herein. A cell line comprising the cell is provided.

[0028] Another aspect described herein is a method for producing a hepatic stellate molecule according to any of the methods described herein. A pharmaceutical composition is provided that includes the cell or population thereof and a pharma- ceutically acceptable carrier.

[0029] Another aspect described herein is a method for treating or preventing liver disease, comprising: The present invention relates to a method for treating a subject in need thereof, comprising administering to the subject a cell produced by any of the methods described herein, or a medicament comprising the generated cells described herein. The present invention provides a method for treating a disease comprising administering any one of the compositions.

[0030] Another aspect described herein is a method for reducing fibrosis in a subject, comprising administering to said subject a therapeutically effective amount of The subject can be administered any of the following: or a pharmaceutical composition comprising the cells produced as described herein. The present invention provides a method for administering any one of the following compounds to a subject:

[0031] Another aspect provided herein is a method for treating liver disease in a subject, comprising administering to the subject a ) WISP1 and / or Acta2 levels (e.g., mRNA levels) compared to a reference level The aim of this study is to identify subjects with increased levels of IL-1, miRNA, and protein levels as having liver disease. and (b) administering to a subject having liver disease a compound that inhibits WISP1. administering an antibody or antibody reagent that inhibits the inflammatory process.

[0032] Another aspect provided herein is a method for treating liver disease in a subject, comprising administering to the subject a ) subjects with increased WISP1 and / or Acta2 levels compared to the reference levels, (b) receiving results of an assay that identifies the individual as having liver disease; and The method includes administering to a subject an agent that inhibits WISP1.

[0033] definition For convenience, certain terms and expressions used in the specification, examples, and appended claims will be The meanings of the following words and phrases are provided below. Unless otherwise stated or the context suggests otherwise, Unless otherwise specified, the following terms and phrases have the meanings given below. Definitions may apply only to specific implementations. It is provided to aid in the explanation of the embodiments and is not intended to limit the claimed technology. It is not intended that the scope of the present technology be limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein are defined by the principles of the art. It has the same meaning as commonly understood by a person skilled in the art to which the art belongs. If there is an apparent discrepancy between the usage of a term in this document and its definition provided herein, Definitions provided in the specification shall prevail.

[0034] Definitions of common terms in immunology and molecular biology can be found in the following references: Merck Manual of Diagnosis and Therapy, 19th Edition, published by Merck Sharp & Dohme Corp., 2011 (ISBN 978-0-911910-19-3);Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Cell Biology and Molecular Medicine, published by Blac kwell Science Ltd., 1999-2012 (ISBN 9783527600908);およびRobert A. Meyers (ed.) , Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8);Immunology by Werner Luttma nn, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 978 0815345305);Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (I SBN-1449659055);Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Ha rbor, N.Y., USA (2012) (ISBN 1936113414);Davis et al., Basic Methods in Molecul ar Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460 149X);Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.) Elsevier, 2013 (I SBN 0124199542);Current Protocols in Molecular Biology (CPMB), Frederick M. Aus ubel (ed.), John Wiley and Sons, 2014 (ISBN 047150338X, 9780471503385), Current Protocols in Protein Science (CPPS), John E. Coligan (ed.), John Wiley and Sons, Inc., 2005; and Current Protocols in Immunology (CPI) (John E. Coligan, ADA M Kruisbeek, David H Margulies, Ethan M Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737). and are incorporated herein by reference in their entireties.

[0035] As used herein, "treat," "treatment," "treating," or "improving" refers to The term "good" refers to a therapeutic treatment, the purpose of which is to treat or reduce the severity of a disease associated with a liver disorder, such as liver fibrosis. The term "treatment" refers to reversing, ameliorating, ameliorating, inhibiting, slowing, or stopping the progression or severity of a condition. The term "treating" refers to treating at least one adverse effect or symptom of liver disease, e.g. reducing or alleviating jaundice, variceal bleeding, reducing fibrosis, scarring, and ascites If one or more symptoms or clinical markers are reduced, treatment is generally Alternatively, a treatment is "effective" if the progression of the disease is reduced or halted. That is, "treatment" does not only mean improving symptoms or markers, but also means curing the disease. The progression or worsening of symptoms compared with what would be expected in the absence of treatment It also includes ceasing, or at least slowing, or reversing. Or desirable clinical outcomes include, but are not limited to, reduction in one or more symptoms. Palliation, reduction in extent of disease, stable disease (i.e. not worsening), or slowing of disease progression or slowing of progression, improvement or remission of the disease state, remission (whether partial or total) and / or reduced mortality, which may be detectable or undetectable. The term "treatment" of a disease means relief from the symptoms or side effects of the disease (e.g., This includes providing medical care (including palliative care).

[0036] As used herein, "prevent" or "prevention" refers to a methodology (e.g., The term "compounds" refers to any methodology in which a disease state does not result from the action of the compound (e.g., administration of a composition as described herein). In one aspect, prevention means that the disease will not become established to the extent that it does in untreated controls. It is understood that prevention of a disease may therefore include preventing a subject from developing the disease. The likelihood is evaluated in untreated subjects (e.g., subjects who have not been treated with a method or composition described herein). It includes reducing the amount of

[0037] As used herein, the terms "administer" and "inject" are used interchangeably herein. In a situation where a cell, e.g., a hepatic stellate cell or an agent described in claim 1 is placed in a subject, the placement comprising: Introduce a desired effect (e.g., a decrease in WISP1 levels or activity) by introducing The cells or agents are at least partially localized to a desired site, such as the liver or a region thereof. In situations where the term is used interchangeably, The agents or cells described herein are delivered agents, cells, or components of cells. Any suitable method that results in delivery of the compound to a desired location in a subject, where at least a portion of the compound remains operable. The cells can be administered by any suitable route. The survival period of the cells after administration to the subject is short, lasting only a few hours. It may be short, for example, from 24 hours to a few days, or it may be as long as several years. In some embodiments, the term "administering" refers to one or more Administration refers to administering a pharmaceutical composition containing an agent or cell of the invention to a subject in need thereof. Direct injection into a subject (e.g., administered directly to target cells), subcutaneous injection, intramuscular injection, oral, Administration can be local or systemic. It's fine.

[0038] The terms "patient," "subject," and "individual" are used interchangeably herein. The term "subject" refers to an animal, particularly a human, to whom treatment, including prophylactic treatment, is provided. As used herein, this refers to humans and non-human animals. The term "mammal" is used interchangeably herein and refers to all vertebrates, e.g., non-vertebrates. Human primates (especially higher primates), sheep, dogs, rodents (e.g., mice or rats), Mammals such as rats, guinea pigs, goats, pigs, cats, rabbits, and cows, and chickens In one embodiment, the subject is a human. In an embodiment, the subject is an experimental animal or a surrogate animal as a disease model. In this study, the subjects were companion animals (e.g., dogs, cats, rats, guinea pigs, hamsters, etc.) The subjects were domesticated animals, including guinea pigs (stars, etc.). Subjects were previously treated for liver disease. Subjects may have had liver disease or may never have been treated for liver disease. They may have been diagnosed with liver disease or may never have been diagnosed with liver disease.

[0039] The terms "reduce," "reduced," "reduce," or "inhibit" all refer to As used herein, a characteristic, level, or other parameter is decreased or decreased by a statistically significant amount. In some embodiments, "reduce" is used to mean "Reduce," or "reduce," or "inhibit" typically refers to a reduction in a level relative to a reference level (e.g., means a reduction of at least 10% compared to the absence of a given treatment, e.g. , at least about 10%, 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%, at least about 98%, at least about 99%, or more As used herein, "reduce" or "inhibit" can include a significant decrease. It does not include complete inhibition or reduction compared to a reference level. The reduction is preferably 100% inhibition compared to that of individuals without the given disorder. It may be decreased to a level considered to be within the normal range. For example, The inhibition is to inhibit WISP1 activity or to increase WISP1 protein levels. The aim is to reduce it.

[0040] The words "increased," "increasing," "enhancing," or "activating" All terms used herein generally refer to a characteristic, level, or other parameter that is statistically For the avoidance of doubt, the term "increase" is used to mean a significant increase in The terms "increased," "increasing," "enhancing," or "activating" is an increase of at least 10%, e.g., at least about 20%, compared to the reference level. or at least about 30%, or at least about 40%, or at least about 50% , or at least about 60%, or at least about 70%, or at least about 8 0%, or an increase of at least about 90%, or up to 100%, including a 100% increase; or any increase between 10 and 100% compared to the reference level, or At least about 2-fold, or at least about 3-fold, or at least about 4-fold, or less At least about a 5-fold increase, or at least about a 10-fold increase, or at least about a 20-fold increase, or at least about a 50-fold increase, at least about a 100-fold increase, at least about a 1000-fold increase, Or it could mean a larger increase.

[0041] As used herein, a "reference level" refers to a normal, otherwise unaffected A cell population or tissue (e.g., a biological sample obtained from a healthy subject, or A biological sample obtained from a subject, e.g., a sample obtained from a patient before he or she has been diagnosed with liver disease. Biological samples that have not been exposed to the agents or compositions disclosed herein. This refers to a biological sample.

[0042] As used herein, a "suitable control" is an untreated, otherwise identical cell or A population (e.g., a population that has not been contacted with an agent or composition described herein or a population that has not been contacted with an agent or composition described herein) (biological samples that were not contacted in the same manner, e.g., for a different period of time, compared to the control cells) Refers to...

[0043] The term "pharmaceutical acceptable" means a compound that can be administered to a subject (e.g., a mammal or can refer to compounds and compositions that can be administered to humans.

[0044] As used herein, the term "pharmaceutical acceptable carrier" refers to a compound that is combined with an active ingredient. The combination allows the components to retain their biological activity and be non-reactive with the subject's immune system. Examples include, but are not limited to, any material or substance that is Any of the standard pharmaceutical carriers, such as phosphate buffered saline solution, oil / water emulsions, etc. "Pharmaceutically acceptable carriers" include emulsions of various types of humectants, and various types of wetting agents. The term excludes tissue culture medium. Non-limiting examples of pharmaceutical carriers include nanoparticles. , microparticles, liposomes, polymer microspheres, or polymer-drug conjugates. Examples include particle or polymer based vehicles such as jugates.

[0045] As used herein, "WNT1-induced signaling pathway protein 1" or The term "WISP1" or "CCN4" refers to the functions of cell adhesion, migration, proliferation, differentiation, and The matrix metalloproteinases encoded by the WISP1 gene have numerous diverse cellular functions, including survival. In the liver, WISP1 is activated in myofibroblasts. WISP1 is secreted by hepatic stellate cells (HSCs) when the hepatocytes are exposed to oxidative stress. It influences HSCs by accelerating collagen activation and secretion via , promoting fibrosis. CCN4, WISP1c, WISP1i, WISP1tc, WIS The sequence of WISP1, also known as P1-OT1 and WISP1-UT1, is For example, human WISP1 (NCBI gene ID: 8840) polyclonal antibody Peptides (e.g., NCBI reference sequence: NP_001191798.1) and mRNA (For example, NCBI reference sequence: NM_001204869.1). WISP1 is Refers to human WISP1, including its naturally occurring variants, molecules, and alleles. WISP1 can be expressed in, for example, mice, rats, rabbits, dogs, cats, cows, and cattle. The nucleic acid sequence of SEQ ID NO:5 refers to WISP1 from mammals, such as horses, hamsters, and pigs. The nucleic acid sequence encoding the

[0046] As used herein, the term "WISP1 activity" refers to the cellular function of WISP1. For example, WISP1 accelerates collagen activation and secretion in HSCs. Promoting fibrosis and attenuating p53-mediated apoptosis, WISP1 has been shown to mediate fibrosis in other cell types In this study, WISP1 was shown to inhibit TNF-induced cell death. It may refer to increased collagen deposition by alpha smooth muscle cells. This may refer to the induction of actin expression or the expression of certain inflammatory cytokines such as IL-6. There is a match.

[0047] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to ribonucleic acid, nucleic acid sequence, Any molecule incorporating units of oxyribonucleic acid or analogs thereof, preferably Refers to a polymer molecule. Nucleic acids can be either single-stranded or double-stranded. Single-stranded nucleic acids The nucleic acid may be one of the nucleic acid strands of a denatured double-stranded DNA. Alternatively, the single-stranded nucleic acid may be any In one embodiment, the nucleic acid is DNA. In another embodiment, the nucleic acid may be RNA. Suitable DNA includes, for example: It may include genomic DNA or cDNA. Suitable RNA includes, for example, mRNA. It may include.

[0048] The term "agent" as used herein includes, but is not limited to, a subgroup. means any compound or substance, such as a molecule, nucleic acid, polypeptide, peptide, drug, or ion. "Agents" include, but are not limited to, synthetic and naturally occurring proteins. The moiety may be any chemical entity or moiety, including proteinaceous and non-proteinaceous entities. In this embodiment, the agent is a nucleic acid, a nucleic acid analog, a protein, an antibody, a peptide, an aptamer, oligomers, oligomers of nucleic acids, amino acids, or carbohydrates, including but not limited to proteins. Proteins, oligonucleotides, ribozymes, DNAzymes, glycoproteins, siRNA, liposomes Proteins, aptamers, and modifications and combinations thereof. In certain embodiments, the agent is a small molecule having a chemical moiety. These include macrolides, leptomycins, and related natural products or their analogs. The compounds include unsubstituted or substituted alkyl, aromatic, or heterocyclyl moieties. The entities may be known to have a desired activity and / or property, or may be a variety of The compound may be selected from a library of compounds.

[0049] The agents may be molecules from one or more chemical classes, e.g., organic molecules. Examples of the agent include organometallic molecules, inorganic molecules, and gene sequences. Fusion proteins, chimeric proteins derived from one or more proteins (e.g., related Domain switching or homologous recombination of functionally important regions of related or different molecules ), synthetic proteins, or other proteins containing substitutions, deletions, insertions, and other variants. It may also be a mutant form of the protein.

[0050] As used herein, an "antibody" refers to an antibody that is an IgG, IgM, IgA, IgD, or IgM antibody. gE molecule, or antigen-specific antibody fragments thereof (including but not limited to Fab, F( ab')2, Fv, disulfide-linked Fv, scFv, single domain antibodies, closed codons Closed conformation multispecific antibody, This refers to any species that naturally produces antibodies. Serum, B cells, haematopoietic cells, whether derived from the human body or produced by recombinant DNA technology, Whether isolated from hybridoma, transfectoma, yeast, or bacteria No question.

[0051] In another example, an antibody contains two heavy (H) chain variable regions and two light (L) chain variable regions. Mm. V H A region (e.g., a portion of an immunoglobulin polypeptide) may be any of the regions described elsewhere herein. V H Note that this is not the same as a segment. H Area and V L The area is , interspersed with more conserved regions termed "framework regions" ("FR"). These can be further subdivided into regions of hypervariability called complementarity determining regions ("CDRs"). The extent of the framework regions and CDRs has been precisely defined (Kabat, EA , et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 and C See Hothia, C. et al. (1987) J. Mol. Biol. 196:901-917; these publications are incorporated by reference. (The entire contents of which are incorporated herein by reference.) H and V L Typically, there are three It consists of four CDRs and four FRs, which are arranged as follows from the amino terminus to the carboxy terminus: Arranged in the order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR 4. As used herein, the terms "protein" and "polypeptide" refer to As used herein, a peptide bond between the alpha-amino and carboxy groups of adjacent residues are used synonymously to designate a series of amino acid residues connected together by

[0052] As used herein, the term "antibody reagent" refers to a reagent that contains at least one immunoglobulin The antibody comprises a phospho- or immunoglobulin variable domain sequence and specifically binds to a given antigen. Antibody reagents refer to polypeptides that bind to antibodies or antigen-binding domains of antibodies. In some embodiments of any of the aspects, the antibody reagent may comprise a polypeptide comprising The antibody is a monoclonal antibody or a polypeptide containing the antigen-binding domain of a monoclonal antibody. For example, the antibody may comprise a heavy (H) chain variable region (herein referred to as VH) and a peptide. and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody may have two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" includes antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDRs, and dorsal fragments. Derivative antibody (dAb) fragments (e.g., de Wildt et al., Eur J. Immunol. 1996; 26(3):6 29-39, which is incorporated herein by reference in its entirety) and The term "antibody" includes whole antibodies. Antibodies may be IgA, IgG, IgE, IgD, or IgM (e.g., The antibody may have the structural features of any of the mouse Any source including rabbit, pig, rat, and primates (human and non-human primates) The antibody may be derived from a human or a humanized antibody. Antibody reagents include antibody fragments, humanized antibodies, and chimeric antibodies. Good too.

[0053] The terms "protein" and "polypeptide" refer to any molecule, regardless of its size or function. In addition, modified amino acids (e.g., phosphorylation, glycation, glycosylation, etc.) and amino acid analogs "Protein" and "polypeptide" refer to a polymer of amino acids that contains a specific amino acid sequence. Often used in reference to large polypeptides, the term "peptide" can also refer to small polypeptides. Although often used in reference to peptides, the usage of such terms in the art is The terms "protein" and "polypeptide" refer to gene products and When referring to fragments thereof, they are used synonymously herein. The peptide or protein may be a gene product, a naturally occurring protein, a homologue, allologs, paralogs, fragments, and other equivalents, variants, fragments, and and analogs.

[0054] In various embodiments described herein, any of the specific polypeptides described Variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants It is further intended that variants, alternates, and / or conservative substitution variants are encompassed. With respect to amino acid sequences, one of skill in the art can easily identify single amino acids or low amino acids in the encoded sequence. Modifying the percentage of amino acids in a nucleic acid sequence, peptide sequence, polypeptide sequence, or Individual substitutions, deletions, or additions to a protein sequence are modifications that alter the chemical structure of amino acids. "Conservative modifications" refer to those modifications which result in the substitution of a similar amino acid with a similar amino acid and which retain the desired activity of the polypeptide. Such conservatively modified variants are within the scope of the present disclosure. In addition to the polymorphic variants, interspecies homologs, and alleles that are consistent with the Does not exclude.

[0055] As used herein, an "antigen" is a molecule that binds to the binding site of an antibody. In other words, antigens can bind to antibody ligands and enhance antibody responses in vivo. Antigens can be polypeptides, proteins, nucleic acids, or other molecules, or their The term "antigenic determinant" can be more specifically defined by an antigen-binding molecule. refers to the epitope on an antigen that is recognized by the antigen-binding site of said molecule.

[0056] As used herein, the term "affinity" refers to an interaction, e.g., for an antigen. This refers to the strength of antibody binding and is known as the dissociation constant (K D ) can be quantitatively expressed as The affinity is a measure of the strength of binding between an antigen-binding molecule (such as an antibody reagent described herein) and the relevant antigen. Avidity is a measure of the affinity of an antigenic determinant to its antigen-binding site on an antigen-binding molecule. The binding affinity of an antigen to an antigen-binding molecule is related to both its affinity for the antigen and the number of relevant binding sites present on the molecule. Typically, an antigen binding protein (such as an antibody reagent described herein) is administered at least 10 -5 ~10 -12 moles / liter or less, preferably 10 -7 ~10 -12 moles / liter or less, more preferably 10 -8 ~10 -12 Dissociation constant K in moles / liter D in (That is, 10 5 ~10 12 Liters / mole or greater, preferably 10 7 ~10 12 liters / mole or greater, more preferably 10 8 ~1012 R Binding constant in t / mol (K A ) by binding to their cognate or specific antigens. 10 -4 Any K greater than mol / liter D value (or 10 4 M -1 Lower than Any K A values) are generally considered to represent non-specific binding. K of biological interactions that are considered to be specific D Typically, 10 -10 M(0 .1nM)~10 -5 The stronger the interaction, the higher its K D Preferably, the binding sites on the antibody reagents described herein are less than 500 nM less than 10 nM, such as less than 200 nM, more preferably less than 500 pM The antibody will bind to the desired antigen with affinity. Specific binding can be determined, for example, by Scatchard analysis and / or radioimmunoassay (RIA). competitive assays such as enzyme immunoassays (EIA), enzyme immunoassays (EIA), and sandwich competition assays Binding assays, as well as different variants thereof, which are known per se in the art, It may be determined in any suitable manner known per se, including other techniques as described herein. It is possible.

[0057] As used herein, the term "specific binding" or "specificity" refers to the binding of two molecules to one another. The first entity refers to a chemical interaction between molecules, compounds, cells, and / or particles. binds to a second target entity with greater specificity and affinity than it binds to a third entity that is a target of In some embodiments of any of the aspects, specific binding is to a third, non-target entity. At least 10-fold, at least 50-fold, at least 100-fold, A second target object that is at least 500-fold larger, at least 1000-fold larger, or even larger. It can refer to the affinity of a first entity for a body. Thus, as used herein, In the present case, "selectively binds" or "specifically binds" refers to an agent ( For example, an antibody reagent is directed to a target, such as a peptide that includes the amino acid sequence of a given antigen. , 10 -5 M (10000nM) or less, e.g., 10 -6 M or less Full, 10 -7 M or less, 10 -8 M or less, 10 -9 M or Less than 10 -10 M or less, 10 -11 M or less, or 10 - 12 M or less K D For example, the agents described herein may be 10 to the first peptide containing the antigen -5 M or lower K D Combined with another If the agent does not bind to the randomly selected peptide, then the agent is specifically associated with the first peptide. Specific binding can be achieved, for example, by determining the affinity and avidity of the agent. The selection of suitable cells and and / or titrating the agent in a peptide binding assay. The method can be used to determine appropriate conditions under which an agent selectively binds to a target.

[0058] The term "expression", where applicable, includes, but is not limited to, for example, transcription, Transcript processing, translation, and protein folding, modification, and processing Responsible for the production of RNA and proteins, including ribosomal RNA, and secreting proteins when appropriate "Expression products" refer to cellular processes. A gene is a polypeptide obtained by translation of mRNA transcribed from a nucleic acid. The term refers to a compound that, when operably linked to appropriate regulatory sequences, can be used in vitro or in vivo. A gene is a nucleic acid sequence (DNA) that is transcribed into RNA in vivo. The regions preceding and following the lead region, e.g., the 5' untranslated (5'UTR) or "leader" sequence and The 3'UTR or "trailer" sequences, as well as between the individual coding segments (exons) The gene may or may not contain intervening sequences (introns).

[0059] As used herein, the term "contacting" when used in reference to a cell or organ The term refers to a process that allows physical contact between a cell and an agent, surface, hormone, etc. By introducing drugs, surfaces, hormones, etc. into cells, and by using miRNA, polypeptides, The expression vector may be a gene construct or vector that allows expression of an agent, such as another expression product, in a cell. The term "genetic modification" encompasses the introduction of any element into a gene that expresses an agent. The cells that are infected are "in contact" with the agent, and so are their cell progeny that express the agent. should be understood.

[0060] The terms "statistically significant" or "significantly" refer to statistical significance and generally refer to 2 Means difference of 2 standard deviations (SD) or greater.

[0061] As used herein, the term "comprising" means any combination of the terms used in the present specification. "Comprises" means that in addition to the specified elements, other elements may also be present. The use of "ng" indicates inclusion rather than limitation.

[0062] The term "consisting of" refers to a composition, method, or and their respective components, and any The element is excluded.

[0063] As used herein, "consisting essentially of" means The term "required" refers to an element required for a given embodiment. The presence of additional elements that do not materially affect the basic novel or functional characteristics of the form. is permitted.

[0064] The singular terms "a," "an," and "the" are used where the context clearly indicates otherwise. Similarly, the word "or" includes plural referents unless the context makes it clear. Unless otherwise indicated, "and" is intended to be inclusive. The tests may use methods and materials similar or equivalent to those described herein. However, suitable methods and materials are described below. The abbreviation "eg" refers to latex. It comes from the Latin word exempli gratia and is used here to give a non-limiting example. Thus, the abbreviation "eg" means "for example." Synonymous with the term.

[0065] Further, unless otherwise required by context, the singular forms "a," "the," and "the" shall include the plural. The form includes the singular.

[0066] Also, as used herein, "and / or" means one or more of the associated listed items. When interpreted as a plural, any and all possible combinations, as well as alternatives ("or"); refers to and encompasses the absence of a combination.

[0067] Furthermore, when referring to measurable values ​​such as amounts, doses, times, and temperatures of the compositions of the present invention As used herein, the term "about" means any range within the range of ±20%, ±10%, ±5%, ± It is meant to encompass variability of 1%, ±0.5%, or even ±0.1%. or amounts of ingredients or reaction conditions used herein unless otherwise indicated. All numerical values ​​expressed herein are understood to be modified in all instances by the term "about." The term "about" when used in connection with percentages means ± It can mean 1%.

[0068] Unless otherwise explained, all technical and scientific terms used herein are defined by the principles of the present disclosure. The terms have the same meaning as commonly understood by one of ordinary skill in the art to which they pertain.

[0069] The present disclosure is not limited to the particular methodology, protocols, and reagents described herein. It should be understood that the terms "terminology" and "terminology used" are not intended to be limiting, and may themselves vary. The terminology is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present disclosure. It is not intended that the scope of the present disclosure be limited solely by the claims.

[0070] All patents and other publications identified may be used, for example, in connection with this disclosure. This publication is hereby incorporated by reference to describe and disclose the methodology therein. Such publications are hereby expressly incorporated by reference herein for their disclosure prior to the filing date of this application. In this regard, even if there is prior disclosure or No part of the present invention is granted to the inventors without their prior written consent. No admission as to the date or content of such documents is made. All statements made herein are based on information available to the applicants and are current as of the date of such document. does not constitute any endorsement as to the accuracy of the information or content. [Brief description of the drawings]

[0071] [Figure 1] Schematic diagram of the research plan: Aim 1. Explore the mechanisms and functions of miR-15a and miR-412, Aim 2. Examine their therapeutic potential, and Aim 3. Elucidate the function of WISP1, a known target of miR-15a. HSC is the term for hepatic stellate cells. [Diagram 2] FIG. 1 is a schematic diagram of the experimental flow in which a genome-wide microRNA mimic library was screened to identify candidates that revert activated hepatic stellate cells to a quiescent state. [Diagram 3] Activated mouse hepatic stellate cells (HSCs) reverted to quiescence as demonstrated by the reformation of Nile Red-staining positive lipid droplets when miR-15a or miR-412 were overexpressed (top row). Activated human HSCs also reverted to quiescence as demonstrated by the reformation of lipid droplets when miR-15a or the human orthologues of miR-412 were overexpressed (bottom row). [Figure 4A]Figure 4 shows that miR-15a or miR-412 delivery to activated hepatic stellate cells (HSCs) resulted in changes in morphology towards quiescent ones. Figure 4A shows that HSCs were reduced in size by 10-100 fold as all pictures were taken using the same magnification (same scale bar for all panels). Figure 4B shows that forced expression of miR-15a or miR-412 in initially activated HSCs downregulated alpha smooth muscle actin (Acta2) and alpha type I collagen (Col1a1) as measured using qPCR. Data are presented as mean + / - SD. [Figure 4B] Same as above. [Diagram 5] Figure 1 shows that miR-15a or miR-412 transfected hepatic stellate cells (HSCs) have a functional phenotype. Activated HSCs treated with miR-15a or miR-412 did not produce steatosis in co-cultured hepatocytes. Activated HSCs treated with negative control microRNAs induced steatosis in co-cultured hepatocytes. qHSC, quiescent hepatic stellate cells; Ac-HSC, activated hepatic stellate cells; miR-Neg, microRNA negative control. [Figure 6] Figure 1 shows that HepG2 cells co-cultured with activated human HSCs receiving miR-15a or miR-412 had reduced expression of proinflammatory cytokines (left panel). Huh7 cells co-cultured with activated human HSCs receiving miR-15a or miR-412 had reduced expression of some proinflammatory cytokines (right panel). Data are presented as mean + / - SD. [Figure 7] (B) Endogenous miR-15a and miR-412 had decreased expression levels in activated primary HSCs compared to quiescent HSCs, although not significantly so for miR-15a. Data are presented as mean + / - SD. [Figure 8]Schematic of the schedule for CCl4 challenge and cell therapy injections (Figure). Top row: Quiescent-like HSCs reprogrammed with miR-15a or miR-412 injected into the spleen and transplanted into the liver, evidenced by liver producing GFP signal incorporated into vectors driving miRNA expression. Middle row: CCl4-challenged mice receiving quiescent-like HSCs had reduced hepatic apoptosis and inflammation, evidenced using H&E staining. Bottom row: CCl4-challenged mice receiving quiescent-like HSCs had reduced liver fibrosis stained with Sirius Red. Relative levels of fibrosis were quantified. CCl4, CCl4 gavage with no HSC injection; HSC control, CCl4 gavage and HSC injection with empty GFP-vector; HSC miR-15a, CCl4 gavage and HSC injection with miR-15a-GFP-vector; HSC miR-412, CCl4 gavage and HSC injection with miR-412-GFP-vector. [Figure 9] FIG. 1 shows that cell therapy with reprogrammed quiescent-like HSCs resulted in decreased expression of alpha 1 collagen type I (Col1a1) in whole liver as measured by qPCR in mice challenged with CCl4. Data are presented as mean + / - SD. [Figure 10] FIG. 2 shows that hepatic stellate cells in human primary biliary cholangitis expressed WISP1, which colocalized with alpha-smooth muscle actin (Acta2). [Figure 11] Figure 1 shows that miR-15a mimics co-transfected with reporters containing either one of the two predicted WISP1 target sequences reduced luciferase expression, while reporters containing mutant sequences were unaffected, indicating that miR-15a binds to both WISP1 target sequences. Data are presented as mean + / - SD (**P<0.01; ***P<0.001). [Figure 12]Quiescent HSCs at low and high magnification (left column). High magnification view clearly shows a few lipid droplets fluorescent green with BODIPY staining (inset). Right column: Activated HSCs at low and high magnification. Activated cells are larger, lack lipid droplets, and do not stain with BODIPY (inset). HSC, hepatic stellate cells. [Figure 13] Comparison of mice fed a standard diet (left column) and a CDAHFD (right column). Mice fed a CDAHFD develop early NASH at 3 weeks, showing increased body size, macroscopic and microscopic hepatic steatosis, and minimal fibrosis as shown by Sirius red staining. CDAHFD, choline-deficient L-amino acid-limited high-fat diet. [Figure 14] Schematic diagram of experiments showing co-culture of healthy hepatocytes (Hep) with either control hepatic stellate cells (HSC) from unchallenged mice or HSC from CDAHFD-challenged NASH mice. Top row: Hepatocytes co-cultured with HSC from control mice showed very few lipid droplets stained with BODIPY. Bottom row: Hepatocytes co-cultured with HSC from NASH mice showed significantly more lipid droplets. CDAHFD, choline-deficient, L-amino acid-limited, high-fat diet. [Figure 15] FIG. 1 shows that hepatocytes co-cultured with hepatic stellate cells (HSCs) from NASH mice expressed higher levels of several inflammatory cytokines and chemoattractants compared to those co-cultured with control HSCs, as measured by qPCR. Data are presented as mean + / - SD. [Figure 16] Figure 1 shows that induction of fat accumulation in hepatocytes (Hep) can be achieved when conditioned medium from NASH hepatic stellate cells (HSC) is applied to normal hepatocytes. Top row: No steatosis was induced when quiescent HSC (qHSC) medium was applied to healthy hepatocytes. Bottom row: NASH-HSC medium initially induced steatosis in healthy hepatocytes. Lipid droplets were stained with BODIPY. [Figure 17]FIG. 1 shows that delivery of miR-15a or miR-412 to activated hepatic stellate cells (AcHSCs) induced the reformation of lipid droplets that were retinoid positive as evidenced by fluorescence under UV light, consistent with that in quiescent hepatic stellate cells (qHSCs). [Figure 18] Figure 1: RNA sequencing data analyzed using multidimensional scaling shows that quiescent-like HSCs that received miR-15a or miR-412 have an overall transcriptional profile 40-50% closer to quiescent HSCs than to activated HSCs. HSC, hepatic stellate cells. [Figure 19] Figure 1 shows that miR-15a or miR-412 transfected hepatic stellate cells (HSCs) have a functional phenotype. Top row: Activated HSCs treated with miR-15a or miR-412 did not produce steatosis in co-cultured hepatocytes. Activated HSCs treated with a negative control microRNA induced steatosis in co-cultured hepatocytes. qHSC, quiescent hepatic stellate cells; Ac-HSC, activated hepatic stellate cells; miR-Neg, microRNA negative control. Bottom row: HSCs taken from the CDAHFD model of NASH induced steatosis in co-cultured hepatocytes. These same HSCs infected with lentiviruses expressing miR-15a or miR-412 assert their ability to induce steatosis in adjacent hepatocytes. [Figure 20] Figure 1 shows that overlap of the set of genes whose mRNA levels were decreased in hepatic stellate cells after receiving either miR-15a or miR-412 with the set of potential direct targets based on the prediction algorithm produced a target candidate set that was likely to contain true miRNA targets. Genes in the target candidate set were further filtered by selecting those that were part of the Tgf beta or Pdgf signaling pathways. [Figure 21]Figure 1 shows that CRISPR technology can be used in primary hepatic stellate cells. The feasibility of using this technology in primary cells was verified by delivering a long non-coding RNA Digit deletion vector, homozygous knock-in is confirmed using the appearance of a novel PCR band, one allele with the puromycin construct and another with the neomycin construct. Ctl, control construct; KI, knock-in construct. [Figure 22] Protein blots of >100 cytokines, chemokines and extracellular matrix proteins show that HSCs from CDAHFD-fed mice induced NASH with elevated WISP1 secretion compared with those from healthy mice. CM, conditioned medium. [Diagram 23] FIG. 1 shows that activated hepatic stellate cells (AcHSC) expressed nearly 30-fold higher WISP1 than quiescent hepatic stellate cells (qHSC). Hepatocytes (Hep) also expressed WISP1, but significantly less than activated hepatic stellate cells. Lv, whole liver. Data are presented as mean + / - SD. [Figure 24] Conditioned medium from hepatic stellate cells overexpressing WISP1 induced steatosis in hepatocytes from healthy mice.CM, conditioned medium. [Diagram 25] FIG. 1 is a schematic diagram showing that WISP1 is involved in human disease. [Figure 26] FIG. 2 is a schematic showing that WISP1 is a member of the CCN family of secreted matricellular proteins. [Figure 27] FIG. 2 is a schematic diagram showing that hepatic stellate cells are a key driver of liver fibrosis. [Figure 28] FIG. 1 is a schematic diagram showing hepatic stellate cell (HSC) activation. [Figure 29A]Figure 29 shows that WISP1 is highly upregulated in activated HSCs and is a direct target of miR-15a. (Figure 29A) WISP1 secreted by activated hepatic stellate cells of NASH mice. (Figure 29B) mrR-15a mimics co-transfected with a reporter containing the predicted WISP1 target sequence reduced luciferase expression. [Figure 29B] Same as above. [Figures 30A-30C] Figure 30: WISP1 and Yap 1 reciprocally activate each other. (Figure 30A) WISP1 and YAP mRNA were increased by rc-WISP1 treatment in qHSCs. (Figure 30B) YAP1 activation was induced by rc-WISP1 treatment in qHSCs. (Figure 30C) WISP1 expression was increased by YAP1 overexpression in miRNA-reprogrammed HSCs. [Fig. 31A-31B] 31A-31B are tables showing existing literature supporting the role of WISP inhibition in liver and lung fibrosis. (FIG. 31A) Anti-WISP1 mAb attenuated CCl4-induced liver fibrosis. (FIG. 31B) Anti-WISP1 mAb attenuated bleomycin-induced lung fibrosis. [Fig. 32A-32B] Figure 32 shows that WISP1 neutralizing antibody attenuates bile duct fibrosis. (Figure 32A) In vivo treatment with a neutralizing antibody against WISP1 reduced bile duct ligation (BDL)-induced liver fibrosis. (Figure 32B) Collagen expression in the indicated conditions. [Fig. 33A-33B] Figure 33 shows that WISP1 is secreted in common and rare human fibrotic liver diseases. (Figure 33A) WISP1 was upregulated in HSCs of NASH. (Figure 33B) WISP1 was upregulated in HSCs of several rare fibrotic liver diseases. [Fig. 34A-34B] Figure 34 shows that WISP1 is a novel secreted fibrotic driver for liver inflammation and fibrosis. (Figure 34A) Activated HSCs from NAFLD mice secrete WISP1. (Figure 34B) WISP1-overexpressing HSCs induce steatosis in healthy co-cultured primary hepatocytes (BODIPY line). [Diagram 35] FIG. 1 shows that Rc WISP1 treatment accelerates HSC proliferation as measured by ki67. [Diagram 36] FIG. 1 shows sequence alignment of human and mouse miR-15a and miR-412. [Fig. 37A-37B] Figure 37 shows that WISP1 expressed by HSCs has self-activating autocrine and pro-steatotic paracrine effects. Figure 37A shows HSC proliferation and activation induced by recombinant WISP1 treatment in mice. Figure 37B shows immunohistochemistry of control and RcWisp1. [Fig. 38A-38B] Figure 38 shows that Rc WISP1 and Wisp IgG treat NASH-HSCs derived from a choline-deficient L-amino acid-limited high-fat diet model induced steatosis (CDAHFD) and prevent fibrosis. 1 μg / ml WISP1 antibody was incubated in conditioned medium for 1 hour at 37°C before application to HSCs. Figure 38A shows the timeline of treatment and sampling. Figure 38B shows cell counts of control, Rc WISP1-treated and Ab WISP1-treated cells. Figure 38C shows Acta2 mRNA expression of qHSC, control, Rc WISP1-treated and Ab WISP1-treated cells. Figure 38D shows Col1a1 mRNA expression of qHSC, control, Rc WISP1-treated and Ab WISP1-treated cells. Figure 38E shows immunohistochemistry of control, Rc WISP1-treated and Ab WISP1-treated cells. FIG. 38F shows quantification of the percentage of BODIPY stained area in control, Rc WISP1-treated, and Ab WISP1-treated cells. [Fig. 38C-38D] Same as above. [Fig. 38E-38F] Same as above. [Figure 39A]39A and 39B demonstrate that WISP1 regulates HSC migration. Figure 39A shows the images of HSC migration in the presence and absence of Rc-WISP1 and WISP1 IgG. Figure 39B shows the relative wound area of ​​HSC treated with Rc-WISP1 and WISP1 IgG compared with control HSC. HSC treated with WISP1 IgG shows similar relative wound area to control HSC within 24 hours, confirming that WISP1 IgG can prevent HSC activation and migration. [Figure 39B] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0072] Liver diseases (e.g., primary biliary cholangitis (PBC)) are characterized by the progressive destruction of the intrahepatic bile ducts, gallbladder obstruction, and cholangitis. This can lead to bile duct stasis, periportal inflammation, and ultimately bile duct fibrosis leading to cirrhotic end-stage liver disease. Liver diseases such as PBC may manifest as autoimmune disorders of unknown etiology. , both genetic and environmental factors are likely to cause liver disease.

[0073] WISP1 is upregulated by hepatic stellate cells (HSCs) in the liver when activated on myofibroblasts. WISP1 is a protein secreted by the IL-1 receptor and is expressed by the IL-1 receptor, which is involved in the autocrine system. Affects HSCs by accelerating activation and secretion of VEGF, promoting fibrosis In particular, liver diseases (e.g., primary biliary cholangitis, autoimmune hepatitis, alpha 1- Antitrypsin deficiency, non-human disease, myoalcholic steatohepatitis Fibroblasts from rheumatoid arthritis, rheumatoid arthritis, and scleroderma highly express WISP1. Therefore, this indicates that inhibition of WISP1 is a strategy for treating liver diseases. .

[0074] Hepatic stellate cells are found in the perisinusoidal space (also known as the space of Disse) of the liver. These are fat-storing pericytes. Quiescent HSCs are distinguished by the presence of lipid droplets in the cytoplasm. When the liver is injured, stellate cells can become activated. Activated HSCs proliferate and produce cells. Activated HSCs promote collagen and scar formation. They become profibrogenic myofibroblasts that secrete ether.

[0075] The methods described herein show that WISP1 is upregulated in HSCs. Inhibition of P1 by the microRNAs miR-15a and miR-412 Inhibition of NF-kappaB1 expression, independently of activated NF-kappaB1 expression, is a cell type that plays a central role in the progression of hepatic fibrosis. Induce hepatic stellate cell (HSC) quiescence. Furthermore, miR-15a directly upregulates WISP1. Directly target and inhibit the profibrogenic function of this protein in activated HSCs WISP1 inhibitors promote HSC quiescence using microRNAs and their targets Therefore, it may be a useful therapy for controlling progressive liver fibrosis in liver diseases such as PBC. be.

[0076] Treatment and / or prevention of liver disease The methods and compositions described herein are intended to provide a method for treating and / or preventing liver disease in a subject. Exemplary liver diseases include, but are not limited to, Alagille syndrome. ;Alcohol-related liver disease;Alpha 1-antitrypsin deficiency;Autoimmune hepatitis;Benign Liver tumor; Biliary atresia; Liver cirrhosis; Crigler-Najjar syndrome; Galactosemia; Zirbe Hemochromatosis; Hepatic encephalopathy; Hepatitis A; Hepatitis B; Hepatitis C; Hepatorenal syndrome Group; Intrahepatic cholestasis of pregnancy (ICP); Lysosomal acid lipase deficiency (LAL-D); Liver cyst; Liver cancer; Neonatal jaundice; Nonalcoholic fatty liver disease; Nonalcoholic fatty liver inflammation; primary biliary cholangitis (PBC); primary sclerosing cholangitis (PSC); progressive familial intrahepatic These include cholestasis (PFIC); Reye's syndrome; glycogen storage disease type I; scleroderma; and Wilson's disease. In one embodiment, the liver disease is primary biliary cholangitis, autoimmune hepatitis, alpha hepatitis, 1-antitrypsin deficiency, nonalcoholic steatohepatitis, and scleroderma.

[0077] In one embodiment, the methods described herein are directed to treating liver failure, e.g., a decrease in liver synthetic and metabolic function. In one embodiment, the methods described herein are used to treat a subject having a deficiency. fulminant or severe acute liver failure (e.g., onset in <8 weeks in previously healthy individuals) hyperacute liver failure (e.g., <1 in previously healthy individuals); liver failure with encephalopathy, which develops over 4 days); acute liver failure (e.g., in previously healthy individuals) chronic liver failure (e.g., without encephalopathy) or acute exacerbation of chronic hepatitis (e.g., chronic liver failure with the development of encephalopathy) The skilled practitioner will be able to administer the drug in a manner consistent with standard procedures, e.g. Child-Pugh score (total bilirubin, albumin, INR, ascites, and hepatic encephalopathy) Combined index), MELD score (serum bilirubin, creatinine, and INR were used PELD score (similar to MELD but for pediatric patients), or The METAVIR score (which assesses the level of fibrosis in a sample) can be used to assess liver disease (e.g. For example, the severity of liver disease (liver failure) can be assessed.

[0078] METAVIR In one embodiment, the methods described herein are used to treat a subject with liver fibrosis. Liver fibrosis can be assessed by an experienced clinician using, for example, the METAVIR score. The METAVIR score is a combination of two scores: a fibrosis score and an activity score. The fibrosis score records the amount of inflammation in the liver (intensity of inflammation / degradation of tissue). For example, F0: no fibrosis; F1: portal vein fibrosis without septa F2: Portal fibrosis with few septa; F3: Multiple septa without cirrhosis; F4: Hepatic Cirrhosis. The activity score is a prediction of how rapidly the degree of fibrosis will progress. For example, A0: no activity; A1: mild activity; A2: moderate activity; and A3: severe activity. In one embodiment, the subject treated with the methods described herein is a patient with F1, F2, F3, or F4, and / or a METAVIR score of A1, A2, or A3.

[0079] In another aspect, provided herein is a method for treating or preventing liver disease, comprising: The method includes administering to a subject in need thereof an antibody or antibody reagent that inhibits WISP1. The law is stated.

[0080] In one aspect, provided herein is a method for treating or preventing liver disease, comprising administering to a subject a therapeutically effective amount of Methods are described that include administering to a subject in need thereof an agent that inhibits WISP1. There are.

[0081] In various embodiments, WISP1 is inhibited in the target cell. Target cells include liver cells that highly express WISP1 and lead to disease states, such as liver disease; For example, increased levels of WISP compared to a suitable control, e.g., healthy non-diseased liver cells. 1. Those skilled in the art can use standard techniques to detect and analyze the expression of the . Using a GFP-based assay or Western blotting, WISP1 in cells The mRNA or protein levels of can be assessed.

[0082] In another embodiment, the target cells are hepatic stellate cells (HSCs). HSCs are found in the peri-sinusoidal cells of the liver. Those skilled in the art will recognize pericytes, which are found in the peripheral space of the retina. Using visualization of lipid droplets within the cytoplasm, we can determine whether a cell is an HSC or not. In one embodiment, the HSCs are quiescent. Quiescent HSCs can be cultured or transfected as described by those of skill in the art. Active HS can be identified by the presence of lipid droplets in the cytoplasm of non-proliferating cells. C is known to those skilled in the art to promote cell proliferation, reduction of lipid droplets in the cytoplasm, and / or collagen synthesis. To identify the secretion of inflammatory mediators that promote scar formation and can be done.

[0083] In one embodiment, the target cell is a fibroblast, e.g., a liver fibroblast. In the present study, the target cells are myofibroblasts. Myofibroblasts are a type of cell that is produced by the synthesis of fibroblasts and smooth muscle cells. Fibroblasts and myofibroblasts, for example, are known to those skilled in the art as For example, microscopy may reveal fibroblast markers or fibroblast and smooth muscle cell markers, respectively. By selecting a cell marker, the cells can be easily identified.

[0084] In one embodiment, the target cell is a mammalian cell, preferably a human cell.

[0085] In another aspect, provided herein is a method for treating liver disease, comprising: (a) administering to a subject a therapeutically effective amount of The levels of WISP1 and / or Yap, Col1a1, and Acta2 in biological samples (b) comparing the measured value in (a) with a reference level; (c) comparing the measured value in (a) with a reference level Compared with (a), WISP1 and / or Yap, Col1a1, and Acta2 were increased. (d) identifying a subject having liver disease as having liver disease; and (e) administering to the subject a WI Methods are described that include administering an antibody or antibody reagent that inhibits SP1.

[0086] In yet another aspect, provided herein is a method for treating liver disease, comprising: (a) administering to a patient a therapeutically effective amount of Levels of WISP1 and / or Yap, Col1a1, and Acta2 in elephant biological samples (b) measuring the level of the measurement in (a); (c) comparing the measurement in (a) to a reference level; (a) WISP1 and / or Yap, Col1a1, and Acta2 levels compared with (b) (d) identifying a subject having an increased level of hepatic disease as having liver disease; and Methods are described that include administering to a subject an agent that inhibits WISP1.

[0087] For measuring WISP1 and / or Yap, Col1a1, and Acta2 levels Assays include, but are not limited to, WISP1 and / or Yap, Co PCR-based assays to assess l1a1 and Acta2 mRNA levels, and We assessed WISP1 and / or Yap, Col1a1, and Acta2 protein levels. In one embodiment, the method includes western blotting to evaluate the expression of WISP1 and and / or Yap, Col1a1, and Acta2 levels were significantly lower than those of the control group. 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 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%, 36%, 37%, 38%, 39%, 40%, 41%, 42% , 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52% , 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62% , 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72% , 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82% , 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92% , 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater or at least 1x, 2x, 3x, 4x, 5x, 6x, 7x, 8x, 9x, 10x , 11x, 12x, 13x, 14x, 15x, 16x, 17x, 18x, 19x, 20x , 21, 22, 23, 24, 25 or more times increased. In an embodiment, WISP1 and / or Yap, Col1a1, Acta2 levels are ISP1 and / or Yap, Col1a1, and Acta2 mRNA levels. In an embodiment, WISP1 and / or Yap, Col1a1, Acta2 levels are , WISP1 and / or Yap, Col1a1, Acta2 protein levels. .

[0088] As used herein, a "reference level" refers to a level measured in a healthy subject, e.g., a subject without liver disease. refers to an otherwise identical biological sample from a subject without

[0089] In one embodiment, the methods described herein further comprise, prior to (a), obtaining a biological sample from the subject. As used herein, a biological sample includes a blood sample, a tissue sample, samples, buffy coat samples (e.g., containing high levels of white blood cells and platelets after centrifugation) A biological sample may be a fraction of an anticoagulated blood sample containing IgG, a serum sample, or a liver biopsy sample. The material can be obtained using any suitable technique known in the art. For example, Tissue samples can be obtained by biopsy, and blood samples can be obtained by finger prick or intravenous blood draw. can be obtained from.

[0090] In various embodiments, the biological sample is from a patient who has previously been diagnosed with liver disease or has previously been diagnosed with liver disease. In another embodiment, the biological sample is obtained from a subject who has not been diagnosed with liver disease. A subject suspected of having liver disease, e.g., a subject having at least one risk factor for liver disease, e.g. , taken from subjects having increased alcohol intake compared to normal intake.

[0091] Another aspect provided herein is a method for treating liver disease in a subject, comprising administering to the subject a ) subjects with increased WISP1 and / or Acta2 levels compared to the reference levels, (b) receiving results of an assay that identifies the individual as having liver disease; and The method includes administering to a subject an antibody or antibody reagent that inhibits WISP1.

[0092] Another aspect provided herein is a method for treating liver disease in a subject, comprising administering to the subject a ) WISP1, Yap, Col1a1, and / or Acta2 compared to reference levels receiving results of an assay that identifies subjects with increased levels as having liver disease; and (b) administering to a subject having liver disease an agent that inhibits WISP1. The method includes:

[0093] WISP1, Yap (NCBI gene ID 10413), Col1a1 (NCBI gene ID 10413), Gene ID 1277), and / or Acta2 (NCBI gene ID 59) levels Assays for measuring WISP1 and / or Based on PCR to assess Yap, Col1a1, and Acta2 mRNA levels Assays for WISP1 and / or Yap, Col1a1, and Acta2 proteins Assays (e.g., Western blotting) to assess protein levels. For example, WISP1 and / or Yap, Col1a1, and Acta2 mRNA levels PCR-based assays to assess WISP1 and / or Yap, C Western blotting to assess ol1a1, Acta2 protein levels) The present invention relates to a method for treating a patient having a disease, comprising administering an agent (e.g., an antibody or antibody reagent) that inhibits WISP1. Alternatively, the assay can be performed by another individual (i.e., a WISP not by a practitioner administering an agent (e.g., an antibody or antibody reagent) that inhibits 1 The results of the assay may be transmitted by any means, e.g., via mail carrier, telephone Communications (e.g., facsimile), electronic communications (e.g., electronic medical records, electronic mail In one embodiment, the treatment can be received upon receipt of the results of the assay. At any time after the receipt of the data (e.g., at least 1 second, 1 minute, 1 hour, 1 day, 1 The subject is administered the compound after one or more weeks, one month, one year, or longer.

[0094] In one embodiment, WISP1 and / or Yap, Col1a1, Acta2 levels is at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% lower than the reference level. %, 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%, 36%, 37%, 38% , 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48% , 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58% , 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68% , 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78% , 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88% , 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% , 99% or more, or at least 1x, 2x, 3x, 4x, 5x , 6x, 7x, 8x, 9x, 10x, 11x, 12x, 13x, 14x, 15x, 16x , 17x, 18x, 19x, 20x, 21x, 22x, 23x, 24x, 25x, or In one embodiment, WISP1 and / or Yap, Co l1a1, Acta2 levels were significantly higher than those of WISP1 and / or Yap, Col1a1, and Acta In another embodiment, WISP1 and / or Yap, Col1a1, Acta2 levels were significantly correlated with WISP1 and / or Yap, Col1a1, Acta2 protein levels.

[0095] In another aspect, provided herein is a method for treating or preventing liver disease, comprising: any of the cells produced using the methods described herein, The present invention relates to a method for administering a pharmaceutical composition comprising cells produced using the methods described herein. The present invention relates to a method for producing a fluororesin comprising the steps of:

[0096] In another aspect provided herein, an antibody generated using the methods described herein. or using any of the antibody reagents, agents, cells, compositions, and pharmaceutical compositions described herein. In one aspect, the method described herein can be used to treat or reduce liver fibrosis in a subject. The present invention relates to a method for reducing fibrosis, e.g., liver fibrosis, comprising administering to a subject in need thereof: Any of the cells produced using the methods described herein or the methods described herein. The method includes administering to the subject a pharmaceutical composition comprising cells produced using the method. It is listed.

[0097] In one embodiment, the subject has previously been diagnosed with liver disease. The subject has been diagnosed with liver disease prior to administration of the agent. Using standard techniques in the art, subjects can be diagnosed with liver disease. For example, blood tests called liver function tests; CT scans, ultrasound, or MRI scans. or tissue biopsy. Liver function tests include the measurement of liver-specific enzymes, e.g. , alanine transaminase (ALT), aspartate aminotransferase ( AST), alkaline phosphatase (ALP), albumin, or bilirubin levels ALT, AST, ALP, albumin, or bilirubin Even a slight increase in the levels of β-lactamase can indicate liver disease. The tests described herein (e.g., Child-Pugh score, MET AVIR score, and PELD and MELD scores).

[0098] In another embodiment, the agents described herein inhibit the WISP1 splice variant of a target cell. Exemplary WISP1 splice variants include, but are not limited to, However, WISP1v, WISP1vx, and WISP1 delta exons 3-4 were not identified. Can be obtained.

[0099] In another embodiment, inhibiting WISP1 is inhibiting WISP1 activity. SP1 activity is any currently known activity of the function of the WISP1 gene or gene product. For example, WISP1 may be a serotype that is related to H In SCs, it accelerates collagen activation and secretion to promote fibrosis and inhibits p53-mediated adhesion In other cell types, it attenuates apoptosis and inhibits TNF-induced cell death. In terms of morphology, WISP1 activity is at least 5%, 10%, 15%, or greater compared to an appropriate control. 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or greater To be harmed.

[0100] As used herein, a "suitable control" refers to an agent or composition described herein. refers to the level of WISP1 activity in otherwise identical samples that have not been exposed to or the level of WISP1 activity in the subject prior to administration of the agent or composition. In particular, a suitable control would be a measurement of WISP1 activity in a healthy subject, e.g., an individual without liver disease. Those skilled in the art will appreciate that, for example, activation and activation of collagen in HSCs Use a functional readout of WISP1 activity by measuring / assessing / quantifying its secretion Thus, the activity of WISP1 can be determined.

[0101] In another embodiment, inhibition of WISP1 reduces WISP1 levels in a cell, e.g., In another embodiment, the WI SP1 levels were at least 5%, 10%, 15%, 20%, 25% higher than appropriate controls %, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75 %, 80%, 85%, 90%, 95%, 99% or greater inhibition. As used herein, a "suitable control" is a control that is Levels or effects of WISP1 in otherwise identical samples that were not exposed The level of WISP1 in a subject prior to administration of the agent or composition. The control is the level of WISP1 in healthy subjects, e.g., individuals without liver disease. Those skilled in the art may, for example, measure collagen activation and secretion in HSCs. To determine / assess the activity of WISP1 using a functional readout of WISP1 activity Those skilled in the art can determine the protein levels and mRNA levels of WISP1. A levels can be measured, for example, by Western blotting or PCR-based assays, respectively. can be evaluated / measured using

[0102] Agent In one embodiment, an agent that inhibits WISP1 is administered to a subject having or at risk of having liver disease. In one embodiment, the agent is a small molecule, an antibody, a peptide, a genomic DNA, or a polypeptide. These include aggregation systems, viral vectors, miRNA, and siRNA.

[0103] The agents described herein, for example, upon administration, can increase the presence, amount, or concentration of WISP1 in cells. A compound is considered effective in inhibiting WISP1 if it inhibits the activity and / or levels of the compound. do.

[0104] The agent can, for example, inhibit the transcription or translation of WISP1 in a cell. The agent may inhibit the activity of WISP1 in a cell (e.g., the expression of WISP1). or the activity can be altered (e.g. the activity no longer occurs, No longer occurs properly (e.g., compared to wild-type WISP1 activity) or is reduced (occurs at a given speed).

[0105] In one embodiment, the agent excludes miRNA412 and miRNA15a. In this embodiment, the agent is any miRNA412 or miRNA15a mimic. exclude.

[0106] The agent can function directly in the form in which it is administered. The present invention relates to a method for the introduction of a nucleic acid sequence into a cell and expression of the nucleic acid and / or protein of WISP1 in the cell. It produces something that inhibits WISP1, such as its transcription, which results in the production of a protein inhibitor. In some embodiments, the construct may be modified to produce a polypeptide that can be used intracellularly. The therapeutic agent may be any agent, including, but not limited to, synthetic and naturally occurring non-proteinaceous entities. In certain embodiments, the agent is a chemical entity or moiety having Small molecules. For example, chemical moieties include macrolides, leptomycins, and related Unsubstituted or substituted alkyl, aromatic, or aryl groups, including natural products or analogs thereof The heterocyclyl moiety is selected from the group consisting of aryl, ... may be known or may be identified from a library of diverse compounds.

[0107] In various embodiments, the agent is a small molecule that inhibits WISP1. When used herein, the term "small molecule" includes, but is not limited to, peptides, peptidomimetics, and the like. mimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, ptamers, nucleotides, nucleotide analogs, less than 10,000 grams per mole Organic or inorganic compounds with molecular weights of less than 10 ... (including compounds having a molecular weight of less than about 5,000 grams per mole), compounds, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole; Organic or inorganic compounds having a molecular weight of less than about 500 grams per mole, as well as other compounds such as those Compounds such as amines, esters, and other pharma- ceutically acceptable forms thereof may also be used. This refers to a chemical agent.

[0108] Methods for screening small molecules are known in the art, e.g., for example, a WISP1 polypeptide, to inhibit WISP1 activity or levels. This can be used to identify small molecules that are effective.

[0109] One embodiment provided herein is any of the agents that inhibit WISP1 described herein. In one embodiment, the composition further comprises a pharma- ceutical acceptable carrier. In one embodiment, the composition is a pharmaceutical composition.

[0110] Polypeptides that inhibit WISP1 In one embodiment, provided herein is a method for treating or preventing liver disease, comprising administering to said subject a The subject is provided with a polypeptide or such that inhibits WISP1 in a target cell. Methods are described that include administering a nucleic acid encoding the polypeptide.

[0111] The term "WISP1 binding polypeptide" refers to a polypeptide that binds to a desired antigen of interest (e.g., WISP1 P1 polypeptide) and linked to a linker or immunoglobulin constant domain. Ig-like proteins comprising one or more of the antigen-binding domains described herein linked to the Ig-like proteins. A binding protein refers to a polypeptide that is a bivariate domain in some embodiments. The "linker polypeptide" may be a main (DVD-Ig) binding protein. Contains two or more amino acid residues joined by peptide bonds, Such linker polypeptides are used to link multiple antigen-binding moieties. Peptides are well known in the art (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; see Poljak (1994) Structure 2: 1121-1123). Brin constant domain refers to a heavy or light chain constant domain. Human IgG heavy and light chains The amino acid sequences of constant domains are known in the art (see, e.g., U.S. Pat. App. Pub. No. 2003 / 013331). See sequence numbers 197, 198, 199, and 200 in Patent Publication No. 2016 / 0200813. (This document is incorporated herein by reference in its entirety for illustrative purposes only).

[0112] In some embodiments, the polypeptide that inhibits WISP1 is heterologous. As used herein, "heterologous" refers to a host cell, e.g., a cell that expresses a heterologous polypeptide. Refers to a polypeptide that is not normally produced in the host cell, but rather is derived from an organism other than the host cell. For example, the WISP1 inhibitors used herein can be derived from, for example, bacterial cells, e.g. For example, it is expressed in mammalian cells.

[0113] In some embodiments, the polypeptide that inhibits WISP1 is selected from the group consisting of SEQ ID NOs: 1-4 or 6. and at least 50%, at least 60%, at least 70%, at least 80%, As used herein, the term "sequence number" refers to a sequence that is at least 90%, or 100% identical to the sequence of the sequence of the invention. The term "homology" or "homologous" as used herein refers to the alignment of sequences and, if necessary, After introducing gaps as necessary to achieve the maximum percent sequence identity, the target chromosome or A nucleotide or amino acid residue that is identical to a nucleotide or amino acid residue of a corresponding sequence on a polypeptide. It is defined as a percentage of nucleotides or amino acid residues. Alignment to determine percent amino acid sequence identity can be performed using, for example, BLAST , BLAST-2, ALIGN, ClustalW2, or Megalign (DNA Using publicly available computer software such as STAR software This can be accomplished in a variety of ways that are within the skill of the art. Any number of alignments required to achieve maximum alignment over the entire length of the sequences under consideration. Determining appropriate parameters, including algorithms, for aligning sequences In some embodiments, for example, a WISP1 binding fragment or polypeptide can be A nucleic acid or amino acid sequence (e.g., a DNA, RNA, or amino acid sequence) is a sequence The corresponding native or unedited nucleic acid sequence (e.g., genomic sequence) or amino acid sequence of WISP1. Sequence and at least 70%, at least 75%, at least 80%, at least 85%, At least 90%, at least 91%, at least 92%, at least 93%, at least At least 94%, at least 95%, at least 96%, at least 97%, at least 98% A sequence is considered "homologous" if it is at least 99% identical or more will be done.

[0114] In various embodiments described herein, any of the specific polypeptides described Variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants It is further intended that variants, alternates, and / or conservative substitution variants are encompassed. With respect to amino acid sequences, one of skill in the art can easily identify single amino acids or a small percentage of the encoded sequence. The nucleic acid sequence, peptide sequence, polypeptide sequence, or protein sequence may be modified to change the amino acid sequence at the Individual substitutions, deletions, or additions to a protein sequence are considered to be modifications of chemically similar amino acids. "Conservatively modified variants" are used to provide amino acid substitutions that retain the desired activity of the polypeptide. It will be recognized that such conservatively modified variants are consistent with this disclosure. In addition to, and excluding, polymorphic variants, interspecies homologs, and alleles that do not have.

[0115] A given amino acid may be replaced by a residue having similar physicochemical characteristics, For example, one aliphatic residue is replaced by another (Ile, Val, Leu, or Ala for each other), or one polar residue is substituted for another (Ly between Arg and Glu, or between Asp and Gln and Asn, etc. Such conservative substitutions, for example, substitutions of entire regions having similar hydrophobicity characteristics, are well known. The polypeptides containing the complementary amino acid substitutions are tested in any one of the assays described herein. The desired activity, e.g., ligand-mediated receptor activity and the natural or reference polypeptide, are then tested. It can be confirmed that the specificity of the tide is maintained.

[0116] Amino acids can be grouped according to the similarity of the properties of their side chains (A.L. Lehninger, in Biochemistry, second ed., pp. 73-75, Worth Publishers, New York. (1975)): (1) Nonpolar: Ala (A), Val (V), Leu (L), Ile (I), Pro(P), Phe(F), Trp(W), Met(M); (2) Uncharged polar: Gly (G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), G In(Q); (3) acidic: Asp(D), Glu(E); (4) basic: Lys(K), Arg (R), His (H). Alternatively, naturally occurring residues are selected based on common side chain properties. They can be divided into groups based on their amino acid sequence: (1) hydrophobic: norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln (3) acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) chain alignment Residues affecting tropism: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Conservative substitutions would involve exchanging a member of one of these classes for another. Specific conservative substitutions include, for example, the following: Ala to Gly. or Ser;Arg to Lys;Asn to Gln or His;Asp to Glu ;Cys to Ser;Gln to Asn;Glu to Asp;Gly to Ala or P ro;His to Asn or Gln;Ile to Leu or Val;Leu to Ile to Val; Lys to Arg, Gln to Glu; Met to Le u to Tyr or Ile;Phe to Met, Leu or Tyr;Se r to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or changes Phe to Val, Ile, or Leu.

[0117] In some embodiments, a polypeptide described herein (or such a polypeptide) The nucleic acid encoding the polypeptide may be a functional fragment of one of the amino acid sequences described herein. As used herein, a "functional fragment" refers to any fragment known in the art or described herein. and at least one wild-type reference polypeptide having an activity according to the assay described below in the literature. A functional fragment is a fragment or segment of a peptide that retains 50% of the amino acid sequence of the peptide. It may include conservative substitutions of the disclosed sequences.

[0118] In some embodiments, the polypeptides described herein are It may be a variant of the peptide or molecule. In some embodiments, the variant is Conservatively modified variants. Conservatively substituted variants are, for example, variants of the naturally occurring nucleotide sequence A "variant" as referred to herein can be obtained by mutation of a Substantially homologous to a natural or reference polypeptide, but with one or more deletions, insertions, or The amino acid sequence may differ from that of a native or reference polypeptide due to substitutions or substitutions. The DNA sequence encoding the variant polypeptide is a polypeptide having a natural or or a reference DNA sequence, one or more additions, deletions, or deletions of nucleotides. A variant protein or polypeptide that contains substitutions but retains the activity of the non-variant polypeptide. includes sequences encoding fragments thereof. Induction techniques are known in the art and can be applied by one of ordinary skill in the art.

[0119] A variant amino acid or DNA sequence has at least 80% similarity to a native or reference sequence. At least 90%, at least 91%, at least 92%, at least 93%, at least At least 94%, at least 95%, at least 96%, at least 97%, at least 98% , may be at least 99% identical. The degree of homology between the native sequence and the mutant sequence The percent identity (%) is widely used for this purpose, e.g., on the World Wide Web. Freely available computer programs used (e.g., the default BLAS It can be determined by comparing two sequences using the BLAST or BLASTn Cut.

[0120] Alterations to the native amino acid sequence can be accomplished by any of a number of techniques known in the art. The mutation can be, for example, to allow ligation to a fragment of the native sequence. An oligonucleotide containing the mutated sequence flanked by restriction sites By synthesizing the nucleotide sequence, it is possible to introduce the nucleotide sequence into a specific gene locus. After ligation, the resulting The resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, an oligonucleotide-directed site-specific mutagenesis procedure can be used to A modified nucleotide sequence having a particular codon altered according to the substitution, deletion, or insertion identified. The nucleotide sequence can be provided by any of the following methods. Techniques for making such modifications are well established. For example, those disclosed by Walder et al. Gene 42:133, 1986);Bauer et al. (Gene 37:73, 1985);Craik (BioTechniques, Januar y 1985, 12-19);Smith et al. (Genetic Engineering: Principles and Methods, Plenu M Press, 1981); and U.S. Pat. No. 462, these documents are incorporated herein by reference in their entireties. In addition, any cysteine ​​residues that are not involved in maintaining the correct conformation of the polypeptide are not included in the To improve the oxidative stability of the molecule and prevent aberrant cross-linking, it is commonly substituted with serine. Conversely, cysteine ​​bond(s) may be added to a polypeptide to improve its stability. , or may promote oligomerization.

[0121] Antibody that blocks WISP1 In one aspect of any of the present embodiments, the present disclosure provides a method for treating or preventing liver disease. The method of claim 1, further comprising administering to a subject in need thereof an antibody that inhibits WISP1 in a target cell. Alternatively, methods are described that include administering an antibody reagent.

[0122] In various embodiments, the agents described herein include antibodies or The antigen-binding fragment or antibody reagent thereof.

[0123] In another embodiment, the antibody or antibody reagent that inhibits WISP1 is In another embodiment, the antibody or antibody reagent specifically binds to SEQ ID NOs: 1-4 or or specifically binds to the amino acid sequence of SEQ ID NO:6.

[0124] As used herein, the term "antibody reagent" refers to a reagent that contains at least one immunoglobulin The antibody comprises a phospho- or immunoglobulin variable domain sequence and specifically binds to a given antigen. Antibody reagents refer to polypeptides that bind to antibodies or antigen-binding domains of antibodies. In some embodiments of any of the aspects, the antibody reagent may comprise a polypeptide comprising The antibody is a monoclonal antibody or a polypeptide containing the antigen-binding domain of a monoclonal antibody. For example, the antibody may comprise a heavy (H) chain variable region (referred to herein as V H and abbreviated) and the light (L) chain variable region (abbreviated herein as V L (abbreviated as In another example, an antibody may have two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody reagent" includes antigen-binding fragments of antibodies (e.g., single chain antibodies, Fab and and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDRs, and dorsal fragments. Derivative antibody (dAb) fragments (e.g., de Wildt et al., Eur J. Immunol. 1996; 26(3):6 29-39, which is incorporated herein by reference in its entirety) and The present invention includes all antibodies.

[0125] Antibodies may be IgA, IgG, IgE, IgD, or IgM (and their subtypes). Antibodies may have the structural features of mouse, rabbit, porcine, ovine, from any source, including goats, rats, and primates (human and non-human primates). The antibody may be a primatized antibody. The antibody may be a midibody, a nanobody, an immunoglobulin, or an antibody that is a primatized antibody. Also included are intrabodies, humanized antibodies, and chimeric antibodies.

[0126] In one embodiment of any of the aspects, the antibody described herein is a humanized monoclonal antibody. An antibody or antigen-binding fragment thereof, or an antibody reagent. In another embodiment, the antibody is a humanized antibody. In another embodiment, the humanized antibody is a humanized polyclonal antibody. In yet another embodiment, the humanized antibody is intended for therapeutic use. That is why.

[0127] The anti-WISP1 antibodies described herein are monospecific or monoclonal antibodies. The term "monospecific antibody" refers to an antibody that is directed against a particular target, e.g., epitope. Refers to an antibody that displays a single binding specificity and affinity. The term is also used to refer to a "monoclonal antibody." or "monoclonal antibody composition," as used herein, refers to any antibody The term "antibody" refers to a preparation of antibodies or fragments thereof of single molecular composition, regardless of how they are produced.

[0128] As used herein, the term "humanized antibody" refers to an antibody produced from a non-human species (e.g., mouse). , rat, sheep, or goat), but V H and / or V L At least a portion of the sequence is made more "human-like," i.e. It refers to antibodies that have been altered to more closely resemble human germline variable sequences. "Humanized" antibodies are antibodies that have been genetically engineered or engineered to contain minimal sequence derived from non-human immunoglobulin. is a form of engineered chimeric antibody. In most cases, humanized antibodies are Residues derived from the hypervariable regions of mouse IgG1 have the desired specificity, affinity, and potency. , derived from hypervariable regions of non-human species (donor antibodies), such as rat, rabbit, or non-human primates. Human immunoglobulin (recipient antibody or acceptor antibody) in which the corresponding residues have been replaced by those of the In some cases, the Fv framework region of a human immunoglobulin (Fv ... In addition, humanized antibodies are characterized in that all R residues are replaced by the corresponding non-human residues. These modifications may include residues that are not found in the recipient antibody or in the donor antibody. Humanization is a process used to further refine antibody performance. Generally, a humanized antibody contains at least one Typically, the antibody will contain substantially all of the two variable domains, including the hypervariable loops. All or substantially all of the FR regions correspond to the hypervariable loops of a non-human immunoglobulin. All or substantially all of the regions are FR regions of a human immunoglobulin sequence. A humanized antibody may optionally contain an immunoglobulin constant region (Fc), typically a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Pr See, e.g., Esta, Curr. Op. Struct. Biol. 2:593-596 (1992). In such cases, they are called "composite human antibodies" or "deimmunized antibodies." "Non-modified antibodies" are designed to reduce or eliminate T cell epitopes from the variable domains. A specific type of engineered or humanized antibody is designed.

[0129] A humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Such non-human amino acid residues are sometimes called "import" residues. Most often, these are taken from "import" variable domains. By substituting rodent CDRs or CDR sequences for the corresponding sequences in a human antibody, This can be performed essentially according to the method of Winter and colleagues (Jones et al. , Nature, 321:522-525 (1986);Riechmann et al., Nature, 332:323-327 (1988);Verh Oeyen et al., Science, 239:1534-1536 (1988)). Thus, such humanized antibodies shows that a significantly smaller portion of the intact human variable domains are replaced by corresponding ones derived from non-human species. It is a chimeric antibody in which the sequence is replaced by the sequence (U.S. Pat. No. 4,816,567). In practice, humanized antibodies typically contain some CDR residues and possibly some FR residues. These are human antibodies in which residues are substituted by residues from analogous sites in rodent antibodies.

[0130] In certain embodiments, the anti-WISP1 antibody is an intrabody. Antibodies are intracellular antibodies that functionally bind to targets within cells (see, generally, Hood et al., Immunology). ology, Benjamin, NY, 2ND ed. (1984), Harlow and Lane, Antibodies. A Laboratory Manual, Cold Spring Harbor Laboratory (1988);Hunkapiller and Hood, Nature, 323 , 15-16 (1986); and Rondon and Marasco, Annu Rev Microbiol, 51:257-83 (1997); U.S. Patent No. 6,004,940; and U.S. Patent No. 5,581,829 (See, e.g., U.S. Pat. No. 6,333,363; these documents are incorporated herein by reference in their entireties). Methods for producing labodies are described, for example, in WO 2002 / 086096. Such techniques are well known to those skilled in the art, such as those described in Lett. Antibodies are generally at least about 1 mM. K of M D , more usually at least about 300 μM, typically at least about 10 μM, Typically at least about 30 μM, preferably at least about 10 μM, more preferably at least K of at least about 3 μM or better D The two are then joined as follows:

[0131] In one embodiment, the anti-WISP1 antibody is a neutralizing antibody. One antibody is a non-neutralizing antibody.

[0132] In some embodiments, the anti-WISP1 antibody is chimeric. The term "chimeric" when used in the context of an antibody or an antibody-encoding sequence refers to a heterologous Characterized by two or more segments or parts derived from different animal species For example, the variable region of a chimeric antibody is a monoclonal antibody made from a mouse monoclonal antibody. The immunoglobulin constant region is derived from a non-human mammalian antibody, such as a cloned antibody, and the immunoglobulin constant region is derived from a human immunoglobulin. The variable segments of a chimeric antibody are typically derived from immunoglobulin molecules. At least a portion of the constant region (Fc), typically at least a portion of a human immunoglobulin The human constant region DNA sequence is expressed in immortalized B cells (WO 87 / 0267 1, which is incorporated herein by reference in its entirety). Antibodies can be isolated from various human cells according to well-known procedures. The heavy chain constant region may comprise both a CH1 region, a hinge region, a C It may contain the H2 region, the CH3 region, and optionally the CH4 region. For example, the CH2 domain may be deleted or eliminated to achieve the desired antigen specificity. Genes derived from antibody molecules of one mouse or other species can be transformed into human antibodies with the appropriate biological activity. By splicing together genes derived from antibody molecules, "chimeric antibodies" are produced. Techniques developed for this purpose are known in the art (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984);Take da et al., Nature 314:452-454 (1985); these publications are incorporated by reference in their entireties. (Incorporated herein).

[0133] In some embodiments of the compositions and methods described herein, the WISP1 binding domain comprises a variable light chain sequence, a variable heavy chain sequence, or both.

[0134] As will be appreciated by those skilled in the art, each heavy chain of a full-length antibody comprises a heavy chain variable domain (referred to herein as a HCVR or V H The heavy chain constant region is composed of Three domains: C H 1. C H 2, and C H Each light chain is composed of a light-chain variable domain. In this specification, LCVR is V L The light chain constant region is composed of the light The chain constant region consists of one domain, C L It consists of V H Area and V L The area is Complementarity determining regions (CDRs) are interspersed with more conserved regions called framework regions (FRs). Each V can be further divided into hypervariable regions called CDRs. H and V L teeth It is composed of three CDRs and four FRs, which are The CDRs are arranged in the following order toward the C-terminus: FR1, CDR1, FR2, CDR2, and FR3. , CDR3, FR4. This structure is well known to those skilled in the art. The chains are usually They are connected to each other via a junction.

[0135] As used herein, "complementarity determining regions" ("CDRs" i.e., CDR1, CDR2, CDR3, CDR4, CDR5, CDR6, CDR7, CDR8, CDR9, CDR10, CDR11, CDR12, CDR13, CDR14, CDR15, CDR16, CDR17, CDR18, CDR19, CDR19, CDR110, CDR19, CDR The terms CDR1, CDR2, and CDR3) refer to those sequences whose presence is necessary for specific antigen binding. The amino acid residues of a heavy or light chain variable domain are typically It has three CDR regions identified as DR1, CDR2, and CDR3. The SDR is a set of amino acids derived from the "complementarity determining region" as defined by Kabat. Residues (i.e., approximately residues 24-34 (L1), 50-56 (L2), and 60-64 (L3) of the light chain variable domain) 2), and 89-97 (L3) and 31-35 (H1) of the heavy chain variable domain, 50 ~65 (H2), and 95~102 (H3); Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or residues derived from the "hypervariable loops" ( Approximately, residues 26-32 (L1), 50-52 (L2), and and 91-96 (L3) and 26-32 (H1), 53-55 ( H2), and 96-101 (H3); Chothia and Lesk J. Mol. Biol. 196:901-917 ( In some instances, the complementarity determining region may comprise a sequence as defined by Kabat et al., 1987). The amino acids may be from both the CDR regions and the hypervariable loops. The term "CDR set" as used herein refers to a set of CDRs capable of binding to an antigen. It refers to a group of three CDRs that occur in a single heavy or light chain variable region. The boundary is defined differently in different ways. al, Sequences of Proteins of Immunological Interest (National Institutes of Hea lth, Bethesda, Md. (1987) and (1991)) is a unique antibody variable region that can be applied to any antibody variable region. It not only provides a simple residue numbering system, but also provides the precise residue boundaries that define the three CDRs. Such CDRs are sometimes referred to as Kabat CDRs. Coworkers (Chothia & Lesk, J. Mol. Biol., 196:901-917 (1987) and Chothia et al., Na (1989) reported that certain subportions within the Kabat CDRs are Despite the large amount of diversity at the row level, they adopt nearly identical peptide backbone conformations. These sub-parts are L1, L2, and L3, or H1, H2, and and H3, with "L" and "H" referring to the light and heavy chain regions, respectively. These regions are sometimes called Chothia CDRs and are related to the Kabat CDRs. Other boundaries that define the CDRs that overlap with the Kabat CDRs are lan (FASEB. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732 -45 (1996)). Still other CDR boundary definitions may be used that strictly adhere to one of the above methods. It is not necessary to follow the sequence closely; specific residues or groups of residues or even entire CDRs may be involved in antigen binding. Shorten or extend the boundaries in light of predictions or experimental findings that do not significantly affect Although the 3D CDRs can be lengthened, they will still overlap with the Kabat CDRs. In some embodiments, the CDRs are "complementarity determining regions" as defined by IMGT. The compositions and methods used herein can be described as including amino acid residues derived from The methods and procedures may use CDRs defined according to any of these methods. However, in a preferred embodiment, the CDRs defined by IMGT or Abysis are used. Nevertheless, the boundaries of the CDRs may refer to either of these numbering conventions. It is clear if you do this.

[0136] The immunoglobulin constant (C) domain is a heavy (C H ) chain or light (C L ) chain constant domain The amino acid sequences of mouse and human IgG heavy and light chain constant domains are described in the art. With respect to the heavy chain, in some embodiments of the aspects described herein, The heavy chains of the antibodies described herein may be alpha (α), delta (Δ), epsilon (ε), gamma ( In some embodiments of the aspects described herein, the heavy chain may be a γ (gamma) or mu (mu) heavy chain. In the study, the heavy chains of the antibodies described are human alpha (α), delta (Δ), epsilon ( The human constant region sequence may comprise a human ε, gamma (γ), or mu (μ) heavy chain. Non-limiting examples are described in the art, e.g., U.S. Pat. See, e.g., and Kabat EA et al. (1991) supra.

[0137] Thus, in embodiments of the compositions and methods described herein, anti-WISP1 is a non- It is composed of an IgG framework.

[0138] As used herein, the terms "donor" and "donor antibody" refer to one or more refers to an antibody providing multiple CDRs. In an exemplary embodiment, the donor antibody is An antibody derived from a species different from the antibody from which the work region is obtained or derived. In embodiments, the donor antibody is of a different isotype than the acceptor antibody. In the context of a humanized antibody, the term "donor antibody" refers to a humanized antibody that provides one or more CDRs. This refers to a non-human antibody.

[0139] As used herein, the terms "acceptor" and "acceptor antibody" At least 80% of the amino acid sequence of one or more of the framework regions is At least 85%, at least 90%, at least 95%, at least 98%, or 100% The term "acceptor" refers to an antibody or an encoding nucleic acid sequence that is provided. In some embodiments, the term "acceptor" refers to an antibody or an encoding nucleic acid sequence that is provided. The term refers to antibody amino acid sequences that provide or encode the constant region. In another embodiment, the term "acceptor" refers to a polypeptide that includes framework and constant regions. The term "antibody amino acid sequence" refers to an antibody amino acid sequence that provides or encodes one or more of the antibody domains. In embodiments, the term "acceptor" refers to one or more of the amino acids in the framework region. At least 80%, preferably at least 85%, at least 90%, Provide or encode at least 95%, at least 98%, or 100% human antibody vectors. According to this embodiment, the acceptor is one of the human antibodies. or at least one, at least two, or at least three that do not occur at multiple specific locations , at least 4, at least 5, or at least 10 amino acid residues. The acceptor framework regions and / or the acceptor constant regions may be Germline antibody genes, mature antibody genes, functional antibodies (e.g., antibodies known in the art, the antibody may be, for example, derived from, an antibody in development, or a commercially available antibody; or can be derived from them.

[0140] Human heavy and light chain acceptor sequences are known in the art. The human heavy and light chain acceptor sequences are available from V-base (the v base.mrc-cpe.cam.ac.uk / ) or IMGT (trademark ) of the international IMMUNOGENETICS INFOR MATION SYSTEM(TM) (on the World Wide Web at imgt.cines. Found in fr / textes / IMGTrepertoire / LocusGenes / In another embodiment of the technology disclosed herein, In one embodiment, the human heavy and light chain acceptor sequences are described in U.S. Patent Application Publication No. 2011 / 023. The sequences are selected from those described in Tables 3 and 4 of US Pat. No. 80800. , which is incorporated herein by reference in its entirety.

[0141] The compositions and methods described herein, in some embodiments, comprise "antigen-binding fragments" of antibodies. " or "antigen-binding portion" of an antibody. The term "ISP1-binding fragment" refers to a fragment that specifically binds to an antigen (e.g., WISP1). Refers to one or more fragments of an antibody that retain their potency.

[0142] The antigen-binding function of an antibody can be performed by fragments of a full-length antibody. An embodiment of the fragment is a bispecific, bivalent domain (DVD-Ig) format. The antibody may be incorporated into a multispecific or multi-specific format, with two or more The "antigen-binding portion" of an antibody can specifically bind to many different antigens. Non-limiting examples of antigen-binding fragments encompassed within the term include: (i)V L , V H , C L , and C H Fab fragment, a monovalent fragment consisting of one domain; ii) bivalent, comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) a F(ab')2 fragment, which is a V H and C H Fd fragment consisting of one domain (iv) V of a single arm of the antibody L and V H (v) an Fv fragment consisting of a single domain; dAb fragments containing the variable domains (Ward et al. (1989) Nature, 341: 544-546; International Publication No. and (vi) isolated complementarity determining regions (CD R). In addition, the two domains of the Fv fragment, V L and V H are due to separate genes However, they have been engineered using recombinant methods to L Area and V H The regions are paired and a single protein chain forming a monovalent molecule (known as single-chain Fv (scFv)). The amides may be joined by synthetic linkers that allow the amides to be fabricated in a multi-step fashion (e.g., Bi rd et al. (1988) Science 242: 423-426; and Huston et al. (1988) Proc. Natl. Acad (See, for example, . Sci. USA 85: 5879-5883.) Such single chain antibodies also provide the "antigen binding" function of antibodies. Other forms such as diabodies are intended to be encompassed within the term "sexual moiety." Also encompassed are single chain antibodies.

[0143] In some embodiments, the antibody reagent is a bispecific monoclonal antibody.

[0144] Diabody is V H and V L The domains are expressed on a single polypeptide chain, but A linker that is too short to allow pairing between the two domains on the same chain is used, This forces the domain to pair with a complementary domain on another chain, forming two antigen binding domains. It is a bivalent, bispecific antibody in which a synthesis site is engineered (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448; Poljak (1994) Structure 2: 1121-1123); Kont ermann and Dubel eds., Antibody Engineering, Springer-Verlag, NY (2001), p. 79 0 (ISBN 3-540-41354-5). In addition, single chain antibodies can be prepared by combining a complementary light chain polypeptide. A pair of tandem Fv segments (V) that together form a pair of antigen-binding regions H - C H 1-V H -C H 1) (Zapata et al. (1995) Protein 1) in Eng. 8(10): 1057-1062; and U.S. Pat. No. 5,641,870.

[0145] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region can be generated by papain digestion of an intact antibody. It may be an Fc region or a variant Fc region. The region generally consists of two constant domains: H 2 domain and C H Contains 3 domains, C by choice H It contains 4 domains. The amino acid residues in the Fc portion are replaced to produce antibody effectors. Changing the function is known in the art (see U.S. Pat. No. 5,648,260). The Fc portion of an antibody has several important effects. target functions, e.g., cytokine induction, antibody-dependent cellular cytotoxicity (ADCC), phagocytosis, complement Cell-dependent cytotoxicity (CDC) and half-life / clearance of antibodies and antigen-antibody complexes In some cases, such effector functions are desirable for therapeutic antibodies. While some of these treatments are desirable, in other cases they may be unnecessary or even harmful depending on the therapeutic objectives. Certain human IgG isotypes, particularly IgG1 and IgG3, each have Fcγ It mediates ADCC and CDC by binding to the receptor and complement C1q. The FcRn receptor is a key component that determines the circulating half-life of an antibody. In some embodiments, at least one amino acid residue of the constant region of the antibody, e.g., the Fc region of the antibody, is The antibody effector functions are altered.

[0146] The present invention provides an antibody or antigen-binding fragment that specifically binds to WISP1, as described herein. The nucleic acid sequence may also be a DNA sequence encoding a homologous mammalian (e.g., mouse) Instead of the sequences, coding sequences for human heavy and light chain constant domains or framework regions are By using a sequence (U.S. Pat. No. 4,816,567; Morrison, et al., Pr oc. Natl. Acad. Sci. USA, 81:6851 (1984)), or as described elsewhere herein. The immunoglobulin coding sequence may be modified to include all or part of the coding sequence for a non-immunoglobulin polypeptide. The amino acid sequence may be modified by covalent conjugation to a nucleotide sequence.

[0147] Such non-immunoglobulin polypeptides can be used in place of the constant domains of an antibody. Often, such a non-immunoglobulin polypeptide is linked to one antigen-binding site of an antibody. to generate a single antigen with specificity for a single antigen of interest. and a second antigen-binding site having specificity for a different antigen of interest. Monomeric bivalent antibodies may also be generated.

[0148] In some embodiments of the aspects described herein, the antibody or WISP1-binding fragment thereof , one, two, three, or four of the framework regions of the heavy chain variable region sequence from which it is derived. is at least 75%, 80%, 85%, 90%, 95%, or 100% identical to In one embodiment, the heavy chain variable region sequence includes one, two, three, or four of the framework regions of the heavy chain variable region sequence. In some embodiments of the described aspects, the heavy chain variable domain is derived from the amino acid sequence. The work region consists of the amino acid sequence, but can contain up to 10 amino acid substitutions, deletions, and modifications. and / or insertions, preferably up to 10 amino acid substitutions. In some embodiments of the aspect, the heavy chain variable framework region derived from the amino acid sequence , the amino acid sequence, The amino acid residues are similar to those of the corresponding non-human, primate, or human heavy chain variable framework regions. In some embodiments of the aspects described herein, the amino acid is substituted with an amino acid found at position The antibody or antigen-binding fragment is a human or primate antibody V H One, two, Three or all four V's H The heavy chains described herein further comprise framework regions. The primate or human heavy chain framework of the antibody selected for use with the CDR sequences The region may, for example, have at least 70% identity to the heavy chain framework region of the non-human parent antibody. You may do so.

[0149] In some embodiments of the aspects described herein, the primate or human heavy chain framework region The amino acid residues in the heavy chain framework region of any of the antibodies described herein may be identical to those in the heavy chain framework region of any of the antibodies described herein. At least 75% identity, at least 80% identity, at least 85% identity (or The heavy chain framework regions of natural primates or humans have a higher identity than the heavy chain framework regions of natural antibodies. In a specific embodiment, the antibody or antigen-binding fragment is selected from the group consisting of the human heavy chain variable subfamily. One, two, three, or all four from (e.g., one of subfamilies 1-7) Teno V H It further comprises a framework region.

[0150] In some such embodiments of the aspects described herein, the antibody or its WISP1 binding domain The synthetic fragment may be a fragment that contains one, two, or three of the framework regions of the light chain variable region sequence from which it is derived. , or four and at least 75%, 80%, 85%, 90%, 95%, or 100% One, two, three, or four of the framework regions of the light chain variable region sequence are identical. In some embodiments of the aspects described herein, the light chain polypeptide derived from the amino acid sequence includes The variant framework region consists of the amino acid sequence, but may have up to 10 amino acid substitutions. There are deletions and / or insertions, preferably up to 10 amino acid substitutions. In some embodiments of the described aspects, the light chain variable framework derived from the amino acid sequence is The target region is composed of the amino acid sequence 1, 2, 3, 4, 5, 6, 7, 8, 9, or Ten amino acid residues are selected from the corresponding non-human, primate, or human light chain variable framework amino acid sequences. The amino acid is replaced with an amino acid found in the analogous position in the region. In embodiments, the antibody or antigen-binding fragment is a V L 1 derived from One, two, three, or all four V's L It further comprises a framework region. The primate or human light chain CDR sequences of the antibodies selected for use with the light chain CDR sequences described herein are For example, the framework region may have at least 70% identical structure to the light chain framework region of the non-human parent antibody. They may have the same identity.

[0151] In some embodiments of the aspects described herein, the primate or human light chain framework region The amino acid residues in this region correspond to the light chain framework region of any of the antibodies described herein, as well as at least At least 75% identity, at least 80% identity, at least 85% identity (or The light chain framework regions of natural primates or humans have a higher identity than the light chain framework regions of natural primates or humans. In some embodiments, the antibody or antigen-binding fragment is selected from the human light chain variable kappa subfamily. One, two, three, or all four V's from Lee L Further developing the framework area In some embodiments, the antibody or antigen-binding fragment comprises a human light chain variable lambda subunit. One, two, three, or all four VL framework regions derived from myelin are added. Included.

[0152] In some embodiments of the aspects described herein, the V H (for example, CDR1, CDR2, or CDR3) region and / or V L (e.g., CDR1, The position of one or more CDRs along the CDR2 or CDR3 region was varied. That is, as long as immunospecific binding to the antigen of interest is maintained (e.g., substantially For example, at least 50%, at least 60%, or at least at least 70%, at least 80%, at least 90%, at least 95% are maintained , 1, 2, 3, 4, 5, or 6 amino acid positions shorter or longer. For example, in some embodiments, the positions defining the CDRs may be varied. , i.e., so long as immunospecific binding to the antigen of interest is maintained (e.g., substantially , at least 50%, at least 60%, at least 70%, or 0%, at least 80%, at least 90%, at least 95% are maintained), CDR The N-terminal and / or C-terminal boundaries of any one of the antibodies described herein may be By shifting by 1, 2, 3, 4, 5, or 6 amino acids compared to the R position In other embodiments, the antibodies described herein may have a length of at least 100 nm, or may be shorter or longer. V H (e.g., CDR1, CDR2, or CDR3) regions and / or V L (example For example, one or more CDRs along the CDR1, CDR2, or CDR3 regions. The length may be any length so long as immunospecific binding to the antigen of interest is maintained (e.g., substantially At least 50%, at least 60%, at least 70% of the binding of the original antibody from which it is derived %, at least 80%, at least 90%, at least 95% are maintained), 1, 2, It may vary by 3, 4, 5, or more amino acids (e.g., (It may be shorter or longer).

[0153] In some embodiments of the aspects described herein, one, two, or more Natural mutations (e.g., amino acid substitutions) may be added to the Fc region or The fragment (e.g. according to the Kabat numbering system (e.g. Kabat's EU Index) The CH2 domain (residues 231-340 of human IgG1) and / or CH 3 domain (residues 341-447 of human IgG1) and / or the hinge region) such as serum half-life, complement fixation, Fc receptor binding, and / or antigen-dependent cellular cytotoxicity. One or more functional properties of the antibody are altered.

[0154] In some embodiments of the aspects described herein, the method is similar to that described in, for example, U.S. Pat. As described in the specification, one, two or more mutations (e.g., amine or a substitution of cysteine ​​residues in the hinge region, such that the number of cysteine ​​residues in the hinge region is altered (e.g., increased or decreased). (C) Fc region (C H 1 domain) into the hinge region. H 1 Domain The number of cysteine ​​residues in the hinge region of the polypeptide can be altered to facilitate, for example, assembly of the light and heavy chains. The antibody may be further modified to facilitate the synthesis or may alter the stability of the antibody (e.g., increased or decreased). (This may be done.)

[0155] In some embodiments of the aspects described herein, one, two, or more Natural mutations (e.g., amino acid substitutions) may be added to the Fc region of the antibodies described herein or to their antigen-binding domain. Synthetic fragments (e.g., according to the Kabat numbering system (e.g., Kabat's EU Index) By numbering, C H 2 domain (residues 231-340 of human IgG1) and / or C H 3 domain (residues 341-447 of human IgG1) and / or the hinge region) The antibody binds to Fc receptors (e.g., activating Fc receptors) on the surface of effector cells. Increase or decrease the affinity of antibodies to Fc receptors. Mutations in the Fc region of an antibody or fragment thereof that cause the Techniques for introducing Fc receptors or fragments thereof are known to those skilled in the art. Abnormalities in the Fc receptors of antibodies can be made to alter the affinity of the antibody for Examples of mutations are described, for example, in Smith P et al., (2012) PNAS 109: 6181-6186, U.S. Pat. No. 37,056, and International Publication No. WO 02 / 060919; International Publication Pamphlet of International Publication No. 98 / 23289 and Pamphlet of International Publication No. 97 / 34631 No. 6,399,413, which is incorporated herein by reference.

[0156] The term "CDR-grafted antibody" refers to an antibody that has one or more of the human CDRs (e.g., CDR3). and antibodies having human heavy and light chain variable regions in which the CDR sequences are replaced by mouse CDR sequences. , which contains heavy and light chain variable region sequences from one species, but H and / or V L of Refers to an antibody in which one or more sequences of the CDR region are replaced with the CDR sequence of another species. The CDR-grafted antibodies described herein are H and / or V L One or more of the CDR regions a human antibody in which one or more of the CDR sequences have been replaced with the CDR sequences of a non-human antibody described herein. The heavy and light chain variable region sequences are derived from

[0157] The anti-WISP1 antibodies described herein may be engineered to express specific binding domains for therapeutic use. The specificity of the binding can be, for example, one or The antigen (e.g., WISP1 or The polypeptide fragments thereof can be assayed by competitive assays. To select an agent, antibody, or other ligand that specifically binds to SP1, various Any suitable immunoassay format is suitable. Specific binding can be determined, for example, by the binding of an agent ( For example, the affinity and avidity of the polypeptide or anti-WISP1 antibody and the action The effect of the binding assay may be influenced by the concentration of the agent. The agents described herein can be used to assess whether they are effective in treating WI infections using any suitable method, such as by titrating the agent. Suitable conditions for selectively binding to SP1 can be determined.

[0158] As used herein, the term "key" residues refers to the specific amino acid sequence of an antibody, particularly a humanized antibody. Variables that have a greater effect than others on the binding specificity and / or affinity of an antibody This refers to a specific residue in a domain. Key residues include, but are not limited to, the following: These may include one or more of the following: residues adjacent to the CDRs, potential glycosylation site (which may be either N- or O-glycosylation site), rare residue, interaction with antigen residues that can interact with the CDRs; canonical residues nical residue, contact residue between heavy chain variable domain and light chain variable domain, Bernier zone (Vernier zone) The residues in the 2nd chromosome (nearer zone) and the Chothia definition of the variable heavy chain CDR and the first heavy chain frame Residues that lie in the region of overlap between the framework's Kabat rules.

[0159] The anti-WISP1 antibodies described herein may be genetically engineered antibodies. As used in this document, the term "genetically engineered" means something that has been manipulated by human hand. For example, a locus refers to an embodiment in which the sequences of the genes that are naturally present together at the locus are Two or more unlinked sequences may be mutually exclusive at the engineered locus. If the gene has been manipulated by humans to directly link the gene to the target gene, it is considered to be "genetically engineered." For example, in some embodiments of the invention, the engineered locus is Contains a variety of Ig sequences with non-natural V segments, all of which are found in nature. are not found at the same locus or in that order at the natural locus. As is common practice and will be understood by those skilled in the art, genetically engineered polynucleotides nucleotides (and / or progeny of cells or animals containing such polynucleotides) and duplication typically still occurs even though the actual operations are performed on the previous entity. These are then called "genetically engineered."

[0160] The WISP1 binding polypeptide, antibody, antibody reagent, or antigen-binding portion thereof, Covalent Linkage of an Antigen-Binding Portion of an Antibody with One or More Other Proteins or Peptides A member of a larger immunoadhesion molecule that is formed by covalent or noncovalent association. Examples of such immune adhesion molecules include streptavidin, streptomycin, and streptomycin. Using the avidin core region to create tetrameric scFv molecules (Kipriyanov et al. (1995) Human Antibod. Hybridomas 6:93-101), as well as cysteine ​​residues, marker peptides, Create bivalent biotinylated scFv molecules using peptides and a C-terminal polyhistidine tag (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions such as ab and F(ab')2 fragments are prepared by the papain or glycerol extraction of whole antibodies, respectively. It can be prepared from whole antibodies using conventional techniques, such as pepsin digestion. Antibodies, antigen-binding portions thereof, and immunoadhesion molecules can be produced using standard recombinant DNA techniques. Alternatively, target binding proteins, such as the antigen-binding portion of an antibody, can be obtained by It may be part of the variable domain (DVD-Ig).

[0161] Therapeutic and / or specific for any particular target antigen (e.g., WISP1) Certain antibodies and antibody reagents can be easily identified by those skilled in the art from known antibodies or antibody reagents, e.g. FDA approved therapeutic antibody reagents and / or their catalogues according to target specificity These antibodies are readily selected from a variety of commercially available antibody reagents.

[0162] In another embodiment, the anti-WISP1 antibody is any anti-WISP1 antibody known in the art. or any anti-WISP1 antibody yet to be discovered. Exemplary anti-WISP1 antibodies available from Abcam include, but are not limited to, Anti-WISP1 antibodies (e.g., ab60114; ab65943), RND System MS (e.g., mab1680), and Sigma-Aldrich (e.g., SAB 2501114).

[0163] In another embodiment, the anti-WISP1 antibody is selected from Table 1 (Table of Antibodies) below. In one embodiment, the anti-WISP1 antibody has any one of the amino acid sequences set forth in SEQ ID NOs: 12 to 120. Includes 1.

[0164] [Table 1-1]

[0165] [Table 1-2]

[0166] In another embodiment, the anti-WISP1 antibody is any known or yet to be discovered antibody. The antibody is a humanized anti-WISP1 antibody derived from a non-human anti-WISP1 antibody.

[0167] In one embodiment, the antibody or antibody reagent is a nucleic acid sequence encoding WISP1 (sequence It binds to the amino acid sequence corresponding to number 1).

[0168] In another embodiment, the anti-WISP1 antibody or antibody reagent comprises an amino acid sequence comprising the sequence of SEQ ID NO:1. or binds to the sequence of SEQ ID NO: 1 at least 70%, at least 80%, or %, at least 85%, at least 90%, at least 95%, at least 96%, at least at least 97%, at least 98%, at least 99% or higher sequence identity In one embodiment, the anti-WISP1 antibody or The antibody reagent binds to an amino acid sequence comprising the entire sequence of SEQ ID NO:1. The antibody or antibody reagent binds to an amino acid sequence comprising a fragment of the sequence of SEQ ID NO:1, the fragment comprising: It binds to its target, e.g., WISP1, and inhibits WISP1 levels and / or activity. It is enough to drip.

[0169] In certain embodiments, the antibodies or antibody reagents are directed to various human WISP1 isoforms. The amino acid sequence corresponding to the amino acid sequence encoding the Combine.

[0170] In another embodiment, the anti-WISP1 antibody or antibody reagent comprises the sequence of SEQ ID NO: 2, 3, or 4. or binds to an amino acid sequence comprising the sequence of SEQ ID NO: 2, 3, or 4 and at least at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or a sequence having a higher sequence identity than that of the amino acid sequence. In one embodiment, the anti-WISP1 antibody or antibody reagent comprises the entire sequence of SEQ ID NO: 2, 3, or 4. In another embodiment, the antibody or antibody reagent binds to an amino acid sequence comprising SEQ ID NO:2, 3, or 4, and the fragment binds to its target, e.g., WI is sufficient to bind SP1 and result in inhibition of WISP1 levels and / or activity .

[0171] In another embodiment, the antibody or antibody reagent comprises the amino acid sequence encoding mouse WISP1. The sequence binds to the amino acid sequence corresponding to the sequence (SEQ ID NO:6).

[0172] In another embodiment, the antibody or antibody reagent binds to an amino acid sequence comprising the sequence of SEQ ID NO:6. or at least 70%, at least 80%, at least At least 85%, at least 90%, at least 95%, at least 96%, at least 97% , at least 98%, at least 99%, or more sequence identity. In one embodiment, the anti-WISP1 antibody or antibody reagent binds to an amino acid sequence comprising the , which binds to an amino acid sequence comprising the entire sequence of SEQ ID NO:6. The antibody reagent binds to an amino acid sequence that includes a fragment of the sequence of SEQ ID NO:6, and the fragment binds to its target, For example, to bind to WISP1 and cause inhibition of WISP1 levels and / or activity. That's enough.

[0173] Thus, in some embodiments, the present specification provides variable heavy chains and and one or more variable domains comprising one or more CDRs encoded by the light chain sequence. Humanized antibodies containing the ribozyme-binding domain have been described.

[0174] In another embodiment, the antibody or antibody reagent comprises at least one of SEQ ID NOs: 12-120. In another embodiment, the antibody or antibody comprises an amino acid sequence having at least 70% homology thereto. The reagent CDR has at least 70% homology to any one of SEQ ID NOs: 12 to 120. In another embodiment, the antibody or antibody reagent comprises an amino acid sequence selected from SEQ ID NOs: 12-120. The amino acid sequence has at least 90% homology to any one of the following:

[0175] In other words, in some embodiments, an antibody or antibody reagent that inhibits WISP1 is Sequence numbers 1 to 4, 6, or sequence numbers 12 to 120 and at least 50%, at least 60 %, at least 70%, at least 80%, at least 90%, or 100% homologous be.

[0176] In other embodiments, for example, the nucleic acid sequence of a WISP1 binding fragment or an anti-WISP1 antibody Or the amino acid sequence (e.g., DNA, RNA, or amino acid sequence) is a sequence that is The corresponding native or unedited nucleic acid sequence (e.g., genomic sequence) of an antibody that specifically binds to SP1 sequence) or amino acid sequence and at least 50%, at least 60%, at least 70%, At least 75%, at least 80%, at least 85%, at least 90%, at least At least 91%, at least 92%, at least 93%, at least 94%, at least 95% , at least 96%, at least 97%, at least 98%, at least 99%, or If there is a higher degree of identity, it is considered to be "homologous."

[0177] One aspect provided herein is an anti-WISP1 antibody or antibody reagent described herein. In one embodiment, the composition further comprises a pharma- ceutically acceptable carrier. In one embodiment, the composition is a pharmaceutical composition.

[0178] Nucleic acids that inhibit WISP1 In one embodiment, the agent that inhibits WISP1 is an antisense oligonucleotide. As used herein, an "antisense oligonucleotide" refers to a It refers to a synthetic nucleic acid sequence that is complementary to a DNA sequence or an mRNA sequence, such as the sequence of a NA. Sense oligonucleotides typically bind to a target and inhibit transcription, translation, or splicing. Blocks expression of DNA or RNA targets by silencing expression at the signaling level. The antisense oligonucleotides of the present invention are designed to inhibit the activity of the gene under cellular conditions. A complementary nucleic acid sequence designed to hybridize to a gene, e.g., WISP1. Therefore, the target is sufficiently complementary, i.e., sufficiently abundant in the context of the cellular environment. An oligonucleotide that hybridizes well and with sufficient specificity to exert the desired effect. For example, an antisense oligonucleotide that inhibits WISP1 is selected. A human WISP1 gene (e.g., SEQ ID NO: 5) or a mouse WISP1 gene (e.g., SEQ ID NO: At least 5, at least 10, or at least 12 sequences complementary to a portion of the coding sequence of At least 15, at least 20, at least 25, at least 30, or more The nucleic acid may contain as many bases as possible.

[0179] In one embodiment, WISP1 can be expressed as, but not limited to, a zinc finger nucleic acid. Any of the following systems, including nucleases, TALENS, meganucleases, and CRISPR / Cas systems A genome editing system is used to delete from the genome of the cell. Genome editing is used to integrate nucleic acids encoding guide RNAs into the genome of cells The system is not a CRISPR / Cas system, which allows the use of small amounts of Cas enzymes / proteins. In the present specification, Cas enzymes can be used to prevent undesirable cell death in cells that are retained. Each of the genes and sgRNAs is expressed under the control of a different inducible promoter. It is also contemplated that this allows for the transient expression of each to prevent such interference. do.

[0180] A nucleic acid encoding one or more sgRNAs and an RNA-guided endonuclease is provided. Adenovirus-associated nucleic acids may be administered in vivo, respectively. The use of vectors (AAV, adenovirus associated vectors) is specifically contemplated. Both components of the genome editing / fragmentation system (e.g., sgRNA, RNA-guided endonuclease) Other vectors for co-delivery include Epstein-Barr and human immunodeficiency viruses. (HIV), hepatitis B virus (HBV), and other lentiviral vectors. Each of the components of the RNA-guided genome editing system (e.g., sgRNA and endonuclease) are delivered in separate vectors, as known in the art or as described herein. It is possible.

[0181] In one embodiment, the agent inhibits WISP1 by RNA inhibition. The inhibitor of expression may be an inhibitory nucleic acid. The inhibitory nucleic acid is an inhibitory RNA (iRNA). The RNAi may be single stranded. It may be double-stranded or double-stranded.

[0182] iRNA can be siRNA, shRNA, endogenous microRNA (miRNA), or In one embodiment, the iRNA described herein can be an artificial miRNA. For example, it results in inhibition of expression and / or activity of WISP1. In some embodiments, the agent is an siRNA that inhibits WISP1. In some embodiments, the agent is an shRNA that inhibits WISP1.

[0183] Those skilled in the art can, for example, use publicly available design tools to design and construct a polypeptide that targets WISP1. One could design siRNAs, shRNAs, or miRNAs that target the si RNA, shRNA, or miRNA are typically delivered by Dharmacon (Rafami). (St. Louis, MO, USA) or Sigma Aldrich (St. Louis, MO, USA). It is produced by companies such as the US State of New York.

[0184] In some embodiments of any of the present aspects, the iRNA may be a dsRNA. sRNA hybridizes to form a double-stranded structure under conditions in which dsRNA will be used. It contains two RNA strands that are sufficiently complementary to form a single strand of dsRNA. The (antisense strand) is substantially complementary to the target sequence, typically completely complementary. The target sequence is determined by the sequence of the mRNA that is formed during expression of the target. The other strand (sense strand) contains a region complementary to the antisense strand, so that When combined under suitable conditions, the two strands hybridize to form a duplex structure.

[0185] The RNA of the iRNA may be chemically modified to enhance stability or other beneficial features. The nucleic acids featured in the present invention can be synthesized by the method described in "Current protocols in nucleic acid synthesis". acid chemistry," Beaucage, SL et al. (Edrs.), John Wiley & Sons, Inc., New York The compounds can be synthesized and synthesized by methods well established in the art, such as those described in 1996, 1997, NY, USA. and / or modified, this document is incorporated herein by reference.

[0186] In one embodiment, the agent is a miRNA that inhibits WISP1. are small non-coding RNAs with an average length of 22 nucleotides. These molecules are by binding to a complementary sequence, usually located in the 3' untranslated (3'UTR) region of an mRNA molecule. They act by promoting the degradation of target mRNA or the inhibition of mRNA translation. The interaction of microRNA with mRNA is due to imperfect Watson-Crick base pairing. A 6- to 8-nucleotide region of a microRNA that directs sequence-specific binding to RNA In mammals, there are over 900 species Many microRNAs are known to be expressed in the human genome. Based on their sequence, similar microR miRNAs can be used to identify "clusters" of NAs within cells, e.g. miRNAs can be expressed as nucleic acids that are expressed in cells. For example, it may be encoded as naked DNA or may be contained within a vector. It may be encoded by a nucleic acid that is

[0187] In one embodiment, the agent that down-modulates WISP1 is miRNA-15a miRNA-15a regulates gene expression in several organs, including the liver. miRNA-15a is a non-coding RNA that is involved in the regulation of the miRNA-15a gene. miRNA-15a, e.g., miRBase accession number MI0000069, and mouse miRNA-15a, for example, is known under the miRBase accession number MI0000564. Human miRNA-15a comprises the sequence of SEQ ID NO: 8. miRNA-15a is a naturally occurring Human miRNA-15a, including its present variants, molecules, and their alleles, was For example, miRNA-15a can refer to a mouse miRNA having the sequence of SEQ ID NO: 10. It may be miRNA-15a.

[0188] In one embodiment, the agent, e.g., miRNA-15A, corresponds to the sequence of SEQ ID NO:8. having the sequence SEQ ID NO: 8 or comprising the sequence SEQ ID NO: 8 and at least At least 80%, at least 85%, at least 90%, at least 91%, at least 92% %, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, at least 99%, or at least 100% of the sequences Includes sequences that have identity.

[0189] In one embodiment, the agent, e.g., miRNA-15a, corresponds to the sequence of SEQ ID NO: 10. or comprising the sequence of SEQ ID NO: 10, or comprising the sequence of SEQ ID NO: 10; At least 80%, at least 85%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or at least 100% The present invention includes sequences having sequence identity of:

[0190] In one embodiment, the agent that down-modulates WISP1 is miRNA-412. The miRNA-412 sequence is similar to many other sequences, including human miRNA-41 2, e.g., miRBase accession number MI0001464, and mouse miRNA-41 2, for example, miRBase accession number MI0001164. miRNA-412 comprises the sequence of SEQ ID NO: 9. miRNA-412 is a naturally occurring variant , molecules, and their alleles. For example, miRNA-15a is a mouse miRNA-41 having the sequence of SEQ ID NO: 11. It may be 2.

[0191] In one embodiment, the agent, e.g., miRNA-412, corresponds to the sequence of SEQ ID NO:9. having the sequence SEQ ID NO: 9 or comprising the sequence SEQ ID NO: 9 and at least At least 80%, at least 85%, at least 90%, at least 91%, at least 92% %, at least 93%, at least 94%, at least 95%, at least 96%, at least at least 97%, at least 98%, at least 99%, or at least 100% of the sequences Includes sequences that have identity.

[0192] In one embodiment, the agent, e.g., miRNA-412, corresponds to the sequence of SEQ ID NO:11. or comprising the sequence of SEQ ID NO: 11, or having the sequence of SEQ ID NO: 11; At least 80%, at least 85%, at least 90%, at least 91%, at least At least 92%, at least 93%, at least 94%, at least 95%, at least 96% , at least 97%, at least 98%, at least 99%, or at least 100% The present invention includes sequences having sequence identity of:

[0193] The agent may be, for example, an RNAi molecule (e.g., siRNA or miRNA) This can result in gene silencing of the target gene (e.g., WISP1). This involves measuring the intracellular mRNA levels of the target relative to those found in the cells in the absence of the agent. At least about 5%, about 10%, about 20%, about 30%, about 40%, about 5%, or about 0%, approximately 60%, approximately 70%, approximately 80%, approximately 90%, approximately 95%, approximately 99%, approximately 100% decrease In one preferred embodiment, the mRNA level is at least about 70%, about 80%, %, about 90%, about 95%, about 99%, about 100%. Transfect cells with iRNA, shRNA, or miRNA and perform Western blot analysis. By using a sequencing or PCR-based assay, genes found in cells (e.g., By detecting ISP1 levels, siRNA, shRNA, or miRNA can be , for example, to easily evaluate whether a compound is effective in targeting WISP1 and downregulating it. It will be possible.

[0194] The agent may be contained in a vector, and thus may further comprise a vector. Many such vectors are available which are useful for transferring exogenous genes into target mammalian cells. Vectors can be episomal, e.g., plasmids, It may be a virus-derived vector, such as tomegalovirus, adenovirus, or By homologous recombination or random integration, e.g., MMLV, HIV-1, ALV, etc. The vector may be integrated into the target cell genome by a retrovirus-derived vector. In certain embodiments, combinations of retroviruses and appropriate packaging cell lines also find use. In such cases, the capsid protein may be functional for infection of a target cell. Typically, the cells and virus are incubated in culture medium for at least about 24 hours. Then, over a short period of time, for some applications, for example, 24 to 73 hours, Allow the cells to grow in culture medium for at least 2 weeks and then separate. The cells may be allowed to grow for 5 weeks or longer before analysing. The retroviral vectors used are "defective," meaning they contain the viral vectors required for productive infection. The vector cannot produce viral proteins. Growth in stocks is necessary.

[0195] The term "vector" as used herein refers to a vector that is used for delivery to a host cell. or a nucleic acid construct designed for transfer between different host cells. In some cases, the vector may be viral or non-viral. The term "vector" refers to any vector capable of replication and transmission when associated with the proper control elements. It includes any genetic element capable of transferring a gene sequence into a cell. Examples include, but are not limited to, cloning vectors, expression vectors, and plasmids. , phages, transposons, cosmids, artificial chromosomes, viruses, virions, etc. It is possible.

[0196] As used herein, the term "expression vector" refers to a transcription factor located on a vector. RNA or polypeptides (e.g., The term refers to a vector that directs the expression of a gene that is a target gene (e.g., a WISP1 inhibitor). The sequence to be expressed must In many cases, but not all, the expression vector will be heterologous to the cell. For example, the expression vector may contain two replication systems. Thus, the expression vector can be transfected in two organisms, e.g., human cells for expression. It can be maintained in a prokaryotic host for cloning and amplification. The term "expression", where applicable, includes, but is not limited to, for example, transcription. , transcript processing, translation and protein folding, modification, and processing For RNA and protein production, including cloning, and protein secretion when required "Expression product" refers to the cellular process involved in a gene. and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to an in vitro gene that is operably linked to appropriate regulatory sequences. Genetics refers to a nucleic acid sequence (DNA) that can be transcribed into RNA either in vivo or in vivo. The offspring may be derived from regions preceding or following the coding region, such as the 5' untranslated (5'UTR) or "leader" The sequences and 3'UTR or "trailer" sequences, as well as the individual coding segments ( It may or may not contain intervening sequences (introns) between the transcription factors.

[0197] Integrative vectors, the RNA / DNA they deliver is permanently integrated into the host cell chromosomes. Non-integrating vectors remain episomal, meaning that the vectors contained therein This means that the nucleic acid contained therein is never integrated into the host cell chromosome. Examples of vectors that can be integrated include retroviral vectors, lentiviral vectors, and hybridomas. Examples of vectors include hybrid adenovirus vectors, and herpes simplex virus vectors.

[0198] One example of a non-integrating vector is a non-integrating viral vector. Virus vectors are integrative retrovirus vectors because their genomes do not integrate into the host DNA. The risk posed by viruses is eliminated. One example is the Epstein-Barr oriP / nuclear antibody The EBNA1 vector is capable of restricting self-replication. It is known to function in mammalian cells. It is derived from the Epstein-Barr virus. It contains two elements, oriP and EBNA1, which act as the EBNA1 protein. The binding of the protein to the viral replicon region oriP is essential for the propagation of the plasmid in mammalian cells. maintains a relatively long-term episomal presence. oriP / EBNA1 vectors This particular feature makes it ideal for generating iPSCs that do not integrate. Viral vectors include adenovirus vectors and adeno-associated virus (AAV) vectors. -It is.

[0199] Another non-integrating viral vector is the RNA Sendai virus vector, which F-deficient Sendai can produce proteins without entering the nucleus of infected cells. Viral vectors persist in the cytoplasm of infected cells for a few passages, but rapidly disappear. and is completely lost after a few passages (e.g., 10 passages).

[0200] Another example of a non-integrating vector is a minicircle vector. The plasmid backbone is released and the eukaryotic promoter and the cDNA to be expressed are inserted into the Only A remains as a circularized vector.

[0201] As used herein, the term "viral vector" refers to any vector of at least some type of viral origin. It contains at least one element and has the ability to be packaged into a viral vector particle. Viral vectors refer to nucleic acid vector constructs that contain a gene encoding a non-essential viral gene. The vector can contain a nucleic acid encoding a polypeptide as described herein. and / or the particles can be used in vitro or in vivo to transfer nucleic acids into cells. Many forms of viral vectors are known in the art and can be used either in vivo or in vivo. It is well known in the field.

[0202] Genetically engineered hepatic stellate cells (HSCs) In one aspect, the present disclosure provides a method for the treatment of hepatic stellate cells or hepatic stellate cells expressing an agent that inhibits WISP1. The method for generating a population of cells comprising treating the cells with an inhibitor of WISP1 as described herein. and for a time sufficient to allow expression of the agent. Methods are described that include culturing the cells over a period of time.

[0203] In one embodiment, the cell is a quiescent cell. The method for identifying a quiescent cell is described herein. It is stated above in the book.

[0204] In one embodiment, the contacting comprises contacting the cell with an agent or a vector encoding the agent. For example, contacting can include, but is not limited to, transduction, nucleofection, ion, electroporation, direct injection (e.g., into HSCs), and / or transcription. A person skilled in the art can easily implement such a method using techniques known in the art. The cells can be contacted with the agents described herein using any of the techniques described herein.

[0205] In one embodiment, the agent is a miRNA. RNA-15a or miRNA412.

[0206] In one embodiment, the cells are cultured under standard culture conditions in 20% (weight / volume) fetal bovine serum. (FBS) and 1% (wt / vol) penicillin / streptomycin supplemented DAL They are cultured in Becko's modified Eagle's medium (DMEM).

[0207] In one embodiment, the cells are incubated for at least 1 hour to allow expression of the agent. In another embodiment, the cells are cultured with at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 1 7, 18, 19, 20, 21, 22, 23, 24, 36, 48, 60, 72, 96, 12 The cells are cultured for 0, 144 hours, or longer. Standard techniques in the art, such as PCR-based assays for detecting miRNA expression, can be used. The assay can be used to determine whether an agent is expressed in the cells after culturing.

[0208] In one embodiment, the cells transiently express the agent. The agent may, for example, be integrated into the genome of a cell such that the cell's progeny express the agent. .

[0209] One embodiment provides an agent that inhibits WISP1 produced using the methods described herein. The present invention provides a cell line comprising HSCs expressing an agent that inhibits WISP1. The compound may be, for example, pharma- tically acceptable for administration to a subject in need of treatment for liver disease. The compound may be present in a carrier.

[0210] Another aspect is an agent that inhibits WISP1 produced using the methods described herein. The present invention provides a pharmaceutical composition comprising a population of HSCs expressing the gene and a pharma- ceutical acceptable carrier.

[0211] Compositions and pharmaceutical compositions In one aspect, the present specification describes a composition comprising any of the agents described herein. In one aspect, the present disclosure provides a pharmaceutical composition comprising any of the agents described herein. The composition is described.

[0212] In another aspect, the present disclosure provides a composition comprising an antibody or antibody reagent that inhibits WISP1. In another aspect, the present invention provides an antibody or antibody that inhibits WISP1. A pharmaceutical composition comprising the agent is described.

[0213] In one embodiment, the compositions or pharmaceutical compositions described herein comprise at least two, three or more For example, the composition may contain one, two, three, four, five or more of the agents described herein. For example, the composition may include an siRNA that inhibits WISP1 and an anti-WISP1 antibody reagent. Alternatively, the composition may comprise two anti-WISP1 antibody reagents.

[0214] In one embodiment of any of this aspect, the composition is formulated for the treatment or prevention of liver disease. In order to clinically use the methods described herein, the WISP 1. An agent that inhibits 1 (e.g., an antibody, an antibody reagent, or a WISP1-binding fragment thereof) Administration of the compound may be parenteral, e.g., intravenous; mucosal, e.g., intranasal; ophthalmic, or other modes of administration. In some embodiments, the compound may be formulated into a pharmaceutical composition or formulation for The agents described herein may be any pharma- ceutically acceptable salt that provides effective treatment in a subject. The present invention can be administered together with a suitable carrier compound, material, or composition. Pharmaceutical formulations for use in the methods described herein include antibodies or The antigen-binding fragment is combined with one or more pharma- ceutically acceptable moieties. This is also fine.

[0215] The term "pharmaceutical acceptable" means, within the scope of sound medical judgment, a substance that is not excessively toxic, irritating, or toxic to humans or animals. Reasonable benefit / risk with no extreme effects, allergic reactions, or other problems or complications Compounds, materials, compositions suitable for use in contact with human and animal tissues that meet the above criteria. The term "pharmaceutically acceptable carrier" refers to a pharmaceutical composition, composition, and / or dosage form. When used in, as a liquid or solid filler, diluent, excipient, solvent, vehicle, encapsulating material, Manufacturing aids (e.g., lubricants, magnesium talc, calcium or zinc stearate or stearic acid), or the stability, solubility, or or a solvent encapsulating material that is involved in maintaining activity. Each carrier must be compatible with the other ingredients of the formulation and not deleterious to the patient. "Acceptable" in the sense of being "excipient," "carrier," or "pharmaceutical "Compound acceptable carrier" and like terms are used interchangeably herein.

[0216] Therapeutic preparations of the WISP1 agonists or inhibitors described herein may be prepared in a form having a desired purity. The antibody or antigen-binding fragment is optionally combined with a pharma- ceutically acceptable carrier, excipient, or stabilizer. Fixed dose (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)) By mixing, it can be prepared for storage in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are suitable for use in the formulation at the dosages and concentrations employed. Non-toxic to the petients, including: phosphates, citrates, and other Buffers such as organic acids; antioxidants including ascorbic acid and methionine; preservatives (octadecanoic acid, Decyldimethylbenzylammonium chloride;Hexamethonium chloride;Benzalkonium chloride Benzethonium chloride; phenol, butyl or benzyl alco Alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight ( a polypeptide of less than about 10 residues; serum albumin, gelatin, or immunoglobulin Proteins such as; hydrophilic polymers such as polyvinylpyrrolidone; glycine, glutamine , asparagine, histidine, arginine, or lysine; glucose monosaccharides, disaccharides, and other carbohydrates, including mannose, or dextrin; EDT Chelating agents such as A; sucrose, mannitol, trehalose, or sorbitol any sugar; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes) and / or TWEEN™, PLURONICS™, or polyethylene An exemplary lyophilized antibody formulation is: The method is described in WO 97 / 04801, which is incorporated herein by reference. It is expressly incorporated herein.

[0217] Optionally, but preferably, formulations containing the compositions described herein are A physiologically acceptable salt, typically sodium chloride, for example, is preferably used at approximately physiological concentrations. Optionally, the formulations of the present invention may contain a pharma- ceutically acceptable preservative. In some embodiments, the preservative concentration is typically in the range of 0.1-2.0% volume / volume. Suitable preservatives include those known in the pharmaceutical art. Cholesterol, phenol, m-cresol, methylparaben, and propylparaben are Optionally, the formulation of the present invention contains a pharmaceutical agent at a concentration of 0.005 to 0.02%. The composition may contain a surfactant acceptable to the pharmaceutical industry.

[0218] Also, the agents described herein (e.g., antibodies, antibody reagents, and their WISP1 Therapeutic formulations of compositions containing the agonist, agonist, and / or agonist (binding fragment) may contain more than one agonist, agonist, and / or agonist as required for the particular indication being treated. Contains multiple active compounds, preferably those with complementary activities that do not adversely affect each other. Alternatively, the composition may comprise, for example, a cytotoxic agent, a cytokine, or a growth inhibitor. Such molecules preferably are effective for their intended purpose. The compounds are present in effective combination amounts.

[0219] Also, the active ingredient of the therapeutic formulation of the composition containing the agent described herein is a colloid drug. Delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, etc.) nanoparticles, and nanocapsules) or macroemulsions, e.g. by coacervation. Microcapsules prepared by the fluoropolymerization technique or by interfacial polymerization, for example, Hydroxymethylcellulose or gelatin microcapsules and poly(methylmethacrylate) Such techniques may be described in Remingto et al. and other related art documents, as disclosed in Osol, A. Ed. (1980).

[0220] In some embodiments, the pharmaceutical composition further comprises a lipid vehicle. Examples of the molecules include, but are not limited to, liposomes, micelles, exosomes, and lipid emulsions. John, and lipid-drug conjugates.

[0221] In some embodiments, the pharmaceutical composition further comprises a particle or polymer-based vehicle. Exemplary particle or polymer-based vehicles include, but are not limited to, Nanoparticles, microparticles, polymer microspheres, or polymer-drug conjugates Examples include

[0222] In some embodiments, sustained release preparations can be used. Suitable examples of sustained release preparations The antibody or antigen-binding fragment may be a semipermeable matrix of a solid hydrophobic polymer containing the antibody or antigen-binding fragment. matrices in the form of shaped articles, e.g. films or microcapsules Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl-methacrylate), or poly(vinyl alcohol)); Polylactide (U.S. Pat. No. 3,773,919), L-glutamic acid and y-ethylene glycol Copolymer of ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, LUPRON DEPOT™ (composed of lactic acid-glycolic acid copolymer and leuprolide acetate) Degradable lactic acid-co-glycolic acid polymers, such as injectable microspheres, and poly- D-(-)-3-hydroxybutyric acid. Ethylene-vinyl acetate and lactate-glycol Polymers such as cholic acid allow for release of molecules for periods longer than 100 days. Certain hydrogels release proteins over a shorter period of time. If it remains in the body for an extended period of time, it may denature or become unstable as a result of exposure to moisture at 37°C. Aggregation may occur, resulting in loss of biological activity and altered immunogenicity. Depending on the mechanism, rational strategies for stabilization can be devised. For example, If it turns out that this is due to intermolecular S--S bond formation by thio-disulfide exchange, Stabilization involves modification of sulfhydryl residues, lyophilization from acidic solution, control of water content, and appropriate This can be achieved by using suitable additives and developing specific polymer matrix compositions. can.

[0223] The methods described herein include therapeutic agents for in vivo administration, such as parenteral administration. The formulation can be sterilized, which can be accomplished by filtration through sterile filtration membranes, or other methods known to those of skill in the art. This can be easily achieved by the method described above.

[0224] Administration In some embodiments, the methods described herein comprise administering to a subject a subject, comprising administering to a subject a subject, the subject being unable to obtain WISP1 as described herein. A subject having or diagnosed as having liver disease, comprising administering an agent that inhibits A subject with liver disease may be treated with a method that, if administered by a physician, is known to be effective in treating the condition. Symptoms of liver disease that characterize the disease and aid in diagnosis: and / or complications are well known in the art and include, but are not limited to, fatigue, These include weight loss, pain, yellowing of the skin and / or eyes, and dark urine. Tests that may aid in the diagnosis of liver disease include, but are not limited to, blood tests. These include CT, noninvasive imaging, and / or tissue biopsy. Family history of liver disease should also be considered. to determine whether a person is likely to have the condition or to make a diagnosis of liver disease. This will help you.

[0225] The agents and compositions described herein (e.g., those that inhibit WISP1) can be used to treat liver disease. In some embodiments, the method may be administered to a subject who has or has been diagnosed with a disease. The methods described herein include administering to a subject an effective amount of a compound that is capable of reducing at least one symptom of liver disease. As used herein, "treatment of at least one patient with liver disease" includes administering to a subject an agent that inhibits the progression of the disease. "Alleviating at least one symptom" means any condition or symptom associated with liver disease (e.g., , fatigue, weight loss, pain, yellowing of the skin and / or eyes, or dark urine) Such reduction, as measured by any standard technique, should be comparable to that observed in equivalent untreated controls. Compared to at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99%, or greater. Various means for administering the agent to the body are known to those of skill in the art. In one embodiment, the agent is administered systemically. In one embodiment, the agent is administered intravenously or locally (e.g., to the liver). In one embodiment, the agent is administered continuously, at intervals, or sporadically. The route of administration of an agent depends on the type of agent being delivered (e.g., miRNA, cells, or The method will be optimized for RNAi and can be determined by the skilled practitioner. Cut.

[0226] The agents and pharmaceutical compositions described herein may be administered in any suitable manner that provides an effective treatment for a subject. The compound may be administered to a subject in need thereof by any suitable route. In this case, the terms "administer" and "introduce" are used interchangeably and are used interchangeably herein. Agents or pharmaceutical compositions (e.g., antibodies, antibody reagents, or WISP1-binding fragments thereof) at a desired site, such as a site of infection or cancer, to produce the desired effect. and a method or pathway that results in at least partial localization of such an agent in a subject. The term "pharmaceutical composition" refers to the placement of an agent or pharmaceutical composition in a subject's body, either systemically or at a desired site. The composition can be administered to a subject by any mode of administration that delivers the composition to a surface or target. Examples of administration include, but are not limited to, injection, infusion, instillation, and inhalation. As long as the various agents can be protected from inactivation in the intestine, oral administration is possible. Forms of injection are also contemplated. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intravesical, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, These include intrathecal, intrathecal, intraspinal, intracerebrospinal, and intrasternal injections and infusions.

[0227] The agents (e.g., antibodies, antibody reagents, and WISP1-binding fragments thereof) are of good quality. Formulated, dosed, and administered to comply with good medical practice Factors to consider in this context include the particular disorder being treated, the specific the specific subject, the clinical condition of the individual subject, the cause of the disorder, the site of delivery of the agent, the method of administration, the schedule of administration These factors include the dosage schedule, the dosage regimen, and other factors known to the health care practitioner. A "therapeutically effective amount" of an agent is constrained by such considerations and is intended to be an effective amount for ameliorating, treating, or preventing cancer. to prolong progression-free survival; or to improve liver disease It refers to the minimum amount required to treat or prevent the onset or recurrence of a disease. For example, In some embodiments, the agent is optionally used to prevent or treat an infection. The drug is formulated with one or more additional therapeutic agents that are used. An effective amount of an agent is determined by the amount of the agent (e.g., antibodies and WISP1-binding fragments thereof) present in the formulation. The amount of the drug that is administered will depend on the type of disorder or treatment, and other factors discussed above. These are generally administered in the same dosages and by the same routes of administration as used herein above, and is used at approximately 1-99% of the dosage previously used.

[0228] Dosage As described herein, when treating liver disease, an agent (e.g., an antibody, an antibody reagent, or a WISP1-binding fragment thereof) can be administered in the form of a medicament as defined above. The type of liver disease to be treated, the severity and course of the disease, whether the agent is preventive or therapeutic whether the drug is being administered for a therapeutic purpose, previous therapeutic indications, the subject's clinical history and response to the agent The treatment will depend on the response of the patient to the drug, as well as the discretion of the attending physician. In combination therapy regimens, the agents described herein are preferably administered to a subject over a period of time. and the one or more additional therapeutic agents are administered in a therapeutically effective or synergistic amount.

[0229] As used herein, a "unit dosage form" refers to a suitable single dosage for administration. Thus, the unit dosage form may be a therapeutic agent that is placed in a delivery device, such as a syringe or an intravenous infusion bag. In one embodiment, the unit dosage form is administered in a single dose. In embodiments, more than one unit dosage form may be administered simultaneously.

[0230] Dosages of the agents described herein may be determined by a physician and, if necessary, monitored. The duration and frequency of treatment may be adjusted according to the therapeutic effect achieved. If so, we will continue to evaluate the efficacy and safety of the treatment to determine when it provides therapeutic benefit and to inject additional cells. Whether to administer treatment, discontinue treatment, restart treatment, or change the treatment regimen Typically, the subject is monitored to determine whether any changes in dosage occur. However, the amount should not be so large as to cause adverse side effects such as cytokine release syndrome. Generally, dosage will vary according to the age, condition, and sex of the patient, and will be understood by those skilled in the art. Dosage can be determined by the individual physician if any complications arise. It can also be adjusted.

[0231] Dosage ranges for therapeutic agents depend on potency and are large enough to produce the desired effect. Dosages should not be so large as to cause unacceptable adverse side effects. Generally, dosage will vary according to the age, condition, and sex of the patient. Dosage can be adjusted according to individual needs and if any complications arise. It can also be adjusted by a physician. In some embodiments, the dosage is 0.001 mg / kg body weight. In some embodiments, the dose range is from 5 μg / kg to 100 mg / kg body weight. g body weight to 100 μg / kg body weight. Alternatively, the dose range can be titrated to achieve serum levels of 1 In the case of systemic administration, the subject may be administered, for example, , 0.1mg / kg, 0.5mg / kg, 1.0mg / kg, 2.0mg / kg, 2.5 mg / kg, 5mg / kg, 7.5mg / kg, 10mg / kg, 15mg / kg, 20 mg / kg, 25mg / kg, 30mg / kg, 40mg / kg, 50mg / kg, and The doses may be administered in therapeutic amounts, such as one or more. can be administered in multiple separate doses or by continuous infusion. For repeated administration over a longer period, treatment may be administered according to the condition, e.g., as described above. or methods known in the art, until the liver disease is treated. However, other dosage regimens may be useful.

[0232] Combination Therapy In one embodiment, the agents or compositions described herein are used as monotherapy. In one embodiment, the agents described herein are administered in combination with other known agents and therapies for liver disease. As used herein, "administered in combination" refers to If so, two (or more) different treatments may be administered during the course of the subject's disorder. is delivered to a subject, e.g., the subject has been diagnosed with a disorder (liver disease). 2 after the treatment has been administered and before the disorder has been cured or resolved or treatment has been discontinued for other reasons. In some embodiments, the delivery of one or more therapies is , which is still occurring at the time delivery of the second treatment begins, thus overlapping in terms of duration of administration. This is sometimes referred to herein as "simultaneous" or "concurrent delivery." In other embodiments, the delivery of one treatment is terminated before the delivery of the other treatment is initiated. In some embodiments in either case, the treatment is more effective due to the combined administration. For example, the second treatment is more effective and, for example, less of the second treatment may be equally effective. or the second treatment is administered in the absence of the first treatment. The symptoms are reduced to a greater extent than was the case with the first treatment, or a similar situation is seen. In some embodiments, the result of the delivery is a reduction in symptoms or other parameters associated with the disorder. is greater than that observed with one treatment delivered in the absence of the other. The effects of the two treatments may be partially additive or fully additive. The result of the delivery may be that the effect of the first treatment delivered is greater than or equal to the effect of the second treatment. It may be that the two treatments are still detectable at the time they are delivered. The agent and at least one additional therapy may be administered simultaneously, in the same or separate compositions. In the case of sequential administration, the agents described herein can be administered in a single dose or sequentially. The agent may be administered first, followed by the additional agent, or the order of administration may be reversed. The agent and / or other therapeutic agent, procedure, or modality may be administered for the duration of the active disorder. The agent may be administered during periods of remission or less active disease. It may be administered prior to treatment, concomitantly with treatment, following treatment, or during remission of the disorder.

[0233] Therapeutic agents currently used to treat liver disease include, but are not limited to, , ursodeoxycholic acid (UDCA, ursodiol, INN, NAN, AAN, and (also known as USAN), cholestyramine, stanozolol, and naltrexone , rifampicin, pioglitazone, metformin, rosiglitazone, lobeglitazone, Retinol esters, vitamin A, liver dialysis, or liver transplantation, or any other known in the art In one embodiment, the agent described herein is any other treatment for a liver disease in which Alternatively, the composition is not administered in combination with another therapy. The agent or composition may be ursodeoxycholic acid (UDCA, ursodiol, INN, NA N, AAN, or USAN), cholestyramine, stanozolol , naltrexone, rifampicin, pioglitazone, metformin, rosiglitazone, lobeglitazone, retinol esters, vitamin A, liver dialysis, or liver transplantation, or It is not administered in conjunction with any other treatments for liver disease known in the art.

[0234] When administered in combination, the agent or composition and the additional agent (e.g., 2 or 3 agents) or all may include the amount or dosage of each agent used individually at an amount or dosage that is higher, lower, or the same as in monotherapy, for example In certain embodiments, the agent, an additional agent (e.g., a second or a third agent), or any dose or dosage may vary depending on each agent used individually. The amount or dosage of the drug is lower (e.g., at least 20%, at least 30%, In other embodiments, the desired effect (e.g., liver function) is achieved by at least 40%, or at least 50%. an agent, an additional agent (e.g., a second or third agent) that provides a therapeutic effect (treatment of a disease) or all amounts or dosages required individually to achieve the same therapeutic effect lower than the amount or dosage of each agent (e.g., at least 20%, at least 30%, At least 40%, or at least 50%).

[0235] Parenteral Dosage Forms Parenteral dosage forms of the agents described herein include, but are not limited to, subcutaneous, intravenous (IV), The drug may be administered to a subject by a variety of routes, including intramuscular (including intravenous injection), intramuscular, and intra-arterial. Administration of parenteral dosage forms typically bypasses the patient's natural defenses against contaminants. Therefore, parenteral dosage forms are preferably sterile or must be sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, injectable solutions. liquids, dry products that can be dissolved or suspended in a pharma- ceutically acceptable vehicle for injection, injectable Includes suspensions, controlled release parenteral dosage forms, and emulsions.

[0236] The terms "parenteral administration" and "parenterally administered" as used herein Refers to modes of administration other than enteral and topical administration, usually by injection. The terms "peripheral administration," "peripheral administration," and "peripherally administered" are used herein. When administered directly to a target site, tissue, or organ, such as a tumor site, refers to the administration of a therapeutic agent other than a vasodilator, such that the therapeutic agent enters the subject's circulatory system and is therefore metabolized and and other similar processes. In another embodiment, the agent is For example, it may be administered locally by direct injection, which may be repeated periodically.

[0237] Suitable vehicles that can be used to provide parenteral dosage forms of the present disclosure are known to those skilled in the art. Examples include, but are not limited to: sterile water; Water for injection; saline solution; glucose solution; sodium chloride injection injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, etc. and aqueous vehicles such as lactated Ringer's injection; water-miscible vehicles such as polyethylene glycol, and propylene glycol; In addition, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, and isopropyl myristate. and non-aqueous vehicles such as isopropyl, and benzyl benzoate.

[0238] The duration of therapy using the methods described herein may be as long as medically indicated or as long as necessary. The administration should be continued until a desired therapeutic effect (e.g., as described herein) is achieved. In certain embodiments, administration of the antibodies or antigen-binding fragments described herein is months, 2 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years for a period of one year, four years, five years, ten years, twenty years, or up to the number of years of the eligible person's life. This will continue.

[0239] Controlled and Delayed Release Dosage Forms In some embodiments of the aspects described herein, the agent or composition is administered by a controlled release means or Ideally, the drug will be administered to a subject by slow release or delayed release means. The use of a well-designed controlled release preparation treats the condition in the shortest possible time using the smallest possible amount of drug substance. The advantages of controlled release formulations include: These include: 1) prolonged activity of the drug; 2) reduced frequency of administration; 3) improved patient 4) reduced total drug use; 5) localized or reduced systemic side effects; 6) minimized drug accumulation; 7) reduced blood concentration 8) improved therapeutic efficacy; 9) enhanced or lost drug activity. and 10) improving the rate at which the disease or condition is controlled. Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lanc A controlled release formulation provides a controlled release formulation that provides a controlled onset of action, duration of action, and It can be used to control plasma levels within the therapeutic window, and peak blood concentrations. In particular, the use of controlled-release or extended-release dosage forms or formulations can help prevent underdosing of the drug (i.e. The potential side effects may result from both dosing below the minimum therapeutic level (below the minimum therapeutic level) and dosing above the toxic level of the drug. Maximum efficacy of the agent is achieved while minimizing potential adverse effects and safety concerns. This can be guaranteed.

[0240] Various known controlled or extended release dosage forms, formulations, and devices are described herein. The present invention can be adapted for use with any of the following agents, including but not limited to: See, but not limited to, U.S. Pat. Nos. 3,845,770 and 3,916,899. ;Specification No. 3,536,809;Specification No. 3,598,123;No. 4,008,71 Specification No. 9; Specification No. 5674,533; Specification No. 5,059,595; No. 5,59 Specification No. 1,767; Specification No. 5,120,548; Specification No. 5,073,543; Specification No. 5,639,476; Specification No. 5,354,556; No. 5,733,566 No. 6,365,185; and No. 6,365,185. Each of the above references is incorporated herein by reference in its entirety. Such dosage forms include, for example, Hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, immersion Osmotic pressure system (OROS® (Alza Corporation, Mountain View, CA) , California, USA), multi-layer coating, microparticles, liposomes, or macrophages. microspheres, or combinations thereof, to provide sustained or controlled release of one or more active ingredients. or controlled release can be provided at various rates to provide the desired release profile. In addition, ion exchange materials can be used to prepare immobilized adsorbed salt forms of the compounds of the present disclosure. and thus provide controlled delivery of the drug. Examples include, but are not limited to, DUOLITE® A568 and DU OLITE® AP143 (Rohm & Haas, Spring House, Pencil (Vanessa, USA).

[0241] efficacy For example, the efficacy of the agents described herein for treating liver disease can be determined by one of skill in the art. However, treatment, as that term is used herein, may be defined as: If one or more of the signs or symptoms of liver disease are modified in a beneficial manner, other clinical If the symptoms observed in the patient are improved or further improved, or if the desired response is For example, at least 0% induction after treatment with the methods described herein is considered to be "effective." Efficacy can be, for example, the efficacy of a condition treated according to the methods described herein. Markers, indicators, symptoms, and / or incidence rates, or any other suitable measurable by measuring important parameters such as fatigue, pain, weight loss, or dark urine. Efficacy can be assessed by the need for hospitalization or medical intervention (i.e., progression of symptoms). It can also be measured by the lack of deterioration of the individual as assessed by Methods for measuring such indicators are known to those of skill in the art and / or are described herein. It is described in.

[0242] Efficacy can be evaluated in animal models, e.g., mouse models, of the conditions described herein, or in In some cases, the efficacy and safety of the compound can be evaluated in appropriate animal models of liver disease. When using the drug, efficacy of treatment is evaluated by markers such as jaundice, fatigue, nausea, vomiting, urine color, and abdominal pain. It is clear when a statistically significant change is observed in the

[0243] The term "effective amount" as used herein refers to a person who has or is believed to have liver disease. A compound that can be administered to a diagnosed subject to alleviate at least one or more symptoms of the disease. "Treatment" refers to the amount of an agent or composition described herein that is required to achieve a therapeutic effect. The term "therapeutically effective amount" refers to a dose that provides a particular anti-liver disease effect when administered to a typical subject. Effective amount, as used herein, refers to an amount of an agent or composition sufficient to , delaying the onset of disease symptoms or modifying the course of disease symptoms in various circumstances; (e.g., slowing the progression of liver disease) or reversing symptoms of the disease (e.g., The composition will contain an amount of agent sufficient to cure or halt the symptoms of the disease. Thus, specifying an exact "effective amount" is generally not practical. Even in a given case, an appropriate "effective amount" can be determined by one of ordinary skill in the art using no more than routine experimentation. It can be determined.

[0244] In one embodiment, the agent or composition is administered continuously (e.g., at a constant rate over a period of time). Continuous administration of the agent can be achieved, for example, by epidermal patches, continuous release formulations, or Alternatively, this can be accomplished by an on-body injector.

[0245] In one embodiment, the agent or composition is administered at intervals (e.g., over a given period of time). The drug is administered at various levels depending on the dosage.

[0246] Effective doses, toxicity, and therapeutic efficacy are determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage can be assessed depending on the dosage form employed and the route of administration used. The dose ratio between toxic and therapeutic effects is the therapeutic index, LD50 / E D50 ratio. Compositions and methods that exhibit large therapeutic indices are preferred The therapeutically effective dose can be estimated initially from cell culture assays. The dose can be formulated in a suitable formulation and determined in cell culture or in an appropriate animal model. Circulating plasma concentrations containing the determined IC50 (i.e., the concentration of agent at which half-maximal inhibition of symptoms is achieved) A range of concentrations can be achieved. Plasma levels can be measured, for example, by high performance liquid chromatography. The effect of any particular dosage can be measured by a suitable bioassay, For example, it can be monitored by measuring liver function or blood tests, among others. Dosage can then be determined and adjusted, if necessary, to the observed therapeutic effect. Cut.

[0247] The invention provided herein can be further described in the following numbered paragraphs. do. 1. A method for treating or preventing liver disease, comprising administering to a subject in need thereof a WI A method comprising administering an antibody or antibody reagent that inhibits SP1. 2. Liver disease includes primary biliary cholangitis, autoimmune hepatitis, and alpha-1 antitrypsin deficiency. 1. The method according to claim 1, wherein the patient is selected from the group consisting of hepatitis C, nonalcoholic fatty liver disease, and scleroderma. Method of posting. 3. WISP1 is made up of WISP1v, WISP1vx, and WISP1 delta exon 3 to 4. method. 4. Antibodies or antibody reagents that inhibit WISP1 include mab1680, AF1680, and S Selected from the group consisting of AB2501114, ab60114, and ab65943 3. The method according to any one of paragraphs 1 to 3. 5. The amino acid sequence of the antibody or antibody reagent is SEQ ID NO: 1 to 4, 6, or 12 to 120. 5. The method according to any one of paragraphs 1 to 4, which has at least 70% homology to any one of Law. 6. The method according to any one of paragraphs 1 to 5, wherein WISP1 is inhibited in target cells. method. 7. The method of any one of paragraphs 1 to 6, wherein the target cell is a mammalian cell. 8. Any of paragraphs 1-7, wherein the target cells are hepatic stellate cells, fibroblasts, or myofibroblasts. The method according to any one of the above. 9. The method of any one of paragraphs 1-8, wherein the hepatic stellate cells are in a quiescent state. 10. The antibody or antibody reagent is administered by direct injection, subcutaneous injection, intramuscular injection, or intranasal administration. 10. The method of any one of paragraphs 1 to 9, wherein the 11. Inhibition of WISP1 is to inhibit WISP1 activity or to inhibit WISP 11. The method according to any one of paragraphs 1 to 10, wherein the method comprises reducing the level of a protein. . 12. WISP1 activity is at least 50% and at least 6% greater than appropriate controls. 0%, at least 70%, at least 80%, at least 90%, or greater 12. The method of any one of paragraphs 1 to 11, wherein the inhibition of 13. The level of WISP1 is at least 50% greater than that of an appropriate control, at least 60%, at least 70%, at least 80%, at least 90% or more 13. The method of any one of paragraphs 1 to 12, wherein the concentration of β-aminobutyric acid in the blood is significantly reduced. 14. A method for the treatment of WISP1 comprising administering to a subject a therapeutically acceptable carrier comprising administering to said subject an antibody or antibody reagent that inhibits WISP1 and a pharma- ceutically acceptable carrier. composition. 15. Antibodies or antibody reagents that inhibit WISP1 include mab1680, AF1680, SAB2501114, ab60114, and ab65943. The composition according to any one of paragraphs 1 to 14. 16. The amino acid sequence of the antibody or antibody reagent is selected from SEQ ID NOs: 1-4, 6, or 12-12 15. Any one of paragraphs 1 to 15 having at least 70% homology to any one of paragraphs 1 to 15. Composition of. 17. Any of paragraphs 1 to 16 that is formulated to treat or prevent liver disease The composition described in 1. 18. A method for treating liver disease in a subject, comprising: a. WISP1 and / or Yap, Col1a1, Act in a biological sample of a subject Detecting the level of a2, b. Comparing the measurement in (a) to a reference level; c. WISP1 and / or Yap, Col1a1 in (a) compared to reference levels Identifying subjects with increased Acta2 as having liver disease; and d. Administering an antibody or antibody reagent that inhibits WISP1 to a subject with liver disease. The method includes: 19. Paragraphs 1 through 18, further including obtaining a biological sample from the subject, prior to (a). The method according to any one of the preceding claims. 20. Liver diseases include primary biliary cholangitis, autoimmune hepatitis, and alpha-1 antitrypsin Any one of paragraphs 1 to 19, which is deficiency, nonalcoholic steatohepatitis, or scleroderma The method described above. 21. The biological sample is a blood sample, tissue, buffy coat, serum, or tissue; 21. The method according to any one of paragraphs 1 to 20. 22. Antibodies or antibody reagents that inhibit WISP1 include mab1680, AF1680, SAB2501114, ab60114, and ab65943. The method according to any one of paragraphs 1 to 21. 23. The amino acid sequence of the antibody or antibody reagent is selected from SEQ ID NOs: 1-4, 6, or 12-12. 22. Any one of paragraphs 1 to 22 having at least 70% homology to any one of paragraphs 1 to 22. How to. 24. A method for treating or preventing liver disease, comprising administering to a subject in need thereof a A method comprising administering an agent that inhibits ISP1. 25. Liver diseases include primary biliary cholangitis, autoimmune hepatitis, and alpha-1 antitrypsin Paragraphs 1 to 5, wherein the patient is selected from the group consisting of rheumatoid arthritis, nonalcoholic steatohepatitis, and scleroderma. 24. The method according to any one of claims 1 to 24. 26. WISP1 is divided into WISP1v, WISP1vx, and WISP1 Delta Exo. Any of paragraphs 1 to 25, wherein the splice variant is selected from the group consisting of splice variants 3 to 4. or one of the methods described above. 27. The method according to any one of paragraphs 1 to 26, wherein WISP1 is inhibited in target cells. Method of posting. 28. Agents that inhibit WISP1 include small molecules, antibodies or antibody reagents, peptides, and genomic DNA. The nucleic acid sequence is selected from the group consisting of a genome editing system, a viral vector, miRNA, and siRNA. 28. The method according to any one of paragraphs 1 to 27. 29. The microRNA is microRNA15a or miRNA412, 29. The method according to any one of 1 to 28. 30. Antibodies or antibody reagents that inhibit WISP1 include mab1680, AF1680, SAB2501114, ab60114, and ab65943. 30. The method according to any one of paragraphs 1 to 29. 31. The amino acid sequence of the antibody or antibody reagent is selected from SEQ ID NOs: 1-4, 6, or 12-12. 30. Any one of paragraphs 1 to 30 having at least 70% homology to any one of paragraphs 1 to 30. How to. 32. The agent is administered by direct injection, subcutaneous injection, intramuscular injection, or intranasal administration; 32. The method according to any one of paragraphs 1 to 31. 33. Inhibition of WISP1 is to inhibit WISP1 activity or to inhibit WISP 33. The method according to any one of paragraphs 1 to 32, wherein the method comprises reducing the level of a protein. . 34. WISP1 activity is at least 50% and at least 6% greater than appropriate controls. 0%, at least 70%, at least 80%, at least 90%, or greater 34. The method of any one of paragraphs 1 to 33, wherein the inhibition of 35. The level of WISP1 is at least 50% greater than that of an appropriate control, 60%, at least 70%, at least 80%, at least 90% or more 35. The method of any one of paragraphs 1 to 34, wherein the concentration of 36. A composition comprising an agent that inhibits WISP1 and a pharma- ceutically acceptable carrier. 37. Agents that inhibit WISP1 include small molecules, antibodies or antibody reagents, peptides, and genomic DNA. The nucleic acid sequence is selected from the group consisting of a genome editing system, a viral vector, miRNA, and siRNA. 37. The composition according to any one of paragraphs 1 to 36. 38. The microRNA is microRNA15a or miRNA412, The composition according to any one of 1 to 37. 39. A method for treating liver disease in a subject, comprising: a. WISP1 and / or Yap, Col1a1, Act in a biological sample of a subject Detecting the level of a2, b. Comparing the measurement in (a) to a reference level; c. WISP1 and / or Yap, Col1a1 in (a) compared to reference levels Identifying subjects with increased Acta2 as having liver disease; and d. Administering an agent that inhibits WISP1 to a subject with liver disease. The method includes: 40. Before (a), paragraphs 1 to 39, further including obtaining a biological sample from the subject The method according to any one of the preceding claims. 41. Liver diseases include primary biliary cholangitis, autoimmune hepatitis, and alpha-1 antitrypsin Any one of paragraphs 1 to 40, which is deficiency, nonalcoholic steatohepatitis, or scleroderma The method described above. 42. The biological sample is a blood sample, tissue, buffy coat, serum, or tissue; 42. The method according to any one of paragraphs 1 to 41. 43. Agents that inhibit WISP1 include small molecules, antibodies or antibody reagents, peptides, and genomic DNA. The nucleic acid sequence is selected from the group consisting of a genome editing system, a viral vector, miRNA, and siRNA. 43. The method according to any one of paragraphs 1 to 42. 44. The microRNA is microRNA15a or miRNA412, 44. The method according to any one of 1 to 43. 45. Antibodies or antibody reagents that inhibit WISP1 include mab1680, AF1680, SAB2501114, ab60114, and ab65943. The method according to any one of paragraphs 1 to 44. 46. ​​The amino acid sequence of the antibody or antibody reagent is SEQ ID NO: 1-4, 6, or 12-12 45. Any one of paragraphs 1 to 45 having at least 70% homology to any one of paragraphs 1 to 45. Method of posting. 47. Genetically engineered hepatic stellate cells or cell populations expressing an agent that inhibits WISP1 A method for generating a population of WISP1-associated proteins comprising contacting a cell with an agent that inhibits WISP1. and culturing the cells for a sufficient time to allow expression of the agent. How to do it. 48. The method of any one of paragraphs 1 to 47, wherein the cells are in a quiescent state. 49. Contacting includes contacting the cell with an agent or a vector encoding the agent. 49. The method of any one of paragraphs 1 to 48, comprising: 50. Contact can be transduction, nucleofection, electroporation, or direct injection. and / or transfection, Law. 51. Agents that inhibit WISP1 include small molecules, antibodies or antibody reagents, peptides, and genomic DNA. The nucleic acid sequence is selected from the group consisting of a genome editing system, a viral vector, miRNA, and siRNA. 51. The method according to any one of paragraphs 1 to 50. 52. The microRNA is microRNA15a or miRNA412, 1. The method according to any one of 1 to 51. 53. Antibodies or antibody reagents that inhibit WISP1 include mab1680, AF1680, SAB2501114, ab60114, and ab65943. The method according to any one of paragraphs 1 to 52. 54. The amino acid sequence of the antibody or antibody reagent is SEQ ID NO: 1-4, 6, or 12-12 53. Any one of paragraphs 1 to 53 having at least 70% homology to any one of paragraphs 1 to 53. Method of posting. 55. Cells containing hepatic stellate cells produced by the method according to any one of paragraphs 46 to 53. Cell line. 56. Hepatic stellate cells or a method thereof produced by the method according to any one of paragraphs 1 to 55. A pharmaceutical composition comprising the population and a pharma- ceutically acceptable carrier. 57. A method for treating or preventing liver disease, comprising administering to a subject in need thereof, A cell produced by the method according to any one of paragraphs 1 to 56. or the pharmaceutical composition according to any one of paragraphs 1 to 56. and 58. A method for reducing fibrosis in a subject, comprising administering to a subject in need thereof a therapeutic agent comprising administering to said subject ... 57. A cell produced by the method according to any one of paragraphs 1 to 57. or the pharmaceutical composition according to any one of paragraphs 1 to 57. Methods including: 59. A method for treating liver disease in a subject, comprising: a. WISP1 and / or Yap, Col1a1, Acta compared to reference levels receiving results of an assay that identifies a subject having an increased IL-2 as having liver disease; and To b. Administering an antibody or antibody reagent that inhibits WISP1 to a subject with liver disease. The method includes: 60. A method for treating liver disease in a subject, comprising: a. WISP1 and / or Yap, Col1a1, Acta compared to reference levels receiving results of an assay that identifies a subject having an increased IL-2 as having liver disease; and To b. Administering to a subject having liver disease an agent that inhibits WISP1. The method includes: EXAMPLES

[0248] [Example 1] Treatment for progressive liver disease In response to an unmet need for liver disease treatment, the present invention provides a method for treating liver disease comprising administering to the liver In activated hepatic stellate cells (HSCs), a cell type that plays a central role in the progression of fibrosis in MicroRNAs miR-15a and miR-412 that independently induce quiescence are described Furthermore, miR-15a inhibits its profibrogenic function in activated HSCs. Using microRNAs and their targets to directly target WISP1 By promoting HSC quiescence, microRNAs mediate progressive hepatic apoptosis in PBC Without being bound by any particular theory, , miR-15a and miR-412 induce quiescence in HSCs, and then miR It is predicted that this will enable elucidation of the function of WISP1, a known target of -15a.

[0249] The studies described herein demonstrated that miR-15a or miR-412-induced Quiescent-like HSCs ameliorated liver injury and fibrosis in CCl4-challenged mice The results show that miR-15a and miR-412 in the CCl4 model Given the beneficial effects of , mouse treatment studies have been conducted in a cholestatic fibrosis model, bile duct ligation (BIL). DL) and 3,5-diethoxycarbonyl-1,4-dihydrocollidine (dihydrocholl Three different miRNA delivery systems were tested: Obtain: 1) Use quiescent-like HSCs that constitutively express miR-15a or miR-412 2) cell therapy to induce miR-15a or miR-412 expression in the tail vein and 3) injecting miR-15a or miR-15b packaged in a lipid-based carrier. or a chemically modified mimic of miR-412 was injected into the tail vein. The mice may be used to analyze the results.

[0250] The studies described herein demonstrate that MiR-15a exerts its profibrogenic function in HSCs. We show that WISP1 can be directly targeted to block BDL and DDC In a challenge setting, the liver phenotype of WISP1 null mice can be defined. The severity of liver injury and HSC dysfunction in mice will be assessed and compared with that in wild-type mice. Mouse models of cholestatic fibrosis may be treated with WISP1 blocking antibodies and then This can be used to analyze the results.

[0251] Primary biliary cholangitis (PBC), formerly known as primary biliary cirrhosis, is a condition caused by the Leading to progressive destruction of the bile ducts, cholestasis, periportal inflammation and ultimately biliary fibrosis It is an insidious liver disease that can lead to cirrhotic end-stage liver disease. Both genetic and environmental factors are involved. Autoimmune diseases with unclear pathogenesis, thought to interact to cause disease Interestingly, PBC is more prevalent in men than women, with a male to female ratio of 9:1. It affects women more than men. The age-standardized incidence of PBC per million people per year in the United States is The incidence rate is 45 in women and 7 in men, and the prevalence rate per million is 654 in women and 7 in men. There were 121 men.[1]

[0252] The hydrophilic bile salt ursodeoxycholic acid (UDCA) still has no survival benefit in PBC. It is the only drug with a proven effect. Improvement in histological and biochemical parameters was observed. It has been shown to slow disease progression; its exact mechanism of action is unknown. However, it is believed to protect bile duct cells against toxic bile salts. Thirty to forty percent of CA-treated individuals still experience disease progression in the setting of a suboptimal response.[2] A number of different agents are under clinical trial to fill this treatment gap. Some of the well-known experimental drugs, such as obeticholic acid, have significant side effects and are not known to have any effect on life extension. There is a lack of evidence.[3]

[0253] Like most chronic liver diseases, PBC progresses from inflammation to fibrosis. During the process, hepatic stellate cells (HSCs) are thought to play a central role in liver fibrosis. HSCs exist in two forms: in healthy individuals, they are quiescent and In the activated state, the retinal cells are characterized by multiple retinoid-rich lipid droplets. Upon exposure to fibrosis, they lose their lipid droplets and become pro-fibrotic myofibroblasts, producing collagen and HSCs secrete mediators that promote hepatic fibrosis and scar formation [7, 8]. Although the importance of is well established, much remains unknown about this cell type. is.

[0254] One of the multiple gaps in our knowledge of HSCs is determining their activation state. The current understanding of the role of microRNAs (miRNAs) in They are non-coding genes, usually 22 nucleotides in length, that play a key role in all biological and pathological processes. They are involved in physiological processes that induce degradation or translation inhibition of those mRNAs. Downregulation of specific coding genes by imperfect base pairing with complementary sequences within the NA target Each miRNA can regulate many different coding genes, while each target gene is involved in the regulation of the genome. It can be regulated by many different miRNAs that constitute a complex layer of gene regulatory networks. [9] The experiments described herein demonstrate that miRNAs play a role in cell proliferation and reprogramming. [10-12]. Nevertheless, HSC activation or quiescence The role of miRNAs in reverting to the normal state has not been fully investigated.

[0255] Although the group characterized the global miRNA expression patterns in HSCs, However, the functional significance of their expression status remains largely unknown [13–15]. Instead of relying on large-scale expression profiling, functional screening is a promising approach to identify activating H This effort was developed to systematically identify miRNAs that induce SCs to return to a quiescent state. We identified miR-15a and miR-412 through co-localization. R-412 has not previously been studied in the context of hepatic stellate cells. However, miR-1 Family 5 inhibits the Tgf beta pathway in the heart to potentially attenuate fibrosis

[16] It was found that these miRNAs or their downstream targets Promoting HSC quiescence can prevent the progression of fibrosis in PBC. The aim of this study was to investigate the role of miR-15a, miR-412 and their direct targets in the treatment of PBC. The objective of this study is to evaluate the effectiveness of treatment in

[0256] result The unbiased functional screen described herein allows for the identification of microbial pathogens that grow on plastic surfaces. This takes advantage of the innate tendency of HSCs to become activated when they They lose lipid droplets, which are abundant in quiescent HSCs. Using this useful phenotypic dichotomy, miRNAs that make activated HSCs more quiescent lead to lipid droplet reformation in the cytoplasm The researchers explored whether miRNAs play a role in organ development, carcinogenesis, or cell proliferation by tracking the levels of these genes. Simultaneous expression of multiple coding genes that affect the entire cellular program, such as cell reprogramming. It is well known that even a single miRNA can mediate transdifferentiation, as it is known that miRNAs can sometimes be targeted

[17] . It was hoped that it would be possible to induce

[0257] To identify these miRNAs that promote the reversion of activated HSCs to a quiescent state, we After activated HSCs received individual miRNA mimics derived from a genome-wide miRNA library Next, we scanned for the reappearance of intracellular lipid droplets containing retinoids as a marker of quiescence ( In fact, HSCs in some wells were reconstituted within 3 days and stained positive for BODIPY. This initial screen revealed 15 primary hits. For the study, the miRNAs miR-15a and miR-412 were selected based on the presence of their human orthologues. These were selected based on their localization and their ability to repopulate lipid droplets in both mouse and human HSCs. The newly formed lipid droplets were UV-induced, consistent with those in quiescent HSCs. The results were positive for retinoids, as evidenced by fluorescence under UV light (data not shown). The overall size of the transfected HSCs was reduced by 10-100 fold and they became quiescent. Furthermore, forced expression of miR-15a or miR-412 inhibited the activity of α-smooth muscle actin (Acta2) and α-type I collagen (Col 1 a1) down-regulated the two most important gene markers (Figure 4).

[0258] For more comprehensive expression analysis, deep RNA sequencing was used to identify miR-15a or miR-412-received quiescent-like HSCs were 40–50% closer to quiescent HSCs than activated cells. We demonstrated that the IL-16 gene had a global transcriptional profile that was significantly different from that of the control (data not shown). HSCs that were rendered quiescent by miR-15a or miR-412 in the first place were considered to be true quiescent HSCs. The ex vivo experiments demonstrated that quiescent-like HSCs were able to express healthy Demonstration that the two cell types did not induce steatosis in hepatocytes when co-cultured In contrast, activated HSCs that were not treated with the candidate miRNAs showed that they When cultured, it induced hepatocyte steatosis (Figure 5). These revertant HSCs were derived from co-cultured human hepatocellular carcinoma cell lines HepG2 and Huh7. These results suggest that miR-1 could reduce the expression of inflammatory cytokines (Figure 6). As expected, endogenous miR-15a was not significantly different from miR-15a (Figure 7). and miR-412 have decreased expression levels in activated HSCs compared with quiescent HSCs miR-412 converts activated HSCs into a quiescent state under activation-promoting culture conditions. This and other important ex vivo observations are Traditional CCl4 mouse model (oral gavage 100 ul, 40% CCl4 twice a week) ) to attenuate the levels of liver pathology in vivo. Whether or not it can be delivered, specifically expressing either miR-15a or miR-412 Can HSCs maintain a quiescence-like state even in diseased livers that contain activation-promoting signals? This allowed us to examine whether staphylococci are involved in the regulation of inflammatory bowel disease through cell contact and soluble mediators. Inhibitory-like HSCs mediate the expression of other signaling pathways in the liver that attenuate those signals that promote inflammation or fibrosis. can induce the following cell types:

[0259] There are various methods to deliver miRNA to live mice, including injection. The HSCs selected for this purpose constitutively expressed miR-15a or miR-412. The reprogrammed HSCs were then cultured in a quiescent state ex vivo for 4 weeks. The HSCs were injected once at week 3 of CCl4 challenge. The results were confirmed by visualization in hepatocytes that emitted GFP signals and expressed miRNAs. We inserted the ggyBac vector

[18] (Figure 8). More importantly, we identified quiescent-like HSCs Mice treated with α-aminobutyric acid showed increased hepatic ballooning, apoptosis, inflammation and fibrosis. The mice had reduced hepatic inflammation (Figure 8) as well as histology showing reduced hepatic collagen expression (Figure 9).

[0260] These two miRNA candidates downregulate HSC genes that promote liver inflammation and fibrosis. Many of the genes that are blocked by miR-15a and miR-412 are thought to regulate Although there are probably other genes, WISP1 is an experimentally validated direct target of miR-15a. Activated HSCs express WISP1 at approximately 30-fold higher levels than quiescent HSCs (data not shown). (not shown), HSCs in human PBCs express WISP1 more abundantly than those in normal liver. Finally, miR-15a contained two potential sites within its 3'-UTR. It is predicted that miR-15a targets WISP1, which contains the miR-15a binding sequence. Constitutively active luciferase reporters that encode putative target sequences or their mutant variants One of these reporters was cloned after the miR-15a mimic. When cotransfected with luciferase, the reporter containing the wild-type sequence miR-15a reduced both enzyme expression, whereas the reporter containing the mutant sequence did not. We have shown that WISP1 binds to the target sequence (Figure 11). It is an important target gene for miR-15a to achieve part of its action in this regard.

[0261] Examination of miR-15a and miR-412 in a mouse model of cholangitis Delivering miR-15a or miR-412 is a treatment for PBC: In view of the surprising results described herein, the use of miR-15a and We extended our in vivo experiments to further evaluate the potential of miR-412 and These miRNAs may be involved in the regulation of HSC function alone or more broadly in other cell types. To assess whether they exert their functions, miRNAs are being used as a virological agent instead of cell therapy. All in vivo treatments can be delivered systemically using vectors or mimetics. Regarding the experiment, the efficacy was evaluated by performing PCR of profibrotic genes in the liver, H Histological examination including staining with E and Sirius red, liver hydroxyproline assay, etc. and plasma ALT and alkaline phosphatase measurements. obtain.

[0262] Each model of PBC has its strengths and weaknesses in representing human PBC. We investigated miRNA candidates and inhibition of WISP1 that may result in reduced fibrosis and inflammation. To investigate the pathogenesis of PBC, a model that steadily progresses to bile duct fibrosis at a relatively early stage is required. Most genetic models of hepatitis do not develop liver fibrosis or develop it very slowly.

[19] For this reason, surgically induced bile duct ligation (BDL) and 3,5-diethoxyquin Chemically induced fibrillation by dicarbonyl-1,4-dihydrocollidine (DDC) Two cholangitis models that result in significant levels of cholestatic fibrosis are available. All experiments were performed at 3 weeks for the BDL model due to the time required for onset of symptoms. The DDC model can then be terminated after 4 weeks [20-22].

[0263] Using mesenchymal stem cells and macrophages, several groups have demonstrated that hepatic fibrosis Cell therapy has already been attempted with some success.

[23] However, the experimental will be the first to utilize intentionally genetically engineered HSCs to prevent liver fibrosis. Although the in vivo results of treatment with cholangitis are compelling, the efficacy of this cell therapy in a mouse model of cholangitis remains unclear. Examining the effect of the method is important considering that only the CCl4 model has been examined so far. For all cell therapy experiments, injections are made into the spleen to obtain quiescent-like HSCs. 500,000 were reprogrammed with miR-15a or miR-412 All appropriate controls, e.g., untreated and unreprogrammed A control group treated with fresh HSCs may also be included.

[0264] Mice challenged with BDL for 21 days typically develop significant fibrosis. Reprogrammed HSCs were cultured at 7 and 14 days during the 21-day course of BDL challenge. All mice are sacrificed on day 21. Mice challenged with the reprogrammed H SC can be injected on days 14 and 21 during the 28 day course of DDC challenge. All mice are sacrificed on day 28.

[0265] The first experiment involved H cells reprogrammed with miR-15a or miR-412. SC injection significantly improved collagen expression and overall liver injury in CC14-induced liver fibrosis. The researchers demonstrated that delivering miRNA via cell therapy can reduce systemic levels of miRNA. has shown promise, but other delivery methods should be explored. The first step is to inject lentiviruses expressing miRNAs. miR-15a or miR-15a on days 7 and 14 during the 21-day course of L challenge 412 can be expressed in the tail vein. All mice are sacrificed on day 21. The virus expressed miR-1 on days 14 and 21 during the 28-day course of DDC challenge. All mice were sacrificed on day 28. will be done.

[0266] An alternative delivery method is a lipid-based carrier (MaxSuppressor™ by B More stable, chemically modified IgGs packaged in 100 Scientific Injection of miRNA mimics (Exiqon and Invitrogen) The feasibility can be examined [24, 25]. By avoiding lentiviruses, this The method or variations thereof have the potential for use in humans. The expression of miR-15a or miR-412 was assessed on days 7 and 14 of a 21-day course of All mice are sacrificed on day 21. The body was cultured to express miR-15a and miR-21 at days 14 and 21 during the 28-day course of DDC challenge. All mice were sacrificed on day 28. Be killed.

[0267] Because it is difficult to induce liver fibrosis in female mice, male mice were used in this study. It is further useful for the in vivo treatment studies described in the specification. The alpha 1 type I collagen level was calculated using the α-β ... A 50% reduction in was measured at a p-value of 0.01 and 90% power. It has been shown that the method can be used to detect miRNA with 0.0001% standard deviation. One of the biggest challenges in HSC treatment is to minimize off-target effects. The goal is to develop a delivery system that specifically targets this cell type. For example, two promising HSC delivery systems have been developed recently: p7 5 Neurotrophin Receptor Peptide (p75NTRp)-Tagged Adenovirus and A The AV6 vector [26, 27].

[0268] Evaluating WISP1 function in a mouse model of cholangitis MiR-15a targets Wispl to promote inflammation and fibrosis in PBC:WI SP1 is a transcription factor that regulates the transcription of Cyr61 (Ccn1), Ctgf (Ccn2), Nov (Ccn3), and WI. Matrix including SP1(Ccn4), Wisp2(Ccn5) and Wisp3(Ccn6) Ctgf is a member of the Ccn family of hepatocyte proteins. It has already been established as an important profibrotic factor

[28] .

[0269] Interestingly, WISP1 is upregulated in human idiopathic pulmonary fibrosis. was found, and bleomycin-challenged mice were treated with a neutralizing WISP1 antibody. In addition, small animal studies have shown that blocking WISP1 resulted in attenuation of pulmonary fibrosis. showed that blocking CCl4-induced liver fibrosis was reversed

[30] . The role of WISP1 in PBC has not been studied in detail, particularly in the context of PBC.

[0270] WISP1 is a direct target of miR-15a, but its effects on HSC activation status and The role of WISP1 in bone formation and in the pathogenesis of PBC remains unclear. This was generated to demonstrate the role of WISP1 in the liver, but its function in the liver has not been studied.

[31] These mice were fertile and had no overt liver phenotype. These mutant alleles were obtained from the University of Tokyo, which ensures good mutant mouse deriving. Frozen sperm and embryos at the MMRRC facility in Davis, California is stored inside.

[0271] The phenotype can be assessed by the overall severity of liver pathology. WISP1 null mice , can be challenged with BDL and DDC. The resulting liver damage can be compared to that of wild-type mice. Given the well-known profibrogenic role of Wispl, WISP1 null mice show reduced hepatic fibrosis These results are predicted to have a reduction in blood glucose levels and possibly a reduction in inflammation. For the study, liver phenotype was assessed by PCR, H&E and scintigraphy of profibrotic genes in the liver. Histological examination including Ruth red staining, liver hydroxyproline assay and plasma A This can be assessed by carrying out measurements of LT and alkaline phosphatase. The functional phenotype of HSCs harvested from these mouse models of biliary fibrosis was assessed. Male wild-type and WISP1 null mice can be challenged with BDL. They can be sacrificed on day 21. Male wild-type and WISP1 null mice were cultured at 100-200°C for 12-3 days after DDC. They can be sacrificed on day 28.

[0272] HSCs were then challenged with BDL for 21 days and DDC for 28 days. These HSCs can be isolated from mice and then analyzed to see whether they are capable of inducing inflammatory mediators. The results are shown in Table 1. These results can be compared to those of wild-type HSCs.

[0273] Blocking WISP1 with a neutralizing antibody abrogates bleomycin-induced

[29] . Similarly, blocking Wispl inhibits fibrosis in PBC. The mouse model of cholangitis was able to reduce the levels of inflammation and fibrosis caused by W They can be treated with ISP1 antibody and assessed for liver phenotype.

[0274] Mice challenged with BDL for 21 days typically develop significant fibrosis. The treatment group was administered a commercially available WISP that was previously used to treat bleomycin-induced pulmonary fibrosis. 1 antibody on days 7 and 14 (R&D Systems)

[29] . A control group can receive IgG. All mice can be sacrificed on day 21. Mice challenged with DDC for 28 days typically develop significant fibrosis. ISP1 blocking antibodies were administered on days 14 and 21 during the 28-day course of DDC challenge. All mice will be sacrificed on day 28.

[0275] As described herein, WISP1 is a potent regulator of inflammation and fibrosis in vivo. Inflammatory and profibrotic mediators that can be neutralized with antibodies to reduce their levels Therefore, blocking WISP1 may also reverse bile duct fibrosis. Therefore, by analyzing the conditioned medium of activated HSCs, Secreted paracrine or autocrine mediators can be identified. Further detailed analysis of the culture medium will allow for mass spectrometry to identify other mediators that are drug targets. It can be analyzed through analysis.

[0276] References Example 1:

[0277] [Table 2-1]

[0278] [Table 2-2]

[0279] [Table 2-3]

[0280] [Example 2] Treatments for Nonalcoholic Fatty Liver Disease (NAFLD) Nonalcoholic fatty liver disease (NAFLD) is expected to become an endemic disease in developed countries over the next 5 to 10 years. It may be the most common cause of advanced liver disease, liver transplants and hepatocellular carcinoma. In the absence of approved medications, management relies on reducing risk factors that may lead to NAFLD. Therefore, developing effective treatments for NAFLD is of utmost importance. It is essential.

[0281] Activated hepatic stellate cells (HSCs) have been shown to play a central role in the progression of liver fibrosis. It is well known and the data presented herein support the conclusion that the microRNAs miR-15a and We demonstrate that miR-412 and miR-413 independently revert activated HSCs to a quiescent state. miR-15a blocks its pro-adipogenic and pro-inflammatory functions in activated HSCs Without being bound by any particular theory, miR-15a directly targets WISP1. and miR-412, which in turn can suppress fibrosis progression in NAFLD. It was hypothesized that this would promote quiescence.

[0282] With the advent of highly effective antiviral treatments for hepatitis B and C, non-alcoholic Non-fatty liver disease (NAFLD) has become the most serious liver disorder. It is considered the most common liver disease in the developed world [1], and by 2025, It is predicted that this disease will become the leading cause of liver disease, liver transplantation, and hepatocellular carcinoma [2, 3]. 64 million people are affected due to the high prevalence of risk factors including diabetes and hyperlipidemia. This is especially prevalent in wealthy countries such as the United States, where it is estimated that 100,000 people are killed in the 2010–2011 nuclear accident. The economic burden of LD is enormous, estimated at approximately $103 billion in annual direct medical costs. [4] The disease begins as fatty liver, but can progress to nonalcoholic steatohepatitis (NASH), This may progress to fibrosis and ultimately to cirrhotic liver failure or hepatocellular carcinoma. Currently, cancer is primarily managed by reducing risk factors. Dans are often difficult to achieve, and even excluding them does not guarantee improvement. [5] NAFLD has become the most common cause of liver-related morbidity and is currently being managed by Given the lack of drugs that can treat this condition, better treatment options are needed. This is the first step towards developing novel treatments for NAFLD.

[0283] NAFLD progresses in a stepwise manner through steatosis, inflammation and fibrosis. During the metabolic cascade, hepatic stellate cells (HSCs) play a central role in liver fibrosis. It is believed that HSCs exist in two forms. In healthy individuals, they are They are quiescent and characterized by multiple retinoid-rich lipid droplets (Figure 12). However, upon activation, they lose lipid droplets and become profibrotic myofibroblasts. They secrete mediators that promote collagen and scar formation (Figure 12) [9, 10]. .

[0284] The importance of HSCs in liver fibrosis is well established, but little is known about this cell type. For example, there are no genes that can revert activated HSCs to a quiescent state. HSCs are also involved in early NAFLD as well as fibrosis, promoting steatosis and inflammation. If HSCs contribute to steatohepatitis or fibrosis, the key factors involved in these processes What are the key mediators? Answering these questions and gaining better biological knowledge will This may provide new ideas for managing NAFLD.

[0285] One of the many gaps in current knowledge is the role of microRNAs in determining HSC activation status. The role of miRNAs in HSCs is particularly poorly understood. We profiled the global miRNA expression patterns in the human retinal sarcoma and the functional significance of their expression status. The significance of miRNAs remains largely unclear [11–13]. Accordingly, studies have focused on the function of miRNAs. We first focused on determining the key miRNAs that influence the activation state of HSCs. Instead of relying on large-scale expression profiling, we used functional screening and To systematically identify miRNAs that induce activated HSCs to revert to a quiescent state miR-15a and miR-412 were identified through this effort. Promoting HSC quiescence through these miRNAs or their downstream targets may be a promising strategy for N It can prevent the progression of fibrosis in AFLD.

[0286] Finally, activated HSCs that cause fibrosis are not unique to NAFLD. Liver disease involves HSCs that promote fibrosis for several years before reaching cirrhosis. Therefore, if a therapeutic agent could slow the rate of fibrosis progression by targeting HSCs, NA This approach can be used in other liver diseases besides FLD. The potential impact is enormous. Patients with primary biliary cirrhosis or autoimmune disease with established medical therapy may be at increased risk of developing pulmonary tuberculosis. Even for diseases such as immune hepatitis, additional agents that inhibit fibrosis progression are needed to This can significantly improve the ability to manage chronic diseases. In view of both the central role that HSCs play in the progression of fibrosis, The significance of the research being carried out is far-reaching.

[0287] Several compounds are in clinical trials for the treatment of NAFLD, with the primary goal being None of this is by preventing or reversing HSC activation [5, 14, 15]. The first major innovation was the use of Hb as a way to prevent progressive liver injury and fibrosis. The in vivo study described herein began with an attempt to control the activation state of SCs. In tro screening has demonstrated the viability of HSCs that are activated when grown on plastic surfaces. When HSCs are activated, they are abundant in quiescent HSCs. This useful phenotypic dichotomy can be used to further characterize activated HSCs. Furthermore, we tracked the levels of lipid droplets reformed in the cytoplasm by expressing quiescent miRNAs. We explored whether miRNAs play important roles in cellular regulation, such as organ development, carcinogenesis, or cell reprogramming. Simultaneous targeting of multiple coding genes to affect the entire cellular program is currently being investigated. Given that it is known that even a single miRNA can induce this transdifferentiation,

[16] Indeed, through unbiased investigations, as demonstrated by the reappearance of intracellular lipid droplets, Several miRNA candidates that may revert HSCs from an activated state to a quiescent state were identified. A subset of these miRNAs was identified that correlated with gene expression patterns, proliferation rate, and and other phenotypic changes toward HSC quiescence also occurred, including effects on nearby hepatocytes. This innovation, utilizing miRNAs to manage HSC activation status, may help prevent liver fibrosis. This could serve as the basis for developing an entire category of treatments to prevent disease.

[0288] The second major innovation comes in the form of a novel cell therapy that uses quiescent-like HSCs. The lean and follow-up in vitro assays were performed using miR-15a or miR- Transient transfection with a mimic of 412 was performed, but no significant results were observed in HSCs. PiggyBac transfection can be integrated into the genome to constitutively express miRNAs. When splice vectors were then used

[17] , they were able to differentiate into morphology, expression and function. The cells were thought to undergo sustained transdifferentiation into a quiescent-like state accompanied by dramatic changes in These reprogrammed HSCs were then challenged with 0014. When injected into the liver, they engrafted in the liver and ameliorated the levels of liver injury and fibrosis. Other groups have attempted cell therapy for cirrhosis using mesenchymal stem cells or macrophages. However, genetically engineered HSCs have been used as a treatment for liver fibrosis or NASH

[18] . This is the first time it has been used.

[0289] The main recent innovations are technical in nature. HSCs have been shown to reduce liver fibrosis in NASH. If the main drivers are already known, it is possible to subvert their effects. However, this effort faces a fundamental challenge: how to investigate HSC function and mechanisms of action? One of the biggest obstacles is the lack of easy-to-use assays to assess functional phenotypes and mechanisms of action. Allows for good in vitro assays of HSC interactions with other cell types To address this need, we developed primary mouse HSC and hepatocyte models. A co-culture system using IL-1 and IL-2 reconstitutes the liver microenvironment in close proximity of these two cell types. Furthermore, by combining this technology with a mouse model of NASH, Thus, the effect of NASH-HSCs on nearby liver cells can be reliably measured. Choline-deficient L-amino acid-limited high-fat diet-derived NASH-HSC (CDAHFD model) (Figure 13) induced steatosis in co-cultured hepatocytes from healthy mice and increased inflammatory cytokines. This signaling was observed in the co-culture. In this study, two cell types could be separated by a transwell, which requires cell-cell contact. It does not require hepatocyte differentiation, but refers to the secretion of soluble factor(s) by HSCs. The induction of fat accumulation was observed when conditioned medium from cultured NASH-HSCs was co-cultured. Even when applied to normal hepatocytes, the results could be reproduced (Figure 16). , an HSC-secreted mediator that induces steatosis and inflammatory cytokines in hepatocytes This allows for the identification of HS in NAFLD using a simple but robust ex vivo system. Mechanistic and drug discovery experiments by modeling the interactions between C and hepatocytes Techniques have been developed to accommodate this, particularly the use of primary cells instead of cell lines. This co-culture system allows us to characterize the pathophysiological microenvironment of these two cell types in vivo.

[20] Co-culture systems allow these cells to be harvested and cultured in a culture vessel to more accurately simulate the environment in which they grow. This can be done by improving existing protocols for These technical enhancements are the basis for the development of novel cellular markers that maximize quality while minimizing costs. These methods are useful for publishing consistent and reproducible results.

[0290] The mechanism and extent by which miR-15a and miR-412 affect HSC activation definition Hepatic stellate cells (HSCs) make up only 5% to 15% of all cells in the liver, but they are the most abundant cells in the liver. The profound profibrotic effects of IL-1 following activation in chronic liver disease are well recognized. Several studies have demonstrated differential m in activated versus quiescent HSCs. Although we have demonstrated iRNA expression [11-13], the function of miRNAs during the activation process has not been The role of miRNAs in reverting activated HSCs back to quiescence remains largely unknown. have identified a poorly understood role for HSCs in the resolution of liver inflammation and fibrosis. This is a possible fate [9, 25].

[0291] To identify those miRNAs that promote reversion of activated HSCs toward quiescence In this study, activated HSCs were treated with individual miRNA mimics derived from a genome-wide miRNA library. After the first 1 h of incubation, we performed unbiased analyses to look for the reappearance of retinoid-containing intracellular lipid droplets, a marker of quiescence. A functional screening test was designed (Figure 2). The experiment was carried out in a 96-well plate in which activated HSCs were cultured. Individual miRNAs were transfected into each well. When transfected, HSCs in some wells showed BODIPY-positive lipid droplets within 3 days. This initial screen yielded 15 primary hits. The miRNAs miR-15a and miR-412 were extracted from human Presence of orthologs and ability to repopulate lipid droplets in both mouse and human HSCs The newly formed lipid droplets were selected based on the force (Figure 3). and was positive for retinoids as evidenced by fluorescence under ultraviolet light (Figure 17). The overall size of transfected HSCs was reduced 10-100 fold and they became quiescent. Furthermore, forced expression of miR-15a or miR-412 increased the activation The two most important gene markers, alpha smooth muscle actin (Acta2) and alpha type 1 (Col1a1) was downregulated (Fig. 4 ).

[0292] For more comprehensive expression analysis, deep RNA sequencing was used to identify miR-15a or m The total number of quiescent-like HSCs exposed to iR-412 was 40–50% closer to quiescent HSCs than to activated cells. We demonstrated that miRNAs had a specific transcription profile (Figure 18). HSCs rendered quiescent by miR-15a or miR-412 resembled truly quiescent HSCs. Ex vivo experiments demonstrated that the two cell types were co-cultured We demonstrated that quiescent-like HSCs did not induce steatosis in healthy hepatocytes. In contrast, activated HSCs or NASH-HSCs not treated with the candidate miRNAs induced hepatocyte hypersteatosis when they were co-cultured (Figure 19). As expected, endogenous miR15a and miR-412 has decreased expression levels in activated HSCs compared with quiescent HSCs. (Figure 7).

[0293] miR-15a and miR-412 have not been studied in the context of hepatic stellate cells. However, the miR-15 family mediates Tgf-mediated signaling in the heart that may attenuate fibrosis. It has been found to inhibit the IL-1 pathway

[26] and is an important tumor suppressor in chronic lymphocytic leukemia. miR-412 is also known to be a factor in the regulation of There is no literature on it, and therefore its function is completely unknown. are short, usually 22 nucleotides in length, non-coding genes that are found in all biological They are involved in metabolic and pathological processes. Downstream target coding genes by imperfect base pairing with complementary sequences within the mRNA target While each miRNA can regulate many different coding genes, The gene is regulated by many different miRNAs that constitute complex layers of gene regulatory networks. miRNAs directly target approximately 50% of all mammalian coding genes, have demonstrated their widespread gene regulation

[29] . It has been shown that HSC activity is essential for the programming of HSCs [30-32]. The role of miRNAs in activation or reversion to quiescence has not been investigated.

[0294] As demonstrated herein, miR-15a and miR-4 in HSCs The quiescence-promoting effects and mechanism of action of miR-12 are compelling. a, but for both miR-15a and miR-412 There may be other related targets of interest. Numerous prediction algorithms are based on sequence complementarity. We predict potential direct targets of miRNAs in are not real targets that can withstand experimental validation. Therefore, RNA-Seq of HSCs is To define expression profiles that could identify true direct targets of miR-15a and miR-412 To investigate the effect of acetylcholine on the activation of acetylcholine, various activation conditions were performed.

[0295] Three types of HSCs were deep sequenced: 1) freshly harvested quiescent HSCs from healthy mice; 2) activated HSCs that had been passaged multiple times in cell culture dishes; and 3) miR- miR-15a or miR-412 induces activation and quiescence Reverted quiescent-like HSCs. Quiescent-like HSCs were treated with miR-15a or miR-412 mimics. The results showed that any coding regions were positive at the mRNA level, as determined by sequencing just 3 days after either The reduction of the gene expression could be the result of direct miRNA targeting. A set of genes whose mRNA levels were decreased after treatment with either miR-412 or miR-412 We overlaid the set of potential direct targets based on a predictive algorithm on the 10-item set

[33] (Figure 20). This new set of common genes from the two parent sets (hereafter referred to as the target candidate set) ) are more likely to contain the true direct target.

[0296] The target candidate set still has too many genes to analyze, so we will not be able to determine whether HSC activation Part of the two best-known signaling pathways, Tgf beta and Pdgf, that promote We selected candidate target genes that are known to be involved in the regulation of these two pathways. When used to select genes that are part of the Supplementary notes were left for miR-15a and miR-412 (Figure 20)

[34] . The gene for the mutant miR was cloned after a constitutively active luciferase gene. This can be tested by constructing reporters that contain or do not contain NA binding sequences. One of these reporter constructs and miRNA mimics was then transfected into 293 cells or primary HSCs. The co-transfected miRNA can be co-transfected into either If a gene that can anneal to this sequence and block translation of the luciferase protein is found, The mutant binding sequence may indicate that the gene is a true direct target of the miRNA tested. This should prevent NA from annealing and allow near-normal expression of luciferase. This provides further support that the coding gene sequences examined are bona fide miRNA targets.23 Highly likely target candidate gene sequences were identified in both real and mutated forms. This experiment is time consuming because the genes need to be cloned into a ferase vector. However, finding direct targets will elucidate the mechanism of action of miRNAs.

[32]

[0297] To date, the efficacy of delivering miR-15a or miR-412 to activated HSCs has been reported. The results are described herein. To this end, definitive loss of function experiments were performed for the first time using previously described CRISPR technology. This can be accomplished by deleting these genes in 3T3 HSCs

[35] .

[0298] The feasibility of using this technology in the first generation HSC is due to the lack of digits already available. This was verified by delivering a deletion vector to target HSCs (Figure 21)

[35] . Strains can be generated: 1) miR-15a null, 2) miR-412 null, and 3) miR-412 null. iR-15a / miR-412 null. Overexpression of both of these miRNAs results in HSC quiescence. Since their deletion promotes an activated phenotype, especially miR-15a / miR It is predicted that miRNAs may play a role in multiple functional overlaps in the R-412 double null strain. Lack of a distinct phenotype is also possible, as activation is well known to be a multifactorial disease. The levels will be analyzed by RNA-Seq to reveal their global expression, as well as activation and Morphology and proliferation can be determined by comparing both live and quiescent HSCs. Activated wild-type HSCs can be assessed while cultured in situ and Matrigel. It is known that the knockout mice can return to a quiescent state on Matrigel

[36] . The phenotype of mouse HSCs can be characterized in this environment. If the -412 deletion contributes to HSC activation, this effect would be consistent with the quiescent state in Matrigel. The interaction between hepatocytes and miRNA-deficient HSCs was investigated using a co-culture system. Activated HSCs can be assessed by measuring the amount of inflammatory cells in the co-cultured hepatocytes, which become steatotic. miRNA-deficient HSCs express mediators that activate wild-type HSCs. It may also be evaluated that they have a pro-steatosis phenotype that is even greater than that of SC. Conversely, reconstitution of miR-15a or miR-412 significantly reduced the expression of miR-15a and miR-412 in the respective deletion strains. Reconstitution of deleted miRNAs has already been demonstrated in clones that can be tested for and rescue the wild-type phenotype. miRNA-expressing piggyBac vectors were screened and used for gain-of-function experiments. This can be achieved by transfecting

[0299] miR-15a or miR-412 were expressed individually, but not in the same HSCs. Co-expression of these miRs is also specifically contemplated herein. Delivery of just one of the NAs shifts the entire gene expression pattern from active to quiescent. They can then shift approximately halfway back along the transcription axis that connects them (Figure 11). Overexpression of miRNAs was more effective than expression of either miRNA alone. Gain-of-function experiments can revert activated HSCs toward a near-quiescent state in single pigs. Clone both miRNAs into a yBac vector and transduce it into activated HSCs. The piggyBac vector can be used to express miRNA genes. Both miRs are expressed constitutively, allowing their integration into the offspring genome.

[17] HSCs that co-express NA were characterized by global gene expression, morphology, and Including proliferation using the MTT assay and interaction with hepatocytes using this co-culture method. , using similar parameters described herein to characterize other HSC lines. The present type can be determined.

[0300] The phenotype of primary HSCs expressing both miRNAs more closely resembled quiescent HSCs. If so, cell therapy could be used to treat HSCs by overexpressing a single miRNA, which could be used to treat CCl4-induced This HSC line can be tested in the setting of liver fibrosis and diet-induced NASH. Compared with those observed using HSC lines expressing either miR-412 or miR-412 alone Furthermore, it can prevent or reduce liver injury and fibrosis.

[0301] Delivery of miR-15a or miR-412 to mouse models of liver fibrosis or NAFLD Our inspection Treating HSCs from a dietary model of NASH with miR-15a or miR-412 and restore them to the quiescent state of hepatocytes (Figure 12). 15a and miR-412 inhibit activated HSCs even in culture conditions that promote activation These important ex vivo observations are consistent with the traditional CC In particular, we found that the expression of either miR-15a or miR-412 in the l4 mouse model HSCs expressing IL-1 can maintain a quiescent state even in diseased livers that have activation-promoting signals. Therefore, it is possible to demonstrate that these miRNAs can be used in vivo to attenuate the levels of liver pathology. This allows for the examination of whether mediators can be delivered via cell contact and soluble mediators. Through this, quiescent-like HSCs attenuate those signals that promote steatosis, inflammation, or fibrosis. Other cell types in the liver can be induced to participate in the

[0302] There are various methods for delivering miRNA to live mice, including injections. HSCs are quiescent by constitutively expressing miR-15a or miR-412. This will allow the feasibility of novel cell therapies for liver disease to be investigated. The engineered HSCs were further examined using a 4-week CCl4 challenge. The study was carried out by injecting reprogrammed HSCs once during the third week of The HSCs were transplanted into the liver and inserted into a piggyBac vector expressing miRNA. This was confirmed by visualization of GFP signaling in hepatocytes

[17] . (Figure 8). More importantly, mice treated with quiescent-like HSCs showed Histology showing reduced ballooning, apoptosis, inflammation and fibrosis (Figure 8), and low In light of these observations, Experiments have been carried out using additional mouse models of liver fibrosis and NASH to investigate the pathogenesis of fatty liver, inflammation and fibrosis. Further evaluate the potential of miR-15a and miR-412 in treating fibrosis These miRNAs may be expanded to mediate the expression of HSCs alone or in other cells. To assess whether miRNAs exert their functions more broadly across the cytoplasmic domain, can be delivered systemically using viral vectors or mimetics as an alternative to cell therapy. For all in vivo treatment experiments, efficacy was assessed by comparing the expression of profibrogenic genes in the liver. PCR, histological examination including H&E and Sirius red staining, and liver hydroxyproline This can be assessed by performing an ALT assay and measuring plasma ALT.

[0303] Using mesenchymal stem cells and macrophages, several groups have had some success. Cell therapy for liver fibrosis has already been performed using fibroblasts

[18] . However, these experiments were limited to the extent that the liver fibrosis This is the first time that HSCs have been purposefully engineered to prevent or reverse a disease. HSCs derived from congenic mice and 500,000 cells were incubated in CCl4 for 4 weeks. The initial results were convincing, but there were no improvements in liver histology. The benefit was from prevention of liver damage resulting from 4 weeks of CCl4 administration or from the onset of CCl4 challenge. It is unclear whether this is due to a reversal of the damage that occurred during the first 3 weeks. The effect of the treatment is quite understandable given that only the CCl4 model has been used so far. Finally, the beneficial effects of cell therapy may be increased with more than one injection. To answer these important questions, we developed the CCl4 model of liver fibrosis (oral gavage The CDAHFD model of NASH was administered 100 ul of 40% CCl4 twice a week. For all cell therapy experiments, 500,000 quiescent cells can be used. HSCs can be reprogrammed using miR-15a or miR-412. groups that received treatment with untreated or unreprogrammed HSCs All appropriate controls may be included.

[0304] To examine whether quiescent-like HSCs can prevent or reverse liver fibrosis For all CCs, two separate experiments with different cell injection schedules can be used. Administration of CCl4 can be by oral gavage twice a week. The challenged mice typically develop progressive fibrosis. may be injected at weeks 2, 4, and 6 of the CCl4 challenge over the course of an 8 week period. All mice can be sacrificed at the end of the eighth week. To test whether reprogrammed HCC could reverse liver fibrosis, SC were injected at weeks 9 and 11 after the 8-week course of CCl4 challenge was completed. All mice can be sacrificed at the end of the 12th week. The CDAHFD model showed significant NASH by week 3 and NASH by week 9. In the fifth week, patients developed stage 2 fibrosis (on a scale of 0 to 4)

[19] . One or three injections may be completed at weeks 3, 5, and 7. All mice were At the end of the period, the animals may be slaughtered.

[0305] Activated human HSCs received the human orthologues of miR-15a or miR-412 It has been previously shown that cells can subsequently revert to a quiescent-like state (Fig. 8). Re-formation of tinoid-positive lipid droplets in co-cultured human hepatocellular carcinoma cell lines HepG2 and Hu It was possible to reduce the expression of inflammatory cytokines from h7 onwards (Figure 6). As a bridge to translate discoveries into disease, primary human HSCs express miR15a and miR-412 To determine the therapeutic potential of CCL4 in treating a CCL4 mouse model of liver fibrosis CCl4 can be used to treat severe complex necrosis factor-positive mice selected to prevent immune rejection of human cells. Gavage twice a week to induce liver fibrosis in immune-deficient SCID mice A CCL4-based liver fibrosis model was also developed by other groups using SCID mice. Fibrosis prevention study: Human HSCs have been successfully generated in SCID mice. The mice may be injected at weeks 2, 4, and 6 during the CCL4 challenge over the course of an 8 week period. Fibrosis reversion study: Reprogrammed human HSCs suppress pre-existing liver fibrillation compared to controls To test whether the disease can be reversed, HSCs are After the 4 challenge is completed, mice can be injected on weeks 9 and 11. They can be slaughtered at the end of the second week.

[0306] The first experiment involved H cells reprogrammed with miR-15a or miR-412. Injecting SCs significantly reduced collagen expression and overall liver injury in CCl4-induced liver fibrosis. One method for delivering miRNA is by injecting miR The aim of this study was to inject lentivirus expressing miR-1 in the CCl4 fibrosis study. Lentiviruses expressing either miR-5a or miR-412 were induced by CCl4 challenge All mice were injected into the tail vein at weeks 2, 4, and 6 over the course of an 8 week period. Patients may be sacrificed at the end of week 8. CDAHFD NASH Study: miR-15a or Lentiviruses expressing either miR-412 or miR-412 increased the expression of β-terminal β-terminal β-terminal cytoplasmic markers during 9 weeks of CDAHFD. Mice may be injected at weeks 3, 5 and 7. All mice may be sacrificed at the end of week 8. .

[0307] An alternative delivery method is a lipid-based carrier (MaxSuppressor™ by B More stable chemically modified m Implementation of injecting iRNA mimics (Exiqon and Invitrogen) The possibility of this approach could be examined [38, 39]. By avoiding lentiviruses, The method, or a variation thereof, has the potential for use in humans. Lipid vehicles containing either miR-15a or miR-412 were administered intracellularly over an 8-week period. Injections were made into the tail vein at weeks 2, 4, and 6 during Cl4 challenge. Patients may be sacrificed at the end of week 8. CDAHFD NASH Study: miR-15a or Lipid vehicles containing either miR-412 or miR-131 were administered intracellularly during the course of 9 weeks on a CDAHFD. The mice may be injected at weeks 3, 5 and 7 of the study. All mice may be sacrificed at week 9.

[0308] Defining the function of WISP1 in fatty liver, inflammation and fibrosis Given that miRNAs generally function by targeting multiple coding genes, Previously, these two candidates down-regulate HSC genes that promote liver inflammation and fibrosis. Many genes are blocked by miR-15a and miR-412. WISP1 is the first experimentally validated direct inhibitor of miR-15a. WISP1 was identified as a target of CDAHFD-induced NASH-HSC conditioned medium. It was first identified as one of several proteins that were up-secreted in >100 representative cytokines, chemokines and extracellular matrix proteins were detected using a cytokine array blot containing The analysis showed that activated HSCs expressed WISP1 at approximately 30-fold higher levels than quiescent HSCs. WISP1 was found to be involved in the transcriptional regulation of Cyr61 (Ccn1), Ctgf (Ccn2), and Nov(Ccn3), WISP1(Ccn4), Wisp2(Ccn5) and Wisp 3 (Ccn6), a member of the Ccn family of matricellular proteins Ctgf has already been established as an important profibrotic factor in the liver

[42] . Interestingly, WISP1 is upregulated in human idiopathic pulmonary fibrosis. We found that neutralizing WISP1 antibodies were used to treat bleomycin-challenged mice. In addition, small animal studies have shown that blocking WISP1 showed that CCl4-induced liver fibrosis was reversed

[44] . The role of WISP1 has not been studied in detail, especially in the context of NASH.

[0309] Given that CDAHFD-induced NASH-HSCs promote steatosis in co-cultured hepatocytes, Thus, WISP1 may contribute to this phenotype. When WISP1 was overexpressed in HSCs from healthy mice, these cells Conditioned medium derived from the cells initially produced steatosis in healthy primary hepatocytes (Figure 24). miR-15a contains two potential miR-15a binding sequences within its 3'-UTR. It is predicted that the putative target sequence of WISP1 is Mutation variants are cloned behind a constitutively active luciferase reporter gene One of these reporters was co-transfected with a miR-15a mimic. In the presence of the mutant sequence, the reporter containing the wild-type sequence reduced luciferase expression, whereas the reporter containing the mutant sequence reduced luciferase expression. We demonstrated that miR-15a binds to both WISP1 target sequences without the use of a reporter. Considering these data and the published literature, WISP1 These genes may be important target genes for miR-15a to achieve some of its effects in HSCs. WISP1 may be a potential drug target for managing NASH. The function of α-amyloides in α-amyloides is discussed herein as a potential therapeutic target for NASH. will be done.

[0310] WISP1 is a direct target of miR-15a, but its effects on HSC activation status and The role of WISP1 in bone marrow regrowth and NAFLD pathogenesis remains unclear. This study was generated to demonstrate the role of WISP1 in the formation of gliomas, but its function in the liver is unknown. These mice were fertile and had a pronounced liver phenotype. These mutant alleles are from the University of California, San Diego, which ensures good mutant mouse derivation. Sperm and sperm are frozen at the MMRRC facility in Davis, California. First, the function of WISP1 is conserved in the W by harvesting ISP1 null mice and characterizing their phenotypes ex vivo This can be elucidated in HSCs.

[0311] Quiescent wild-type HSCs become activated after 7–10 days of culture on plastic. The RNA-seq data presented here demonstrates a global genetic difference between these two groups. Demonstrating clear differences in gene expression (Figure 11). Parallel experiments demonstrated that WISP1 deletion, even in a proactivating environment, altered the global expression profile. These can be used to assess whether the markers change the HSC quiescence state. After isolating HSCs from ISP1 null mice, the cells were incubated for 1 day (quiescent) or 28 days. They can be cultured in plastic dishes for a period of time (wild type activation). The expression of the offspring can be determined through RNA-seq and compared to that of wild-type HSCs. Global comparisons can be performed using multidimensional scaling analysis to identify differentially expressed The genes can be identified for detailed analysis.

[0312] Cell morphology, proliferation and migration can be compared while cultured on plastic. The morphology of the cells was determined by their size, the extent of cellular processes, the presence of lipid droplets, and whether they were UV-fluorescent or retinoids. Indications of idiopathic stores and quiescence can be determined using parameters such as Proliferation is reduced in WISP1 null HSCs, as well as in quiescent HSCs and PCNs. A and Ki-67 immunostaining. It can also be performed to quantify cell proliferation rates as previously described

[32] . Preliminary data and published literature indicate that WISP1 is a profibrotic gene. Therefore, deletion of WISP1 induces quiescence in HSCs, possibly It can be promoted even while being cultured on plastic dishes. Finally, knockout HS Reconstitution of WISP1 in C. can rescue the wild-type phenotype. To reconstitute 1, transfect WISP1-expressing piggyBac vectors. This can be achieved by transfecting the cells with the WISP1 expression vector. WISP1 null HSCs were used in untransfected WISP1 null cells. The antibodies can be characterized using the same in vitro assays used.

[0313] The interaction of WISP1 null HSCs with hepatocytes was investigated using the co-culture system described herein. Activated wild-type HSCs induce co-cultured hepatocytes to become fatty (Figure 14). WISP1-overexpressing HSCs caused nearby hepatocytes to become more severely fatty. (Figure 24). WISP1 null HSCs may have an attenuated adipose-promoting phenotype. The readouts for the co-culture assay were Bodipy staining for lipid droplets and inflammation. This involves PCR of co-cultured hepatocytes to measure the expression of sex factors.

[0314] The phenotype can be assessed by the overall severity of liver pathology. WISP1 null mice used CCl4 and CD40 to model toxin-induced liver fibrosis and NASH, respectively. The resulting WISP1 null mice can be challenged with an AHFD. Liver injury can be assessed using that of wild-type mice. WIS in other organs, including the lungs Given the well-known profibrotic role of P1, WISP1 null mice exhibited reduced liver fibrosis. and possibly even a reduction in steatosis and inflammation. For in vivo experiments, the liver phenotype was determined by PC of profibrogenic genes in the liver. R, histological examination including H&E and Sirius red staining, liver hydroxyproline uptake Finally, liver disease can be assessed by measuring serum ALT levels and plasma ALT levels. The functional phenotype of HSCs harvested from these mouse models can be determined.

[0315] CCl4 fibrosis model: Male wild-type and WISP1 null mice were gavaged with CCl4. They may receive CCl4 for 8 weeks. The CDAHFD NASH model was Wild-type and WISP1 null mice can be fed for 9 weeks. DAHFD, 6 weeks) and end-stage NASH with stage 2 fibrosis (CDAHFD, 9 weeks) To define the role of WISP1 in the development of aging (weeks), half of the mice were The other half can be slaughtered at the end of the 9th week.

[0316] HSCs were obtained from mice challenged with CCl4 for 4 weeks and CDAHFD for 3 weeks. These HSCs can be isolated from mice, where they develop steatosis and express inflammatory factors. The results are consistent with those of the same challenge. The results can be compared to those of wild-type HSCs that have undergone immunization.

[0317] Blocking WISP1 with a neutralizing antibody reduces bleomycin-induced

[43] . Similarly, neutralizing WISP1 reduced the damage caused by NASH. The mouse model of NASH was treated with WISP1 antibody. and the liver phenotype can be assessed after 6 and 9 weeks of treatment.

[0318] CDAHFD NASH model: Wild-type mice can be fed a CDAHFD for 9 weeks. Treatment groups were treated with either a commercially available antibody (R&D Systems)

[43] or an antibody

[46] . The control group received either IgG or IgG1, administered by injection every week. To examine the effect of WISP1 blockade in moderate and end-stage NASH, respectively Half the mice can be sacrificed at the end of the sixth week and the other half at the end of the ninth week.

[0319] In addition, we analyzed the conditioned medium of NASH-derived HSCs to identify secreted paracrine or oligamine Further characterization of NASH-HSC conditioned medium through mass spectrometry Further analysis could identify other mediators that may be potential drug targets. The co-culture system described herein is useful for studying other liver diseases when a representative mouse model is available. For example, the interaction of hepatocytes with HSCs in alcohol-fed mice can be used to Alcoholic liver disease can be modeled by isolating cell types in the liver

[0047] .

[0320] References Example 1 / 2:

[0321] [Table 3-1]

[0322] [Table 3-2]

[0323] [Table 3-3]

[0324] [Table 3-4]

[0325] [Table 3-5]

[0326] [Example 3] HSC Isolation Protocol The following protocol describes the present invention for isolating HSCs, e.g., mod-Hep / HSCs. It is used in the experiments described in the specification. 1. Cannulate the Portal Vein with an Angiocatheter or Butterfly Needle 2. Perfuse the liver with solution at 6 mL / min at 37°C: 30mL HBSS(Ca 2+ and Mg 2+ (not including the peritoneal cavity), immediately after the start of perfusion, IVC disconnection 30mL 0.05% collagenase [0.5mg / mL-->50mL, 50 0 ul of 50 mg / mL collagenase stock solution] (e.g., Collagenase B ;Roche 11088815001) 3. After perfusion, carefully remove the liver (especially the gallbladder) and remove the Glisson's capsule. 4. Transfer the liver to a sterile beaker and cut into very thin pieces 5. Add the remaining 20 mL of 0.05% collagenase and incubate at 37°C. Hold for 30 min [add DNAse (10ug / mL)] (e.g., DNaseI; Ro che 10104159001) 6. Pipette up and down and filter using a cell strainer (70um) in a Petri dish. Add S+DNAse (10ug / mL) to 45mL 7. Centrifuge at 50xg for 3 min to remove hepatocytes, and then transfer the supernatant to a fresh Transfer to a tube 8. Precipitate by centrifugation at 635 g for 10 min. Wash with 9.45mL PBS+DNAse (10ug / mL), x635g, 10 Centrifuge for 1 min 10. Filter the cell suspension through a 70um cell strainer Wash with 11.45mL PBS+DNAse (10ug / mL), x635g, 1 Centrifuge for 0 minutes 12. Aspirate the supernatant and add PBS to the pellet until the total volume is 6.5 mL. 13. Add 3.5 mL Percoll (9 parts Percoll, 1 part 10xPBS) to a total volume of 10 mL. Add 100 ml of PBS, mix, and transfer to a 15 mL tube (e.g., Percoll pl us, GE Healthcare 17-5445-02) 14. Carefully add 1 mL of PBS to the top of the column. 15. Centrifuge the column at room temperature for 30 minutes at 1130 g (acceleration 9, brake 0). 16. After centrifugation, HSCs are in the layer between PBS and 35% Percoll. 17. Aspirate the HSCs using a micropipette and wash them with 10 mL DMEM. Centrifuge for x635g x6 minutes 18. Aspirate the Supernatant 19. Resuspend pellet and plate in DMEM + 20% FBS + 1% P / S.

[0327] [Example 4] WISP1-inhibiting antibodies for the treatment and prevention of liver disease - Patents.com Antibodies that specifically bind and inhibit WISP1 have been shown to be effective in treating primary biliary cholangitis (PBC) and It can be used to treat and prevent liver diseases such as autoimmune hepatitis (AIH).

[0328] Specifically, anti-(ant)-WISP1 IgG is a promising candidate for inhibiting WISP1. 1 / IgG4 antibody and IgG1 ADCC). Strategies used to engineer, but are not limited to, Fc Genetic engineering; bispecific antibody technology that allows for the simultaneous inhibition of two anti-fibrotic targets; and (1) by sequestering the entity into a catalytic drug by driving a targeted catabolic reaction; (2) drastically reducing CoG, dosing levels and dosing frequency; and (3) (4) increasing the availability of subcutaneous medications, and (5) eliminating the need to completely block the availability of subcutaneous medications; One example is the sweeping technique.

[0329] To evaluate the ability of anti-WISP1 antibodies to inhibit WISP1, the antibodies were incubated on fresh HSCs. The WISP1 antibody (Ab WISP1 or WISP IgG, RND Sy stems, 1 μg / mL) was incubated with conditioned medium for 1 h at 37°C and then incubated for 1 h at 37°C. As a negative control, HSCs were incubated with conditioned medium alone. As a positive control, recombinant WISP1 (Rc-WISP1, 100 ng / ml) was used instead of the antibody. L) was also added to HSCs. HSC samples were evaluated 48 hours after treatment (Figure 38A). ISP1, e.g., WISP1-CCN4, is a gene identified under accession number O54774. Rc-WISP1 is obtained from a cellular line and is commercially available from R&D Systems. In HSCs, the cell number (Figure 38B), A cta2 mRNA expression (Fig. 38C) and Col1a1 mRNA expression (Fig. 38D) A significant increase was found, indicating that HSCs were activated in the presence of recombinant WISP1. Surprisingly, the commercially available anti-WISP1 IgG (Ab Wisp1) Incubation with anti-WISP1 prevented HSC activation in HSCs; c Compared with HSCs incubated with WISP1, 1 h after incubation On days 1 and 2, cell number (Fig. 38B), Acta2 mRNA expression (Fig. 38C) and This resulted in a significant reduction in Col1a1 mRNA expression (Figure 38D).

[0330] Next, anti-WISP1 IgG was administered to mice treated with choline-deficient L-amino acid-limited high-fat diet-induced steatosis (C The effects of hepatitis C on liver disease, e.g. A hallmark of NALFD and viral hepatitis is an increase in lipid droplets in diseased liver cells To assess the presence of lipid droplets, BODIPY staining is used. As expected, CDAHFD cells treated with control IgG showed increased BODIPY staining (Figure 38 E and 38F). In contrast, CDAHFD cells treated with anti-WISP1 IgG The BODIPY staining was reduced in the control and healthy cells (Figures 38E and 38F). Thus, the data presented herein demonstrate that anti-WISP1 IgG inhibits diseased liver cells. This shows that they can be restored to healthy liver cells.

[0331] Finally, to demonstrate the ability of anti-WISP1 IgG to regulate HSC migration, we cultured HSCs in The cells were cultured to form a monolayer and then wounded (Figures 39A-B). The cells were cultured at 90% confluence in a 60 mm dish at 4 °C for 1 h. A single linear scratch was made in a sterile A 200 pipette tip was used to dislodge the cells from the suspension to remove any cell debris. Wash with PBS and incubate with recombinant Wisp1 protein (100 ng / mL, R&D Sy stems, Minneapolis, MN) and monoclonal Wisp1 antibody (1 (μg / mL, R&D Systems, Minneapolis, MN) for 24 h. Wound closure or cell migration was assessed when a scratch was introduced using an inverted microscope. The area between the wound edges in the dish at each time point was recorded on the image. The data were calculated using a standard template placed over the wound area compared to the initial wound area. Wound area was expressed as a percentage. Wound area was measured using publicly available ImageJ (NIH) software. This was determined using software.

[0332] Following injury, HSCs were incubated with either Rc WISP1 or anti-WISP1 IgG. HSCs incubated with rc WISP1 showed a marked increase in phenotype, especially at 12:00. At later time points, they showed more rapid migration compared to control or anti-WISP1 IgG-treated HSCs. In contrast, HSCs incubated with anti-WISP1 IgG showed no significant changes compared to control treatment. Anti-WISP1 IgG resulted in slow migration similar to that of transplanted HSCs (Figures 39A-B). This prevented HSC migration after injury compared to HSCs treated with Rc-WISP1. The results showed that antibodies that inhibit WISP1 can prevent HSC activation and migration, These have been identified to make them useful agents for treating liver disease.

[0333] array SEQ ID NO:1 is the human WNT1-induced signaling pathway protein 1 isoform 1 precursor The amino acid sequence encoding the target protein.

[0334] [ka]

[0335] SEQ ID NO:2 is the human WNT1-induced signaling pathway protein 1 isoform 2 precursor The amino acid sequence encoding the target protein.

[0336] [ka]

[0337] SEQ ID NO:3 is the human WNT1-induced signaling pathway protein 1 isoform 3 precursor The amino acid sequence encoding the target protein.

[0338] [ka]

[0339] SEQ ID NO:4 is the human WNT1-induced signaling pathway protein 1 isoform 4 precursor The amino acid sequence encoding the target protein.

[0340] [ka]

[0341] SEQ ID NO:5 is human WNT1-inducible signaling pathway protein 1 (WISP1), a transcription factor-dependent transcription factor (TGF-D1) inhibitor. Transcript variant 1 is the nucleotide sequence of the mRNA.

[0342] [ka] [ka] [ka]

[0343] SEQ ID NO:6 is the amino acid sequence of mouse WNT1-induced signaling pathway protein 1 precursor. The sequence is [mouse (Mus musculus)].

[0344] [ka]

[0345] SEQ ID NO:7 is mouse WNT1-inducible signaling pathway protein 1 (Wisp1); Transcript variant 1, nucleotide sequence of mRNA [mouse (Mus musculus)].

[0346] [ka] [ka] [ka]

[0347] SEQ ID NO: 8 is the nucleotide sequence of human MiR 15a.

[0348] [ka]

[0349] SEQ ID NO:9 is the nucleotide sequence of human Mir-412.

[0350] [ka]

[0351] SEQ ID NO: 10 is the nucleotide sequence of mouse MiR 15a.

[0352] [ka]

[0353] SEQ ID NO:11 is the nucleotide sequence of mouse Mir-412.

[0354] [ka]

[0355] SEQ ID NOs: 12 to 31 are Rattus heavy chain complementarity determining regions for IgG. It is.

[0356] [Table 4-1]

[0357] [Table 4-2]

[0358] SEQ ID NOs: 32 to 51 are Rattus light chain complementarity determining regions for IgG It is.

[0359] [Table 5]

[0360] SEQ ID NOs: 52-71 are Ovis aries (sheep) heavy chain complementarity sequences for IgG This is the decision area.

[0361] [Table 6]

[0362] SEQ ID NOs: 72-90 are Ovis aries (sheep) light chain complementarity sequences for IgG This is the decision area.

[0363] [Table 7]

[0364] SEQ ID NOs: 91-110 are Capra hircus (goat) heavy chain complementarity for IgG This is the decision area.

[0365] [Table 8]

[0366] SEQ ID NOs: 111-121 are Leporidae (rabbit) heavy chains for IgG Complementarity determining region.

[0367] [Table 9]

[0368] SEQ ID NOs: 122-141 are Leporidae (rabbit) light chains for IgG Complementarity determining region.

[0369] [Table 10]

Claims

1. A pharmaceutical composition for treating or preventing liver disease in a subject in need thereof, comprising: the pharmaceutical composition comprises an antibody or antibody fragment that binds to and inhibits WNT1-induced signaling pathway protein 1 (WISP1); The liver disease is primary biliary cholangitis (PBC), non-alcoholic steatohepatitis (NASH), non-alcoholic fatty liver disease (NAFLD) or primary sclerosing cholangitis (PSC); The pharmaceutical composition.

2. The pharmaceutical composition described in claim 1, wherein WISP1 is a splice variant selected from the group consisting of WISP1v, WISP1vx, and WISP1 delta exons 3-4.

3. The pharmaceutical composition of claim 1, wherein the antibody or antibody fragment is selected from the group consisting of mab1680, AF1680, SAB2501114, ab60114, and ab65943.

4. The pharmaceutical composition described in claim 1, wherein WISP1 is inhibited in target cells.

5. The pharmaceutical composition described in claim 4, wherein the target cells are hepatic stellate cells, fibroblasts, or myofibroblasts.

6. The pharmaceutical composition of claim 1, wherein the antibody or antibody fragment inhibits the activity of WISP1 and / or reduces WISP1 protein levels.

7. The pharmaceutical composition of claim 6, wherein the activity of WISP1 is inhibited by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to a suitable control.

8. The pharmaceutical composition of claim 6, wherein the WISP1 protein level is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more compared to a suitable control.

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