Methods for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis

Abstract

The present invention relates to a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

Description

Technical Field

[0001] The present invention relates to a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

Background Art

[0002] Cholestasis is characterized by a decrease or stasis of bile flow in the liver. This can be caused either by obstruction of the bile ducts outside the liver or by intrahepatic defects in bile synthesis or circulation. Etiologies include drug poisoning, alcoholic or viral hepatitis, biliary atresia, gallstones, and genetic diseases. Cholestatic liver diseases are a significant personal and economic burden for patients and society, and liver dysfunction due to cholestasis accounts for approximately 10% of all liver transplants performed in Europe. 1 This is due to the disconcertingly high number of effective treatment options being lost for cholestatic diseases such as primary sclerosing cholangitis (PSC) or primary biliary cirrhosis (PBC). Currently, the first choice of treatment involves the administration of ursodeoxycholic acid (UDCA), an immunomodulatory and bicarbonate secretion-stimulating drug. This improves the survival rate without transplantation in approximately 60% of PBC patients, but shows only limited efficacy in PSC patients. 2 New treatment approaches focus on immunomodulatory strategies 3~5 , changes in the microbiome 6、7 or activation of FGF19 / FXR signaling aimed at anti-fibrotic effects and improvement of bile acid excretion and detoxification. 8~10 Therefore, some groups have used PPAR agonists for the treatment of cholestasis to increase the excretion of toxic bile acids. 11~13has been proposed. Currently, the treatment of chronic cholestatic diseases is difficult and often ineffective. New treatments for cholestasis and / or fibrosis associated with cholestasis, and / or fibrosis associated with cholestasis are needed to meet high medical needs. Summary of the Invention

[0003] Unexpectedly, it has been found by the inventors of the present application that claudin 3 inhibition is a possible treatment approach for treating cholestasis and / or fibrosis associated with cholestasis. This can be achieved by using siRNA targeting claudin 3, which can be expected to recover cholestatic liver injury. In view of such unexpected findings, the inventors provide the present invention in the following aspects.

[0004] In a first aspect, the present invention provides an agent that inhibits the expression and / or activity of claudin 3 for use in a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

[0005] In a second aspect, the present invention provides a composition comprising an agent that inhibits the expression and / or activity of claudin 3 for use in a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis, and a pharmaceutically acceptable carrier.

[0006] In a third aspect, the present invention provides a dosage form for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis, comprising an agent that inhibits the expression and / or activity of claudin 3, or a composition comprising the agent, and a pharmaceutically acceptable carrier.

[0007] In a fourth aspect, the present invention provides siRNA targeting claudin 3. Brief Description of the Drawings

[0008]

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Best Mode for Carrying Out the Invention

[0009] As outlined above, the present invention relates to a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

[0010] Accordingly, in a first aspect, the present invention provides an agent that inhibits the expression and / or activity of claudin 3 for use in a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis.

[0011] For the purpose of interpreting this specification, the following definitions apply and, whenever appropriate, terms used in the singular shall also include the plural and vice versa. It should be understood that the technical terms used in this specification are for the purpose of describing particular embodiments only and are not intended to be limiting. "Comprising", "having", and "including" should be construed as open-ended terms (i.e., meaning "including but not limited to") unless otherwise specified.

[0012] It should be understood that any feature, integer, property, agent, compound, chemical moiety or group described in conjunction with a particular aspect, embodiment or example of the invention is applicable to any other aspect, embodiment or example described herein, unless inconsistent. All of the features disclosed in this specification (including any of the accompanying claims, abstract and drawings) and / or all of the steps of any method or process so disclosed may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. The invention is not limited to the details of any of the foregoing embodiments. The invention extends to any novel, or any novel combination of features disclosed in this specification (including any of the accompanying claims, abstract and drawings) or to any novel, or any novel combination of steps of any method or process so disclosed.

[0013] As used herein, the term "cholestasis" refers to any disease in which the release of bile from the liver is blocked. The blockage can occur within the liver (intrahepatic cholestasis) or within the bile ducts (extrahepatic cholestasis). Physiologically, "cholestasis" means an impairment of the bile flow and an insufficiency in the secretion of the inorganic and organic components of bile. In particular, cholestasis results from molecular and ultrastructural changes that impair the entry of organic small molecules, inorganic salts, proteins, and ultimately water into the bile ducts. Clinically, the physical findings of jaundice and pruritus are accompanied by an increase in the serum concentrations of bilirubin, bile salts, and alkaline phosphatase (ALP). Cholestasis can be caused by extrahepatic problems such as gallstones or tumors that block the flow of bile outside the liver. However, it can also have intrahepatic causes such as viral diseases, genetic disorders, and bile duct strictures. Bile constitutes a major route for removing bilirubin, which is poorly soluble in water for excretion into urine, excess cholesterol (both as free cholesterol and as bile salts), and foreign substances. The basic driving factor for bile formation is the hepatocyte secretion of bile salts into the canaliculi, which causes the secretion of phosphatidylcholine and cholesterol from the hepatocytes. The fluid secretion by hepatocytes and by the downstream bile duct cells that line the bile tree together contribute to the several liters of bile secreted by the human liver per day. Bile facilitates the digestion and absorption of lipids from the intestine. Since bile formation requires well-functioning hepatocytes and an intact bile tree, this process is easily disrupted.

[0014] The term "fibrosis associated with cholestasis" refers to any fibrotic liver disease associated with cholestasis and / or initially caused by hepatic cholestasis.

[0015] As used herein, the term "agent that inhibits the expression and / or activity of Claudin 3" refers to any biological or chemical agent that enables inhibition of the expression and / or activity of Claudin 3 by reducing or interrupting the interaction of Claudin 3 or its gene with other biomolecules, such as, but not limited to, protein-protein interactions, ligand-receptor interactions, or protein-nucleic acid interactions. Such agents include, but are not limited to, antibodies, protein-binding agents, nucleic acid molecules, small molecules, recombinant proteins, peptides, aptamers, avimers and protein-binding derivatives, or fragments thereof. The activity of Claudin 3 can be inhibited by, for example, an antibody that binds to Claudin 3, such as an antibody that binds to at least one of the extracellular domains of Claudin 3, or by a toxin that binds to Claudin 3. The expression of Claudin 3 can be inhibited by, for example, a DNA targeting agent (e.g., CRISPR system, TALE, zinc finger protein) or an RNA targeting agent (e.g., inhibitory nucleic acid molecule). Inhibition of the expression of Claudin 3 includes a decrease in expression of at least 10%, preferably at least 40%, in the presence of the agent as compared to the expression of Claudin 3 without the agent. Inhibition of the activity of Claudin 3 includes a decrease in activity of at least 10%, preferably at least 40%, in the presence of the agent as compared to the activity of Claudin 3 without the agent.

[0016] Claudin, as referred to herein, is a family of integral membrane proteins that constitute TJs, which act as a permeability barrier and are major cell-cell adhesion sites that confer polarity to epithelial cells by defining the boundary between the apical and basolateral membrane domains. Currently, the mammalian claudin family contains 27 proteins, and many alternatively spliced claudin proteins are expressed in various tissues. Over the past decade, the crystal structures of this protein family have been gradually elucidated. Claudin is a four-pass transmembrane protein that contains four transmembrane domains (TM1-4), an intracellular N-terminus and C-terminus, and two extracellular loops (ECL1 and ECL2). ECL1 contains four β-strands and an extracellular helix (ECH), and ECL2 contains β-strands and a transmembrane three-domain that is exposed on the cell surface. ECLs are involved in the formation of interactions between claudin strands and determine the gating function of claudin-based TJs by two variable regions. Claudin 3 was originally called rat ventral prostate 1 protein (RVP1) and Clostridium perfringens enterotoxin receptor 2 (CPETR2). It was reclassified as claudin 3 based on cDNA similarity to claudin 1 and claudin 2 and antibody studies showing that it is expressed at tight junctions. The term "claudin 3" as used herein refers to human claudin 3 having a mammalian, preferably Uniprot (www.uniprot.org) identifier Uniprot. For the human sequence as shown in SEQ ID NO: 4, it is O15551:

Number

[0017] As used herein, the term "toxin that binds to claudin 3" refers to a peptide that binds to claudin 3, such as CPE (Clostridium perfringens enterotoxin) or a C-terminal fragment of Clostridium perfringens enterotoxin (cCPE), or a fragment or variant thereof, as shown, for example, in doi:10.1074 / jbc.M111.312165. "Fragment or variant thereof" related to CPE means that the fragment or variant (cCPE analog, derivative or mutant) is capable of binding to the extracellular domain of claudin 3 in order to inhibit claudin 3 bound to another protein. Such variants include naturally occurring allelic variants and non-naturally occurring variants. CPE is the main virulence determinant of C. perfringen.

[0018] An "antibody", also synonymously referred to as an "immunoglobulin" (Ig), generally consists of four polypeptide chains, including two heavy (H) chains and two light (L) chains, and thus is a multimeric protein or an Ig homolog equivalent thereto (e.g., the nanobody of camelids containing only heavy chains, a single domain antibody (dAb) that can be derived from either a heavy or a light chain), including full-length functional variants, variants or derivatives thereof (including murine antibodies, chimeric antibodies, humanized antibodies and fully human antibodies that retain the essential epitope-binding characteristics of the Ig molecule), including bispecific, bispecific, multispecific and dual variable domain immunoglobulins, and the immunoglobulin molecule can be of any class (e.g., IgG, IgE, IgM, IgD, IgA and IgY) or subclass (e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2) and allotype. The antibodies used in the present invention are preferably monoclonal antibodies or fragments thereof, including modified antibody formats and antibody mimetics, particularly human or humanized antibodies.

[0019] As used herein, "antibody fragment" refers to a molecule comprising at least one polypeptide chain derived from an antibody that is not full-length and includes, but is not limited to, alone or in combination, (i) a Fab fragment, which is a monovalent fragment consisting of the variable light (VL), variable heavy (VH), constant light (CL), and constant heavy 1 (CH1) domains; (ii) an F(ab')2 fragment, which is a divalent fragment comprising two Fab fragments linked by a disulfide bridge in the hinge region; (iii) the heavy-chain portion of a Fab (Fa) fragment consisting of the VH and CH1 domains; (iv) a variable fragment (Fv) fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a domain antibody (dAb) fragment comprising a single variable domain; (vi) an isolated complementarity determining region (CDR); (vii) a single chain FvFragment (scFv); (viii) a diabody, which is a bivalent and bispecific antibody in which the VH and VL domains are expressed on a single polypeptide chain but using a linker that is too short to allow pairing between the two domains on the same chain, thereby allowing pairing of the complementary domains with domains on another chain to form two antigen-binding sites; (ix) a linear antibody comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) that form a pair of antigen-binding regions together with a complementary light-chain polypeptide; and (x) other non-full-length portions of immunoglobulin heavy and / or light chains, or variants, mutants, or derivatives thereof.

[0020] As used herein, the term "modified antibody format" includes antibody-drug conjugates, polyalkylene oxide-modified scFv, monobody, diabody, camelid antibody, domain antibody, bispecific or trispecific antibody, two IgG structures joined by IgA or J chain and secretory component, shark antibody, New World primate framework + non-New World primate CDR, IgG4 antibody having a removed hinge region, IgG having two additional binding sites modified to CH3 domain, antibody having a modified Fc region to enhance affinity for Fcγ receptor, dimerization construct containing CH3+VL+VH, and the like.

[0021] As used herein, the term "antibody mimetic" refers to proteins that do not belong to the immunoglobulin family, and further non-proteins such as aptamers or synthetic polymers. Some types have an antibody-like β-sheet structure. Potential advantages of "antibody mimetics" or "alternative scaffolds" over antibodies are good solubility, higher tissue penetration, higher stability to heat and enzymes, and relatively low manufacturing costs.

[0022] As used herein, the term "binding" or "bound" relates to a covalent or non-covalent bond of a molecule to another molecule. Covalent bonds include the formation of covalent bonds. Non-covalent bonds include p-p (aromatic) interactions, van der Waals interactions, H-bonding interactions, and ionic interactions. Complexes containing covalent bonds of the present invention are, for example, N-acetylgalactosamine (GalNAc) siRNA complexes, and siRNAs such as siRNAs targeting claudin 3 are covalently bound to N-acetylgalactosamine (GalNAc), preferably covalently bound to 1 to 5 moieties of N-acetylgalactosamine (GalNAc), more preferably covalently bound to the 3 moiety of N-acetylgalactosamine (GalNAc). Complexes containing non-covalent bonds of the present invention are, for example, lipid nanoparticles, liposomes or adenoviruses containing silencing RNAs targeting claudin 3.

[0023] The terms "nucleic acid", "nucleic acid sequence", "nucleic acid molecule", "polynucleic acid sequence", "nucleotide sequence", and "nucleotide acid sequence" are used interchangeably herein and have the same meaning herein, and preferably refer to DNA or RNA. In some embodiments, a nucleic acid sequence is a polymer comprising or consisting of nucleotide monomers that are covalently bonded to each other by phosphodiester bonds of a sugar / phosphate backbone. The term "nucleic acid sequence" also encompasses modified nucleic acid sequences, DNA, or RNA such as base modifications, sugar modifications, or backbone modifications.

[0024] The term "nucleic acid targeting a gene or mRNA", as used herein, refers to at least one nucleic acid sequence encoding or comprising a nucleic acid, such as small interfering RNA (siRNA), short or small hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), and long non-coding RNA (lncRNA). Small interfering RNA (siRNA) can bind to a target gene or target messenger RNA (mRNA). In some embodiments, the siRNA used herein may be processed from dsRNA or shRNA. Thus, as used herein, the term "siRNA" may include siRNA according to the present invention and molecules that can be generated in mammalian cells by the RNA interference pathway by the siRNA according to the present invention, particularly dsRNA molecules. RNA may be made by synthetic chemistry and enzymatic methodologies known to those skilled in the art, or by the use of recombinant techniques, or isolated from natural sources, or made by combinations thereof. RNA may optionally contain non-natural and naturally occurring nucleoside modifications known in the art, such as N1-methylpseudouridine, also referred to as methylpseudouridine for example. In some embodiments, the nucleic acid targeting a gene or mRNA of the present invention comprises multiple copies of siRNA that can target one mRNA.

[0025] As used herein, the term "siRNA that binds to Claudin 3" relates to small interfering RNA (siRNA) capable of binding to the target messenger RNA (mRNA) of Claudin 3, and the siRNA used herein may comprise a double-stranded RNA (dsRNA) region, a hairpin structure, a loop structure, or any combination thereof. In some embodiments, the siRNA may comprise at least one shRNA, at least one dsRNA region, or at least one loop structure. In some embodiments, the siRNA may be processed from dsRNA or shRNA. In some embodiments, the siRNA may be processed or cleaved from shRNA by an endogenous protein such as Dicer. In some embodiments, the hairpin structure or loop structure may be cleaved or removed from the siRNA. For example, the hairpin structure or loop structure of shRNA may be cleaved or removed. In some embodiments, the RNA described herein may be made by synthetic, chemical, or enzymatic methodologies known to those skilled in the art, may be made by recombinant techniques known to those skilled in the art, may be isolated from natural sources, or may be made by any combination thereof. The RNA may comprise modified or unmodified nucleotides, or mixtures thereof. For example, the RNA may optionally comprise chemical and naturally occurring nucleoside modifications known in the art. In some embodiments, the siRNA may comprise a nucleic acid sequence comprising a sense siRNA strand. In some embodiments, the siRNA may comprise a nucleic acid sequence comprising an antisense siRNA strand. In some embodiments, the siRNA may comprise a nucleic acid sequence comprising a sense siRNA strand and a nucleic acid sequence comprising an antisense siRNA strand.

[0026] The term "compound that promotes the delivery of an agent to the liver" or "compound that promotes the delivery of siRNA to the liver", as used herein, relates to, for example, sugars, lipid nanoparticles, liposomes or adenoviruses. A compound that promotes the delivery of siRNA to the liver is, for example, N-acetylgalactosamine (GalNAc), which is preferred.

[0027] The terms "individual", "subject" or "patient" are used interchangeably herein. In certain embodiments, the subject is a mammal. Mammals include, but are not limited to, primates (including humans and non-human primates). In preferred embodiments, the subject is a human.

[0028] The term "pharmaceutically acceptable carrier" as used herein refers to a carrier that is commensurate with a reasonable benefit / risk ratio and is suitable for use in humans and / or animals without undue adverse side effects (such as toxicity, irritation and allergic reactions). A "carrier" can be a solvent, suspending agent or vehicle for delivering the agent to a subject.

[0029] As used herein, the term "about" refers to + / - 10% of a given measured value.

[0030] Accordingly, in a first aspect, the present invention provides an agent that inhibits the expression and / or activity of claudin 3 for use in a method of preventing, delaying the progression of or treating cholestasis and / or fibrosis associated with cholestasis.

[0031] An agent that inhibits the expression and / or activity of claudin 3 In one embodiment, the agent that inhibits the expression and / or activity of claudin 3 is an siRNA that targets claudin 3 or an antibody or fragment thereof that binds to claudin 3.

[0032] In one embodiment, the agent that inhibits the expression and / or activity of claudin 3 is an agent that inhibits the expression of claudin 3.

[0033] In yet another embodiment, the agent that inhibits the expression of Claudin 3 is a nucleic acid that targets the gene or mRNA encoding Claudin 3.

[0034] The nucleic acid that targets the gene encoding Claudin 3 or the mRNA encoding Claudin 3 can be, for example, at least one nucleic acid sequence that encodes or contains a nucleic acid such as small interfering RNA (siRNA), short hairpin RNA or small hairpin RNA (shRNA), microRNA (miRNA), piwi-interacting RNA (piRNA), and long non-coding RNA (IncRNA). Preferably, the agent that inhibits the expression of Claudin 3 is small interfering RNA (siRNA) that targets Claudin 3, that is, siRNA that can bind to the gene encoding Claudin 3 or the target messenger RNA (mRNA) encoding Claudin 3, and more preferably small interfering RNA (siRNA) that can bind to the target messenger RNA (mRNA) encoding Claudin 3. Preferably, the siRNA that targets Claudin 3 contains 10 to 50, more preferably 15 to 40, and even more preferably 17 to 24 nucleotides. In certain embodiments, the siRNA according to the present invention contains 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, or 27 nucleotides.

[0035] In an even more preferred embodiment, it includes siRNA containing a sequence as shown in SEQ ID NO: 1 (sense strand) that targets Claudin 3, siRNA containing a sequence as shown in SEQ ID NO: 2 (sense strand), siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99%, or 100% identical to the siRNA containing the sequence as shown in SEQ ID NO: 1, and siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99%, or 100% identical to the siRNA containing the sequence as shown in SEQ ID NO: 2.

[0036] In a preferred embodiment, the siRNA targeting Claudin 3 is characterized by a sequence reverse complementary to SEQ ID NO: 1 or a sequence reverse complementary to SEQ ID NO: 2. Therefore, in one embodiment, the sequence reverse complementary to SEQ ID NO: 1 is a sequence as shown in SEQ ID NO: 5 (antisense strand), and the sequence reverse complementary to SEQ ID NO: 2 is a sequence as shown in SEQ ID NO: 6 (antisense strand).

[0037] In a particular preferred embodiment, the siRNA targeting Claudin 3 is an siRNA comprising a sequence as shown in any of SEQ ID NOs: 1, 2, 5, 6 or 33 - 168, or an siRNA consisting of an siRNA comprising a sequence as shown in any of SEQ ID NOs: 1, 2, 5, 6 or 33 - 168 and being at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical, to the siRNA. The siRNA is selected from the group consisting of such siRNAs.

[0038] In a particular more preferred embodiment, the siRNA targeting Claudin 3 is an siRNA comprising a sequence as shown in any of SEQ ID NOs: 33 - 168, or an siRNA consisting of an siRNA comprising a sequence as shown in any of SEQ ID NOs: 33 - 168 and being at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical, to the siRNA. The siRNA is selected from the group consisting of such siRNAs.

[0039] In a particular even more preferred embodiment, the siRNA targeting Claudin 3 is an siRNA comprising a sequence as shown in any of SEQ ID NOs: 33 - 100, or an siRNA consisting of an siRNA comprising a sequence as shown in any of SEQ ID NOs: 33 - 100 and being at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical, to the siRNA. The siRNA is selected from the group consisting of such siRNAs.

[0040] In a more specific preferred embodiment, the siRNA targeting Claudin 3 is an siRNA comprising a sequence as shown in any of SEQ ID NO: 1, 2, 33 to 66 or 101 to 134, or an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to an siRNA comprising a sequence as shown in any of SEQ ID NO: 1, 2, 33 to 66 or 101 to 134, and is selected from the group consisting of such siRNAs.

[0041] In a more specifically preferred embodiment, the siRNA targeting Claudin 3 is an siRNA comprising a sequence as shown in any of SEQ ID NO: 33 to 66 or 101 to 134, or an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to an siRNA comprising a sequence as shown in any of SEQ ID NO: 33 to 66 or 101 to 134, and is selected from the group consisting of such siRNAs.

[0042] In a more preferred embodiment, the siRNA targeting Claudin 3 is an siRNA comprising a sequence as shown in any of SEQ ID NO: 33 to 66, or an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to an siRNA comprising a sequence as shown in any of SEQ ID NO: 33 to 66, and is selected from the group consisting of such siRNAs.

[0043] In the context of this specification, the terms "sequence identity" and "percentage of sequence identity" refer to values determined by comparing two aligned sequences. Methods for aligning sequences for comparison are well known in the art. Alignment of sequences for comparison may be carried out by the local homology algorithm of Smith and Waterman, Adv. Appl. Math. 2:482 (1981), by the global alignment algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Nat. Acad. Sci. 85:2444 (1988), or by computerized implementations of these algorithms including, but not limited to, CLUSTAL, GAP, BESTFIT, BLAST, FASTA, and TFASTA. Software for performing BLAST analysis is publicly available through the National Center for Biotechnology-Information (http: / / blast.ncbi.nlm.nih.gov / ).

[0044] In one embodiment, the agent that inhibits the expression and / or activity of claudin 3 is an agent that inhibits the activity of claudin 3.

[0045] In a further embodiment, the agent that inhibits the activity of claudin 3 is selected from the group consisting of an antibody or a fragment thereof that binds to claudin 3 and a toxin that binds to claudin 3. Preferably, the toxin that binds to claudin 3 is Clostridium perfringens enterotoxin (CPE) or a fragment or variant thereof.

[0046] In a preferred embodiment, the agent that inhibits the activity of Claudin 3 is an antibody or a fragment thereof that binds to Claudin 3, preferably a monoclonal antibody or a fragment thereof that binds to Claudin 3, and even more preferably an antibody or a fragment thereof that binds to the extracellular domain of Claudin 3, particularly a monoclonal antibody or a fragment thereof that binds to the extracellular domain of Claudin 3. In a further preferred embodiment, the antibody or a fragment thereof that binds to Claudin 3 is a human or humanized antibody, more preferably a monoclonal human antibody. In a specific preferred embodiment, the antibody or a fragment thereof that binds to Claudin 3 comprises a light chain containing the amino acid sequence as set forth in SEQ ID NO: 169, and / or a heavy chain containing the amino acid sequence as set forth in SEQ ID NO: 170.

[0047] In a more specific preferred embodiment, the agent that inhibits the activity of Claudin 3 is an antibody or a fragment thereof, preferably a monoclonal antibody or a fragment thereof that binds to extracellular loop 1 and / or 2 (ECL1 and / or ECL2) of Claudin 3.

[0048] In one embodiment, the agent that inhibits the expression and / or activity of Claudin 3 is conjugated to a compound that promotes the delivery of the agent to the liver. The compound that promotes the delivery of the agent to the liver can be, for example, N-acetylgalactosamine (GalNAc), lipid nanoparticles, liposomes or adenoviruses.

[0049] In a preferred embodiment, the antibody or a fragment thereof that binds to Claudin 3 is conjugated to a compound that promotes the delivery of the antibody to the liver, and even more preferably, the antibody or a fragment thereof that binds to Claudin 3 is an N-acetylgalactosamine (GalNAc)-antibody conjugate.

[0050] In a preferred embodiment, the siRNA targeting Claudin 3 is conjugated to a compound that promotes the delivery of the siRNA to the liver. In a more preferred embodiment, the agent is an N-acetylgalactosamine (GalNAc)-siRNA conjugate, i.e., a conjugate of the siRNA targeting Claudin 3 and N-acetylgalactosamine (GalNAc). Typically, such a conjugate contains 1 to 5, preferably 3 moieties of GalNAc covalently bound to one part of the siRNA.

[0051] Composition As outlined above, the present invention also relates to an agent that inhibits the expression and / or activity of Claudin 3 for use in a method of preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis, and a composition comprising the agent and optionally a pharmaceutically acceptable carrier.

[0052] The term "composition" generally refers to a fixed-dose combination (FDC) containing a compound that inhibits the expression and / or activity of Claudin 3 in a single dosage form having a predetermined combination of each dosage.

[0053] The composition may also be further used as an additional therapy. As used herein, "additional" or "additional therapy" means a collection of reagents for use in therapy, and the subject receiving the therapy starts a first therapy regimen of one or more reagents in addition to a first treatment regimen of one or more different reagents before starting a second treatment regimen of one or more reagents, but as a result, not all of the reagents used in the therapy are started simultaneously.

[0054] The amount of the agent that inhibits the expression and / or activity of Claudin 3 administered varies according to the specific circumstances surrounding the case, including, for example, the specific agent that inhibits the expression and / or activity of Claudin 3 being administered, the route of administration, the condition being treated, the target area being treated, and the subject or host being treated, and depends on factors such as the specific agent, the disease state and its severity.

[0055] In one embodiment, the present invention provides a composition comprising an agent that inhibits the expression and / or activity of claudin-3, wherein the agent that inhibits the expression and / or activity of claudin-3 is present in a therapeutically effective amount.

[0056] As used herein, the expression "effective amount" or "therapeutically effective amount" refers to an amount capable of causing one or more of the following effects in a subject receiving the composition of the present invention: (i) dilution and / or detoxification of bile containing a greater amount of water than before the start of treatment, (ii) reduction in the concentration of liver and / or blood bile acids, (iii) reduction in the value of alkaline phosphatase (ALP), a bile stasis marker in the blood, (iv) reduction in tissue necrosis in the liver, (v) improvement in quality of life, (vi) extension of asymptomatic survival period, (vii) extension of asymptomatic survival period after liver transplantation, (viii) recovery of bile stasis symptoms such as jaundice or pruritus, (ix) increase in the survival rate of patients with bile stasis and / or fibrosis associated with bile stasis.

[0057] Determination of the therapeutically effective amount is well within the ability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0058] In one embodiment, the present invention provides a composition comprising an agent that inhibits the expression and / or activity of claudin-3, wherein the amount of the agent that inhibits the expression and / or activity of claudin-3 in the composition is from about 0.1 mg to about 10 g.

[0059] Formulation and Administration The composition according to the present invention is preferably suitable for subcutaneous administration, intravenous administration or oral administration to a subject, and comprises a therapeutically effective amount of an active ingredient and one or more suitable pharmaceutically acceptable carriers.

[0060] Preferred compositions, such as pharmaceutical compositions, can be formulated in a conventional manner using one or more pharmaceutically acceptable inert ingredients, such as a pharmaceutical carrier that facilitates processing the active compound into a preparation that can be pharmaceutically used. Appropriate formulations depend on the chosen route of administration, and an overview of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999). Unless otherwise indicated, the compositions according to the invention are prepared in a manner known per se, for example by conventional mixing, granulating, coating, dissolving or lyophilization processes. When preparing compositions for oral dosage forms, any of the usual pharmaceutical media, such as water, glycols, oils, alcohols, and carriers, diluents, granulating agents, lubricants, binders, disintegrants, etc., such as starch, sugar or microcrystalline cellulose, may be utilized.

[0061] In one embodiment, the present invention provides a composition comprising an agent that inhibits the expression and / or activity of claudin 3, and at least one pharmaceutically acceptable carrier, wherein the composition is a solution.

[0062] In one embodiment, the present invention provides a composition comprising an agent that inhibits the expression and / or activity of claudin 3, and at least one pharmaceutically acceptable carrier, wherein the composition is a tablet or capsule, preferably a tablet.

[0063] Administration regimen Exemplary treatment regimens require once-daily, twice-daily, three-times-daily, once every two days, twice weekly, or once weekly administration. The compositions of the present invention are typically administered multiple times. The interval between single administrations can be, for example, less than one day, daily, once every two days, twice weekly, or once weekly. The compositions of the present invention may be administered as a continuous, uninterrupted treatment. The compositions of the present invention may also be administered in a regimen in which the subject undergoes a cycle of treatment interrupted by a drug withdrawal period or a non-treatment period. Thus, the compositions of the present invention may be administered according to the selected intervals described above over a continuous period such as one week or a portion thereof, over two weeks, over three weeks, over four weeks, over five weeks, or over six weeks, and may be discontinued over a period of one week or a portion thereof, over two weeks, over three weeks, over four weeks, over five weeks, over six weeks. The composition of the treatment interval and the non-treatment interval is called a cycle. The cycle may be repeated one or more times. Two or more different cycles may be used in combination to repeat the treatment one or more times. The intervals can also be irregular, as indicated by measuring the blood level of the agent that inhibits the expression and / or activity of claudin 3 in the patient. In a preferred embodiment, the composition according to the present invention is administered once daily. In an exemplary treatment, the agent that inhibits the expression and / or activity of claudin 3 can be administered at 0.1 mg to 10 g per day.

[0064] Use of an agent or a composition thereof that inhibits the expression and / or activity of claudin 3 for the prevention, delay in progression, or treatment of cholestasis and / or fibrosis associated with cholestasis The present invention provides an agent or a composition thereof that inhibits the expression and / or activity of claudin 3 as described herein for use in a method of preventing, delaying the progression, or treating cholestasis and / or fibrosis associated with cholestasis in a subject.

[0065] Also provided is the use of an agent or a composition thereof that inhibits the expression and / or activity of claudin 3 as described herein for the manufacture of an agent for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis in a subject.

[0066] Also provided is the use of an agent or a composition thereof that inhibits the expression and / or activity of claudin 3 as described herein for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis in a subject.

[0067] Also provided is a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis in a subject, the method comprising administering to the subject a therapeutically effective amount of an agent that inhibits the expression and / or activity of claudin 3, or a therapeutically effective amount of a composition thereof, as described herein.

[0068] As used herein, the terms "treat" / "treating" include (1) delaying the onset of clinical symptoms of a condition, disorder or disease that develops in an animal, specifically a mammal, particularly a human, where the animal is suffering from or is susceptible to the condition, disorder or disease but has not yet experienced or manifested the clinical or latent symptoms of the condition, disorder or disease, (2) inhibiting the condition, disorder or disease (e.g., in the case of a disease or managed treatment, suppressing, reducing or delaying the recurrence, onset of at least one of its clinical or latent symptoms) and / or (3) alleviating the disease (i.e., producing a remission of the condition, disorder or disease, or at least one of its clinical or latent symptoms). The benefit to the patient being treated is either statistically significant or at least perceptible to the patient or the physician. However, it will be understood that when an agent is administered to a patient treating a disease, the outcome may not always be an effective treatment.

[0069] As used herein, "delay in progression" means an increase in the time from the symptoms of cholestasis and / or fibrosis associated with cholestasis to the worsening of those symptoms, and includes converting or inhibiting the progression of the disease. "Inhibition" of the progression of a disease or disease complication in a subject means preventing or reducing the progression of the disease and / or disease complication in the subject.

[0070] Preventive treatment includes prophylactic treatment. In prophylactic use, the compositions of the invention are administered to a subject suspected of having or at risk of developing cholestasis and / or fibrosis associated with cholestasis. In therapeutic use, the composition is administered to a subject, such as a patient already suffering from cholestasis and / or fibrosis associated with cholestasis, in an amount sufficient to cure or at least partially suppress the symptoms of the disease. The amount effective for such use will vary depending on the severity and course of the disease, previous therapy, the health status of the subject, the response to the drug, and the judgment of the treating physician.

[0071] If the subject's condition does not improve, the compositions of the invention may be administered chronically over an extended period, including the entire lifespan of the subject, to remit the symptoms of the subject's disease or condition, or otherwise control or limit them.

[0072] If the subject's status improves, the composition may be administered continuously, or alternatively the dose of the drug administered may be temporarily decreased, or temporarily interrupted (i.e., a drug holiday) for a specified length of time.

[0073] When improvement in the patient's condition occurs, a maintenance dose of the composition of the invention is administered as needed. Subsequently, the dose and / or frequency of administration, or both, are optionally decreased as a function of the symptoms to a level at which the improved disease is maintained.

[0074] In one embodiment, the cholestasis is intrahepatic cholestasis or extrahepatic cholestasis, preferably intrahepatic cholestasis. In a preferred embodiment, the cholestasis is intrahepatic cholestasis, and the intrahepatic cholestasis is acute intrahepatic cholestasis or chronic intrahepatic cholestasis. In an even more preferred embodiment, the cholestasis is chronic intrahepatic cholestasis, preferably chronic intrahepatic cholestasis selected from the group consisting of primary biliary cholangitis (PBC) and primary sclerosing cholangitis (PSC).

[0075] In one embodiment, the cholestasis is a disease selected from the group consisting of obstructive cholestasis, non-obstructive cholestasis, biliary tract diseases and defects in biliary tract function, more preferably biliary tract diseases, particularly malignant biliary tract obstruction.

[0076] In a preferred embodiment, the cholestasis and the fibrosis associated with cholestasis are selected from the group consisting of chronic intrahepatic cholestasis and biliary tract diseases.

[0077] In a more preferred embodiment, the cholestasis and the fibrosis associated with cholestasis are selected from the group consisting of primary biliary cholangitis (PBC), primary sclerosing cholangitis (PSC) and malignant biliary tract obstruction.

[0078] Obstructive cholestasis can be associated with or caused by common bile duct obstruction caused by gallstones, pancreatic cancer, cholangiocarcinoma, stricture of the common bile duct, biliary atresia such as extrahepatic biliary atresia, sludge or something like gallstones, or bile duct cysts with sludge and / or concentrated bile / mucus plugs.

[0079] Non-obstructive cholestasis may be associated with or caused by viral hepatitis (hepatitis B, hepatitis C), toxic effects of drugs or nutrition, tumor-associated syndromes such as Hodgkin's lymphoma, Wilson's disease, familial intrahepatic cholestasis, intrahepatic cholestasis of pregnancy, infiltrative diseases such as amyloidosis or metastatic cancer, and / or cirrhosis (regardless of cause), bacterial infections such as infections with Gram-negative enteric bacteria, syphilis, Listeria, Toxoplasma, or sepsis or endotoxemia caused by bacterial infections, viral infections with cytomegalovirus, herpes viruses (including simplex virus, varicella-zoster virus, parvovirus B19, adenovirus), rubella, reovirus, and enterovirus.

[0080] Biliary tract diseases may be associated with or caused by primary biliary cholangitis, primary sclerosing cholangitis, graft-versus-host disease (acute and chronic), transplant rejection (acute and chronic), infarction of the biliary tree secondary to hepatic artery occlusion in transplantation, vanishing bile duct syndrome caused by toxic effects of drugs such as ibuprofen or chlorpromazine.

[0081] Defects in biliary function may be associated with or caused by α-1-antitrypsin accumulation disease, cystic fibrosis, galactosemia, tyrosinemia, fatty acid metabolism disorders, lipid storage diseases, glycogenosis, peroxisomal diseases, bile acid formation disorders, PFIC1, PFIC2, PFIC3 (PFIC, progressive familial intrahepatic cholestasis), paucity of bile ducts or asymptomatic absence of biliary syndromes such as Alagille syndrome.

[0082] In a preferred embodiment, cholestasis is obstructive cholestasis associated with or caused by stricture of the intrahepatic bile ducts, biliary atresia such as extrahepatic biliary atresia, and / or intrahepatic bile duct obstruction caused by things such as biliary sludge or gallstones, preferably obstructive cholestasis associated with or caused by stricture of the intrahepatic bile ducts, non-obstructive cholestasis associated with or caused by the toxic effects of drugs, Wilson's disease and / or familial intrahepatic cholestasis, primary biliary cholangitis, primary sclerosing cholangitis, and / or syndromes of vanishing bile ducts associated with or caused by the toxic effects of drugs such as ibuprofen or chlorpromazine, and diseases associated with or caused by these biliary diseases, and cholestasis is a disease selected from the group consisting of defects in bile acid formation disorders (PFIC1, PFIC2, PFIC3) or caused by these defects in bile duct function.

[0083] In a more preferred embodiment, cholestasis is obstructive cholestasis. In an even more preferred embodiment, cholestasis is obstructive cholestasis associated with or caused by stricture of the intrahepatic bile ducts or biliary atresia such as extrahepatic biliary atresia.

[0084] In one embodiment, the fibrosis associated with cholestasis is a disease induced by at least partially unresolved cholestasis, preferably the fibrosis associated with cholestasis is a liver disease induced by at least partially unresolved cholestasis.

[0085] Fibrosis associated with cholestasis usually is selected from the group consisting of fibrosis resulting from hepatitis A virus infection associated with cholestasis, fibrosis resulting from hepatitis B virus infection associated with cholestasis, fibrosis resulting from hepatitis C virus infection associated with cholestasis, fibrosis resulting from Epstein-Barr virus infection associated with cholestasis, fibrosis resulting from hemochromatosis associated with cholestasis, fibrosis resulting from autoimmune hepatitis associated with cholestasis, fibrosis resulting from secondary biliary cholangitis associated with cholestasis, fibrotic fibrosis resulting from alcoholic liver disease associated with cholestasis and / or clinical jaundice, fibrosis resulting from drug-induced liver disease associated with cholestasis, fibrosis caused by a metabolic syndrome (e.g., glucose metabolism disorder, fat accumulation or lipid metabolism disorder) associated with cholestasis, non-alcoholic fatty liver disease (NAFLD) associated with cholestasis, liver fibrosis caused by dysregulation of iron and copper homeostasis associated with non-alcoholic fatty liver disease associated with cholestasis, liver fibrosis resulting from non-alcoholic steatohepatitis (NASH) associated with cholestasis, fibrosis resulting from radiation-induced liver disease associated with cholestasis, fibrosis resulting from Wilson's disease associated with cholestasis, fibrosis resulting from α1-antitrypsin deficiency associated with cholestasis, and fibrosis resulting from sclerosing cholangitis syndrome associated with cholestasis. Preferably, the present invention provides a treatment for fibrosis causing metabolic diseases such as NAFLD or NASH, particularly a treatment for fibrosis causing liver diseases using an agent as described herein.

[0086] In a further aspect, the present invention provides a dosage form for preventing, delaying the progression or treating cholestasis and / or fibrosis associated with cholestasis, which comprises an agent that inhibits the expression or activity of claudin 3, or a composition containing the agent, and optionally a pharmaceutically acceptable carrier.

[0087] In a further aspect, the present invention provides siRNAs that target Claudin 3. In a preferred embodiment, the present invention provides siRNAs comprising a sequence as set forth in SEQ ID NO: 1, siRNAs comprising a sequence as set forth in SEQ ID NO: 2, siRNAs that are at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising a sequence as set forth in SEQ ID NO: 1, and siRNAs that are at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising a sequence as set forth in SEQ ID NO: 2, and provides siRNAs that target Claudin 3, which are selected from the group consisting of these siRNAs.

[0088] In yet another embodiment, the present invention provides siRNAs comprising a sequence as set forth in SEQ ID NO: 5, siRNAs comprising a sequence as set forth in SEQ ID NO: 6, siRNAs that are at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising a sequence as set forth in SEQ ID NO: 5, and siRNAs that are at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising a sequence as set forth in SEQ ID NO: 6, and provides siRNAs that target Claudin 3, which are selected from the group consisting of these siRNAs.

[0089] In a preferred embodiment, the present invention provides siRNAs comprising a sequence as set forth in any one of SEQ ID NO: 1, 2, 5, 6 or 33 - 168, or siRNAs that are at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising a sequence as set forth in any one of SEQ ID NO: 1, 2, 5, 6 or 33 - 168, and provides siRNAs that target Claudin 3, which are selected from the group consisting of these siRNAs.

[0090] In a more preferred embodiment, the present invention provides an siRNA targeting claudin 3, which is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NOs: 33 to 168, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity, to an siRNA comprising a sequence as shown in any one of SEQ ID NOs: 33 to 168.

[0091] In an even more preferred embodiment, the present invention provides an siRNA targeting claudin 3, which is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NOs: 33 to 100, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity, to an siRNA comprising a sequence as shown in any one of SEQ ID NOs: 33 to 100.

[0092] In a specific embodiment, the present invention provides an siRNA targeting claudin 3, which is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NOs: 1, 2, 33 to 66 or 101 to 134, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity, to an siRNA comprising a sequence as shown in any one of SEQ ID NOs: 1, 2, 33 to 66 or 101 to 134.

[0093] In a more specific embodiment, the present invention provides an siRNA targeting claudin 3, which is selected from the group consisting of an siRNA comprising a sequence as shown in any one of SEQ ID NOs: 33 to 66 or 101 to 134, or an siRNA having at least 95% identity, more preferably 96%, 97%, 98%, 99% or 100% identity, to an siRNA comprising a sequence as shown in any one of SEQ ID NOs: 33 to 66 or 101 to 134.

[0094] In an even more specific embodiment, the present invention provides an siRNA that targets claudin 3 and comprises a sequence as set forth in any of SEQ ID NOs: 33 to 66, or an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to an siRNA comprising a sequence as set forth in any of SEQ ID NOs: 33 to 66, and is selected from the group consisting of such siRNAs.

[0095] In a further aspect, the present invention provides an antibody or a fragment thereof that binds to claudin 3. In one embodiment, the antibody or a fragment thereof that binds to claudin 3 is a human antibody. In a preferred embodiment, the antibody or a fragment thereof that binds to claudin 3 comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 169 and / or a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 170. In a further embodiment, the antibody or a fragment thereof that binds to claudin 3 is conjugated to a compound that promotes delivery of the antibody to the liver. In a more preferred embodiment, the antibody or a fragment thereof that binds to claudin 3 is an N-acetylgalactosamine (GalNAc)-antibody conjugate. A particularly preferred antibody is a human antibody that binds to claudin 3, and more specifically, a human antibody that binds to claudin 3 and comprises a light chain comprising an amino acid sequence as set forth in SEQ ID NO: 169 and / or a heavy chain comprising an amino acid sequence as set forth in SEQ ID NO: 170, and is an N-acetylgalactosamine (GalNAc)-antibody conjugate.

Examples

[0096] The examples of the present invention are intended to illustrate the present invention without limiting it.

[0097] Materials and Methods Animal Housing and Ethical Approval 12- to 18-week-old C57BL / 6J background CLDN3 + / + or CLDN3 - / -Mice (about 18 - 22 g) were maintained in a 12 - hour light - cycle room and fed a standard chow diet for experimental animals. All mouse experiments were conducted in accordance with good animal - care guidelines as defined by the Animal Experiment Committee and complied with the National Center for the Replacement, Refinement and Reduction of Animals (NC3Rs) guidelines for the replacement, refinement and reduction of animals in research (https: / / www.nc3rs.org.uk / arrive - guidelines), and were approved by the Veterinary Office of the Canton of Bern (permit BE37 / 20).

[0098] Bile flow and passage of FITC - dextran into bile The integrity of the paracellular tight junctions of hepatocyte tight junctions was measured as previously described. 14 . Quantification of blood to bile passage of FITC - dextran (40 kDa) was measured. In anesthetized mice, the common bile duct was ligated and a cannula was inserted into the gallbladder. Subsequently, bile was collected for 20 minutes to determine bile flow. Then, 400 μL of FITC - dextran (40 kDa, 25 mg / mL in saline) was injected into the inferior vena cava. Bile was collected by cannulation in 3 - minute fractions over 40 minutes. The liver was surgically excised and weighed. Bile was diluted 1 / 50 with water, and FITC fluorescence was measured at an excitation of 489 nm / emission of 534 nm using a Tecan Spark® plate reader. Data are reported as relative fluorescent units (RFU) per μL of bile per minute per gram of liver.

[0099] Quantification of FITC - dextran uptake In anesthetized mice, the common bile duct was ligated and 300 μL of FITC-dextran (40 kDa, 6.25 mg / mL in saline) was injected via the tail vein. FITC fluorescence was acquired by time-lapse imaging using an IVIS® Spectrum system (Perkin Elmer). One image was acquired every 30 seconds for a total of 45 minutes. Regions of interest with the same area size were drawn around the liver. The FITC signal intensity was quantified in each image using the software of the device. Data were reported as total radiant efficiency [p / s] / [μW / cm 2 . Using the same experimental setup, the livers of the mice were harvested 20 minutes after FITC-dextran (40 kDa) injection and frozen sections (25 μm) were prepared. The sections were stained with DAPI (D9542, diluted 1:2000, Sigma-Aldrich) for 15 minutes and mounted with a fluorescence mounting medium (H-1000, Vectorlabs, Burlingame, CA). Confocal Z-stack images were acquired in the periportal liver region to visualize the uptake of FITC-dextran (LSM710, Zeiss, 20× objective lens, Oberkochen, Germany). The FITC signal was quantified in a complete Z-stack of the same area and thickness using ImageJ software (version 1.48, National Institutes of Health, Bethesda, MD). Data were reported as total signal intensity (total density).

[0100] Bile duct ligation The BDL procedure was performed as previously described 15. Briefly described, analgesia was administered to anesthetized mice with buprenorphine (0.1 μg / g body weight, Temgesic®), and laparotomy was performed. The common bile duct was exposed, doubly ligated using 7-0 silk (Sofsilk™, Covidien / Medtronic, Dublin, Ireland), and the tissue between the ligatures was transected. The laparotomy was closed using 6-0 Prolene® suture (Ethicon, Bridgewater, New Jersey, United States). Based on the animal's health scoring, buprenorphine analgesia was repeated if necessary.

[0101] Hematoxylin & Eosin staining Liver paraffin sections were deparaffinized, stained with hematoxylin (Merck, catalog number HX43078349) for 6 minutes, and differentiated with HCL-ALC (1:1) by performing 3 immersions. The slides were incubated in eosin (Fluka Chemical Corp, catalog number 45240) for 3 minutes, followed by dehydration and mounting with Eukitt® (Kindler).

[0102] Sirius Red staining and quantification The slides were dewaxed, hydrated, and placed in pre-warmed Bouin's fixative (Sigma-Aldrich, catalog number HT-10-1-32) at 54°C for 20 minutes. After washing with running water, the slides were stained with Weigert's iron hematoxylin (Sigma-Aldrich, catalog number HAT10-79) for 5 minutes. After washing with running tap water for 5 minutes, the slides were decolorized using HCl-EtOH (5 mL of 37% HCl in 1 liter of 70% EtOH) and then further washed in water for 5 minutes. The slides were then stained with picrosirius (0.5 g in 1 liter of Bouin's solution, Sigma-Aldrich catalog number 365548) for 5 minutes. Subsequently, the slides were washed twice for 5 minutes each in 0.5% acetic acid diluted with water, dehydrated, and mounted with Eukitt (Kindler). Images were acquired using a slide scanner (Panoramic 250 Flash III, 3DHISTECH), and the staining was quantified using ImageJ (v1.52n, National Institutes of Health, Bethesda, MD). Data analysis was performed blindly. For each mouse liver, 12 randomly selected regions of 10x magnified images of the same size were quantified. Only unmodified images were used, and all five liver lobes were included in the analysis range. The deconvolution setting "Azan Mallory" was used to separate the sirius red signal from other channels. In the red channel, a threshold was set. After converting the threshold-adjusted image to an 8-bit image, the signal intensity was measured as the total density. The results were expressed as the average total density per 2 mm

[0103] Masson's trichrome staining and quantification Masson trichrome staining. Paraffin-embedded liver tissues were dewaxed and placed in Bouin's fixative (HT10-1-32, Sigma-Aldrich) at 56 °C for 10 minutes. The slides were washed with tap water and distilled H2O, and the slides were stained with hematoxylin (HT10-79, Sigma-Aldrich) for 5 minutes. After washing with running tap water and distilled H2O, the slides were decolorized once with HCl-alcohol (1:1) and rinsed again with distilled H2O. Next, the slides were placed in a Biebrich scarlet acid fuchsin solution (HT151-250mL, Sigma-Aldrich) diluted 1:2 in 1% acetic acid (K45741563 425, Dr. Grogg Chemie, Stettlen, Switzerland) for 1 minute. The slides were rinsed and stained with phosphomolybdic-phosphotungstic acid (HT153-250ML and HT152-250ML, Sigma) 1:1 for 5 minutes. Then the slides were stained with aniline blue (HT154-250ML, Sigma) for 20 minutes. After the final rinse, the slides were placed in 0.75% acetic acid for dehydration and mounted with Eukitt (Kindler). Images were acquired using a slide scanner (Panoramic 250 Flash III, 3DHISTECH), and the staining was quantified with ImageJ (v1.52n, National Institutes of Health, Bethesda, MD). For each mouse liver, six randomly selected regions of 7-fold magnified images of the same size were quantified. Only unmodified images were used. Using the deconvolution setting "Masson trichrome staining", the blue collagen signal was separated from the other channels. In the blue channel, a threshold was set. After converting the threshold-adjusted image to an 8-bit image, the signal intensity was measured as the total density. The results are presented as the average total density.

[0104] Quantification of tissue necrosis Sections of liver tissues before and after BDL were stained with hematoxylin / eosin, and images were taken using a bright-field microscope (Panoramic 250 Flash III, 3DHISTECH). Sections for quantification were 75 mm per individual 2covered the average surface. The necrotic parenchymal area was manually outlined. Data were reported as the percentage of the necrotic area across the entire section.

[0105] Measurement of serum bilirubin, ALT, AST, ALP, cholesterol, and total bilirubin Liver injury markers ALT and AST were measured on a Cobas 8000 modular analyzer using module C502 (Roche, Switzerland). ALP and total bilirubin were also measured on the Cobas 8000 using module C702 (Roche, Switzerland). All measurements were performed according to the manufacturer's instructions.

[0106] Quantification of renal bile plugs Kidneys from BDL at 7 days were completely sectioned thinly, and the tissues were stained with hematoxylin / eosin. Images were taken using a bright-field microscope (Panoramic 250 Flash III, 3DHISTECH). Using ImageJ software, bile plugs stained yellow from pink hematoxylin / eosin staining were deconvolved. Yellow was converted to an 8-bit black-and-white image, and the signal intensity was measured as the total density. At least 10 different images covering the renal tissue of 3,034 mm 2 were taken per mouse and the results were averaged. Data were reported as the total density per 1 mm of renal tissue. 2

[0107] Scoring of periportal liver edema The entire hematoxylin- and eosin-stained liver sections were scored for periportal edema based on an arbitrary score from 0 (no edema) to 10 (the maximum amount of edema observed). Only the periportal area was considered. Periportal edema was defined as prominent laminae with swollen connective tissue and an expanded extracellular matrix around the bile ducts.

[0108] Evaluation of bacterial translocation The liver and spleen were harvested under sterile conditions 2 or 7 days after BDL. After weighing the tissues, sterile PBS and stainless steel beads were added to the samples, and then the tissues were homogenized at 30 Hz for 5 minutes using a Tissuelyser (Quiagen). The samples were then seeded onto lysogeny broth (LB) agar plates and cultured under aerobic conditions (37 °C for 48 h) as previously described 16 After incubation, bacterial colonies were manually counted and the results were obtained as colony-forming units normalized to the organ weight. For data visualization, the obtained CFU / g were normalized using log transformation.

[0109] Flow cytometry The antibodies used for fluorescence-activated cell sorting are listed below. The liver was placed in Roswell Park Memorial Institute (RPMI) 1640 medium supplemented with GlutaMAX and digested at 37 °C for 25 minutes using collagenase D (Merck), 0.05% collagenase IV (Worthington Biochemical), DNase I (Sigma-Aldrich), and dispase. The digested liver tissue was poured through a 100 μm cell strainer (BD Falcon) and washed in 50 mL RPMI at 4 °C. After centrifugation at 300 g for 5 minutes, the supernatant was discarded and the pellet was resuspended in 30 mL of RPMI. After repeating the centrifugation at 300 g for 5 minutes to discard the supernatant, 5 mL of red blood cell (RBC) lysis buffer was added per sample, filtered through a 40 μm cell strainer, and incubated at room temperature. After incubation, the cell suspension was spun down and washed in 5 mL of PBS (Gibco) containing 3% fetal bovine serum, 2 mM EDTA, and HEPES (staining buffer). The isolated non-parenchymal cells were then incubated in the dark at 4 °C for 20 minutes with purified anti-CD16 / CD32 and the dead cell labeling reagent eFluor506 (Thermo Fisher Scientific) diluted in PBS to block non-specific binding antibodies and exclude dead cells. After the first staining and washing of the cells, the samples were stained with primary antibodies (Table 1) diluted in the staining buffer in the dark at 4 °C for 20 minutes. After the final washing step at 300 g for 5 minutes, the samples were resuspended in the staining buffer and fixed by adding IC Fixation buffer (eBioscience). The fixed samples were evaluated using a flow cytometer (BD LSR Fortessa; BD Pharmingen, Inc, San Diego, CA) using the corresponding BD FACS Diva software. Data analysis was performed using FlowJo software (Treestar, Inc, Ashland, OR).

[0110] FACS antibody [Table 1]

[0111] Measurement of total bile acids in serum and liver tissue samples Total bile acid values were measured using an assay kit purchased from Crystal Chem (catalog number 80470). Bile, serum, and total liver tissue samples were processed according to the manufacturer's instructions. Final absorbance levels were measured on a Tecan Tecan Spark (registered trademark) plate reader.

[0112] Bile acid quantification using LC-MS / MS The method applied was the one recently described 17 Briefly, for bile acid quantification, 25 μL of serum samples were diluted 1:4 with water and the standards were subjected to protein precipitation by adding a mixture of 900 μL of 2-propanol and deuterated internal standard. Extraction was performed at 4 °C for 30 min with continuous shaking, followed by centrifugation at 16000 g for 10 min. The supernatant was transferred to a new test tube, evaporated to dryness and reconstituted with 100 μL of methanol:water (1:1, v / v). For liver sample extraction, 900 μL of chloroform:methanol:water (1:3:1, v / v / v) and 100 μL of internal standard mixture were added to a Precellys tube containing beads and 30 ± 5 mg of liver tissue. Samples were homogenized using a Precellys tissue homogenizer and centrifuged at 16000 g for 10 min at 20 °C. The supernatant was transferred to a new test tube and the procedure was repeated by adding 800 μL of extraction solvent. After evaporation to dryness, samples were resuspended with 200 μL of methanol:water (1:1, v / v). The injection volume was 3 μL in both cases. LC-MS / MS consisted of an Agilent 1290 UPLC coupled to an Agilent 6490 triple quadrupole mass spectrometer equipped with an electrospray ionization source (Agilent Technologies, Basel, Switzerland). Chromatographic separation of bile acids was performed on a reversed-phase column (ACQUITY UPLC BEH C18, 1.7 mm, 2.1 μm, 150 mm, Waters, Wexford, Ireland) 18was achieved using.

[0113] ANIT poisoning The α-naphthylisothiocyanate (ANIT) poisoning protocol is derived from previous publications. 19,20 . In the acute poisoning model, ANIT (Sigma-Aldrich, St. Louis, Missouri, US) was dissolved in corn oil and orally administered at a dose of 60 mg / kg body weight. The same volume of corn oil was given to control animals. For the 2-day or 10-day models, a single dose or two doses were administered. In the chronic injury model, a custom solid diet containing 0.1% ANIT (Granovit, Lucens, Switzerland) was given ad libitum. A custom solid diet containing the same amount of corn oil was given to the control group. The groups were fed the custom solid diet for a total of 4 weeks.

[0114] Immunohistochemistry Paraffin-embedded liver tissues were sectioned at a thickness of 6 μm for conventional imaging or 30 μm for confocal z-stack imaging. The slides were deparaffinized and hydrated in a series of xylene and ethanol. Antigen retrieval was performed by heat-induced epitope retrieval, and the slides were cooked in citrate buffer (Sigma-Aldrich, catalog number C9999) at pH 6.0 for 10 minutes at 95°C. Nonspecific antibody binding was blocked using a protein blocking solution (Dako, catalog number X0909) for 1 hour at room temperature. Antibodies were prepared in the following dilution of antibody diluent (Dako, catalog number S3022). Primary antibody: Cytokeratin 7 (Novus Biologicals, catalog number NBP1-88080), 1:200. Secondary antibody: Polyclonal rabbit anti-goat immunoglobulin / HRP (Dako, catalog number P0449). Streptavidin-peroxidase (BioConcept, catalog number 71-00-38) and DAB (Sigma-Aldrich, catalog number D4293-50SET) were used for the development of immunohistochemical staining.

[0115] The primary antibody was incubated with gentle stirring overnight at 4°C inside a humid chamber. The slides were washed in PBS-Tween-20 [0.5%] (Sigma-Aldrich, catalog number P1379) for 20 minutes and incubated with the secondary antibody and DAPI (Sigma-Aldrich, catalog number D9542, 1:2000 dilution) for 90 minutes in the dark. After final washing in PBS-Tween20 [0.5%], the slides were mounted. Before incubation with the first antibody, red blood cells were quenched in 5% H2O2 for 10 minutes, and staining was developed after application of the second antibody by incubating with streptavidin-peroxidase for 30 minutes and with DAB for 1 minute.

[0116] Western blot All proteins were extracted from liver tissues or cultured cells using RIPA lysis buffer and TissueLyser (Qiagen, TissueLyserII). The lysates were centrifuged at 20000g for 15 minutes, and the supernatants were aliquoted. Protein concentrations were quantified using the Bradford assay (Bio-Rad, catalog number 5000006) and a microplate reader. Equal amounts of protein per sample were separated by SDS-PAGE under reducing conditions using a precast gel (Bio-Rad, catalog number 456-1094). The proteins were then transferred onto a nitrocellulose membrane (Biorad, catalog number 170-4158). The membrane was blocked with 5% w / v non-fat dry milk in PBS for 1 hour at room temperature. The primary antibodies were diluted in the blocking medium and incubated overnight at 4°C. Primary antibodies: CLDN3 (Novus Biologicals, catalog number NBP1-35668), 1:1000; anti-β-actin-peroxidase (Sigma-Aldrich, catalog number A3854), 1:50000; secondary antibody: anti-rabbit HRP (Dako, catalog number P0448), 1:2000.

[0117] After incubation with the primary antibody, the membrane was washed three times for 5 minutes each in PBS-Tween-20 [0.1%]. The secondary antibody was diluted using 5% w / v non-fat dry milk in PBS, and the membrane was incubated at room temperature for 1 hour, followed by a total of 30 minutes of three washing steps. Enhanced chemiluminescence solution (Perkin Elmer, catalog number NEL105001EA) was added for 1 minute to generate a signal. A film combined with a developer (AGFA, CURIX60) was used to visualize the bands. The exact band size was estimated with the aid of a standard protein ladder (Biorad, catalog number 161-0374).

[0118] Human siRNA candidate screening Candidate siRNAs targeting human claudin 3 mRNA were first identified bioinformatically (human reference sequence used: NM_001306.4). All possible siRNAs were generated and then scored for specificity, cross-reactivity, activity, and off-targets (Axolabs, Kulmbach, Germany). The best candidates containing 2'-fluoro modification or 2'O-methyl modification for stabilization, as well as phosphorothioate linkers at the indicated positions, were selected and synthesized (see Figure 6). The synthesized candidate siRNAs were then transfected into a hepatocellular carcinoma cell line (ATCC catalog number CRL-8024) of human PLC hepatocytes using Lipofectamine-3000. The siRNAs were used at a final concentration of 50 nM. Two days after transfection, RNA was isolated from the cell culture, and the values of the remaining human claudin 3 mRNA were quantified using RT-qPCR according to the following protocol. Thirty-four siRNAs with knockdown efficacy of 50% or more were selected for further testing (Table 6). Table 7 shows the same identified human claudin 3 siRNA sequences without chemically stabilizing modifications.

[0119] RT-qPCR mRNA expression analysis RNA was extracted from snap-frozen tissues or cell cultures using NucleoZOL (Macherey-Nagel, catalog number 740404.200), and cDNA was generated from either 500 μg of tissue RNA using the Omniscript Reverse Transcription Enzyme Kit (Qiagen, catalog number 205113). Real-time qPCR was performed using either SYBR Green or Taqman-based assays according to the manufacturer's instructions on a QuantStudio™ 7 Flex thermocycler (Applied Biosystems, Foster City, CA). Primer sequences are listed below.

[0120] Primer sequences (m; mouse, F; forward, R; reverse)

Table 2

[0121] Antibody production and sequences Expi293 cells were used to generate a recombinant mouse IgG2Aκ anti-Claudin 3 antibody (UB-VS003). The antibody consists of a heavy chain (SEQ ID NO: 170) and a light chain (SEQ ID NO: 169) as shown below.

[0122] When Expi293 cells were transfected with the expression vector, the cells efficiently produced and secreted the recombinant antibody. The cell supernatant was then purified using Protein A resin (Repligen, catalog number PRI-0005). Light chain (SEQ ID NO: 169)

Number

Number

[0123] Primary cell and cell line culture methods Liver samples were obtained from patients undergoing liver resection at the University Hospital in Basel and Bern, Switzerland. Human hepatocytes as well as mouse hepatocytes from C57BL / 6 and C57BL / 6 Cldn3 - / - Mouse hepatocytes from mice were isolated from liver specimens according to a two-step enzyme perfusion protocol (doi: 10.1172 / JCI115207). The viability of the isolated hepatocytes was determined by trypan blue exclusion, and only preparations with a viability exceeding 90% were used. Hepatocytes were seeded onto tissue culture plastic coated with rat tail collagen in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, allowed to attach for 1 - 2 hours, and then washed twice with phosphate-buffered saline (PBS) to remove any remaining non-viable cells from the culture. Hepatocytes were cultured for 24 hours prior to use in arginine-free Williams E medium supplemented with insulin (0.015 IU / mL), hydrocortisone (5 μmol / L), penicillin (100 IU / mL), streptomycin (100 μg / mL), glutamine (2 mmol / L), and ornithine (0.4 mmol / L).

[0124] SNU-449 cells (ATCC#2234) were grown in RPMI1640 medium supplemented with 10% fetal bovine serum, 100 U / mL of penicillin, and 100 mg / mL of streptomycin. Hepa1-6 (ATCC#CRL-1830) was grown in DMEM supplemented with 10% fetal bovine serum, 100 U / mL of penicillin, and 100 mg / mL of streptomycin.

[0125] Flow cytometry for human Claudin 3 antibody testing The binding ability of the Cldn3 antibody was determined at 4°C for 30 minutes at 5.10 5Evaluated by serially diluting a stock solution of anti-claudin 3 antibody (0.5 mg / mL) in facs buffer exposed to individual human hepatocytes or SNU449 cells. The cells were then rinsed with PBS and exposed to cyanine 5 conjugate secondary anti-mouse IgG (Life technologies, #A10524) at 1:200 for 30 minutes at 4°C in the dark, followed by rinsing with PBS. Finally, cell data was acquired with a SORP LSRII (BD Pharmingen Inc., San Diego, CA). Flow cytometry analysis was performed using FlowJo software (Treestar, Inc, Ashland, OR).

[0126] Evaluation of the binding ability of the Cldn3 antibody To determine the binding ability of the anti-claudin 3 antibody, serial dilutions of the stock solution (0.5 mg / mL) were prepared in FACS buffer. The diluted antibody solution was then exposed to 5.10 5 individual human hepatocytes or SNU449 cells for 30 minutes at 4°C.

[0127] After the incubation period, the cells were rinsed with PBS to remove unbound antibody. Subsequently, secondary FITC anti-mouse IgG (reference) was applied to the cells at a dilution of 1:200. The secondary antibody was allowed to bind for 30 minutes at 4°C in the dark. The cells were then washed with PBS to remove any excess secondary antibody.

[0128] Cell data was acquired using a SORP LSRII flow cytometer (BD Pharmingen Inc., San Diego, CA). The acquired data was then analyzed for flow cytometry using FlowJo software (Treestar, Inc, Ashland, OR).

[0129] Functionalization of GalNAc-anti-Cldn3 antibody The antibody was first buffer-exchanged using a 7K Zeba size-exclusion column to effectively remove any unwanted buffer components and ensure compatibility with subsequent reactions.

[0130] To functionalize the antibody, it was reacted with 25 equivalents of DBCO-PEG4-NHS (BroadPharm #BP-22288). The antibody solution in PBS was incubated overnight at room temperature to allow for efficient conjugation of the antibody with DBCO-PEG4-NHS.

[0131] After the conjugation reaction, the functionalized antibody was purified using a PBS-equilibrated 7K Zeba size-exclusion column. This purification step served to separate the conjugated antibody from any unreacted DBCO-PEG4-NHS and other impurities, yielding a purified functionalized antibody preparation.

[0132] Next, the purified functionalized antibody was reacted with 100 equivalents of TreblerGalNAc-azide (primetech #0079). This reaction promoted the attachment of the galactose N-acetyl (GalNAc) moiety to the functionalized antibody, thereby enabling targeted delivery to the GalNAc receptor.

[0133] After the conjugation reaction with GalNAc-azide, the antibody was purified again, this time using a PBS-equilibrated 40K Zeba size-exclusion column. This purification step further separated the conjugated antibody from any unreacted GalNAc-azide and other remaining impurities, yielding a purified functionalized antibody that was available for downstream applications.

[0134] Cldn3 KD using GalNAc anti-Cldn3 antibody Human hepatocytes in a 12-well plate were treated with various concentrations of GalNAc-anti-Cldn3 antibody. Specifically, the cells were treated with antibodies at concentrations of 0 nM, 1 nM, 10 nM, and 100 nM for a period of 24 hours. After the 24-hour treatment period, proteins were extracted from the treated cells using 50 μL of RIPA lysis buffer.

[0135] The extracted protein was quantified using a Bio-Rad Protein assay system (Bio-Rad Laboratory, Melville, NY).

[0136] Western blot for testing the efficacy of human Claudin 3 inhibitory antibody The protein lysate was boiled in Laemmli buffer and run on a BIO-RAD Mini-Protean TGXTM (10 - 20% Ready Gel Tris-HCl gel system, 12-well comb #456 - 1095 and 10-well comb #456 - 1094) at 120 volts for 90 minutes, and transferred to a BIO-RAD Trans-Blot Turbo Mini or Midi PVDF membrane (Mini PVDF Transfer Packs #170-4156 and Midi PVDF Transfer Packs #170-4159) by semi-dry transfer (Trans-Blot Turbo Transfer System BIO-RAD). The protein was detected using the following primary antibodies: Claudin 3 (1:300) (Novus Biologicals, catalog number NBP1-35668) β-actin HRP-conjugated (1:5000) (Sigma-Aldrich, catalog number 2228, RRID; AB_476697). The antibodies were diluted with 5% milk and incubated overnight at 4°C. The following secondary antibody was used: HRP-conjugated anti-rabbit (1:2000) (Dako, #P0448, RRID: AB_2617138). The membrane was then washed and protein expression was analyzed by chemiluminescence (Western Lightning Plus-ECL Perkin Elmer) using a Fusion-Fx (Vilber).

[0137] Bile autofluorescence measurement Bile obtained from the gallbladder was diluted 1:50 with water. Autofluorescence was measured at an excitation of 489 nm / emission of 534 nm using a Tecan Spark® plate reader.

[0138] Bulk RNA sequencing of tissue samples Total RNA was extracted from the liver using NucleoZOL and quantified with a bioanalyzer. The sequencing run settings included TruSeq Stranded mRNA with paired-end reads. The read length was set to 50 and the multiplex level was 1.

[0139] RNA-seq alignment The fastq files were aligned to the mouse reference genome mm10 with hisat2 and converted to bam files using samTOOLS. The read count matrix was generated from the bam files using the featurecounts function of the R package Rsubread.

[0140] Dimensionality reduction Principal component analysis: The read count matrix was normalized to reads per million (RPM) and log-transformed (f(x) = (1 + log(x))). The principal components were calculated using the R function prcomp. The data was shown using ggplot2. Heatmap: The read count matrix was normalized to RPM. When comparing gene expression across various conditions, the inventors normalized all genes from 0 - 1 where f(x_i) = (x_i - min(x_i)) / (max(x_i) - min(x_i)), where max(x_i) is the maximum value of gene i across all conditions (similarly for min(x_i)). When comparing gene expression across cell types, the inventors normalized all cell types from 0 - 1 where f(x_i) = (x_i - min(x_i)) / (max(x_i) - min(x_i)), where max(x_i) is the maximum value of gene i across all conditions.

[0141] RNA-seq differential expression The optimally expressed genes were calculated using the R package DESeq2. Genes with p-values less than 0.05 adjusted by the false discovery rate were considered statistically significant for further analysis.

[0142] Enrichment analysis To determine the pathways associated with genes, the inventors used Metascape. For statistically significant genes, if more than 2000 were present in the list, the inventors adopted the top 2000 selected by fold change.

[0143] GalNAc siRNA construct and GalNAc siRNA injection The following siRNA sequences were prepared (synthetic nomenclature: n = 2’OMe-RNA, Nf = 2’-fluoro-RNA, s = phosphorothioate):

[0144] Claudin 3-targeting siRNA1: · Sense strand sequence (5’-3’): csusAfcCfaGfcAfgUfcGfaUfgAfaAf (SEQ ID NO: 1) · Antisense strand sequence (5’-3’): UfsUfsuCfaUfcGfaCfuGfcUfgGfuAfgsusu (SEQ ID NO: 5)

[0145] Claudin 3-targeting siRNA2: · Sense strand sequence (5’-3’): gsasAfaCfgGfgCfcAfuUfuCfaUfaAf (SEQ ID NO: 2) · Antisense strand sequence (5’-3’): UfsUfsaUfgAfaAfuGfgCfcCfgUfuUfcsusu (SEQ ID NO: 6)

[0146] AHSA1 control siRNA · Sense strand sequence (5’-3’): uscsUfcGfuGfgCfcUfuAfaUfgAfaAf (SEQ ID NO: 3)

[0147] The above siRNA was synthesized by Axolab, Kulmbach, Germany. All siRNAs were designed from the mouse Cldn3 mRNA sequence (NM_009902.4). The bases in the siRNA were modified using 2'-fluoro and / or 2'O-methyl residues and phosphorothioate linkages to enhance chemical stability. Claudin 3 target siRNA1 (sense strand sequence), Claudin 3 target siRNA2 (sense strand sequence), and AHSA1 control siRNA (sense strand sequence) were conjugated to a branched GalNAc cluster (CPG, Primetech), and the siRNA was purified by HPLC and lyophilized. For in vivo use, the GalNAc siRNA was diluted with saline. The siRNA was injected subcutaneously at a concentration of 10 mg / KG body weight. Mice were pre-treated with siRNA 2 or 4 days before the experiment.

[0148] Results Claudin 3 knockout results in disorders of bile acid metabolism, bile dilution, increased bile flow, and impaired uptake of the tracer FITC dextran.

[0149] Cldn3 + / + and Cldn3 - / - mouse 27 By analyzing the RNAseq dataset published by the inventors comparing with that of Cldn3 knockout mice, it was found that bile acid and bile salt metabolism are present in the top gene pathways that are expressed at lower levels in the absence of claudin 3 (Figure 1A). Differential gene expression analysis revealed that genes such as Cyp27a1, which has an important function in BA synthesis, were hardly expressed (Figure 1B). The inventors confirmed this result for Cyp27a1 and showed that the expression of Cyp7b1 in the liver was also low using RT-qPCR in Cldn3 + / + knockout mice (Figure 1C). The inventors questioned whether the suppressed BA synthesis was affecting the amount of total bile acids (TBA) in bile. The bile TBA value of Cldn3 + / + knockout mice averaged 4194 μmol / L, while that of Cldn3 - / -Mouse bile contained only 3932 μmol / L (P<0.01) (Figure 1D). Bile has been described as having autofluorescence properties that can be mostly attributed to the abundance of bilirubin. 28 We measured bile autofluorescence with 489 nm excitation and 534 nm emission. Cldn3 + / + Bile had 499.7 relative fluorescence units (RFU) / μL of bile / g of liver, but Cldn3 - / - Bile autofluorescence was only 14.4 RFU / μL of bile / g of liver and was surprisingly dramatically lower (P = 0.0079) (Figure 1E), indicating that bile is diluted with respect to bilirubin.

[0150] Next, we aimed to determine the bile flow, as well as the uptake and paracellular permeability of the blood-bile barrier in Cldn3 - / - mice. We used the experimental setup previously described 14 . Briefly, we performed bile duct ligation (BDL) and inserted a cannula into the gallbladder (Figure 1F). Bile was collected over a 20-minute period to determine the bile flow (Figure 1G). We found that the bile flow was very fast, at 3.7 μL / min / g of liver in Cldn3 + / + mice compared to only 0.8 μL / min / g of liver in Cldn3 - / - mice (P = 0.0036) (Figure 1G).

[0151] In the same setup, we tested the paracellular barrier by intravenous (IV) injection of the fluorescent tracer FITC-dextran and then collecting bile at 3-minute intervals (Figure 1H). We found that FITC-dextran was gradually taken up and secreted into bile starting at 3 minutes, but the abundance of FITC-dextran was consistently low in Cldn3 - / - mice (Figure 1H). This indicates that the hepatocyte paracellular barrier is not primarily impaired for FITC-dextran passage.

[0152] Next, the present inventors investigated Cldn3 - / - We further verified our hypothesis that FITC-dextran (40 kDa) uptake is impaired in mice. We performed BDL and injected FITC-dextran (40 kDa, 6.25 mg / mL, 300 μL volume) via the tail vein. In anesthetized mice undergoing laparotomy, we continuously imaged FITC-dextran fluorescence for 45 min using an IVIS® Spectrum in vivo imaging system (PerkinElmer). To analyze FITC-dextran intensity in the liver, we selected regions of interest of the same area size and determined the FITC signal using the instrument's software. 20 min after injection, Cldn3 showed a lower intensity FITC signal. - / - Quantification of the FITC signal over time demonstrated that FITC-dextran upregulates Cldn3. + / + It is rapidly taken up by the liver, but Cldn3 - / - We showed that Cldn3 was not taken up by the liver (Figure 1J). Using the same experimental setup, livers were harvested 20 min after injection and 25 μm thick cryosections were prepared. Cryosections were stained with Dapi for 15 min and then confocal Z-stack imaging was performed (LSM710, Zeiss, 20x objective, Oberkochen, Germany). Quantification of FITC fluorescence across the Z-stack revealed that Cldn3 - / - The liver showed significantly less FITC-dextran uptake 20 min after injection (P=0.0049, n=3 / 4, Student's t test).

[0153] Taken together, our data indicate that loss of claudin 3 impairs BA synthesis, resulting in bile that is diluted with total bile acids and bilirubin. - / - Bile flow was faster in mice, and intrahepatocyte paracellular permeability was not impaired for the passage of FITC-dextran, except for Cldn3. - / - The liver showed decreased uptake and bile / or secretion of FITC-dextran (40 kDa).

[0154] Protection from obstructive cholestatic liver injury by the absence of Claudin 3 The fact that bile acids are considered a major cause of the pathophysiology of cholestatic liver diseases 29 led the inventors to subsequently suspect the results of bile dilution of Cldn3 in cholestasis. The inventors first applied an extrahepatic obstructive cholestasis model. Cldn3 - / - mice and Cldn3 + / + mice were subjected to BDL for either 2 days, 7 days or 12 days. Macroscopic photographs showed no difference as expected in the control group. However, after BDL, the inventors observed a significant difference in the liver appearance. Wild-type animals exhibited the expected BDL phenotype, including dark green bile and visible necrotic spots in the liver tissue (Figure 2A). These features were absent in the Cldn3 - / - group, indicating a different bile composition and a significant recovery of necrosis. - / - Histological analysis of liver tissue showed no difference in the control group with normal liver morphology (Figure 2B). After BDL, wild-type animals developed tissue necrosis as a typical feature of obstructive liver injury (Figure 2B and Figure 2C), with increasing severity as cholestasis progressed. Notably, mice lacking Claudin 3 showed little necrosis (Figure 2B and Figure 2C). Therefore, the values of clinical liver injury markers were significantly lower in the absence of Claudin 3. Seven days after BDL, the alanine aminotransferase (ALT) value was only 176 U / L in Cldn3

[0155] mice, compared to an average of 702 U / L in Cldn3 - / - mice (Figure 2D). The Aspartate transaminase (AST) value was 882 U / L in Cldn3 + / + mice 7 days after BDL, and 25 + / + mice in Cldn3 - / -In mice, it was only 242 U / L (Figure 2E). The alkaline phosphatase (ALP) value was significantly different 12 days after BDL, and in Cldn3 + / + mice, it was on average 822.5 U / L, and in Cldn3 - / - mice, it was 452.0 U / L (Figure 2F).

[0156] Bacterial translocation to the liver, an indicator of tissue damage, has been previously described to occur upon BDL 16 . The inventors seeded liver homogenates after BDL and counted the number of colony-forming units (CFU). Quantification showed that the number of bacteria translocated to the liver was significantly lower in Cldn3 - / - livers 2 days after BDL compared to wild-type livers (P = 0.01) (Figure 2G). Seven days after BDL, the amount of CFU was similar between groups.

[0157] As a final indicator of inflammation and injury, the inventors used fluorescence-activated cell sorting (FACS) to quantify the frequency of immune cell appearance 7 days after BDL. The inventors found that the frequency of inflammatory monocytes was significantly lower, while the frequency of cytoprotective and inflammation-resolving T cells was high in cholestatic Cldn3 - / - livers (Figure 2H). In Cldn3 - / - livers, there was also a tendency for a lower frequency of neutrophils. Other analyzed populations, including B cells and Kupffer cells, were not different.

[0158] In summary, the inventors' results show that loss of claudin 3 results in a marked recovery of inflammation and necrosis, protecting the mouse liver from injury in this obstructive cholestasis model.

[0159] Decrease in bile acid concentration in the liver and gallbladder bile after BDL Next, the inventors further investigated the mechanism that causes the protective effect of Claudin-3 loss. The composition of bile acids changed slightly in Cldn3 - / - mice 27、30 under normal conditions. The inventors hypothesized that the recovery of injury could be explained by a significant decrease in bile acid levels. Therefore, the inventors analyzed the concentration and composition of liver bile acids in the BDL model.

[0160] Seven days after BDL, the bile collected from the gallbladders of Cldn3 + / + mice was yellow or dark green, while the bile of Cldn3 - / - was yellow (Figure 3A, Cldn3 - / - bile appears darker in the grayscale image). The TBA value in the gallbladder bile collected 7 days after BDL was 3435 μmol / L in Cldn3 + / + mice and 2611 μmol / L in Cldn3 - / - mice (P < 0.01) (Figure 3B). The liver TBA at 2 days after BDL was on average 308.3 nmol / g in Cldn3 + / + mice and only 191.8 nmol / g in the liver of Cldn3 - / - (p < 0.01) (Figure 3C). Together with a higher amount of liver injury (Figure 2), the liver TBA value increased after 7 days and was significantly lower at 447.2 nmol / g on average in Cldn3 + / + mice and 226.2 nmol / g in Cldn3 - / - mice (p < 0.05) (Figure 3C).

[0161] Interestingly, the difference in TBA values was reversed when serum was tested (Figure 3D). Two days after BDL, the average serum TBA value was 109.1 μmol / L in Cldn3 + / + mice and 125.6 μmol / L in Cldn3 - / - mice (p < 0.05) (Figure 3D). The serum TBA value increased on the 7th day and the difference between groups became larger. After 7 days, Cldn3 + / + serum contained 689.2 μmol / L, while Cldn3 - / -The serum contained significantly more TBA at 774.1 μmol / L (p < 0.05) (Figure 3D). Notably, there was no significant difference in liver, serum, or bile TBA at day 12 (Figures 3B - 3D). The inventors further analyzed the serum by checking bilirubin levels, and Cldn3 - / - decreased in mice 2 days after BDL but not at other time points (Figure 3E).

[0162] The inventors also checked for a potential negative effect of a slight increase in serum bile acid levels in the kidneys of Cldn3 - / - mice. The inventors quantified the number of biliary plugs (also referred to as "bile cylinders") in the kidneys and found no difference between Cldn3 + / + and Cldn3 - / - mice 7 days after BDL (Figure 3F).

[0163] Overall, the data demonstrate that claudin 3 - deficient animals have diluted bile even after induction of cholestasis. The inventors found significantly lower total bile acid values in the liver and gallbladder but higher values in the serum. The reduction of the hepatobiliary system from bile acid - related cytotoxicity could potentially explain the recovery observed in liver injury.

[0164] Claudin 3 deletion does not mainly change the composition of the bile acid pool under normal or cholestatic conditions During cholestasis, the increased hydrophobicity of bile can potentially contribute significantly to tissue damage depending on the composition of bile with individual bile acid (BA) subtypes. The inventors questioned whether claudin 3-deficient mice have different BA pools regardless of the presence or absence of cholestasis. Therefore, the inventors used liquid chromatography-mass spectrometry (LC-MS / MS) to determine the concentrations of individual BA types in the liver and serum 7 days after BDL in control animals (Figure 4 and Table 1). The inventors observed that conjugated muricholic acid T-αMCA, T-βMCA, T-ωMCA, and tauro-cholic acid (TCA) were the bile acid species present in the largest amounts by far in the liver bile acid pool (Figure 4A-4C and Table 1). By reporting the amount of bile acid as nanograms / milligram of liver tissue, it was shown that liver bile acid values increased dramatically 7 days after BDL (Figure 4A). By LC-MS / MS analysis, the inventors further confirmed their previous observation that liver bile acid values decreased in the liver 7 days after BDL (Figure 3C and Figure 4A). - / - The inventors' previous observation that liver bile acid values decreased in the liver was further confirmed (Figure 3C and Figure 4A).

[0165] The inventors next calculated the percentage of bile acid subtypes in the total liver bile acid pool. When comparing the bile acid subtypes of Cldn3 - / - with Cldn3 + / + , there were slightly small differences such as a slightly lower percentage of T-αMCA in the control liver tissue of Cldn3 - / - mice (Figure 4B and Table 1). Seven days after BDL, there was no difference in the liver bile acid pool composition between Cldn3 + / + and Cldn3 - / - . The data showed that the diversity of the total liver bile acid pool decreased favorably for the main bile acid subtypes when comparing control with 7 days after BDL (Figure 4B and Figure 4C and Table 1).

[0166] Next, the inventors analyzed serum bile acids. Quantification of bile acids in μmol / L showed that Cldn3 7 days after BDL- / - It showed significantly higher values in the serum (Figure 4D), confirming the inventors' previous results (Figure 3D). The calculation of the percentage of bile acid subtypes from the total bile acid pool was performed for Cldn3 + / + and Cldn3 - / - and revealed no difference when compared to control serum (Figure 4E and Table 1). However, 7 days after BDL, the inventors observed slight differences in Cldn3− / − serum, such as a slightly higher percentage for T-ωMCA and slightly lower percentages for GCA, αMCA, and CDCA (Figure 4F and Table 1). The diversity of the serum bile composition decreased 7 days after BDL when compared to the control (Figure 4E and Figure 4F). The inventors observed that the diversity of the serum bile composition changed favorably to TCA and T-βMCA, as previously confirmed in liver tissue (Figure 4B and Figure 4C).

[0167] In summary, the analysis of liver and plasma bile acids confirmed the previous finding that Cldn3 - / - mice decreased liver bile acids and increased plasma bile acid levels. The inventors were unable to find a significant difference in the bile acid pool composition when comparing Cldn3 + / + and Cldn3 - / - liver or serum.

[0168]

Table 3

[0169] Biliary stasis Cldn3 - / - Lower expression of BA synthesis and importer genes in the liver Cldn3 - / - The observation that bile was diluted and the BAs contained before and after BDL decreased raised the question of whether BA synthesis and / or transport was altered. The inventors analyzed Cldn3 + / + and Cldn3 - / -Sequenced the transcriptome of liver tissue and performed RT-qPCR on important genes to answer this question. Differential gene expression analysis showed that there were 168 up-regulated genes and 255 down-regulated genes in the liver (P<0.05) (Figure 5A). - / - It was shown that there were 168 up-regulated genes and 255 down-regulated genes in the liver (P<0.05) (Figure 5A).

[0170] The inventors first performed Metascape analysis and obtained an overview of the most significantly different gene pathways when comparing Cldn3 - / - and Cldn3 + / + . (Tables 3 and 4). (Figure 5A). The pathways that were more highly expressed in the Cldn3 - / - liver were metabolic-related pathways including amino acid catabolism, fatty acid transport, and carnitine metabolism. Interestingly, the bile stasis-protective PPAR signaling pathway 2、11、13 was also highly expressed in the Cldn3 - / - liver (Figure 3). After confirming less tissue damage and immune cell infiltration (Figure 2B - Figure 2H), the inventors found that several gene pathways were related to inflammation and immune responses that were significantly less expressed in the Cldn3 - / - liver after BDL. This induced leukocyte migration and adhesion, biosynthesis of reactive oxygen species, biosynthesis of tumor necrosis factor, and neutrophil degranulation, which were expressed lower in the absence of claudin 3 (Table 4). Importantly, the analysis showed that bile acid and bile salt metabolism was one of the upper pathways that were less expressed in Cldn3 - / - mice (Table 4). Important bile synthesis genes with low expression included Cyp7b1, Cyp27a1, Cyp3a11, Akr1b7, Akr1c6, and others (Figure 5A). The inventors confirmed this finding by qPCR for Cyp7b1 and Cyp27a1, and they were significantly less expressed in the Cldn3 - / - liver at the control and 2 days after BDL (Figure 5B). Only Cyp7a1 showed a tendency to be lowly expressed 2 days after BDL. Cholesterol, as a precursor of bile acids, did not differ in blood levels in Cldn3 - / - mice (Figure 5C).

[0171]

Table 4

[0172]

Table 5

[0173] The inventors also confirmed the expression of BA transporters. As expected, the expression of the basolateral BA importers Oatp1a1, Oatp1b2, and Ntcp was downregulated in response to BDL in wild-type animals, but Oatp1a1 / Oatp1b2 was significantly more decreased in the liver 2 days after BDL (Figure 5D). The basolateral BA exporter Ost1-β was more highly expressed in the liver than in the control Cldn3 - / - in accordance with our previous findings. - / - Ost1-β expression was strongly upregulated in response to BDL, which was the same in Cldn3 27 and Cldn3 + / + liver (Figure 5E). The apical anion and bile acid exporter Mpr3 was slightly upregulated 7 days after BDL in Cldn3 - / - but not in Cldn3 + / + liver (Figure 5E). Finally, the apical bile duct phospholipid exporter Mdr2 was not significantly regulated overall after BDL (Figure 5E). - / - Overall, gene expression analysis confirmed a decrease in inflammation and showed that BA synthesis was less expressed. Consistent with our observations on the uptake of tracers of impaired bile acid size (Figures 1H–1L), we found impairment of the expression of bile acid importers in Cldn3

[0174] liver. - / - Cldn3

[0175] Cldn3 - / - mice increased periportal edema but did not change in the tubule reaction. Cldn3 - / - To understand the recovery of cholestatic injury in mice, the inventors described in more detail the tissue morphology of wild-type and claudin 3-deficient mice after BDL. Claudin 3 is highly expressed in cholangiocytes 27 and the loss of claudin 3 has been previously associated with disruption of the paracellular barrier to water 30、31 Therefore, the inventors scored hematoxylin and eosin-stained liver sections for edema in the periportal zone. Cldn3 + / + The mean periportal edema score in animals was 2.8 compared to 6 at 2 days after BDL in Cldn3 - / - (P < 0.05) (Figures 6A, 6B). The inventors also observed high ductal edema in knockout mice at day 7. The edema score was highest at 12 days after BDL but did not differ significantly between groups

[0176] In summary, histological analysis showed that Cldn3 - / - increased periportal edema after BDL as a possible result of a leaky water barrier in the liver

[0177] Cldn12 - / - Animals were not protected from cholestatic liver injury Next, the inventors questioned whether cholestatic protection could also be obtained by knocking out another claudin family tight junction protein. The inventors have recently described the cell-type specific expression of liver tight junction genes and found that Cldn12 is expressed in mouse hepatocytes 27 . Therefore, the inventors subjected C57BL / 6 mice with global claudin 12 loss to BDL. Cldn12 + / + and Cldn12 - / - livers also developed similarly dilated and darkly stained gallbladders 2 days after BDL (Figure 7A). Quantification of the necrotic tissue area showed that both groups had similar levels of tissue necrosis (Figures 7A, 7B). Finally, liver concentration or serum TBA was Cldn12- / - There was no change in mice (FIGS. 7C and 7D). These results indicate that the loss of Claudin 12 does not cause a change in bile acid levels and does not restore cholestatic liver injury.

[0178] Protection from intrahepatic cholestasis by loss of Claudin 3 The expected results from the BDL model raised the question of whether Claudin 3 is also beneficial in intrahepatic cholestatic diseases. Therefore, the inventors challenged Cldn3 mice with an established model of oral administration of α-naphthylisothiocyanate (ANIT) - / - and challenged the mice 32 .

[0179] First, the inventors tested an acute intrahepatic cholestasis model. The inventors challenged mice with a single dose of ANIT [60 mg / KG body weight] and analyzed the tissues 2 days later (FIG. 8A). In wild-type mice, a predicted phenotype of cholestatic liver injury was induced, with macroscopic and microscopically visible tissue necrosis (FIGS. 8B - 8D), high levels of clinical liver injury markers (FIG. 8E), and elevated bilirubin and bile acid levels in the tissues and serum (FIGS. 8E and 8F). Notably, there was no tissue necrosis in the liver of Cldn3 - / - mice (FIGS. 8B - 8D). The bile contained in the gallbladder was normally light yellow (appearing brighter / whiter in the grayscale figure) in Cldn3 - / - mice. This was in contrast to the dark green color in ANIT-challenged Cldn3 + / + (FIG. 8B, appearing darker / blacker in the grayscale figure). Serum ALT, ALP, and bilirubin levels were significantly lower (P < 0.05) in Cldn3 - / - animals, and there was also a similar tendency for AST levels to be lower (FIG. 8E). Evidence of the absence of cholestatic injury was also a significantly lower BA value. The total BA in the liver was on average 188 nmol / g in Cldn3 + / + but was - / -It was only 87 nmol / g in the liver (P < 0.001) (Figure 8F). The difference in serum BA levels was also prominent. In Cldn3 + / + it was 412 μmol / L on average, but in Cldn3 - / - mice it was only 46 μmol / L (P < 0.0001) (Figure 8F).

[0180] The inventors questioned whether ANIT was similarly metabolized by Cldn3 + / + and Cldn3 - / - in the liver. Therefore, the expression of the genes glutathione S-transferase alpha 1 (Gsta1) and Cyp4a12, which are involved in glutathione metabolism and drug processing, was confirmed. As expected, Gsta1 was upregulated 2 days after ANIT, but in Cldn3 - / - the liver it was hardly upregulated (Figure 8G). Cyp4a14 was similarly downregulated in both groups in response to ANIT. When presenting negative feedback against cholestasis, the important BA synthase Cyp7b1 was downregulated as expected in Cldn3 + / + mice. However, this was not the case when claudin 3 was absent, indicating that less bile acids induced feedback and showed protection from cholestasis (Figure 8G). The loss of claudin 3 was also investigated to confirm whether it protects from fibrosis that can be induced by long-term intrahepatic cholestasis. The inventors subjected mice to two subsequent doses over a total period of 10 days (Figure 8H). Quantification of the collagen intensity of Masson's trichrome-stained liver sections showed that Cldn3 - / - liver contained significantly less collagen than wild-type mice (Figures 8I and 8J).

[0181] Finally, a model in which 0.1% ANIT was fed ad libitum for 4 weeks was used to test the protective effect of Claudin 3 loss in chronic intrahepatic cholestasis (Figure 8K). Mice in the control group were given a solid diet containing the same volume of corn oil instead. As expected, mice in the control group did not develop any tissue necrosis, and clinical injury markers and bilirubin / cholesterol values remained normal (Figures 8L - 8O). Four weeks after ANIT feeding, Cldn3 + / + mice developed clear signs of ductular reaction and tissue necrosis, which was not the case in Cldn3 - / - tissues (Figure 8L). Quantification of tissue necrosis showed that the liver had a total necrotic area of 0.24% on average, while Cldn3 + / + liver had only 0.066% (P < 0.001) (Figure 8M). The ALP value as a clinical marker of cholestatic liver injury was 448 U / L in ANIT-fed Cldn3 - / - mice, but only 211 U / L in the corresponding Cldn3 + / + group (P < 0.01) (Figure 8N). AST and ALT values did not differ between ANIT-fed groups (Figure 8N). Bilirubin values did not differ, but cholesterol values were significantly lower in Cldn3 - / - serum (P < 0.05) (Figure 8O). - / - Next, the inventors quantified the amount of fibrotic liver tissue by staining with Sirius red. Mice fed corn oil did not show fibrosis as indicated by minimal Sirius red-stained tissue (Figure 8P). Cldn3

[0182] mice fed 0.1% ANIT for 4 weeks developed fibrosis as indicated by extensive collagen deposition in Sirius red-stained sections, which was not the case in Cldn3 + / + mice (Figure 8P). Quantification of the Sirius red signal (Figure 8Q) showed that, compared with ANIT-fed Cldn3 - / - mice, ANIT-fed Cldn3 - / - mice had - / -It was confirmed that the mice did not develop any liver fibrosis as indicated by a significantly low Sirius red signal (P = 0.0018, Student's t-test).

[0183] In summary, experiments using acute and chronic intrahepatic cholestasis models show that Cldn3 - / - is protected from cholestatic liver injury and fibrosis associated with cholestasis.

[0184] GalNAc siRNA targeting of Claudin 3 achieves a modest recovery of cholestatic injury The promising results obtained by the present inventors in Cldn3 - / - mice suggest that targeting of Claudin 3 may be beneficial for cholestasis therapy. To prove this concept, the present inventors used GalNAc siRNA to knockdown (KD) Claudin 3. The advantages of this technology are to generate stable KD of the target, to be subcutaneously administrable, to target only hepatocytes, and to be already used clinically 33 are. The present inventors first screened 12 candidate siRNAs in vitro using primary mouse hepatocytes. An siRNA (SEQ ID NO: 3, sense strand) targeting an activator of heat shock protein ATPase 1 (AHSA1) was used as a negative control in subsequent experiments. Western blot showed that candidate 9 and candidate 12 produced strong KD of Claudin 3 when the cells were treated at a concentration of 50 nmol. Therefore, the present inventors continued to use candidate 9 (hereinafter referred to as "siRNA1"; SEQ ID NO: 1, sense strand) and candidate 12 (hereinafter referred to as "siRNA2"; SEQ ID NO: 2, sense strand) in an obstructive cholestasis model using the BDL technique.

[0185] C57BL / 6 wild-type mice were subcutaneously injected with GalNAc siRNA1 or siRNA2 at 10 mg / kg body weight. Two days after the injection, BDL was performed. Then, two days after the surgery, tissues were collected and analyzed for claudin 3 values and markers of cholestatic liver injury (Figure 9A). The in vivo KD efficacy of siRNA for claudin 3 was approximately 40 - 50% in terms of mRNA and protein values, but there were considerable individual differences in efficacy (Figure 9B and Figure 9C). Nevertheless, KD of claudin 3 resulted in significantly decreased ALP (Figure 9D and Table 5). The ALP value of control mice with BDL averaged 1582 U / L, while the averages of siRNA1- or siRNA2-receiving animals were 713 U / L and 866 U / L, respectively (P < 0.01 and P < 0.05, respectively) (Figure 9D and Table 5). Similarly, ALT and AST values were decreased compared to the control group (Figure 9D and Table 5). Recovery of tissue injury in the claudin 3 knockdown group was confirmed by representative H&E-stained liver tissue sections, but the effect was not very prominent and the variability among individuals remained high (Figure 9E).

[0186]

Table 6

[0187] Next, the inventors tested siRNA1, the most promising siRNA candidate, in an intrahepatic cholestasis model using ANIT. The inventors subcutaneously injected either control siRNA AHSA1 or claudin 3-targeting siRNA1 [60 mg / kg body weight] four days before oral gavage of ANIT (Figure 9F). Two days after ANIT gavage, tissues and sera were collected to quantify liver injury. The inventors found that the main clinical blood marker of cholestasis, i.e., the ALP value, was significantly lower in animals treated with anti-claudin 3 siRNA1 (Figure 9G and Table 6). Therefore, histology of liver tissue showed less necrosis in animals that received anti-claudin 3 siRNA1 compared to the AHSA1 control siRNA (Figure 9H).

[0188]

Table 7

[0189] In summary, the inventors have shown that recovery of extrahepatic or intrahepatic cholestatic liver injury occurred by targeting Claudin 3 with GalNAc siRNA knockdown.

[0190] Screening, synthesis and evaluation of siRNA targeting human Claudin 3 As a first step to translate these findings to human patients, the inventors next screened for siRNA sequences that efficiently knockdown human Claudin 3. In a bioinformatics pre-selection (Axolabs, Kulmbach, Germany), siRNAs were scored for target specificity, intra- and inter-species cross-reactivity and activity. The best siRNA candidates were then synthesized (Axolabs, Kulmbach, Germany). The siRNA sequences contained either 2'-fluoro or 2'O-methyl modified chemically stabilized additions at the positions shown in Table 6, or phosphorothioate linkers.

[0191] Next, the synthesized siRNA candidates were evaluated for efficiency to knockdown human Claudin 3 in cultures of human liver cell line PLC (ATCC catalog number CRL-8024). Human cell cultures were treated with siRNA at a final concentration of 50 nM and RNA was extracted 2 days after treatment. Finally, RT-qPCR was used to quantify the amount of residual human Claudin 3 mRNA after siRNA treatment. Table 6 lists all human siRNA sequences that knockdown more than 50% of human Claudin 3 mRNA. Table 7 shows the same identified human Claudin 3 siRNA sequences without chemically stabilizing modifications.

[0192]

Table 8

[0193]

Table 9

[0194] The experiment was conducted twice and equivalent results were obtained. In summary, the inventors identified 34 siRNA sequences that knockdown human Claudin 3 with more than 50% efficacy. The siRNA that works best is further used here for confirmation and risk removal studies after a typical drug development process.

[0195] Depletion of human Claudin 3 protein using monoclonal antibodies To demonstrate that human Claudin 3 inhibition can be achieved with another pharmaceutical modality, the inventors generated a human Claudin 3 antibody (light chain as shown in SEQ ID NO: 169 and heavy chain as shown in SEQ ID NO: 170) that binds to GalNAc and functionalized it. The inventors' Claudin 3 antibody efficiently bound to human Claudin 3 in liver cell lines and inhibited the target in a dose-dependent manner. This is described in more detail below.

[0196] Binding of anti-Claudin 3 antibody UB-VS003 to human and mouse cells Figure 10 represents the binding analysis of anti-Claudin 3 antibody UB-VS003 to human SNU449 cells and hepatocytes, and mouse Hepa1-6 cells. The results demonstrate a consistent trend in antibody binding for all cell types.

[0197] For human SNU449 cells and hepatocytes, the control sample showed no detectable binding (0%). However, a progressive increase in binding was observed as the antibody concentration increased. At a dilution of 1 / 400, the binding percentage was approximately 3%. This increased to 7% at a dilution of 1 / 200, 12% at 1 / 100, and reached a maximum of 25% at a 1 / 50 dilution.

[0198] In the case of mouse Hepa1-6 cells, no binding was observed in the control sample or at a 1 / 400 dilution. However, a significant increase in binding was observed with increasing antibody concentration. At a 1 / 200 dilution, approximately 5% of the cells showed binding, which increased to 7% at a 1 / 100 dilution and up to 10% at a 1 / 50 dilution.

[0199] Furthermore, a comparison was made between wild-type (WT) mouse hepatocytes and claudin 3 knockout (KO). The background signal (without antibody) was similar between WT and KO hepatocytes, accounting for approximately 2 / 3% of the total cells. When the anti-claudin 3 antibody was added, the background signal increased to 3% in KO hepatocytes, while in WT hepatocytes, the signal increased to 10%.

[0200] Overall, these results demonstrate specific binding of the anti-claudin 3 antibody to both human and mouse cells with a dose-dependent increase in binding. In addition, the comparison between WT and KO hepatocytes suggests claudin 3-dependent binding of the UB-VS003 antibody.

[0201] GalNAc-functionalized UB-VS003 induces claudin 3 knockdown. Figure 11 shows Western blot analysis representing the expression levels of claudin 3 and β-actin as a loading control. The ratio of claudin 3 to β-actin was quantified to evaluate the relative abundance of the claudin 3 protein.

[0202] In the control sample, the ratio of claudin 3 to β-actin was set at 100%, indicating that claudin 3 was fully expressed under normal conditions. After treatment with GalNAc anti-claudin 3 antibody for 24 hours, a dose-dependent decrease in the claudin 3 / β-actin ratio was observed, reflecting a decrease in the claudin 3 protein value.

[0203] At a concentration of 1 nM of the GalNAc anti-claudin 3 antibody, the claudin 3 / β-actin ratio decreased by approximately 45% compared to the control sample. This decrease became more pronounced as the antibody concentration further increased. At 30 nM, the ratio decreased to approximately 30%, and at 100 nM, the maximum concentration tested, the ratio decreased to approximately 10%.

[0204] These findings demonstrate that the addition of the GalNAc anti-claudin 3 antibody resulted in a dose-dependent decrease in the expression level of claudin 3 protein. This suggests that the functionalized antibody efficiently induced the lysosomal degradation of claudin 3 in human hepatocytes.

[0205] In summary, human claudin 3 GalNAc siRNA and the human claudin 3 GalNAc antibody UB-VS003 could be efficiently used to deplete human claudin 3. Both modalities are currently being further used in preclinical and clinical studies for the purpose of treating cholestasis and / or fibrosis associated with cholestasis.

[0206] References:

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Claims

Claim 1 An agent that inhibits the expression and / or activity of Claudin 3 for use in a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis. Claim 2 The agent for use according to claim 1, wherein the agent that inhibits the expression and / or activity of Claudin 3 is an siRNA targeting Claudin 3 or an antibody or fragment thereof that binds to Claudin 3. Claim 3 The agent for use according to claim 1, wherein the agent inhibits the expression of Claudin 3. Claim 4 The agent for use according to claim 3, wherein the agent that inhibits the expression of Claudin 3 is a nucleic acid targeting the gene or mRNA encoding Claudin 3. Claim 5 The agent for use according to claim 3, wherein the agent that inhibits the expression of Claudin 3 is an siRNA targeting Claudin 3. Claim 6 The agent for use according to claim 2 or 5, wherein the siRNA targeting Claudin 3 is bound to a compound that promotes delivery of the siRNA to the liver. Claim 7 The agent for use according to claim 2 or 5, wherein the siRNA targeting Claudin 3 is an N-acetylgalactosamine (GalNAc) siRNA complex. Claim 8 The agent for use according to any one of claims 5 to 7, wherein the siRNA is an siRNA comprising a sequence shown in any of SEQ ID NOs: 1, 2, 5, 6, or 33 to 168, or an siRNA consisting of an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99%, or 100% identical to an siRNA comprising a sequence shown in any of SEQ ID NOs: 1, 2, 5, 6, or 33 to 168, and is selected from the group consisting of such siRNAs. Claim 9 The agent for use according to any one of claims 5 to 7, wherein the siRNA is an siRNA comprising a sequence shown in any of SEQ ID NOs: 33 to 168, or an siRNA consisting of an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99%, or 100% identical to an siRNA comprising a sequence shown in any of SEQ ID NOs: 33 to 168, and is selected from the group consisting of such siRNAs. Claim 10 The substance for use according to any one of claims 5 to 7, wherein the siRNA is an siRNA comprising a sequence shown in any of SEQ ID NOs: 33 to 100, or an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to an siRNA comprising a sequence shown in any of SEQ ID NOs: 33 to 100, and is selected from the group consisting of such siRNAs.

11. The substance for use according to any one of claims 5 to 7, wherein the siRNA is an siRNA comprising the sequence shown in SEQ ID NO: 1, an siRNA comprising the sequence shown in SEQ ID NO: 2, an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence shown in SEQ ID NO: 1, and an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence shown in SEQ ID NO: 2, and is selected from the group consisting of such siRNAs.

12. The substance for use according to claim 1, wherein the substance inhibits the activity of claudin 3.

13. The substance for use according to claim 12, wherein the substance that inhibits the activity of claudin 3 is selected from the group consisting of an antibody or a fragment thereof that binds to claudin 3 and a toxin that binds to claudin 3.

14. The substance for use according to claim 13, wherein the toxin that binds to claudin 3 is Clostridium perfringens enterotoxin (CPE) or a fragment or variant thereof.

15. The substance for use according to claim 12, wherein the substance that inhibits the activity of claudin 3 is an antibody or a fragment thereof that binds to claudin 3.

16. The substance for use according to claim 15, wherein the antibody or fragment thereof that binds to claudin 3 comprises a light chain comprising the amino acid sequence shown in SEQ ID NO: 169 and / or a heavy chain comprising the amino acid sequence shown in SEQ ID NO:

170.

17. The substance for use according to claim 15 or 16, wherein the antibody or fragment thereof that binds to claudin 3 is bound to a compound that promotes delivery of the antibody to the liver.

18. An agent for use according to claim 15 or 16, wherein the antibody or fragment thereof that binds to Claudin 3 is an N-acetylgalactosamine (GalNAc) - antibody complex.

19. An agent for use according to any one of claims 1 to 18, wherein the cholestasis is a disease selected from the group consisting of obstructive cholestasis, non - obstructive cholestasis, bile duct diseases, and defects in bile duct function.

20. A composition for use in a method for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis, comprising an agent according to any one of claims 1 to 18 and optionally a pharmaceutically acceptable carrier.

21. A pharmaceutical formulation for preventing, delaying the progression of, or treating cholestasis and / or fibrosis associated with cholestasis, comprising an agent according to any one of claims 1 to 18 or a composition comprising the agent according to claim 20, and optionally a pharmaceutically acceptable carrier.

22. An siRNA targeting Claudin 3.

23. An siRNA targeting Claudin 3, selected from the group consisting of an siRNA comprising a sequence shown in any of SEQ ID NO: 1, 2, 5, 6, or 33 - 168, or an siRNA consisting of an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99%, or 100% identical to an siRNA comprising a sequence shown in any of SEQ ID NO: 1, 2, 5, 6, or 33 - 168.

24. An siRNA targeting Claudin 3, selected from the group consisting of an siRNA comprising a sequence shown in any of SEQ ID NO: 33 - 168, or an siRNA consisting of an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99%, or 100% identical to an siRNA comprising a sequence shown in any of SEQ ID NO: 33 - 168.

25. An siRNA targeting Claudin 3, selected from the group consisting of an siRNA comprising a sequence shown in any of SEQ ID NO: 33 - 100, or an siRNA consisting of an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99%, or 100% identical to an siRNA comprising a sequence shown in any of SEQ ID NO: 33 - 100.

26. An siRNA targeting Claudin 3, selected from the group consisting of an siRNA comprising the sequence shown in SEQ ID NO: 1, an siRNA comprising the sequence shown in SEQ ID NO: 2, an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence shown in SEQ ID NO: 1, and an siRNA that is at least 95% identical, more preferably 96%, 97%, 98%, 99% or 100% identical to the siRNA comprising the sequence shown in SEQ ID NO:

2.

27. An antibody or a fragment thereof that binds to Claudin 3.

28. The antibody or fragment thereof according to claim 27, wherein the antibody or fragment thereof that binds to Claudin 3 is a human antibody.

29. The antibody or fragment thereof according to claim 27 or 28, wherein the antibody or fragment thereof that binds to Claudin 3 comprises a light chain comprising the amino acid sequence shown in SEQ ID NO: 169 and / or a heavy chain comprising the amino acid sequence shown in SEQ ID NO:

170.

30. The antibody or fragment thereof according to any one of claims 27 to 29, wherein the antibody or fragment thereof that binds to Claudin 3 is bound to a compound that promotes delivery of the antibody to the liver.

31. The antibody or fragment thereof according to any one of claims 27 to 29, wherein the antibody or fragment thereof that binds to Claudin 3 is an N-acetylgalactosamine (GalNAc) - antibody conjugate.

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