Compositions and methods for treatment of liver disease

Abstract

The present invention relates to compositions and methods for the treatment of liver disease. Described herein are methods and compositions for treating liver disease. Aspects of the invention relate to administering to a subject an agent that inhibits WISP1. Another aspect of the invention relates to administering to a subject an HSC expressing an agent that inhibits WISP1.

Description

[0001] This application is a divisional application of the patent application "Compositions and Methods for the Treatment of Liver Diseases" with an application date of October 18, 2019, and an application number of 201980083971.8 (International Application No. PCT / US2019 / 056910).

[0002] Cross - reference to related applications

[0003] This application claims the benefit of U.S. Provisional Application No. 62 / 747,903, filed on October 19, 2018, under 35 U.S.C.§119(e), the content of which is incorporated herein by reference in its entirety. Technical field

[0004] The field of the present invention relates to the treatment of liver diseases. Background art

[0005] The liver is a very important organ that filters metabolites, synthesizes proteins, and can produce biochemicals required for digestion. Specifically, the liver produces bile to break down fats and emulsify lipids. Chronic progressive liver diseases can lead to the progressive destruction of bile ducts in the liver, which can cause bile accumulation, severe inflammation, scarring, and fibrosis. When scar tissue replaces healthy liver tissue, liver function becomes increasingly impaired. For some liver diseases (e.g., primary biliary cholangitis (PBC)), there is only one drug, ursodeoxycholic acid (UDCA), that can improve survival rates. Unfortunately, approximately 40% of patients treated with UDCA show an inadequate response to treatment. Therefore, more effective therapies are needed for the treatment of liver diseases such as PBC. Summary of the invention

[0006] The invention described herein relates in part to the discovery that the inhibition of WISP1 by miRNA - 15a, miRNA - 412, and anti - WISP1 antibodies induces quiescence in activated hepatic stellate cells (HSCs), which are the cell type that plays a central role in the progression of liver fibrosis. It is also shown herein that miR - 15a and WISP1 IgG can directly target WISP1 to inhibit the profibrotic function of proteins in activated HSCs.

[0007] Accordingly, one aspect described herein is a method for treating or preventing liver diseases, which comprises administering to a subject in need an antibody or an antibody agent that inhibits WISP1.

[0008] In one embodiment in 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 tumor; biliary atresia; cirrhosis; Crigler-Najjar syndrome; galactosemia; Gilbert syndrome; hemochromatosis; hepatic encephalopathy; hepatitis A; hepatitis B; hepatitis C; hepatorenal syndrome; intrahepatic cholestasis of pregnancy (ICP); lysosomal acid lipase deficiency (LAL-D); liver cyst; liver cancer; neonatal jaundice; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis; primary biliary cholangitis (PBC); primary sclerosing cholangitis (PSC); progressive familial intrahepatic cholestasis (PFIC); Reye's syndrome; type I glycogen storage disease; scleroderma; and Wilson's disease.

[0009] In one embodiment in any aspect, WISP1 is a splice variant selected from the group consisting of: WISP1v, WISP1vx, and WISP1 exon 3-4 delta.

[0010] In one embodiment in any aspect, the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of: mab1680, AF1680, SAB2501114, ab60114, and ab65943.

[0011] In one embodiment in any aspect, the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

[0012] In one embodiment in any aspect, the subject is a mammal.

[0013] In one embodiment in any aspect, WISP1 is inhibited in the target cell. In one embodiment in any aspect, the target cell is a mammalian cell. In one embodiment in any aspect, the target cell is a hepatic stellate cell, a fibroblast, or a myofibroblast. In one embodiment in any aspect, the hepatic stellate cell is quiescent.

[0014] In one embodiment in any aspect, the antibody or antibody agent is administered by direct injection, subcutaneous injection, intramuscular injection, oral administration, transdermal or nasal administration.

[0015] In one embodiment in any aspect, inhibiting WISP1 is inhibiting WISP1 activity or reducing WISP1 protein level. In one embodiment in any aspect, compared to a suitable control, the activity of WISP1 is inhibited by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more. In one embodiment in any aspect, compared to a suitable control, the level of WISP1 is reduced by at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or more.

[0016] Another aspect described herein provides a composition comprising an antibody or antibody agent that inhibits WISP1, and a pharmaceutically acceptable carrier.

[0017] In one embodiment in any aspect, the composition is formulated for treating or preventing liver disease.

[0018] Another aspect described herein provides a method for treating a subject with liver disease, the method comprising: (a) detecting the levels of WISP1 and / or Yap, Col1a1, Acta2 in a biological sample of the subject; (b) comparing the determination result of (a) with a reference level; (c) identifying a subject having increased WISP1 and / or Yap, Col1a1, Acta2 compared to the reference level in (a) as having liver disease; and (d) administering to the subject having liver disease an antibody or antibody agent that inhibits WISP1.

[0019] In one embodiment in any aspect, the method further comprises obtaining a biological sample from the subject before (a).

[0020] In one embodiment in any aspect, the biological sample is a blood sample, tissue, buffy coat, serum, or tissue.

[0021] Yet another aspect described herein provides a method for treating or preventing liver disease, which comprises administering to a subject in need a reagent that inhibits WISP1.

[0022] In one embodiment in any aspect, the reagent that inhibits WISP1 is selected from the group consisting of: small molecules, antibodies or antibody agents, peptides, genome editing systems, viral vectors, miRNAs, lncRNAs, mRNAs, and siRNAs. In one embodiment in any aspect, the microRNA is miRNA15a or miRNA412.

[0023] In one embodiment in any aspect, the reagent is administered by direct injection, subcutaneous injection, intramuscular injection, oral administration, transdermal or nasal administration.

[0024] Another aspect described herein provides a composition comprising an agent that inhibits WISP1 and a pharmaceutically acceptable carrier.

[0025] Another aspect described herein provides a method of treating liver disease in a subject, comprising: (a) detecting the levels of WISP1 and / or Acta2 in a biological sample of the subject; (b) comparing the result of the determination in (a) with a reference level; (c) identifying a subject having increased WISP1 and / or Acta2 compared to the reference level in (a) as having liver disease; and (d) administering to the subject having liver disease an agent that inhibits WISP1.

[0026] Yet another aspect described herein provides a method of generating an engineered hepatic stellate cell or a population thereof that expresses an agent that inhibits WISP1, comprising contacting the cell with an agent that inhibits WISP1 and culturing the cell for a sufficient time to allow expression of the agent.

[0027] In one embodiment of any aspect, the cell is quiescent.

[0028] In one embodiment of any aspect, the contacting comprises contacting the cell with the agent or a vector encoding the agent. In one embodiment of any aspect, the contacting comprises transduction, nucleofection, electroporation, direct injection, and / or transfection.

[0029] Another aspect described herein provides a cell line comprising hepatic stellate cells generated by any method described herein.

[0030] Another aspect described herein provides a pharmaceutical composition comprising hepatic stellate cells or a population thereof generated by any method described herein and a pharmaceutically acceptable carrier.

[0031] Another aspect described herein provides a method of treating or preventing liver disease, comprising administering to a subject in need thereof a cell generated by any method described herein, a cell generated as described herein, or a pharmaceutical composition comprising a cell generated as described herein.

[0032] Another aspect described herein provides a method of reducing fibrosis in a subject, the method comprising: administering to a subject in need thereof a cell generated by any method described herein, a cell generated as described herein, or a pharmaceutical composition comprising a cell generated as described herein.

[0033] Another aspect provided herein is a method of treating a subject having liver disease, comprising: (a) receiving the results of a test that identifies a subject having increased levels of WISP1 and / or Acta2 (e.g., mRNA, miRNA, protein levels, etc.) compared to a reference level as having liver disease; and (b) administering to the subject having liver disease an antibody or an antibody agent that inhibits WISP1.

[0034] Another aspect provided herein is a method of treating a subject having liver disease, comprising: (a) receiving the results of a test that identifies a subject having increased levels of WISP1 and / or Acta2 compared to a reference level as having liver disease; and (b) administering to the subject having liver disease a reagent agent that inhibits WISP1.

[0035] Definitions

[0036] For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise indicated or implied from the context, the following terms and phrases include the meanings provided below. The definitions are provided to aid in describing particular embodiments and are not intended to limit the claimed technology, as the scope of the technology is limited only by the claims. Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this technology belongs. If there is an apparent conflict between the usage of a term in the art and its definition provided herein, the definition provided in the specification will control.

[0037] Definitions of common terms in immunology and molecular biology can be found in: The 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 Blackwell Science Ltd., 1999 - 2012 (ISBN 9783527600908); and 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 Luttmann, published by Elsevier, 2006; Janeway's Immunobiology, Kenneth Murphy, Allan Mowat, Casey Weaver (eds.), Taylor & Francis Limited, 2014 (ISBN 0815345305, 9780815345305); Lewin's Genes XI, published by Jones & Bartlett Publishers, 2014 (ISBN-1449659055); Michael Richard Green and Joseph Sambrook, Molecular Cloning: A Laboratory Manual, 4th Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., USA (2012) (ISBN 1936113414); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing, Inc., New York, USA (2012) (ISBN 044460149X); Laboratory Methods in Enzymology: DNA, Jon Lorsch (ed.), Elsevier, 2013 (ISBN 0124199542); Current Protocols in Molecular Biology (CPMB), Frederick M. Ausubel (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, ADAM Kruisbeek, David H. Margulies, Ethan M. Shevach, Warren Strobe, (eds.) John Wiley and Sons, Inc., 2003 (ISBN 0471142735, 9780471142737), the contents of which are incorporated herein by reference in their entirety.

[0038] As used herein, the terms “treat,” “treatment,” “treating,” or “ameliorate” refer to a therapeutic treatment wherein the object is to reverse, alleviate, improve, inhibit, slow down, or stop the progression or severity of a condition associated with a liver disease such as, for example, liver fibrosis. The term “treatment” includes reducing or alleviating at least one adverse effect or symptom of a liver disease, such as jaundice, variceal bleeding, reduction of fibrosis, scarring, and ascites. A treatment is generally “effective” if one or more symptoms or clinical markers are reduced. Optionally, a treatment is “effective” if the progression of the disease is reduced or stopped. That is, “treatment” includes not only an improvement in symptoms or markers, but also a halt, or at least a slowing or reversal, of the progression or worsening of symptoms as compared to what would be expected in the absence of treatment. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of one or more symptoms, diminution of the degree of the disease, stabilization (i.e., non-worsening) of the disease state, delay or slowing of disease progression, amelioration or palliation of the disease state, remission (partial or total), and / or reduced mortality, whether detectable or not. The term “treatment” of a disease also includes providing relief of the symptoms or side effects of the disease (including palliative therapy).

[0039] As used herein, "preventing" or "prevention" refers to any methodology by which a disease state does not occur due to the action of a methodology (e.g., administration of a composition as described herein). In one aspect, it is understood that prevention can also mean that the disease does not reach the degree that occurs in an untreated control. Thus, prevention of a disease includes a reduction in the likelihood that a subject will develop the disease relative to an untreated subject (e.g., a subject not treated with the methods or compositions described herein).

[0040] As used herein, the terms "administer" and "inject" may be used interchangeably in the context of a method or route that causes a cell or reagent introduced to be at least partially localized at a desired location such as the liver or a region thereof, such that a desired effect(s) (e.g., reduced WISP1 level or activity) is produced. The reagents or cells described herein can be administered by any suitable route that results in delivery to a desired location in a subject, where at least a portion of the reagent, cell, or component of the cell delivered is maintained in a viable state at the desired location. After administration to a subject, the survival period of the cells can be as short as a few hours, e.g., 24 hours, to several days, up to several years, i.e., long term. In some embodiments, the term "administer" refers to the administration of a pharmaceutical composition comprising one or more reagents or cells. Administration can be accomplished by direct injection (e.g., directly to a target cell), subcutaneous injection, intramuscular injection, oral, or nasal delivery to a subject in need. Administration can be local or systemic.

[0041] The terms "patient", "subject", and "individual" may be used interchangeably herein and refer to an animal, particularly a human, to whom treatment, including prophylactic treatment, is provided. The term "subject" as used herein refers to human and non-human animals. The terms "non-human animal" and "non-human mammal" may be used interchangeably herein and include all vertebrates, such as mammals, such as non-human primates (particularly higher primates), sheep, dogs, rodents (e.g., mice or rats), guinea pigs, goats, pigs, cats, rabbits, cows, and non-mammals, such as chickens, amphibians, reptiles, etc. In one embodiment, the subject is a human. In another embodiment, the subject is an experimental animal or an animal substitute as a disease model. In another embodiment, the subject is a domestic animal including companion animals (e.g., dogs, cats, rats, guinea pigs, hamsters, etc.). The subject may have previously received treatment for liver disease or may never have received treatment for liver disease. The subject may have previously been diagnosed with liver disease or may never have been diagnosed with liver disease.

[0042] As used herein, all terms “reduce,” “reduced,” “reducing,” or “inhibit” mean a reduction or decrease in a property, level, or other parameter by a statistically significant amount. In some embodiments, “reduced,” “reducing” or “reduction” or “inhibit” generally means a reduction of at least 10% compared to a reference level (e.g., in the absence of a given treatment), and can include, for example, a reduction of 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” does not include complete inhibition or reduction compared to a reference level. “Complete inhibition” is 100% inhibition compared to a reference level. Reduction can preferably be as low as an acceptable level within the normal range for an individual without a given condition. For example, inhibiting WISP1 is inhibiting WISP1 activity or reducing WISP1 protein level.

[0043] As used herein, all terms “increased,” “increase,” or “enhanced” or “activate” generally mean an increase in a property, level, or other parameter by a statistically significant amount; for the avoidance of any doubt, the terms “increased,” “increase,” or “enhanced” or “activate” mean an increase of at least 10% compared to a reference level, e.g., an increase of at least about 20%, 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 80%, or at least about 90% or up to and including a 100% increase or any increase between 10 - 100% compared to a reference level, or an increase of at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold or at least about 10-fold increase, at least about 20-fold increase, at least about 50-fold increase, at least about 100-fold increase, at least about 1000-fold increase or more.

[0044] As used herein, “reference level” means a normal, otherwise unaffected cell population or tissue (e.g., a biological sample obtained from a healthy subject, or a biological sample obtained from a subject at a previous time point, e.g., a biological sample obtained from a patient before being diagnosed with liver disease, or a biological sample not contacted with a reagent or composition disclosed herein).

[0045] As used herein, “suitable control” means an untreated, otherwise identical cell or population (e.g., a biological sample not contacted with a reagent or composition described herein, or not contacted in the same manner, e.g., for a different duration compared to non-control cells).

[0046] The term "pharmaceutically acceptable" can refer to compounds and compositions that can be administered to a subject (e.g., a mammal or a human) without undue toxicity.

[0047] As used herein, the term "pharmaceutically acceptable carrier" can include any material or substance that, when combined with an active ingredient, allows the ingredient to retain its biological activity and does not react with the subject's immune system. Examples include, but are not limited to, any standard pharmaceutical carrier such as phosphate buffered saline solution, emulsions such as oil / water emulsions, and various wetting agents. The term "pharmaceutically acceptable carrier" does not include tissue culture medium. Non-limiting examples of pharmaceutical carriers include particulate or polymer-based carriers such as nanoparticles, microparticles, liposomes, polymeric microspheres, or polymer-drug conjugates.

[0048] As used herein, the term "WNT1 inducible signaling pathway protein 1" or "WISP1" or "CCN4" is a stromal cell protein encoded by the WISP1 gene and has many different cellular functions including cell adhesion, migration, proliferation, differentiation, and survival. In the liver, WISP1 is secreted by hepatic stellate cells (HSCs) which activate into myofibroblasts. Through an autocrine system, WISP1 also affects HSCs by accelerating the activation and secretion of collagen to promote fibrosis. The sequences of WISP1 (also known as CCN4, WISP1c, WISP1i, WISP1tc, WISP1-OT1, and WISP1-UT1) are known for many species, e.g., human WISP1 (NCBI Gene ID: 8840): polypeptide (e.g., NCBI Reference Sequence: NP_001191798.1) and mRNA (e.g., NCBI Reference Sequence: NM_001204869.1). WISP1 can refer to human WISP1, including its naturally occurring variants, molecules, and alleles. WISP1 refers to mammalian WISP1 such as, for example, mouse, rat, rabbit, dog, cat, bovine, equine, and porcine. The nucleic sequence of SEQ ID NO:5 includes the nucleic sequence encoding WISP1.

[0049] As used herein, the term "WISP1 activity" refers to the cellular functions of WISP1, e.g., WISP1 accelerates the activation and secretion of collagen to promote fibrosis of HSCs, and attenuates p53-mediated apoptosis, and WISP1 can inhibit TNF-induced cell death in other cell types. For example, an increase in WISP1 activity can refer to an increase in collagen deposition in cells. WISP1 activity can refer to the induction of α smooth muscle actin expression or the expression of some pro-inflammatory cytokines such as IL-6.

[0050] As used herein, the term "nucleic acid" or "nucleic acid sequence" refers to any molecule, preferably a polymeric molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or analogs thereof. The nucleic acid can be single-stranded or double-stranded. A single-stranded nucleic acid can be one strand of a denatured double-stranded DNA. Optionally, it can be a single-stranded nucleic acid that does not originate from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable DNA can include, for example, genomic DNA or cDNA. Suitable RNA can include, for example, mRNA.

[0051] As used herein, the term "reagent" refers to any compound or substance, such as, but not limited to, small molecules, nucleic acids, polypeptides, peptides, drugs, ions, etc. A "reagent" can be any chemical substance, entity, or moiety, including but not limited to synthetic and naturally occurring proteinaceous and non-proteinaceous entities. In some embodiments, the reagent is a nucleic acid, nucleic acid analog, protein, antibody, peptide, aptamer, oligomer of nucleic acid, amino acid, or carbohydrate, including but not limited to proteins, oligonucleotides, ribozymes, deoxyribozymes, glycoproteins, siRNA, lipoproteins, aptamers, and their modifications and combinations, etc. In certain embodiments, the reagent is a small molecule having a chemical moiety. For example, the chemical moiety includes an unsubstituted or substituted alkyl, aromatic, or heterocyclic moiety, including macrolides, rapamycins, and their related natural products or analogs thereof. The compound can be known to have a desired activity and / or property, or can be selected from a library of different compounds.

[0052] The reagent can be a molecule from one or more chemical classes, such as organic molecules, which can include organometallic molecules, inorganic molecules, gene sequences, etc. The reagent can also be a fusion protein, chimeric protein (e.g., domain swapping or homologous recombination of functional domains of related or different molecules), synthetic protein, or other protein variants including substitutions, deletions, insertions, and other variants from one or more proteins.

[0053] As used herein, "antibody" refers to an IgG, IgM, IgA, IgD, or IgE molecule or an antigen-specific antibody fragment thereof (including but not limited to, Fab, F(ab')2, Fv, disulphide-linked Fv, scFv, single-domain antibody, locked conformation multispecific antibody, disulphide-linked scfv, diabody), whether derived from any substance that naturally produces antibodies, or produced by recombinant DNA technology; whether isolated from serum, B cells, hybridomas, transfectomas, yeast, or bacteria.

[0054] In another example, the antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. It should be noted that V HRegions (e.g., portions of immunoglobulin polypeptides that are not identical to the V H segments, as described elsewhere herein). The V H and V L regions can be further subdivided into hypervariable regions called "complementary determining regions" ("CDRs") interspersed with more conserved regions called "framework regions" ("FRs"). The ranges of the framework regions and CDRs have been precisely defined (see, Kabat, E.A. et al. (1991) Sequences of Proteins of Immunological Interest, 5th ed., U.S. Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; which are incorporated herein by reference in their entirety). Each V H and V L is typically composed of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues joined to one another by peptide bonds between the α-amino and carboxyl groups of adjacent residues.

[0055] As used herein, the term "antibody agent" refers to a polypeptide that comprises at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds a given antigen. Antibody agents can include antibodies or polypeptides comprising antigen-binding domains of antibodies. In some embodiments in any aspect, the antibody agent can include a monoclonal antibody or a polypeptide comprising the antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In another example, an antibody includes two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody agent" includes antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDRs, and domain antibody (dAb) fragments (see, e.g., Wildt et al., Eur J. Immunol. 1996;26(3):629-39; which is incorporated herein by reference in its entirety)) as well as intact antibodies. Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, or IgM (and their subtypes and combinations). Antibodies can be from any source, including mice, rabbits, pigs, rats, and primates (human and non-human primates) and primatized antibodies. Antibodies also include midbodies, nanobodies, humanized antibodies, chimeric antibodies, etc. Antibody agents can be antibody fragments.

[0056] The terms "protein" and "polypeptide" refer to polymers of amino acids regardless of their size or function, including modified amino acids (e.g., phosphorylated, glycosylated, glycated, etc.) and amino acid analogs. "Protein" and "polypeptide" are generally used to refer to relatively large polypeptides, while the term "peptide" is generally used to refer to small polypeptides, but these terms are used interchangeably in the art. When referring to gene products and their fragments, the terms "protein" and "polypeptide" can be used interchangeably herein. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments and other equivalents, variants, fragments, and analogs of the foregoing.

[0057] In the various embodiments described herein, it is also contemplated to include variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any of the specific polypeptides described. For amino acid sequences, those skilled in the art will recognize that single substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that change a single amino acid or a small percentage of amino acids in the coding sequence are "conservatively modified variants", where the change results in the amino acid being replaced by a chemically similar amino acid and the desired polypeptide activity is retained. Such conservatively modified variants are in addition to polymorphic variants, interspecies homologs, and alleles that are consistent with the present disclosure and do not exclude these.

[0058] As used herein, an "antigen" is a molecule that binds through a binding site on an antibody. Typically, an antigen binds through an antibody ligand and is capable of eliciting an antibody response in vivo. An antigen can be a polypeptide, protein, nucleic acid, or other molecule or a portion thereof. The term "epitope" refers to an epitope on an antigen that is recognized by an antigen-binding molecule, more specifically by the antigen-binding site of the molecule.

[0059] As used herein, the term "affinity" refers to the strength of an interaction such as an antibody binding an antigen, and can be quantitatively expressed as the dissociation constant (K D ). Avidity is a measure of the strength of the binding between an antigen-binding molecule (such as an antibody agent described herein) and a relevant antigen. Avidity is related to the affinity between the epitope and the antigen-binding site on the antigen-binding molecule and the number of relevant binding sites present on the antigen-binding molecule. Typically, an antigen-binding protein (such as an antibody agent described herein) will bind to its cognate or specific antigen with a dissociation constant (10 -5 to 10 -12 mol / L or less, preferably 10 -7 to 10 -12 mol / L or less and more preferably 10 -8 to 10 -12 mol / L of K D (i.e., having a binding constant (K 5 to 10 12 L / mol or more, preferably 10 7 to 10 12 L / mol or more and more preferably 10 8 to 10 12 L / mol of K A ). Any K -4 value greater than 10 D mol / L (or any K 4 M -1 less than 10 Avalues) are generally considered to indicate non - specific binding. For a K of a biological interaction that is considered significant (e.g., specific) D is typically in the range of 10 -10 M (0.1 nM) to 10 -5 M (10000 nM). The stronger the interaction, the lower its K D value. Preferably, the binding site on the antibody agents described herein will bind to the desired antigen with an affinity of less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 500 pM. Specific binding of the antibody agent to the antigen or epitope can be determined by any suitable method known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassay (RIA), enzyme immunoassay (EIA), and sandwich competition assay, and its various variants known per se in the art; and other techniques mentioned herein.

[0060] As used herein, the term "specific binding" or "specificity" refers to a chemical interaction between two molecules, compounds, cells, and / or particles, wherein the first entity binds to the second target entity with a higher specificity and affinity than it binds to a third entity that is a non - target entity. In some embodiments in any aspect, specific binding can mean that the affinity of the first entity for the second target entity is at least 10 - fold, at least 50 - fold, at least 100 - fold, at least 500 - fold, at least 1000 - fold, or more than its affinity for the third non - target entity. Thus, as used herein, "selectively bind" or "specifically bind" refers to the ability of a reagent (e.g., an antibody agent) described herein to bind to a target such as a peptide, including, for example, the amino acid sequence of a given antigen, with a K D of 10 -5 M (10000 nM) or less, such as 10 -6 M or less, 10 -7 M or less, 10 -8 M or less, 10 -9 M or less, 10 -10 M or less, 10 -11 M or less, or 10 -12 M or less. For example, if a reagent described herein binds to a first peptide comprising an antigen with a K -5 of 10 D M or less but does not bind to another randomly selected peptide, the reagent is said to specifically bind the first peptide. Specific binding can be affected by, for example, the affinity and avidity of the reagent and the concentration of the reagent. One of ordinary skill in the art can use any suitable method, such as titration of the reagent in a suitable cell and / or peptide binding assays, to determine the appropriate conditions for the reagent to selectively bind to the target.

[0061] The term "expression" refers to cellular processes involved in the production of RNA and proteins, and where appropriate, secreted proteins, including but not limited to, for example, transcription, transcript processing, translation, and protein folding, modification, and handling. "Expression products" include RNA transcribed from a gene, and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" refers to a nucleic acid sequence that is transcribed (DNA) into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions before and after the coding region, for example, 5' untranslated (5' UTR) or "leader" sequences and 3' UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0062] As used herein, the term "contacting", when used with respect to a cell or an organ, includes introducing a reagent, surface, hormone, etc. into the cell in a manner that allows physical contact of the cell with the reagent, surface, hormone, etc., and introducing elements such as a gene construct or vector that allows expression of a reagent such as mRNA, polypeptide, or other expression products in the cell. It is understood that a cell modified to express a reagent is "contacted" with the reagent, as are the progeny of the cell expressing the reagent.

[0063] The term "statistically significant" or "significantly" means statistically significant and generally refers to a difference of two standard deviations (2SD) or greater.

[0064] As used herein, the term "comprising" means that other elements may also be present in addition to the recited elements. The use of "comprising" indicates inclusion rather than limitation.

[0065] The term "consisting of" refers to the compositions, methods, and their respective components as described herein, which do not include any element not recited in the description of the embodiment.

[0066] As used herein, the term "consisting essentially of" refers to those elements required for a given embodiment. The term permits the presence of other elements that do not substantially affect the basic and novel or functional characteristics (one or more) of the embodiment of the invention.

[0067] Unless the context clearly indicates otherwise, the singular terms "a", "an", and "the" include plural referents. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the following describes suitable methods and materials. The abbreviation "e.g." is derived from the Latin exempli gratia and is used herein to denote non-limiting examples. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0068] Furthermore, unless the context requires otherwise, singular terms shall include the plural and plural terms shall include the singular.

[0069] Also as used herein, "and / or" means and includes any and all possible combinations of one or more of the associated listed items, as well as the absence of combinations when interpreted in the alternative ("or").

[0070] In addition, when referring to measurable values such as amounts, doses, times, and temperatures of the compositions of the present invention, for example, the term "about" as used herein means including variations of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of a particular amount. Except in the operating examples, or otherwise indicated, all numbers expressing amounts of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about". When used with percentages, the term "about" may be ±1% of the average value.

[0071] Unless otherwise noted, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains.

[0072] It is to be understood that the present disclosure is not limited to the specific methods, protocols, and agents, etc. described herein and may be varied. The terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the present disclosure, the scope of which is defined only by the claims.

[0073] All patents and other publications identified are hereby expressly incorporated by reference for the purpose of describing and disclosing, for example, the methods described in such publications that may be used in conjunction with the present disclosure. These publications are provided only for their disclosure prior to the filing date of the present application. In this regard, nothing should be construed as an admission that the inventors are not entitled to antedate such disclosure by virtue of prior disclosure or for any other reason. All statements as to the date or content of these documents are based on information available to the applicant and do not constitute any admission as to the correctness of the dates or content of these documents. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 Schematic showing the research plan: Aim 1 to explore the mechanisms and functions of miR-15a and miR-412, Aim 2 to test their therapeutic potential, and Aim 3 to illustrate the function of the known miR-15a target WISP1. HSC is the term used for hepatic stellate cells.

[0075] Figure 2 Schematic showing the experimental procedure of screening a genome-wide microRNA mimic library to identify candidates that will revert activated hepatic stellate cells towards quiescence.

[0076] Figure 3 Shown is the reversion of activated mouse hepatic stellate cells (HSCs) towards quiescence, as illustrated by the reformation of Nile Red-stained positive lipid droplets, upon overexpression of miR-15a or miR-412 (top row). Figure 3 Also shown is the reversion of activated human HSCs towards quiescence, as illustrated by the reformation of lipid droplets, upon overexpression of the human orthologs of miR-15a or miR-412 (bottom row).

[0077] Figure 4A and Figure 4B Shown is that delivery of miR-15a or miR-412 to activated hepatic stellate cells (HSCs) results in morphological changes towards quiescence. Figure 4A Shown is a 10 - 100-fold decrease in the size of HSCs, with all photographs taken at the same magnification (the same scale bar is used for all figures). Figure 4B Shown is that forced expression of miR-15a or miR-412 in initially activated HSCs, as determined by qPCR, downregulates α-smooth muscle actin (Acta2) and α-1 type I collagen (Col1a1). Data are represented as mean + / − SD.

[0078] Figure 5 Shown is that miR-15a- or miR-412-transfected hepatic stellate cells (HSCs) have a functional phenotype. Activated HSCs treated with miR-15a or miR-412 do not cause steatosis in co-cultured hepatocytes. Activated HSCs treated with a negative control microRNA induce steatosis in co-cultured hepatocytes. qHSC, quiescent hepatic stellate cells; Ac-HSC, activated hepatic stellate cells; miR-Neg, microRNA negative control.

[0079] Figure 6HepG2 cells co-cultured with human HSCs activated to receive miR-15a or miR-412 showed reduced expression of pro-inflammatory cytokines (left panel). Huh7 cells co-cultured with human HSCs activated to receive miR-15a or miR-412 showed reduced expression of the same pro-inflammatory cytokines (right panel). Data are represented as mean + / - SD.

[0080] Figure 7 Endogenous miR-15a or miR-412 showed reduced expression levels in activated primary HSCs compared to quiescent HSCs, although not significantly for miR-15a. Data are represented as mean + / - SD.

[0081] Figure 8 Schematic (diagram) showing the schedule of CCl4 challenge and cell therapy injection. Top row: Quiescent-like HSCs reprogrammed with miR-15a or miR-412 injected into the spleen engrafted in the liver, demonstrated by GFP signal from the vector incorporated in the liver to drive miRNA expression. Middle row: Mice challenged with CCl4 receiving quiescent-like HSCs had reduced hepatocyte apoptosis and inflammation, demonstrated by H&E staining. Bottom row: Mice challenged with CCl4 receiving quiescent-like HSCs had reduced liver fibrosis stained with Sirius Red. Quantification of the relative level of fibrosis. CCl4, CCl4 gavaged without injecting HSCs; HSC control, CCl4 gavaged and injected with HSCs with empty GFP vector; HSC miR-15a, CCl4 gavaged and injected with HSCs with miR-15a-GFP vector; HSC miR-412, CCl4 gavaged and injected with HSCs with miR-412-GFP vector.

[0082] Figure 9 Cell therapy with reprogrammed quiescent-like HSCs resulted in reduced expression of alpha-1 type I collagen (Col1a1) throughout the liver in mice challenged with CCl4, determined by qPCR. Data are represented as mean + / - SD.

[0083] Figure 10 Hepatic stellate cells in human primary biliary cholangitis express WISP1 that co-localizes with alpha smooth muscle actin (Acta2).

[0084] Figure 11 miR-15a mimics co-transfected with a reporter containing either of two predicted WISP1 target sequences reduced luciferase expression, while it did not affect the reporter containing the mutant sequence, indicating that miR-15a binds to the two WISP1 target sequences. Data are represented as mean + / - SD (**P<0.01; ***P<0.001).

[0085] Figure 12 Show quiescent HSCs (left column) at low and high magnifications. The high-magnification view clearly shows several lipid droplets fluorescing green by BODIPY staining (inset). Right column: Activated HSCs at low and high magnifications. Activated cells are very large, lack lipid droplets, and are not stained by BODIPY (inset). HSC, hepatic stellate cell.

[0086] Figure 13 Show a comparison of mice fed a standard diet (left column) and those fed a CDAHFD (right column). Mice fed a CDAHFD develop early NASH at three weeks, showing increased body size, gross and microscopic fatty liver, and fibrosis traced by Sirius red staining. CDAHFD, choline-deficient L-amino acid-defined high-fat diet.

[0087] Figure 14 Show a schematic of an experiment depicting co-culture of healthy hepatocytes (Hep) with control hepatic stellate cells (HSCs) from unchallenged mice or HSCs from CDAHFD-challenged NASH mice. Top row: Hepatocytes co-cultured with HSCs from control mice show very few lipid droplets stained by BODIPY. Bottom row: Hepatocytes co-cultured with HSCs from NASH mice show significantly more lipid droplets. CDAHFD, choline-deficient L-amino acid-defined high-fat diet.

[0088] Figure 15 Show that hepatocytes co-cultured with hepatic stellate cells (HSCs) from NASH mice express higher levels of several inflammatory cytokines and chemoattractants than when they are co-cultured with control HSCs, as determined by qPCR. Data are presented as mean + / − SD.

[0089] Figure 16 Show that induction of lipid accumulation in hepatocytes (Hep) can be achieved when conditioned medium from NASH-hepatic stellate cells (HSCs) is applied to normal hepatocytes. Top row: When quiescent HSC (qHSC) medium is applied to healthy hepatocytes, steatosis is not induced. Bottom row: NASH-HSC medium induces steatosis in initially healthy hepatocytes. Lipid droplets are stained by BODIPY.

[0090] Figure 17 Show that delivery of miR-15a or miR-412 to activated hepatic stellate cells (AcHSCs) induces re-formation of lipid droplets positive for retinoid, as demonstrated by fluorescence under ultraviolet light, consistent with those in quiescent hepatic stellate cells (qHSCs).

[0091] Figure 18 RNA sequencing data analyzed by multidimensional scaling shows that quiescent-like HSCs receiving miR-15a or miR-412 have a global transcriptional profile that is 4D-50% closer to quiescent HSCs than activated cells. HSC, hepatic stellate cell.

[0092] Figure 19 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 do not cause steatosis in co-cultured hepatocytes. Activated HSCs treated with a negative control microRNA induce steatosis in co-cultured hepatocytes. qHSC, quiescent hepatic stellate cell: Ac-HSC, activated hepatic stellate cell; miR-Neg, microRNA negative control. Bottom row: HSCs harvested from the CDAHFD model of NASH induce steatosis in co-cultured hepatocytes. These same HSCs infected with lentiviruses expressing miR-15a or miR-412 bet their ability to induce steatosis in adjacent hepatocytes.

[0093] Figure 20 Shows that after receiving miR-15a or miR-412, covering the set of genes with reduced mRNA levels in hepatic stellate cells with a set of potential direct targets based on a prediction algorithm yields a set of target candidates that is more likely to contain true miRNA targets. Genes in the set of target candidates are further screened by selecting those that are part of the Tgf-β or Pdgf signaling pathways.

[0094] Figure 21 Shows that CRISPR technology can be used on primary hepatic stellate cells. The feasibility of using the technology on primary cells is verified by delivering a long non-coding RNA Digit deletion vector. Homozygous knock-in is demonstrated by the presence of a new PCR band, one allele with a puromycin construct and the other with a neomycin construct. Ctl, control construct; KI, knock-in construct.

[0095] Figure 22 Shows western blots of >100 cytokines, chemokines, and extracellular matrix proteins, showing that HSCs from mice with CDAHFD induce up-secreted WISP1 on NASH compared to HSCs from healthy mice. CM, conditioned medium.

[0096] Figure 23Activated hepatic stellate cells (AcHSCs) were shown to express approximately 30-fold higher levels of WISP1 than quiescent hepatic stellate cells (qHSCs). Hepatocytes (Heps) also expressed WISP1, but at significantly lower levels than activated hepatic stellate cells. Lv, whole liver. Data are represented as mean + / - SD.

[0097] Figure 24 Conditioned medium from hepatic stellate cells overexpressing WISP1 was shown to induce steatosis in hepatocytes harvested from healthy mice. CM, conditioned medium.

[0098] Figure 25 Is a schematic diagram showing WISP1 involved in human diseases.

[0099] Figure 26 Is a schematic diagram showing that WISP1 is a member of the CCN family of secreted matrix cell proteins.

[0100] Figure 27 Is a schematic diagram showing that hepatic stellate cells are key drivers of liver fibrosis.

[0101] Figure 28 Is a schematic diagram showing the activation of hepatic stellate cells (HSCs).

[0102] Figure 29A and 29B Show that WISP1 is highly upregulated in activated HSCs and is a direct target of miR-15a. ( Figure 29A ) WISP1 is secreted by activated hepatic stellate cells in NASH mice. ( Figure 29B ) Luciferase expression was reduced by co-transfection of miR-15a mimics with a reporter containing the predicted WISP1 target sequence.

[0103] Figure 30A - 30C Show that WISP1 and Yap 1 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 HSCs reprogrammed by miRNA.

[0104] Figure 31A and 31B Show existing literature supporting the role of WISP inhibition in liver and lung fibrosis. ( Figure 31A ) Anti-WISP1 mAb attenuated CCl4-induced liver fibrosis. ( Figure 31B ) Anti-WISP1 mAb attenuated bleomycin-induced lung fibrosis.

[0105] Figure 32A and 32B Showing that neutralizing antibodies against WISP1 attenuate biliary fibrosis.( Figure 32A ) Reduction of bile duct ligation (BDL)-induced pulmonary fibrosis by in vivo treatment with neutralizing antibodies against WISP1.( Figure 32B ) Collagen expression in the specified conditions.

[0106] Figure 33A and 33B Showing that WISP1 is secreted in common and rare human fibrotic liver diseases.( Figure 33A ) WISP1 is upregulated in HSCs of NASH.( Figure 33B ) WISP1 is upregulated in HSCs of several rare fibrotic liver diseases.

[0107] Figure 34A and 34B Showing that WISP1 is a newly secreted fibrogenic driver for liver inflammation and fibrosis.( Figure 34A ) Activated HSCs from NAFLD mice secrete WISP1.( Figure 34B ) WISP1 overexpressing HSCs induce steatosis (BODIPY staining) in healthy co-cultured primary hepatocytes.

[0108] Figure 35 Showing that Rc WISP1 treatment accelerates HSC proliferation, as determined by ki67.

[0109] Figure 36 Showing the sequence alignment of human and mouse miR-15a and miR-412.

[0110] Figure 37A and 37B Showing that WISP1 expressed by HSCs has autocrine and paracrine effects of self-activation and promoting steatosis. Figure 37A Showing the induction of HSC proliferation and activation by recombinant WISP1 treatment in mice. Figure 37B Showing the immunohistochemistry of control and Rc Wisp1.

[0111] Figure 38A - 38F Showing that Rc WISP1 and Wisp IgG treatment of NASH-HSCs from a choline-deficient L-amino acid-defined high-fat diet model-induced steatosis (CDAHFD) and prevent fibrosis. Before administration to HSCs, 1 μg / ml WISP1 antibody was cultured in conditioned medium at 37 °C for 1 hour. Figure 38A Showing the timeline of treatment and sampling. Figure 38B Showing the cell numbers of control, Rc WISP1-treated, and Ab WISP1-treated cells. Figure 38CShow Acta2 mRNA expression in qHSC, control, Rc WISP1-treated, and Ab WISP1-treated cells. Figure 38D Show Col1a1 mRNA expression in qHSC, control, Rc WISP1-treated, and Ab WISP1-treated cells. Figure 38E Show immunohistochemistry in control, Rc WISP1-treated, and Ab WISP1-treated cells. Figure 38F Show quantification of the BODIPY stained area fraction in control, Rc WISP1-treated, and Ab WISP1-treated cells.

[0112] Figure 39A and 39B Show that WISP1 regulates HSC migration. Figure 39A Show images of HSC migration in the presence and absence of Rc-WISP1 and WISP1IgG. Figure 39B Show the relative wound area of HSC treated with Rc-WISP1 and WISP1 IgG compared to control HSC. HSC treated with WISP1 IgG showed a relative wound area similar to control HSC within 24 hours, demonstrating that WISP1 IgG can prevent HSC activation and migration. Detailed implementation

[0113] Liver diseases (e.g., primary biliary cholangitis (PBC)) can lead to progressive destruction of intrahepatic bile ducts, cholestasis, periportal inflammation, and ultimately biliary fibrosis, ending in cirrhotic end-stage liver disease. Liver diseases such as PBC can present as autoimmune diseases of unknown pathogenesis, although both genetic and environmental factors may contribute to liver diseases.

[0114] WISP1 is a protein secreted by hepatic stellate cells (HSCs) of the liver, which activate into myofibroblasts. Through an autocrine system, WISP1 also affects HSCs by accelerating the activation and secretion of collagen to promote fibrosis. WISP is highly expressed in fibroblasts in liver diseases (e.g., primary biliary cholangitis, autoimmune hepatitis, α1-antitrypsin deficiency, non-human diseases, non-alcoholic steatohepatitis, and scleroderma). Therefore, it is shown that inhibiting WISP1 is a strategy for treating liver diseases.

[0115] Hepatic stellate cells are the fat-storing pericytes of the liver and are found in the perisinusoidal space (also known as the space of Disse). Quiescent HSCs are identified by the presence of cytoplasmic lipid droplets. When the liver is damaged, stellate cells can become activated. Activated HSCs proliferate and have reduced lipid droplets in the cytoplasm. Activated HSCs become profibrotic myofibroblasts that secrete collagen and mediators that promote scar formation.

[0116] The methods described herein show that WISP1 is upregulated in HSCs. When WISP1 is inhibited by microRNAs, miR-15a and miR-412, this inhibition independently induces quiescence of activated hepatic stellate cells (HSCs), which are the cell type that plays an important role in the progression of liver fibrosis. In addition, miR-15a directly targets WISP1 to inhibit the profibrotic function of the protein in activated HSCs. By using microRNAs and their targets to promote HSC quiescence, WISP1 inhibitors are useful therapies for controlling progressive liver fibrosis in liver diseases such as PBC.

[0117] Treatment and / or prevention of liver diseases

[0118] The methods and compositions described herein are used for the treatment and / or prevention of liver diseases in a subject. Exemplary liver diseases include, but are not limited to, Alagille syndrome; alcohol-related liver disease; α-1 antitrypsin deficiency; autoimmune hepatitis; benign liver tumors; biliary atresia; cirrhosis; Crigler-Najjar syndrome; galactosemia; Gilbert syndrome; hemochromatosis; hepatic encephalopathy; hepatitis A; hepatitis B; hepatitis C; hepatorenal syndrome; intrahepatic cholestasis of pregnancy (ICP); lysosomal acid lipase deficiency (LAL-D); liver cysts; liver cancer; neonatal jaundice; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis; primary biliary cholangitis (PBC); primary sclerosing cholangitis (PSC); progressive familial intrahepatic cholestasis (PFIC); Reye's syndrome; type I glycogen storage disease; scleroderma; and Wilson's disease. In some embodiments, the liver disease is primary biliary cholangitis, autoimmune hepatitis, α1 antitrypsin deficiency, non-alcoholic steatohepatitis, and scleroderma.

[0119] In one embodiment, the methods described herein are used to treat a subject suffering from liver failure, such as failure of liver synthetic and metabolic functions. In one embodiment, the methods described herein are used to treat a subject suffering from fulminant or severe acute liver failure (e.g., development of liver failure with encephalopathy in a previously healthy individual within < 8 weeks); hyperacute liver failure (e.g., development of liver failure with encephalopathy in a previously healthy individual within < 14 days); acute liver failure (development of liver failure with encephalopathy in a previously healthy individual within < 26 weeks); chronic liver failure (e.g., liver failure without encephalopathy); or acute-on-chronic liver failure (e.g., chronic liver failure with development of encephalopathy). A skilled artisan can use standard methods to evaluate the severity of liver disease (e.g., liver failure), such as the Child-Pugh score (which is a composite of total bilirubin, albumin, INR, ascites, and hepatic encephalopathy), the MELD score (which uses serum bilirubin, creatinine, and INR), or the PELD score (similar to MELD but for pediatric patients), or the METAVIR score (which evaluates the level of fibrosis in a sample. METAVIR

[0120] In one embodiment, the methods described herein are used to treat a subject suffering from liver fibrosis. Liver fibrosis can be diagnosed by a skilled clinician using, for example, the METAVIR score, etc. The METAVIR score provides two scores, a fibrosis score and an activity score. The fibrosis score is used to describe the amount of inflammation in the liver (intensity of inflammation / decomposition of the tissue), e.g., F0: no fibrosis; F1: partial fibrosis, no septa; F2: partial fibrosis, few septa; F3: many septa, no cirrhosis; F4: cirrhosis. The activity score is a prediction of the rapidity of progression of the degree of fibrosis, e.g., A0: no activity; A1: mild activity; A2: moderate activity; and A3: severe activity. In one embodiment, the subject treated with the methods described herein has a METAVIR score of F1, F2, F3, or F4 and / or A1, A2, A3.

[0121] In another aspect, the present disclosure describes methods of treating or preventing liver disease, the methods comprising: administering to a subject in need thereof an antibody or an antibody agent that inhibits WISP1.

[0122] In one aspect, the present disclosure describes methods for treating or preventing liver disease, comprising administering to a subject in need thereof an agent that inhibits WISP1.

[0123] In various embodiments, WISP1 is inhibited in target cells. In one embodiment, the target cells are hepatocytes that highly express WISP1 and result in a disease state such as liver disease. For example, hepatocytes with increased WISP1 levels compared to a suitable control such as healthy, non-diseased hepatocytes. Those skilled in the art can use standard techniques to evaluate the mRNA or protein levels of WISP1 in cells, using PCR-based assays or immunoblotting, respectively.

[0124] In another embodiment, the target cells are hepatic stellate cells (HSCs). HSCs are pericytes found in the perisinusoidal space of the liver. Those skilled in the art can determine whether a cell is an HSC using, for example, selective staining with gold chloride or visualization of lipid droplets in the cytoplasm. In one embodiment, the HSCs are quiescent. Quiescent HSCs can be identified by those skilled in the art by the presence of lipid droplets in the cytoplasm of non-proliferating cells. Activated HSCs can be identified by those skilled in the art by evaluating cell proliferation, reduced lipid droplets within the cytoplasm, and / or secretion of collagen and mediators that promote scar formation.

[0125] In one embodiment, the target cells are fibroblasts, such as hepatic fibroblasts. In one embodiment, the target cells are myofibroblasts. Myofibroblasts have the characteristics of both fibroblasts and smooth muscle cells. Fibroblasts and myofibroblasts can be readily identified by those skilled in the art, for example, by separately selecting fibroblast or fibroblast and smooth muscle cell markers through microscopy.

[0126] In one embodiment, the target cells are mammalian cells, preferably human cells.

[0127] In another aspect, a method of treating liver disease is described herein, which includes (a) determining the levels of WISP1 and / or Yap, Col1a1, Acta2 in a biological sample of a subject; (b) comparing the determination result of (a) with a reference level; (c) identifying a subject having increased WISP1 and / or Yap, Col1a1, Acta2 compared to the reference level in (a) as having liver disease; and (d) administering an antibody or an antibody agent that inhibits WISP1 to the subject having liver disease.

[0128] In still another aspect, a method of treating liver disease is described herein, which includes (a) determining the levels of WISP1 and / or Yap, Col1a1, Acta2 in a biological sample of a subject; (b) comparing the determination result of (a) with a reference level; (c) identifying a subject having increased WISP1 and / or Yap, Col1a1, Acta2 compared to the reference level in (a) as having liver disease; and (d) administering a reagent that inhibits WISP1 to the subject having liver disease.

[0129] Assays for determining the levels of WISP1 and / or Yap, Col1a1, Acta2 include, but are not limited to, PCR-based assays to evaluate the levels of WISP1 and / or Yap, Col1a1, Acta2 mRNA, or immunoblotting to evaluate the levels of WISP1 and / or Yap, Col1a1, Acta2 protein. In one embodiment, compared to a reference level, the levels of WISP1 and / or Yap, Col1a1, Acta2 are increased by at least 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 more, or at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold or more. In one embodiment, the levels of WISP1 and / or Yap, Col1a1, Acta2 are the levels of WISP1 and / or Yap, Col1a1, Acta2 mRNA. In another embodiment, the levels of WISP1 and / or Yap, Col1a1, Acta2 are the levels of WISP1 and / or Yap, Col1a1, Acta2 protein.

[0130] As used herein, "reference level" refers to a biological sample from a healthy subject, such as a subject without liver disease, that is otherwise identical.

[0131] In one embodiment, the methods described herein further include obtaining a biological sample from a subject prior to (a). As used herein, a biological sample refers to a blood sample, a tissue sample, a buffy coat sample (e.g., a portion of an anticoagulated blood sample containing high levels of white blood cells and platelets after centrifugation, a serum sample, or a liver biopsy sample). Biological samples can be obtained using generally suitable techniques known in the art. For example, tissue samples can be obtained by biopsy, and blood samples can be obtained from finger pricks or intravenous blood draws.

[0132] In various embodiments, the biological sample is taken from a subject who has previously been diagnosed with liver disease or who has not been diagnosed with liver disease. In another embodiment, the biological sample is taken from a subject suspected of having liver disease, e.g., a subject who has at least one risk factor for liver disease, such as increased alcohol intake compared to normal intake.

[0133] Another aspect provided herein is a method for treating liver disease in a subject, comprising (a) receiving the results of a test that identifies a subject having increased levels of WISP1 and / or Acta2 compared to a reference level as having liver disease; and (b) administering to the subject having liver disease an antibody or an antibody agent that inhibits WISP1.

[0134] Another aspect provided herein is a method for treating liver disease in a subject, comprising (a) receiving the results of a test that identifies a subject having increased levels of WISP1, Yap, Col1a1, and / or Acta2 compared to a reference level as having liver disease; and (b) administering to the subject having liver disease a reagent agent that inhibits WISP1.

[0135] Assays for determining the levels of WISP1, Yap (NCBI Gene ID 10413), Col1a1 (NCBI Gene ID 1277), and / or Acta2 (NCBI Gene ID 59) include, but are not limited to, PCR-based assays to evaluate the levels of WISP1 and / or Yap, Col1a1, Acta2 mRNA, or immunoblotting to evaluate the levels of WISP1 and / or Yap, Col1a1, Acta2 protein. The assays (e.g., PCR-based assays to evaluate the levels of WISP1 and / or Yap, Col1a1, Acta2 mRNA, or immunoblotting to evaluate the levels of WISP1 and / or Yap, Col1a1, Acta2 protein) can be performed by a skilled technician administering a reagent that inhibits WISP1 (e.g., an antibody or an antibody agent). Optionally, the assay can be performed by another individual (i.e., without a practitioner administering a reagent that inhibits WISP1 (e.g., an antibody or an antibody agent)). The results of the assay can be received in any manner, e.g., by a postal courier, telephone transmission (e.g., fax), electronic transmission (e.g., electronic medical record, electronic letter (e-mail), etc.). In one embodiment, treatment is administered to the subject at any time (e.g., at least 1 second, 1 minute, 1 hour, 1 day, 1 week, 1 month, 1 year, or longer) after receiving the assay results.

[0136] In one embodiment, compared to a reference level, the levels of WISP1 and / or Yap, Col1a1, Acta2 are increased by at least 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 more, or at least 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 11-fold, 12-fold, 13-fold, 14-fold, 15-fold, 16-fold, 17-fold, 18-fold, 19-fold, 20-fold, 21-fold, 22-fold, 23-fold, 24-fold, 25-fold or more. In one embodiment, the levels of WISP1 and / or Yap, Col1a1, Acta2 are the levels of WISP1 and / or Yap, Col1a1, Acta2 mRNA. In another embodiment, the levels of WISP1 and / or Yap, Col1a1, Acta2 are the levels of WISP1 and / or Yap, Col1a1, Acta2 protein.

[0137] In another aspect, methods for treating or preventing liver diseases are described herein, which comprise administering to a subject in need thereof any cell produced using the methods described herein, or any pharmaceutical composition comprising a cell produced using the methods described herein.

[0138] In another aspect provided herein, any antibody or antibody agent, reagent, cell, component of the pharmaceutical composition described herein produced using the methods described herein can be used to treat or reduce fibrosis of the liver of a subject. In one aspect described herein, a method for reducing fibrosis such as liver fibrosis comprises administering to a subject in need thereof any cell produced using the methods described herein, or any pharmaceutical composition comprising a cell produced using the methods described herein.

[0139] In one embodiment, the subject has been previously diagnosed with liver disease. In another embodiment, the subject is diagnosed with liver disease prior to administration of the reagent. A subject with liver disease can be diagnosed by those skilled in the art using, for example, standard techniques in the art. For example, a blood test known as a liver function test; non-invasive imaging such as CT scan, ultrasound, or MRI scan; or a tissue biopsy. A liver function test, a blood test, evaluates the levels of liver-specific enzymes such as alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), albumin, or bilirubin. Even a slight increase in the levels of ALT, AST, ALP, albumin, or bilirubin can indicate liver disease. Other tests for evaluating liver disease are described herein (e.g., Child-Pugh score, METAVIR score, and PELD and MELD scores).

[0140] In another embodiment, the reagent WISP1 described herein is a splicing variant that is inhibited in target cells. Exemplary WISP1 splicing variants include, but are not limited to, WISP1v, WISP1vx, and WISP1δ exons 3-4.

[0141] In another embodiment, inhibiting WISP1 is inhibiting WISP1 activity. WISP1 activity can be any currently known activity, or an activity of a yet-to-be-discovered function of the WISP1 gene or gene product. For example, WISP1 accelerates the activation and secretion of collagen to promote fibrosis of HSCs, and attenuates p53-mediated apoptosis and inhibits TNF-induced cell death in other cell types. In another embodiment, the activity of WISP1 is inhibited by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to a suitable control.

[0142] As used herein, "suitable control" refers to the level of WISP1 activity in an otherwise identical sample that is not contacted with the reagent or composition described herein, or the level of WISP1 activity in a subject prior to administration of the reagent or composition. Additionally, a suitable control can be, for example, the level of WISP1 activity in a healthy subject such as an individual without liver disease. Those skilled in the art can use a functional readout of WISP1 activity to determine the activity of WISP1, for example, by measuring / evaluating / quantifying the activity and secretion of collagen in HSCs.

[0143] In another embodiment, inhibiting WISP1 is inhibiting the level of WISP1 in a cell, e.g., the gene expression level or the gene product level. In another embodiment, the level of WISP1 is inhibited by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99% or more compared to a suitable control. As used herein, a "suitable control" is the level of WISP1 in an otherwise identical sample that is not contacted with the reagents or compositions described herein, or the level of WISP1 in a subject prior to administration of the reagent or composition. Additionally, a suitable control can be, for example, the level of WISP1 in a healthy subject such as an individual without liver disease. One of ordinary skill in the art can use a functional readout of WISP1 activity to determine the activity of WISP1, e.g., by assaying / evaluating the activity and secretion of collagen in HSCs. One of ordinary skill in the art can evaluate / assay the protein and mRNA levels of WISP1, e.g., using immunoblotting or PCR-based assays, respectively.

[0144] Reagent

[0145] In one aspect, a reagent that inhibits WISP1 is administered to a subject having liver disease or at risk of having liver disease. In one embodiment, the reagent is a small molecule, an antibody, a peptide, a genome editing system, a viral vector, an miRNA, and an siRNA.

[0146] A reagent described herein is considered effective for inhibiting WISP1 if, for example, in the case of administration, the reagent inhibits the presence, amount, activity, and / or level of WISP1 in a cell.

[0147] The reagent can inhibit, for example, the transcription or translation of WISP1 in a cell. The reagent can inhibit or alter the activity of WISP1 in a cell (e.g., the expression of WISP) (e.g., such that the activity no longer occurs, no longer occurs correctly (e.g., compared to wild-type WISP1 activity), or occurs at a reduced rate).

[0148] In one embodiment, the reagent does not include miRNA412 and miRNA15a. In another embodiment, the reagent does not include any miRNA412 or miRNA 15a mimics.

[0149] The reagent can act directly in the form in which it is administered. Optionally, the reagent can be modified or utilized intracellularly to produce something that inhibits WISP1. For example, a nucleic acid sequence can be introduced into a cell and its transcription causes the production of nucleic acid and / or protein inhibitors of intracellular WISP1. In some embodiments, the reagent is any chemical entity or moiety, including but not limited to synthetic and naturally occurring non-protein entities. In certain embodiments, the reagent is a small molecule having a chemical moiety. For example, the chemical moiety includes unsubstituted or substituted alkyl, aromatic, or heterocyclic moieties including macrolides, rapamycins, and their related natural products or analogs. The reagent can be known to have the desired activity and / or property, or can be identified from a library of different compounds.

[0150] In various embodiments, the reagent is a small molecule that inhibits WISP1. As used herein, the term "small molecule" refers to a chemical reagent that can include but is not limited to peptides, peptidomimetics, amino acids, amino acid analogs, polynucleotides, polynucleotide analogs, aptamers, nucleotides, nucleotide analogs, organic or inorganic compounds having a molecular weight of less than about 10,000 grams per mole (e.g., including heteroorganic and organometallic compounds), organic or inorganic compounds having a molecular weight of less than about 5,000 grams per mole, organic or inorganic compounds having a molecular weight of less than about 1,000 grams per mole, inorganic or organic compounds having a molecular weight of less than about 500 grams per mole, and salts, esters, and other pharmaceutically acceptable forms of such compounds.

[0151] Given a desired target (e.g., a WISP1 polypeptide), methods for screening small molecules are known in the art and can be used to identify small molecules that are effective, for example, in inhibiting WISP activity or level.

[0152] One aspect provided herein is a composition comprising any reagent that inhibits WISP1 as described herein. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In one embodiment, the composition is a pharmaceutical composition.

[0153] A polypeptide that inhibits WISP1

[0154] In one embodiment, methods for treating or preventing liver disease are described herein, including administering to a subject in need thereof a polypeptide in which WISP1 is inhibited in target cells or a nucleic acid encoding such a polypeptide.

[0155] The term "WISP1-binding polypeptide" refers to a polypeptide (e.g., a WISP1 polypeptide) that specifically binds to a desired antigen of interest and is an Ig-like protein that comprises one or more antigen-binding domains described herein linked to a linker or an immunoglobulin constant domain. In some embodiments, the binding protein can be a dual variable domain (DVD-Ig) binding protein. "Linker polypeptide" includes one or more amino acid residues joined by peptide bonds and is used to link one or more antigen-binding moieties. Such linker polypeptides are known in the art (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123). Immunoglobulin constant domain refers to a heavy chain or light chain constant domain. The amino acid sequences of human IgG heavy chain and light chain constant domains are known in the art (e.g., see SEQ ID NOs: 197, 198, 199, and 200 of U.S. Application 2016 / 0200813, which are incorporated herein by reference in their entirety as representative examples).

[0156] In some embodiments, the polypeptide that inhibits WISP1 is heterologous. As used herein, "heterologous" means a polypeptide that is not normally produced by a host cell, such as a cell that expresses the heterologous polypeptide, but is derived from an organism different from the host cell. For example, the WISP1 inhibitor used herein is derived from, for example, a bacterial cell and is expressed in, for example, a mammalian cell.

[0157] In some embodiments, the polypeptide that inhibits WISP1 is at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% homologous to SEQ ID NOs: 1-4 or 6. As used herein, the term "homology" or "homologous" as used herein is defined as the percentage of nucleotide or amino acid residues that are identical to the nucleotide or amino acid residues in the corresponding sequence on a target chromosome or polypeptide after aligning the sequences as needed and introducing gaps to achieve the maximum percentage sequence identity. For the purpose of determining the percentage homology of nucleotide or amino acid sequences, the alignment can be achieved in a variety of ways within the skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, ClustalW2, or Megalign (DNASTAR) software. Those skilled in the art can determine the appropriate parameters for aligning sequences, including any algorithms required to achieve the maximum alignment over the full length of the sequences to be compared. In some embodiments, a nucleic acid or amino acid sequence (e.g., a DNA, RNA, or amino acid sequence) such as a WISP1 binding fragment or polypeptide is considered "homologous" when the sequence has 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 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to the native or unedited nucleic acid sequence (e.g., genomic sequence) or amino acid sequence of the corresponding WISP1.

[0158] In the various embodiments described herein, it is also contemplated to include variants (naturally occurring or otherwise), alleles, homologs, conservatively modified variants, and / or conservatively substituted variants of any of the specific polypeptides described. With respect to amino acid sequences, those skilled in the art will recognize that single substitutions, deletions, or additions to a nucleic acid, peptide, polypeptide, or protein sequence that change a single amino acid or a small percentage of amino acids in the coding sequence are "conservatively modified variants" where the change results in the substitution of an amino acid with a chemically similar amino acid and maintains the desired activity of the polypeptide. Such conservatively modified variants are in addition to and do not exclude polymorphic variants, interspecies homologs, and alleles that are consistent with the present disclosure.

[0159] A given amino acid can be replaced by residues having similar physicochemical characteristics. For example, one aliphatic residue can be substituted for another (e.g., Ile, Val, Leu, or Ala for one another), or one polar residue can be substituted for another (e.g., Lys and Arg; Glu and Asp; or Gln and Asn). Other such conservative substitutions are known, such as substitutions of regions of entities having similar hydrophobic characteristics. Polypeptides containing conservative amino acid substitutions can be examined in any of the assays described herein to demonstrate that desired activities, such as ligand-mediated receptor activity and the specificity of a native or reference polypeptide, are retained.

[0160] Amino acids can be grouped according to similarities in the nature of their side chains (as in A.L. Lehninger, in Biochemistry, 2nd 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), Gln (Q); (3) acidic: Asp (D), Glu (E); (4) basic: Lys (K), Arg (R), His (H). Optionally, naturally occurring residues can be grouped based on common side chain properties: (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) residues affecting chain orientation: Gly, Pro; (6) aromatic: Trp, Tyr, Phe. Nonconservative substitutions would require the exchange of a member of one of these classes for another. Specific conservative substitutions include, for example: Ala to Gly or to Ser; Arg to Lys; Asn to Gln or to His; Asp to Glu; Cys to Ser; Gln to Asn; Glu to Asp; Gly to Ala or to Pro; His to Asn or to Gln; Ile to Leu or to Val; Leu to Ile or to Val; Lys to Arg, to Gln or to Glu; Met to Leu, to Tyr or to Ile; Phe to Met, to Leu or to Tyr; Ser to Thr; Thr to Ser; Trp to Tyr; Tyr to Trp; and / or Phe to Val, to Ile or to Leu.

[0161] In some embodiments, the polypeptides (or nucleic acids encoding such polypeptides) described herein can be functional fragments of one of the amino acid sequences described herein. As used herein, a "functional fragment" is a fragment or segment of a peptide that retains at least 50% of the activity of the wild-type reference polypeptide according to assays known in the art or described hereinafter herein. Functional fragments can include conservative substitutions of the sequences disclosed herein.

[0162] In some embodiments, the polypeptides described herein can be variants of the polypeptides or molecules described herein. In some embodiments, the variants are conservatively modified variants. For example, conservatively substituted variants can be obtained by mutation of the native nucleotide sequence. As used herein, a "variant" is a polypeptide having an amino acid sequence that is substantially homologous to a native or reference polypeptide but that has an amino acid sequence different from the amino acid sequence of the native or reference polypeptide due to one or more deletions, insertions, or substitutions. When compared to the native or reference DNA sequence, the DNA sequence encoding the variant polypeptide comprises a sequence having one or more additions, deletions, or substitutions of nucleotides, but that encodes a variant protein or fragment thereof that retains the activity of the non-variant polypeptide. Various PCR-based site-directed mutagenesis methods are known in the art and can be applied by those skilled in the art.

[0163] Variant amino acid or DNA sequences can have at least 80%, at least 90%, 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% identity to the native or reference sequence. The degree of homology (percent identity) between the native and mutant sequences can be determined, for example, by comparing the two sequences using computer programs that are freely available on the World Wide Web and commonly used for this purpose (e.g., BLASTp or BLASTn with default settings).

[0164] Alterations of native amino acid sequences can be accomplished by any of a number of techniques known in the art. For example, mutations are introduced at specific loci by synthesizing oligonucleotides containing the mutant sequence, flanked by restriction sites that permit ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide a nucleotide sequence having altered specific codons according to the desired substitution, deletion, or insertion. Techniques for making such alterations are well established and include, for example, those disclosed by: Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos. 4,518,584 and 4,737,462, which are incorporated herein by reference in their entirety. Any cysteine residues not involved in maintaining the proper conformation of the polypeptide can be substituted, generally with serine, to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Conversely, cysteine bond(s) can be added to the polypeptide to improve its stability or promote oligomerization.

[0165] Antibodies that inhibit WISP1

[0166] In one aspect of any of the embodiments, methods of treating or preventing liver disease are described herein, the methods comprising administering to a subject in need thereof an antibody or antibody agent in which WISP1 is inhibited in target cells.

[0167] In various embodiments, the reagents described herein are antibodies or antigen-binding fragments thereof, or antibody agents that are specific for WISP1.

[0168] In another embodiment, an antibody or antibody agent that inhibits WISP1 specifically binds to the WISP1 polypeptide. In another embodiment, the antibody or antibody agent specifically binds to the amino acid sequences SEQ ID NO: 1-4, or SEQ ID NO: 6.

[0169] As used herein, the term "antibody agent" refers to a polypeptide that includes at least one immunoglobulin variable domain or immunoglobulin variable domain sequence and specifically binds to a given antigen. Antibody agents can include antibodies or polypeptides containing the antigen-binding domain of an antibody. In some embodiments of any aspect, the antibody agent can include a monoclonal antibody or a polypeptide containing the antigen-binding domain of a monoclonal antibody. For example, an antibody can include a heavy (H) chain variable region (abbreviated herein as VH ) and the variable regions of the light (L) chain (abbreviated herein as V L ). In another example, the antibody comprises two variable regions of the heavy (H) chain and two variable regions of the light (L) chain. The term "antibody agent" includes antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, CDRs, and domain antibody (dAb) fragments (see, e.g., de Wildt et al., Eur J. Immunol. 1996; 26(3): 629-39; which is incorporated herein by reference in its entirety)) and intact antibodies.

[0170] Antibodies can have the structural characteristics of IgA, IgG, IgE, IgD, or IgM (and their subtypes and combinations). Antibodies can be from any source, including mice, rabbits, pigs, sheep, goats, rats, and primates (human and non-human primates) and primatized antibodies. Antibodies also include intermediate antibodies, nanobodies, intrabodies, humanized antibodies, and chimeric antibodies, etc.

[0171] In one embodiment of any aspect, the antibodies described herein are humanized, monoclonal antibodies or their antigen-binding fragments, or antibody agents. In another embodiment, the humanized antibody is a humanized monoclonal antibody. In another embodiment, the humanized antibody is a humanized polyclonal antibody. In yet another embodiment, the humanized antibody is for therapeutic use.

[0172] The anti-WISP1 antibodies described herein can be monospecific antibodies or monoclonal antibodies. The term "monospecific antibody" refers to an antibody that exhibits single binding specificity and affinity for a particular target, such as an epitope. As used herein, the term includes "monoclonal antibody" or "monoclonal antibody composition", which refers to a preparation of an antibody or its fragments of a single molecular composition, regardless of how the antibody is produced.

[0173] As used herein, the term "humanized antibody" refers to an antibody that comprises heavy and light chain variable domain sequences from non-human species (e.g., mice, rats, sheep, or goats), but wherein at least part of the V H and / or V LThe sequences have been changed to more "human-like" antibodies, i.e., antibodies more similar to the variable sequences of the human germ line. Thus, "humanized" antibodies are in the form of chimeric antibodies that have been engineered or designed to include minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient or acceptor antibodies) in which the residues of the hypervariable regions from the recipient are replaced by the residues of the hypervariable regions from a non-human species such as a mouse, rat, rabbit, or non-human primate (donor antibody) with the desired specificity, affinity, and capacity. In some cases, the Fv framework region (FR) residues of the human immunoglobulin are replaced by the corresponding non-human residues. In addition, humanized antibodies can include residues not found in the recipient antibody or the donor antibody. These modifications can be made to further improve antibody performance. Generally, a humanized antibody will substantially include at least one, and usually two, variable domains in which all or substantially all of the hypervariable loops correspond to those of the non-human immunoglobulin and all or substantially all of the FR regions are those of the human immunoglobulin sequence. A humanized antibody will also optionally include at least a portion of the immunoglobulin constant region (Fc), usually the immunoglobulin constant region of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). As used herein, "composite human antibody" or "deimmunized antibody" is a specific type of engineered or humanized antibody designed to reduce or eliminate T cell epitopes from the variable domains.

[0174] A humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are typically referred to as "import" residues and are typically taken from an "import" variable domain. Humanization can be carried out essentially according to the methods of Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by replacing the corresponding sequences of a human antibody with rodent CDRs or CDR sequences. Thus, such humanized antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than the entire human variable domain is replaced by the corresponding sequences from a non-human species. In practice, a humanized antibody is typically a human antibody in which some CDR residues and possibly some FR residues are replaced by residues from an analogous site in a rodent antibody.

[0175] In certain embodiments, the anti-WISP1 antibody is an intracellular antibody. An intracellular antibody is an antibody that functionally binds a target within a cell (generally, see, Hood et al., Immunology, Benjamin, N.Y., 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 Number 6,004,940; and U.S. Patent No. 5,581,829; which are incorporated herein by reference in their entirety). Methods for producing intracellular antibodies are known to those of skill in the art, for example, as described in WO 2002 / 086096. The antibody will typically bind with a KD of at least about 1 mM, more typically at least about 300 μM, typically at least about 10 μM, more typically at least about 30 μM, preferably at least about 10 μM, and more preferably at least about 3 μM or better.

[0176] In one embodiment, the anti-WISP1 antibody is a neutralizing antibody. In one embodiment, the anti-WISP1 antibody is a non-neutralizing antibody.

[0177] In some embodiments, the anti-WISP1 antibody is chimeric. As used herein, the term "chimeric", as used in the context of an antibody, or a sequence encoding an antibody, refers to an immunoglobulin molecule characterized as comprising two or more segments or portions derived from different animal species. For example, the variable region of a chimeric antibody is derived from a non-human mammalian antibody, such as a murine monoclonal antibody, and the immunoglobulin constant region is derived from a human immunoglobulin molecule. The variable fragment of the chimeric antibody is typically linked to at least a portion of the immunoglobulin constant region (Fc), typically the immunoglobulin constant region (Fc) of a human immunoglobulin. Human constant region DNA sequences can be isolated according to known procedures from a variety of human cells, such as immortalized B cells (WO 87 / 02671; which is incorporated herein by reference in its entirety). The antibody can comprise both the light chain and heavy chain constant regions. The heavy chain constant region can include the CH1, hinge region, CH2, CH3, and sometimes the CH4 regions. For therapeutic purposes, the CH2 domain can be deleted or omitted. Techniques known in the art have been developed for the production of "chimeric antibodies" (see Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature 314:452-454 (1985); which are incorporated herein by reference in their entirety), for example, by splicing together the genes of antibody molecules from mice, or other species, specific for a suitable antigen with the genes of human antibody molecules of suitable biological activity.

[0178] 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.

[0179] As will be understood by those skilled in the art, in a full-length antibody, each heavy chain is composed of a heavy chain variable domain (abbreviated herein as HCVR or V H ) and a heavy chain constant region. The heavy chain constant region is composed of three domains: C H 1, C H 2, and C H 3. Each light chain is composed of a light chain variable domain (abbreviated herein as HCVR or V L ) and a light chain constant region. The light chain constant region is composed of one domain C L . V H and V L regions can be further subdivided into hypervariable regions, called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each V H and V LIt consists of three CDRs and four FRs, arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure is known to those skilled in the art. The chains are usually linked to each other by disulfide bonds.

[0180] As used herein, the term "complementary determining region" ("CDR"), i.e., CDR1, CDR2, and CDR3, refers to the amino acid residues of the variable domain of a heavy or light chain that are required for specific antigen binding. Each variable domain typically has three CDR regions identified as CDR1, CDR2, and CDR3. Each complementary determining region may include amino acid residues from the "complementary determining regions" defined by Kabat (i.e., approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) in the light chain variable domain and 31-35 (H1), 50-65 (H2), and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from "hypervariable loops" (i.e., approximately residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in the light chain variable domain and 26-32 (H1), 53-55 (H2), and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). In some cases, the complementary determining region may include amino acids from both the CDR regions defined according to Kabat and the hypervariable loops. The term "CDR set" as used herein refers to the group of three CDRs present in a single heavy or light chain variable region capable of binding an antigen. The exact boundaries of these CDRs have been defined differently according to different systems. The system described by Kabat (Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md. (1987) and (1991)) not only provides a clear residue numbering system applicable to any variable region of an antibody, but also provides precise residue boundaries that define the three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia and coworkers (Chothia & Lesk, J. Mol. Biol, 196:901-917 (1987) and Chothia et al., Nature 342:877-883 (1989)) found that although there are significant differences at the amino acid sequence level, certain subportions in the Kabat CDRs adopt nearly identical peptide backbone conformations. These subportions are designated as L1, L2, and L3 or H1, H2, and H3, where "L" and "H" designate the light and heavy chain regions, respectively.These regions may be referred to as Chothia CDRs, which have boundaries that overlap with Kabat CDRs. Other boundaries defining CDRs that overlap with Kabat CDRs have been described by Padlan (FASEB. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45 (1996)). Other CDR boundary definitions may not strictly follow one of the above systems, but will still overlap with Kabat CDRs, although they may be shortened or lengthened based on specific residues or groups of residues or even entire CDRs that do not significantly affect antigen-binding prediction or experimental results. In some embodiments, a CDR may also be described as including amino acid residues from the "complementary determining regions" defined by IMGT. The compositions and methods used herein may utilize CDRs defined according to any of these systems, although preferred embodiments use CDRs defined by IMGT or Abysis. However, with reference to any of these numbering conventions, the boundaries of the CDRs are clear.

[0181] The immunoglobulin constant (C) domain refers to the heavy (C H ) or light (C L ) chain constant domain. Amino acid sequences of murine and human IgG heavy and light chain constant domains are known in the art. With respect to the heavy chain, in some embodiments of the aspects described herein, the heavy chain of the antibodies described herein can be an alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. In some embodiments of the aspects described herein, the heavy chain of the antibodies described can be a human alpha (α), delta (δ), epsilon (ε), gamma (γ), or mu (μ) heavy chain. Non-limiting examples of human constant region sequences have been described in the art, e.g., see U.S. Patent No. 5,693,780 and Kabat E A et al., (1991) as described above.

[0182] Thus, in embodiments of the compositions and methods described herein, the anti-WISP1 is composed of a non-IgG framework.

[0183] As used herein, the terms "donor" and "donor antibody" refer to an antibody that provides one or more CDRs. In an exemplary embodiment, the donor antibody is an antibody from a species different from the species from which the framework region is obtained or derived. In some embodiments, the donor antibody is a different isotype from the receptor antibody. In the context of humanized antibodies, the term "donor antibody" refers to a non-human antibody that provides one or more CDRs.

[0184] As used herein, the terms "acceptor" and "acceptor antibody" refer to an antibody or nucleic acid sequence encoding the same that provides at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or at least 100% of the amino acid sequence of one or more framework regions. In some embodiments, the term "acceptor" refers to an antibody amino acid or nucleic acid sequence encoding the same that provides a constant region(s). In yet another embodiment, the term "acceptor" refers to an antibody amino acid or nucleic acid sequence encoding one or more framework regions and a constant region(s) that provides one or more framework regions and a constant region(s). In certain embodiments, the term "acceptor" refers to a human antibody amino acid or nucleic acid sequence that provides or encodes at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or 100% of the amino acid sequence of one or more framework regions. According to this embodiment, the acceptor may comprise at least 1, at least 2, at least 3, at least 4, at least 5, or at least 10 amino acid residues at one or more specific positions not present in human antibodies. The acceptor framework region and / or acceptor constant region(s) may be, for example, derived from or obtained from germline antibody genes, mature antibody genes, functional antibodies (e.g., antibodies known in the art, antibodies under development, or commercially available antibodies).

[0185] Human heavy and light chain acceptor sequences are known in the art. In some embodiments, the human heavy and light chain acceptor sequences are selected from the sequences listed in V-base (found at vbase.mrc-cpe.cam.ac.uk / on the World Wide Web) or in IMGT TM the international ImMunoGeneTics Information System TM (found at imgt.cines.fr / textes / IMGTrepertoire / LocusGenes / on the World Wide Web). In another embodiment of the technology disclosed herein, the human heavy and light chain acceptor sequences are selected from the sequences described in Tables 3 and 4 of U.S. Patent Publication No. 2011 / 0280800, which is incorporated herein by reference in its entirety.

[0186] In some embodiments, the compositions and methods described herein may include an "antigen-binding fragment" or "antigen-binding portion" of an antibody. The term "antigen-binding fragment" or "WISP1-binding fragment" of an antibody refers to one or more fragments of an antibody that retain the ability to specifically bind to an antigen (e.g., WISP1).

[0187] The antigen-binding function of an antibody can be performed by a fragment of the full-length antigen. Such antibody fragment embodiments can be incorporated into bispecific, dual-specific, or multispecific forms, such as the dual variable domain (DVD-Ig) form; specifically binding to two or more antigens. Non-limiting examples of antigen-binding fragments included in the term "antigen-binding portion" of an antibody include (i) Fab fragments, monovalent fragments consisting of V L and V H , C L , and C H 1 domains; (ii) F(ab')2 fragments, divalent fragments including two Fab fragments linked by a disulfide bond in the hinge region; (iii) Fd fragments, consisting of V H and C H 1 domains; (iv) Fv fragments, consisting of V L and V H domains of a single arm of an antibody; (v) dAb fragments (Ward et al. (1989) Nature, 341:544-546; PCT Publication No. WO 90 / 05144), which include a single variable domain; and (vi) isolated complementarity-determining regions (CDRs). In addition, although the two domains V L and V H of an Fv fragment are encoded by their respective genes, they can be joined using recombinant methods, by a synthetic linker that enables them to be made into a single protein chain in which the V L and V H regions pair to form a monovalent molecule (referred to as single-chain Fv (scFv); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be included in the term "antigen-binding portion" of an antibody. Other forms of single-chain antibodies, such as diabodies, are also included.

[0188] In some embodiments, the antibody agent is a bispecific monoclonal antibody.

[0189] A diabody is a bivalent bispecific antibody in which V H and V LThe domains are expressed on a single polypeptide chain, but the linker used is too short to pair between the two domains on the same chain, forcing the domains to pair with complementary domains on another chain and generating two antigen-binding sites (see, e.g., Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448; Poljak (1994) Structure 2:1121-1123); Kontermann and Dubel, eds., Antibody Engineering, Springer-Verlag, N.Y. (2001), p. 790 (ISBN 3-540-41354-5). In addition, single-chain antibodies also include "linear antibodies" which include tandem Fv fragment pairs (V H -C H 1-V H -C H 1), which together with a complementary light chain polypeptide form pairs of antigen-binding regions (Zapata et al. (1995) Protein Eng. 8(10):1057-1062; and U.S. Patent No. 5,641,870).

[0190] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain, which can be generated by papain digestion of a whole antibody. The Fc region can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin generally includes two constant domains, the C H 2 domain, and the C H 3 domain, and optionally includes the C H 4 domain. It is known in the art to replace amino acid residues in the Fc portion to alter antibody effector functions (U.S. Patent Nos. 5,648,260 and 5,624,821). The Fc portion of an antibody mediates several important effector functions, e.g., cytokine induction, antibody-dependent cell cytotoxicity (ADCC), phagocytosis, complement-dependent cytotoxicity (CDC), and the half-life / clearance rate of the antibody and antigen-antibody complex. In some cases, these effector functions are desirable for therapeutic antibodies, but in other cases, depending on the therapeutic purpose, they are unnecessary or even harmful. Certain human IgG isotypes, particularly IgG1 and IgG3, mediate ADCC and CDC by binding to Fcγ receptors and complement C1q, respectively. The neonatal Fc receptor (FcRn) is a key component in determining the circulating half-life of an antibody. In yet another embodiment, at least one amino acid residue in the constant region of the antibody, e.g., the Fc region of the antibody, is replaced such that the effector function of the antibody is altered.

[0191] A DNA sequence encoding an antibody or antigen-binding fragment that specifically binds to WISP1 as described herein. The nucleic acid sequence may also be modified, for example, by substituting the coding sequences of the human heavy and light chain constant domains or framework regions with homologous mammalian (e.g., murine) sequences (U.S. Patent No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851 (1984)), or by covalently linking the coding sequence of all or part of a non-immunoglobulin polypeptide to the immunoglobulin coding sequence, also as described elsewhere herein.

[0192] Such non-immunoglobulin polypeptides may replace the constant domains of an antibody, or they may replace the variable domains of one antigen-binding site of an antibody to produce a chimeric bivalent antibody that includes one antigen-binding site having specificity for an antigen of one target and another antigen-binding site having specificity for a different antigen of the target.

[0193] In some embodiments of the aspects described herein, the antibody or its WISP1-binding fragment includes one, two, three, or four framework regions of a heavy chain variable region sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% identity to one, two, three, or four framework regions of the heavy chain variable region sequence from which it is derived. In some embodiments of the aspects described herein, the heavy chain variable framework region derived from the amino acid sequence consists of the amino acid sequence, but with up to 10 amino acid substitutions, deletions, and / or insertions, preferably up to 10 amino acid substitutions. In some embodiments of the aspects described herein, the heavy chain variable framework region derived from the amino acid sequence consists of the amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues substituted with amino acids found at similar positions in the corresponding non-human, primate, or human heavy chain variable framework region. In some embodiments of the aspects described herein, the antibody or antigen-binding fragment also includes one, two, three, or all four V H of a human or primate antibody H framework regions. The primate or human heavy chain framework regions of the antibody selected for use with the heavy chain CDR sequences described herein may have, for example, at least 70% identity to the heavy chain framework region of the non-human parental antibody.

[0194] In some embodiments of aspects described herein, the primate or human heavy chain framework region amino acid residues are from a native primate or human antibody heavy chain framework region having at least 75%, at least 80%, at least 85% (or more) identity to the heavy chain framework region of any antibody described herein. In certain embodiments, the antibody or antigen-binding fragment further comprises one, two, three, or all four V H framework regions from a human heavy chain variable subgroup (e.g., one of subgroups 1 to 7).

[0195] In some such embodiments of aspects described herein, the antibody or its WISP1-binding fragment comprises one, two, three, or four framework regions of a light chain variable region sequence having at least 75%, 80%, 85%, 90%, 95%, or 100% identity to one, two, three, or four framework regions of the light chain variable region sequence from which it is derived. In some embodiments of aspects described herein, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence, but with up to 10 amino acid substitutions, deletions, and / or insertions, preferably up to 10 amino acid substitutions. In some embodiments of aspects described herein, the light chain variable framework region derived from the amino acid sequence consists of the amino acid sequence having 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues substituted with amino acids found at similar positions in the corresponding non-human, primate, or human light chain variable framework region. In some embodiments of aspects described herein, the antibody or antigen-binding fragment further comprises one, two, three, or all four V L framework regions from a V L of a human or primate antibody. The primate or human light chain framework region of the antibody selected for use with the light chain CDR sequences described herein can have, for example, at least 70% identity to the light chain framework region of the non-human parental antibody.

[0196] In some embodiments of aspects described herein, the primate or human light chain framework region amino acid residues are from a native primate or human antibody light chain framework region having at least 75%, at least 80%, at least 85% (or more) identity to the light chain framework region of any antibody described herein. In some embodiments, the antibody or antigen-binding fragment further comprises one, two, three, or all four V L framework regions from a human light chain variable kappa subgroup. In some embodiments, the antibody or antigen-binding fragment further comprises one, two, three, or all four V L framework regions from a human light chain variable lambda subgroup.

[0197] In some embodiments of aspects described herein, one or more CDRs are along the V of the antibodies described herein H (e.g., CDR1, CDR2, or CDR3) and / or V L (e.g., CDR1, CDR2, or CDR3) regions may be altered, i.e., shorter or longer, by one, two, three, four, five, or six amino acid positions, so long as immunologically specific binding to the antigen of interest is maintained (e.g., substantially maintaining at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding from the original antibody from which it is derived). For example, in some embodiments, the positions defining the CDR may be altered, i.e., shorter or longer, by shifting the N-terminal and / or C-terminal boundaries of the CDR by one, two, three, four, five, or six amino acids relative to the CDR positions of any of the antibodies described herein, so long as immunologically specific binding to the target antigen is maintained (e.g., substantially maintaining, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding from the original antibody from which it is derived). In other embodiments, one or more CDRs are along the V of the antibodies described herein H (e.g., CDR1, CDR2, or CDR3) and / or V L (e.g., CDR1, CDR2, or CDR3) regions may be altered (e.g., shorter or longer) by one, two, three, four, five, or more amino acids, so long as immunologically specific binding to the target antigen is maintained (e.g., substantially maintaining, e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% of the binding from the original antibody from which it is derived).

[0198] In some embodiments of aspects described herein, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region or fragment thereof of any of the antibodies described herein (e.g., CH2 domain (residues 231 - 340 of human IgG1) and / or CH3 domain (residues 341 - 447 of human IgG1) and / or hinge region, numbered according to the Kabat numbering system (e.g., EU index in Kabat)) to alter one or more functional properties of the antibody, e.g., serum half-life, complement binding, Fc receptor binding, and / or antigen-dependent cell cytotoxicity.

[0199] In some embodiments of aspects described herein, one, two or more mutations (e.g., amino acid substitutions) are introduced into the Fc region (C Hthe hinge region of the [domain 1] such that the number of cysteine residues in the hinge region is altered (e.g., increased or decreased), as described, for example, in U.S. Patent No. 5,677,425. C H The number of cysteine residues in the hinge region of the [domain 1] can be altered to, for example, facilitate assembly of the light and heavy chains, or to alter (e.g., increase or decrease) the stability of the antibody.

[0200] In some embodiments of the aspects described herein, one, two, or more mutations (e.g., amino acid substitutions) are introduced into the Fc region of the antibodies described herein or antigen-binding fragments thereof (e.g., 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, numbered according to the Kabat numbering system (e.g., EU index in Kabat)) to increase or decrease the affinity of the antibody for Fc receptors on the surface of effector cells. Mutations in the Fc region of an antibody or fragments thereof that decrease or increase the affinity of the antibody for Fc receptors and techniques for introducing such mutations into the Fc receptor or fragments thereof are known to those of skill in the art. Examples of mutations of the Fc receptor of an antibody to alter the affinity of the antibody for the Fc receptor are described, for example, in Smith P et al. (2012) PNAS 109:6181 - 6186, U.S. Patent No. 6,737,056, and International Publication No. WO 02 / 060919; WO 98 / 23289; and WO 97 / 34631, which are incorporated herein by reference.

[0201] The term "CDR-grafted antibody" refers to an antibody that includes heavy and light chain variable region sequences from one species, but in which the sequence of one or more CDR regions of V H and / or V L is replaced with CDR sequences from another species, such as an antibody having human heavy and light chain variable regions in which one or more human CDRs (e.g., CDR3) have been replaced with mouse CDR sequences. The CDR-grafted antibodies described herein include heavy and light chain variable region sequences of a human antibody in which the sequence of one or more CDR regions of V H and / or V L is replaced with CDR sequences of a non-human antibody described herein.

[0202] The anti-WISP1 antibodies described herein can be engineered to improve binding specificity or pharmacokinetic properties for therapeutic use. Binding specificity can be assayed, for example, by competition assays using the antigen (e.g., WISP1 or a polypeptide fragment thereof) compared to competition with one or more unrelated or different antigens. A variety of immunoassay formats are suitable for selecting antigens, antibodies, or other ligands that specifically bind WISP1. Specific binding can be affected, for example, by the affinity and avidity of the reagent (e.g., polypeptide or anti-WISP1 antibody) described herein and the concentration of the reagent. One of ordinary skill in the art can determine the appropriate conditions for the reagent described herein to selectively bind WISP1 using any suitable method, such as titration of the reagent in a suitable binding assay.

[0203] As used herein, the term "critical" residue refers to certain residues within the variable domain that have a greater impact on the binding specificity and / or affinity of an antibody, particularly a humanized antibody, than other residues. Critical residues include, but are not limited to, one or more of the following: residues adjacent to the CDRs, potential glycosylation sites (which can be either N- or O-glycosylation sites), rare residues, residues capable of interacting with the antigen, residues capable of interacting with the CDRs, canonical residues, contact residues between the heavy chain variable domain and the light chain variable domain, residues in the Vernier zone, and residues in the region of overlap between the Chothia definition of the variable heavy chain CDR and the Kabat definition of the first heavy chain framework.

[0204] The anti-WISP1 antibodies described herein can be engineered antibodies. As used herein, the term "engineered" refers to aspects that are manually operated by a human. For example, a locus is considered "engineered" when two or more sequences that are not naturally linked together in that order at the locus are manually operated by a human to be directly linked to each other at the engineered locus. For example, in some embodiments of the present invention, the engineered locus includes various Ig sequences having non-natural V segments, all of which are found in nature but not in the same locus or in the order found in a locus in nature. As is commonly practiced and understood by those of ordinary skill in the art, progeny or copies of engineered polynucleotides (and / or cells or animals comprising such polynucleotides) are generally still referred to as "engineered," even if the actual manipulation was performed on a previous entity.

[0205] A WISP-conjugated polypeptide, antibody, antibody agent, or antigen-binding portion thereof can be part of a larger immunoadhesion molecule or a molecular composition formed by covalent or non-covalent binding of the antibody antigen-binding portion to one or more other proteins or peptides. Examples of such immunoadhesion molecules include using a streptavidin core region to create a tetrameric scFv molecule (Kipriyanov et al. (1995) Human Antibod. Hybridomas 6:93-101) and using cysteine residues, a marker peptide, and a C-terminal polyhistidine tag to create divalent and biotinylated scFv molecules (Kipriyanov et al. (1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab and F(ab').sub.2 fragments, can be prepared from intact antibodies using conventional techniques such as papain or pepsin digestion of intact antibodies, respectively. In addition, antibodies, their antigen-binding portions, and immunoadhesion molecules can be obtained using standard recombinant DNA techniques. The target-binding protein, such as the antigen-binding portion of an antibody, can also be part of a dual variable domain (DVD-Ig).

[0206] Antibodies and antibody agents that are therapeutic and / or specific for any particular target antigen (e.g., WISP1) can be readily selected by those skilled in the art from known antibodies or antibody agents, such as from FDA-approved therapeutic antibody agents and / or commercially available antibody agents listed in catalogs according to their targeting specificities.

[0207] In another embodiment, the anti-WISP1 antibody is any known anti-WISP1 antibody in the art or any anti-WISP1 antibody yet to be discovered. Exemplary anti-WISP1 antibodies known in the art include, but are not limited to, the anti-WISP1 antibodies sold by Abcam (e.g., ab60114; ab65943), the anti-WISP1 antibodies sold by RND Systems (e.g., mab1680), and the anti-WISP1 antibodies sold by Sigma-Aldrich (e.g., SAB2501114).

[0208] In another embodiment, the anti-WISP1 antibody is selected from Table 1 (Table of Antibodies) below. In another embodiment, the anti-WISP1 antibody comprises any one of the amino acid sequences of SEQ ID NOs: 12-120.

[0209] Table 1: Table of Antibodies (anti-WISP1)

[0210]

[0211]

[0212]

[0213] *The sequence was obtained from AbYsis of <abysis.org> available on the World Wide Web.

[0214] In another embodiment, the anti-WISP1 antibody is a humanized anti-WISP1 antibody derived from any known or yet to be discovered non-human anti-WISP1 antibody.

[0215] In one embodiment, the antibody or antibody agent binds to an amino acid sequence corresponding to the amino acid sequence encoding WISP1 (SEQ ID NO:1).

[0216] In another embodiment, the anti-WISP1 antibody or antibody agent binds to an amino acid sequence comprising the sequence of SEQ ID NO:1; or binds to an amino acid sequence comprising a sequence having 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 more sequence identity to the sequence of SEQ ID NO:1. In one embodiment, the anti-WISP1 antibody or antibody agent binds to an amino acid sequence comprising the entire sequence of SEQ ID NO:1. In another embodiment, the antibody or antibody agent binds to an amino acid sequence of a fragment of the sequence comprising SEQ ID NO:1, wherein the fragment is sufficient to bind its target, such as WISP1, and cause inhibition of WISP1 level and / or activity.

[0217] In certain embodiments, the antibody or antibody agent binds to an amino acid sequence corresponding to the amino acid sequences encoding various human WISP1 isoforms (SEQ ID NO:2, 3, or 4).

[0218] In another embodiment, the anti-WISP1 antibody or antibody agent binds to an amino acid sequence comprising the sequence of SEQ ID NO:2, 3, or 4; or binds to an amino acid sequence comprising a sequence having 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 more sequence identity to the sequence of SEQ ID NO:2, 3, or 4. In one embodiment, the anti-WISP1 antibody or antibody agent binds to an amino acid sequence comprising the entire sequence of SEQ ID NO:2, 3, or 4. In another embodiment, the antibody or antibody agent binds to an amino acid sequence of a fragment of the sequence comprising SEQ ID NO:2, 3, or 4, wherein the fragment is sufficient to bind its target, such as WISP1, and cause inhibition of WISP1 level and / or activity.

[0219] In another embodiment, the antibody or antibody agent binds to an amino acid sequence corresponding to the amino acid sequence of murine WISP1 (SEQ ID NO:6).

[0220] In another embodiment, the antibody or antibody agent binds to an amino acid sequence comprising the sequence of SEQ ID NO:6; or binds to an amino acid sequence comprising a sequence having 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 more sequence identity to the sequence of SEQ ID NO:6. In one embodiment, the anti-WISP1 antibody or antibody agent binds to an amino acid sequence comprising the entire sequence of SEQ ID NO:6. In another embodiment, the antibody or antibody agent binds to an amino acid sequence comprising a fragment of the sequence of SEQ ID NO:6, wherein the fragment is sufficient to bind its target, such as WISP1, and cause inhibition of WISP1 level and / or activity.

[0221] Thus, in some embodiments, described herein are humanized antibodies comprising one or more variable domains, the variable domains comprising one or more CDRs encoded by the variable heavy and light chain sequences of SEQ ID NOs:12 - 120.

[0222] In another embodiment, the antibody or antibody agent comprises an amino acid sequence having at least 70% homology to any of SEQ ID NOs:12 - 120. In another embodiment, the antibody or antibody agent CDR comprises an amino acid sequence having at least 70% homology to any of SEQ ID NOs:12 - 120. In another embodiment, the antibody or antibody agent comprises an amino acid sequence having at least 90% homology to any of SEQ ID NOs:12 - 120.

[0223] Stated another way, in some embodiments, the antibody or antibody agent that inhibits WISP1 has at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or 100% homology to SEQ ID NO 1 - 4, 6, or SEQ ID NOs:12 - 120.

[0224] In other embodiments, a nucleic acid or amino acid sequence (e.g., a DNA, RNA, or amino acid sequence) such as a WISP1 binding fragment or an anti-WISP1 antibody is considered "homologous" when the sequence has at least 50%, at least 60%, 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 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more identity to the native or unedited nucleic acid sequence (e.g., genomic sequence) or amino acid sequence of an antibody that specifically binds WISP1.

[0225] One aspect provided herein is a composition comprising any of the anti-WISP1 antibodies or antibody agents described herein. In one embodiment, the composition further comprises a pharmaceutically acceptable carrier. In one embodiment, the composition is a pharmaceutical composition.

[0226] Nucleic acids that inhibit WISP1

[0227] In one embodiment, the agent that inhibits WISP1 is an antisense oligonucleotide. As used herein, "antisense oligonucleotide" refers to a synthetic nucleic acid sequence that is complementary to a DNA or mRNA sequence, such as the DNA and mRNA sequences of a microRNA sequence. Antisense oligonucleotides are typically designed to block the expression of a DNA or RNA target by binding to the target and preventing expression at the transcriptional, antisense, or splicing levels. The antisense oligonucleotides of the present invention are complementary nucleic acid sequences designed to hybridize to a gene such as WISP1 under cellular conditions. Thus, an oligonucleotide sufficient to be complementary to the target is selected, i.e., one that hybridizes well enough and has sufficient specificity in the context of the cellular environment to provide the desired effect. For example, an antisense oligonucleotide that inhibits WISP1 may comprise at least 5, at least 10, at least 15, at least 20, at least 25, at least 30 or more bases that are complementary to a portion of the coding sequence of the human WISP1 gene (e.g., SEQ ID NO:5) or the mouse WISP1 gene (e.g., SEQ ID NO:7).

[0228] In one embodiment, WISP1 is removed from the cell genome using any genome editing system, including but not limited to zinc finger nucleases, TALENs, meganucleases, and CRISPR / Cas systems. In one embodiment, the genome editing system used to introduce nucleic acids encoding one or more guide RNAs into the cell genome is not a CRISPR / Cas system; this can prevent unwanted cell death in cells that maintain low levels of Cas enzyme / protein. It is also contemplated herein that either the Cas enzyme or the sgRNA is expressed under the control of a different inducible promoter, allowing for their respective transient expression to prevent such interference.

[0229] When nucleic acids encoding one or more sgRNAs and nucleic acids encoding RNA-guided endonucleases each require in vivo administration, the use of adeno-associated virus (AAV) vectors is specifically contemplated. Other vectors for delivering nucleic acids to both components of the genome editing / fragmenting system (e.g., sgRNA, RNA-guided endonuclease) include lentiviral vectors, such as Epstein-Barr, human immunodeficiency virus (HIV), and hepatitis B virus (HBV). The components of the RNA-guided genome editing system (e.g., sgRNA and endonuclease) can be delivered in their respective vectors known in the art or as described herein.

[0230] In one embodiment, the reagent inhibits WISP1 by RNA interference. An inhibitor of the expression of a given gene can be an inhibitory nucleic acid. In some embodiments of any aspect, the inhibitory nucleic acid is inhibitory RNA (iRNA). RNAi can be single-stranded or double-stranded.

[0231] The iRNA can be siRNA, shRNA, endogenous microRNA (miRNA), or artificial miRNA. In one embodiment, the iRNA described herein affects the inhibition of the expression and / or activity of a target such as WISP1. In some embodiments of any aspect, the reagent is an siRNA that inhibits WISP1. In some embodiments of any aspect, the reagent is an shRNA that inhibits WISP1.

[0232] One of ordinary skill in the art will be able to design siRNA, shRNA, or miRNA to target WISP1, for example, using publicly available design tools. Companies such as Dharmacon (Layfayette, CO) or Sigma Aldrich (St. Louis, MO) typically manufacture siRNA, shRNA, or miRNA.

[0233] In some embodiments in any aspect, the iRNA can be dsRNA. The dsRNA includes two RNA strands that are sufficiently complementary to hybridize under conditions of use of the dsRNA to form a duplex structure. One strand of the dsRNA (the antisense strand) includes a complementary region that is substantially complementary, and typically fully complementary, to the target sequence. The target sequence can be derived from the sequence of an mRNA formed during expression of the target. The other strand (the sense strand) includes a region that is complementary to the antisense strand such that when bound under appropriate conditions, the two strands hybridize and form a duplex structure.

[0234] The RNA of the iRNA can be chemically modified to enhance stability or other beneficial properties. Specific nucleic acids of the invention can be synthesized and / or modified by well-established methods in the art, such as those described in “Current protocols in nucleic acid chemistry,” Beaucage, S.L. et al. (Eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference.

[0235] In one embodiment, the agent is a miRNA that inhibits WISP1. MicroRNAs are small non-coding RNAs with an average length of 22 nucleotides. These molecules act by binding to complementary sequences in mRNA molecules, typically in the 3' untranslated (3'UTR) region, thereby promoting targeted mRNA degradation or inhibiting mRNA translation. The interaction between microRNA and mRNA is mediated by what is called a “seed sequence,” which is a 6-8 nucleotide region of a microRNA that directs sequence-specific binding to mRNA by imperfect Watson-Crick base pairing. Over 900 microRNAs are known to be expressed in mammals. Most of these can be grouped into families based on their seed sequences, thus identifying “clusters” of similar microRNAs. The miRNA can be expressed in cells, such as naked DNA. The miRNA can be encoded by a nucleic acid expressed in cells, such as naked DNA, or can be encoded by a nucleic acid contained within a vector.

[0236] In one embodiment, the reagent that downregulates WISP1 is miRNA-15a. miRNA-15a is a non-coding RNA that regulates gene expression in many organs including the liver. The miRNA-15a sequence is known for many species, such as human miRNA-15a, e.g., miRBase accession number MI0000069, and mouse miRNA-15a, e.g., miRBase accession number MI0000564. Human miRNA-15a includes the sequence of SEQ ID NO:8. miRNA-15a may refer to human miRNA-15a, including its naturally occurring variants, molecules, and alleles. For example, miRNA-15a may be mouse miRNA-15a, having the sequence of SEQ ID NO:10.

[0237] In one embodiment, a reagent such as miRNA-15A has a sequence corresponding to the sequence of SEQ ID NO:8; or includes the sequence of SEQ ID NO:8; or includes a sequence having 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 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:8.

[0238] In one embodiment, a reagent such as miRNA-15a has a sequence corresponding to the sequence of SEQ ID NO:10; or includes the sequence of SEQ ID NO:10; or includes a sequence having 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 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:10.

[0239] In one embodiment, the reagent that downregulates WISP1 is miRNA-412. miRNA-412 is known for many species, such as human miRNA-412, e.g., miRBase accession number MI0001464, and mouse miRNA-412, e.g., miRBase accession number MI0001164. Human miRNA-412 includes the sequence of SEQ ID NO:9. miRNA-412 may refer to human miRNA-15a, including its naturally occurring variants, molecules, and alleles. For example, miRNA-15a may be mouse miRNA-412, having the sequence of SEQ ID NO:11.

[0240] In one embodiment, a reagent such as miRNA-412 has a sequence corresponding to the sequence of SEQ ID NO:9; or comprises the sequence of SEQ ID NO:9; or comprises a sequence having 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 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:9.

[0241] In one embodiment, a reagent such as miRNA-412 has a sequence corresponding to the sequence of SEQ ID NO:11; or comprises the sequence of SEQ ID NO:11; or comprises a sequence having 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 97%, at least 98%, at least 99%, or at least 100% sequence identity to the sequence of SEQ ID NO:11.

[0242] The reagent can cause gene silencing of a target gene (e.g., WISP1), such as caused by an RNAi molecule (e.g., siRNA or miRNA). This results in at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% reduction in the mRNA level in the cell for the target as compared to the mRNA level in cells without the reagent. In a preferred embodiment, the mRNA level is reduced by at least about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%. A person skilled in the art can readily evaluate whether an siRNA, shRNA, or miRNA is effective in targeting, e.g., WISP1 for its downregulation, for example by transfecting the siRNA, shRNA, or miRNA into cells and detecting the level of the gene (e.g., WISP1) found in the cells by immunoblotting or a PCR-based assay.

[0243] The reagent can be included in a vector and thus further includes the vector. Many such vectors that can be used to transfer foreign genes into target mammalian cells are available. The vector can be episomal, such as a plasmid, for example, vectors derived from viruses such as cytomegalovirus, adenovirus, etc., or can be integrated into the target cell genome by homologous recombination or random integration, for example, vectors derived from retroviruses such as MMLV, HIV-1, ALV, etc. In some embodiments, a combination of a retrovirus and a suitable packaging cell line can also find use where the capsid protein has the function of infecting the target cell. Generally, cells and viruses are cultured in a medium for at least about 24 hours. Then in some applications, before analysis, the cells are allowed to grow in the medium at short time intervals, such as 24 - 73 hours, or grow for at least two weeks, and can be allowed to grow for more than five weeks. Commonly used retroviral vectors are "defective", i.e., unable to produce the viral proteins required for productive infection. Replication of the vector requires growth in a packaging cell line.

[0244] As used herein, the term "vector" refers to a nucleic acid construct designed for delivery to a host cell or for transfer between different host cells. As used herein, a vector can be viral or non-viral. The term "vector" includes any genetic element that is capable of replicating when associated with appropriate control elements and can transfer a gene sequence to a cell. Vectors can include, but are not limited to, cloning vectors, expression vectors, plasmids, phages, transposons, cosmids, artificial chromosomes, viruses, virus particles, etc.

[0245] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide (e.g., a WISP1 inhibitor) from a nucleic acid sequence contained therein ligated to transcriptional regulatory sequences on the vector. The expressed sequence will generally but not necessarily be heterologous to the cell. An expression vector can contain additional elements, for example, an expression vector can have two replication systems, thereby allowing it to be maintained in two organisms, such as expression in human cells and cloning and propagation in a prokaryotic host. The term "expression" refers to the cellular processes involved in the production of RNA and protein and, as needed, the secretion of the protein, including but not limited to, for example, transcription, transcript processing, translation, and protein folding, modification, and processing, where applicable. "Expression product" includes RNA transcribed from a gene, and a polypeptide obtained by translation of the mRNA transcribed from the gene. The term "gene" refers to a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include regions before and after the coding region, for example, 5' untranslated (5'UTR) or "leader" sequences and 3'UTR or "trailer" sequences, as well as intervening sequences (introns) between individual coding segments (exons).

[0246] Integrating vectors enable the RNA / DNA they deliver to be permanently incorporated into the host cell chromosome. Non-integrating vectors maintain episomes, which means that the nucleic acids contained therein are never integrated into the host cell chromosome. Examples of integrating vectors include retroviral vectors, lentiviral vectors, hybrid adenoviral vectors, and herpes simplex virus vectors.

[0247] An example of a non-integrating vector is a non-integrating viral vector. Non-integrating viral vectors eliminate the risks posed by integrating retroviruses because they do not incorporate their genomes into the host DNA. An example is the Epstein-Barr oriP / Nuclear Antigen-1 (“EBNA1”) vector, which is capable of restricted self-replication and is known to function in mammalian cells. As containing two elements from the Epstein-Barr virus, oriP and EBNA1, the binding of the EBNA1 protein to the viral replicon region oriP maintains the episomal presence of the plasmid for a relatively long term in mammalian cells. This particular feature of the oriP / EBNA1 vector makes it ideal for the generation of integration-free iPSCs. Another non-integrating viral vector is the adenoviral vector and the adeno-associated virus (AAV) vector.

[0248] Another non-integrating viral vector is the RNA Sendai viral vector, which can produce proteins without entering the nucleus of the infected cell. The F-deficient Sendai viral vector stays in the cytoplasm of the infected cell for a few generations but is rapidly diluted and completely lost after a few generations (e.g., 10 generations).

[0249] Another example of a non-integrating vector is the minicircle vector. Minicircle vectors are circular vectors in which the plasmid backbone has been released, leaving only the eukaryotic promoter and cDNA(s) to be expressed.

[0250] As used herein, the term “viral vector” refers to a nucleic acid vector construct that includes at least one virus-derived element and has the ability to be packaged into viral vector particles. The viral vector may contain nucleic acids encoding the polypeptides described herein in place of non-essential viral genes. The vector and / or particle can be used for the purpose of transferring nucleic acids into cells in vitro or in vivo. Many forms of viral vectors are known in the art.

[0251] Engineered hepatic stellate cells (HSCs)

[0252] In one aspect, methods are described herein for producing hepatic stellate cells or a population thereof that express an agent that inhibits WISP1, the method comprising contacting the cells with any of the agents that inhibit WISP1 described herein, and culturing the cells for a sufficient time to allow expression of the agent.

[0253] In one embodiment, the cells are quiescent cells. Methods for identifying quiescent cells were described above.

[0254] In one embodiment, contacting comprises bringing the cells into contact with a reagent or a vector encoding the reagent. For example, contacting can include, but is not limited to, transduction, nucleofection, electroporation, direct injection (e.g., into HSCs), and / or transfection. Those skilled in the art can use techniques known in the art to bring the cells into contact with the reagents described herein.

[0255] In one embodiment, the reagent is a miRNA. In one embodiment, the miRNA is miRNA-15a or miRNA412.

[0256] In one embodiment, the cells are cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 20% (weight / volume) fetal bovine serum (FBS) and 1% (weight / volume) penicillin / streptomycin under standard culture conditions.

[0257] In one embodiment, the cells are cultured for at least 1 hour to allow expression of the reagent. In another embodiment, the cells are cultured for at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 36, 48, 60, 72, 96, 120, 144 or more hours to fully support expression of the reagent. Those skilled in the art can use standard techniques in the art, such as PCR-based assays to detect miRNA expression, to determine whether the reagent is expressed in the cells after culturing.

[0258] In one embodiment, the cells transiently express the reagent. In another embodiment, expression of the reagent is integrated into the genome of the cells, such that the progeny of the cells express the reagent.

[0259] In one aspect, there is provided a cell line comprising HSCs expressing a reagent that inhibits WISP1 produced by the methods described herein. The HSCs expressing a reagent that inhibits WISP1 can be in a pharmaceutically acceptable carrier, e.g., for administration to a subject in need thereof, e.g., for liver disease.

[0260] In another aspect, there is provided a pharmaceutical composition comprising a population of HSCs expressing a reagent that inhibits WISP1 produced by the methods described herein and a pharmaceutically acceptable carrier.

[0261] Compositions and pharmaceutical compositions

[0262] In one aspect, the present disclosure describes a composition comprising any of the reagents described herein. In one aspect, the present disclosure describes a pharmaceutical composition comprising any of the reagents described herein.

[0263] In another aspect, the present disclosure describes a composition comprising an antibody or an antibody agent that inhibits WISP1. In another aspect, the present disclosure describes a pharmaceutical composition comprising an antibody or an antibody agent that inhibits WISP1.

[0264] In one embodiment, the composition or pharmaceutical composition described herein may comprise at least 2, 3, 4, 5, or more of the reagents described herein. For example, the composition may comprise an siRNA that inhibits WISP1 and an anti-WISP1 antibody agent. Optionally, the composition may comprise two anti-WISP1 antibody agents.

[0265] In one embodiment of any aspect, the composition is formulated for the treatment or prevention of liver diseases. For the clinical use of the methods described herein, the administration of the reagent that inhibits WISP1 (e.g., an antibody, an antibody agent, or a WISP1-binding fragment thereof) described herein may comprise a pharmaceutical composition or a pharmaceutical formulation formulated for parenteral administration, e.g., intravenous; mucosal, e.g., intranasal; ocular, or other modes of administration. In some embodiments, the reagent described herein may be administered together with any pharmaceutically acceptable carrier compound, material, or composition that elicits an effective treatment in a subject. Accordingly, a pharmaceutical formulation for the methods described herein may comprise a combination of the antibody or its antigen-binding fragment described herein with one or more pharmaceutically acceptable ingredients.

[0266] The phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms within the scope of sound medical judgment that are suitable for contact with human and animal tissues without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. The phrase "pharmaceutically acceptable carrier" as used herein refers to a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, medium, encapsulating material, manufacturing aid (e.g., lubricant, talc magnesium, calcium stearate or zinc stearate, or stearic acid), or solvent encapsulating material, involved in maintaining the stability, solubility, or activity of the antibody or its antigen-binding fragment. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. The terms "excipient", "carrier", or "pharmaceutically acceptable carrier" and the like are used interchangeably herein.

[0267] The therapeutic formulations of the reagents or inhibitors of WISP1 described herein can be prepared for storage by mixing an antibody or antigen-binding fragment of desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the form of a lyophilized formulation or an aqueous solution. Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosages and concentrations employed, and include, for example, buffers such as phosphates, citrates, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as cetrimonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butanol, or benzyl alcohol; alkyl parabens such as methyl paraben or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants such as TWEEN, PLURONICS, or polyethylene glycol (PEG). Exemplary lyophilized antibody formulations are described in WO 97 / 04801, which is hereby incorporated by reference in its entirety.

[0268] Optionally but preferably, the formulations containing the compositions described herein contain pharmaceutically acceptable salts, typically, for example, sodium chloride, and preferably at about physiological concentrations. Optionally, the formulations of the present invention may contain pharmaceutically acceptable preservatives. In some embodiments, the preservative concentration ranges from 0.1 to 2.0%, typically v / v. Suitable preservatives include those known in the pharmaceutical art. Benzyl alcohol, phenol, m-cresol, methyl paraben, and propyl paraben are examples of preservatives. Optionally, the formulations of the present invention may include a pharmaceutically acceptable surfactant at a concentration of from 0.005 to 0.02%.

[0269] The therapeutic formulations of the compositions comprising the reagents (e.g., antibodies, antibody agents, and their WISP-binding fragments) described herein may also contain, depending on the particular indication to be treated, more than one active compound, preferably those having complementary activities that do not adversely affect each other. Optionally, for example, the composition may contain a cytotoxic agent, a cytokine, or a growth inhibitor, for example. Such molecules are present in a combination with an amount effective for the intended purpose.

[0270] The active ingredients of the therapeutic formulations of the compositions comprising the reagents described herein are encapsulated in microcapsules, which are prepared, for example, by coacervation techniques or interfacial polymerization of methylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or in macroemulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

[0271] In some embodiments, the pharmaceutical composition further includes a liquid carrier. Exemplary liquid carriers include, but are not limited to, liposomes, micelles, exosomes, fat emulsions, and liquid-drug complexes.

[0272] In some embodiments, the pharmaceutical composition further includes a particulate or polymer-based carrier. Exemplary particulate or polymer-based carriers include, but are not limited to, nanoparticles, microparticles, polymeric microspheres, or polymer-drug conjugates.

[0273] In some embodiments, sustained-release formulations may be used. Suitable examples of sustained-release formulations include semipermeable matrices of solid hydrophobic polymers containing an antibody or antigen-binding fragment, wherein the matrix is in the form of a shaped article such as a membrane or a microcapsule. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), or poly(vinyl alcohol)), polylactic acid (U.S. Patent No. 3,773,919), L-glutamic acid and y-ethyl L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid polymers such as LUPRON DEPOT TM(Injectable microspheres composed of lactic acid-glycolic acid polymers and leuprorelin acetate), and poly-D-(-)-3-hydroxybutyric acid. Although polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid, for example, are capable of releasing molecules for over 100 days, certain hydrogels release proteins over a shorter time period. When encapsulated antibodies remain in the body for a long time, as a result of exposure to humidity at 37 °C, they can deform or aggregate, leading to loss of biological activity and possible changes in immunogenicity. Rational strategies can be designed for stabilization depending on the mechanisms involved. For example, if the aggregation mechanism is found to be intermolecular S--S bond formation via thio-disulfide exchange, stabilization can be achieved by modifying thiol residues, lyophilization from acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0274] In the methods described herein, therapeutic formulations for in vivo administration, such as for parenteral administration, can be sterile, which can be readily achieved by filtration through a sterile filtration membrane or other methods known to those skilled in the art.

[0275] Administration

[0276] In some embodiments, the methods described herein involve treating a subject having or diagnosed as having a liver disease, comprising administering a reagent known to WISP1 described herein. A subject having a liver disease can be identified by a physician using current methods for diagnosing the condition. Symptoms and / or complications of liver disease that characterize liver disease and aid in diagnosis are known in the art and include, but are not limited to, fatigue, weight loss, pain, yellowing of the skin and / or eyes, and dark urine. Tests that can aid in the diagnosis of, for example, liver disease include, but are not limited to, example blood tests, non-invasive imaging, and / or tissue biopsies. A family history of liver disease will also aid in determining whether a subject is predisposed to the condition or aid in the diagnosis of liver disease.

[0277] The reagents and compositions described herein (e.g., those that inhibit WISP1) can be administered to a subject having or diagnosed with liver disease. In some embodiments, the methods described herein include administering to a subject an effective amount of a reagent to alleviate at least one symptom of liver disease. As used herein, "alleviating at least one symptom of liver disease" is improving 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 is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more as determined by any standard technique as compared to an equivalent untreated control. Various methods for administering the reagents and compositions described herein are known to those of skill in the art. In one embodiment, the reagent is administered systemically or locally (e.g., administered to the liver). In one embodiment, the reagent is administered intravenously. In one embodiment, the reagent is administered continuously, intermittently, or sporadically. The route of administration of the reagent will be optimized for the type of reagent to be delivered (e.g., miRNA, cell, or RNAi) and can be determined by a skilled artisan.

[0278] The reagents and pharmaceutical compositions described herein can be administered to a subject in need thereof by any suitable route that results in effective treatment in the subject. As used herein, the terms "administer" and "introduce" are used interchangeably and refer to a method or route by which the reagent or pharmaceutical composition described herein (e.g., an antibody, an antibody agent, or a WISP1-binding fragment thereof) is placed in a subject by causing at least partial localization of such reagent at a desired site such as, for example, at the site of an infection or cancer, such that the desired effect(s) is produced. The reagent or pharmaceutical composition can be administered to a subject by any mode of administration that delivers the reagent systemically or to a desired surface or target and can include, but is not limited to, injection, infusion, instillation, and inhalation administration. Oral administration forms are also contemplated to the extent that they can protect various reagents from inactivation in the gut. "Injection" includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratracheal, subcutaneous, subepidermal, intraarticular, subcapsular, subarachnoid, intraspinal, intracerebro spinal, and intrasternal injection and infusion.

[0279] Reagents (e.g., antibodies, antibody agents, and their WISP1-binding fragments) can be formulated, dosed, and administered in a manner consistent with good medical practice. Factors to be considered in this context include the particular disease being treated, the particular subject being treated, the clinical condition of the individual subject, the cause of the disorder, the site of delivery of the reagent, the method of administration, the administration schedule, and other factors known to the physician. The “therapeutically effective amount” of the reagent to be administered is governed by such considerations and refers to the minimum amount necessary to ameliorate, treat, or stabilize cancer; increase the time to progression (progression-free survival period) or treat or prevent the occurrence or recurrence of liver disease. In some embodiments, for example, the reagent is optionally formulated with one or more additional therapeutic agents currently used to prevent or treat infection. The effective amount of such other reagents depends on the amount of the reagent (e.g., antibody and its WISP1-binding fragment) present in the formulation, the type of disorder or treatment, and the other factors discussed above. These are typically used at the same dose and in the manner of administration as previously used herein or at about 1 to 99% of the dose heretofore employed.

[0280] Dose

[0281] For the treatment of liver disease, as described herein, the appropriate dose of the reagent (e.g., antibody, antibody agent, or its WISP1-binding fragment) will depend on the liver disease to be treated, as defined above, the severity and course of the disease, whether the reagent is administered for prophylactic or therapeutic purposes, previous treatment indications, the clinical history of the subject and response to the reagent, and the judgment of the attending physician. The reagent is administered to the subject either as a single dose or in a series of treatments. In a combination treatment regimen, the reagent and one or more additional therapeutic agents described herein are administered in therapeutically effective or synergistic amounts.

[0282] As used herein, “unit dosage form” refers to a dosage suitable for single administration. By way of example, the unit dosage form may be the amount of the therapeutic agent placed in a delivery device such as a syringe or an intravenous drip bag. In one embodiment, the unit dosage form is administered as a single administration. In another embodiment, more than one unit dosage form may be administered simultaneously.

[0283] The dose of the reagent as described herein can be determined by a physician and adjusted as needed to suit the observed effects of the treatment. With regard to the duration and frequency of treatment, a skilled clinician typically monitors the subject to determine when the treatment provides a therapeutic benefit and to determine whether to administer additional cells, discontinue treatment, restart treatment, or make other changes to the treatment regimen. The dose should not be so large as to cause adverse side effects, such as cytokine release syndrome. Generally, the dose will vary with the age, condition, and sex of the patient and can be determined by those skilled in the art. In the event of any complications, the dose can also be adjusted by the individual physician.

[0284] The dosage range of the therapeutic agent depends on potency and includes an amount large enough to produce the desired effect. The dosage should not be so large as to cause unacceptable adverse side effects. Generally, the dosage will vary with the age, condition, and sex of the patient and can be determined by one of ordinary skill in the art. In any case of complication, the dosage can also be adjusted by the individual physician. In some embodiments, the dosage range is from 0.001 mg / kg body weight to 100 mg / kg body weight. In some embodiments, the dosage range is from 5 μg / kg body weight to 100 μg / kg body weight. Optionally, the dosage range can be titrated to maintain serum levels between 1 μg / mL and 1000 μg / mL. For systemic administration, a subject can be administered a therapeutically effective amount, such as, for example, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 7.5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg or more. These dosages can be administered by one or more separate administrations or by continuous infusion. For repeated administration over several days or longer, depending on the condition, treatment can continue until, for example, the liver disease is treated, as determined by the methods described above or known in the art. However, other dosage regimens may also be useful.

[0285] Combination therapy

[0286] In one embodiment, the reagent or composition described herein is used as a monotherapy. In one embodiment, the reagent described herein can be used in combination with other known reagents and therapies for liver disease. As used herein, "combination" administration refers to the delivery of two (or more) different treatments to a subject during the subject's affliction with a disorder, e.g., between the time the subject has been diagnosed with a disorder (liver disease) and the disorder has been cured or eliminated or treatment has stopped for other reasons, the delivery of two or more treatments. In some embodiments, at the start of the delivery of the second treatment, the delivery of one treatment is still occurring, such that there is an overlap in administration. This is sometimes referred to herein as "simultaneous" or "parallel delivery". In other embodiments, the delivery of one treatment stops before the start of the delivery of the other treatment. In some embodiments of either case, the treatment is more effective due to the combination administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, or a similar situation is seen with the first treatment, compared to what would be seen if the second treatment were administered in the absence of the first treatment. In some embodiments, the delivery results in a reduction in symptoms, or other parameters associated with the disorder are higher than those observed when the first treatment is delivered in the absence of the other treatment. The effects of the two treatments can be partially cumulative, fully cumulative, or greater than cumulative. The delivery can be such that when the second treatment is delivered, the effect of the first treatment delivered is still detectable. The reagent described herein and at least one additional therapy can be administered simultaneously, or sequentially, in the same or in separate compositions. For sequential administration, the reagent described herein can be administered first, the additional reagent can be administered second, or the order of administration can be the reverse. The reagent and / or other therapeutic agent, process, or modality can be administered during the course of an active disorder, or can be administered during remission or less active disease. The reagent can be administered before another treatment, concurrently with the treatment, after the treatment, or during remission of the disorder.

[0287] Current therapies for treating liver diseases include, but are not limited to: ursodeoxycholic acid (UDCA, also known as ursodiol, INN, NAN, AAN, or USAN), cholestyramine, stanozolol, naltrexone, rifampicin, pioglitazone, metformin, rosiglitazone, lobeglitazone, retinyl esters, vitamin A, liver dialysis, or liver transplantation or any other treatment for liver diseases known in the art. In one embodiment, the reagent or composition described herein is not administered in combination with another therapy. Specifically, the reagent or composition described herein is not administered in combination with: ursodeoxycholic acid (UDCA, also known as ursodiol, INN, NAN, AAN, or USAN), cholestyramine, stanozolol, naltrexone, rifampicin, pioglitazone, metformin, rosiglitazone, lobeglitazone, retinyl esters, vitamin A, liver dialysis, or liver transplantation or any other treatment for liver diseases known in the art.

[0288] When administered in combination, the reagent or composition and the additional reagent(s) (e.g., a second or third reagent), or all, can be administered in an amount or dose that is higher than, lower than, or the same as the amount or dose of each individual reagent when used alone, such as in monotherapy. In certain embodiments, the amount or dose of the reagent, the additional reagent(s) (e.g., a second or third reagent), or all is lower than (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) the amount or dose of each individual reagent when used alone. In other embodiments, the amount or dose of the reagent, the additional reagent(s) (e.g., a second or third reagent), or all that results in the desired effect (e.g., treatment of liver disease) is lower than (e.g., at least 20%, at least 30%, at least 40%, or at least 50%) the amount or dose of each individual reagent required to achieve the same therapeutic effect when used alone.

[0289] Parenteral dosage forms

[0290] The parenteral dosage forms of the reagent described herein can be administered to a subject by various routes, including but not limited to, subcutaneous, intravenous (including bolus injection), intramuscular, and intra-arterial. Since the administration of parenteral dosage forms generally bypasses the patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or can be sterilized prior to administration to the patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products ready to be dissolved or dispersed in a pharmaceutically acceptable carrier for injection, suspensions ready for injection, controlled-release parenteral dosage forms, and emulsions.

[0291] As used herein, the phrase "parenteral administration" or "administering parenterally" refers to forms of administration other than enteral and topical administration, typically by injection. As used herein, the phrases "systemic administration", "administering systemically", "peripheral administration", and "administering peripherally" refer to the administration of a therapeutic agent other than directly to a target site, tissue, or organ such as, for example, a tumor site, such that it enters the circulatory system of a subject and thereby undergoes metabolism and other similar processes. In other embodiments, when the condition permits, the reagent is administered locally, such as by direct injection, and the injection can be repeated periodically.

[0292] Suitable carriers for providing the parenteral dosage forms of the present disclosure are known to those of skill in the art. Examples include, but are not limited to: sterile water; water for injection USP; saline solutions; glucose solutions; aqueous carriers such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-soluble carriers such as, but not limited to, ethanol, polyethylene glycol, and propanol; and non-aqueous carriers such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate.

[0293] The duration of therapy using the methods described herein will extend as long as medically indicated or until a desired therapeutic effect (e.g., those described herein) is achieved. In certain embodiments, the administration of the antibodies or antigen-binding fragments described herein continues for 1 month, 2 months, 4 months, 6 months, 8 months, 10 months, 1 year, 2 years, 3 years, 4 years, 5 years, 10 years, 20 years, or up to several years of the subject's lifespan.

[0294] Controlled-release and delayed-release dosage forms

[0295] In some embodiments of the aspects described herein, a reagent or composition is administered to a subject by a controlled release or delayed release manner. Ideally, the use of an optimally designed controlled release formulation in drug therapy is characterized by curing or controlling a condition with the least amount of drug in the smallest amount of time. The advantages of controlled release dosage forms include: 1) extending the activity of the drug; 2) reducing the dosing frequency; 3) increasing patient compliance; 4) using less total drug; 5) reducing local or systemic side effects; 6) minimizing drug accumulation; 7) reducing blood level fluctuations; 8) improving the efficacy of treatment; 9) reducing the potentiation or loss of drug activity; and 10) improving the rate of control of a disease or condition (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)). Controlled release formulations can be used to control the onset of action, duration of action, plasma levels within the therapeutic window, and peak blood level of the compounds of formula (I). In particular, controlled release or sustained release dosage forms or formulations can be used to ensure the maximum potency of the reagent is achieved while minimizing potential adverse reactions and safety issues that can occur due to underdosing of the drug (i.e., below the minimum therapeutic level) and exceeding the toxic level of the drug.

[0296] A variety of known controlled release or sustained release dosage forms, formulations, and devices can be employed for any of the reagents described herein. Examples include, but are not limited to, those described in the following: U.S. Patent Nos.: 3,845,770; 3,916,899; 3,536,809; 3,598,123; 4,008,719; 5674,533; 5,059,595; 5,591,767; 5,120,548; 5,073,543; 5,639,476; 5,354,556; 5,733,566; and 6,365,185, each of which is incorporated herein by reference in its entirety. These dosage forms can be used to provide slow release or controlled release of one or more active ingredients, using various proportions of, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, osmotic membranes, osmotic systems (e.g., (Alza Corporation, Mountain View, Calif. USA)), multilayer coatings, microparticles, liposomes, or microspheres or combinations thereof to provide the desired release performance. In addition, ion exchange materials can be used to prepare immobilized, adsorbed salt forms of the disclosed compounds, thereby affecting the controlled delivery of the drug. Examples of specific anion exchangers include, but are not limited to A568 and AP143 (Rohm & Haas, Spring House, Pa. USA).

[0297] Efficacy

[0298] The efficacy of the reagents described herein, such as for the treatment of liver disease, can be determined by a skilled physician. However, as used herein, if one or more markers or symptoms of liver disease are altered in a beneficial manner, other clinically acceptable symptoms are improved, or even alleviated, or a desired response is induced, such as being induced by at least 10% after treatment according to the methods described herein, the treatment is considered to be an "effective treatment". Efficacy can be evaluated by, for example, measuring markers, metrics, symptoms, and / or incidence of the condition being treated according to the methods described herein, or any other suitable measurable parameter such as fatigue, pain, weight loss, or dark urine. Efficacy can also be determined by the lack of deterioration of the individual being evaluated in the hospital or the need for medical intervention (i.e., progression of symptoms). Methods for measuring these metrics are known to those of skill in the art and / or described herein.

[0299] Efficacy can be evaluated in animal models of the conditions described herein, for example, a mouse model of liver disease or a suitable animal model as appropriate. When using an experimental animal model, the efficacy of the treatment is confirmed when a statistically significant change is observed in markers such as jaundice, fatigue, nausea, vomiting, urine color, abdominal pain, etc.

[0300] As used herein, the term "effective amount" refers to the amount of the reagent or composition described herein that can be administered to a subject having or diagnosed with liver disease and in need of alleviating at least one or more symptoms of the disease. Thus, the term "therapeutically effective amount" refers to the amount of the reagent or composition that, when administered to a typical subject, is sufficient to provide a specific anti-liver disease effect. In various cases, the effective amount as used herein can also include an amount of the reagent sufficient to delay the development of symptoms of the disease, alter the course of symptoms of the disease (e.g., slow the progression of liver disease), or reverse the symptoms of the disease (e.g., correct or stop the symptoms of the disease). Thus, an exact "effective amount" generally cannot be specified. However, for any given situation, the "effective amount" can be determined by those of ordinary skill in the art using only routine experimentation as needed.

[0301] In one embodiment, the reagent or composition is administered continuously (e.g., at a constant level over a period of time). Continuous administration of the reagent can be, for example, by an epidermal patch, a sustained-release dosage form, or an on-body injector.

[0302] In one embodiment, the reagent or composition is administered at intervals (e.g., at various levels over a given period of time).

[0303] Effective amounts, toxicity, and therapeutic efficacy can be evaluated by standard pharmaceutical procedures in cell cultures or experimental animals. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The dose ratio between toxicity and therapeutic effect is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods with a large therapeutic index are preferred. The therapeutically effective dose can initially be estimated from cell culture assays. Moreover, the dosage can be formulated in animal models to achieve a circulating plasma concentration range of the IC50 (i.e., the concentration of the agent that achieves half the maximal inhibition of symptoms) as determined in cell culture or in a suitable animal model. The levels in the plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dosage can be monitored by appropriate bioassays, where, for example, liver function or blood work is determined. The dosage can be determined by a physician and adjusted as needed to suit the observed therapeutic effect.

[0304] The invention provided herein may be further described in the following paragraphs:

[0305] 1. A method of treating or preventing a liver disease, the method comprising: administering to a subject in need thereof an antibody or antibody agent that inhibits WISP1.

[0306] 2. The method according to paragraph 1, wherein the liver disease is selected from the group consisting of: primary biliary cholangitis, autoimmune hepatitis, alpha-1 antitrypsin deficiency, non-alcoholic steatohepatitis, and scleroderma.

[0307] 3. The method according to any preceding paragraph, wherein the WISP1 is a splice variant selected from the group consisting of: WISP1v, WISP1vx, and WISP1 delta exon 3-4.

[0308] 4. The method according to any preceding paragraph, wherein the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of: mab1680, AF1680, SAB2501114, ab60114, and ab65943.

[0309] 5. The method according to any preceding paragraph, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

[0310] 6. The method according to any preceding paragraph, wherein WISP1 is inhibited in a target cell.

[0311] 7. The method according to any preceding paragraph, wherein the target cell is a mammalian cell.

[0312] 8. A method according to any of the preceding paragraphs, wherein the target cell is a hepatic stellate cell, a fibroblast, or a myofibroblast.

[0313] 9. A method according to any of the preceding paragraphs, wherein the hepatic stellate cell is quiescent.

[0314] 10. A method according to any of the preceding paragraphs, wherein the antibody or antibody agent is administered by direct injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0315] 11. A method according to any of the preceding paragraphs, wherein inhibiting WISP1 is inhibiting WISP1 activity or reducing the WISP1 protein level.

[0316] 12. A method according to any of the preceding paragraphs, 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.

[0317] 13. A method according to any of the preceding paragraphs, wherein the level of WISP1 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.

[0318] 14. A composition comprising an antibody or antibody agent that inhibits WISP1 and a pharmaceutically acceptable carrier.

[0319] 15. A composition according to any of the preceding paragraphs, wherein the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of mab1680, AF1680, SAB2501114, ab60114, and ab65943.

[0320] 16. A composition according to any of the preceding paragraphs, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

[0321] 17. A composition according to any of the preceding paragraphs, wherein the composition is configured to treat or prevent liver diseases.

[0322] 18. A method of treating a liver disease in a subject, the method comprising:

[0323] a. Detecting the levels of WISP1 and / or Yap, Col1a1, Acta2 in a biological sample of the subject;

[0324] b. Comparing the measurement result of (a) with a reference level;

[0325] c. Identifying a subject having increased WISP1 and / or Yap, Col1a1, Acta2 compared to a reference level in (a) as having liver disease; and

[0326] d. Administering to a subject having liver disease an antibody or antibody agent that inhibits WISP1.

[0327] 19. The method according to any of the preceding paragraphs, further comprising obtaining a biological sample from the subject before (a).

[0328] 20. The method according to any of the preceding paragraphs, wherein the liver disease is primary biliary cholangitis, autoimmune hepatitis, α1 - antitrypsin deficiency, non - alcoholic steatohepatitis, or scleroderma.

[0329] 21. The method according to any of the preceding paragraphs, wherein the biological sample is a blood sample, tissue, buffy coat, serum, or tissue.

[0330] 22. The method according to any of the preceding paragraphs, wherein the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of mab1680, AF1680, SAB2501114, ab60114, and ab65943.

[0331] 23. The method according to any of the preceding paragraphs, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1 - 4, 6, or 12 - 120.

[0332] 24. A method for treating or preventing liver disease, the method comprising: administering to a subject in need a reagent that inhibits WISP1.

[0333] 25. The method according to any of the preceding paragraphs, wherein the liver disease is selected from the group consisting of primary biliary cholangitis, autoimmune hepatitis, α1 - antitrypsin deficiency, non - alcoholic steatohepatitis, and scleroderma.

[0334] 26. The method according to any of the preceding paragraphs, wherein the WISP1 is a splice variant selected from the group consisting of WISP1v, WISP1vx, and WISP1 exon 3 - 4.

[0335] 27. The method according to any of the preceding paragraphs, wherein the WISP1 is inhibited in target cells.

[0336] 28. The method according to any of the preceding paragraphs, wherein the reagent that inhibits WISP1 is selected from the group consisting of small molecules, antibodies or antibody agents, peptides, genome editing systems, viral vectors, miRNAs, and siRNAs.

[0337] 29. A method according to any of the preceding paragraphs, wherein the microRNA is microRNA15a or miRNA412.

[0338] 30. A method according to any of the preceding paragraphs, wherein the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of mab1680, AF1680, SAB2501114, ab60114, and ab65943.

[0339] 31. A method according to any of the preceding paragraphs, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

[0340] 32. A method according to any of the preceding paragraphs, wherein the reagent is administered by direct injection, subcutaneous injection, intramuscular injection, or nasal administration.

[0341] 33. A method according to any of the preceding paragraphs, wherein inhibiting WISP1 is inhibiting WISP1 activity or reducing WISP1 protein level.

[0342] 34. A method according to any of the preceding paragraphs, 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.

[0343] 35. A method according to any of the preceding paragraphs, wherein the level of WISP1 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.

[0344] 36. A composition comprising a reagent that inhibits WISP1 and a pharmaceutically acceptable carrier.

[0345] 37. A composition according to any of the preceding paragraphs, wherein the reagent that inhibits WISP1 is selected from the group consisting of small molecules, antibodies or antibody agents, peptides, genome editing systems, viral vectors, microRNAs, and siRNAs.

[0346] 38. A composition according to any of the preceding paragraphs, wherein the microRNA is microRNA15a or miRNA412.

[0347] 39. A method of treating liver disease in a subject, the method comprising:

[0348] a. Detecting the levels of WISP1 and / or Yap, Col1a1, Acta2 in a biological sample of the subject;

[0349] b. Compare the measurement result in (a) with a reference level;

[0350] c. Identify subjects in (a) with increased WISP1 and / or Yap, Col1a1, Acta2 compared to a control level for subjects with liver disease; and

[0351] d. Administer an agent that inhibits WISP1 to a subject with liver disease.

[0352] 40. The method according to any of the preceding paragraphs, further comprising obtaining a biological sample from a subject prior to (a).

[0353] 41. The method according to any of the preceding paragraphs, wherein the liver disease is primary biliary cholangitis, autoimmune hepatitis, α1 - antitrypsin deficiency, non - alcoholic steatohepatitis, or scleroderma.

[0354] 42. The method according to any of the preceding paragraphs, wherein the biological sample is a blood sample, tissue, buffy coat, serum, or tissue.

[0355] 43. The method according to any of the preceding paragraphs, wherein the agent that inhibits WISP1 is selected from the group consisting of small molecules, antibodies or antibody agents, peptides, genome editing systems, viral vectors, miRNAs, and siRNAs.

[0356] 44. The method according to any of the preceding paragraphs, wherein the microRNA is microRNA15a or miRNA412.

[0357] 45. The method according to any of the preceding paragraphs, wherein the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of mab1680, AF1680, SAB2501114, ab60114, and ab65943.

[0358] 46. The method according to any of the preceding paragraphs, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1 - 4, 6, or 12 - 120.

[0359] 47. A method of producing an engineered hepatic stellate cell or a population thereof that expresses an agent that inhibits WISP1, the method comprising: contacting the cell with an agent that inhibits WISP1, and culturing the cell for a sufficient length of time to allow expression of the agent.

[0360] 48. The method according to any of the preceding paragraphs, wherein the cell is quiescent.

[0361] 49. The method according to any of the preceding paragraphs, wherein the contacting comprises contacting the cell with the agent or a vector encoding the agent.

[0362] 50. According to the method described in any of the preceding paragraphs, wherein the contacting comprises transduction, nuclear transfection, electroporation, direct injection, and / or transfection.

[0363] 51. According to the method described in any of the preceding paragraphs, wherein the agent that inhibits WISP1 is selected from the group consisting of: small molecules, antibodies or antibody agents, peptides, genome editing systems, viral vectors, miRNAs, and siRNAs.

[0364] 52. According to the method described in any of the preceding paragraphs, wherein the microRNA is microRNA15a or miRNA412.

[0365] 53. According to the method described in any of the preceding paragraphs, wherein the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of: mab1680, AF1680, SAB2501114, ab60114, and ab65943.

[0366] 54. According to the method described in any of the preceding paragraphs, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

[0367] 55. A cell line comprising hepatic stellate cells produced by the method described in any one of paragraphs 46-53.

[0368] 56. A pharmaceutical composition comprising hepatic stellate cells or a population thereof produced by the method described in any of the preceding paragraphs, and a pharmaceutically acceptable carrier.

[0369] 57. A method of treating or preventing liver disease, the method comprising: administering to a subject in need thereof the cells produced by the method described in any of the preceding paragraphs, the cells described in any of the preceding paragraphs, or the pharmaceutical composition described in any of the preceding paragraphs.

[0370] 58. A method of reducing fibrosis in a subject, the method comprising: administering to a subject in need thereof the cells produced by the method described in any of the preceding paragraphs, the cells described in any of the preceding paragraphs, or the pharmaceutical composition described in any of the preceding paragraphs.

[0371] 59. A method of treating liver disease in a subject, the method comprising:

[0372] a. receiving the results of a test that identifies a subject having increased WISP1 and / or Yap, Col1a1, Acta2 compared to a reference level as having liver disease; and

[0373] b. administering to the subject having liver disease an antibody or antibody agent that inhibits WISP1.

[0374] 60. A method for treating liver disease in a subject, the method comprising:

[0375] a. receiving the results of a test that identifies subjects with increased WISP1 and / or Yap, Col1a1, Acta2 compared to a reference level as having liver disease; and

[0376] b. administering a reagent agent that inhibits WISP1 to a subject with liver disease.

[0377] Examples

[0378] Example 1: Treatment of progressive liver disease

[0379] In response to an unmet need for liver disease therapies, the independent induction of the quiescent microRNAs miR-15a and miR-412 in activated hepatic stellate cells (HSCs) is described herein, which are the cell type that plays a central role in the progression of liver fibrosis. In addition, miR-15a directly targets WISP1 to inhibit its profibrotic function in activated HSCs. By using microRNAs and their targets to promote HSC quiescence, microRNAs can be useful therapies for controlling progressive liver fibrosis in PBC ( Figure 1 ). Without being bound by a particular theory, it is expected that miR-15a and miR-412 induce quiescence in HSCs, which may subsequently account for the function of miR-15a known to target WISP1.

[0380] The work described herein shows that quiescence-like HSCs induced by miR-15a or miR-412 result in improved liver injury and fibrosis in CCl4-challenged mice. Given the beneficial effects of miR-15a or miR-412 in the CCl4 model, mouse treatment studies can be extended using the cholestatic fibrosis model, bile duct ligation (BDL), and 3,5-diethoxycarbony1-1,4-dihydrochollidine (DDC). Three different miRNA delivery systems can be tested: 1) cell therapy using quiescence-like HSCs constitutively expressing miR-15a or miR-412, 2) injection of lentivirus expressing miR-15a or miR-412 into the tail vein, and 3) injection of chemically modified mimics of miR-15a or miR-412 packaged in a lipid-based carrier into the tail vein. Control and treated mice can be used to analyze the results.

[0381] The work described herein shows that MiR-15a can directly target WISP1 to inhibit its profibrotic function in HSCs. The hepatic phenotype of WISP1-null mice in the context of BDL and DDC challenge can be defined. The severity of liver injury and HSC dysfunction after challenge can be evaluated and compared to those of wild-type mice. A murine model of cholestatic fibrosis can be treated with a WISP1-blocking antibody and then used to analyze the results.

[0382] Primary biliary cholangitis (PBC), formerly known as primary biliary cirrhosis, is a latent liver disease that results in the progressive destruction of intrahepatic bile ducts, cholestasis, periportal inflammation, and ultimately biliary fibrosis, ending in end-stage liver disease of cirrhosis. Although genetic and environmental factors may interact to present the disease, it is an autoimmune disease of unknown pathogenesis. Interestingly, PBC affects women far more than men, with a female-to-male disease ratio of 9:1. In the United States, the age-adjusted incidence of PBC per million person-years is 45 for women and 7 for men, while the prevalence per million is 654 for women and 121 for men [1].

[0383] The hydrophilic bile salt ursodeoxycholic acid (UDCA) remains the only drug with a proven survival benefit in PBC. It has been shown to delay disease progression as demonstrated by improved histological and biochemical parameters. Although the exact mechanism of its action is unknown, it appears to protect cholangiocytes against toxic bile salts. Unfortunately, in the case of suboptimal response, 30%-40% of UDCA-treated individuals still experience disease progression [2]. To fill this therapeutic gap, many different agents have been in clinical trials, but some important investigational drugs such as obeticholic acid still have significant side effects and lack evidence of survival benefit [3].

[0384] Like most chronic liver diseases, PBC progresses from inflammation to fibrosis. In this pathological cascade, hepatic stellate cells (HSCs) are thought to play a central role in liver fibrogenesis [4-6]. HSCs exist in two forms. In healthy individuals, they are quiescent, characterized by multiple vitamin A-rich lipid droplets. However, once activated, they lose their lipid droplets and become profibrotic myofibroblasts, secreting collagen and mediators that promote scar formation [7, 8]. Although the importance of HSCs in liver fibrosis has been well established, much remains unknown about this cell type.

[0385] Among the many gaps in knowledge regarding HSCs is the current understanding of the role of microRNAs (miRNAs) in determining HSC activation status. miRNAs are short, non-coding genes, typically 22 nucleotides in length and involved in all biological and pathological processes. They downregulate specific coding genes by imperfect base pairing with complementary sequences within their mRNA targets to induce either destruction or translational repression. Each miRNA can regulate many different coding genes, while each target gene can be regulated by many different miRNAs, constituting a complex layer of the gene regulatory network [9]. The experiments described herein have shown that miRNAs are essential for cell proliferation and reprogramming [10-12]. However, the role of miRNAs in HSC activation or reversion to quiescence has not been thoroughly explored.

[0386] Although global miRNA expression patterns have been described in HSCs in various groups, the functional significance of their expression status remains largely unknown [13-15]. In response, instead of relying on large-scale expression profiling, functional screens have been developed to systematically identify miRNAs that force activated HSCs to return to quiescence, and through this effort, miR-15a and miR-412 were identified. miR-15a and miR-412 have not been previously studied in the context of hepatic stellate cells. Subsequently, the miR-15 family was found to inhibit the Tgf-β pathway in the heart to potentially attenuate fibrosis

[16] . Promotion of HSC quiescence through these miRNAs or their downstream targets may prevent fibrosis progression in PBC. The aim of this study was to evaluate the therapeutic efficacy of miR-15a, miR-412, and their direct targets in the treatment of PBC.

[0387] Results

[0388] The unbiased functional screen described herein takes advantage of the natural tendency of HSCs to become activated when grown on plastic surfaces. As HSCs become activated, they lose the lipid droplets that are abundant in quiescent HSCs. Using this useful phenotypic dichotomy, miRNAs that force activated HSCs to become more quiescent-like were searched for by tracking the levels of lipid droplets reforming in the cytoplasm. Since miRNAs are known to simultaneously target multiple coding genes to affect whole-cell programs such as organ development, carcinogenesis, or cell reprogramming

[17] , it was anticipated that even a single miRNA could induce transdifferentiation.

[0389] To identify those miRNAs that promote the reversion of activated HSCs to quiescence, after activated HSCs received single miRNA mimics from a whole-genome miRNA library, the intracellular lipid droplet reappearance with retinoids was scanned as a marker of quiescence ( Figure 2)。In fact, HSCs in some of the pores showed re-formed Bodipy-stained positive lipid droplets within three days. This initial screening yielded 15 primary hits. Based on the presence of human orthologs and the ability to re-form lipid droplets in mouse and human HSCs ( Figure 3 ), the miRNAs miR-15a and miR-412 were selected for further study. The newly formed lipid droplets were shown to be retinoid-positive by fluorescence under ultraviolet light, which was consistent with those in quiescent HSCs (data not shown). The overall size of the transfected HSCs decreased 10 - 100-fold and became more quiescent-like (Figure 4). In addition, the forced expression of miR-15a or miR-412 downregulated two of the most important gene markers of activation, alpha-smooth muscle actin (Acta2) and alpha-1 type I collagen (Col1a1) (Figure 4).

[0390] For more comprehensive expression analysis, deep RNA sequencing showed that the received miR-15a or miR-412 had a global transcriptional profile that was closer to that of quiescent-like HSCs than activated cells by 40 - 50% (data not shown). Most importantly, HSCs that became quiescent-like by miR-15a or miR-412 had a functional phenotype similar to that of truly quiescent HSCs. Ex vivo experiments showed that when the two cell types were co-cultured, quiescent-like HSCs did not induce steatosis in healthy hepatocytes. In contrast, activated HSCs not treated with the candidate miRNAs induced hepatocyte steatosis when they were co-cultured ( Figure 5 ). In addition, these reversed HSCs were able to reduce the expression of pro-inflammatory cytokines from co-cultured human hepatocellular carcinoma cell lines HepG2 and Huh7 ( Figure 6 ). Finally, as expected, endogenous miR-15a and miR-412 had reduced expression levels in activated HSCs compared to quiescent HSCs, although not significantly for miR-15a ( Figure 7 ), and miR-41 seemed to promote the reversal of activated HSCs towards quiescence in forced activation culture conditions. This and other key ex vivo observations allowed for the following tests: whether these miRNAs could be delivered in vivo to attenuate the level of liver pathology in the traditional CCl4 mouse model (oral gavage of 100 ul of 40% CCl4, twice a week), especially since HSCs expressing miR-15a or miR-412 could maintain a quiescent-like state even in a diseased liver with activation-promoting signals. Through cell contact and soluble mediators, quiescent-like HSCs could induce other cell types in the liver to inhibit their pro-inflammatory or fibrotic signals.

[0391] Despite the different methods of delivering miRNA to live mice, the HSCs selected for injection were made quiescent-like ex vivo by constitutively expressing miR-15a or miR-412. The reprogrammed HSCs were injected once during the third week of four-week CCl4 challenge. Engraftment of the injected HSCs onto the liver was confirmed by visualization of the GFP signal emitted within hepatocytes, inserting the piggyBac vector expressing miRNA

[18] ( Figure 8 ). More importantly, mice treated with quiescent-like HSCs had histology showing reduced ballooning, apoptosis, inflammation, and fibrosis in the liver( Figure 8 ), and less hepatic collagen expression( Figure 9 ).

[0392] These two miRNA candidates may downregulate HSC genes that promote liver inflammation and fibrosis. Although many genes may be inhibited by miR-15a and miR-412, WISP1 has been experimentally verified as a direct target of miR-15a. WISP1 expressed by activated HSCs is almost 30-fold that of quiescent HSC levels (data not shown), and WISP1 expressed by HSCs in human PBC is much higher than that in normal liver( Figure 10 ). Finally, miR-15a was predicted to target WISP1 which contains two potential miR-15a binding sequences within the 3'-UTR. Therefore, the putative target sequences or their mutant variants were cloned downstream of a constitutively activated luciferase reporter gene. When one of these reporters was co-transfected with a miR-15a mimic, the reporter with the wild-type sequence had reduced luciferase expression, while the reporter with the mutant sequence did not have reduced luciferase expression, indicating that miR-15a binds to both WISP1 target sequences( Figure 11 ). WISP1 is a key target gene for some of the actions of miR-15a in HSCs.

[0393] Testing miR-15a and miR-412 in a murine model of cholangitis

[0394] Delivery of miR - 15a or miR - 412 is a therapy for PBC : Given the surprising results described herein, in vivo experiments were extended to further evaluate the potential of miR-15a and miR-412 in treating PBC. To evaluate whether these miRNAs exert their functions only through HSCs or more broadly through other cell types, systemic delivery of miRNAs using viral vectors or mimics can be used instead of cell therapy. For all in vivo treatment experiments, efficacy can be evaluated by performing the following: PCR of profibrotic genes in the liver, histology including H&E and Sirius red staining, hepatic hydroxyproline assay, and determination of plasma ALT and alkaline phosphatase.

[0395] Each model of PBC has advantages and disadvantages in representing human PBC. For the purposes of this study, a model was needed that would consistently progress to biliary fibrosis relatively quickly to test miRNA candidates and inhibition of WISP that could lead to reduced fibrosis and inflammation. Most genetic models of PBC do not result in liver fibrosis or do so very slowly

[19] . Therefore, two models of cholangitis that reliably achieve fibrosis by induction via bile duct ligation (BDL) surgery and chemical induction by 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) can be used. All experiments can be terminated after 3 weeks for the BDL model and after 4 weeks for the DDC model, as these take time to develop significant levels of cholestatic fibrosis [20-22].

[0396] Using mesenchymal stem cells and macrophages, multiple groups have readily attempted cell therapy for liver fibrosis and achieved some success

[23] . However, the experiments described herein are the first to utilize purposefully engineered HSCs to prevent liver fibrosis. Although the initial in vivo treatment is compelling, considering that only the CCl4 model has been examined to date, it is useful to examine the effect of this cell therapy in a murine model of cholangitis. For all cell therapy experiments, 500,000 quiescent-like HSCs reprogrammed by miR-15a or miR-412 can be injected into the spleen. All appropriate controls can also include, for example, an untreated control group and a control group treated with non-reprogrammed HSCs.

[0397] Mice challenged with BDL for 21 days typically develop significant fibrosis. During the 21-day course of BDL challenge, reprogrammed HSCs can be injected on days 7 and 14. All mice are sacrificed on day 21. Mice challenged with DDC for 28 days typically develop significant fibrosis. During the 28-day course of DDC challenge, reprogrammed HSCs can be injected on days 14 and 21. All mice are sacrificed on day 28.

[0398] Initial experiments showed that injection of HSCs reprogrammed by miR-15a or miR-412 could reduce collagen expression and the overall level of liver injury in CCl4-induced liver fibrosis. Although delivery of miRNA by cell therapy has shown promise, other delivery methods can be explored. One method is injection of lentivirus expressing miRNA. On days 7 and 14 during the 21-day course of BDL challenge, lentivirus can express miR-15a or miR-412 into the tail vein. All mice are sacrificed on day 21. On days 14 and 21 during the 28-day course of DDC challenge, lentivirus can express miR-15a or miR-412. All mice are sacrificed on day 28.

[0399] As another delivery method, the feasibility of more stable, chemically modified miRNA mimics (Exiqon and Invitrogen) encapsulated in a lipid-based carrier (MaxSuppressorTM from B100 Scientific) can be tested [24, 25]. By avoiding lentiviruses, this method or its variants have the potential for use in humans. On days 7 and 14 during the 21-day course of BDL challenge, carriers containing miR-15a or miR-412 can be injected into the tail vein. All mice are sacrificed on day 21. On days 14 and 21 during the 28-day course of DDC challenge, carriers containing miR-15a or miR-412 can be injected. All mice are sacrificed on day 28.

[0400] Since it is difficult to induce liver fibrosis in female mice, male mice may be preferred for the in vivo treatment studies described herein. Power calculation shows that 8 mice per group are required to detect a 50% reduction in α1 type I collagen levels in assays at a p-value of 0.01 and 90% power, with a 20% standard deviation. One of the greatest challenges in treating HSCs with miRNA is to develop a delivery system that specifically targets this cell type to minimize off-target effects. The delivery system can be optimized to improve specificity. For example, two recently developed promising HSC delivery systems are the p75 neurotrophin receptor peptide (p75NTRp)-labeled adenovirus and the AAV6 vector [26, 27].

[0401] Evaluate the function of WISP1 in a murine model of cholangitis

[0402] MiR - 15a targets Wispl and promotes inflammation and fibrosis in PBC: WISP1 is a member of the Ccn family of matricellular proteins, which includes Cyr61 (Ccn1), Ctgf (Ccn2), Nov (Ccn3), WISP1 (Ccn4), Wisp2 (Ccn5), and Wisp3 (Ccn6). Ctgf has been established as an important profibrotic factor in the liver

[28] .

[0403] Interestingly, WISP1 has been found to be upregulated in human congenital pulmonary fibrosis, and treatment with a neutralizing WISP1 antibody attenuates bleomycin-induced pulmonary fibrosis

[29] . In addition, small animal studies have shown that blocking WISP1 improves CCl4-induced liver fibrosis

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

[0404] Although WISP1 is a direct target of miR-15a, its effect on the activated state of HSCs and its role in the pathogenesis of PBC are unknown. A WISP1-null mouse line has been generated to illustrate the role of WISP1 in bone formation, but its function in the liver has not been studied

[31] . These mice are fertile and do not have an obvious liver phenotype. Their mutant alleles are preserved in frozen sperm and embryos in the MMRRC facility in University of California at Davis, which ensures a successful source of mutant mice.

[0405] The phenotype can be evaluated by the overall severity of liver pathology. WISP1-null mice can be challenged with BDL and DDC. The resulting liver injury sustained by WISP1-null mice can be compared with that of wild-type mice. Given the known profibrotic role of Wisp1 in other organs including the lung, WISP1-null mice are expected to have reduced liver fibrosis and possibly even reduced inflammation. For all in vivo experiments, the liver phenotype can be evaluated by performing the following: PCR of profibrotic genes in the liver, histology including H&E and Sirius red staining, liver hydroxyproline assay, and measurement of plasma ALT and alkaline phosphatase. Finally, the functional phenotype of HSCs harvested from these murine models of biliary fibrosis can be evaluated. Male wild-type and WISP1-null mice can be challenged with BDL. They are sacrificed on day 21. Male wild-type and WISP1-null mice can be challenged with DDC. They are sacrificed on day 28.

[0406] Then, HSCs can be isolated from mice challenged with BDL for 21 days and DDC for 28 days. These HSCs can be co-cultured with healthy hepatocytes to determine whether pro-inflammatory mediators are induced. The results can be compared with those of wild-type HSCs that have undergone the same challenge.

[0407] Blocking WISP1 with neutralizing antibodies attenuates bleomycin-induced fibrosis in the mouse lung

[29] . Similarly, blocking Wisp1 can reduce the levels of inflammation and fibrosis that occur in PBC. A murine model of cholangitis can be treated with a WISP1 antibody and the liver phenotype can be evaluated.

[0408] Mice challenged with BDL for 21 days typically develop significant fibrosis. The treatment group can receive a commercially available WISP1 antibody (R&D Systems) that has been previously used to treat bleomycin-induced pulmonary fibrosis on days 7 and 14. The control group can receive IgG. All mice are sacrificed on day 21. Mice challenged with DDC for 28 days typically develop significant fibrosis. A WISP1-blocking antibody can be injected on days 14 and 21 during the 28-day course of DDC challenge. All mice are sacrificed on day 28.

[0409] As described herein, WISP1 is a pro-inflammatory and profibrotic mediator that can be neutralized by an antibody to reduce the levels of inflammation and fibrosis in the body. Thus, blocking WISP1 is also expected to improve biliary fibrosis. Accordingly, paracrine or autocrine mediators can be identified by analyzing the conditioned medium of activated HSCs. A more detailed analysis of the conditioned medium of activated HSCs can be performed by mass spectrometry to identify other mediators as drug targets.:

[0410] References - Example 1:

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[0415] 4. Friedman SL (2008) Mechanisms of hepatic fibrogenesis.

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[0447] doi:10.1074 / jbc.M114.628818.

[0448] Example 2: Treatment of Non-Alcoholic Fatty Liver Disease (NAFLD)

[0449] Non-alcoholic fatty liver disease (NAFLD) has become the most common cause of end-stage liver disease, liver transplantation, and hepatocellular carcinoma in developed countries in the next 5-10 years. Currently, since there are no FDA-approved drugs, reducing the risk factors that can lead to NAFLD is the main management approach. Therefore, developing effective therapies for NAFLD is extremely important.

[0450] Activated hepatic stellate cells (HSCs) are known to play a key role in the progression of liver fibrosis, and the data presented herein show that microRNAs miR-15a and miR-412 independently restore activated HSCs back to quiescence. In addition, miR-15a directly targets WISP1 to inhibit its pro-steatotic and pro-inflammatory functions in activated HSCs. Without wishing to be limited by a particular theory, it is speculated that miR-15a and miR-412 promote quiescence in HSCs, which may subsequently inhibit the progression of fibrosis in NAFLD.

[0451] With the advent of highly effective antiviral therapies against hepatitis B and C, non-alcoholic fatty liver disease (NAFLD) has become the most prevalent liver disease. NAFLD is currently considered the most common liver disease in developed countries [1], and is projected to become the leading cause of end-stage liver disease, liver transplantation, and hepatocellular carcinoma by 2025 [2, 3]. The disease is particularly common in affluent countries such as the United States, where 64 million people are estimated to be affected due to the high prevalence of risk factors including obesity, diabetes, and hyperlipidemia. In addition, the economic burden of NAFLD is staggering, with direct medical costs estimated at approximately $103 billion per year [4]. The disease begins as hepatic steatosis, but can progress to non-alcoholic steatohepatitis (NASH), fibrosis, and ultimately cirrhotic liver failure or hepatocellular carcinoma. NAFLD is currently managed primarily by reducing risk factors. However, these measures are often difficult to achieve, and even eliminating them does not ensure improvement [5]. Given that NAFLD is becoming the most common cause of liver-related morbidity and there are currently no drugs to manage it, better treatment options are needed. As described herein, this is the first step in developing new therapies for NAFLD.

[0452] NAFLD progresses in a stepwise manner through steatosis, inflammation, and fibrosis. In this pathological cascade, hepatic stellate cells (HSCs) are thought to play a central role in liver fibrogenesis [6-8]. HSCs exist in two forms. In healthy individuals, they are quiescent, characterized by multiple vitamin A-rich lipid droplets ( Figure 12 ). However, once activated, they lose their lipid droplets and become pro-fibrotic myofibroblasts, secreting collagens and mediators that promote scarring ( Figure 12 ) [9, 10].

[0453] Although the importance of HSCs in liver fibrosis has been well established, much remains unknown about this cell type. For example, are there genes that can reverse the activation of HSCs back to a quiescent state? Are HSCs also involved in early NAFLD, promoting steatosis and inflammation, and not just fibrosis? If HSCs contribute to steatohepatitis or fibrosis, what are the key mediators involved in these processes? Answering these questions and gaining better biological insights could lead to new ideas for managing NAFLD.

[0454] Among the many gaps in current knowledge, the understanding of the role of microRNAs (miRNAs) in determining the activated state of HSCs is particularly poor. Although global miRNA expression patterns in HSCs have been described in various groups, the functional significance of their expression states remains largely unclear [11 - 13]. In response, studies have focused on first determining miRNA function and exploring key miRNAs that affect the activated state of HSCs. Instead of relying on broad expression profiling, functional screens and assays have been developed to systematically identify miRNAs that force activated HSCs to return to quiescence, and through this effort, miR - 15a and miR - 412 have been identified. Promotion of HSC quiescence through these miRNAs or their downstream targets may prevent the progression of fibrosis in NAFLD.

[0455] Ultimately, activated HSCs that lead to fibrosis are not unique to NAFLD. Most chronic liver diseases involve HSC - promoted fibrosis for several years before cirrhosis is reached. Thus, if therapeutic agents can reduce the rate of fibrosis progression by targeting HSCs, this approach could be used in liver diseases other than NAFLD. The potential impact of such a therapy is substantial. Even for diseases such as primary biliary cirrhosis or autoimmune hepatitis for which established drug therapies exist, additional agents that inhibit fibrosis progression could significantly improve the ability to manage these chronic diseases. In summary, given the importance of NAFLD and the central role that HSCs play in its fibrosis progression, the importance of the studies described in this article is broad.

[0456] Although several compounds have been used in human trials to treat NAFLD, none of them have the primary goal of preventing or reversing HSC activation [5, 14, 15]. In response, the first major innovation began with the attempt to control the activated state of HSCs as a means of preventing progressive liver injury and fibrosis. The in vitro screen described in this article takes advantage of the natural tendency of HSCs to become activated when grown on plastic surfaces. As HSCs become activated, they lose the lipid droplets that are abundant in quiescent HSCs ( Figure 12 ). Taking advantage of this useful phenotypic dichotomy, miRNAs that force activated HSCs to become more quiescent - like were sought by tracking the levels of lipid droplets that reform in the cytoplasm. Since miRNAs are known to simultaneously target multiple coding genes to affect entire cellular programs such as organ development, carcinogenesis, or cell reprogramming

[16] , it is reasonable to assume that even a single miRNA could induce this trans - differentiation. Indeed, through unbiased investigation, several miRNA candidates were identified that appear to reverse HSCs from the activated state back to quiescence, as indicated by the reappearance of intracellular lipid droplets ( Figure 3)。 These subsets of miRNAs also result in other phenotypic changes towards HSC quiescence, including gene expression patterns, proliferation rates, and effects on neighboring hepatocytes. This innovation of using miRNAs to control the HSC activation state has laid the foundation for developing an entirely new class of therapies to prevent liver fibrosis.

[0457] The second major innovation is in the form of a new cell therapy using quiescent-like HSCs. Although initial screening and subsequent in vitro assays were done using transient transfection of mimics of miR-15a or miR-412, when using a piggyBac transposon vector that can integrate into the genome to constitutively express either miRNA in HSCs

[17] , they appear to undergo permanent transdifferentiation towards a quiescent-like state, with dramatic changes in morphology, expression, and function. Strikingly, when these reprogrammed HSCs were injected into mice challenged with 0014, they engrafted in the liver and increased the levels of liver injury and fibrosis. Other groups have used mesenchymal stem cells or macrophages to attempt cell therapy in cirrhosis

[18] , but this is the first time engineered HSCs have been used as a therapy for liver fibrosis or NASH.

[0458] The last major innovation is technical. If HSCs are known to be the major drivers of liver fibrosis in NASH, their actions can be disrupted. However, there are fundamental challenges to this endeavor. One of the biggest obstacles hindering the study of HSC function and mechanism of action is the lack of a good in vitro model of the interaction between HSCs and other cell types that would allow simple assays to evaluate functional phenotypes and mechanisms of action. To meet this need, a co-culture system using primary mouse HSCs and hepatocytes was developed to reconstruct the liver microenvironment in which these two cell types are in very close proximity. Additionally, by combining this technique with a mouse model of NASH, the effects of NASH-HSCs on neighboring hepatocytes can be easily determined. Interestingly, NASH-HSCs from a choline-deficient, L-amino acid-defined high-fat diet (CDAHFD model ( Figure 13 ) induce steatosis and stimulate the expression of pro-inflammatory cytokines in co-cultured hepatocytes harvested from healthy mice ( Figure 14 、 15 )

[19] . This signaling does not require cell-cell contact, as the two cell types in co-culture can be separated by a transwell, implying secretion of soluble factor(s) by the HSCs. Indeed, even when conditioned medium from cultured NASH-HSCs is applied to normal hepatocytes without co-culture, induction of fat accumulation in the hepatocytes can be reproduced ( Figure 16)。Therefore, the system allows the identification of mediators secreted by HSCs that induce steatosis and pro-inflammatory cytokines in hepatocytes. By using a simple, yet robust, ex vivo system to model the interaction between HSCs and hepatocytes in NAFLD, a technique has been established that can be used for mechanistic and drug development experiments. In particular, by using primary cells instead of cell lines, this co-culture system more faithfully mimics the in vivo pathophysiological microenvironment of these two cell types

[20] . The co-culture system is feasible, maximizing quality while minimizing effort by modifying existing protocols for harvesting and culturing these cells

[21] . These techniques are useful for delivering consistent and reproducible results using primary cells.

[0459] Determine the mechanisms and extent to which miR-15a and miR-412 affect HSC activation.

[0460] Hepatic stellate cells (HSCs) account for only 5% to 15% of all cells in the liver, but their large profibrotic effects after activation have been well recognized in progressive liver diseases [6, 7, 23, 24]. Although several studies have shown different miRNA expression in activated versus quiescent HSCs [11 - 13], the functions of miRNAs during the activation process remain largely unclear. Even less is known about the role of miRNAs in the reversal of activated HSCs back to quiescence, which is a possible fate of HSCs in the resolution of liver inflammation and fibrosis [9, 25].

[0461] To identify those miRNAs that promote the reversal of activated HSCs towards quiescence, an unbiased functional screen was designed to look for the reappearance of intracellular lipid droplets of retinoid, which is a marker of quiescence, after activated HSCs received a single miRNA mimic from a whole-genome miRNA library ( Figure 2 ). The investigation was conducted in 96-well plates, culturing activated HSCs with few lipid droplets. Once a single miRNA was transfected into each well, HSCs in some wells showed the reformation of Bodipy-stained positive lipid droplets within three days. This initial screen yielded 15 primary hits. The miRNAs miR-15a and miR-412 were selected for further investigation, based on the presence of human orthologs in mouse and human HSCs and the ability to reform lipid droplets ( Figure 3 ). The newly formed lipid droplets were shown to be retinoid positive by fluorescence under ultraviolet light, which was consistent with those in quiescent HSCs ( Figure 17 ). The overall size of the transfected HSCs decreased by 10 - 100 fold and became more quiescent-like (Figure 4). In addition, the forced expression of miR-15a or miR-412 downregulated two of the most important gene markers of activation, alpha smooth muscle actin (Acta2) and alpha-1 type I collagen (Col1a1) (Figure 4).

[0462] For more comprehensive expression profiling, deep RNA sequencing revealed that quiescent-like HSCs receiving miR-15a or miR-412 had a global transcriptional profile that was 40-50% closer to that of quiescent HSCs than activated cells Figure 18 ). Most strikingly, HSCs that became quiescent-like by miR-15a or miR-412 had a functional phenotype similar to that of truly quiescent HSCs. Ex vivo experiments showed that when the two cell types were co-cultured, 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 steatosis when co-cultured Figure 19 ). In addition, these reverted HSCs were able to reduce the expression of pro-inflammatory cytokines from co-cultured human hepatocellular carcinoma cell lines HepG2 and Huh7 Figure 6 ). Finally, as expected, endogenous miR-15a and miR-412 had reduced expression levels in activated HSCs compared to quiescent HSCs, although not significantly for miR-15a Figure 7 ).

[0463] Neither miR-15a nor miR-412 has been studied in the context of hepatic stellate cells. However, the miR-15a family has been found to inhibit the Tgf-β pathway in the heart to potentially attenuate fibrosis

[26] , and is also known as a key tumor suppressor in chronic lymphocytic leukemia [27, 28]. In contrast, there is no specific literature on miR-412, and thus its function is completely unknown. miRNAs are generally short, non-coding genes, typically 22 nucleotides in length and involved in all biological and pathological processes. They downregulate target coding genes by inducing degradation or translational repression through imperfect base pairing with complementary sequences within their miRNA targets. Each miRNA can regulate many different coding genes, while each target gene can be regulated by many different miRNAs, constituting a complex layer of the gene regulatory network. miRNAs directly target approximately 50% of all mammalian coding genes, indicating their broad influence in gene regulation

[29] . miRNAs have been shown to be essential for cell proliferation and reprogramming [30-32]. Nevertheless, the role of miRNAs in HSC activation or reversion to quiescence remains unexplored.

[0464] As described herein, are the remarkable quiescence-promoting effects and mechanisms of action of miRNA-15a and miR-412 in HSCs. WISP1 was identified as a direct target of miR-15a, but there may be other related targets for both miR-15a and miR-412. Many prediction algorithms predict potential direct targets of miRNAs based on sequence complementarity, but most of these predicted targets are not true targets that can withstand experimental verification. Therefore, RNA-Seq of HSCs in various activation states was performed to determine the expression profiles that could identify the true direct targets of miR-15a and miR-412.

[0465] Deep sequencing was performed on three types of HSCs: 1) quiescent HSCs freshly harvested from healthy mice, 2) activated HSCs that had been passaged multiple times in cell culture dishes, and 3) quiescent-like HSCs that had been reversed from the activated state to the quiescent state by receiving miR-15a or miR-412. Since the quiescent-like HSCs were sequenced only three days after receiving miR-15a or miR-412, any decrease in the mRNA levels of any coding gene could be the result of direct miRNA targeting. Therefore, the set of genes with reduced mRNA levels after receiving miR-15a or miR-412 overlapped with the set of potential direct targets based on the prediction algorithm [

[33] ] Figure 20 . This new set of common genes derived from the two parental sets (henceforth referred to as the target candidate set) has a higher likelihood of containing true direct targets.

[0466] Since the target candidate set still has too many genes to analyze, target candidate genes that are known to be part of two of the most well-known signaling pathways that promote HSC activation, those of Tgf-β and Pdgf, were selected. When PANTHER analysis was used to filter genes that are part of these two pathways, miR-15a and miR-412 retained 12 and 11 target candidates, respectively Figure 20 . These genes can be tested by constructing reporters with or without mutant miRNA binding sequences cloned behind a constitutively active luciferase gene. One of these reporter constructs and a miRNA mimic can be co-transfected into 293 cells or primary HSCs. If the co-transfected miRNA can anneal to this sequence and inhibit the translation of the luciferase protein, this can indicate that the coding gene is a true direct target of the tested miRNA. The mutant binding sequence can prevent miRNA annealing and allow almost normal expression of luciferase, further supporting that the tested coding gene sequence is a true miRNA target. It is predicted that this experiment will be time-consuming because the sequences of 23 high-potential target candidate genes in both the true and mutant forms need to be cloned into the luciferase vector. However, searching for direct targets is irreplaceable in elucidating the mechanism of action of miRNAs [

[32] ].

[0467] So far, this article has described the effect of delivering miR-15a or miR-412 to activated HSCs. To further understand the functions of miR-15a and miR-412, definitive loss-of-function experiments can be done by deleting these genes in primary HSCs using CRISPR technology as previously described

[35] .

[0468] The feasibility of using this technology on primary HSCs has been verified by delivering a digit deletion vector used in the past ( Figure 21 )

[35] . Three targeted HSC lines can be generated: 1) miR-15a-null, 2) miR-412-null, and 3) miR-15a / miR-412-null. Since overexpression of either of these miRNAs promotes HSC quiescence, deletion of them is expected to result in an activated phenotype, especially in the miR-15a / miR-412 double-null line. The lack of a unique phenotype may also be because miRNAs are known to have any functional redundancy. The activation level can be determined by defining their overall expression by RNA-Seq and comparing it to both activated and quiescent HSCs. Morphology and proliferation can be evaluated while culturing on plastic and Matrigel. Activated wild-type HSCs are known to revert back to quiescence on Matrigel

[36] , and the phenotype of knockout HSCs can be characterized in this environment. If deletion of miR-15a or miR-412 contributes to HSC activation, this effect can overcome the quiescence-promoting effect of Matrigel. The interaction of miRNA-deleted HSCs with hepatocytes can be evaluated by a co-culture system. Activated HSCs cause co-cultured hepatocytes to become steatotic and express pro-inflammatory mediators. miRNA-deleted HSCs can also be evaluated to have an even higher steatogenic phenotype than the steatogenic phenotype of activated wild-type HSCs. Finally, reconstituted miR-15a or miR-412, which can be evaluated in separate deletion lines, can rescue the wild-type phenotype. Reconstitution of the deleted miRNA can be achieved by transfecting a miRNA expression piggyBac vector that has been cloned and used in gain-of-function experiments.

[0469] Although miR-15a or miR-412 has been expressed individually, this article also specifically anticipates expressing them together in the same HSCs. In addition, delivering only one of these miRNAs can shift the overall gene expression model along the transcriptional axis connecting them from the activated state by about half back to quiescence ( Figure 11)。Overexpression of two miRNAs can reverse activated HSCs back to quiescence, even closer than expression of either miRNA alone. This gain-of-function experiment can be conducted by cloning the two miRNAs in a single piggyBac vector and transfecting them into activated HSCs. The piggyBac vector allows integration of the miRNA genes into the genome and constitutive expression of them

[17] . HSCs co-expressing the two miRNAs can be phenotyped with similar parameters as described herein to characterize other HSC lines, including global gene expression using RNA-Seq, morphology, proliferation using the MTT assay, and interaction with hepatocytes using this co-culture method.

[0470] If the phenotype of primary HSCs expressing the two miRNAs is even closer to that of similar quiescent HSCs, cell therapy can be tested in the context of CCl4-induced liver fibrosis and diet-induced NASH, with HSCs overexpressing a single miRNA. This HSC line can prevent or reduce liver injury and fibrosis, even more than that observed with HSC lines expressing miR-15a or miR-412 alone.

[0471] Test delivery of miR-15a or miR-412 to mouse models of liver fibrosis or NAFLD

[0472] Test HSCs from a diet model of NASH with miR-15a or miR-412 and reverse them back to quiescence in hepatocytes ( Figure 12 )。In addition, miR-15a and miR-412 appear to reverse even HSCs that are forcibly activated back to quiescence, even in promoting activation culture conditions. These key in vitro observations allow testing whether these miRNAs can be delivered in vivo to attenuate the level of liver pathology in the traditional CCl4 mouse model, especially since HSCs expressing miR-15a or miR-412 maintain a quiescence-like state even in the diseased liver with activation-promoting signals. Through cell contact and soluble mediators, quiescence-like HSCs can induce other cell types in the liver to suppress their signals promoting steatosis, inflammation, or fibrosis.

[0473] Although there are different methods to deliver miRNAs to live mice, injected HSCs become quiescence-like by constitutively expressing miR-15a or miR-412. Therefore, the feasibility of a new cell therapy for liver disease is further tested using HSCs that have been engineered to a quiescence-like state. This experiment is conducted by injecting the reprogrammed HSCs once during the third week of a four-week CCl4 challenge. The injected HSCs engraft on the liver, visually confirmed by GFP signal emission in hepatocytes that have inserted the GFP signal from the miRNA-expressing piggyBac vector

[17] ( Figure 8)。More importantly, mice treated with quiescent-like HSCs had histology showing reduced ballooning, apoptosis, inflammation, and fibrosis in the liver ( Figure 8 ) and less hepatic collagen expression ( Figure 9 ). Given this observation, in vivo experiments can be extended to further evaluate the potential of miR-15a and miR-412 in treating hepatic steatosis, inflammation, and fibrosis using additional mouse models of liver fibrosis and NASH. To evaluate whether these miRNAs function only through HSCs or more broadly through other cell types, viral vectors or mimics can be used to deliver the miRNAs systemically instead of cell therapy. For all in vivo treatment experiments, efficacy can be evaluated by performing: PCR of profibrotic genes in the liver, histology including H&E and Sirius red staining, liver hydroxyproline assays, and measurement of plasma ALT.

[0474] Using mesenchymal stem cells and macrophages, multiple groups have performed cell therapy for liver fibrosis and had some success

[18] . However, these experiments were the first to use purposefully engineered HSCs to prevent or reverse liver fibrosis. The HSCs were derived from isolated, syngeneic mice, and 500,000 cells were injected once in the third week of a four-week course of CCl4. Although the initial results were striking, it was unclear whether the improvement in liver morphology was due to prevention of liver injury induced in the fourth week of CCl4 administration or reversal of injury sustained in the first three weeks of CCl4 induction. Considering that only the CCl4 model has been used to date, the role of this cell therapy in NASH is not well understood. Ultimately, the beneficial effects of cell therapy may increase with more than one injection. To answer these important questions, in addition to the CDAHFD model of NASH, the CCl4 model of liver fibrosis (oral gavage of 100 ul of 40% CCl4, twice a week)

[19] can also be used. For all cell therapy experiments, 500,000 quiescent-like HSCs can be reprogrammed with miR-15a or miR-412. All appropriate controls can be included, such as untreated or groups treated with non-reprogrammed HSCs.

[0475] To test whether quiescent-like HSCs can prevent or reverse liver fibrosis, two separate experiments with different cell injection schedules can be used. All CCl4 administrations can be performed by twice-weekly oral gavage. Mice challenged with CCl4 for 8 weeks typically develop advanced fibrosis. Reprogrammed HSCs can be injected at weeks 2, 4, and 6 during the 8-week course of CCl4 challenge. All mice can be sacrificed at the end of week 8. To test whether cell therapy can reverse pre-existing liver fibrosis compared to controls, HSCs can be injected at weeks 9 and 11 after the 8-week course of CCl4 challenge has been completed. All mice can be sacrificed at the end of week 12. The choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD) model develops significant NASH at week 3 and stage 2 fibrosis (on a 0 - 4 scale) at week 9

[19] . A single injection of cell therapy can be completed at week 5 or three injections can be completed at weeks 3, 5, and 7. All mice can be sacrificed at the end of week 9.

[0476] It has previously been shown that activated human HSCs can be reversed to a quiescent-like state after receiving the human orthologs of miR-15a or miR-412 ( Figure 8 ). These reversed cells reform vitamin A-positive lipid droplets and are able to reduce pro-inflammatory cytokine expression from co-cultured human hepatocellular carcinoma cell lines HepG2 and Huh7 ( Figure 6 ). As a bridge to translate discoveries for human therapies, primary human HSCs can be used in a CCl4 mouse model of liver fibrosis to determine the therapeutic potential of miR15a and miR-412. CCl4 can be gavaged twice a week to induce liver fibrosis in severe combined immunodeficient mice (SCID), which are selected to prevent immune rejection of human cells. Other groups have successfully generated CCl4-based liver fibrosis models with SCID mice

[37] . Fibrosis prevention study : Human HSCs can be injected at weeks 2, 4, and 6 during the 8-week course of CCl4 challenge in SCID mice. Fibrosis reversal study : To test whether reprogrammed human HSCs can reverse pre-existing liver fibrosis compared to controls, HSCs can be injected at weeks 9 and 11 after the 8-week course of CCl4 challenge has been completed. All mice can be sacrificed at the end of week 12.

[0477] Initial experiments showed that injecting HSCs reprogrammed with miR-15a or miR-412 can reduce collagen expression and the overall level of liver injury in CCl4-induced liver fibrosis. One method of delivering miRNA is to inject lentivirus expressing the miRNA. CCl4 fibrosis study : Lentivirus expressing miR-15a or miR-412 can be injected into the tail vein at weeks 2, 4, and 6 during the 8-week course of CCl4 challenge. All mice can be sacrificed at the end of week 8.CDAHFD NASH study : Lentiviruses expressing miR-15a or miR-412 can be injected at weeks 3, 5, and 7 of the 9-week CDAHFD. All mice can be sacrificed at the end of week 9.

[0478] As another delivery method, the feasibility of injecting more stable, chemically modified miRNA mimics (Exiqon and Invitrogen) packaged in lipid-based carriers (MaxSuppressorTM from B100 Scientific) can be tested [38, 39]. By avoiding lentiviruses, this method or its variants have the potential for use in humans. CCl4 fibrosis study : Lipid carriers containing miR-15a or miR-412 can be injected into the tail vein at weeks 2, 4, and 6 during the 8-week CCl4 challenge. All mice can be sacrificed at the end of week 8. CDAHFD NASH study : Lipid carriers containing miR-15a or miR-412 can be injected at weeks 3, 5, and 7 of the 9-week CDAHFD. All mice can be sacrificed at the end of week 9.

[0479] Determine the function of WISP1 in hepatic steatosis, inflammation, and fibrosis

[0480] It is known that miRNAs exert general functions by targeting multiple coding genes, and these two candidates may downregulate HSC genes that promote liver inflammation and fibrosis. Although miR-15a and miR-412 may inhibit many genes, WISP1 has emerged as the first experimentally verified direct target of miR-15a. WISP1 was first identified as one of the multiple up-secreted proteins in the CDAHFD-induced NASH-HSC conditioned medium detected using a cytokine array blot that retains >100 canonical cytokines, chemokines, and extracellular matrix proteins ( Figure 22 ). More specific analysis showed that WISP1 expressed by activated HSCs was almost 30-fold the level of quiescent HSCs ( Figure 23)。WISP is a member of the Ccn family of matricellular proteins, which includes Cyr61 (Ccn1), Ctgf (Ccn2), Nov (Ccn3), WISP1 (Ccn4), Wisp2 (Ccn5), and Wisp3 (Ccn6). Ctgf has been recognized as an important profibrotic factor in the liver

[42] . Interestingly, WISP1 has been found to be upregulated in human idiopathic pulmonary fibrosis, and treatment of bleomycin-challenged mice with neutralizing WISP1 antibody results in attenuation of pulmonary fibrosis

[43] . In addition, small animal studies have shown that blockade of WISP1 improves CCl4-induced liver fibrosis

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

[0481] It is known that CDAHFD-induced NASH-HSCs promote steatosis in co-cultured hepatocytes ( Figure 14 、 16 ), and WISP1 may contribute to this phenotype. Indeed, when WISP1 was overexpressed in HSCs harvested from healthy mice, conditioned medium from these cells resulted in steatosis in initially healthy primary hepatocytes ( Figure 24 ). In addition, miR-15a was predicted to target WISP1, which contains two potential miR-15a binding sequences in its 3'-UTR. Therefore, the putative target sequences or their mutant variants were cloned downstream of a constitutively active luciferase reporter gene. When one of these reporters was co-transfected with miR-15a mimics, the reporter with the wild-type sequence had reduced luciferase expression, while the reporter with the mutant sequence did not, indicating that miR-15a binds to the two WISP1 target sequences ( Figure 11 ). Given these data and the published literature, WISP1 may be a key target gene for completing some of the actions of miR-15a in HSCs and may also be a drug target for managing NASH. The hepatic function of WISP1 is herein predicted to be a potential therapeutic target for NASH.

[0482] Although WISP1 is a direct target of miR-15a, its effect on the activated state of HSCs and its role in the pathogenesis of NAFLD are unclear. A WISP1-null mouse line has been generated to demonstrate the role of WISP1 in bone formation, but its function in the liver has not been studied

[45] . These mice are fertile and do not have an obvious liver phenotype. Their mutant alleles are preserved in frozen sperm and embryos in the MMRRC facility in University of California at Davis, which ensures a successful source of mutant mice (see attached file). First, HSCs were harvested from WISP1-null mice and their phenotypes were characterized ex vivo to demonstrate the function of WISP1 in HSCs.

[0483] Wild-type HSCs in the quiescent state became activated after 7-10 days of culture on plastic, and the RNA-seq data shown herein indicate a significant difference in the overall gene expression between these two groups ( Figure 11 ). Parallel experiments using WISP1-null HSCs can be used to evaluate whether WISP1 deletion alters the overall expression profile towards the overall expression profile of HSC quiescence, even in an activation-promoting environment. After isolating HSCs from WISP1-null mice, the cells can be cultured on plastic dishes for 1 day (quiescent) or 28 days (activated in wild-type). Their overall gene expression can be determined by RNA-seq and the results can be compared with those of wild-type HSCs. The overall comparison can be accomplished by multidimensional scaling analysis, and differentially expressed genes can be identified for detailed analysis.

[0484] Cell morphology, proliferation, and migration can be compared while culturing on plastic. Morphology can be determined using parameters such as size, degree of cell processes, presence of lipid droplets, and whether they are UV-fluorescent positive, which is a marker of retinoid and quiescence. Proliferation can be examined by PCNA and Ki-67 to prevent WISP1-null HSCs from having reduced proliferation, similar to quiescent HSCs. An MTT assay can also be performed as done in the past to quantify the cell proliferation rate

[32] . Since the initial data shown herein and published literature indicate that WISP1 is a profibrotic gene, deletion of WISP1 may promote quiescence in HSCs, perhaps even when cultured on plastic dishes. Finally, reconstitution of WISP1 in knockout HSCs can rescue the wild-type phenotype. Reconstitution of WISP1 can be achieved by transfection of a WISP1-expressing piggyBac vector. WISP1-null HSCs transfected with the WISP1 expression vector can be characterized by the same in vitro assays used for untransfected WISP1-null cells.

[0485] The interaction of WISP1-null HSCs with hepatocytes can be evaluated by the co-culture system described herein. Activated wild-type HSCs cause co-cultured hepatocytes to become steatotic ( Figure 14 ). HSCs overexpressing WISP1 induce nearby hepatocytes to become even more severely steatotic ( Figure 24 ). WISP1-null HSCs may have a diminished steatogenic phenotype. Readouts of the co-culture assays include Bodipy staining for lipid droplets and PCR of co-cultured hepatocytes to determine the expression of pro-inflammatory factors.

[0486] The phenotype can be evaluated by the overall severity of liver pathology. WISP1-null mice can be challenged with CCl4 and CDAHFD to model toxin-induced liver fibrosis and NASH, respectively. The resulting liver injury sustained by WISP1-null mice can be evaluated against that of wild-type mice. Given the profibrotic role of WISP1 in other organs including the lung, it is expected that WISP1-null mice will have reduced liver fibrosis and possibly even reduced steatosis and inflammation. For all in vivo experiments, liver phenotypes can be evaluated by: PCR of profibrotic genes in the liver, histology including H&E and Sirius red staining, hydroxyproline assays, and measurement of plasma ALT. Finally, the functional phenotype of HSCs harvested from these murine models of liver disease can be determined.

[0487] CCl4 fibrosis model: Male wild-type and WISP1-null mice can be challenged by twice-weekly CCl4 gavage. They can receive CCl4 for 8 weeks and be sacrificed at the end of week 8. CDAHFD NASH model: Wild-type and WISP1-null mice can be fed CDAHFD for 9 weeks. Half of the mice can be sacrificed at the end of week 6 and the other half at the end of week 9 to define the role of WISP1 in moderate NASH with stage 2 fibrosis (6 weeks of CDAHFD) and end-stage NASH (9 weeks of CDAHFD).

[0488] HSCs can be isolated from mice challenged with CCl4 for 4 weeks and CDAHFD for 3 weeks. These HSCs can be co-cultured with healthy hepatocytes to determine whether they develop steatosis and express pro-inflammatory factors. The results can be compared to those of wild-type HSCs subjected to the same challenge.

[0489] Blocking WISP1 with neutralizing antibodies attenuates bleomycin-induced injury in the murine lung

[43] . Similarly, neutralizing WISP1 can reduce the levels of inflammation and fibrosis that occur in NASH. A murine model of NASH can be treated with WISP1 antibody and liver phenotypes evaluated after 6 and 9 weeks of treatment.

[0490] CDAHFD NASH model: Wild-type mice can be fed a CDAHFD for 9 weeks. The treatment groups can receive commercially available antibodies (R&D Systems)

[43] or antibodies injected weekly

[46] . The control groups can receive IgG. Half of the mice can be sacrificed at the end of week 6 and the other half at the end of week 9 to test the effects of WISP1 blockade in moderately and end-stage NASH with fibrosis, respectively.

[0491] In addition, conditioned media from NASH HSCs can be analyzed to identify autocrine or paracrine factors. More detailed analysis of NASH-HSC conditioned media by mass spectrometry can identify other mediators that may be drug targets. Finally, the co-culture system described herein can be used to study other liver diseases, provided they are typical mouse models. For example, by isolating cell types from alcohol-fed mice, the interaction between hepatocytes and HSCs can be modeled in alcoholic liver disease

[47] .

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[0548] WISP1 / CCN4: a potential target for inhibiting prostate cancer growth and spread to bone. PLoS One 8:e71709. doi:10.1371 / journal.pone.0071709

[0549] 47. Hamarneh SR, Kim BM, Kaliannan K, Morrison SA, Tantillo TJ, Tao Q, Mohamed MMR, Ramirez JM, Karas A, Liu W, Hu D, Teshager A, Gul SS, Economopoulos KP, Bhan AK, Maio MS, Choi MY and Hodin RA

[0550] (2017) Intestinal Alkaline Phosphatase Attenuates Alcohol-Induced Hepatosteatosis in Mice. Dig Dis Sci. doi:10.1007 / s10620-017-4576-0 Example 3 - HSC Isolation Protocol

[0551] The following protocol was used for the experiments described herein to isolate HSCs, e.g., mod-Hep / HSCs.

[0552] 1. Cannulate the portal vein using an indwelling catheter or a butterfly needle.

[0553] 2. Perfuse the liver at 6 mL / min with a solution at 37 °C:

[0554] 30 mL HBSS (without Ca 2+ and Mg 2+ ), and shortly after the start of perfusion, cut the IVC

[0555] 30 mL of 0.05% collagenase [0.5 mg / mL --> for 50 mL, 500 ul of 50 mg / mL collagenase stock solution] (e.g., collagenase B; Roche 11088815001).

[0556] 3. After perfusion, carefully excise the liver (especially the gallbladder) and remove Glisson’s capsule.

[0557] 4. Transfer the liver to a sterile beaker and cut into very thin slices.

[0558] 5. Add the remaining 20 mL of 0.05% collagenase and incubate at 37 °C in an incubator for 30 minutes [add DNAse (10 ug / mL)] (e.g., DNase I; Roche 10104159001).

[0559] 6. Pipette up and down and filter through a cell strainer (70um) in a Petri dish. Add PBS + DNAse (10ug / mL) to make up to 45mL.

[0560] 7. Remove hepatocytes by centrifugation at x 50g for 3 minutes and transfer the supernatant to a new tube.

[0561] 8. Form a pellet by centrifugation at x 635g for 10 minutes.

[0562] 9. Wash with 45mL PBS + DNAse (10ug / mL) and centrifuge at x 635g for 10 minutes.

[0563] 10. Filter the cell suspension through a 70um cell strainer.

[0564] 11. Wash with 45mL PBS + DNAse (10ug / mL) and centrifuge at x 635g for 10 minutes.

[0565] 12. Aspirate the supernatant. Add PBS to the pellet to a total volume of 6.5mL.

[0566] 13. Add 3.5mL of percoll (9 parts percoll, 1 part 10x PBS) to a total volume of 10mL, mix, and transfer to a 15mL tube (e.g., Percoll; Percoll plus, GE Healthcare 17-5445-02).

[0567] 14. Carefully add 1mL of PBS to the top of the column.

[0568] 15. Centrifuge the column at x 1130g for 30 minutes at room temperature (acceleration 9, brake 0).

[0569] 16. After centrifugation, HSCs are in the layer between PBS and 35% percoll.

[0570] 17. Aspirate HSCs with a micropipette and wash with 10mL DMEM, centrifuge at x 635g for 6 minutes.

[0571] 18. Aspirate the supernatant.

[0572] 19. Resuspend the pellet and place in DMEM + 20% FBS + 1% P / S.

[0573] Example 4: Antibodies that inhibit WISP1 for the treatment and prevention of liver diseases

[0574] Antibodies that specifically bind to and inhibit WISP1 can be used to treat or prevent liver diseases such as primary biliary cholangitis (PBC) and autoimmune hepatitis (AIH).

[0575] Specifically, lead candidates for inhibiting WISP1 include ant-WISP1 IgG1 / IgG4 antibodies and IgG1ADCC). Strategies for engineering anti-WISP1 antibodies include, but are not limited to: Fc engineering for extended half-life; bispecific antibody technology capable of simultaneously inhibiting two antifibrotic targets; and sweeping technology, which (1) converts the antibody from a sequestering entity to a catalytic drug by driving target catabolism, (2) significantly reduces CoG, dose levels, and dosing frequency, (3) increases the likelihood of subcutaneous dosing, and (4) eliminates the need for full blocking potential.

[0576] To evaluate the ability of anti-WISP1 antibodies to inhibit WISP1, the antibodies were tested on fresh HSCs. The WISP1 antibody (Ab WISP1 or WISPIgG, RND Systems, 1 μg / mL) was incubated with conditioned medium at 37 °C for 1 hour and then applied to the HSCs. As a negative control, the HSCs were incubated with conditioned medium only. As a positive control, recombinant WISP1 (Rc-WISP1, 100 ng / mL) was also added to the HSCs instead of the antibody. The HSC samples were evaluated 48 hours after treatment ( Figure 38A ). Rc-WISP1 is, for example, WISP1-CCN4, obtained from the cell line identified as accession number O54774 and commercially available from R&D Systems. Rc-WISP1 was found to significantly increase the cell number ( Figure 38B ), Acta2 mRNA expression in HSCs ( Figure 38C ), and Col1a1 mRNA expression ( Figure 38D ) on days 1 and 2 after culture, indicating that HSCs become activated in the presence of recombinant WISP1. Surprisingly, commercially available anti-WISP1 IgG (Ab Wisp1) blocked HSC activation in HSCs; culture with anti-WISP1 led to a significant decrease in the cell number ( Figure 38B ), Acta2 mRNA expression ( Figure 38C ), and Col1a1 mRNA expression ( Figure 38D ) on days 1 and 2 after culture compared to HSCs cultured with rc WISP1.

[0577] Then, anti-WISP1 IgG was tested on primary hepatocytes isolated from a choline-deficient, L-amino acid-defined, high-fat diet-induced steatosis (CDAHFD) mouse model. Markers of liver diseases such as NALFD and viral hepatitis are an increase in lipid droplets in diseased hepatocytes. To evaluate the presence of lipid droplets, BODIPY staining was employed. As expected, CDAHFD cells treated with control IgG had increased BODIPY staining ( Figure 38E and 38F ). In contrast, CDAHFD cells treated with anti-WISP1 IgG had decreased BODIPY staining, similar to control, healthy cells ( Figure 38E and 38F ). Thus, the data presented herein show that anti-WISP1 IgG can restore diseased hepatocytes to healthy hepatocytes.

[0578] Finally, to show the ability of anti-WISP1 IgG to regulate HSC migration, HSCs were cultured to form a monolayer and then damaged ( Figure 39A - Figure 39B ). Cells were cultured at 90% confluence in 60 mm dishes in growth medium. A single linear scratch wound was created using a sterile P200 pipette tip. Cells were washed with PBS to remove floating cell debris and recombinant Wisp1 protein (100 ng / mL, R&D Systems, Minneapolis, MN) and monoclonal Wisp1 antibody (1 μg / mL, R&D Systems, Minneapolis, MN) were added again for 24 hours. Wound closure or cell migration was photographed using an inverted microscope at the time of introducing the scratch wound, or 12 and 24 hours after damage. The area between the wound edges in the dish at each time was determined using a standard template placed on the image. Data are represented as the scratch area (percentage) relative to the initial scratch area. The scratch area was determined using publicly available ImageJ (NIH) software.

[0579] After damage, HSCs were cultured with Rc WISP1 or anti-WISP1 IgG. Compared to control or anti-WISP1 IgG-treated HSCs, HSCs cultured with rc WISP1 showed more rapid migration, especially after the 12-hour time point. In contrast, HSCs cultured with anti-WISP1 IgG resulted in slower migration, similar to control-treated HSCs ( Figure 39A - 39B ). Compared to HSCs treated with Rc-WISP1, anti-WISP1 IgG prevented HSC migration after damage. This result confirms that antibodies that inhibit WISP1 can prevent HSC activation and migration, making them useful reagents for treating liver diseases.

[0580] Sequence

[0581] SEQ ID NO:1 is the amino acid sequence encoding human WNT1 inducible signaling pathway protein 1 isoform 1 precursor.

[0582]

[0583] SEQ ID NO:2 is the amino acid sequence encoding human WNT1 inducible signaling pathway protein 1 isoform 2 precursor.

[0584]

[0585] SEQ ID NO:3 is the amino acid sequence encoding human WNT1 inducible signaling pathway protein 1 isoform 3 precursor.

[0586]

[0587] SEQ ID NO:4 is the amino acid sequence encoding human WNT1 inducible signaling pathway protein 1 isoform 4 precursor.

[0588]

[0589] SEQ ID NO:5 is the nucleotide sequence of human WNT1 inducible signaling pathway protein 1 (WISP1), transcript variant 1, mRNA.

[0590]

[0591]

[0592]

[0593] SEQ ID NO:6 is the amino acid sequence of mouse WNT1 inducible signaling pathway protein 1 precursor [Mus musculus].

[0594]

[0595] SEQ ID NO:7 is the nucleotide sequence of mouse WNT1 inducible signaling pathway protein 1 (Wisp1), transcript variant 1, mRNA [Mus musculus].

[0596]

[0597]

[0598]

[0599] SEQ ID NO:8 is the nucleotide sequence of human MiR 15a.

[0600]

[0601] SEQ ID NO:9 is the nucleotide sequence of human Mir-412.

[0602]

[0603] SEQ ID NO:10 is the nucleotide sequence of mouse MiR 15a.

[0604]

[0605] SEQ ID NO:11 is the nucleotide sequence of mouse Mir-412.

[0606]

[0607] SEQ ID NOs:12 - 31 are the heavy chain complementarity-determining regions of Rattus for IgG.

[0608]

[0609]

[0610] SEQ ID NOs:32 - 51 are the light chain complementarity-determining regions of Rattus for IgG.

[0611]

[0612]

[0613] SEQ ID NOs:52 - 71 are the heavy chain complementarity-determining regions of Ovis aries for IgG.

[0614]

[0615] SEQ ID NOs:72 - 90 are the light chain complementarity-determining regions of Ovis aries for IgG.

[0616]

[0617]

[0618] SEQ ID NOs:91 - 110 are the heavy chain complementarity-determining regions of Capra hircus for IgG.

[0619]

[0620] SEQ ID NOs: 111-121 are the rabbit (Leporidae) heavy chain complementarity determining regions for IgG.

[0621]

[0622]

[0623] SEQ ID NOs: 122-141 are the rabbit light chain complementarity determining regions for IgG.

[0624]

Claims

1. A method for treating or preventing liver diseases, the method comprising: Administer an antibody or antibody agent that inhibits WISP1 to a subject in need thereof.

2. The method according to claim 1, wherein the liver disease is selected from the group consisting of: Alagille syndrome; alcohol-related liver disease; alpha-1 antitrypsin deficiency; autoimmune hepatitis; benign liver tumor; biliary atresia; cirrhosis; Crigler-Najjar syndrome; galactosemia; Gilbert syndrome; hemochromatosis; hepatic encephalopathy; hepatitis A; hepatitis B; hepatitis C; hepatorenal syndrome; intrahepatic cholestasis of pregnancy (ICP); lysosomal acid lipase deficiency (LAL-D); liver cyst; liver cancer; neonatal jaundice; non-alcoholic fatty liver disease; non-alcoholic steatohepatitis; primary biliary cholangitis (PBC); primary sclerosing cholangitis (PSC); progressive familial intrahepatic cholestasis (PFIC); Reye's syndrome; type I glycogen storage disease; scleroderma; and Wilson's disease.

3. The method according to claim 1, wherein the WISP1 is a splice variant selected from the group consisting of: WISP1v, WISP1vx, and WISP1 exon 3-4 delta.

4. The method according to claim 1, wherein the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of: mab1680, AF1680, SAB2501114, ab60114, and ab65943.

5. The method according to claim 1, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

6. The method according to claim 1, wherein WISP1 is inhibited in the target cell.

7. The method according to claim 1, wherein the target cell is a mammalian cell.

8. The method according to claim 1, wherein the target cell is a hepatic stellate cell, a fibroblast, or a myofibroblast.

9. The method according to claim 6, wherein the hepatic stellate cell is quiescent.

10. The method according to claim 1, wherein the antibody or antibody agent is administered by direct injection, intravenous delivery, subcutaneous injection, intramuscular injection, transdermal, oral, or nasal administration.

11. The method according to claim 1, wherein inhibiting WISP1 is inhibiting WISP1 activity or reducing the WISP1 protein level.

12. The method according to claim 11, 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.

13. The method according to claim 11, wherein the level of WISP1 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.

14. A composition comprising an antibody or antibody agent that inhibits WISP1 and a pharmaceutically acceptable carrier.

15. The composition according to claim 14, wherein the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of mab1680, AF1680, SAB2501114, ab60114, and ab65943.

16. The composition according to claim 14, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

17. The composition according to claim 14, wherein the composition is formulated for the treatment or prevention of liver disease.

18. A method for treating liver disease in a subject, the method comprising: a. Detecting the levels of WISP1 and / or Acta2, Col1a1 in a biological sample of the subject; b. Comparing the measurement result of (a) with a reference level; c. Identifying a subject having increased WISP1 and / or Yap, Acta2, Col1a1 compared to the reference level in (a) as having liver disease; and d. Administering to the subject having liver disease an antibody or antibody agent that inhibits WISP1.

19. The method according to claim 18, further comprising obtaining a biological sample from the subject before (a).

20. The method according to claim 18, wherein the liver disease is primary biliary cholangitis, autoimmune hepatitis, α1-antitrypsin deficiency, non-alcoholic steatohepatitis, or scleroderma.

21. The method according to claim 18, wherein the biological sample is a blood sample, tissue, buffy coat, serum, or tissue.

22. The method according to claim 18, wherein the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of mab1680, AF1680, SAB2501114, ab60114, and ab65943.

23. The method according to claim 18, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

24. A method for treating or preventing liver diseases, the method comprising: Administering to a subject in need a reagent that inhibits WISP1.

25. The method according to claim 24, wherein the liver disease is selected from the group consisting of primary biliary cholangitis, autoimmune hepatitis, α1-antitrypsin deficiency, non-alcoholic steatohepatitis, and scleroderma.

26. The method according to claim 24, wherein the WISP1 is a splice variant selected from the group consisting of WISP1v, WISP1vx, and WISP1 exon 3-4 deletion.

27. The method according to claim 24, wherein the reagent that inhibits WISP1 is selected from the group consisting of small molecules, antibodies or antibody agents, peptides, genome editing systems, viral vectors, miRNAs, and siRNAs.

28. The method according to claim 27, wherein the microRNA is microRNA15a or miRNA412.

29. The method according to claim 27, wherein the antibody or antibody agent that inhibits WISP1 is selected from the group consisting of mab1680, AF1680, SAB2501114, ab60114, and ab65943.

30. The method according to claim 27, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

31. The method according to claim 24, wherein the reagent is administered by direct injection, subcutaneous injection, intramuscular injection, or nasal administration.

32. The method according to claim 24, wherein inhibiting WISP1 is inhibiting WISP1 activity or reducing the WISP1 protein level.

33. The method according to claim 32, 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.

34. The method according to claim 32, wherein the level of WISP1 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.

35. The method according to claim 24, wherein WISP1 is inhibited in target cells.

36. A composition comprising a reagent that inhibits WISP1 and a pharmaceutically acceptable carrier.

37. The composition according to claim 36, wherein the reagent that inhibits WISP1 is selected from the group consisting of small molecules, antibodies or antibody agents, peptides, genome editing systems, viral vectors, miRNAs, and siRNAs.

38. The composition according to claim 37, wherein the microRNA is microRNA15a or miRNA412.

39. A method of treating liver disease in a subject, the method comprising: a. Detecting the levels of WISP1 and / or Yap, Col1a1, Acta2 in a biological sample of the subject; b. Comparing the measurement result of (a) with a reference level; c. Identifying a subject having increased WISP1 and / or Yap, Col1a1, Acta2 compared to the reference level in (a) as having liver disease; and d. Administering a reagent that inhibits WISP1 to the subject having liver disease.

40. The method according to claim 39, further comprising obtaining a biological sample from the subject before (a).

41. The method according to claim 39, wherein the liver disease is primary biliary cholangitis, autoimmune hepatitis, α1-antitrypsin deficiency, non-alcoholic steatohepatitis, or scleroderma.

42. The method according to claim 39, wherein the biological sample is a blood sample, tissue, buffy coat, serum, or tissue.

43. The method according to claim 39, wherein the reagent for inhibiting WISP1 is selected from the group consisting of: small molecules, antibodies or antibody agents, peptides, genome editing systems, viral vectors, miRNAs, and siRNAs.

44. The method according to claim 43, wherein the microRNA is microRNA15a or miRNA412.

45. The method according to claim 43, wherein the antibody or antibody agent for inhibiting WISP1 is selected from the group consisting of: mab1680, AF1680, SAB2501114, ab60114, and ab65943.

46. The method according to claim 43, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

47. A method of engineering hepatic stellate cells or a population thereof that produce an agent that inhibits the expression of WISP1, the method comprising: Contact the cell with a reagent for inhibiting WISP1, and culture the cell for a sufficient time to allow expression of the reagent.

48. The method according to claim 47, wherein the cell is quiescent.

49. The method according to claim 47, wherein the contacting comprises contacting the cell with the reagent or a vector encoding the reagent.

50. The method according to claim 47, wherein the contacting comprises transduction, nucleofection, electroporation, direct injection, and / or transfection.

51. The method according to claim 47, wherein the reagent for inhibiting WISP1 is selected from the group consisting of: small molecules, antibodies or antibody agents, peptides, genome editing systems, viral vectors, miRNAs, and siRNAs.

52. The method according to claim 51, wherein the microRNA is microRNA15a or miRNA412.

53. The method according to claim 51, wherein the antibody or antibody agent for inhibiting WISP1 is selected from the group consisting of: mab1680, AF1680, SAB2501114, ab60114, and ab65943.

54. The method according to claim 51, wherein the amino acid sequence of the antibody or antibody agent has at least 70% homology with any one of SEQ ID NO: 1-4, 6, or 12-120.

55. A cell line comprising hepatic stellate cells produced by the method according to any one of claims 47-54.

56. A pharmaceutical composition comprising hepatic stellate cells or a population thereof produced by the method according to any one of claims 47-54, and a pharmaceutically acceptable carrier.

57. A method for treating or preventing liver diseases, the method comprising: Administer to a subject in need a cell produced by the method according to any one of claims 47-54, the cell according to claim 55, or the pharmaceutical composition according to claim 56.

58. A method of reducing fibrosis in a subject, the method comprising: Administer to a subject in need a cell produced by the method according to any one of claims 47-54, the cell according to claim 55, or the pharmaceutical composition according to claim 56.

59. A method for treating liver disease in a subject, the method comprising: a. receiving the results of a test that identifies as having liver disease a subject having increased WISP1 and / or Yap, Acta2, or Col1a1 compared to a reference level; and b. administering to the subject having liver disease an antibody or antibody agent that inhibits WISP1.

60. A method for treating liver disease in a subject, the method comprising: a. receiving the results of a test that identifies as having liver disease a subject having increased WISP1 and / or Yap, Col1a1, Acta2 compared to a reference level; and b. administering to the subject having liver disease a reagent agent that inhibits WISP1.

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